Use of small molecule compounds targeting tim-3 in tumor immunotherapy

Abstract

The application discloses application of a small-molecule compound targeting TIM-3 in tumor immunotherapy and relates to the technical field of immunology and antitumor drugs. The small-molecule compounds DTS and CT are safe and verified marketed drugs, and it is found that the small-molecule compounds can bind to TIM-3 protein and regulate T cell exhaustion related to TIM-3. + Experiments prove that DTS and CT can effectively inhibit CD8 T cell exhaustion, promote expansion of exhausted precursor T cells, and comprehensively enhance T cell antitumor immune function. In a melanoma model, the compounds significantly inhibit tumor growth and reduce the proportion of tumor-infiltrating T cell exhaustion. The application uses the strategy of "new use of old drugs", provides a TIM-3 small-molecule inhibitor with high safety, oral administration, strong penetration and low cost, effectively solves the clinical transformation predicament of existing antibody drugs and newly synthesized small molecules, and provides a new scheme for rapid transformation of tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the fields of immunology and antitumor drug technology, and in particular to the application of small molecule compounds targeting TIM-3 in tumor immunotherapy. Background Technology

[0002] Immune checkpoint blockade (ICB) therapy has become a recognized and effective treatment for cancer, following surgery, radiotherapy, and chemotherapy. Therapeutic antibodies targeting programmed death receptor 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have induced relatively durable anti-tumor responses in various tumor types by restoring the effector function of exhausted T cells. However, a large proportion of cancer patients do not respond well to PD-1 or CTLA-4 antibody therapy; therefore, identifying novel immune checkpoint molecules as therapeutic targets is crucial.

[0003] T cell immunoglobulin and mucin domain-3 (TIM-3) is involved in T cell terminal differentiation and exhaustion during chronic viral infection and tumor progression, and is one of the immune checkpoints that has received widespread attention after PD-1 / CTLA-4. Studies have shown that TIM-3 plays a crucial role in the depletion of CD8+ cells. + High expression of TIM-3 on the surface of T cells, and antibodies targeting TIM-3 can reverse T cell exhaustion and promote tumor regression. Therefore, targeting TIM-3 has become an important strategy for reversing T cell exhaustion and enhancing anti-tumor immunity.

[0004] For the TIM-3 target, current technologies mainly focus on antibody drug development. Anti-TIM-3 monoclonal antibodies such as Sabatolimab (MBG453), Cobolimab (TSR-022), and LY3321367 have entered different stages of clinical trials, attempting to reverse depleted T cell function and enhance the body's anti-tumor immune response by blocking the interaction between TIM-3 and its ligands (Galectin-9, HMGB1, PtdSer, CEACAM1, etc.).

[0005] In the field of small molecule TIM-3 inhibitors, selective small molecule inhibitors that directly target TIM-3 are still in the exploratory stage, with few public reports. Existing research mainly focuses on the discovery and optimization of newly synthesized lead compounds.

[0006] The existing technology still has the following limitations and shortcomings: (1) Inherent limitations of macromolecular antibody drugs: The molecular weight of monoclonal antibody immune checkpoint inhibitors is usually around 150 kDa, which has the following shortcomings: poor penetration into tumor tissue, making it difficult to effectively penetrate the dense extracellular matrix of solid tumors; limited administration methods, requiring intravenous injection, resulting in poor patient compliance; high preparation costs, relying on mammalian cell expression systems; immunogenicity risks, which may induce anti-drug antibodies (ADA) and serious immune-related adverse events (such as immune pneumonia, colitis, hepatitis, etc.).

[0007] (2) The clinical translation dilemma of existing TIM-3 antibodies: Anti-TIM-3 antibodies such as Sabatolimab (MBG453) have entered the clinical research stage and are being explored for combination therapy in indications such as MDS, AML and solid tumors. However, the efficacy of anti-TIM-3 monoclonal antibody monotherapy in the field of solid tumors is still limited, suggesting that the strategy of simply blocking the interaction between the extracellular domain of TIM-3 and its ligand may be incomplete at the mechanistic level and cannot fully regulate the TIM-3-mediated intracellular signaling cascade. At the same time, antibody macromolecular drugs have inherent limitations such as being unable to be administered orally, difficulty in penetrating the blood-brain barrier, and high preparation costs. There is an urgent need to develop small molecule TIM-3 targeted drugs with complementary advantages.

[0008] (3) Limitations of existing small molecule compounds: Most of the publicly reported TIM-3 small molecule inhibitors are newly synthesized lead compounds, which require complete toxicological studies, pharmacokinetic studies and phase I and phase II clinical trials to be carried out from scratch. The clinical translation cycle is long (usually more than 10 years), with high risks and high costs, and it is difficult to achieve clinical application in the short term.

[0009] (4) Lack of targeted regulation of exhausted precursor T cells in existing technologies: Existing technologies generally lack targeting of TCF-1 + PD-1 + TIM-3 low The ability of depleted precursor T cells (Tpex) to expand and maintain is insufficient, and the persistence of T cell immune function reconstruction is inadequate, leaving some patients at risk of relapse even after obtaining an initial response.

[0010] In summary, existing technologies in the field of TIM-3-targeted tumor immunotherapy suffer from core drawbacks, including poor penetration of large-molecule antibodies, limited administration routes, high manufacturing costs, immunogenicity risks, and difficulties in clinical translation. While existing small-molecule TIM-3 inhibitors have potential advantages in tissue penetration, ease of administration, and production cost, they are all newly synthesized compounds with limited safety evaluation data and long translation cycles. Therefore, there is an urgent need to develop a compound with well-verified safety, that specifically binds to TIM-3, and combines the advantages of small molecules to reverse T cell exhaustion, improve the immune function of the tumor microenvironment, and rapidly enter clinical translation. Summary of the Invention

[0011] In view of this, the purpose of the present invention is to provide an application of a small molecule compound targeting TIM-3 in tumor immunotherapy, so as to solve the problems existing in the prior art.

[0012] In a first aspect, the present invention provides the use of the compounds dinoprost tromethamine (DTS) and / or carboprost tromethamine (CT), or pharmaceutically acceptable salts thereof, in the preparation of a medicament targeting the TIM-3 protein.

[0013] Specifically, the structure of DTS is as follows: Drug name: Dinoprost tromethamine salt (DTS); The structure of a CT scanner is shown below: Drug name: Carboprost tromethamine (CT).

[0014] Furthermore, the drug targeting the TIM-3 protein is a TIM-3 specific inhibitor, wherein the compound specifically binds to the TIM-3 protein and blocks the interaction between the TIM-3 protein and its ligands (Galectin-9, HMGB1, PtdSer, CEACAM1).

[0015] In a second aspect, the present invention provides the use of the compounds dinoprost tromethamine and / or carboprost tromethamine, or pharmaceutically acceptable salts thereof, in the preparation of an antitumor drug targeting the TIM-3 protein, wherein the tumor is a solid tumor that highly expresses TIM-3.

[0016] Furthermore, the tumor is a melanoma.

[0017] Furthermore, in the antitumor drug, the therapeutically effective dose of the compound is 1 mg / kg to 5 mg / kg.

[0018] Furthermore, the antitumor drug also includes pharmaceutically acceptable excipients.

[0019] Furthermore, the dosage forms of the antitumor drugs include, but are not limited to, oral formulations (tablets, capsules), injectable formulations, etc.

[0020] Furthermore, the routes of administration of the antitumor drugs include intraperitoneal injection, oral administration, and intravenous injection.

[0021] In a third aspect, the present invention provides the compounds dinoprost tromethamine and / or carboprost tromethamine, or pharmaceutically acceptable salts thereof, in the preparation of CD8-promoting proteins by targeting TIM-3. + Application in drugs that promote T cell proliferation.

[0022] Furthermore, the promotion of CD8 + T cell proliferation includes promoting the depletion precursor CD8. + T cell proliferation and / or suppression of CD8 + T cell depletion.

[0023] Promote CD8 + T cell proliferation specifically involves downregulating TIM-3-positive CD8+ cells. + T cell ratio; promote CD8 + T cell proliferation and cell viability, and increased CD8 + The proportion of T cells.

[0024] The depletion precursor CD8 + T cell proliferation specifically involves upregulating TCF-1-positive CD8+ cells. + T cell ratio.

[0025] In a fourth aspect, the present invention provides the compounds dinoprost tromethamine and / or carboprost tromethamine, or pharmaceutically acceptable salts thereof, in the preparation of CD8 enhanced by targeting the TIM-3 protein. + Application in drugs that enhance the anti-tumor effect of T cells.

[0026] Furthermore, the compound enhances CD8 + T cells secrete TNF-α, Granzyme B, and IFN-γ, thereby enhancing CD8+. + T-cell anti-tumor effect.

[0027] In a fifth aspect, the present invention provides the use of dinoprost tromethamine, carboprost tromethamine, in combination with a PD-1 / PD-L1 inhibitor, or dinoprost tromethamine, carboprost tromethamine, in combination with a CTLA-4 inhibitor, in any of the following: (1) Application in the preparation of drugs for treating melanoma; (2) Application in the preparation of drugs that inhibit the proliferation of melanoma cells; (3) Application in the preparation of drugs that promote melanoma cell apoptosis; (4) Preparation of CD8, a precursor that promotes depletion + Application in drugs for T cell proliferation; (5) Preparation of CD8 inhibitors + Applications in drugs that deplete T cells; (6) Preparation of enhanced CD8 + Application in drugs that enhance the anti-tumor effect of T cells.

[0028] This invention adopts a "drug repurposing" strategy. Dinoprost tromethamine (DTS) and carboprost tromethamine (CT) already have certain clinical or pharmaceutical safety data, which can significantly shorten the research and development cycle, reduce the risk of clinical translation, and fill the gap in the safety data of TIM-3 targeted small molecules in the existing technology.

[0029] This invention employs a drug retargeting strategy, and the discovered active compounds have existing pharmaceutical research or clinical safety data as a foundation. This is expected to reduce some repetitive safety evaluation work, shorten the research and development cycle, reduce translational risks, and accelerate the translation of drugs from basic research to clinical application.

[0030] The compounds DTS and CT discovered in this invention both belong to the prostaglandin class of structure-related compounds. Comparing their functions under the same target background provides a unique perspective for structure-activity relationship analysis and candidate compound optimization. Compared with the approach of conducting research on only a single compound, this invention can provide more information for the structure-activity relationship analysis of compounds with similar structures.

[0031] The TIM-3-targeting small molecule compounds DTS and CT provided by this invention have molecular weights much smaller than those of monoclonal antibodies, exhibiting good tissue permeability. They can effectively penetrate the dense tumor microenvironment of solid tumors, establishing sufficient effective concentrations locally, thereby exerting a more complete immunomodulatory effect.

[0032] The small molecule compounds involved in this invention can be prepared by chemical synthesis methods, which are relatively simple and have low production costs, thus helping to improve drug accessibility and benefit a wider range of patients.

[0033] The small molecule compounds DTS and CT provided by this invention have the potential to be developed into oral formulations, which can significantly improve patient convenience and compliance, and broaden clinical application scenarios.

[0034] The small molecule compounds DTS and CT of this invention can bind to the TIM-3 protein and effectively inhibit CD8 in T cell culture systems by regulating the TIM-3-mediated signaling pathway. + T cell depletion process, promoting TCF-1 + PD-1 + TIM-3 low The efficient expansion of exhausted precursor T cells (Tpex) enhances the secretion levels of effector molecules TNF-α, Granzyme B, and IFN-γ.

[0035] The small molecule compounds DTS and CT target TIM-3 in this invention, which have the potential to synergize with existing PD-1 / PD-L1 inhibitors and CTLA-4 inhibitors. This provides a new combination therapy to overcome the clinical difficulties of insufficient efficacy and non-response in some patients caused by existing single immune checkpoint blockade therapy, and is expected to further improve the objective response rate and survival benefit of patients with melanoma and other tumors.

[0036] The compounds DTS and CT of this invention are small molecule non-protein substances. Compared with antibody drugs, they have a lower risk of inducing anti-drug antibodies (ADA). At the same time, these compounds have been approved by the FDA and their safety has been fully validated in clinical trials. They are expected to reduce the risk of immunogenicity associated with antibody drugs and have a good safety basis.

[0037] The compound of this invention can bind to the TIM-3 protein and effectively inhibit CD8 in T cell culture systems by regulating the TIM-3-mediated signaling pathway. + The process of T cell depletion promotes the efficient expansion of depleted precursor T cells (Tpex) and enhances the secretion levels of effector molecules TNF-α, Granzyme B, and IFN-γ, thereby achieving more precise and in-depth T cell immune function reconstruction at the level of cell subset regulation, making up for the shortcomings of existing antibody strategies in this regard.

[0038] In summary, this invention provides a novel, low-cost, safe, and clinically translational technical solution for the immunotherapy of melanoma and other tumors by offering a small molecule compound with fully validated safety that targets TIM-3, thereby achieving effective intervention in T cell depletion and functional remodeling of the tumor microenvironment.

[0039] This invention utilizes a highly efficient virtual screening platform based on the TIM-3 protein structure to discover small molecule compounds with TIM-3 binding activity from the TargetMol compound library. The specific technical steps are as follows: I. Discovery of small molecule compounds targeting TIM-3: Virtual screening technology Step 1: Virtual Filtering Using the TIM-3 crystal structure (PDB number: 3KAA) as the acceptor, water molecules and ligands were removed to optimize the protein structure and minimize its energy. The main active site of TIM-3 is the FG-CC' loop region (IgV domain cleft). The predicted active pockets for protein binding are (TRP41, SER42, GLN43, ARG50, ARG93, GLN95, PHE96, PRO97, GLY98, LEU99, MET100, ASN101, ASP102, LYS104). Before docking, the center (X:23, Y:-14, Z:39) and size (12 Å) of the box were set according to the active pockets. For small molecule compound processing, LigPrep was first used to convert compounds from the TargetMol compound library to 3D form. Then, Schrodinger's QikProp was used to perform initial screening of 40,000 compounds using the Lipinski rule to determine their druglikeness and predicted oral bioavailability. We further used the Virtual Screening Workflow module to perform high-throughput virtual docking on small molecule compounds. The top 10% of the initial docking SP scores were retained, resulting in 2750 compounds. Further precise docking XP scores were used to retain the top 1%, resulting in 27 compounds with XP scores above -8.5. After evaluating the MM / GBSA scores of these 27 compounds, we selected 10 compounds with the highest MM / GBSA scores for in vitro experimental verification.

[0040] Step 2: Verification of in vitro binding activity Surface plasmon resonance (SPR) and micro-thermophoresis (MST) experiments were used to detect the binding affinity (kd value) of compounds DTS and CT to recombinant his-labeled TIM-3 protein, thus verifying the binding ability of the compounds to TIM-3.

[0041] II. In vitro functional verification scheme for compounds inhibiting T cell exhaustion Spleens were collected from 6-8 week old OT-I mice or OT-I / TCF-1-GFP double-positive mice (obtained by hybridization of OT-I and TCF-1-GFP mice). After grinding and erythrocyte lysis, the cells were resuspended in complete culture medium containing 10 ng / mL interleukin-2 (IL-2), 1 ng / mL interleukin-7 (IL-7), 50 μM β-mercaptoethanol, 10 ng / mL OVA257-264 peptide, and 10% heat-inactivated fetal bovine serum (FBS), and the cell concentration was adjusted to 1×10⁻⁶. 6The concentration of compounds obtained from the virtual screening was increased to 10 μM during the activation process. An equal volume of dimethyl sulfoxide (DMSO) was added to the control group for preliminary screening. After day 10, exhausted precursor T cells (TCF-1) were analyzed by flow cytometry. + PD-1 + TIM-3 low (i.e., Tpex subset) and terminally exhausted T cells (PD-1) + TIM-3 + The proportion of Tex subsets was used to verify the regulatory effects of the screened compounds on exhausted precursor T cells and the T cell exhaustion process. The screened compounds were used to treat T cells at different concentrations (5 μM, 10 μM, and 20 μM) to verify the optimal concentration for inhibiting T cell exhaustion.

[0042] III. Verification of the effects of compounds on T cell function CD8 cultured + T cells were divided into a control group (DMSO) and an experimental group (supplemented with DTS and CT, final concentration 20 μM). The drugs were continuously administered during T cell culture until day 10. After cell collection, flow cytometry was used to detect the secretion levels of intracellular cytokines TNF-α, IFN-γ, and Granzyme B, comprehensively evaluating the effect of the compounds on enhancing effector T cell function.

[0043] IV. Verification of the effect of compounds enhancing T-cell therapy C57BL / 6J mice were given sublethal whole-body irradiation (4.5 Gy) followed by subcutaneous inoculation with B16-OVA tumors (2 × 10⁻⁶ g / L). 6 (cells / animal). On day 12 after inoculation, CD45.1.2 OT-1 CD8 cells were added. + T cells (2×10) 6 (Cells / mouse) were injected into tumor-bearing mice via tail vein, followed by daily injections of DTS and CT (5 mg / kg) from days 12 to 17. Tumor volume was measured regularly and growth curves were plotted. Body weight changes were monitored to assess safety. Tumor tissue was harvested on day 18, weighed, and CD45.1 levels were measured by flow cytometry. + The proportion of T cell exhaustion was used to verify its effect on enhancing T cell-mediated anti-tumor immune responses.

[0044] The beneficial effects of this invention include at least the following: (1) The present invention adopts the strategy of “repurposing old drugs”. The compounds DTS and CT provided are drugs that have been approved by the FDA and their safety has been fully verified. There is no need to conduct toxicology and Phase I clinical studies from scratch, which greatly shortens the research and development cycle and reduces the risk of transformation. At the same time, it has the comprehensive advantages of small molecule drugs such as oral potential, high tissue penetration, low preparation cost and no non-protein immunogenicity, which effectively solves the clinical application dilemma of existing large molecule antibody drugs and newly synthesized small molecule compounds.

[0045] (2) This invention is the first to demonstrate that DTS and CT can bind to TIM-3 protein and can effectively inhibit CD8. + T cell exhaustion specifically upregulates the proportion of exhausted precursor T cell subsets, overcoming the technical limitations of existing antibody strategies in Tpex-directed amplification regulation, and achieving more durable T cell immune function reconstruction.

[0046] (3) The compound of the present invention can significantly enhance CD8 + The levels of TNF-α, IFN-γ, and Granzyme B secreted by T cells enhance the killing ability of T cells against tumor cells. In vivo experiments have confirmed that the compound can significantly inhibit melanoma growth and reduce the proportion of tumor-infiltrating T cells that are exhausted, demonstrating good in vivo anti-tumor efficacy and safety. Attached Figure Description

[0047] Figure 1 To verify the effect of the screened compounds on CD8 in vitro + Regulation of T cell exhaustion. (A) Schematic diagram of experimental procedure; (B) Flow cytometry selection strategy for exhausted T cells; (C) Expression ratio of T cell surface exhaustion markers (PD-1, TIM-3) after treatment with ten candidate compounds; (D) Expression ratio of T cell surface exhaustion markers (PD-1, TIM-3) after treatment with different concentrations of DTS; (E) Expression ratio of T cell surface exhaustion markers (PD-1, TIM-3) after treatment with different concentrations of CT.

[0048] Figure 2 Adding the depleted precursor CD8 to DTS and CT + Validation of T cells. (A) Flow cytometry box selection strategy for exhausted precursor T cells (Tpex); (B) Expression ratio of exhausted precursor T cells.

[0049] Figure 3 The results of DTS and CT on the in vitro regulation of T cell function. (A) Intracellular TNF-α expression level in T cells; (B) Intracellular INF-γ expression level in T cells; (C) Intracellular Granzyme B expression level in T cells; (D) CD8 expression level after DTS or CT treatment. + Detection of the ability of T cells to kill B16-OVA target cells in vitro.

[0050] Figure 4 To verify the inhibitory effect of DTS or CT on T cell exhaustion in tumors in in vivo experiments. (A) Experimental flowchart; (B) Tumor size; (C) Tumor volume; (D) Expression ratio of exhaustion markers (PD-1, TIM-3) on the surface of tumor-infiltrating T cells.

[0051] Figure 5 The results of SPR and MST affinity assays and molecular docking between DTS or CT and TIM-3 protein are shown. (A) SPR affinity assay results of DTS and TIM-3; (B) Micro-thermophoretic assay results of DTS; (C) Molecular docking results of DTS and TIM-3; (D) SPR affinity assay results of CT and TIM-3; (E) Micro-thermophoretic assay results of CT; (F) Molecular docking results of CT and TIM-3. Detailed Implementation

[0052] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0053] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0054] The following specific embodiments illustrate the solution proposed in this invention: Example 1 1. In vitro experimental verification of the effects of DTS and CT on CD8 + Regulation of T cell exhaustion (1) Experimental methods Spleens from 6-8 week old OT-I mice were collected and placed in a culture dish containing 2% FBS solution. The cells were ground to obtain a cell suspension, which was then collected into 15 mL centrifuge tubes. 3 mL of 2% FBS was added, and the grinding was repeated once. The resulting suspension was centrifuged at 350g for 5 minutes at 4°C, and the supernatant was discarded. The cells were resuspended in 1 mL of erythrocyte lysis buffer and lysed on ice for 5 minutes. Lysis was terminated by adding 4 mL of 2% FBS, and the cells were centrifuged again, discarding the supernatant. Cells were resuspended in 1 mL of complete culture medium (containing 10 ng / mL IL-2, 1 ng / mL IL-7, 50 μM β-mercaptoethanol, 10 ng / mL OVA 257–264 peptide, and 10% heat-inactivated FBS). The cell concentration was adjusted to 1×10⁶ cells / mL. 6 Cells / mL. During activation, 20 μM of compound DTS or CT were added to the culture system, while the control group received only an equal volume of DMSO. After day 10, the ratio of exhausted precursor cells to terminally exhausted T cells was analyzed by flow cytometry.

[0055] (2) Experimental results Figure 1 To verify the inhibitory effects of the 10 screened compounds on T cell exhaustion in vitro, the results showed that T4546 (DTS) and T14865 (CT) had the best inhibitory effects. Subsequent treatment with different concentrations of 5 μM, 10 μM, and 20 μM showed that 20 μM DTS and CT had the best inhibitory effects.

[0056] 2. DTS and CT increase the depletion of the precursor CD8 + T cells (1) Experimental methods Spleens were collected from 6-8 week old OT-1 and TCF-1-GFP double-positive mice (mouses that are double-positive by hybridization of OT-1 and TCF-1 mice). The cells were ground in a culture dish containing 2% FBS solution, and the resulting cell suspension was collected into 15 mL centrifuge tubes. Then, 3 mL of 2% FBS was added, and the grinding was repeated once. The resulting suspension was centrifuged at 350 g for 5 minutes at 4°C, and the supernatant was discarded. The cells were resuspended in 1 mL of erythrocyte lysis buffer and lysed on ice for 5 minutes. Lysis was terminated by adding 4 mL of 2% FBS, and the cells were centrifuged again, discarding the supernatant. The cells were then resuspended in 1 mL of complete culture medium (containing 10 ng / mL IL-2, 1 ng / mL IL-7, 50 μM β-mercaptoethanol, 10 ng / mL OVA 257–264 peptide, and 10% heat-inactivated FBS). The cell concentration was adjusted to 1 × 10^6 cells / mL. 6Cells / mL. During activation, 20 μM of compound DTS or CT were added to the culture system, while the control group received only an equal volume of DMSO. After 10 days of culture, exhausted precursor cells (TCF-1) were analyzed by flow cytometry. + PD-1 + TIM-3 low (i.e., the Tpex subset) and the proportion of exhausted T cells.

[0057] (2) Experimental results Figure 2 Results of increasing the proportion of exhausted precursor T cells (Tpex) with compounds DTS and CT. Experimental results show that treatment of T cells with 20 μM of compounds DTS or CT can significantly increase the proportion of exhausted precursor T cells.

[0058] 3. Regulation of T cell function in vitro by DTS and CT (1) Experimental methods CD8 stimulated by OVA + T cells were divided into a control group (DMSO control) and an experimental group. DTS or CT was added during T cell culture to a final concentration of 20 μM. On day 10, cells were collected, and the secretion levels of intracellular cytokines TNF-α, IFN-γ, and Granzyme B were detected using flow cytometry.

[0059] CD8 without OVA stimulation + T cells were divided into a control group and an experimental group. The experimental group was treated with 20 μM DTS or CT for 48 hours. The resulting T cells were then co-cultured with B16-OVA target cells at a 3:1 ratio for 18 hours. Cells were collected, and 1 mL of PBS was added to each sample. The cells were centrifuged at 350g for 3 minutes, the supernatant was removed, and the cells were washed once more. Each sample was then incubated with 100 μL of 1×binding buffer (containing PI staining solution 1:1000 and 5 μL of APC Annexin V antibody) at room temperature in the dark for 10-15 minutes. Annexin V was analyzed. + The proportion of tumor cells was used to assess the in vitro antitumor ability of T cells.

[0060] (2) Experimental results Figure 3 The results show the effects of compounds DTS and CT on T cell function. The experimental results indicate that, compared to the control group, CD8... + The expression levels of cytokines TNF-α, INF-γ, and Granzyme B in T cells were all elevated. In addition, Annexin V... +Staining results showed that, compared with the control group, the proportion of Annexin V positive cells was significantly increased after DTS or CT treatment, suggesting that DTS or CT treatment can enhance target cell apoptosis. The Annexin V positive proportions in the DTS group and the CT group were similar, suggesting that their effects on enhancing T cell-mediated target cell apoptosis may be comparable.

[0061] 4. In vivo experiments to verify the inhibitory effect of DTS or CT on T cell exhaustion in tumors. (1) Experimental methods C57BL / 6J mice were given sublethal whole-body irradiation (4.5 Gy) followed by subcutaneous inoculation with B16-OVA tumors (2 × 10⁻⁶ g / L). 6 (cells / animal). On day 12 after tumor inoculation, CD45.1.2 OT-1 CD8 were injected. + T cells (2×10) 6 (Cells / mouse) were injected into tumor-bearing mice via tail vein, followed by daily injections of DTS or CT (5 mg / kg) from days 12 to 17. Tumor volume was measured periodically, and growth curves were plotted. Mouse weight changes were monitored to assess safety. Tumor tissue was harvested on day 18, weighed, and CD45.1 levels were measured by flow cytometry. + PD-1 in T cells + TIM-3 + Cell ratio.

[0062] (2) Experimental results Figure 4 To evaluate the effects of DTS or CT on the in vivo inhibition of T cell exhaustion in a B16-OVA tumor-bearing model. Compared with the Mock group, infusion of activated OT-1 T cells partially inhibited tumor growth; however, combined DTS treatment further suppressed tumor growth, significantly reducing the final tumor size, with effects similar to the combined CT group. Flow cytometry results showed that the Mock group had tumor infiltration of CD8... + PD-1 in T cells + TIM-3 + The proportion of double-positive cells was high, while DTS or CT treatment significantly reduced this proportion, suggesting that these two compounds may work by reversing CD8. + T cell depletion and improvement of their functional state can enhance T cell-mediated anti-tumor immune responses.

[0063] 5. Results of DTS or CT affinity assays and molecular docking with TIM-3 protein SPR and MST. (1) Experimental methods To determine whether the compound directly binds to TIM-3 and functions as a TIM-3 inhibitor, this invention used commercially available TIM-3 protein to perform surface plasmon resonance (SPR), microthermophoresis (MST), and molecular docking experiments.

[0064] (2) Experimental results The results are as follows Figure 5 As shown in Figures A and 5D, DTS binds to TIM-3 at a concentration limit (kd) of 5.48 μM, while CT binds at 9.72 μM. Both exhibit concentration-dependent binding to TIM-3 protein in SPR experiments, and both demonstrate good binding affinity. Microthermophoresis (MST) results show that DTS binds to TIM-3 at a kd of 1.42 μM, and CT binds to TIM-3 at a kd of 9.83 μM. Figure 5 B, 5E), and MST experiments further demonstrated that both DTS and CT have good binding ability with TIM-3. Furthermore, molecular docking of the two compounds with the TIM-3 protein structure showed that both could bind to the active pocket of TIM-3, and simultaneously interact with the amino acid residues GLN43, SER42, ASP102, and MET100 of TIM-3 via hydrogen bonding, and form salt bridges with calcium ions (CA119). This indicates a strong intermolecular interaction between the two compounds and TIM-3, and the interaction sites are key sites within the active pocket, overlapping with binding sites previously identified through virtual screening, thus increasing the hit probability. Figure 5 C represents the docking result of TIM-3 and DTS molecules. Figure 5 F represents the docking results between TIM-3 and CT molecules. These results demonstrate that TIM-3 is the target of both compounds.

[0065] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0066] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0067] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The use of the compounds dinoprost tromethamine and / or carboprost tromethamine, or pharmaceutically acceptable salts thereof, in the preparation of medicaments targeting the TIM-3 protein.

2. The application according to claim 1, characterized in that, The compound binds to the TIM-3 protein and regulates TIM-3-related T cell exhaustion.

3. The use of compounds dinoprost tromethamine and / or carboprost tromethamine, or pharmaceutically acceptable salts thereof, in the preparation of antitumor drugs targeting TIM-3 protein, characterized in that, The tumor is a solid tumor that highly expresses TIM-3.

4. The application according to claim 3, characterized in that, The tumor is melanoma.

5. The application according to claim 3 or 4, characterized in that, In the aforementioned antitumor drug, the therapeutically effective dose of the compound is 0.1 mg / kg to 10 mg / kg.

6. The compounds dinoprost tromethamine and / or carboprost tromethamine, or pharmaceutically acceptable salts thereof, are used in the preparation of CD8-promoting compounds by targeting the TIM-3 protein. + Application in drugs that promote T cell proliferation.

7. The application according to claim 6, characterized in that, The promotion of CD8 + T cell proliferation includes promoting the depletion precursor CD8. + T cell proliferation and / or suppression of CD8 + T cell depletion.

8. The compounds dinoprost tromethamine and / or carboprost tromethamine, or pharmaceutically acceptable salts thereof, are used in the preparation of CD8 proteins that enhance CD8 by targeting the TIM-3 protein. + Application in drugs that enhance the anti-tumor effect of T cells.

9. The application according to claim 8, characterized in that, The compound enhances CD8 + T cells secrete TNF-α, Granzyme B, and IFN-γ, thereby enhancing CD8+. + T-cell anti-tumor effect.

10. The use of a combination of dinoprost tromethamine, carboprost tromethamine, and a PD-1 / PD-L1 inhibitor, or a combination of dinoprost tromethamine, carboprost tromethamine, and a CTLA-4 inhibitor in any of the following: (1) Application in the preparation of drugs for treating melanoma; (2) Application in the preparation of drugs that inhibit the proliferation of melanoma cells; (3) Application in the preparation of drugs that promote melanoma cell apoptosis; (4) Preparation of CD8, a precursor that promotes depletion + Application in drugs for T cell proliferation; (5) Preparation of CD8 inhibitors + Applications in drugs that deplete T cells; (6) Preparation of enhanced CD8 + Application in drugs that enhance the anti-tumor effect of T cells.

Images