Ligand conjugates and their applications

Abstract

A ligand conjugate, a kit comprising the ligand conjugate, and a method for treating a disease that would benefit from T-effector cell stimulation.

Description

Technical Field

[0001] The present application relates to the fields of cancer treatment and immunomodulation. More specifically, the present application relates to a ligand conjugate, a kit comprising the ligand conjugate, and a method for treating a disease that can benefit from T-effector cell stimulation. Background Art

[0002] OX40 (also known as "CD134" or "TNFRSF4") is an important T cell co-stimulatory molecule and a member of the tumor necrosis factor receptor (TNF) superfamily. The ligand OX40L (also known as CD252) of OX40 usually binds to three OX40 proteins in the form of a trimer to form a hexameric complex, thereby activating downstream signaling pathways such as NF-κB, PI3K, and AKT. Continuous activation of these downstream signals can stimulate the production of cytokines, thereby enhancing the killing ability of effector T cells. A considerable number of anti-OX40 antibodies have been developed for the treatment of cancer and immune regulation.

[0003] TLR9 (Toll-like receptor 9) is a member of the Toll-like receptor family. It is mainly expressed on dendritic cells (DCS) and B cells and is one of the most promising targets in tumor treatment. The ligand naturally recognized by TLR9 is a sequence containing unmethylated CpG in bacterial DNA. Natural and synthetic CpG oligodeoxynucleotides (ODN) can stimulate the body's innate immune response in a TLR9-dependent manner, triggering an immune regulatory cascade reaction, ultimately producing Th1 and proinflammatory cytokines and chemokines, and can also improve the antigen presentation function of dendritic cells, monocytes and macrophages, induce B cell proliferation, stimulate the immune protection activity of NK cells, and induce immune response in the body. CpG ODN is often used as an immunopotentiator in clinical studies because of its good effect in promoting immune response, but CpG ODN is usually used alone in the form of a single chain.

[0004] Although TLR9 is considered a very promising target, TLR9 agonists still have certain limitations in clinical practice. Systemic administration may induce toxic reactions. Although local injection into the tumor can improve the body's tolerance, the drug may be rapidly metabolized from the site of action, resulting in attenuation of efficacy. Therefore, local administration into the tumor once a week or every two weeks is required clinically to maintain the anti-tumor efficacy. In addition, although the results of preclinical and phase II trials of polynucleotide TLR9 agonists are promising, systemic administration of polynucleotide TLR9 agonists did not improve the survival rate of patients with non-small cell lung cancer when combined with chemotherapy regimens.

[0005] Therefore, there is still a need for an improved method for delivering TLR9 agonists to better exert the effects of TLR9 agonists, reduce the pain of patients when administering TLR9 agonists, and improve the therapeutic effect. Summary of the invention

[0006] One aspect of the present application provides a method for treating a disease in a subject, the method comprising administering to the subject (1) a dual or multi-ligand conjugate or a pharmaceutically acceptable salt thereof and (2) a T-effector cell agonist, wherein the dual or multi-ligand conjugate has a payload linked to the ligand portion, the payload is an immunostimulatory sequence, and the disease is a disease that can benefit from T-effector cell agonism.

[0007] In some embodiments, the bi-ligand or multi-ligand conjugate or a pharmaceutically acceptable salt thereof can produce a synergistic effect with a T-effector cell agonist.

[0008] In some embodiments, the bi- or multi-ligand conjugate or a pharmaceutically acceptable salt thereof is capable of upregulating the expression of a target of a T-effector cell agonist.

[0009] In some embodiments, the payload is a TLR9 agonist. In some embodiments, the TLR9 agonist is an oligonucleotide. In some embodiments, the TLR9 agonist is a CpG oligonucleotide containing one or more unmethylated CpG motifs. In some other embodiments, the CpG oligonucleotide comprises any nucleotide sequence of the nucleotides shown in SEQ ID NO: 1-11. In some embodiments, the CpG oligonucleotide comprises the nucleotide sequence shown in SEQ ID NO: 1.

[0010] In some embodiments, the ligand portion of the bi-ligand or multi-ligand conjugate binds to a cell surface molecule. In some embodiments, the bi-ligand or multi-ligand portions of the bi-ligand or multi-ligand conjugate bind to different cell surface molecules, respectively. In some embodiments, the cell surface molecules are highly expressed in cancer cells. In some embodiments, the cell surface molecules are FOLR1 and PSMA, respectively.

[0011] In some embodiments, the bi-ligands or multi-ligands of the bi-ligand or multi-ligand conjugate are directly connected or connected through a spacer; preferably, the spacer is 1 amino acid, 2 amino acids or 3 amino acids; preferably, the spacer is cysteine, lysine, cysteine-lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-glutamate, aspartic acid-aspartic acid, aspartic acid-aspartic acid-lysine or lysine-cysteine-lysine, optionally, the carboxylic acids in the above amino acids are amidated.

[0012] In some embodiments, the ligand portion of the bi- or multi-ligand conjugate has the following structure:

[0013]

[0014] In some embodiments, the ligand portion of the dual-ligand or multi-ligand conjugate is directly connected to the payload or connected through a linker; preferably, the connection is completed through a click reaction; preferably, the ligand portion is connected to the payload through a thiol group.

[0015] In some embodiments, the linker of the dual-ligand or multi-ligand conjugate comprises the following structure: -S-, -S-(CH2)6-O-, -S-(CH2)6-OP(=S)(OH)-, or -S-(CH2)6-OP(=S)(OH)-O-.

[0016] In some embodiments, the ligand conjugate has the following structure:

[0017]

[0018] In some embodiments, the T-effector cell agonist is an antibody. In some embodiments, the T-effector cell agonist is an anti-OX40 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-OX40 antibody is selected from BE0031 of BioXCell, IBI101 of Innovent Biologics, SHR-1806 of Hengrui Medicine, ES102 of Covance Bio, BMS-986178 of Bristol-Myers Squibb, PF-04518600 of Pfizer, ABBV-368 of AbbVie, or TH002 of Youhe Bio. In some embodiments, the anti-OX40 antibody is selected from BE0031 of BioXCell.

[0019] In some embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof and the T-effector cell agonist are administered simultaneously. In some embodiments, the T-effector cell agonist is administered for a period of time after the administration of the ligand conjugate or a pharmaceutically acceptable salt thereof, preferably, the period of time is 1 minute to 3 days. In some embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof is administered for a period of time after the administration of the T-effector cell agonist, preferably, the period of time is 1 minute to 3 days.

[0020] In some embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof is administered by intravenous injection. In some embodiments, the T-effector cell agonist is administered intraperitoneally, intravenously or subcutaneously, preferably, by intraperitoneal injection.

[0021] In some embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof is administered once a week, twice a week, three times a week, once every three days, once every two days, once a day, twice a day, or three times a day; preferably, the administration frequency is once a week; preferably, the administration frequency is twice a week.

[0022] In some embodiments, the T-effector cell agonist is administered once a week, twice a week, three times a week, once every three days, once every two days, once a day, twice a day, or three times a day; preferably, the administration frequency is once a week; preferably, the administration frequency is twice a week.

[0023] In some embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof is administered twice a week; and the T-effector cell agonist is administered twice a week.

[0024] In some embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof is administered at a dose of 1-400 mg per time. In some embodiments, the dosage of the ligand conjugate or a pharmaceutically acceptable salt thereof is gradually reduced over the course of treatment.

[0025] In some embodiments, the T-effector cell agonist is administered at a dose of 1-600 mg per time. In some embodiments, the dosage of the T-effector cell agonist is gradually reduced over the course of treatment.

[0026] In some embodiments, the subject is a mammal, preferably, the subject is a human.

[0027] In some embodiments, the disease that can benefit from a T-effector cell agonist is cancer or an immune disease. In some embodiments, the cancer is selected from the group consisting of liver cancer, breast cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, lung cancer, kidney cancer, leukemia, ovarian cancer, gastric cancer, uterine cancer, endometrial cancer, colon cancer, testicular cancer, skin cancer, prostate cancer, lymphoma, and multiple myeloma. In some embodiments, the immune disease is selected from the group consisting of connective tissue disease, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

[0028] Another aspect of the present application provides a kit comprising (1) a dual or multi-ligand conjugate or a pharmaceutically acceptable salt thereof and (2) a T-effector cell agonist, wherein the dual or multi-ligand conjugate has a payload linked to the ligand portion, wherein the payload is an immunostimulatory sequence.

[0029] In some embodiments, the payload is a TLR9 agonist. In some embodiments, the TLR9 agonist is an oligonucleotide. In some embodiments, the TLR9 agonist is a CpG oligonucleotide containing one or more unmethylated CpG motifs. In some embodiments, the CpG oligonucleotide comprises any nucleotide sequence of the nucleotides shown in SEQ ID NO: 1-11. In some embodiments, the CpG oligonucleotide comprises the nucleotide sequence shown in SEQ ID NO: 1.

[0030] In some embodiments, the ligand portion of the bi-ligand or multi-ligand conjugate binds to a cell surface molecule. In some embodiments, the bi-ligand or multi-ligand portions of the bi-ligand or multi-ligand conjugate bind to different cell surface molecules, respectively. In some embodiments, the cell surface molecules are highly expressed in cancer cells.

[0031] In some embodiments, the ligands of the dual-ligand or multi-ligand conjugate target different cell surface molecules, and the cell surface molecules are FOLR1 and PSMA, respectively.

[0032] In some embodiments, the bi-ligands or multi-ligands of the bi-ligand or multi-ligand conjugate are directly connected or connected through a spacer; preferably, the spacer is 1 amino acid, 2 amino acids or 3 amino acids; preferably, the spacer is cysteine, lysine, cysteine-lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-glutamate, aspartic acid-aspartic acid, aspartic acid-aspartic acid-lysine or lysine-cysteine-lysine, optionally, the carboxylic acids in the above amino acids are amidated.

[0033] In some embodiments, the ligand portion of the bi- or multi-ligand conjugate has the following structure:

[0034]

[0035] In some embodiments, the ligand portion of the dual-ligand or multi-ligand conjugate is directly connected to the payload or connected through a linker; preferably, the connection is completed through a click reaction; preferably, the ligand portion is connected to the payload through a thiol group.

[0036] In some embodiments, the linker of the dual-ligand or multi-ligand conjugate comprises the following structure: -S-, -S-(CH2)6-O-, -S-(CH2)6-OP(=S)(OH)-, or -S-(CH2)6-OP(=S)(OH)-O-.

[0037] In some embodiments, the ligand conjugate has the following structure:

[0038]

[0039] In some embodiments, the T-effector cell agonist is an antibody. In some embodiments, the T-effector cell agonist is an anti-OX40 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-OX40 antibody is selected from BE0031 of BioXCell, IBI101 of Innovent Biologics, SHR-1806 of Hengrui Medicine, ES102 of Covance Bio, BMS-986178 of Bristol-Myers Squibb, PF-04518600 of Pfizer, ABBV-368 of AbbVie, or TH002 of Youhe Bio. In some embodiments, the anti-OX40 antibody is selected from BE0031 of BioXCell.

[0040] In some embodiments, the kit is configured so that the ligand conjugate or a pharmaceutically acceptable salt thereof and the T-effector cell agonist are administered simultaneously. In other embodiments, the kit is configured to administer the T-effector cell agonist after a period of time after the administration of the ligand conjugate or a pharmaceutically acceptable salt thereof, preferably, the period of time is 1 minute to 3 days. In other embodiments, the kit is configured to administer the ligand conjugate or a pharmaceutically acceptable salt thereof after a period of time after the administration of the T-effector cell agonist, preferably, the period of time is 1 minute to 3 days.

[0041] Another aspect of the present application provides a kit of dual-ligand or multi-ligand conjugates or pharmaceutically acceptable salts thereof, wherein the dual-ligand or multi-ligand conjugate comprises a payload connected to a ligand portion targeting a cell surface molecule, wherein the payload is an immunostimulatory sequence.

[0042] In some embodiments, the payload is a TLR9 agonist. In some embodiments, the TLR9 agonist is an oligonucleotide. In some embodiments, the TLR9 agonist is a CpG oligonucleotide containing one or more unmethylated CpG motifs. In some embodiments, the CpG oligonucleotide comprises any one of the nucleotide sequences of the nucleotides shown in SEQ ID NO: 1-11. In some embodiments, the CpG oligonucleotide comprises the nucleotide sequence shown in SEQ ID NO: 1.

[0043] In some embodiments, the dual ligand or multi-ligand portions of the ligand conjugate bind to different cell surface molecules, respectively. In some embodiments, the cell surface molecules are highly expressed in cancer cells. In some embodiments, the cell surface molecules are FOLR1 and PSMA, respectively.

[0044] In some embodiments, at least two ligands of the ligand conjugate are directly connected or connected through a spacer; preferably, the spacer is 1 amino acid, 2 amino acids or 3 amino acids; preferably, the spacer is cysteine, lysine, cysteine-lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-glutamate, aspartic acid-aspartic acid, aspartic acid-aspartic acid-lysine or lysine-cysteine-lysine, optionally, the carboxylic acids in the above amino acids are amidated.

[0045] In some embodiments, the ligand portion of the ligand conjugate has the following structure:

[0046]

[0047] In some embodiments, the ligand portion of the ligand conjugate is directly linked to the payload or linked via a linker; preferably, the linking is accomplished via a click reaction; preferably, the ligand portion is linked to the payload via a thiol group.

[0048] In some embodiments, the linker of the ligand conjugate comprises the following structure: -S-, -S-(CH2)6-O-, -S-(CH2)6-OP(=S)(OH)-, or -S-(CH2)6-OP(=S)(OH)-O-.

[0049] In some embodiments, the ligand conjugate has the following structure:

[0050] BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The graph shows that the livers of mice were enlarged in the groups that received different doses of CR19501 and CR19503 alone.

[0052] Figure 2 The graph shows that the spleen of mice was enlarged in the groups that received different doses of CR19501 and CR19503 alone.

[0053] Figure 3 Shown are the changes in body weight of mice in response to different dosing regimens.

[0054] Figure 4 Shown are the changes in liver weight of mice immediately after the last administration for different dosing regimens.

[0055] Figure 5 Shown are changes in spleen weights of mice immediately after the last administration for different dosing regimens.

[0056] Figure 6 Shown are changes in liver weight of mice after 4 weeks of recovery from the last dose for different dosing regimens.

[0057] Figure 7 Shown are the changes in spleen weight of mice after 4 weeks of recovery from the last administration for different dosing regimens. DETAILED DESCRIPTION OF THE INVENTION

[0058] The following description is only for illustrating the various embodiments of the present application. Therefore, the specific embodiments herein should not be construed as limiting the scope of the application. A person skilled in the art can easily derive a variety of equivalents and modifications based on the purpose of the present invention and the description herein, and it should be understood that such equivalent embodiments are included within the scope of the present invention. All documents cited in this application, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0059] As used herein, the singular form "a," "an," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0060] As used herein, terms such as "comprises," "comprising," "containing," "including," and "having" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term "consisting of" is closed.

[0061] In one aspect, the present application discloses a method for treating a disease in a subject, the method comprising administering to the subject (1) a dual or multi-ligand conjugate or a pharmaceutically acceptable salt thereof and (2) a T-effector cell agonist, wherein the dual or multi-ligand conjugate has a payload linked to a ligand portion, the payload is an immunostimulatory sequence, and the disease is a disease that can benefit from T-effector cell agonism.

[0062] In one aspect, the present application discloses a method for enhancing immune response / enhancing cancer treatment effect / enhancing T-effector cell agonist effect, the method comprising administering to the subject (1) a dual-ligand or multi-ligand conjugate or a pharmaceutically acceptable salt thereof and (2) a T-effector cell agonist, wherein the dual-ligand or multi-ligand conjugate has a payload linked to the ligand portion, and the payload is an immunostimulatory sequence.

[0063] One aspect of the present application discloses a method for enhancing immune response / enhancing cancer treatment effect / enhancing T-effector cell agonist effect, the method comprising administering a dual-ligand or multi-ligand conjugate or a pharmaceutically acceptable salt thereof to the subject, wherein the subject has been administered a T-effector cell agonist, wherein the dual-ligand or multi-ligand conjugate has a payload linked to the ligand portion, and the payload is an immunostimulatory sequence.

[0064] One aspect of the present application discloses the use of a combination of (1) a dual or multi-ligand conjugate or a pharmaceutically acceptable salt thereof and (2) a T-effector cell agonist for use in a method of treating a disease in a subject, the method comprising administering to the subject (1) a dual or multi-ligand conjugate or a pharmaceutically acceptable salt thereof and (2) a T-effector cell agonist, wherein the dual or multi-ligand conjugate has a payload linked to a ligand portion, the payload is an immunostimulatory sequence, and the disease is a disease that can benefit from T-effector cell agonism.

[0065] One aspect of the present application discloses the use of a combination of (1) a dual or multi-ligand conjugate or a pharmaceutically acceptable salt thereof and (2) a T-effector cell agonist, which is used in a method of enhancing immune response / enhancing cancer treatment effect / enhancing the effect of T-effector cell agonist, the method comprising administering to the subject (1) a dual or multi-ligand conjugate or a pharmaceutically acceptable salt thereof and (2) a T-effector cell agonist, wherein the dual or multi-ligand conjugate has a payload linked to the ligand portion, and the payload is an immunostimulatory sequence.

[0066] One aspect of the present application discloses the use of a dual-ligand or multi-ligand conjugate or a pharmaceutically acceptable salt thereof, which is used in a method of enhancing immune response / enhancing cancer treatment effect / enhancing T-effector cell agonist effect, the method comprising administering a dual-ligand or multi-ligand conjugate or a pharmaceutically acceptable salt thereof to the subject, wherein the subject has been administered a T-effector cell agonist, wherein the dual-ligand or multi-ligand conjugate has a payload connected to the ligand portion, and the payload is an immunostimulatory sequence.

[0067] One aspect of the present application discloses the use of a combination of (1) a dual-ligand or multi-ligand conjugate or a pharmaceutically acceptable salt thereof and (2) a T-effector cell agonist in the preparation of a medicament for treating a disease, wherein the dual-ligand or multi-ligand conjugate has a payload linked to a ligand portion, the payload is an immunostimulatory sequence, and the disease is a disease that can benefit from T-effector cell agonism.

[0068] One aspect of the present application discloses the use of a dual-ligand or multi-ligand conjugate or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a disease in a subject, wherein the subject has been treated with a T-effector cell agonist, the dual-ligand or multi-ligand conjugate has a payload linked to the ligand portion, the payload is an immunostimulatory sequence, and the disease is a disease that can benefit from T-effector cell agonism.

[0069] One aspect of the present application discloses the use of a T-effector cell agonist in the preparation of a medicament for treating a disease in a subject, wherein the subject has been administered a dual-ligand or multi-ligand conjugate or a pharmaceutically acceptable salt thereof, wherein the dual-ligand or multi-ligand conjugate has a payload linked to a ligand portion, wherein the payload is an immunostimulatory sequence, and the disease is a disease that can benefit from T-effector cell agonism.

[0070] One aspect of the present application discloses the use of a combination of (1) a dual or multi-ligand conjugate or a pharmaceutically acceptable salt thereof and (2) a T-effector cell agonist in the preparation of a medicament for enhancing immune response / enhancing cancer treatment effect / enhancing the effect of a T-effector cell agonist, wherein the dual or multi-ligand conjugate has a payload linked to the ligand portion, and the payload is an immunostimulatory sequence.

[0071] One aspect of the present application discloses the use of a dual-ligand or multi-ligand conjugate or a pharmaceutically acceptable salt thereof in the preparation of a medicament for enhancing an immune response / enhancing the effect of cancer treatment / enhancing the effect of a T-effector cell agonist, wherein the subject has been administered a T-effector cell agonist, wherein the dual-ligand or multi-ligand conjugate has a payload linked to the ligand portion, and the payload is an immunostimulatory sequence.

[0072] Another aspect of the present application discloses a kit comprising (1) a dual or multi-ligand conjugate or a pharmaceutically acceptable salt thereof and (2) a T-effector cell agonist, wherein the dual or multi-ligand conjugate has a payload linked to the ligand portion, wherein the payload is an immunostimulatory sequence.

[0073] Another aspect of the present application discloses a dual-ligand or multi-ligand conjugate or a pharmaceutically acceptable salt thereof, wherein the dual-ligand or multi-ligand conjugate comprises a payload connected to a ligand portion that targets a cell surface molecule, wherein the payload is an immunostimulatory sequence.

[0074] T-effector cell agonists

[0075] The term "T-effector cell agonist" used in this application refers to any molecule or agent that can bind to T-effector cells and directly cause T-effector cell activation, or indirectly cause T-effector cell activation. T-effector cell agonists can increase T cell effects, prolong the survival time of T cells, inhibit the differentiation and activity of regulatory T cells (Tregs), and enhance the killing ability of effector T cells to enhance immune responses.

[0076] In some embodiments, the T-effector cell agonist is an antibody.

[0077] The term "antibody" as used in this application includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, bispecific antibody or corresponding antigen-binding fragment that binds to a specific antigen. A natural complete antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ and μ, each heavy chain consisting of a variable region (VH) and a first, second, third and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively); mammalian light chains are classified as λ or κ, and each light chain consists of a variable region (VL) and a constant region. The antibody is in the shape of a "Y", in which the stem of the Y consists of the second and third constant regions of two heavy chains bound together by disulfide bonds. Each arm of the Y comprises the variable region and the first constant region of a single heavy chain bound to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in the two chains generally contain three highly variable loops, called complementarity determining regions (CDRs) (the light chain CDRs include LCDR1, LCDR2, and LCDR3, and the heavy chain CDRs include HCDR1, HCDR2, and HCDR3).The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol Biol. 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., Sequences of Proteins of Immunological Relevance. Interest), 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (2nd ed.), Chapter 26, 481-514, (2015)). The three CDRs are inserted between flanking segments (stretches) called framework regions (FRs) (light chain FRs include LFR1, LFR2, LFR3 and LFR4, and heavy chain FRs include HFR1, HFR2, HFR3 and HFR4), which are more highly conserved than CDRs and form a scaffold that supports highly variable loops. The constant regions of the heavy and light chains do not participate in antigen binding, but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are large immunoglobulin A (IgA), IgD, IgE, IgG and IgM, which are characterized by the presence of α, δ, ε, γ and μ heavy chains, respectively.The several major antibody classes are divided into subclasses, such as IgGl (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgAl (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain).

[0078] In some embodiments, antibodies provided herein encompass any antigen binding fragment thereof. The term "antigen binding fragment" used in the present application refers to an antibody fragment formed by a part of an antibody, which comprises one or more CDRs or binds to an antigen but does not comprise any other antibody fragment of a complete native antibody structure. Examples of antigen binding fragments include, but are not limited to, bifunctional antibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), bispecific antibodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and divalent domain antibodies. Antigen binding fragments can be bound to the same antigen as that bound by the parent antibody.

[0079] In some embodiments, the T-effector cell agonist is an anti-OX40 antibody. It is generally believed that T cell activation requires at least two signals. The first signal is that the T cell recognizes the MHC-peptide compound, and various signal stimulations are also required during the recognition process. OX40 (also known as "CD134" or "TNFRSF4") is an important class of T cell co-stimulatory molecules and a member of the tumor necrosis factor receptor (TNF) superfamily. The ligand OX40L (also known as CD252) of OX40 usually binds to three OX40 proteins in the form of a trimer to form a hexameric complex, thereby activating downstream signaling pathways such as NF-κB, PI3K and AKT. Continuous activation of these downstream signals can stimulate the production of cytokines, thereby enhancing the killing ability of effector T cells.

[0080] The term "anti-OX40 antibody" used in this application can be any anti-OX40 antibody or antigen-binding fragment thereof known or unknown. In some embodiments, the anti-OX40 antibody is selected from BE0031 of BioXCell, IBI101 of Innovent Biologics, SHR-1806 of Hengrui Medicine, ES102 of Covance Bio, BMS-986178 of Bristol-Myers Squibb, PF-04518600 of Pfizer, ABBV-368 of AbbVie, or TH002 of Youhe Bio. In some embodiments, the anti-OX40 antibody is BE0031 purchased commercially from BioXCell (clone: ​​OX-86; type: Rat IgG1, κ; purity: greater than 95%; RRID: AB_1107592; molecular weight: 150 kDa; product link: https: / / bxcell.com / product / m-cd134-ox40 / ).

[0081] disease

[0082] The term "disease that can benefit from T-effector cell stimuli" as used in this application refers to a disease in which the symptoms can be alleviated, delayed or cured to a certain extent, and / or the relevant physiological or biochemical parameters are partially or completely restored to normal after the T-effector cells in the body are activated. In some embodiments, the disease that can benefit from T-effector cell agonists is cancer or an immune disease. In some embodiments, the cancer is selected from the group consisting of liver cancer, breast cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, lung cancer, kidney cancer, leukemia, ovarian cancer, gastric cancer, uterine cancer, endometrial cancer, colon cancer, testicular cancer, skin cancer, prostate cancer, lymphoma and multiple myeloma. In some embodiments, the immune disease is selected from the group consisting of connective tissue disease, systemic sclerosis, rheumatoid arthritis and systemic lupus erythematosus.

[0083] Double ligand or multi-ligand conjugate or pharmaceutically acceptable salt thereof

[0084] In some embodiments, the dual ligand or multi-ligand conjugate or its pharmaceutically acceptable salt can produce a synergistic effect with a T-effector cell agonist. In some embodiments, the dual ligand or multi-ligand conjugate or its pharmaceutically acceptable salt can upregulate the expression of the target of the T-effector cell agonist.

[0085] The term "payload" as used in this application refers to a molecule or substance to be delivered to a target cell or tissue. Without limitation, a payload can be any molecule or substance intended to treat or prevent a disease in a subject.

[0086] In some embodiments, the payload is an immunostimulatory sequence.

[0087] "Immunostimulatory sequence" used in this application refers to a class of nucleotides or nucleotide-like sequences, which can activate immune cells after applying the sequence, such as promoting or increasing T effector cells to express costimulatory molecules (such as OX40, 4-1BB and / or CD3, etc.), inducing INF-α production and causing NK cell activation; and / or stimulating the ability of B cells (such as stimulating B cell proliferation and secreting IgM). In some embodiments, the immunostimulatory sequence can promote and increase T effector cells to express OX40.

[0088] In some embodiments, the payload is a TLR9 agonist. TLR9 (Toll-like receptor 9) is a member of the Toll-like receptor family, mainly expressed in dendritic cells (DCS) and B cells, and is one of the most promising targets in tumor treatment. The ligands naturally recognized by TLR9 are sequences containing unmethylated CpG in bacterial DNA. Natural and synthetic CpG oligodeoxynucleotides (ODN) can stimulate the body's innate immune response in a TLR9-dependent manner.

[0089] In some embodiments, the TLR9 agonist is an oligonucleotide. In some embodiments, the TLR9 agonist is a CpG oligonucleotide (ODN) containing one or more non-methylated CpG motifs. In the art, CpG ODN is often used as an immunopotentiator in clinical studies due to its good effect of promoting immune response. But CpG ODN is usually used separately in the form of a single strand. CpG ODN usually has a base length of 9-100, and preferably, CpG ODN has a base length of 18-50. More preferably, CpG ODN has a base length of 20-40, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 bases. In some other embodiments, the CpG oligonucleotide comprises any nucleotide sequence of the nucleotides shown in SEQ ID NO: 1-11. In some embodiments, the CpG oligonucleotide comprises the nucleotide sequence shown in SEQ ID NO:1.

[0090] The term "ligand" used in this application may include a variety of chemical molecules or peptides that have specific binding affinity for a selected target, and the selected target may be a cell surface molecule, such as a cell surface receptor, a cell surface antigen, a cell, a tissue, an organ, etc. In some embodiments, the ligand may specifically bind to a protein or marker expressed on the surface of a target cell (i.e., a "cell surface molecule"). In some embodiments, the ligand of this application is expressed in 10 -6 ~10 -11 M(K dIn some embodiments, the ligand of the present invention binds to a cell surface protein or marker with an affinity of at least 10 -7 , at least 10 -8 , at least 10 -9 M(K d In some embodiments, the ligand of the present invention binds to a cell surface protein or marker with an affinity of less than 10 -6 , less than 10 -7 , less than 10 -8 M(K d In some embodiments, the ligand of the present application is combined with a cell surface protein or a marker with an affinity of at least 2, 3, 4, 5, 6, 8, 10, 20, 50, 100 or more times higher than the affinity of a non-target cell surface protein or a marker. In some embodiments, the ligand of the present application is combined with a cell surface protein or a marker with an affinity of at least 2, 3, 4, 5, 6, 8, 10, 20, 50, 100 or more times higher than the affinity of a non-target cell surface protein or a marker. In some embodiments, the expression of the cell surface protein or a marker of the present application in target cells (e.g., cancer cells) is significantly higher than the expression in normal cells. The term "significant" used in the present application refers to a statistically significant difference, or a significant difference that can be recognized by those skilled in the art.

[0091] The term "ligand moiety" used in this application refers to any molecule or moiety that can be targeted to a target site, target tissue, target organ, target cell or target cell intracellular region (e.g., cell surface molecule) comprising a dual ligand or multi-ligand. In some embodiments, the ligand moiety causes the dual ligand or multi-ligand conjugate comprising the ligand moiety to be distributed more at the target site, target tissue, target organ, target cell or target cell intracellular region compared to a non-target site, non-target tissue, non-target organ, non-target cell or non-target cell intracellular region, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more. In some embodiments, compared to a compound without a ligand portion, a conjugate with the ligand portion or a pharmaceutically acceptable salt thereof is distributed more at the target site, target tissue, target organ, target cell or target cell intracellular region, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more. In some embodiments, the ligand portion can trigger or promote the specific binding of a conjugate containing such a ligand portion to a target molecule, trigger or promote the endocytosis of the conjugate by the target cell, trigger or promote the enrichment of the conjugate around the target cell and / or enter the target cell.

[0092] In some embodiments, the expression level of the cell surface molecule (protein or marker) of the present application in the target cell (e.g., cancer cell) is 2 to 1,000,000 times the expression level in the normal cell, for example, the expression level in the target cell (e.g., cancer cell) is 2 to 10 times, 2 to 100 times, 2 to 1,000 times, 2 to 10,000 times, 2 to 100,000 times, 2 to 1,000,000 times (can be equal to any value in the above numerical range, including the end point of the range). In some embodiments, the expression level of the cell surface receptor in the target cell (e.g., cancer cell) is at least 10 times, or at least 100 times, or at least 1,000 times, or at least 10,000 times, or at least 100,000 times higher than the expression level in the normal cell. In some embodiments, the level of a cell surface molecule on a normal cell is reduced by at least 50%, 60%, 70%, 80%, 90%, 95% or 99% when compared to the level of a cell surface molecule (protein or marker) on a target cell (e.g., a cancer cell). In some embodiments, the cell surface molecule described herein is not detectable in a normal cell.

[0093] In some embodiments, the ligand portion of the bi-ligand or multi-ligand conjugate binds to a cell surface molecule. In some embodiments, the bi-ligand or multi-ligand portions of the bi-ligand or multi-ligand conjugate bind to different cell surface molecules, respectively. In some embodiments, the cell surface molecules are highly expressed in cancer cells. In some embodiments, the cell surface molecules are FOLR1 and PSMA, respectively.

[0094] FOLR1 (Folate Receptor 1) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein that binds folate with nanomolar affinity, thereby promoting receptor-mediated endocytosis. Fast-growing solid malignancies, including ovarian and lung cancer, are dependent on folate for metabolism and nucleic acid synthesis. In some embodiments, the ligand moiety described herein comprises a ligand that targets FOLR1. In some embodiments, the ligand that targets FOLR1 is folate or a folate analog. In some embodiments, the folate analog is selected from 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate, and 5,10-methylenetetrahydrofolate.

[0095] PSMA (prostate-specific membrane antigen) refers to a type II transmembrane glycoprotein present in the membrane of prostate epithelial cells. It is composed of 750 amino acids, including 19 intracellular amino acids, 24 transmembrane amino acids, and 707 extracellular amino acids. PSMA is expressed in normal prostate epithelial cells, but its expression level in prostate cancer cells is much higher. Compared with the traditional prostate-specific antigen used for clinical testing, PSMA is a more sensitive and specific prostate cancer tumor marker, especially in hormone-refractory prostate cancer and prostate cancer metastasis, which are highly expressed. It has high sensitivity and specificity in distinguishing prostate cancer from other types of malignant tumors. At the same time, in a variety of non-prostate solid tumors (such as lung cancer, bladder cancer, gastric cancer, pancreatic cancer, kidney cancer, and colorectal cancer, etc.), PSMA is also highly specifically expressed on tumor vascular endothelial cells.

[0096] In some embodiments, the PMSA-targeting ligand of the present application can be selected from the group consisting of: 2-[[methylhydroxyphosphinyl]methyl]glutaric acid; 2-[[ethylhydroxyphosphinyl]methyl]glutaric acid; 2-[[propylhydroxyphosphinyl]methyl]glutaric acid; 2-[[butylhydroxyphosphinyl]methyl]glutaric acid; 2-[[cyclohexylhydroxyphosphinyl]methyl]glutaric acid; 2-[[phenylhydroxyphosphinyl]methyl]glutaric acid; 2-[[2-(tetrahydrofuranyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[(2-tetrahydropyranyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((4-pyridyl)methyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((2-pyridyl)methyl)hydroxyphosphinyl]methyl]glutaric acid 2-[[(phenylmethyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((2-phenylethyl)methyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((3-phenylpropyl)methyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((3-phenylbutyl)methyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((2-phenylbutyl)methyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[(4-phenylbutyl)hydroxyphosphinyl]methyl]glutaric acid; and 2-[[(aminomethyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[methylhydroxyphosphinyl]oxy]glutaric acid; 2-[[ethylhydroxyphosphinyl]oxy]glutaric acid; 2-[[propylhydroxyphosphinyl]oxy]glutaric acid; 2- [[Butylhydroxyphosphinyl]oxy]glutaric acid; 2-[[phenylhydroxyphosphinyl]oxy]glutaric acid; 2-[[((4-pyridyl)methyl)hydroxyphosphinyl]oxy]glutaric acid; 2-[[((2-pyridyl)methyl)hydroxyphosphinyl]oxy]glutaric acid; 2-[[(phenylmethyl)hydroxyphosphinyl]oxy]glutaric acid; and 2[[((2-phenylethyl)methyl)hydroxyphosphinyl]oxy]glutaric acid; 2-[[(N-hydroxy)carbamoyl]methyl]glutaric acid; 2-[[(N-hydroxy-N-methyl)carbamoyl]methyl]glutaric acid; 2-[[(N-butyl-N-hydroxy)carbamoyl]methyl]glutaric acid; 2-[[(N-benzyl-N-hydroxy)carbamoyl]methyl]glutaric acid; 2-[[(N-hydroxy 2-[[(N-hydroxy-N-2-phenylethyl)carbamoyl]methyl]glutaric acid; 2-[[(N-ethyl-N-hydroxy)carbamoyl]methyl]glutaric acid; 2-[[(N-hydroxy-N-propyl)carbamoyl]methyl]glutaric acid; 2-[[(N-hydroxy-N-3-phenylpropyl)carbamoyl]methyl]glutaric acid; 2-[[(N-hydroxy-N-4-pyridyl)carbamoyl]methyl]glutaric acid; 2-[[(N-hydroxy)amido]methyl]glutaric acid; 2-[[N-hydroxy(methyl)amido]methyl]glutaric acid; 2-[[N-hydroxy(benzyl)amido]methyl]glutaric acid; 2-[[N-hydroxy(phenyl)amido]methyl]glutaric acid;2-[[N-hydroxy(2-phenylethyl)amide]methyl]glutaric acid; 2-[[N-hydroxy(ethyl)amide]methyl]glutaric acid; 2-[[N-hydroxy(propyl)amide]methyl]glutaric acid; 2-[[N-hydroxy(3-phenylpropyl)amide]methyl]glutaric acid; and 2-[[N-hydroxy(4-pyridyl)amide]methyl]glutaric acid; 2-[(sulfinyl)methyl]glutaric acid; 2-[(methylsulfinyl)methyl]glutaric acid; 2-[(ethylsulfinyl)methyl]glutaric acid; 2-[(propylsulfinyl)methyl]glutaric acid; 2-[(butylsulfinyl)methyl]glutaric acid; 2-[( 2-[[(2-phenylethyl)sulfinyl]methyl]glutaric acid; 2-[[(3-phenylpropyl)sulfinyl]methyl]glutaric acid; 2-[[(4-pyridyl)sulfinyl]methyl]glutaric acid; 2-[(benzylsulfinyl)methyl]glutaric acid; 2-[(sulfonyl)methyl]glutaric acid; 2-[(methylsulfonyl)methyl]glutaric acid; 2-[(ethylsulfonyl)methyl]glutaric acid; 2-[(propanesulfonyl)methyl]glutaric acid; 2-[(butanesulfonyl)methyl]glutaric acid; 2-[(phenylsulfonyl]methyl]glutaric acid; 2-[[(2-phenylethyl)sulfonyl] 2-[((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((( ] glutaric acid; 2-[[(3-phenylpropyl)sulfoximino]methyl]glutaric acid; 2-[[(4-pyridyl)sulfoximino]methyl]glutaric acid; and 2-[(benzylsulfoximino)methyl]glutaric acid; N-[methylhydroxyphosphinyl]glutamate; N-[ethylhydroxyphosphinyl]glutamate; N-[propylhydroxyphosphinyl]glutamate; N-[butylhydroxyphosphinyl]glutamate; N-[phenylhydroxyphosphinyl]glutamate; N-[(phenylmethyl)hydroxyphosphinyl]glutamate; N-[((2-phenylethyl)methyl)hydroxyphosphinyl]glutamate; and N-methyl-N-[phenylhydroxyphosphinyl]glutamate. ;

[0097] In some embodiments, the biligands or multi-ligand conjugates of biligands or multi-ligands are directly connected or connected by a spacer. In some embodiments, the spacer is composed of amino acids. In some embodiments, the spacer is composed of natural amino acids or non-natural amino acids. In some embodiments, the spacer is 1 amino acid, 2 amino acids or 3 amino acids. In some embodiments, the spacer is cysteine, lysine, cysteine-lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-glutamic acid, aspartic acid-aspartic acid, aspartic acid-aspartic acid-lysine or lysine-cysteine-lysine, and optionally, the carboxylic acid in the above amino acids is amidated. In some embodiments, the spacer can be cleaved by protease cleavage or reduction cleavage under specific physiological conditions. In some embodiments, the spacer can be cleaved by a protease specifically expressed by a target cell or expressed by a target cell.

[0098] As used in this application, the terms "cleavable" or "cleavage" include metabolic processes or reaction processes performed on the conjugate compounds provided in this application, thereby destroying the linker between the payload and the ligand portion, or the spacer between the ligands to release the free payload or ligand.

[0099] In some embodiments, the ligand portion of the bi- or multi-ligand conjugate has the following structure:

[0100]

[0101] In some embodiments, the ligand portion of the dual-ligand or multi-ligand conjugate is directly connected to the payload or connected through a linker; preferably, the connection is completed through a click reaction; preferably, the ligand portion is connected to the payload through a thiol group. The "click reaction" described in this application is a type of chemical synthesis method with high efficiency and selectivity. The click reaction splices several molecular fragments together by forming an efficient connection unit. The entire reaction process is simple, fast and has few by-products. Some examples of click reactions include, but are not limited to, addition reactions of thiols and olefins, and cyclization reactions of azides and alkynes.

[0102] The term "connector" as used in this application refers to a molecule or moiety that covalently connects a payload to a ligand moiety. A connector includes a functional group for connecting a payload to a ligand moiety. In some embodiments, the functional group may contain two reactive moieties, one for connecting to the payload and the other for connecting to the ligand moiety. In some embodiments, the functional groups are different from one another. In some embodiments, the functional group includes a group containing a sulfhydryl reactive moiety, an amine reactive moiety, or a carboxylic acid reactive moiety. In some embodiments, the functional groups are identical to one another.

[0103] In some embodiments, the linker is sufficiently stable to avoid accidental release of the payload during blood circulation to increase the effective amount of the payload to the target cell or tissue. In some embodiments, the linker can release the payload around or inside the target cell to effectively play an immunostimulatory function.

[0104] In some embodiments, the linker is non-cleavable. The non-cleavable linker used in the present application refers to a linker that remains substantially intact during intracellular metabolism. In some embodiments, the linker is cleavable. In some embodiments, the cleavable functional group is generally stable enough, but it tends to cleave to release the payload when it is close to or aggregated in the target cell, or after entering the target cell. In some embodiments, the cleavage efficiency of the cleavage functional group in the target cell is at least 10, 20, 30, 50, 100 times or more higher than the cleavage efficiency in blood or serum. In some embodiments, the cleavable linker of the present application can be hydrolyzed, enzymatically reacted, or reduced, or cleaved by pH changes. In some embodiments, the linker is cleavable under a specific physiological environment (e.g., under a suitable pH environment).

[0105] In some embodiments, the linker of the dual-ligand or multi-ligand conjugate comprises the following structure: -S-, -S-(CH2)6-O-, -S-(CH2)6-OP(=S)(OH)-, or -S-(CH2)6-OP(=S)(OH)-O-.

[0106] In some embodiments, the ligand conjugate has the following structure:

[0107]

[0108] Application

[0109] In some embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof and the T-effector cell agonist are administered simultaneously.

[0110] In some embodiments, the T-effector cell agonist is administered some time after the administration of the ligand conjugate or a pharmaceutically acceptable salt thereof. In some embodiments, the T-effector cell agonist is administered 1 minute to 3 days after the administration of the ligand conjugate or a pharmaceutically acceptable salt thereof. In some embodiments, the T-effector cell agonist is administered 0.1-48 hours after the administration of the ligand conjugate or a pharmaceutically acceptable salt thereof. In some embodiments, the T-effector cell agonist is administered 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours after administration of the ligand conjugate or a pharmaceutically acceptable salt thereof.

[0111] In some embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof is administered some time after the administration of the T-effector cell agonist. In some embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof is administered 1 minute to 3 days after the administration of the T-effector cell agonist. In some embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof is administered 0.1-48 hours after the administration of the T-effector cell agonist. In some embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof is administered 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours after administration of the T-effector cell agonist.

[0112] The T-effector cell agonists and ligand conjugates provided herein, or pharmaceutically acceptable salts thereof, can be administered by any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous (including intravenous infusion), intramuscular, intravaginal, epidermal, intratumoral, transdermal or intradermal injection) or non-parenteral (e.g., oral, intranasal, transmucosal, intraocular, sublingual, rectal or topical) routes.

[0113] In some embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof is administered by intravenous injection. In some embodiments, the T-effector cell agonist is administered intraperitoneally, intravenously or subcutaneously, preferably, by intraperitoneal injection.

[0114] In some embodiments, the ligand conjugates and / or T-effector cell agonists described herein are administered in a therapeutically effective amount. The term "therapeutically effective amount" used in this application refers to an amount of the ligand conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition that alleviates one or more symptoms of a disease or condition in a subject to a certain extent; an amount that partially or completely restores one or more physiological or biochemical parameters associated with or causing a disease or condition to normal; and / or an amount that reduces the likelihood of onset of a disease or condition.

[0115] The effective amount of the ligand conjugates and / or T-effector cell agonists described herein will depend on various factors known in the art, such as the individual's weight, age, past medical history, current medications, health status, and the possibility of cross-reactions, allergies, sensitivities, and adverse side effects, as well as the route of administration and the extent of disease progression. A person skilled in the art (e.g., a physician or veterinarian) may proportionally reduce or increase the dosage as indicated by these and other circumstances or requirements.

[0116] In some embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof is administered once a week, twice a week, three times a week, once every three days, once every two days, once a day, twice a day, or three times a day; preferably, the administration frequency is once a week; preferably, the administration frequency is twice a week.

[0117] In some embodiments, the T-effector cell agonist is administered once a week, twice a week, three times a week, once every three days, once every two days, once a day, twice a day, or three times a day; preferably, the administration frequency is once a week; preferably, the administration frequency is twice a week.

[0118] In some embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof is administered twice a week; and the T-effector cell agonist is administered twice a week.

[0119] In some embodiments, the ligand conjugate or its pharmaceutically acceptable salt is administered at a dose of 1-400 mg each time. Preferably, it is administered at a dose of 25 mg-150 mg each time. More preferably, it is administered at a dose of 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg or 150 mg each time. In some embodiments, the dosage of the ligand conjugate or its pharmaceutically acceptable salt is gradually reduced over the course of treatment.

[0120] In some embodiments, the T-effector cell agonist is administered at a dose of 1-600 mg per administration, preferably at a dose of 25 mg-250 mg per administration. More preferably, each dose of 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 105mg, 110mg, 115mg, 120mg, 125mg, 130mg, 135mg, 140mg, 145mg, 150mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg, 190mg, 195mg, 200mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg or 250mg is administered. In some embodiments, the dosage of the T-effector cell agonist is gradually reduced over the course of treatment.

[0121] In some embodiments, the amount of the ligand conjugate or its pharmaceutically acceptable salt, or the pharmaceutical composition is sufficient to inhibit the disease or condition in the subject, or preventively inhibit or prevent the onset of the disease or condition. Although the therapeutically effective amount can be changed in different subjects, it generally ranges from 0.01 to 100 mg / kg, such as 0.01 to 90 mg / kg, 0.01 to 80 mg / kg, 0.01 to 70 mg / kg, 0.01 to 60 mg / kg, 0.01 to 50 mg / kg, 0.01 to 40 mg / kg, 0.01 to 30 mg / kg, 0.01 to 20 mg / kg, 0.01 to 10 mg / kg, 0.01 to 5 mg / kg, 0.01 to 4 mg / kg, 0.01 to 3 mg / kg, 0.01 to 2 mg / kg, 0.01 to 1 mg / kg, 0.01 to 0.1 mg / kg. The therapeutically effective amount described herein can be equal to any value within the above numerical range, including the endpoint of the range.

[0122] In some embodiments, the subject is a mammal, preferably, the subject is a human. The term "subject" used in this application refers to humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. The subject can also be livestock, such as cattle, pigs, sheep, poultry and horses, or domestic animals, such as dogs and cats. The object can be male (e.g., male) or female (e.g., female), can be old age, adult, teenager, child or infant. People can be Caucasians, Africans, Asians, Semites or other ethnic backgrounds, or a mixture of these ethnic backgrounds.

[0123] One aspect of the present application discloses a method for preparing a ligand conjugate, the method comprising connecting the ligand portion described herein to a payload. In some embodiments, the ligand portion is directly connected to the payload. In some embodiments, the ligand portion is connected to the payload via a linker.

[0124] One aspect of the present application discloses a pharmaceutical composition, the pharmaceutical composition comprising the ligand conjugate described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition described herein is used for intravenous, subcutaneous, oral, intramuscular or intraventricular administration. In some preferred embodiments, the ligand conjugate or a pharmaceutically acceptable salt thereof or the pharmaceutical composition is administered intravenously.

[0125] The term "pharmaceutically acceptable" as used in this application means that it is suitable for contact with cells of humans and other animals without undue toxicity, irritation, allergic response, etc., within the scope of reasonable medical judgment, and is commensurate with a reasonable benefit / risk ratio.

[0126] The term "pharmaceutically acceptable salt" used in the present application refers to relatively nontoxic, inorganic and organic acid addition salts and base addition salts of the ligand conjugates of the present application. Representative acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, glucoheptonate, lactobionate, sulfamate, malonate, salicylate, propionate, methylene-bis-b-hydroxynaphthoate, gentisate, isethionate, di-p-toluoyl tartrate, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylaminosulfonate and quinic acid lauryl sulfonate etc. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium and aluminum salts. In some embodiments, sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases, including, for example, sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide and zinc hydroxide. Suitable amine base addition salts are prepared from amines having sufficient basicity to form stable salts, and preferably include the following amines commonly used in pharmaceutical chemistry because of their low toxicity and acceptable medical use: ammonia, ethylenediamine, N-methylglucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, trishydroxymethylaminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, diphenylhydroxymethylamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids (e.g., lysine and arginine) and dicyclohexylamine and the like.

[0127] The term "pharmaceutically acceptable carrier" as used in the present application refers to a pharmaceutically acceptable solvent, suspension or any other pharmaceutically inert carrier for delivering the conjugate compound provided herein to a subject, which does not interfere with the structure and properties of the conjugate compound. Some such carriers can formulate the conjugate compound into, for example, tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and pastilles for oral ingestion by a subject. Some such carriers can formulate the conjugate compound into a formulation for injection, infusion or topical administration.

[0128] Pharmaceutically acceptable carriers used in the pharmaceutical compositions provided herein include, but are not limited to, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous carriers (e.g., sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection), non-aqueous carriers (e.g., fixed oils of plant origin, cottonseed oil, corn oil, sesame oil or peanut oil), antimicrobial agents, isotonic agents (e.g., sodium chloride or dextrose), buffers (e.g., phosphate or citric acid buffers), antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending / dispersing agents (e.g., sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifiers (e.g., polysorbate 80 (Tween-80)), diluents, adjuvants, excipients, or non-toxic auxiliary substances, other ingredients known in the art, or various combinations thereof. Suitable ingredients may include, for example, fillers, binders, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants or emulsifiers.

[0129] In some embodiments, the pharmaceutical composition is an injection. The injection includes a sterile aqueous solution or a dispersant, a suspension or an emulsion. In all cases, the injection should be sterile and should be fluid for injection. The injection should remain stable under production and storage conditions, and must prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures and / or vegetable oils. The injection should maintain appropriate fluidity. For example, appropriate fluidity can be maintained by using a coating such as lecithin, by using a surfactant, etc. The effect of preventing microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.

[0130] In some embodiments, the pharmaceutical composition can be used by mixing with one or more other drugs. In some embodiments, the pharmaceutical composition comprises at least one other drug. In some embodiments, the other drug is an anti-tumor drug, a cardiovascular drug, an anti-inflammatory drug, an antiviral drug, a digestive system drug, a nervous system drug, a respiratory system drug, an immune system drug, a dermatological drug, a metabolic drug, etc.

[0131] In some embodiments, the pharmaceutical composition can be administered to a subject in need by a suitable route, including but not limited to oral, injection (e.g., intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural, intraperitoneal injection, etc.), mucosal (e.g., intranasal, oral administration, etc.), sublingual, rectal, transdermal, intraocular, and pulmonary administration. In some embodiments, the pharmaceutical composition can be administered intravenously, subcutaneously, orally, intramuscularly, or intraventricularly. In some embodiments, the pharmaceutical composition can be injected intravenously.

[0132] Another aspect of the present application discloses a method for treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a conjugate compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein. In some embodiments, the method further comprises administering one or more therapeutic agents in combination with the conjugate compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

[0133] Another aspect of the present application discloses use of the conjugate compound described in the present application or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in the present application in the preparation of a drug for treating a disease in a subject.

[0134] The application will be described in more detail below by specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present invention in any way. Those skilled in the art will readily appreciate that multiple non-critical parameters can be changed or modified to produce substantially the same results. Example

[0135] The following examples are intended to better illustrate the present invention and should not be construed as limiting the scope of the present invention. All of the following specific compositions, materials and methods, in whole or in part, are within the scope of the present invention. These specific compositions, materials and methods are not intended to limit the present invention, but are only intended to illustrate that specific embodiments are within the scope of the present invention. Those skilled in the art may develop equivalent compositions, materials and methods without adding creativity and without departing from the scope of the present invention. It should be understood that the various modifications made to the method of the present invention may still be included within the scope of the present invention. The inventor intends to include such changes within the scope of the present invention.

[0136] Example 1 Preparation of ligand conjugates

[0137] 1. Preparation of the ligand

[0138] (1) Preparation and purification of CR19208

[0139]

[0140] Rink Amide-AM resin was loaded into a solid phase reaction column, dichloromethane was added, nitrogen was bubbled into the solvent, the resin was swollen for 30 minutes, and the dichloromethane was drained; the Fmoc protecting group was removed with DBLK, Fmoc-Lys(Dde)-OH, HOBt and HBTU were weighed, dissolved with DMF and dichloromethane, DIPEA was added to the above solution under a 0°C ice water bath, mixed to activate it for 10 minutes, and the solution was added to the above reaction column. After reacting for 2 hours, the solvent was drained and the resin in the reaction column was washed 3 times. Subsequently, the Fmoc protecting group was removed with DBLK. The above operation was repeated, and Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH and Fmoc-Asp(OtBu)-OH were coupled in sequence according to the structure. Then, the Fmoc protecting group was removed with DBLK, and the intermediate CR19607 (structure shown below), HOBt and PyBop were weighed, and DIPEA was added to the above solution under an ice-water bath at 0°C, and it was activated for 10 minutes by mixing. The solution was added to the above reaction column, and the solvent was drained after 2 small tests. The resin in the reaction column was washed 3 times, and the DMF solution of hydrazine hydrate was used for deprotection 2 times. Fmoc-Glu-OtBu, HOBt and HBTU were weighed, and they were dissolved in DMF and dichloromethane, and DIPEA was added to the above solution under an ice-water bath at 0°C, and it was activated for 10 minutes by mixing. The solution was added to the above reaction column, and the solvent was drained after 2 small tests. The resin in the reaction column was washed 3 times. Then, the Fmoc protecting group was removed with DBLK, and pteroic acid, HOAt, EDC.HCl and DIPEA were weighed, dissolved in DMSO, and reacted at 25°C for 20 hours. The resin was cleaved with a cleavage solution and then filtered. The filtrate was poured into MTBE to precipitate a solid, which was washed with MTBE to obtain a crude product. The crude product was prepared by Pre-HPLC to obtain ligand-01 (ie, CR19208).

[0141]

[0142] (2) Preparation and purification of CR20302

[0143]

[0144] Weigh CR19208 (1.0 eq) and CR20301 (5.0 eq) into a 250 mL three-necked flask, add 40 ml of acetonitrile and 50 ml of water to dissolve them, and adjust the pH of the solution to 7-7.5 with saturated NaHCO3 solution in an ice-water bath. Remove the ice-water bath and react at room temperature. After the intermediate control reaction is completed, obtain the CR20302 reaction solution.

[0145] The obtained CR20302 reaction solution was filtered with a Buchner funnel, and the filter residue was rinsed with 2 times the amount of water and the supernatant was combined. The supernatant was filtered with a 0.45 μm organic membrane, and the filtrate was diluted and loaded to prepare the purification system. Mobile phase A: acetonitrile, mobile phase B: 10 mmol / L ammonium bicarbonate aqueous solution, a 50*250 mm chromatographic column (filler: UniSil 10-120C18 Ultra), a flow rate of 50 ml per minute, and a detection wavelength of 220+254 nm were used to collect the product fractions and freeze-dried to obtain CR20302 with a purity of 99.75%, ESI+[M+2H] 2+ :816.76.

[0146] 2. Preparation and Purification of Ligand Conjugates

[0147] Weigh CR19501 (1.0 eq) and CR20302 (2.8 eq) in a 1L three-necked flask, add 300 ml of acetonitrile and 280 ml of water to dissolve them, place in an ice-water bath, control the internal temperature at 10°C, protect with nitrogen and avoid light, and adjust the solution pH to 7.5-8 with a saturated NaHCO3 solution. Remove the ice-water bath and react at low temperature. After the intermediate control reaction is completed, obtain the CR19503 reaction solution.

[0148] The obtained CR19503 reaction solution was filtered with a Buchner funnel (0.45 μm organic membrane), and the filtrate was loaded to prepare the purification system mobile phase A: 100 mmol / L triethylamine aqueous solution, acetic acid was adjusted to pH = 6.0, mobile phase B: ACN, using a 50*250 mm chromatographic column (filler: UniSil 10-120 C18 Ultra), the flow rate was 50 ml per minute, the detection wavelength was 220+254 nm, the product fractions were collected, and the fractions were loaded by salt replacement, the system mobile phase A: acetonitrile, mobile phase B: 20 ​​mmol / L sodium acetate aqueous solution, using a 50*250 mm chromatographic column (filler: UniSil 10-120 C18 Ultra), flow rate of 50 ml per minute, detection wavelength of 220 + 254 nm, A: B = 5:95, wash twice the volume, then use mobile phase A: acetonitrile, mobile phase B: pure water, elute the column, collect the product fractions, freeze-dry to obtain product CR19503, purity 99.71%, ESI-[MH] - :11404.5.

[0149] CR19503

[0150]

[0151] Example 2 Pharmacodynamic Study of Ligand Conjugates in CT26.WT Model-1

[0152] CDX Model: CT26.WT

[0153] Experimental plan: The prepared cells were inoculated subcutaneously on the right side of BALB / c mice. When the tumor volume expanded to about 100 mm 3 The mice were randomly divided into groups, and a model control group and a drug-treated group were set up. The experimental groups and drug-treated groups are shown in Table 1. The test substance OX40 is an anti-OX40 antibody (purchased from BioXCell; Catalog No. BE0031; Clone: ​​OX-86; Type: Rat IgG1, κ; Purity: greater than 95%; RRID: AB_1107592; Molecular weight: 150 kDa; Product link: https: / / bxcell.com / product / m-cd134-ox40 / ), and CR19501 is CpG (the sequence is shown in SEQ ID NO: 1).

[0154] Table 1. Animal experimental groups and dosing regimens

[0155]

[0156] *: 50 μg / 25 μl means that the injection is 25 μl (obtained by dissolving the active substance in PBS) and contains 50 μg of the active substance.

[0157] **: 5 mg / kg means that the anti-OX40 antibody is administered according to the mouse body weight, wherein 5 mg of the antibody is administered per kilogram of body weight.

[0158] ***: 45μg / 0.1ml means that the injection is 0.1ml, which contains 45μg of active substance.

[0159] #: qw*3w means that the drug is administered once a week and the experimental cycle is 3 weeks, that is, the drug is administered a total of 3 times in 1 experimental cycle.

[0160] ##: Single means that the drug is administered only once during the experimental study.

[0161] ###: biw*3w means that the drug is administered twice a week, and the experimental cycle is 3 weeks, that is, a total of 6 doses are administered in 1 experimental cycle.

[0162] After grouping and drug administration, monitoring included tumor growth and the effects of treatment on the normal behavior of animals, including the activity of experimental animals, food and water intake, weight gain or loss, eyes, fur and other abnormalities. After grouping and drug administration, the weight of mice was measured twice a week, and the weight change rate was calculated; at the same time, the long and short diameters of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor growth rate, tumor volume inhibition rate and other indicators were calculated. The tumor volume formula is TV = 0.5a × b 2 , where a is the long diameter of the tumor and b is the short diameter of the tumor.

[0163] Experimental results: As shown in Table 2, when CR19501 was administered as a single drug (Group 2), there was no obvious tumor inhibition effect at a dose of 50μg / 25μl. When the ligand conjugate CR19503 was administered as a single drug (Groups 4, 5 and 6), it had a significant tumor inhibition effect at doses of 45μg / 0.1ml and 75μg / 0.1ml, wherein the relative tumor inhibition rate TGI (%) of Groups 4, 5 and 6 reached 72%, 83% and 75%, respectively. At a dose of 75μg / 0.1ml, the tumor of one mouse achieved complete regression. When the anti-OX40 antibody was administered as a single drug (Group 3), there was no obvious tumor inhibition effect at a dose of 5mg / kg. After CR19501 was used in combination with the anti-OX40 antibody, the efficacy was improved (Group 7, TGI=81%). The combination of ligand conjugate CR19503 and anti-OX40 antibody had a fairly high efficacy (Group 8, TGI=93%; Group 9, TGI=96%), and the tumors of several mice in the group completely regressed.

[0164] Table 2. Growth inhibition effect on CDX model (CT26.WT)

[0165] Group Tumor inhibition rate (%) Group 1 -- Group 2 54 Group 3 57 Group 4 72 Group 5 83 Group 6 75 Group 7 81 Group 8 93 Group 9 96

[0166] Example 3 Pharmacodynamic study of ligand conjugates in CT26.WT model-2

[0167] CDX Model: CT26.WT

[0168] Experimental plan: The prepared cells were inoculated subcutaneously on the right side of BALB / c mice. When the tumor volume expanded to about 85 mm 3 The subjects were randomly divided into a model control group and a drug-treated group. The experimental groups and drug-treated regimens are shown in Table 3.

[0169] Table 3. Animal experimental groups and dosing regimens

[0170]

[0171] After grouping and drug administration, monitoring included tumor growth and the effects of treatment on the normal behavior of animals, including the activity of experimental animals, food and water intake, weight gain or loss, eyes, fur and other abnormalities. After grouping and drug administration, the weight of mice was measured twice a week, and the weight change rate was calculated; at the same time, the long and short diameters of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor growth rate, tumor volume inhibition rate and other indicators were calculated. The tumor volume formula is TV = 0.5a × b 2 , where a is the long diameter of the tumor and b is the short diameter of the tumor.

[0172] Experimental results: As shown in Table 4, the ligand conjugate CR19503, at a dose of 45 μg / mouse and 90 μg / mouse at a once-weekly dosing frequency and combined with anti-OX40 antibody (5 mg / kg dose, twice-weekly dosing frequency), produced significant anti-tumor effects on the tumor growth of the mouse syngeneic transplant colorectal cancer model CT26.WT. Moreover, at the same dose, the anti-tumor effect of the multiple dosing (combined with anti-OX40 antibody) dosing regimen was higher than that of the single dosing (combined with anti-OX40 antibody) dosing regimen.

[0173] Table 4. Growth inhibition effect on CDX model CT26.WT

[0174] Group Tumor inhibition rate (%) Group 1 -- Group 2 50 Group 3 62 Group 4 71 Group 5 41 Group 6 52 Group 7 59

[0175] Example 4 Safety Evaluation Study of Ligand Conjugates in BALB / c Mouse Model-1

[0176] Experimental plan: 70 female mice were randomly divided into the following 7 groups according to their body weight: blank control group, CR19501 low, medium and high dose groups and ligand conjugate CR19503 low, medium and high dose groups. The day of grouping was defined as day 0 after grouping, and drug administration began on day 0 after grouping. Animal grouping and drug administration are detailed in Table 5.

[0177] Table 5. Animal experimental groups and dosing regimen

[0178]

[0179]

[0180] Experimental results: The mice tolerated different doses of CR19501 and ligand conjugate CR19503 well, their body weight increased, and no mice died during the experiment.

[0181] The liver and spleen were removed by autopsy at the end of the experiment. Figure 1 and Figure 2 The results showed that in the CR19501 different dose groups, the liver and spleen of mice were significantly enlarged, which was consistent with the liver and spleen weight analysis results, indicating that CR19501 had certain liver and spleen toxicity and side effects, and had a certain dose-effect relationship (further pathological examination is needed to confirm). In the CR19503 different dose groups, the liver and spleen of mice were significantly enlarged, which was consistent with the liver and spleen weight analysis results, indicating that CR19503 had certain liver and spleen toxicity and side effects, and had a certain dose-effect relationship (further pathological examination is needed to confirm).

[0182] Example 5 Safety Evaluation Study of Ligand Conjugates in BALB / c Mouse Model-2

[0183] Experimental plan: 120 female mice were randomly divided into 15 groups according to their body weight, with 8 mice in each group. The day of grouping was defined as day 0 after grouping. Dosing began on day 0 after grouping. The dosing volume of CR19501 and ligand conjugate CR19503 was 0.1 mL / mouse; the dosing volume of anti-OX40 antibody was adjusted according to the most recent body weight of the mouse, and the dosing volume was 10 μl / g. 120 female mice were randomly divided into groups according to their body weight. The experimental groups and dosing schedule are shown in Table 6.

[0184] Table 6. Animal experimental groups and dosing regimen

[0185]

[0186]

[0187] Experimental results: The overall weight of mice showed an increasing trend (see Figure 3 ). On the 13th day after administration, one mouse in group 8 died. On the 16th day after administration, one mouse in group 15 died. On the 20th day after administration, one mouse in group 6 died; one mouse in group 9 died; and one mouse in group 12 died. The results showed that the ligand conjugate CR19503 had a certain degree of tolerability whether used alone or in combination.

[0188] The results of liver and spleen weight showed that when samples were taken immediately after the last dose, the liver and spleen weights of mice in the single-dose group (group 10) of ligand conjugate CR19503 27.5 μg / mouse and the single-dose group (group 14) of ligand conjugate CR19503 55 μg / mouse combined with anti-OX40 antibody were significantly lower than those in the multiple-dose group. There was no significant difference in the liver and spleen weights of mice in the control group (see Figure 4 and Figure 5 ). When samples were collected 4 weeks after the last administration, the liver and spleen of mice in each administration group had recovered significantly, and there was no significant difference in weight compared with the control group (see Figure 6 and Figure 7 ), the results showed that the toxic side effects of the conjugate compound CR19503 can be restored.

Claims

1. Use of a bi-ligand or multi-ligand conjugate or a pharmaceutically acceptable salt thereof and a T-effector cell agonist in the preparation of a medicament for treating a disease in a subject, wherein the bi-ligand or multi-ligand conjugate has a payload linked to a ligand portion, the payload is an immunostimulatory sequence, the disease is a disease that can benefit from T-effector cell agonism, the disease is a solid tumor, wherein, The ligand portion of the bi-ligand or multi-ligand conjugate binds to cell surface molecules, wherein the cell surface molecules are FOLR1 and PSMA, and wherein the ligand portion comprises folic acid or a folic acid analog targeting FOLR1, and the following structure targeting PSMA: The payload is a TLR9 agonist, wherein the TLR9 agonist is a CpG oligonucleotide containing one or more unmethylated CpG motifs, and The T-effector cell agonist is an anti-OX40 antibody or an antigen-binding fragment thereof.

2. The use according to claim 1, wherein the dual-ligand or multi-ligand conjugate or a pharmaceutically acceptable salt thereof can produce a synergistic effect with a T-effector cell agonist.

3. The use according to claim 1 or 2, wherein the dual-ligand or multi-ligand conjugate or a pharmaceutically acceptable salt thereof can upregulate the expression of a target of a T-effector cell agonist.

4. The use according to claim 1, wherein the CpG oligonucleotide comprises any one of the nucleotide sequences shown in SEQ ID NO: 1-11.

5. The use according to claim 1, wherein the CpG oligonucleotide comprises the nucleotide sequence shown in SEQ ID NO:

1. The use according to claim 1 , wherein the bi-ligand or multi-ligand is directly linked or linked via a spacer.

7. The use according to claim 6, wherein the spacer is 1 amino acid, 2 amino acids or 3 amino acids.

8. The use according to claim 6, wherein the spacer is cysteine, lysine, cysteine-lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-glutamate, aspartate-aspartate, aspartate-aspartate-lysine or lysine-cysteine-lysine.

9. The use according to claim 7 or 8, wherein the carboxylic acid in the amino acid in the spacer is amidated.

10. The use according to claim 9, wherein the ligand portion has the following structure:

11. The use according to claim 1, wherein the ligand moiety is directly linked to the payload or is linked via a linker.

12. The use according to claim 11, wherein the ligation is accomplished by a click reaction.

13. The use according to claim 11, wherein the ligand moiety is linked to the payload via a thiol group.

14. The use according to claim 11, wherein the linker comprises the following structure: -S-, -S-(CH2)6-O-, -S-(CH2)6-OP(=S)(OH)- or -S-(CH2)6-OP(=S)(OH)-O-.

15. The method according to claim 1, wherein the ligand conjugate has the following structure:

16. The use according to claim 1, wherein the anti-OX40 antibody is selected from BE0031 of BioXCell, IBI101 of Innovent Biologics, SHR-1806 of Hengrui Medicine, ES102 of Covance Biopharma, BMS-986178 of Bristol-Myers Squibb, PF-04518600 of Pfizer, ABBV-368 of AbbVie or TH002 of Euhe Biopharma.

17. The use according to claim 1, wherein the anti-OX40 antibody is BE0031 from BioXCell.

18. The use according to claim 1, wherein the ligand conjugate or a pharmaceutically acceptable salt thereof and the T-effector cell agonist are administered simultaneously.

19. The use according to claim 1, wherein the T-effector cell agonist is administered some time after the administration of the ligand conjugate or a pharmaceutically acceptable salt thereof.

20. The use according to claim 19, wherein the period of time is from 1 minute to 3 days.

21. The use according to claim 1, wherein the ligand conjugate or a pharmaceutically acceptable salt thereof is administered some time after the administration of the T-effector cell agonist.

22. The use according to claim 21, wherein the period of time is from 1 minute to 3 days.

23. The use according to claim 1, wherein the ligand conjugate or a pharmaceutically acceptable salt thereof is administered by intravenous injection.

24. The use according to claim 1, wherein the T-effector cell agonist is administered intraperitoneally, intravenously or subcutaneously.

25. The use according to claim 24, wherein the T-effector cell agonist is administered by intraperitoneal injection.

26. The use according to claim 1, wherein the ligand conjugate or a pharmaceutically acceptable salt thereof is administered once a week, twice a week, three times a week, once every three days, once every two days, once a day, twice a day, or three times a day.

27. The use according to claim 26, wherein the administration frequency is once a week.

28. The use according to claim 26, wherein the administration frequency is twice a week.

29. The use according to claim 1, wherein the T-effector cell agonist is administered once a week, twice a week, three times a week, once every three days, once every two days, once a day, twice a day or three times a day.

30. The use according to claim 29, wherein the administration frequency is once a week.

31. The use according to claim 29, wherein the administration frequency is twice a week.

32. The use according to claim 1, wherein the ligand conjugate or a pharmaceutically acceptable salt thereof is administered twice a week; and the T-effector cell agonist is administered twice a week.

33. The use according to claim 1, wherein the ligand conjugate or a pharmaceutically acceptable salt thereof is administered at a dose of 1-400 mg per time.

34. The use according to claim 1, wherein the dosage of the ligand conjugate or a pharmaceutically acceptable salt thereof is gradually reduced over the course of treatment.

35. The use according to claim 1, wherein the T-effector cell agonist is administered at a dose of 1-600 mg per time.

36. The use according to claim 1, wherein the dosage of the T-effector cell agonist administered is gradually reduced over the course of treatment.

37. The use according to claim 1, wherein the subject is a mammal.

38. The use according to claim 37, wherein the subject is a human.

39. The use according to claim 1, wherein the solid tumor is selected from the group consisting of liver cancer, breast cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, lung cancer, kidney cancer, ovarian cancer, stomach cancer, uterine cancer, colon cancer, testicular cancer, skin cancer, and prostate cancer.

40. The use according to claim 1, wherein the solid tumor is endometrial cancer.

41. A kit comprising (1) a bi- or multi-ligand conjugate or a pharmaceutically acceptable salt thereof and (2) a T-effector cell agonist, wherein the bi- or multi-ligand conjugate has a payload linked to a ligand portion, the payload being an immunostimulatory sequence, wherein: The ligand portion of the bi-ligand or multi-ligand conjugate binds to cell surface molecules, wherein the cell surface molecules are FOLR1 and PSMA, and wherein the ligand portion comprises folic acid or a folic acid analog targeting FOLR1, and the following structure targeting PSMA: The payload is a TLR9 agonist, wherein the TLR9 agonist is a CpG oligonucleotide containing one or more unmethylated CpG motifs, and The T-effector cell agonist is an anti-OX40 antibody or an antigen-binding fragment thereof.

42. The kit according to claim 41, wherein the CpG oligonucleotide comprises any one of the nucleotide sequences shown in SEQ ID NOs: 1-11.

43. The kit according to claim 41, wherein the CpG oligonucleotide comprises the nucleotide sequence shown in SEQ ID NO:

1.

44. The kit of claim 41, wherein the bi- or multi-ligands are directly linked or linked via a spacer.

45. The kit of claim 44, wherein the spacer is 1 amino acid, 2 amino acids, or 3 amino acids.

46. ​​The kit of claim 44, wherein the spacer is cysteine, lysine, cysteine-lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-glutamate, aspartate-aspartate, aspartate-aspartate-lysine, or lysine-cysteine-lysine.

47. The kit according to claim 45 or 46, wherein the carboxylic acid in the amino acid in the spacer is amidated.

48. The kit of claim 41, wherein the ligand moiety has the structure:

49. The kit of claim 41, wherein the ligand moiety is linked to the payload directly or through a linker.

50. The kit of claim 49, wherein the ligation is accomplished by a click reaction.

51. The kit of claim 49, wherein the ligand moiety is linked to the payload via a thiol group.

52. The kit of claim 49, wherein the linker comprises the following structure: -S-, -S-(CH2)6-O-, -S-(CH2)6-OP(=S)(OH)-, or -S-(CH2)6-OP(=S)(OH)-O-.

53. The kit of claim 41, wherein the ligand conjugate has the following structure:

54. The kit according to claim 41, wherein the anti-OX40 antibody is selected from BE0031 of BioXCell, IBI101 of Innovent Biologics, SHR-1806 of Hengrui Medicine, ES102 of Covance Biopharmaceuticals, or TH002 of Euhe Biopharmaceuticals.

55. The kit of claim 41, wherein the anti-OX40 antibody is BE0031 from BioXCell.

56. The kit of claim 41, wherein the kit is configured so that the ligand conjugate or a pharmaceutically acceptable salt thereof and the T-effector cell agonist are administered simultaneously.

57. The kit of claim 41, wherein the kit is configured to administer the T-effector cell agonist some time after administration of the ligand conjugate or a pharmaceutically acceptable salt thereof.

58. The kit of claim 57, wherein the period of time is from 1 minute to 3 days.

59. The kit of claim 41, wherein the kit is configured to administer the ligand conjugate or a pharmaceutically acceptable salt thereof some time after administration of the T-effector cell agonist.

60. The kit of claim 59, wherein the period of time is from 1 minute to 3 days.

61. A bi- or multi-ligand conjugate or a pharmaceutically acceptable salt thereof, the bi- or multi-ligand conjugate comprising a payload linked to a ligand portion that targets a cell surface molecule, wherein the payload is an immunostimulatory sequence, wherein, The ligand portion of the bi-ligand or multi-ligand conjugate binds to cell surface molecules, wherein the cell surface molecules are FOLR1 and PSMA, and wherein the ligand portion comprises folic acid or a folic acid analog targeting FOLR1, and the following structure targeting PSMA: and The effective load is a TLR9 agonist, wherein the TLR9 agonist is a CpG oligonucleotide containing one or more unmethylated CpG motifs.

62. The ligand conjugate according to claim 61 or a pharmaceutically acceptable salt thereof, wherein the CpG oligonucleotide comprises any one of the nucleotide sequences shown in SEQ ID NOs: 1-11.

63. The ligand conjugate according to claim 61 or a pharmaceutically acceptable salt thereof, wherein the CpG oligonucleotide comprises the nucleotide sequence shown in SEQ ID NO:

1.

64. The ligand conjugate according to claim 61 or a pharmaceutically acceptable salt thereof, wherein the two ligands in the bi-ligand or multi-ligand are directly linked or linked through a spacer.

65. The ligand conjugate according to claim 64 or a pharmaceutically acceptable salt thereof, wherein the spacer is 1 amino acid, 2 amino acids or 3 amino acids.

66. The ligand conjugate of claim 64, or a pharmaceutically acceptable salt thereof, wherein the spacer is cysteine, lysine, cysteine-lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-glutamate, aspartate-aspartate, aspartate-aspartate-lysine, or lysine-cysteine-lysine.

67. The ligand conjugate according to claim 65 or 66, or a pharmaceutically acceptable salt thereof, wherein the carboxylic acid in the amino acid in the spacer is amidated.

68. The ligand conjugate of claim 61 or a pharmaceutically acceptable salt thereof, wherein the ligand portion has the following structure:

69. The ligand conjugate of claim 61 or a pharmaceutically acceptable salt thereof, wherein the ligand moiety is directly linked to the payload or is linked via a linker.

70. The ligand conjugate according to claim 69 or a pharmaceutically acceptable salt thereof, wherein the connection is accomplished by a click reaction.

71. The ligand conjugate of claim 69, or a pharmaceutically acceptable salt thereof, wherein the ligand moiety is linked to the payload via a thiol group.

72. The ligand conjugate of claim 69 or a pharmaceutically acceptable salt thereof, wherein the linker comprises the following structure: -S-, -S-(CH2)6-O-, -S-(CH2)6-OP(=S)(OH)- or -S-(CH2)6-OP(=S)(OH)-O-.

73. The ligand conjugate according to claim 61 or a pharmaceutically acceptable salt thereof, wherein the ligand conjugate has the following structure:

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