Medicaments for enhancing the efficacy of immunotherapeutic treatments and their use for treating tumors

Abstract

The application provides a medicine for enhancing the efficacy of immunotherapy, provides application of the medicine in preparation of a medicine for enhancing the efficacy of immunotherapy, and further provides a method for enhancing the efficacy of an immune checkpoint inhibitor. Compared with the use of an immune checkpoint inhibitor alone, the medicine provided by the application can significantly improve the anti-tumor efficacy in combination of the immune checkpoint inhibitor and an anti-tumor experiment: improve the response rate of immunotherapy, reduce the tumor volume, improve the survival period, and solve the technical problems of low general response rate and unsatisfactory treatment effect in the current treatment of immune checkpoint inhibitors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to immunotherapy drugs, in particular to a drug for enhancing the efficacy of immunotherapy and application thereof in treating tumors. BACKGROUND

[0002] Malignant tumor is one of the biggest problems that needs to be solved in the world at present. At present, the number of people suffering from malignant tumor is still increasing all over the world, and the number of patients who die of cancer every year is incalculable. Therefore, how to develop new cancer treatment strategies and how to improve the efficacy of existing treatment plans have become the primary problems we face.

[0003] Treating cancer through the immune system is an effective and less side-effect treatment method. With the proposal of immune checkpoint blockade (ICB), drugs targeting immune checkpoints (such as programmed cell death protein 1 (PD-1) and its ligand PD-L1) have emerged, such as Nivolumab, Pembrolizumab, etc. Researches on treating tumors from the perspective of immune checkpoints are emerging in an endless stream. Immune checkpoint blockade is currently a hot research field as part of cancer immunotherapy.

[0004] Although research on ICB therapy is hot, and related drugs are successively put on the market, in a considerable number of patients, ICB therapy still has problems such as poor efficacy and low response rate. The currently marketed immune checkpoint inhibitors are mainly CTLA-4 inhibitors and PD-1 inhibitors (PD-1 / PD-L1 inhibitors), among which PD-1 inhibitors (PD-1 / PD-L1 inhibitors) include PD-1 antibodies (PD-1 inhibitors) and PD-L1 antibodies (PD-L1 inhibitors), that is, PD-1, PD-L1 inhibitors belong to immune checkpoint inhibitors.

[0005] Immune checkpoint inhibitor therapy is the most commonly used immunotherapy scheme at present, and is approved for clinical treatment of melanoma, renal cancer, lung cancer, head and neck cancer, bladder cancer and other tumors. Immune checkpoints are immunosuppressive pathways, which are crucial for maintaining self-tolerance and controlling the duration and range of immune responses in peripheral tissues.

[0006] The research of cancer immunotherapy is developing rapidly, and in 2018, Professor Tasuku Honjo and Professor James Allison, who discovered PD-1 and CTLA-4, were awarded the Nobel Prize in Physiology or Medicine. At present, ICB therapy is considered a promising clinical treatment for curing cancer. However, most patients with solid tumors do not respond to immune checkpoint inhibitor therapy, and it is generally believed that the average response rate is only about 20%, and in some cancers, it is even as low as 10% (highly invasive cancer). At present, the biggest difficulty in the clinical application of immune checkpoint inhibitors is the low overall remission rate.

[0007] The present application provides a drug for enhancing the efficacy of immunotherapy, and a method for enhancing the anti-tumor efficacy of immune checkpoint inhibitors. SUMMARY

[0008] The present application provides a drug for enhancing the efficacy of immunotherapy, and a method for enhancing the anti-tumor efficacy of immune checkpoint inhibitors.

[0009] The technical solution of the present application is that the drug for enhancing the efficacy of immunotherapy provided by the present application is thyroid stimulating hormone (TSH) or / and a substance that can increase the level of thyroid stimulating hormone (TSH). The method for enhancing the anti-tumor efficacy of immune checkpoint inhibitors provided by the present application is that immune checkpoint inhibitors are combined with thyroid stimulating hormone (TSH) or / and a substance that can increase the level of thyroid stimulating hormone (TSH) for anti-tumor treatment; or the application of immune checkpoint inhibitors combined with increasing the level of thyroid stimulating hormone (TSH) in vivo.

[0010] The substance capable of increasing the level of thyroid stimulating hormone (TSH) can be a substance capable of acting on the hypothalamic-pituitary-thyroid axis to increase the level of thyroid stimulating hormone (TSH) in the body. The substance capable of increasing the level of thyroid stimulating hormone (TSH) can be thyroid hormone releasing hormone (TRH) or thyroid hormone releasing hormone analog. The thyroid hormone releasing hormone analog is a substance other than thyroid hormone releasing hormone (TRH) capable of causing the release of thyroid stimulating hormone (TSH). The thyroid hormone releasing hormone (TRH) can be Protirelin in particular. The thyroid hormone releasing hormone analog can be Taltirelin, Taltirelin Tetrahydrate, Montirelin, Azetirelin, Orotirelin or RX 77368 in particular. The thyroid stimulating hormone (TSH) can be natural human thyroid stimulating hormone or recombinant human thyroid stimulating hormone (rhTSH), and the recombinant human thyroid stimulating hormone (rhTSH) can be Thyrogen in particular.

[0011] The immunotherapy can be treatment using an immune checkpoint inhibitor, and in particular can be anti-tumor treatment using an immune checkpoint inhibitor. The tumor can be a solid tumor, including but not limited to skin tumors, liver tumors, kidney tumors, lung tumors, pancreatic tumors, breast tumors, colon tumors or stomach tumors, and the skin tumors can include but are not limited to melanoma, squamous cell carcinoma, basal cell carcinoma, etc., and the tumor can be a malignant tumor in a solid tumor. The immunotherapy drug can be an immune checkpoint inhibitor, and in particular can be one or two or all of Anti-PD-1 antibody, Anti-PD-L1 antibody and Anti-CTLA-4 antibody.

[0012] The present application has found that thyroid hormone releasing hormone (TRH), thyroid hormone releasing hormone analog (TRH analog) and thyroid stimulating hormone (TSH), i.e. substances capable of increasing the level of thyroid stimulating hormone (TSH) in the body, have the effect of enhancing the efficacy of immunotherapy, and when combined with an immune checkpoint inhibitor, the response rate of the immune checkpoint inhibitor can be improved, thereby solving the technical problem of low response rate and poor treatment effect for most patients in the prior art. Accordingly, thyroid hormone releasing hormone (TRH), thyroid hormone releasing hormone analog (TRH analog) and thyroid stimulating hormone (TSH), as well as substances capable of increasing the level of thyroid stimulating hormone (TSH) in the body, can be used to prepare a drug for enhancing the efficacy of immunotherapy or a drug for enhancing the efficacy of an immunotherapy drug, i.e. in the preparation of a drug for enhancing the efficacy of immunotherapy or in the preparation of a drug for enhancing the efficacy of an immunotherapy drug.

[0013] Thyrotropin releasing hormone analogues (TRH analogues) refer to substances having similar structure to TRH and having thyrotropin releasing effect or having similar thyrotropin releasing hormone effect (i.e. substances similar in function to TRH). Similar in function means that they can substitute thyrotropin releasing hormone (TRH) to some extent to exert effects, and can produce strong and lasting multiple effects on central nervous system (CNS) through TRH receptors, such as pituitary hormone release excitement, etc., for example, causing the release of TSH.

[0014] Protirelin: We noticed that some literature uses Protirelin as TRH, i.e. using Protirelin is regarded as using natural TRH, such as:

[0015] (1) English Wikipedia entry for Thyrotropin releasing hormone considers Protirelin as a pharmaceutical form of TRH;

[0016] (2) Book Hormones (Asher Ornoy, Corinna Weber- in Drugs During Pregnancy and Lactation (Third Edition), 2015) describes in 2.15.1 Hypothalamic releasing hormones: "The synthetic substance protirelin was used to test the equivalent of natural hypothalamic thyrotropin releasing hormone (TRH)";

[0017] (3) Literature Protirelin (TRH) A Potent Neuromodulator With Therapeutic Potential (doi: 10.1001 / archinte.1984.00350180050006) uses Protirelin as TRH;

[0018] Some databases also classify Protirelin as an analogue of TRH, such as:

[0019] (1) Inxight Drugs database of the National Institutes of Health (NIH) of the United States: Protirelin is a pharmaceutically available synthetic analogue of the endogenous peptide thyrotropin releasing hormone (TRH).

[0020] (2) DrugBank ( https: / / go.drugbank.com / drugs / DB09421 ): Protirelin is a synthetic analogue of thyrotropin releasing hormone.

[0021] TRH analogues, specifically, include but are not limited to the following substances:

[0022] Taltirelin, a TRH analogue, is a synthetic thyrotropin-releasing hormone (TRH) analogue that mimics the physiological effects of TRH, causing the release of thyroid-stimulating hormone, but with a longer half-life and a longer duration of effect.

[0023] The TRH analogue Taltirelin Tetrahydrate also possesses the aforementioned functions of Taltirelin.

[0024] Montirelin (NS-3, CG-3703), an analogue of TRH, is more potent and has a longer duration of action than TRH.

[0025] In addition, there are some TRH analogs, such as Orotirelin (CG-3509), Azetirelin, RX 77368, etc. Some TRH analogs have not been approved for marketing and are still in the animal experiment or clinical trial stage. This invention has found that they have the effect of enhancing the efficacy of immunotherapy, and can enhance the anti-tumor therapeutic effect when used in combination with immune checkpoint inhibitors.

[0026] Taltirelin is a TRH analogue, an agonist of the TRH receptor, indicated for spinocerebellar degeneration and neurological disorders. It was first approved for marketing in Japan as an oral medication in September 2000. Therefore, this invention first uses a marketed TRH analogue as an example. Specifically, Examples 1 and 2 of this patent application detail the pharmacodynamic experiments of Taltirelin in combination with an immune checkpoint inhibitor; Example 4 of this patent application describes the RNA-seq sequencing results of tumor tissue after the pharmacodynamic experiments of Taltirelin in combination with an immune checkpoint inhibitor.

[0027] The immune checkpoint inhibitors described in this invention can specifically be one or more of the following: Anti-PD-1 antibody, Anti-CTLA-4 antibody, and Anti-PD-L1 antibody, used in combination.

[0028] The present invention describes a pharmacodynamic experiment of tatirelin, a thyrotropin-releasing hormone analog, synergistically with an immune checkpoint inhibitor in antitumor activity. The pharmacodynamic experiment shows that tatirelin can greatly enhance the antitumor therapeutic effect of the immune checkpoint inhibitor.

[0029] The Anti-PD-1 is combined with thyroid-stimulating hormone releasing hormone (TRH), thyroid-stimulating hormone (TSH) and thyroid hormone (TH) respectively in the anti-tumor pharmacodynamic experiment of embodiment 3, and the pharmacodynamic experiment shows that thyroid-stimulating hormone releasing hormone (TRH) and thyroid-stimulating hormone (TSH) can greatly improve the anti-tumor effect of the immune checkpoint inhibitor, and thyroid hormone (TH) has no such effect.

[0030] The results of RNA-seq sequencing of the mouse tumor tissue taken after the end of the pharmacodynamic experiment in embodiment 2 are recorded in embodiment 4, and the results show that triptorelin combined with the immune checkpoint inhibitor can indeed activate the anti-tumor related pathways compared with the immune checkpoint inhibitor alone, thereby achieving the effect of improving the anti-tumor effect.

[0031] Combined with the results in the embodiments (triptorelin, TRH and TSH can synergize with the immune checkpoint inhibitor to resist tumors, and TH has no such effect), and the negative feedback regulation between thyroid-stimulating hormone releasing hormone (TRH), thyroid-stimulating hormone (TSH) and thyroid hormone (TH) (as described in the specification Figure 15 ), the thyroid-stimulating hormone releasing hormone analog (triptorelin) is similar to the thyroid-stimulating hormone releasing hormone (TRH), and both can increase the hormone level of the downstream thyroid-stimulating hormone (TSH), so that the drugs or hormones that can improve the thyroid-stimulating hormone (TSH) can synergize with the immune checkpoint inhibitor to resist tumors to a certain extent.

[0032] The method for improving the level of thyroid-stimulating hormone (TSH) in vivo can be directly administering thyroid-stimulating hormone (TSH) to the patient, or administering a substance that can improve the level of thyroid-stimulating hormone (TSH) in vivo to the patient. The substance that can improve the level of thyroid-stimulating hormone (TSH) in vivo can improve the hormone level of thyroid-stimulating hormone (TSH) by acting on the hypothalamic-pituitary-thyroid axis. The substance that can improve the level of thyroid-stimulating hormone (TSH) in vivo can be thyroid-stimulating hormone releasing hormone (TRH), thyroid-stimulating hormone releasing hormone analog (TRH analog) and the like.

[0033] The present application experiment reveals that thyrotropin releasing hormone analogue (TRH analogue), thyrotropin releasing hormone (TRH) and thyrotropin (TSH), i.e. substances capable of increasing the level of thyrotropin (TSH) in vivo, can all increase the hormone level of thyrotropin (TSH) in vivo, thereby synergistically enhancing the effect of immune checkpoint inhibitor anti-tumor therapy, significantly reducing tumor volume, significantly improving survival period, and improving patient survival. The research results of the present application show that thyrotropin (TSH) and substances capable of increasing the level of TSH hormone in vivo (including but not limited to chemical drugs, biological drugs, and hormones, etc.) can significantly improve the response rate and treatment effect of immune checkpoint inhibitors, solving the technical problems of low response rate and unsatisfactory treatment effect of existing immune checkpoint inhibitors. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is a schematic diagram of the administration mode of Example 1, wherein the Tumor bearing marked in the figure represents tumor-bearing, i.e. the day when each group of mice is tumor-bearing on day 0, the arrow marked in the figure represents the administration day, and the administration starts on day 12, Anti-PD-1 is administered 4 times, once every two days, Taltirelin is administered 6 times, once every day, and ip in the figure represents intraperitoneal injection.

[0035] Figure 2 is a curve graph of the change of tumor volume of 4 groups of mice in the pharmacodynamic experiment of Example 1 with treatment days; the curve graph of the change of tumor volume of mice in the PBS group (solvent control group) with treatment days is marked 1 in the figure; the curve graph of the change of tumor volume of mice in the Anti-PD-1 antibody group with treatment days after administration is marked 2 in the figure; the curve graph of the change of tumor volume of mice in the Taltirelin (Taltirelin) group with treatment days after administration is marked 3 in the figure; and the curve graph of the change of tumor volume of mice in the Anti-PD-1+Taltirelin group with treatment days after administration is marked 4 in the figure.

[0036] Figure 3is the tumor volume curve comparison chart of the 4 groups of mice in the pharmacodynamic experiment in Example 1: PBS, Anti-PD-1 antibody, Taltirelin, Anti-PD-1+Taltirelin, that is, the tumor inhibition curve chart, the four asterisks represent that the tumor volume difference between groups is extremely significant (Adjust p-value<0.0001), that is, the pharmacodynamic effect of Anti-PD-1 antibody combined with Taltirelin is extremely significantly better than that of Anti-PD-1 antibody or Taltirelin alone, thereby indicating that Taltirelin has extremely significantly enhanced the anti-tumor ability of immune checkpoint inhibitors; in addition, Taltirelin alone, Anti-PD-1 alone, and PBS group (control group) are significantly different (Adjust p-value<0.01), and the therapeutic effect of Taltirelin alone after the 8th day is slightly better than that of Anti-PD-1 alone, thereby indicating that Taltirelin alone has certain anti-tumor efficacy.

[0037] Figure 4 is the survival curve comparison chart of the 4 groups of mice in the pharmacodynamic experiment in Example 2: PBS, Anti-PD-1 antibody, Taltirelin, Anti-PD-1+Taltirelin, in the figure, "Log-rank (Mantel-Cox) test P value=0.0004" represents that the p-value value of Log-rank (Mantel-Cox) test is 0.0004, indicating that the survival period of Anti-PD-1+Taltirelin group mice is extremely significantly improved relative to other groups, that is, Taltirelin combined with immune checkpoint inhibitors has a significant effect on improving survival.

[0038] Figure 5 is the schematic diagram of the administration method of Example 2, wherein the marked Tumor bearing in the figure represents tumor-bearing, that is, the day when the mice in each group are tumor-bearing on the 0th day, the arrow marked in the figure represents the day of drug administration, drug administration starts on the 7th day, Anti-PD-1 is administered 4 times, once every two days, Taltirelin is administered 6 times, once every day, the tumors of mice in each group are cut on the 18th day for subsequent experiments, and ip in the figure represents intraperitoneal injection.

[0039] Figure 6Figure 2 is a graph showing the change in tumor volume over time for the mice in Example 2, Group 4. Figure 2 shows the tumor volume over time for the mice in the PBS group (solo), the Anti-PD-1 antibody group, the Taltirelin group, and the Anti-PD-1 + Taltirelin group. The Anti-PD-1 + Taltirelin group showed a significant reduction in tumor volume compared to the other groups.

[0040] Figure 7 Figure 3 is a photograph showing the tumors removed from the mice in Example 2, Group 4, after the end of the pharmacodynamic experiment (day 18 after tumor inoculation).

[0041] Figure 8 Figure 4 is a graph showing the tumor volume of the mice in Example 2, Group 4, after the end of the pharmacodynamic experiment. The Kruskal-Wallis H test was used to determine the differences between the four groups. The Mann-Whitney test was used to determine the differences between the Anti-PD-1 + Taltirelin group and the other three groups. The False Discovery Rate BH method was used to correct the results. The Anti-PD-1 + Taltirelin group showed a significant reduction in tumor volume compared to the other groups.

[0042] Figure 9is a tumor volume inhibition curve graph according to the statistical tumor volume size change with the number of days of administration in Example 2, that is, a comparison graph of the tumor volume of each group of mice corresponding to the four groups (PBS, Anti-PD-1 antibody, Taltirelin, Anti-PD-1+Taltirelin) of the efficacy experiment with the number of days of administration, and the asterisk represents the significance of the difference in the tumor volume of mice between groups (*** represents p-value <0.001; **** represents p-value <0.0001), that is, the anti-tumor efficacy of Anti-PD-1 antibody+Taltirelin is extremely significantly better than Anti-PD-1 antibody alone or very significantly better than Taltirelin alone, thus indicating that Taltirelin has a very significant ability to enhance the anti-tumor ability of Anti-PD-1 antibody; in addition, Taltirelin alone, Anti-PD-1 alone, and PBS group (control group) are significantly different (p-value <0.01), and the anti-tumor ability of Taltirelin alone in this experiment is slightly better than the therapeutic effect of Anti-PD-1 group throughout the process (the tumor volume of Taltirelin group in the figure is less than that of Anti-PD-1 group), thus indicating that Taltirelin alone has a certain anti-tumor efficacy, and the results of the above repeated pharmacodynamic experiment and the results of the first pharmacodynamic experiment Figure 3 ) are almost completely consistent, indicating that Taltirelin enhances the anti-tumor effect of immune checkpoint inhibitors and has stability, and Taltirelin alone also exhibits certain stable anti-tumor properties.

[0043] Figure 10 is a schematic diagram of the administration method of Example 3, wherein the Tumor bearing marked in the figure represents tumor-bearing, that is, the day when each group of mice is tumor-bearing on day 0, the arrow marked in the figure represents the number of days of drug administration, and the administration starts on day 9, Anti-PD-1 is administered 4 times, every two days, TRH, TSH, and TH are administered 6 times, every day, ip represents intraperitoneal injection, im represents intramuscular injection, and the tumor is removed on day 23 for subsequent experiments.

[0044] Figure 11is a tumor volume inhibition curve graph according to the statistical tumor volume size change with days in Example 3, the horizontal coordinate in the figure is the number of days after tumor-bearing, and the curve records the curve of the change of the tumor volume of each group of mice from the 8th day to the 20th day after tumor-bearing, that is, the curve comparison of the tumor volume of each group of mice with days corresponding to the 8 groups (PBS, Anti-PD-1 antibody, TSH, TRH, TH, Anti-PD-1+TSH, Anti-PD-1+TRH, Anti-PD-1+TH) of the efficacy experiment, and the asterisk represents the degree of significance of the difference in the tumor volume of mice between two groups (*, p-value <0.05; **, p-value <0.01; ****, p-value <0.0001); on the 17th day after tumor-bearing, all mice in all groups were alive at this time, the efficacy of Anti-PD-1 antibody+TRH / TSH combination was significantly better than that of Anti-PD-1 antibody alone; on the 20th day, the PBS group, the Anti-PD-1 antibody group, and the Anti-PD-1 antibody+TRH / TSH combination group were still all alive at this time, the efficacy of Anti-PD-1 antibody+TRH / TSH was extremely significantly better than that of Anti-PD-1 antibody alone (p-value <0.0001), which indicated that TRH or TSH had extremely significant Anti-PD-1 antibody anti-tumor ability, that is, TRH or TSH could synergize with immune checkpoint inhibitors to resist tumors and greatly improve the effect of immune checkpoint inhibitors on resisting tumors.

[0045] Figure 12 is a photo of the ex vivo tumor of each group of mice after the tumor of the surviving mice was removed on the 23rd day after tumor-bearing in Example 3.

[0046] Figure 13 is a photo of the surviving mice after tumor-bearing in Example 3 on the 16th day and the 21st day after tumor-bearing, as shown on the left side of the figure, all mice in the 8 groups were alive on the 16th day; as shown on the right side of the figure, on the 21st day, the corresponding rectangle grid on the 16th day is “blank”, representing that the mouse has died, and the grid mark “cross” represents that the tumor volume of the mouse exceeds the ethical range, and the mouse has been euthanized.

[0047] Figure 14 is a mouse survival curve drawn according to the survival condition in Example 3, which shows the survival condition of each group of mice from the 0th day to the 23rd day after tumor-bearing, all mice in the TSH+Anti-PD-1 group and the TRH+Anti-PD-1 group were in a survival state on the 23rd day after tumor-bearing, and the mice in the remaining groups all died.

[0048] Figure 15This diagram illustrates the regulatory relationship between thyrotropin-releasing hormone analogs (TRH analogs) or thyrotropin-releasing hormone (TRH), thyroid-stimulating hormone (TSH), and thyroid hormone (TH). The checkmarks in the diagram indicate that TRH, TRH analogs (tatirelin is just one typical example of TRH; other TRH analogs with similar effects are also possible), and TSH ultimately increase TSH levels. Therefore, these three drugs (or hormones) synergistically enhance the anti-tumor effects of immune checkpoint inhibitors, significantly improving their efficacy. The crosses in the diagram indicate that thyroid hormone (TH) did not synergistically enhance the anti-tumor effects of immune checkpoint inhibitors. Therefore, based on the pathway regulation mechanisms of these hormones, it is TSH that effectively enhances the anti-tumor effects of immune checkpoint inhibitors.

[0049] Figure 16 The lower half of the chart is a visualization of the RNA-seq sequencing results from Example 4. The chart shows the expression of genes corresponding to six immune-related gene sets (antigen presentation-related genes, natural killer cell-related genes, TCR signaling pathway, genes related to cytotoxic immune cells provided by the ImmuCellAI tool, antibacterial-related genes, and BCR signaling pathway). Specifically, it is a heatmap showing the expression relationship of genes related to tumor tissues in mice after the mouse drug administration experiment in Example 2 (PBS group, Anti-PD-1 group, Taltirelin group, and Anti-PD-1 combined with Taltirelin group). Blue represents relatively low gene expression levels, and orange represents relatively high gene expression levels. Darker blue indicates lower gene expression levels, and darker orange indicates higher expression levels. Each column in the heatmap represents the expression level of a related gene in a tumor tissue; that is, the color of a small square in the heatmap represents the relative expression level of a gene in the corresponding tumor sample. Above this expression chart is the instruction manual appendix. Figure 7(i.e. tumor graph after the end of administration of Example 2), the tumor sequence of each row in the tumor graph corresponds to the column sequence (from left to right) of each group in the heat map in turn, it can be seen that the expression of the 6 gene sets corresponding to the two tumors with obvious large volume (i.e. the two tumors circled by the red circle in the figure, which are generally considered as "non-response" in the art, i.e. not affected by immunotherapy) in the Anti-PD-1 combined with the administration of the group of heptarelin is at a low level (the column pointed to by the arrow in the figure shows mainly blue), and the expression of the 6 gene sets corresponding to the remaining 8 tumors with obvious small volume (which are generally considered as typical "response" in the art, i.e. affected by immunotherapy) in the combined drug group is at a high level (the corresponding column mainly shows orange); similarly, the expression level of the 6 gene sets corresponding to one tumor with obvious large volume (the third from left to right, circled by the red circle) in the PBS + Anti-PD-1 group is at a low level (the column is obviously deeper in blue).

[0050] Figure 17 is a gray-scale graph of the accompanying drawings of the specification Figure 16 The accompanying drawings of the specification Figure 16 converted into a gray-scale graph cannot reflect the high and low relationship of the expression of each group of genes, therefore the accompanying drawings of the specification Figure 16 provides the original color graph, the accompanying drawings of the specification Figure 17 provides the version of the accompanying drawings of the specification Figure 16 converted into a gray-scale graph.

[0051] Figure 18 is a result graph of HE staining of the internal organs (heart, liver, spleen, lung and kidney) of the 8 groups of mice respectively after the mice were sacrificed on the 23rd day after tumor bearing in Example 3.

[0052] Labeling description: PD1 or PD-1 marked in the specification and drawings both represent Anti-PD-1 antibody. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0054] Example 1

[0055] Pharmacodynamic test of a marketed thyrotropin releasing hormone analogue (heptarelin) combined with Anti-PD-1:

[0056] 1. Model construction and grouping

[0057] 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 Beijing Union Medical College); Taltirelin purchased from Aladdin Reagent Co., Ltd., specification 10 mg; Anti-PD-1 purchased from BioXcell, specification 100 mg.

[0058] Grouping: PBS (control group), Taltirelin (a thyrotropin-releasing hormone analogue), PBS+Anti-PD-1, Taltirelin+Anti-PD-1, a total of 4 groups, 8 mice in each group.

[0059] The administration methods of Taltirelin and Anti-PD-1 are as follows (see the administration schematic diagram in the specification) Figure 1 ) :

[0060] Drug Dose Route of administration Frequency of administration Taltirelin 0.5 mg / kg Intraperitoneal injection Once every 1 day for a total of 6 times Anti-PD-1 100 μg / each Intraperitoneal injection Once every 2 days for a total of 4 times

[0061] 2. Pharmacodynamic experiment

[0062] The tumor volume of the mice was monitored every day after the start of administration. The values in the following table are the tumor volumes (cubic millimeters) of the mice measured in the test experiment, and the italic (asterisk) values represent the data recorded before the mice died.

[0063] PBS group (control group)

[0064]

[0065] Taltirelin administration group

[0066]

[0067]

[0068] PBS+Anti-PD-1 administration group

[0069]

[0070] Taltirelin+Anti-PD-1 combined administration group

[0071]

[0072] 3. Survival time

[0073] Survival time data: The days in the table are calculated from the injection of tumor cells in the mice, i.e. the tumor-bearing day is the 0th day. The data recorded in the following table are used to draw the survival curve as shown in the specification Figure 4 .

[0074]

[0075]

[0076] The following table is the median survival time between groups:

[0077] Summary of survival data PBS PBS + Anti-PD-1 Taltirelin Taltirelin + Anti-PD-1 Median survival 28 days 30.5 days 28 days 43 days

[0078] Survival experiments show that the administration of Taltirelin combined with Anti-PD-1 to mice significantly improves the survival period relative to the other three groups (p-value of Log-rank (Mantel-Cox) test is 0.0004).

[0079] Example 2

[0080] After the efficacy experiment of Example 1, a survival test was performed, and in this Example 2, an efficacy test will also be performed. After the efficacy test, the tumors of the mice will be excised and weighed, and high-throughput sequencing will be performed. The sequencing test is described in Example 4.

[0081] 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 Beijing Union Medical College); Taltirelin purchased from Aladdin Reagent Co., Ltd., specification 10 mg; Anti-PD-1 purchased from BioXcell, specification 100 mg.

[0082] In this efficacy experiment, the following three groups each have 8 mice: PBS group, Taltirelin group (Taltirelin, a thyrotropin-releasing hormone analogue), PBS+Anti-PD-1 group;

[0083] The following combination drug group uses 10 mice for drug administration experiment: Taltirelin+Anti-PD-1.

[0084] The administration methods of Taltirelin and Anti-PD-1 are as follows (see the administration diagram in the instruction manual Figure 5 ) :

[0085] Drug Dose Route of administration Frequency of administration Taltirelin 0.5 mg / kg Intraperitoneal injection Once every 1 day for a total of 6 times Anti-PD-1 100 μg / each Intraperitoneal injection Once every 2 days for a total of 4 times

[0086] 2. After the administration is completed, the tumors of the mice are excised, and photographs are taken. The tumor photographs of the mice in each group are shown in the attached Figure 7 ; The tumor weights of the mice in each group are measured as follows (unit: grams), and the statistical differences between groups are shown in the attached Figure 8 .

[0087]

[0088] 3. Statistical test

[0089] 3.1, Non-parametric test was used: Kruskal-Wallis H test was used to test the difference between the above four groups of data

[0090] Kruskal-Wallis test P value 0.0306 Exact or approximate P value? Approximate P value summary * Do the medians vary significantly (P<0.05)? Yes Number of groups 4 Kruskal-Wallis statistic 8.903

[0091] 3.2, Pairwise test was performed between the combination group and the other three groups: Mann-Whitmey test was used for pairwise significance test

[0092]

[0093]

[0094] 3.3, P value of Mann-Whitmey test in 3.2 above was corrected: False Discovery Rate BH method was used for correction

[0095]

[0096] Conclusion: The overall statistical difference of the four groups of pharmacodynamics is significant. The statistical difference between the combination group and the other three groups is significant. The anti-tumor effect of Taltirelin combined with Anti-PD-1 is significantly better than that of Anti-PD-1 or Taltirelin alone.

[0097] The relevant figures of this example are shown in the attached drawings of the specification Figure 5-9 .

[0098] The dose of Taltirelin in the combination group of this example is 0.5 mg / kg, and the patient can calculate the combined Anti-PD-1 administration according to the dose.

[0099] Example 3: Pharmacodynamics test of thyrotropin releasing hormone (TRH), thyrotropin (TSH), and thyroid hormone (TH) combined with Anti-PD-1 respectively:

[0100] 1. Test materials

[0101] The B16 / F10 mouse model involved is the same as in Example 1, Anti-PD-1 is purchased from Wuhan Furui De Medical Technology Co., Ltd., product name: InVivoMab anti-mouse PD-1 (CD279), specification: 100 mg / branch; TRH is purchased from Shanghai Haoyuan Biomedicine Co., Ltd., product name: Protirelin (Acetate), specification: 10 mg / branch; The TSH used is rhTSH, purchased from Hong Kong Huajian Medical Co., Ltd., product name: Thyrogen (Suitable for progress), specification: 0.9 mg*2 bottles / box; TH is purchased from Shanghai Haoyuan Biomedicine Co., Ltd., product name: L-Thyroxine, specification: 500 mg / branch.

[0102] 2, Group and its administration method, times:

[0103] 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), TH+Anti-PD-1 (8 mice) a total of 8 groups.

[0104] The mice were counted from the day of tumor bearing as day 0, and the administration started on day 9: TRH, TSH, TH were given once every 1 day, that is, on day 9, day 11, day 13, day 15, day 17, day 19, among which TRH, TH were intraperitoneal injection (ip), TSH was intramuscular injection (im); Anti-PD-1 was given once every 2 days, a total of 4 times, that is, on day 9, day 12, day 15, day 18, the administration method was intraperitoneal injection (ip). The surviving mice were sacrificed on day 23 for the following toxicity test.

[0105] Anti-PD-1 administration: 100 μg per mouse each time;

[0106] The TSH used is rhTSH (Thyrogen), the administration dose is: 4 μg per mouse each time;

[0107] Dose instruction: Each vial of Thyrogen contains 1.1 mg of thyrotropin alfa, and the package of the drug is specially marked that the extractable active ingredient of thyrotropin alfa in each vial is 0.9 mg, so the actual active ingredient should be calculated as 0.9 mg per vial. In addition to thyrotropin alfa, each vial contains other excipients: 36 mg of mannitol, 5.1 mg of sodium phosphate and 2.4 mg of sodium chloride, a total of 44.6 mg; the activity value of thyrotropin alfa is 4-12 IU / mg, and in this study, the effective ingredient is calculated according to the median value of 8 IU / mg, the weight of each mouse is calculated according to 20 g, and each mouse is given 4 μg of thyrotropin alfa, which is equivalent to the theoretical dose of 0.032 IU of thyrotropin alfa; that is, 1.6 IU / kg.

[0108] L-Thyroxine: 2 μg per mouse per time;

[0109] Protirelin (acetate): 200 μg per mouse per time.

[0110] 3. Pharmacodynamic test:

[0111] The day before the start of drug administration (the 8th day after tumor inoculation), the tumor volume of the mice was monitored every day thereafter. The values in the table below are the tumor volumes (cubic millimeters) of the mice measured in the experiment. In this pharmacodynamic test, if a mouse in a certain group dies or the tumor volume exceeds the ethical range, the data after that point will be stopped.

[0112] PBS group (control group)

[0113]

[0114] TRH group

[0115]

[0116] TSH group

[0117]

[0118] TH group

[0119]

[0120] Anti-PD-1 group

[0121]

[0122] TRH+Anti-PD-1 group

[0123]

[0124] TSH+Anti-PD-1 group

[0125]

[0126] TH+Anti-PD-1 group

[0127]

[0128] 4、Tumor weight

[0129] After the end of administration, on the 23rd day of tumor-bearing, the tumors of mice were excised, photographed, and the tumor photos of mice in each group were as shown in the attached drawings of the specification. Figure 12 The tumor weights of mice in each group were measured as follows, unit: gram.

[0130]

[0131] 5、Antitumor effect (i.e. drug efficacy)

[0132] Statistical tests were performed on the tumor volumes of mice in each group obtained in the efficacy test of 3. Through two-factor variance analysis, it can be seen that the drug factor and the number of days affect the tumor volume of mice.

[0133] Source of difference Total difference percentage P value P value summary Significant? Time 31.97 <0.0001 **** Yes Drug 8.073 <0.0001 **** Yes

[0134] Through the efficacy test of 3 above, statistical tests were performed on each two of the 8 groups: the statistical test results showed that the antitumor effects of TRH+Anti-PD-1 and TSH+Anti-PD-1 were far superior to Anti-PD-1 alone, and also far superior to TRH or TSH alone. This result revealed that both TRH and TSH have synergistic immunotherapy antitumor effects, which can greatly enhance the antitumor efficacy of immunotherapy.

[0135] The TSH+Anti-PD-1 group (TSH used is rhTSH, Thyrotropin) of this embodiment used 4 μg thyrotropin alfa per mouse per time, which is equivalent to the theoretical dose of 0.032 IU thyrotropin alfa; i.e. 1.6 IU / kg. This dose has a significant synergistic effect with Anti-PD-1, and the clinical dose can be calculated according to the patient's body weight.

[0136] The TRH+Anti-PD-1 group in this example (the TRH used is Protirelin) uses 200 μg of Protirelin per mouse per time, and the mouse weight is calculated at 20 g, which is equivalent to a dose of 10 mg / kg. This dose has a significant synergistic effect with Anti-PD-1, and the dose can be calculated according to the patient's weight for clinical administration.

[0137] 6. Survival of mice

[0138] The survival of mice in each group was recorded after tumor inoculation. Since the next toxicity test was performed on day 23, the mice were collectively sacrificed on day 23, and the survival of the remaining mice that did not die was recorded on day 23. The following table shows the raw data of the survival of mice in each group, and the survival curve graph is shown in FIG. 1. Figure 14

[0139] The following table and the description Figure 13 indicate that all mice in the TRH+PD-1 group and the TSH+PD-1 group survived on day 23, and the rest of the groups had deaths.

[0140]

[0141]

[0142] Example 4: High-throughput sequencing

[0143] The tumor tissues removed from the mice after the efficacy test in Example 2 (as shown in the description Figure 7 ) were subjected to RNA-seq sequencing. After removing the mouse tumor tissues, the tissues were quickly frozen in liquid nitrogen and then stored in a -80°C refrigerator. After being wrapped in dry ice boxes, the samples were sent to Haplox Company for Bulk RNA-seq sequencing. According to the sequencing results, the expression levels of 6 immune-related pathways or gene sets were significantly improved in the combination drug (Anti-PD-1+Taltirelin) group compared to the two single-drug groups (single Anti-PD-1 or single Taltirelin) and the PBS control group (as shown in the description Figure 16 ). This indicates that the combination drug can significantly improve immune cell activity and promote immune responses in the body, thereby better allowing the body's immune cells to kill tumor cells.

[0144] The description Figure 7 indicates that the tumor volume of the combination drug group was significantly larger than that of the other tumors, i.e., the tumors of 2 mice in the combination drug group were not inhibited in growth, and the tumor sizes of the remaining 7 mice were significantly inhibited in growth. The description Figure 16 ​The expression levels of the relevant genes shown in the surface combination group are significantly higher than those of the other tumor tissues. The immune genes in the two volumes are at a relatively very low expression level. It can be considered that the two tumors corresponding to the mice in vivo do not obtain the effect of combined treatment, which may be due to experimental accidental factors, may be due to the difference in the immune level of the mice, and a few mice do not benefit from immunotherapy, or the inherent "two-part phenomenon" of immune checkpoint treatment (that is, as long as the number of immune checkpoint treated mice in the drug efficacy experiment is sufficient, there will always be "responding" and "non-responding" mice); and the expression levels of other immune genes in the tumor tissues of the combined drug group are relatively high. The tumor tissues obtained very significant combined treatment anti-tumor effect. This reflects from another side that Taltirelin combined with Anti-PD-1 can indeed enhance the activity of immune-related genes, and the tumor tissues with significantly enhanced expression of related immune genes benefit from the treatment effect of Taltirelin combined with immune checkpoint inhibitors.

[0145] Example 5: TSH (Thyrogen) gradient drug efficacy test

[0146] 1. Reagent source:

[0147] The B16 / F10 mouse model involved is the same as in Example One, Anti-PD-1 is purchased from Wuhan Furui De Medicine Technology Co., Ltd., the product name is: InVivoMab anti-mouse PD-1 (CD279), the specification is 100 mg / branch; The TSH used is rhTSH, purchased from Hong Kong Huajian Medical Co., Ltd., the product name is: Thyrogen (Suitable for progress), the specification is 0.9 mg*2 bottles / box;

[0148] 2. Grouping of this example:

[0149] PBS (control group, 8 mice), Anti-PD-1 (8 mice), 0.25 μg thyrotropin / time / mouse+Anti-PD-1 (8 mice), 0.5 μg thyrotropin / time / mouse+Anti-PD-1 (8 mice), 1 μg thyrotropin / time / mouse+Anti-PD-1 (8 mice), 2 μg thyrotropin / time / mouse+Anti-PD-1 (8 mice), 3 μg thyrotropin / time / mouse+Anti-PD-1 (8 mice), 4 μg thyrotropin / time / mouse+Anti-PD-1 (8 mice), 6 μg thyrotropin / time / mouse+Anti-PD-1 (8 mice).

[0150] The mice were counted from the day of tumor inoculation as day 0, and the administration was started on day 9: thyrotropin was administered once every 1 day, i.e. on day 9, day 11, day 13, day 15, day 17, and day 19, and the administration was performed by intramuscular injection (im); Anti-PD-1 was administered once every 2 days, a total of 4 times, i.e. on day 9, day 12, day 15, and day 18, and the administration was performed by intraperitoneal injection (ip);

[0151] Anti-PD-1 administration: 100 μg per mouse per time;

[0152] rhTSH (Thyrogen) gradient administration: 0.25 μg, 0.5 μg, 1 μg, 2 μg, 3 μg, 4 μg, and 6 μg per mouse per time;

[0153] Dose specification: each bottle of Thyrogen contains 1.1 mg of thyrotropin alfa (theoretical dose), and the package of the drug is specially marked that the effective component thyrotropin alfa that can be extracted from each bottle is 0.9 mg, so the actual effective component should be calculated as 0.9 mg per bottle, in addition to the thyrotropin alfa, each bottle contains other excipients: 36 mg of mannitol, 5.1 mg of sodium phosphate, and 2.4 mg of sodium chloride, a total of 44.6 mg; the activity value of thyrotropin alfa is 4-12 IU / mg, and in this study, the effective component is calculated according to the median value of 8 IU / mg, and the weight of each mouse is calculated according to 20 g, and each mouse is given 0.25 μg, 0.5 μg, 1 μg, 2 μg, 3 μg, 4 μg, and 6 μg of thyrotropin alfa per time, which is equivalent to the theoretical dose of 0.002 IU, 0.004 IU, 0.008 IU, 0.016 IU, 0.024 IU, 0.032 IU, and 0.048 IU of thyrotropin alfa, and according to the IU / kg calculation, it 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, and 2.4 IU / kg.

[0154] The test results show that the above-mentioned 7 doses are significantly reduced in tumor volume relative to the Anti-PD-1 group alone, and the dose can be calculated according to the corresponding body weight clinically in IU / kg unit, and the Anti-PD-1 administration is synergistic.

[0155] Example 6: Taltirelin (taltirelin) gradient efficacy test

[0156] 1. Reagent source:

[0157] The B16 / F10 mouse model involved is the same as in Example One, Anti-PD-1 is purchased from Wuhan Furui De Biotechnology Co., Ltd., product name: InVivoMab anti-mouse PD-1 (CD279), specification: 10 mg / branch; Taltirelin used is purchased from Aladdin Reagent Co., Ltd., specification: 100 mg;

[0158] 2、This embodiment is grouped:

[0159] 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.

[0160] The mice are counted from the day of tumor bearing as day 0, and the administration starts on day 9: Taltirelin is administered every 1 day, i.e. on day 9, day 11, day 13, day 15, day 17, and day 19, and the administration of Taltirelin to mice is intraperitoneal injection (ip); Anti-PD-1 is administered every 2 days, a total of 4 times, i.e. on day 9, day 12, day 15, and day 18, and the administration is intraperitoneal injection (ip);

[0161] Anti-PD-1 administration: 100 μg per mouse per time;

[0162] Taltirelin gradient administration: mice are calculated according to 20 g, and Taltirelin in the combination groups is 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;

[0163] The test results show that the above-mentioned 7 doses are significantly reduced in tumor volume relative to the Anti-PD-1 alone group, and the dose can be calculated according to the corresponding body weight in mg / kg unit combined with Anti-PD-1 administration.

[0164] Example 7: Protirelin gradient efficacy test

[0165] 1. Reagent source:

[0166] The B16 / F10 mouse model involved was the same as in Example One, Anti-PD-1 was purchased from Wuhan Furui De Medicine Biotechnology Co., Ltd., product name: InVivoMab anti-mouse PD-1 (CD279), specification: 10 mg / branch; TRH used was purchased from Shanghai Haoyuan Biomedicine Co., Ltd., product name: Protirelin (Acetate), specification: 10 mg / branch;

[0167] 2. Grouping of this example:

[0168] PBS (control group, 8 mice), Anti-PD-1 (8 mice), 2 μg / time Protirelin+Anti-PD-1 (8 mice), 5 μg / time Protirelin+Anti-PD-1 (8 mice), 10 μg / time Protirelin+Anti-PD-1 (8 mice), 50 μg / time Protirelin+Anti-PD-1 (8 mice), 100 μg / time Protirelin+Anti-PD-1 (8 mice), 200 μg / time Protirelin+Anti-PD-1 (8 mice), 500 μg / time Protirelin+Anti-PD-1.

[0169] The mice were counted from the day of tumor bearing as day 0, and the drug was administered from day 9: Taltirelin was administered once every 1 day, i.e. on day 9, day 11, day 13, day 15, day 17, day 19, Protirelin was intraperitoneal injection (ip); Anti-PD-1 was administered once every 2 days, a total of 4 times, i.e. on day 9, day 12, day 15, day 18, the administration method was intraperitoneal injection (ip);

[0170] Anti-PD-1 administration: 100 μg per mouse each time;

[0171] Protirelin gradient administration: according to 20g, Protirelin in the combination drug 7 groups is administered at 2ug / time / each, 5ug / time / each, 10ug / time / each, 50ug / time / each, 100ug / time / each, 200ug / time / each, 500ug / time / each respectively; equivalent dose: 0.1mg / kg, 0.25mg / kg, 0.5mg / kg, 2.5mg / kg, 5mg / kg, 10mg / kg, 25mg / kg.

[0172] The test results show that the above 7 doses are significantly reduced in tumor volume relative to the Anti-PD-1 group alone, and the clinical dose can be calculated according to the corresponding body weight and administered in combination with Anti-PD-1.

[0173] Those skilled in the art will readily understand that the above description is only the preferred embodiment of the present application and is not intended to limit the present application, and any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Use of Taltirelin, Thyrotropin Releasing Hormone (TRH), Thyrotropin (TSH) or Protirelin in the manufacture of a medicament for enhancing the efficacy of Anti-PD-1 antibody treatment of melanoma.

2. Use according to claim 1, characterized in that, Said Thyrotropin (TSH) is natural human Thyrotropin or recombinant human Thyrotropin (rhTSH).

3. Use according to claim 2, characterized in that, Said recombinant human Thyrotropin (rhTSH) is Thyrogen.

4. Use of a combination drug in the manufacture of a medicament for treating melanoma, said combination drug comprising Taltirelin, Thyrotropin Releasing Hormone (TRH), Thyrotropin (TSH), Protirelin and Anti-PD-1 antibody, which are administered simultaneously or separately.

5. Use according to claim 4, characterized in that, Said Thyrotropin (TSH) is natural human Thyrotropin or recombinant human Thyrotropin (rhTSH).

6. Use according to claim 5, characterized in that, Said recombinant human Thyrotropin (rhTSH) is Thyrogen.

7. Use of Taltirelin in the manufacture of a medicament for treating melanoma.

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