Method and drug for enhancing Anti-tumor efficacy of immune checkpoint inhibitor, and use thereof

Abstract

Provided are a method and drug for enhancing the anti-tumor efficacy of an immune checkpoint inhibitor, and use thereof in the treatment of tumors. The method for enhancing the anti-tumor efficacy of the immune checkpoint inhibitor is to improve the level of thyroid-stimulating hormone (TSH) in the body of a patient, and comprises combined use of the immune checkpoint inhibitor and TSH or / and a substance having the function of improving the level of TSH in the body. Compared with using the immune checkpoint inhibitor alone, the provided method and drug, when combined with the immune checkpoint inhibitor in an anti-tumor experiment, can significantly improve the anti-tumor efficacy: reducing the tumor volume, improving the survival time, etc., which can solve the current technical problem that the therapeutic effect of treatment with the immune checkpoint inhibitor is generally unsatisfactory.

Description

[Corrected 19.11.2024 according to Rule 91] Methods and drugs for enhancing the anti-tumor efficacy of immune checkpoint inhibitors and their applications Technical Field

[0001] The present invention belongs to the field of medicine and biotechnology, and relates to anti-tumor treatment, in particular to methods and drugs for synergistic treatment with immune checkpoint inhibitors, and specifically to methods and drugs for enhancing the efficacy of immune checkpoint inhibitors in treating tumors and their application in treating tumors. Background Art

[0002] Malignant tumors are one of the biggest challenges facing the world today. The number of people suffering from malignant tumors continues to increase worldwide, and countless patients die from cancer each year. Therefore, developing new cancer treatment strategies and improving the efficacy of existing treatment options have become primary challenges.

[0003] Immune checkpoint inhibitors (ICIs), also known as immune checkpoint blockers (ICBs), are a class of immunotherapy drugs used to treat cancer. They block tumor cells from exploiting the immune system's "checkpoint" mechanism to evade attack. The 2018 Nobel Prize in Physiology or Medicine was awarded to Professors Tasuku Honjo and James Allison, the discoverers of PD-1 and CTLA-4. Currently, ICB therapy is considered a promising clinical treatment for cancer. With the advent of immune checkpoint blockade therapy, drugs targeting immune checkpoints (such as programmed death receptor 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)) have emerged. Currently marketed immune checkpoint inhibitors primarily include PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors, such as nivolumab, pembrolizumab, and ipilimumab. Immune checkpoint inhibitor therapy is currently the most commonly used immunotherapy option and has been approved for the clinical treatment of various tumors, including melanoma, kidney cancer, breast cancer, lung cancer, head and neck cancer, and bladder cancer.

[0004] In recent years, research on immune checkpoints has been burgeoning, and immune checkpoint blockade, as part of cancer immunotherapy, is currently a hot research area. While research on ICB therapy is thriving and related drugs have been released one after another, ICB therapy still suffers from poor efficacy in a significant number of patients. For example, ICB has little to no therapeutic effect on many cancer patients, leaving a large number of patients unable to benefit from ICB therapy.

[0005] Researching and exploring reasonable combination therapies to enhance the therapeutic effects of immune checkpoint inhibitors, that is, to improve the effectiveness of immune checkpoint inhibitors in treating tumors, reduce tumor volume, and improve patient survival rates, is a technical challenge that researchers in this field are eager to solve.

[0006] Summary of the Invention

[0007] The present invention provides a method for enhancing the anti-tumor efficacy of immune checkpoint inhibitors. Another embodiment of the present invention provides a drug for treating tumors. Another embodiment of the present invention provides a drug for enhancing the anti-tumor efficacy of immune checkpoint inhibitors.

[0008] The technical solution for realizing the present invention is:

[0009] The method for enhancing the anti-tumor efficacy of immune checkpoint inhibitors provided by the present invention is to increase the level of thyroid stimulating hormone (TSH) in the patient's body. The method for increasing the level of thyroid stimulating hormone (TSH) in the patient's body is to administer thyroid stimulating hormone (TSH) or / and a drug with the ability to increase the level of thyroid stimulating hormone (TSH) to the patient. The drug administered to the patient with the ability to increase the level of thyroid stimulating hormone (TSH) is thyrotropin-releasing hormone (TRH) or a thyrotropin-releasing hormone analogue that can cause the release of thyroid stimulating hormone (TSH) to increase the level of thyroid stimulating hormone (TSH) in the body. The thyrotropin-releasing hormone analogue is taltirelin, taltirelin tetrahydrate, montirelin, azetirelin, posatirelin, rovatirelin, JTP-2942, NP-654, orotirelin, RX 77368, etc. Increasing the TSH level in the patient's body can be caused by increasing the TRH level in the body. In addition to directly using TRH and TRH analogs, substances that increase the TRH level in the patient's body can also use direct precursors of thyrotropin-releasing hormone (TRH), such as TRH-Gly (TRH-glycine, also known as pE-HPG), TRH precursor peptide, etc.

[0010] The method for enhancing the anti-tumor efficacy of immune checkpoint inhibitors provided by the present invention, wherein the thyroid stimulating hormone (TSH) administered to the patient may be natural human thyroid stimulating hormone or recombinant human thyroid stimulating hormone (rhTSH). The recombinant human thyroid stimulating hormone (rhTSH) may be recombinant human thyroid stimulating hormone alpha (rhTSH alpha), and recombinant human thyroid stimulating hormone alpha (rhTSH alpha) may specifically be Thyrogen. In addition to Thyrogen, SNA001 (SNA001 is a recombinant human thyroid stimulating hormone (rhTSH) drug developed by Zhihe Biotechnology, which can safely and quickly increase human serum thyroid stimulating hormone levels) is also a recombinant human thyroid stimulating hormone (rhTSH) drug, and SNA001 can also enhance the anti-tumor effect of immune checkpoint inhibitors.

[0011] The method for enhancing the anti-tumor efficacy of immune checkpoint inhibitors provided by the present invention can also include administering to the patient a combination of an immune checkpoint inhibitor and thyroid stimulating hormone (TSH) or / and a drug with an increased thyroid stimulating hormone (TSH) level. The immune checkpoint inhibitor can be an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, TIGIT, NKG2A and LAG-3. The drug administered to the patient with an increased thyroid stimulating hormone (TSH) level can be a drug that can act on the hypothalamus-pituitary-thyroid axis to increase the level of thyroid stimulating hormone (TSH) in the body. The drug administered to the patient with an increased thyroid stimulating hormone (TSH) level can be thyrotropin-releasing hormone (TRH) or a thyrotropin-releasing hormone analogue that can cause thyroid stimulating hormone (TSH) to be released, thereby increasing the level of thyroid stimulating hormone (TSH) in the body. The drug thyrotropin-releasing hormone (TRH) administered to the patient with an increased thyroid stimulating hormone (TSH) level can specifically be Protirelin.

[0012] The present invention provides a drug for treating tumors, taltirelin. Research has found that taltirelin has therapeutic effects on tumors. A pharmaceutical formulation for treating tumors can be prepared using taltirelin as an active ingredient in combination with pharmaceutically acceptable excipients. Based on the discovery of taltirelin's therapeutic effects, the present invention provides a method for treating tumors, comprising administering to a patient an effective amount of taltirelin or a drug comprising an effective amount of taltirelin as an active ingredient and pharmaceutically acceptable excipients.

[0013] The present invention also provides a pharmaceutical composition for enhancing the efficacy of immune checkpoint inhibitors in treating tumors, the pharmaceutical composition comprising an immune checkpoint inhibitor and thyroid stimulating hormone (TSH) or / and a substance capable of increasing the level of thyroid stimulating hormone (TSH) in the body, wherein the immune checkpoint inhibitor can be an inhibitor of immune checkpoint molecules selected from PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, TIGIT, NKG2A and LAG-3; the substance capable of increasing the level of thyroid stimulating hormone (TSH) in the body can be thyrotropin-releasing hormone (TRH) or a thyrotropin-releasing hormone analogue that can induce the release of thyroid stimulating hormone (TSH) and thereby increase the level of thyroid stimulating hormone (TSH) in the body, and the thyrotropin-releasing hormone analogue can be taltirelin, taltirelin tetrahydrate, montirelin, azetirelin, posatirelin, rovatirelin, JTP-2942, NP-654, orotirelin, RX 77368, etc.; the thyroid stimulating hormone (TSH) can be natural TSH or rhTSH, the rhTSH can be rhTSH alpha, and the rhTSH alpha can specifically be Thyrogen. The above-mentioned pharmaceutical composition can be combined with pharmaceutically acceptable excipients to prepare a pharmaceutical preparation for treating tumors.

[0014] The present invention also provides the use of thyroid-stimulating hormone (TSH) or / and a substance capable of increasing the level of thyroid-stimulating hormone (TSH) in the body in the preparation of a drug for enhancing the anti-tumor efficacy of immune checkpoint inhibitors, as well as the use of an immune checkpoint inhibitor and thyroid-stimulating hormone (TSH) or / and a substance capable of increasing the level of thyroid-stimulating hormone (TSH) in the body in the preparation of an anti-tumor drug.

[0015] The immune checkpoint inhibitor described in the present invention can be an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, TIGIT, NKG2A and LAG-3. The tumor described in the present invention is a solid tumor or a non-solid tumor. The solid tumor is melanoma, non-small cell lung cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, cervical cancer, small cell lung cancer, mesothelioma, nasopharyngeal carcinoma, renal cell carcinoma, esophageal cancer, head and neck squamous cell carcinoma, Merkel cell carcinoma, cutaneous squamous cell carcinoma, alveolar soft part sarcoma, basal cell carcinoma, biliary tract cancer, bladder cancer, breast cancer, mismatch repair deficient solid tumor, endometrial cancer, multiple myeloma, prostate cancer, gastroesophageal cancer, testicular cancer, thyroid cancer, central nervous system tumor, pancreatic cancer, ovarian cancer, germ cell cancer, bone cancer, sarcoma virus-related cancer and / or high tumor mutation load cancer; the non-solid tumor is leukemia, Hodgkin lymphoma and / or non-Hodgkin lymphoma.

[0016] The present invention has found that thyrotropin-releasing hormone (TRH), thyrotropin-releasing hormone analogs (TRH analogs) and thyroid stimulating hormone (TSH), that is, substances that increase the level of thyroid stimulating hormone (TSH) in the body, have the effect of enhancing the efficacy of immune checkpoint inhibitors in treating tumors. Combining them with immune checkpoint inhibitors can improve the therapeutic effect of immune checkpoint inhibitors, solving the technical problem that most patients who use immune checkpoint inhibitors to treat tumors have unsatisfactory treatment effects in the prior art. Accordingly, thyrotropin-releasing hormone (TRH), thyrotropin-releasing hormone analogs (TRH analogs) and thyroid stimulating hormone (TSH), as well as substances that increase the level of thyroid stimulating hormone (TSH) in the body, can be used to prepare drugs for enhancing the therapeutic efficacy of immune checkpoint inhibitors.

[0017] Thyrotropin-releasing hormone analogs (TRH analogs) refer to substances with a structure similar to TRH and a thyrotropin-releasing effect, or substances with effects similar to thyrotropin-releasing hormone (i.e., substances with similar functions to TRH). Functional similarity means that they can, to a certain extent, replace the effects of thyrotropin-releasing hormone (TRH) and can produce strong and long-lasting multiple effects on the central nervous system (CNS) through TRH receptors, such as stimulating pituitary hormone release, for example, causing the release of TSH. Direct precursors of thyrotropin-releasing hormone (TRH), such as TRH-Gly (TRH-glycine, also known as pE-HPG) and TRH precursor peptide, also fall into this category of analogs.

[0018] References about Protirelin: Some references refer to Protirelin as TRH, i.e., the use of Protirelin is considered as the use of natural TRH. For example: (1) The entry for Thyrotropin releasing hormone on Wikipedia states that Protirelin is the pharmaceutical form of TRH; (2) The book Hormones (Asher Ornoy, Corinna In Drugs During Pregnancy and Lactation (Third Edition), 2015, it is described in 2.15.1 Hypothalamic releasing hormones: “The synthetic substance protirelin is used to perform a test equivalent to natural hypothalamic thyrotropin-releasing hormone (TRH)”; (3) Protirelin (TRH) A Potent Neuromodulator With Therapeutic Potential (doi:10.1001 / archinte.1984.00350180050006) uses protirelin as TRH; some other databases classify protirelin as a TRH analogue, such as: (1) Inxight Drugs database of the National Institutes of Health (NIH): Protirelin is a pharmaceutically available synthetic analogue of the endogenous peptide thyrotropin-releasing hormone (TRH). (2) DrugBank (https: / / go.drugbank.com / drugs / DB09421): Prorelin is a synthetic analog of thyrotropin-releasing hormone.

[0019] TRH analogs: Specifically, TRH analogs include but are not limited to the following substances:

[0020] Taltirelin, a TRH analog, is a synthetic thyrotropin-releasing hormone (TRH) analog that mimics the physiological effects of TRH and can induce the release of thyroid-stimulating hormone, but with a longer half-life and a longer duration of effect. Taltirelin tetrahydrate, a TRH analog, also has the functions of taltirelin mentioned above. Montirelin (NS-3, CG-3703), a TRH analog, is more potent than TRH and has a longer duration of action. In addition, there are some TRH analogs, such as orotirelin (CG-3509), azetirelin, RX 77368, posatirelin, rovatirelin, JTP-2942, NP-654, etc. Some TRH analogs have not been approved for marketing and are still in the animal experiment or clinical trial stage. The present invention has found that it has the effect of enhancing the efficacy of immunotherapy, such as enhancing the anti-tumor therapeutic effect when used in combination with immune checkpoint inhibitors.

[0021] Taltirelin is a TRH analog and a TRH receptor agonist indicated for spinocerebellar degeneration and neurological diseases. It is an oral medication first approved for marketing in Japan in September 2000. Therefore, the present invention first uses a marketed TRH analog as an example. Specifically, Examples 1 and 2 of the present patent application describe in detail the efficacy studies of taltirelin combined with immune checkpoint inhibitors. Example 4 of the present patent application describes the RNA-seq sequencing results of tumor tissues following the efficacy studies of taltirelin combined with immune checkpoint inhibitors.

[0022] Examples 1, 2, 6 and 10 of the present invention describe a pharmacodynamic experiment of a thyrotropin-releasing hormone analogue, taltirelin, in combination with immune checkpoint inhibitors for anti-tumor effects. The pharmacodynamic experiments show that taltirelin can greatly enhance the anti-tumor therapeutic effect of immune checkpoint inhibitors. Example 3 of the present invention describes pharmacodynamic experiments of thyrotropin-releasing hormone (TRH), thyroid stimulating hormone (TSH) and thyroid hormone (TH) in combination with anti-PD-1 for anti-tumor effects. The pharmacodynamic experiments show that thyrotropin-releasing hormone (TRH) and thyroid stimulating hormone (TSH) can greatly enhance the anti-tumor effect of immune checkpoint inhibitors, while thyroid hormone (TH) has no such effect. Example 4 of the present invention describes the results of RNA-seq sequencing of mouse tumor tissues removed after the efficacy experiment in Example 2. The results show that taltirelin combined with immune checkpoint inhibitors can indeed activate anti-tumor-related pathways compared to the use of immune checkpoint inhibitors alone, thereby achieving an enhanced anti-tumor effect. The present invention reveals that taltirelin, TRH, and TSH can all synergize with immune checkpoint inhibitors to enhance the anti-tumor efficacy of immune checkpoint inhibitors, while TH does not have this effect. Based on this, the present invention further reveals that drugs or hormones that can increase thyroid-stimulating hormone (TSH) levels in the body can, to a certain extent, synergize with immune checkpoint inhibitors to achieve anti-tumor effects.

[0023] There is a negative feedback regulation among thyrotropin-releasing hormone (TRH), thyroid-stimulating hormone (TSH), and thyroid hormone (TH) (see Figure 15 of the specification). Thyrotropin-releasing hormone analogues (such as taltirelin) are similar to thyrotropin-releasing hormone (TRH) and can increase the hormone level of its downstream thyroid-stimulating hormone (TSH). Therefore, drugs or hormones that can enhance thyroid-stimulating hormone (TSH) from a mechanistic perspective can, to a certain extent, cooperate with immune checkpoint inhibitors to fight tumors.

[0024] The specific method of increasing the level of thyroid stimulating hormone (TSH) in the body can be to directly administer thyroid stimulating hormone (TSH) to the patient, or to administer to the patient a drug or substance that can increase the level of thyroid stimulating hormone (TSH) in the body. The drug or substance that can increase the level of thyroid stimulating hormone (TSH) in the body acts on the hypothalamus-pituitary-thyroid axis to increase the hormone level of thyroid stimulating hormone (TSH). The drug or substance that can increase the level of thyroid stimulating hormone (TSH) in the body can specifically be thyrotropin-releasing hormone (TRH), thyrotropin-releasing hormone analogues (TRH analogues), etc.

[0025] The present invention experiments reveal that thyrotropin-releasing hormone analogs (TRH analogs), thyrotropin-releasing hormone (TRH) and thyroid stimulating hormone (TSH), that is, substances that can increase the level of thyroid stimulating hormone (TSH) in the body, can all increase the hormone level of thyroid stimulating hormone (TSH) in the body, thereby synergistically enhancing the effect of immune checkpoint inhibitors in anti-tumor treatment, significantly reducing tumor volume, significantly improving survival, and improving patient survival. The research results of the present invention show that thyroid stimulating hormone (TSH) and substances that can increase the level of TSH hormone in the body (including but not limited to chemical drugs, biological drugs, herbal drugs, and hormones, etc.) can significantly improve the anti-tumor therapeutic effect of immune checkpoint inhibitors, solving the technical problem that existing immune checkpoint inhibitors are not ideal in most anti-tumor treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a schematic diagram of the dosing method of Example 1. The day of the first administration is counted as Day 0. The arrow next to the Tumor Bearing marked in the figure points to the tumor-bearing time point of the mice, that is, Day -12 is the day when each group of mice bears tumors. The arrows marked in the figure represent the time points of administration. Dosing began on Day 0. Anti-PD-1 was administered 4 times, with an interval of two days. Taltirelin was administered 6 times, with an interval of one day. In the figure, ip represents intraperitoneal injection.

[0027] Figure 2 is a graph showing changes in tumor volume over treatment days in four groups of mice in the pharmacodynamic experiment in Example 1; the PBS group marked in the figure is the solvent control group; the other groups are the anti-PD-1 group, the taltirelin group, and the anti-PD-1 + taltirelin group, respectively;

[0028] Figure 3 shows the results of the efficacy experiment in Example 1, including four groups: PBS, anti-PD-1 antibody, taltirelin, and the combination of anti-PD-1 and taltirelin, and the corresponding growth curves of tumor volume in mice in each group (data are expressed as mean ± standard deviation); the values ​​shown in the figure are adjusted p-values, which were obtained by two-way analysis of variance followed by Bonferroni multiple comparison test;

[0029] FIG4 shows the survival curves of mice in the four groups of the efficacy experiment in Example 1: PBS, anti-PD-1, taltirelin, and the combination of anti-PD-1 and taltirelin. The p-value = 0.0004 in the figure is based on the results of the Log-rank (Mantel-Cox) test.

[0030] Figure 5 shows a schematic diagram of the dosing regimen of Example 2. The arrow next to the label "Tumor bearing" in the figure indicates the time point when the mice became tumor-bearing, i.e., day -7 is the day when the mice became tumor-bearing. Starting from day 0, i.e., the day when each group of mice began to receive the drug, anti-PD-1 was administered every two days for a total of four doses; taltirelin was administered once a day for a total of six doses. On day 11, the tumors of the mice in each group were removed for subsequent experimental analysis. The term "ip" in the figure indicates the administration regimen via intraperitoneal injection.

[0031] FIG6 shows a graph showing the changes in tumor volume over the number of days of treatment in the four mouse groups in the pharmacodynamic experiment in Example 2; as marked in the figure, the four groups are the PBS group (solvent control group), the anti-PD-1 group, the taltirelin group, and the anti-PD-1 and taltirelin combination treatment group;

[0032] FIG7 shows photos of tumors removed and taken from all experimental mice at the end of the pharmacodynamics experiment in Example 2 (i.e., day 11 after tumor loading);

[0033] Figure 8 shows the relationship between the tumor weights of mice in the four experimental groups (PBS group, anti-PD-1 group, taltirelin group, and anti-PD-1 and taltirelin combined treatment group) measured and plotted at the end of the pharmacodynamic experiment in Example 2 (i.e., 11 days after tumor loading); Kruskal-Wallis H test analysis showed that there were significant differences in tumor volume among the four groups; Mann-Whitney test was further used to compare the combined treatment group with other groups, and the results showed that all comparisons were statistically significant; after correction by the BH method of False Discovery Rate (FDR), all corrected FDR values ​​were less than 0.05, and these values ​​are marked with FDR values ​​on the graph.

[0034] FIG9 shows a tumor inhibition curve diagram showing the relationship between tumor volume change and administration days in Example 2; data are presented as mean ± standard deviation; the values ​​shown in the figure are adjusted p-values, which were obtained by two-way ANOVA followed by Bonferroni multiple comparison test;

[0035] Figure 10 shows the dosing regimen in Example 3. The arrow next to Tumor bearing in the figure points to Day-9, indicating that the mice received tumor-bearing treatment on Day -9. Dosing began on Day 0, with anti-PD-1 administered every two days for a total of four times; while TRH, TSH, and TH were administered once a day for a total of six times. In the figure, ip indicates intraperitoneal injection, and im indicates intramuscular injection. All mice underwent tumor resection on Day 14 for subsequent experiments.

[0036] Figure 11 shows a tumor inhibition curve graph showing the relationship between tumor volume changes and days of administration in Example 3. Tumor volume changes in mice in eight experimental groups (PBS, anti-PD-1, TSH, TRH, TH, anti-PD-1 + TSH, anti-PD-1 + TRH, and anti-PD-1 + TH) were recorded from the day of administration (day 0) to day 11 after administration. The abscissa represents the number of days after tumor loading. Data are presented as mean ± standard deviation, with 8-9 mice in each group. Two-way ANOVA and Bonferroni multiple comparison tests were performed, and the p-values ​​shown in the figure are corrected results.

[0037] FIG12 is a photograph of the in vitro tumors of mice in each group in Example 3, after the surviving mice were sacrificed on the 23rd day after tumor bearing and the tumors of the mice were removed;

[0038] Figure 13 shows photographs of surviving mice taken on days 7 and 13 after the first treatment in Example 3. As shown on the left side of the figure, on day 7, all mice in the eight experimental groups survived. On day 13, as shown on the right side of the figure, solid crosses in the rectangular box indicate dead mice, while dashed crosses indicate mice euthanized due to tumor volumes exceeding ethical limits.

[0039] Figure 14 shows the Kaplan-Meier survival curves of the mice in Example 3, reflecting the survival from the start of drug administration (day 0) to day 13 after tumor loading; among them, all mice in the TSH+anti-PD-1 group and the TRH+anti-PD-1 group survived during this period, while mice in the other groups died;

[0040] Figure 15 shows a schematic diagram of the regulatory relationship between thyrotropin-releasing hormone (TRH), TRH analogs (typically represented by taltirelin, but also applicable to other TRH analogs with similar effects), thyroid-stimulating hormone (TSH), and thyroid hormone (TH); the "check mark" in the figure indicates that TRH, TRH analogs, and TSH can all increase TSH levels, thereby synergistically enhancing the anti-tumor effect of immune checkpoint inhibitors; in contrast, the "cross" in the figure indicates that thyroid hormone (TH) failed to show experimental results showing synergistic enhancement of the anti-tumor effect of immune checkpoint inhibitors;

[0041] FIG16 shows the results of H&E staining of sections of major organs (heart, liver, spleen, lung, and kidney) in Example 3, after the efficacy experiment was completed, i.e., on day 14 after tumor loading, mice were sacrificed; scale bar: 20 μm;

[0042] FIG17 shows the histological observation of major organs (heart, liver, spleen, lung, and kidney) of B16-F10 tumor-bearing mice after a new round of taltirelin efficacy experiments, as shown by H&E staining; scale bar: 20 μm;

[0043] Figure 18 shows the number of immune cells in the tumor tissues of mice in each group: 11 days after treatment, the number of CD8 + T cells, CD8 + GZnB + T cells, CD8 + Tcm cells, CD8 + TEM cells, CD4 + T cells, CD4 + Tcm cells, CD4 + The number of TEM cells, NKT cells, γδT cells, and activated dendritic cells (DCs); data are expressed as mean ± SD (7-10 mice per group, as shown in the figure; one-way ANOVA followed by Bonferroni multiple comparison post-test, the p-value shown in the figure is corrected)

[0044] Figure 19 shows the number of other immune cells in mouse tumor tissues: CD3 + The number of T cells (A) and NK cells (B); data are expressed as mean ± SD (7-10 mice per group; one-way ANOVA followed by Bonferroni multiple comparison post-hoc test; p-values ​​are corrected)

[0045] Figure 20 Detection of BMDCs and CD8 by RT-PCR +Expression of TSH receptor and TRH receptor mRNA in T cells

[0046] Figure 21 shows the cell viability of B16-F10 cells treated with PBS, taltirelin (100 nM, 200 nM, 400 nM, 800 nM, 1600 nM), or rhTSH (10 nM, 20 nM, 40 nM, 80 nM) for 24 hours in a tumor cell killing experiment; data are expressed as mean ± SD (six independent biological samples per group; one-way ANOVA followed by Bonferroni multiple comparison post-test).

[0047] Figure 22 ac is the flow cytometry results: after CD8 + After 5 days of T cell treatment, CFSE was measured by flow cytometry. low CD8 + T cells (A), IFN-γ + CD8 + T cells (B) and GzmB + CD8 + Percentage of T cells (C); Treatment conditions: PBS, taltirelin (500 nM) or TSH (rhTSH, thyrotropin alfa, 20 nM) in the presence or absence of ConA; (ac) Data are expressed as mean ± SD (three biologically independent samples per group; one-way ANOVA followed by Bonferroni multiple comparison post-test; corrected p-values ​​are shown in the figures); Panel d shows that TSH enhances the phagocytic capacity of DCs: the relative phagocytic activity of DIR-labeled BMDCs treated with PBS, taltirelin or TSH on CFSE-labeled B16-F10 cells was determined by flow cytometry after 4 h of co-culture at 37°C; Data are expressed as mean ± SD (three biologically independent samples per group; one-way ANOVA followed by Bonferroni multiple comparison post-test; corrected p-values ​​are shown in the figures); Panel eh shows that TSH enhances antigen presentation: BMDCs treated with PBS, taltirelin or TSH Mean fluorescence intensity (MFI) of CD86 (e), MHC I (f), and MHC II (g) after 24 hours, and in the presence or absence of 10 μg / mL OVA; (h) GzmB expression in OT-I CD8+ T cells after 3 days of co-culture with mature BMDCs. +The percentage of cells was determined by flow cytometry; (eh) Data are expressed as mean ± SD (three independent biological samples per group; one-way ANOVA followed by Bonferroni multiple comparison post-test, the p-values ​​shown in the figure are corrected)

[0048] Figure 23 shows taltirelin activating CD8 + T cells: (A) CD8 T cells were stimulated with PBS, taltirelin (1 nM, 10 nM, 100 nM, 500 nM, 1000 nM), or rhTSH (20 nM) in the presence or absence of ConA (2.5 μg / ml). + T cells 5 days after CFSE low The percentage of cells (C); (B, C) CD8 + IFN-γ in T cells + (B) and GzmB + Percentage of cells (C); Data are expressed as mean ± SD (three independent biological samples per group; one-way ANOVA followed by Tukey's HSD multiple comparison post-test; p-values ​​shown in the figure are corrected)

[0049] Figure 24 shows the expression of PD-1 in CD8 + T cell effects: OT-I CD8 T cells were measured by flow cytometry after 3 days of co-culture with the above mature BMDCs. + PD-1 in T cells + Percentage of cells; data are expressed as mean ± SD (three independent biological samples per group; one-way ANOVA followed by Bonferroni multiple comparison post-test)

[0050] Figure 25 is a schematic diagram of the mechanism by which taltirelin enhances the anti-PD-1 anti-tumor effect: The results of in vitro mechanism experiments show that taltirelin can directly act on CD8 + TRH receptors on T cells promote their proliferation; at the same time, taltirelin effectively increases CD8 + GZMB in T cells + and IFN-γ + The proportion of cells, thereby increasing the CD8 +T cell cytotoxicity; In addition, taltirelin, as an analog of TRH, has similar effects to TRH, negatively feedback regulating TSH levels, that is, taltirelin administration can significantly increase TSH levels in mice; TSH can promote DC maturation by acting on TSH receptors on DCs, and enhance their ability to phagocytize and present tumor antigens by upregulating MHC-I and co-stimulatory molecules, activating CD8 + T cells effectively kill tumors. Through this dual mechanism, taltirelin reshapes the tumor immune environment and significantly enhances the effect of ICB therapy.

[0051] FIG26 is a timetable for an anti-tumor experiment in LLC tumor-bearing mice: The figure shows the dosing schedule for an anti-tumor experiment in LLC tumor-bearing mice: taltirelin was administered every 2 days for a total of 6 doses; anti-PD-1 (100 μg per mouse) was intraperitoneally injected once every 3 days for a total of 4 doses; arrows indicate drug injection times;

[0052] Figure 27 is a drug efficacy experiment: Tumor volume growth curve of LLC tumor-bearing mice treated with taltirelin combined with anti-PD-1 (6-7 mice per group; one-way ANOVA followed by Bonferroni multiple comparison post-test; p-values ​​shown are corrected)

[0053] The lower half of Figure 28 is a visualization of the RNA-seq sequencing results in Example 4; the figure shows the expression of genes corresponding to the six immune-related gene sets (pathways); that is, after the mouse drug administration experiment in Example 2, the expression relationship heat map of the tumor tissue-related genes of each mouse in the PBS group, anti-PD-1 drug administration group, taltirelin drug administration group, and anti-PD-1 combined with taltirelin drug administration group; each column in the heat map represents the expression level of the relevant gene of a tumor tissue; above the expression map is Figure 7 of the specification (i.e., the in vitro tumor map after the drug administration in Example 2), and the order of the tumors in each row in the in vitro tumor map is consistent with the expression heat map of each group. The order of the columns (from left to right) corresponds to the expression of the six gene sets corresponding to the two significantly larger tumors in the anti-PD-1 combined with taltirelin treatment group (i.e., the two circled in the figure, generally considered in the field to be "unresponsive", i.e., almost completely unaffected by immunotherapy) at a low level. The expression of the six gene sets corresponding to the remaining eight significantly smaller tumors in the combination treatment group (generally considered in the field to be typical "responsive", i.e., affected by immunotherapy) at a high level. Similarly, the expression levels of the six gene sets corresponding to the two significantly larger tumors in the PBS + anti-PD-1 group (the two circled in the figure) were at a low level.

[0054] Figure 29: (A) The average tumor weight of the PBS group was used as the baseline (indicated by the red dashed line), and the other three treatment groups were divided into responding and non-responding subgroups; (B) Average tumor growth curves of B16-F10 tumor-bearing mice after treatment; data are expressed as mean ± SD (2-8 mice per group, as indicated in the figure; two-way ANOVA followed by Bonferroni multiple comparison post-test, and the figure shows the corrected p-value). DETAILED DESCRIPTION

[0055] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0056] Example 1: Pharmacodynamic study of a marketed thyrotropin-releasing hormone analogue (taltirelin) combined with anti-PD-1:

[0057] 1. Model building and grouping

[0058] Experimental materials: C57BL / 6J male mice (6-8 weeks old); B16 / F10 subcutaneous tumor model (mouse melanoma cell line B16-F10 was purchased from the Cell Resource Center of Peking Union Medical College); taltirelin was purchased from Aladdin Reagent Co., Ltd., with a specification of 10 mg; anti-PD-1 was purchased from BioXcell, with a specification of 100 mg.

[0059] Grouping: PBS (control group), taltirelin, PBS+anti-PD-1, taltirelin+anti-PD-1, a total of 4 groups, 8 mice in each group.

[0060] The administration of Taltirelin and anti-PD-1 is as follows (see Figure 1 of the manual for a schematic diagram of administration):

[0061] 2. Pharmacodynamics experiments

[0062] After the start of drug administration, the tumor volume of the mice was monitored daily. Tumor growth curves for each group, as shown in Figures 2 and 3 of the accompanying instructions, show that taltirelin alone showed significant differences compared to the PBS control group (adjusted p-value = 0.0020), demonstrating a moderate anti-tumor effect. In contrast, anti-PD-1 alone showed no significant difference compared to the PBS control group in this experiment. The combined therapeutic effect of anti-PD-1 and taltirelin was significantly superior to either monotherapy, indicating that taltirelin significantly enhances the anti-tumor effect of anti-PD-1.

[0063] The experimental results showed that the anti-tumor effect of taltirelin combined with anti-PD-1 was better than that of any single-drug treatment group, and the taltirelin alone treatment group showed a certain anti-tumor effect.

[0064] 3. Survival time

[0065] The following table shows the median survival time between the groups:

[0066] The survival curve is shown in Figure 4 of the accompanying figure. The survival experiment shows that the combination of taltirelin and anti-PD-1 significantly improved the survival of mice compared with the other three groups (the p-value of the Log-rank Mantel-Cox test was 0.0004). This result shows that the combination treatment of taltirelin and immune checkpoint inhibitors (such as anti-PD-1) can significantly improve the survival of mice.

[0067] Example 2

[0068] After completing the efficacy experiment and subsequent survival test in Example 1, we continued with another round of efficacy test in Example 2. This time, after the efficacy test was completed, we removed the tumors of the mice (see Figure 7 of the specification), then weighed the tumors (see Figure 8 of the specification), and performed high-throughput sequencing (bulk RNA-seq) analysis on the tumor tissues.

[0069] The materials used in the experiment include: 6-8 week old C57BL / 6J male mice; B16 / F10 subcutaneous tumor model, the mouse melanoma cell line B16-F10 used was obtained from the Cell Resource Center of Peking Union Medical College; taltirelin was purchased from Aladdin Reagent Co., Ltd., with a specification of 10 mg; anti-PD-1 was purchased from BioXcell, with a specification of 100 mg.

[0070] In this round of efficacy experiments, we set up the following four experimental groups, with the following number of mice in each group: PBS group, taltirelin group, and PBS + anti-PD-1 group, with 8 mice each; and the combination drug group (taltirelin + anti-PD-1) used 10 mice for the experiment.

[0071] The tumor growth curve of the pharmacodynamic experiment of this embodiment is shown in Figure 9 of the specification. After the administration, the mouse tumors were removed and photographed. The photos of the tumors of each group of mice are shown in Figure 7 of the specification; the scatter plot of the tumor weight of each group is shown in Figure 8 of the specification. The anti-tumor effect of the combined use of anti-PD-1 antibodies and taltirelin is significantly better than that of either drug alone. The results of this embodiment once again demonstrate the significant role of taltirelin in enhancing the anti-tumor ability of anti-PD-1 antibodies. In addition, the use of taltirelin or anti-PD-1 antibodies alone also showed significant anti-tumor effects compared with the PBS control group (p-value <0.01), and the overall effect of taltirelin was slightly better than that of anti-PD-1 antibodies, indicating that taltirelin alone has a certain anti-tumor effect. These results are highly consistent with the results of the first pharmacodynamic experiment (Figure 3 of the specification), which proves that the anti-tumor effect of taltirelin in enhancing immune checkpoint inhibitors is stable, and taltirelin itself also exhibits stable anti-tumor properties;

[0072] Based on the mean tumor weight of the PBS group, the taltirelin, anti-PD-1, and combination treatment groups were divided into large and small subgroups. The large tumor volume was defined as the non-responder group (NR), and the small tumor volume was defined as the responder group (R) (Figure 29A). The original four groups were reclassified into seven groups based on response or non-response. Based on the reclassification, a new tumor growth curve was plotted (Figure 29B). The new tumor growth curve more intuitively reflects the comparison between samples that benefited from drug treatment. The mean tumor volume of the eight groups of mice that responded to the combination of taltirelin and anti-PD-1 treatment was only slightly increased compared to the initial mean tumor volume, further demonstrating the significant efficacy of the combination treatment (Figure 29B). In particular, the effect of taltirelin treatment was significantly greater than that of anti-PD-1 treatment (compare the Taltirelin R group and the Anti-PD-1R group in the figure), indicating that taltirelin also has certain anti-tumor properties. However, as shown in FIG29B , the mean weight volume of the non-responsive samples in the taltirelin monotherapy group was almost identical to the mean tumor volume in the PBS group, indicating that the anti-tumor effect of taltirelin was conditional.

[0073] These results clearly demonstrate that the combination therapy is superior to either monotherapy, confirming the significant tumor reduction effect of taltirelin combined with immune checkpoint inhibitors.

[0074] The dosage of taltirelin in the combination drug group of this embodiment is 0.5 mg / kg. Clinically, patients can calculate the dosage based on this dosage and administer the combined anti-PD-1 drug.

[0075] Example 3: Pharmacodynamics test of thyrotropin-releasing hormone (TRH), thyroid-stimulating hormone (TSH), and thyroid hormone (TH) combined with anti-PD-1:

[0076] 1. Test materials

[0077] The B16 / F10 mouse model involved is the same as that in Example 1. Anti-PD-1 was purchased from Wuhan Furuidem Biotechnology Co., Ltd., the product name is: InVivoMab anti-mouse PD-1 (CD279), the specification is 100 mg / vial; TRH was purchased from Shanghai Haoyuan Biotechnology Co., Ltd., the product name is Protirelin (Acetate), the specification is 10 mg / vial; the TSH used is rhTSH, purchased from Hong Kong Huajian Medical Co., Ltd., the product name is: Thyrogen (Shizhejin); the specification is 0.9 mg*2 bottles / box; TH was purchased from Shanghai Haoyuan Biotechnology Co., Ltd., the product name is L-Thyroxine, the specification is 500 mg / vial.

[0078] 2. Grouping, administration method and frequency:

[0079] There were 8 groups in total, including PBS (control group, 8 mice), TRH (8 mice), TSH (9 mice), TH (9 mice), anti-PD-1 (8 mice), TRH+anti-PD-1 (8 mice), TSH+anti-PD-1 (8 mice), and TH+anti-PD-1 (8 mice).

[0080] The day of the first dose was designated as day 0, and the day mice were tumor-bearing was designated day -9. TRH, TSH, and TH were administered every other day, i.e., on days 0, 2, 4, 6, 8, and 10. TRH and TH were administered intraperitoneally (ip), and TSH was administered intramuscularly (im). Anti-PD-1 was administered every two days for a total of four doses, i.e., on days 0, 3, 6, and 9, by intraperitoneal (ip). Surviving mice were sacrificed on day 14 for subsequent toxicity testing.

[0081] Anti-PD-1 administration: 100 μg per mouse per time;

[0082] The TSH used was rhTSH (Thyrogen), and the dosage was 4 μg per mouse per administration. Dosage instructions: Each bottle of Thyrogen contains 1.1 mg of thyrotropin alfa. The packaging specifically states that the extractable active ingredient, thyrotropin alfa, is 0.9 mg per bottle. Therefore, the actual active ingredient should be calculated based on 0.9 mg per bottle. In addition to thyrotropin alfa, each bottle contains other excipients: 36 mg mannitol, 5.1 mg sodium phosphate, and 2.4 mg sodium chloride, for a total of 44.6 mg. The activity value of thyrotropin alfa is 4-12 IU / mg. In this study, the active ingredient was calculated based on a median of 8 IU / mg. Each mouse weighed 20 g, and the 4 μg thyrotropin alfa dose per mouse per administration was equivalent to a theoretical dose of 0.032 IU thyrotropin alfa, or 1.6 IU / kg.

[0083] L-Thyroxine: 2 μg per mouse per dose;

[0084] Protirelin (Acetate): 200 μg was administered to each mouse each time.

[0085] 3. Efficacy test:

[0086] After the start of drug administration, the tumor volume of the mice was monitored every day, and the tumor growth curve is shown in Figure 11 of the specification. In this efficacy experiment, if any mice in a group died or the tumor volume exceeded the ethical limit, the subsequent data recording was stopped.

[0087] 4. Take photos of the tumor after removal:

[0088] After the administration, the mouse tumors were removed and photographed. The photographs are shown in Figure 12 of the specification.

[0089] 5. Tumor inhibition effect (drug efficacy experiment)

[0090] After the dosing experiment, statistical analysis was performed: a two-way analysis of variance revealed the effects of drug and day. The results showed that the anti-tumor effects of TRH + anti-PD-1 and TSH + anti-PD-1 were far superior to those of either drug alone. This result reveals that TRH and TSH have synergistic immunotherapy anti-tumor effects, which can greatly enhance the anti-tumor efficacy of immunotherapy.

[0091] In this example, the TSH + anti-PD-1 group (the TSH used was rhTSH, Thyrotropin) was administered 4 μg of thyrotropin alfa to each mouse each time, which is equivalent to a theoretical dose of 0.032 IU of thyrotropin alfa; i.e., 1.6 IU / kg. This dose has a significant synergistic effect with anti-PD-1, and clinical administration can be calculated based on the patient's weight.

[0092] In this example, the TRH+anti-PD-1 group (the TRH used was Protirelin) was given 200 μg Protirelin per mouse each time. The mouse weight was calculated as 20 g, which was equivalent to a dose of 10 mg / kg. This dose had a significant synergistic effect with anti-PD-1. Clinically, this dose can be calculated based on the patient's weight before administration.

[0093] 6. Mouse survival

[0094] The survival of mice in each group was recorded, and all mice were sacrificed on day 14 for subsequent organ toxicity testing. The survival of the remaining mice was finally recorded on day 13, and the corresponding survival curve is shown in Figure 14. As can be seen from the figure, all mice in the TRH + anti-PD-1 group and the TSH + anti-PD-1 group were alive on day 13, while all other groups had died, indicating that TRH and TSH enhance the anti-tumor effect of anti-PD-1.

[0095] Example 4: High-throughput sequencing

[0096] RNA-seq sequencing was performed on the mouse tumor tissue removed after the efficacy test in Example 2 (as shown in Figure 7 of the specification). The mouse tumor tissue was removed and quickly frozen in liquid nitrogen, then stored in a -80 ° C refrigerator, wrapped in a dry ice box and sent to Haplox for Bulk RNA-seq sequencing. From the sequencing results, the combination drug (anti-PD-1 + taltirelin) group was compared with the two single drug groups (anti-PD-1 alone or taltirelin alone) and the PBS control group. The expression levels of 6 immune-related pathways or gene sets were significantly increased (Figure 28 of the specification). This shows that the combination drug can significantly enhance the activity of immune cells and promote the immune response in the body, thereby better allowing the body's own immune cells to kill tumor cells.

[0097] Figure 7 of the specification shows that the volume of two tumors in the combination group was significantly larger than that of other tumors, that is, the tumors of two mice in the combination group were not inhibited from growing, while the tumors of the remaining seven mice were significantly inhibited from growing. The overall expression levels of the six immune gene-related pathways in the two non-responsive tumors in the combination group were at a low value. The mice corresponding to the two tumors did not respond to the combination treatment. This may be due to experimental accidental factors, or it may be due to differences in the immune levels of the mice, resulting in a small number of mice not benefiting from immunotherapy, or it may be the inherent "dichotomy phenomenon" of immune checkpoint therapy (people in this field should know that as long as there are enough mice receiving immune checkpoint treatment in a drug efficacy experiment, there will always be "responsive" and "non-responsive" mice, and this is not due to poor drug efficacy); while the tumor tissues with relatively high expression levels of other immune genes in the combination group all achieved very significant anti-tumor efficacy of the combination therapy. This reflects from another aspect that taltirelin combined with anti-PD-1 can indeed enhance the activity of immune-related genes, and tumor tissues with significantly elevated expression of related immune genes benefit from the therapeutic effect of taltirelin combined with immune checkpoint inhibitors.

[0098] Example 5: TSH (Thyrogen) Gradient Efficacy Test

[0099] 1. Source of reagents:

[0100] The B16 / F10 mouse model involved was the same as that in Example 1. Anti-PD-1 was purchased from Wuhan Furuidemu Biotechnology Co., Ltd. under the product name InVivoMab anti-mouse PD-1 (CD279), with a specification of 100 mg / vial. The TSH used was rhTSH, purchased from Hong Kong Huajian Medical Co., Ltd. under the product name Thyrogen (Shizhejin), with a specification of 0.9 mg*2 bottles / box.

[0101] 2. Grouping in this embodiment:

[0102] PBS (control group, 8 mice), anti-PD-1 (8 mice), 0.25μg thyrotropin / time / mouse + anti-PD-1 (8 mice), 0.5μg thyrotropin / time / mouse + anti-PD-1 (8 mice), 1μg thyrotropin / time / mouse + anti-PD-1 (8 mice), 2μg thyrotropin / time / mouse + anti-PD-1 (8 mice), 3μg thyrotropin / time / mouse + anti-PD-1 (8 mice), 4μg thyrotropin / time / mouse + anti-PD-1 (8 mice), 6μg thyrotropin / time / mouse + anti-PD-1 (8 mice).

[0103] Mice were counted as day -9 from the day of tumor bearing, and drug administration began on day 0: thyrotropin was administered every other day, i.e., on days 0, 2, 4, 6, 8, and 10, by intramuscular injection (im); anti-PD-1 was administered every 2 days for a total of 4 doses, i.e., on days 0, 3, 6, and 9, by intraperitoneal injection (ip); anti-PD-1 was administered at a dose of 100 μg per mouse each time; rhTSH (Thyrogen) was administered in a gradient manner: 0.25 μg, 0.5 μg, 1 μg, 2 μg, 3 μg, 4 μg, and 6 μg per mouse each time;

[0104] Dosage instructions: Each bottle of Thyrogen contains 1.1 mg of thyrotropin alfa (theoretical dose). The drug packaging specifically states that the extractable active ingredient, thyrotropin alfa, is 0.9 mg per bottle. Therefore, the actual active ingredient should be calculated based on 0.9 mg per bottle. In addition to thyrotropin alfa, each bottle contains other excipients: 36 mg of mannitol, 5.1 mg of sodium phosphate, and 2.4 mg of sodium chloride, for a total of 44.6 mg. The activity value of thyrotropin alfa is 4-12 IU / mg. In this study, the active ingredient was calculated based on the median of 8 IU / mg. Each mouse weighed 20 g, and each mouse was given 0.25 μg, 0.5 μg, 1 μg, 2 μg, 3 μg, 4 μg, and 6 μg of thyrotropin each time. The theoretical dose of thyrotropin alfa, equivalent to 0.002 IU, 0.004 IU, 0.008 IU, 0.016 IU, 0.024 IU, 0.032 IU, 0.048 IU, calculated in IU / kg, is equivalent to 0.1 IU / kg, 0.2 IU / kg, 0.4 IU / kg, 0.8 IU / kg, 1.2 IU / kg, 1.6 IU / kg, 2.4 IU / kg.

[0105] The test results showed that the above seven doses significantly reduced the tumor volume of mice compared with the group using anti-PD-1 alone. Clinically, the dose can be calculated in IU / kg according to the corresponding body weight to coordinate the administration of anti-PD-1.

[0106] Example 6: Taltirelin Gradient Efficacy Test

[0107] 1. Source of reagents:

[0108] The B16 / F10 mouse model involved is the same as that in Example 1. In this example, anti-PD-1 was purchased from BioXCell, the product name is: Anti-mouse PD-1, the specification is 100 mg; the taltirelin used was purchased from Aladdin Reagent Co., Ltd., the specification is 100 mg;

[0109] 2. Grouping in this embodiment:

[0110] PBS (control group, 8 mice), anti-PD-1 (8 mice), 0.01 mg / kg taltirelin + anti-PD-1 (8 mice), 0.05 mg / kg taltirelin + anti-PD-1 (8 mice), 0.1 mg / kg taltirelin + anti-PD-1 (8 mice), 0.2 mg / kg taltirelin + anti-PD-1 (8 mice), 0.5 mg / kg taltirelin + anti-PD-1 (8 mice), 0.8 mg / kg taltirelin + anti-PD-1 (8 mice), 1 mg / kg taltirelin + anti-PD-1.

[0111] Mice were treated with taltirelin every other day, i.e., on days 0, 2, 4, 6, 8, and 10. Anti-PD-1 was administered every other day for a total of four times, i.e., on days 0, 3, 6, and 9.

[0112] Anti-PD-1 administration: 100 μg per mouse per dose; taltirelin gradient administration: based on a 20 g mouse, taltirelin in each combination group was administered at 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 0.8 mg / kg, and 1 mg / kg.

[0113] The experimental results showed that the above seven doses significantly reduced the tumor volume of mice compared with the group using anti-PD-1 alone. Clinically, the dose can be calculated in mg / kg units according to the corresponding body weight and combined with anti-PD-1.

[0114] Example 7: Protirelin Gradient Efficacy Test

[0115] 1. Source of reagents:

[0116] The B16 / F10 mouse model involved is the same as that in Example 1. In this example, anti-PD-1 was purchased from BioXCell, the product name is: Anti-mouse PD-1, the specification is 100 mg; the TRH used was purchased from Shanghai Haoyuan Biopharmaceutical Co., Ltd., the product name is Protirelin (Acetate), the specification is 10 mg / vial;

[0117] 2. Grouping in this embodiment:

[0118] PBS (control group, 8 mice), anti-PD-1 (8 mice), 2μg / time / mouse Protirelin+anti-PD-1 (8 mice), 5μg / time / mouse Protirelin+anti-PD-1 (8 mice), 10μg / time / mouse Protirelin+anti-PD-1 (8 mice), 50μg / time / mouse Protirelin+anti-PD-1 (8 mice), 100μg / time / mouse Protirelin+anti-PD-1 (8 mice), 200μg / time / mouse Protirelin+anti-PD-1 (8 mice), 500μg / time / mouse Protirelin+anti-PD-1.

[0119] Mice were treated with taltirelin every other day, i.e., on days 0, 2, 4, 6, 8, and 10, and protirelin was administered by intraperitoneal injection (ip). Anti-PD-1 was administered every other day for a total of four times, i.e., on days 0, 3, 6, and 9, by intraperitoneal injection (ip).

[0120] Anti-PD-1 administration: 100 μg per mouse each time; Protirelin gradient administration: mice were calculated based on 20 g, and the Protirelin in the 7 combination groups was administered at 2 μg / time / mouse, 5 μg / time / mouse, 10 μg / time / mouse, 50 μg / time / mouse, 100 μg / time / mouse, 200 μg / time / mouse, and 500 μg / time / mouse, respectively; equivalent doses: 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, and 25 mg / kg.

[0121] The experimental results showed that the above seven doses significantly reduced the tumor volume of mice compared with the group using anti-PD-1 alone. Clinically, the dose can be calculated in mg / kg units according to the corresponding body weight and combined with anti-PD-1.

[0122] Example 8: Immune infiltration of tumor tissue after drug efficacy experiment

[0123] To investigate the effect of taltirelin combined with anti-PD-1 therapy on immune infiltration, this study repeated the efficacy experiment in B16-F10 tumor-bearing mice, administering the drug according to Figure 5. In this example, anti-PD-1 was purchased from BioXCell as Anti-mouse PD-1, in a 100 mg dose. Following the dosing experiment, tumor tissue was collected and further analyzed for immune infiltration using flow cytometry.

[0124] Flow cytometry results showed that the combination of taltirelin and anti-PD-1 significantly increased CD3 + T cells, CD8 + T cells, CD8 expressing Granzyme B (GzmB) + T cells, central memory CD8 + T (Tcm) cells, effector memory CD8 + T (TEM) cells, CD4 + T cells, CD4 + Tcm cells, CD4 + The number of TEM cells, natural killer (NK cells), natural killer T (NKT cells), gamma delta T (γδT) cells, and DC cells decreased. These results indicate that taltirelin enhances the anti-tumor ability of anti-PD-1 by enhancing anti-tumor immune response and improving immune cell infiltration in tumors.

[0125] Example 9: Mechanism Experiment

[0126] Since CD8 + The infiltration of T cells and dendritic cells (DCs) increased significantly. We used reverse transcription-polymerase chain reaction (RT-PCR) to detect CD8 + The expression of thyrotropin-releasing hormone receptors (TRH-R1 and TRH-R2) and thyroid hormone receptor (TSH-R) genes in T cells, mouse bone marrow-derived dendritic cells (BMDCs) and B16-F10 tumor cells was investigated. +High expression of TRH-R2 was observed in T cells, while high expression of TSH-R was observed in BMDCs (see Figure 20). Given the high expression of TRH-R by tumor cells, we first evaluated the cytotoxicity of taltirelin and TSH on B16-F10 tumor cells. The results showed that both substances were not toxic to tumor cells (see Figure 21).

[0127] Next, we treated CD8 T cells labeled with carboxyfluorescein succinimidyl ester (CFSE) with taltirelin or TSH. + T cells, and the experiment was conducted in the presence or absence of concanavalin A (conA, a substance that promotes T cell mitosis). Flow cytometry results showed that taltirelin significantly increased the proportion of low CFSE expression components in the presence of conA, indicating that CD8 + The proliferation of T cells was significant (see Figures 22a and 23a). In addition, activated CD8 + The proportion of T cells in the conA plus taltirelin treatment group was significantly higher than that in the control group, which further demonstrated that CD8 + Enhancement of T cell cytotoxicity (see Figures 22b, 22c, 23b, and 23c of the specification). These results emphasize the ability of taltirelin to regulate immune responses by directly promoting the proliferation and activation of T cells.

[0128] Dendritic cells (DCs) are key antigen-presenting cells (APCs), responsible for presenting antigens to T cells and possessing phagocytic activity. Given that DCs express the TSH receptor (TSH-R) on their surface, we investigated the effects of TSH on DC phagocytic and antigen-presenting activity. Flow cytometry results showed that TSH significantly enhanced the phagocytic capacity of bone marrow-derived dendritic cells (BMDCs) toward tumor cells, whereas taltirelin treatment did not significantly alter this effect, suggesting that TSH enhances DC phagocytosis (see Figure 22d in the accompanying figure).

[0129] To evaluate antigen presentation ability, we used ovalbumin (OVA) as a model antigen and co-incubated it with BMDCs pre-treated with taltirelin or TSH. We found that under the action of TSH, the treatment of OVA by BMDCs led to an increase in the expression of CD86, MHC-II and MHC-I (see Figure 22e-g in the specification), which helps to activate T cells. DC promotes T cell activation by presenting antigens to T cells in the form of peptide-MHC (pMHC) complexes and by increasing co-stimulatory molecules. Next, we detected the OT-I CD8 +The results showed that TSH significantly promoted the activation of OT-I CD8 + T cell activation was measured by increased GzmB expression (see Figure 22d in the specification), but there was no significant effect on the expression of the immune checkpoint PD-1 (see Figure 24 in the specification). These findings suggest that taltirelin not only directly acts on CD8 + T cells can also indirectly enhance the antigen presenting function of DC through TSH, further promoting CD8 + The proliferation and activation of T cells can enhance the efficacy of anti-PD-1 therapy.

[0130] Example 10: Pharmacodynamics Experiment in Mouse Lung Cancer Model

[0131] To further determine the anti-tumor efficacy of taltirelin in combination with anti-PD-1 in other cancers, in this example, a combination therapy experiment was performed using C57BL / 6J mice bearing subcutaneous Lewis lung carcinoma (LLC) tumors.

[0132] Experimental materials: C57BL / 6J male mice (6-8 weeks old); B16 / F10 subcutaneous tumor model (mouse melanoma cell line B16-F10 purchased from the Cell Resource Center of Peking Union Medical College); taltirelin in this example was purchased from Med Chem Express Reagent Company, catalog number (HY-B0596); anti-PD-1 was purchased from BioXcell, specification of 100 mg.

[0133] In this efficacy experiment, the number of mice in each of the following three groups was as follows: PBS group (7 mice), taltirelin group (6 mice), PBS + anti-PD-1 group (7 mice); taltirelin + anti-PD-1 (6 mice).

[0134] The administration of Taltirelin and anti-PD-1 is as follows (see Figure 24 in the instructions for administration diagram):

[0135] The experimental results, shown in Figure 25 of the accompanying specification, demonstrate that the anti-tumor efficacy of taltirelin combined with anti-PD-1 therapy in mice with lung cancer is significantly superior to either monotherapy and significantly superior to the control group. This result, combined with the previous examples, demonstrates that taltirelin can synergistically enhance the anti-tumor activity of anti-PD-1 in multiple cancers.

[0136] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of thyroid-stimulating hormone (TSH) or / and substances capable of increasing thyroid-stimulating hormone (TSH) levels in the body in the preparation of drugs for enhancing the anti-tumor efficacy of immune checkpoint inhibitors.

2. Use of immune checkpoint inhibitors in combination with thyroid stimulating hormone (TSH) or / and substances capable of increasing thyroid stimulating hormone (TSH) levels in the body in the preparation of anti-tumor drugs.

3. The use according to claim 1 or 2, characterized in that The substance capable of increasing the level of thyroid stimulating hormone (TSH) is a substance that can act on the hypothalamus-pituitary-thyroid axis to increase the level of thyroid stimulating hormone (TSH) in the body.

4. The use according to claim 1 or 2, characterized in that The substance capable of increasing the level of thyroid stimulating hormone (TSH) is thyrotropin-releasing hormone (TRH) or a thyrotropin-releasing hormone analogue that can induce the release of thyroid stimulating hormone (TSH) and thus increase the level of thyroid stimulating hormone (TSH) in the body.

5. The use according to claim 4, characterized in that The thyrotropin-releasing hormone (TRH) is Protirelin.

6. The use according to claim 4, characterized in that The thyrotropin-releasing hormone analogue is taltirelin, taltirelin tetrahydrate, montirelin, azetirelin, posatirelin, rovatirelin, JTP-2942, NP-654, orotirelin or RX 77368.

7. The use according to claim 1 or 2, characterized in that The immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA and LAG-3.

8. Application of Taltirelin in the preparation of anti-tumor drugs.

9. An anti-tumor drug, characterized in that: The invention comprises taltirelin as an active ingredient and pharmaceutically acceptable excipients.

10. A method for treating tumors, comprising administering to a patient an effective amount of taltirelin or a medicament comprising taltirelin and a pharmaceutically acceptable excipient.

11. A method for treating tumors, comprising administering to a patient a combination of an immune checkpoint inhibitor and thyroid stimulating hormone (TSH) or / and a drug that can elevate thyroid stimulating hormone (TSH) levels.

12. The method for treating tumors according to claim 11, characterized in that: The immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA and LAG-3.

13. The method according to claim 11, characterized in that The drug administered to the patient and capable of increasing the level of thyroid stimulating hormone (TSH) is a drug that can act on the hypothalamus-pituitary-thyroid axis to increase the level of thyroid stimulating hormone (TSH) in the body.

14. The method according to claim 11, characterized in that The drug administered to the patient and capable of increasing the level of thyroid stimulating hormone (TSH) is thyrotropin-releasing hormone (TRH) or a thyrotropin-releasing hormone analogue that can induce the release of thyroid stimulating hormone (TSH) and thus increase the level of thyroid stimulating hormone (TSH) in the body.

15. The method according to claim 14, characterized in that The thyrotropin-releasing hormone (TRH) drug administered to the patient to increase the level of thyroid stimulating hormone (TSH) is Protirelin.

16. The method according to claim 14, characterized in that The thyrotropin-releasing hormone analogue is taltirelin, taltirelin tetrahydrate, montirelin, azetirelin, posatirelin, rovatirelin, JTP-2942, NP-654, orotirelin or RX 77368.

17. A method for enhancing the anti-tumor efficacy of immune checkpoint inhibitors, characterized in that Increase the level of thyroid stimulating hormone (TSH) in the patient's body.

18. The method according to claim 17, characterized in that The method for increasing the level of thyroid stimulating hormone (TSH) in a patient's body is to administer thyroid stimulating hormone (TSH) or / and a drug capable of increasing the level of thyroid stimulating hormone (TSH) to the patient.

19. The method according to claim 18, characterized in that The drug administered to the patient and capable of increasing the level of thyroid stimulating hormone (TSH) is thyrotropin-releasing hormone (TRH) or a thyrotropin-releasing hormone analogue that can induce the release of thyroid stimulating hormone (TSH) and thus increase the level of thyroid stimulating hormone (TSH) in the body.

20. The method according to claim 19, characterized in that The thyrotropin-releasing hormone analogue is taltirelin, taltirelin tetrahydrate, montirelin, azetirelin, posatirelin, rovatirelin, JTP-2942, NP-654, orotirelin or RX 77368.

21. The method according to claim 18, wherein The thyroid stimulating hormone (TSH) administered to the patient is natural human thyroid stimulating hormone or recombinant human thyroid stimulating hormone (rhTSH).

22. The method according to claim 21, characterized in that The recombinant human thyroid stimulating hormone (rhTSH) is recombinant human thyroid stimulating hormone alpha (rhTSH alpha).

23. The method according to claim 22, characterized in that The recombinant human thyroid stimulating hormone alpha (rhTSH alpha) is Thyrogen.

24. A pharmaceutical composition for treating tumors, characterized in that: Contains immune checkpoint inhibitors and thyroid stimulating hormone (TSH) or / and substances that have the effect of increasing thyroid stimulating hormone (TSH) levels in the body.

25. The pharmaceutical composition according to claim 24, characterized in that The immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA and LAG-3.

26. The pharmaceutical composition according to claim 24, characterized in that The substance capable of increasing the level of thyroid stimulating hormone (TSH) in the body is thyrotropin-releasing hormone (TRH) or a thyrotropin-releasing hormone analogue that can induce the release of thyroid stimulating hormone (TSH) and thus increase the level of thyroid stimulating hormone (TSH) in the body.

27. The pharmaceutical composition according to claim 26, characterized in that The thyrotropin-releasing hormone analogue is taltirelin, taltirelin tetrahydrate, montirelin, azetirelin, posatirelin, rovatirelin, JTP-2942, NP-654, orotirelin or RX 77368.

28. The pharmaceutical composition according to claim 24, characterized in that The thyroid stimulating hormone (TSH) is natural TSH or rhTSH.

29. The pharmaceutical composition according to claim 28, characterized in that The rhTSH is rhTSH alpha.

30. The pharmaceutical composition according to claim 29, characterized in that The rhTSH alpha is a thyrogen.

31. A drug for treating tumors, characterized in that: The invention comprises the pharmaceutical composition according to any one of claims 24 to 30 and a pharmaceutically acceptable excipient.

32. The tumor of any one of claims 1, 2, 8, 9, 10, 11, 12, 17, 24 and 31 is a solid tumor or a non-solid tumor.

33. The solid tumor of claim 32 is melanoma, non-small cell lung cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, cervical cancer, small cell lung cancer, mesothelioma, nasopharyngeal carcinoma, renal cell carcinoma, esophageal cancer, head and neck squamous cell carcinoma, Merkel cell carcinoma, cutaneous squamous cell carcinoma, alveolar soft part sarcoma, basal cell carcinoma, biliary tract cancer, bladder cancer, breast cancer, mismatch repair deficient solid tumor, endometrial cancer, multiple myeloma, prostate cancer, gastroesophageal cancer, testicular cancer, thyroid cancer, central nervous system tumor, pancreatic cancer, ovarian cancer, germ cell cancer, bone cancer, sarcoma virus-related cancer and / or high tumor mutation load cancer; the non-solid tumor is leukemia, Hodgkin lymphoma and / or non-Hodgkin lymphoma.

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