Androgen receptor antagonists synergize with radiotherapy agents to enhance the immune response of pd-1 inhibitors to androgen-independent tumors in males

Abstract

The application provides application of an androgen receptor antagonist combined with a PD-1 inhibitor in preparation of a medicine for treating male sex gland tumors in combination with a radiotherapy agent. + The function state of T cells is remodeled, the exhaustion phenotype of tumor-infiltrating CD8 + T cells is improved, and the effect function of the T cells is improved, and the efficacy of the PD-1 inhibitor in immunotherapy of male sex gland tumors is improved.

Description

Technical Field

[0001] This invention relates to the field of therapeutic drugs for non-gonadal tumors, particularly to the field of combination therapeutic drugs for non-gonadal tumors, and even more particularly to the field of combining androgen receptor antagonists with radiotherapy agents and PD-1 inhibitors as therapeutic drugs for male non-gonadal tumors. Background Technology

[0002] Cancer poses a serious threat to human health. Tumors infiltrate CD8 cells. + Terminal exhaustion differentiation of T cells is a key factor limiting tumor immune response and low responsiveness to immune checkpoint therapies such as PD-1 inhibitors. How to inhibit or reverse T cell exhaustion and promote its stem-like differentiation is an important scientific question in the field of tumor immunotherapy.

[0003] Radiotherapy agents can enhance the therapeutic effect of PD-1 inhibitors, but the clinical benefits for patients with advanced refractory tumors are still not optimistic. It is urgent to explore new synergistic strategies for combining radiotherapy agents with immunotherapy to treat relapsed and refractory tumors and to conduct translational research.

[0004] The inventors of this application have pioneered a combined treatment regimen based on PD-1 inhibitors, radiotherapy agents, and granulocyte-macrophage colony-stimulating factors—"Brag Therapy" (see patent CN202110311772.7, selected for the National Health and Wellness Technology Promotion and Application Information Service Platform Technology Candidate Database, No.: RKJS-20240234). Bragg Therapy has shown some efficacy in patients with advanced refractory tumors. Animal experiments related to Bragg Therapy have found that the tumor radiosensitivity of male mice is significantly lower than that of female mice, and this difference appears to be closely related to the androgen / androgen receptor signaling axis. Researchers believe that diseases such as prostate cancer, breast cancer, androgen-dependent hirsutism, androgenic alopecia, uterine fibroids, leiomyomas, endometrial cancer, or endometriosis are related to the effects of androgens and androgen receptors.

[0005] The existing literature, "Research Progress of Combined Immunotherapy in Prostate Cancer," reports that for organ-localized prostate cancer, radical prostatectomy, brachytherapy, and external beam radiation therapy are commonly used, with good treatment results, and some cases even achieving a cure. For locally advanced and metastatic prostate cancer, androgen deprivation therapy is the primary treatment. Because prostate cancer is androgen-driven, treatment lowers testosterone levels, but over time, most patients develop resistance, progressing to castration-resistant prostate cancer. Androgen deprivation therapy can enhance immune infiltration in prostate cancer patients, and its effect is even better when combined with immunotherapy. However, it is worth noting that the effects of androgens on the prostate cancer tumor microenvironment (TME) and immune cell activity are complex. When androgen deprivation therapy is combined with immunotherapy, the combination regimen should be carefully selected, including different drug combinations and different timing of administration, as these can lead to different or even diametrically opposed results. Patent CN201780076363.5 provides a method for treating a subject with prostate cancer, castration-resistant prostate cancer (CRPC), metastatic CRPC (mCRPC), or locally restricted, inoperable CRPC by administering a combination of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 inhibitor, such as atezolizumab) and an anti-androgen (e.g., enzalutamide). Patent CN201480015802.8 provides a method for treating a male human patient with advanced castration-sensitive prostate cancer, castration-refractory prostate cancer, or high-risk localized prostate cancer, the method comprising administering 4-[7-(6-cyano-5-trifluoromethylpyridin-3-yl)-8-oxo-6-thio-5,7-diazaspiro[3.4]oct-5-yl]-2-fluoro-N-methylbenzamide or a pharmaceutically acceptable salt thereof to a male human patient requiring such treatment at a dose ranging from about 30 mg / day to about 480 mg / day. Patent CN202080039623.3 discloses pharmaceutical compositions and combinations that can be used to treat various cancers, such as breast cancer and prostate cancer, comprising an androgen receptor modulator and a second therapeutic agent. The second therapeutic agent is selected from a wide range of options, including modulators of PD-1 or PD-L1, radiotherapy agents emitting alpha particles, etc. However, the combined use of androgen receptor antagonists with immunotherapy is primarily targeted at gonadal tumors.

[0006] The technical problem to be solved by this invention is the effect of androgen receptor antagonists in synergistically enhancing the immune response of immunotherapy agents to non-gonadal tumors, and the mechanism of immune microenvironment regulation. The study aims to investigate how androgen receptor antagonists enhance the synergistic effect of radiotherapy agents combined with immunotherapy by regulating the tumor immune microenvironment and systemic immune response, thereby providing a new treatment option for the clinical combined treatment of male non-gonadal tumors. Summary of the Invention

[0007] The purpose of this invention is to provide the use of androgen receptor antagonists in combination with PD-1 inhibitors in the preparation of medicaments for use in combination with radiotherapy agents to treat male non-gonadal tumors.

[0008] Another object of the present invention is to provide the use of androgen receptor antagonists in combination with PD-1 inhibitors in the preparation of medicaments for use in combination with radiotherapy agents to optimize the immune microenvironment of male non-gonadal tumors.

[0009] Another object of the present invention is to provide the use of androgen receptor antagonists in combination with PD-1 inhibitors in the preparation of medicaments for use in combination with radiotherapy agents to enhance the immune response against androgenic nongonadal tumors.

[0010] Another object of the present invention is to provide the use of androgen receptor antagonists in combination with PD-1 inhibitors in the preparation of medicaments for use in combination with radiotherapy agents to reduce the volume of male non-gonadal tumors.

[0011] Another object of the present invention is to provide an androgen receptor antagonist combined with a PD-1 inhibitor in the preparation of a drug for use in combination with radiotherapy agents to reduce CD8 infiltrating male non-gonadal tumors. + Application in drugs that address the degree of T cell depletion.

[0012] Another object of the present invention is to provide an androgen receptor antagonist combined with a PD-1 inhibitor for use in combination with radiotherapy agents to increase CD45 in male non-gonadal tumors. + Cells, CD3 + T cells and CD8 + Application in drugs that increase the number of T cells per unit tumor mass.

[0013] The androgen receptor antagonist refers to a drug that can block the binding of androgens to their receptors and inhibit the biological effects of androgens.

[0014] Preferably, the androgen receptor antagonist is selected from enzalutamide (MCE, HY-70002), flutamide, bicalutamide, abiraterone, saralasin, etc.

[0015] Non-gonadal tumors refer to tumors that occur in non-reproductive gland tissues;

[0016] Preferably, the non-gonadal tumor is selected from B16 melanoma, MC38 colorectal cancer, CT26 colorectal cancer, LLC1 lung cancer, Pan02 pancreatic cancer, H22 liver cancer, etc.

[0017] Preferably, the radiotherapy agent includes drugs that cause DNA damage, and the radiotherapy agent includes those commonly referred to as gamma rays, X-rays, and / or the targeted delivery of radioactive isotopes to tumor cells.

[0018] Preferably, the radiotherapy agent is administered before the androgen receptor antagonist is administered into or adjacent to the tumor.

[0019] Another object of the present invention is to provide a pharmaceutical combination for treating male non-gonadal tumors, comprising a therapeutically effective amount of an androgen receptor antagonist, a PD-1 inhibitor, and an effective amount of a radiotherapy agent administered to a subject in need thereof, said radiotherapy agent comprising the use of a drug that causes DNA damage, including those commonly referred to as gamma rays, X-rays, and / or the directed delivery of a radioactive isotope to non-gonadal tumor cells.

[0020] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0021] This study found that in a non-gonadal tumor model, radiotherapy can regulate CD8 through the androgen / androgen receptor signaling pathway. + The differentiation status (stem maintenance / exhaustion differentiation) and effector function of T cells affect the therapeutic response to PD-1 inhibitors.

[0022] Radiotherapy drugs can induce CD8 in the microenvironment of non-gonadal tumors. + Upregulation of T cell androgen receptor expression; high expression of androgen receptors promotes tumor infiltration of CD8. + T cells in a depleted, "incapacitated" state are detrimental to PD-1 therapy; combining radiotherapy with androgen receptor antagonists or castration surgery can remodel tumor-infiltrating CD8 cells. + T cell functional status significantly improved tumor-infiltrating CD8 + T-cell exhaustion phenotype and effector function significantly improve the efficacy of PD-1 immunotherapy.

[0023] Female mice treated with radiation therapy were given androgen, which induced CD8+ in B16 melanomas in these mice. + T cells are more prone to exhaustion, exhibiting reduced effector function and a significant increase in tumor volume. Androgen receptor antagonism in radiation-treated male mice significantly reduces CD8+ in B16 melanomas of male mice. + T cell exhaustion was significantly reduced, effector function was significantly enhanced, and tumor volume was significantly reduced.

[0024] In in vitro experiments, the combination of radiotherapy drugs and the androgen receptor antagonist enzalutamide did not inhibit the growth of B16 melanoma cells. This ruled out the effect of the androgen receptor antagonist enzalutamide on the growth of B16 melanoma cells.

[0025] Compared with the sham-operated group of male mice, the castration group of male mice showed a significant reduction in bilateral tumor volume, and the B16 melanomas in this group of mice infiltrated CD8. + The exhaustion level of T cells was significantly reduced, and their effector function was significantly enhanced.

[0026] This study is the first to discover that the synergistic application of radiotherapy drugs, PD-1 inhibitors and androgen receptor antagonists can optimize the immune microenvironment of non-gonadal tumors, enhance the anti-tumor immune response of non-gonadal tumors, thereby improving the treatment effect of non-gonadal tumors and achieving a higher treatment response rate.

[0027] This study is the first to discover that CD45 is present in mouse tumors after synergistic administration of radiotherapy agents, PD-1 inhibitors, and androgen receptor antagonists. + Cells, CD3 + T cells and CD8 + The number of T cells per unit tumor mass was significantly higher in the dual therapy group (radiotherapy + PD-1 inhibitor) than in the dual therapy group.

[0028] This study is the first to find that in a female mouse model, no significant anti-tumor effect or survival advantage was observed by synergistic administration of radiotherapy agents, PD-1 inhibitors and androgen receptor antagonists, which is speculated to be related to the lower androgen levels in female mice. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 Schematic diagram of androgens and radiation therapy;

[0031] Figure 2 Androgen and radiotherapy experiments: Tumor volume and weight on the irradiated and non-irradiated sides;

[0032] Figure 3 Androgen and radiotherapy experiments: T cell exhaustion indicators and effector function indicators on the non-irradiated side;

[0033] Figure 4Androgen and radiotherapy experiments: T cell exhaustion indicators and effector function indicators on the irradiated side;

[0034] Figure 5 Schematic diagram of androgen receptor antagonists and radiotherapy;

[0035] Figure 6 Androgen receptor antagonist and radiotherapy experiment: tumor volume and weight on the irradiated and non-irradiated sides;

[0036] Figure 7 Androgen receptor antagonists and radiotherapy experiments: T cell exhaustion indicators and effector function indicators on the non-irradiated side;

[0037] Figure 8 Androgen receptor antagonists and radiotherapy experiments: T cell exhaustion indicators and effector function indicators on the irradiated side;

[0038] Figure 9 Androgen receptor antagonists and radiotherapy for B16 melanoma plate clone experiment;

[0039] Figure 10 Androgen receptor antagonist-treated LNCaP prostate cancer plate clone assay;

[0040] Figure 11 Schematic diagram of castration surgery combined with radiotherapy and immunotherapy;

[0041] Figure 12 Experimental study of castration surgery combined with radiotherapy and immunotherapy, including tumor volume and weight on the irradiated and unirradiated sides;

[0042] Figure 13 Indicators of T-cell exhaustion on the irradiated and non-irradiated sides in an experiment involving castration combined with radiotherapy and immunotherapy.

[0043] Figure 14 Experimental study of T-cell effector function in the non-irradiated side of castration surgery combined with radiotherapy and immunotherapy;

[0044] Figure 15 Experimental study of T-cell effector function in the irradiated side of castration surgery combined with radiotherapy and immunotherapy;

[0045] Figure 16 Schematic diagram of an experimental study involving androgen receptor antagonists combined with radiotherapy and immunotherapy;

[0046] Figure 17 Experimental study of tumor volume and weight using androgen receptor antagonists combined with radiotherapy and immunotherapy;

[0047] Figure 18 Indicators of T-cell exhaustion on the irradiated side in an experimental study of androgen receptor antagonists combined with radiotherapy and immunotherapy.

[0048] Figure 19 Experimental study of T-cell effector function in the non-irradiated side of androgen receptor antagonists combined with radiotherapy and immunotherapy;

[0049] Figure 20 Experimental study of T-cell effector function indices on the irradiated side in combination with androgen receptor antagonists, radiotherapy, and immunotherapy.

[0050] Figure 21 Experimental study of androgen receptor antagonists combined with radiotherapy and immunotherapy: number of tumor-infiltrating cells per unit mass on the irradiated and non-irradiated sides.

[0051] Figure 22 Tumor volume and survival curves on the irradiated and unirradiated sides of female mice in an experiment involving androgen receptor antagonists combined with radiotherapy and immunotherapy. Detailed Implementation

[0052] Example 1

[0053] The C57BL / 6 mice (6-8 weeks old) used in the experiments were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. All mice met the Specific Pathogen Free (SPF) standard. The experiments involved in this application have been approved by the Institute's Animal Management Committee. The experimental animals were housed at the Suzhou Institute of Systems Medicine Animal Center and met the SPF standard. All animal experimental procedures involved in the experiments complied with the Animal Ethics Committee Regulations of the Institute of Systems Medicine, Chinese Academy of Medical Sciences (ISM-IACUC-0018).

[0054] Female C57BL / 6 mice were randomly divided into a control group (radiotherapy) and an experimental group (radiotherapy + androgens). Each group consisted of 4-5 mice. The control group had 4 mice, and the experimental group had 5 mice.

[0055] On day 0, 2 × 10⁶ B16 melanomas were subcutaneously injected into the right and left back near the groin of the mice. 5 One tumor cell (the B16 melanoma was obtained from the cell bank of the Institute of Systems Medicine, Chinese Academy of Medical Sciences), with a subcutaneous tumor size of approximately 90-100 mm. 3 At that time, the right subcutaneous tumor was irradiated with a single dose of 8 Gy using a Rad Source RS-2000 biological X-ray irradiator for 3 consecutive days. From day 1, the experimental group mice were treated with intraperitoneal injection of androgens (Testosterone propionate, 10 mg / kg, MACKLIN) every 3 days (e.g., Figure 1 (As shown). Tumor growth was monitored and the tumor microenvironment was analyzed by flow cytometry. Samples were collected on the fourth day after radiotherapy. During the experiment, the volume of both tumors was measured every two days, with the tumor volume being the long axis × short axis.2 ÷2.

[0056] Experimental results showed that, regardless of whether the tumor was on the irradiated or unirradiated side, the tumor volume of female mice treated with androgens was significantly increased (e.g., Figure 2 (As shown), and the tumors in this group of mice infiltrated CD8. + T cells are more prone to exhaustion and exhibit reduced effector function (e.g., Figure 3-4 (As shown). Experimental results indicate that androgens cause tumors infiltrating CD8+ in female mice receiving radiotherapy. + T cell depletion leads to reduced effector function.

[0057] Example 2

[0058] Six- to eight-week-old male C57BL / 6 mice were randomly divided into a control group (received radiotherapy) and an experimental group (received radiotherapy plus the androgen receptor antagonist enzalutamide (MCE, HY-70002)). The control group consisted of four mice, and the experimental group consisted of five mice.

[0059] On day 0, mice were subcutaneously injected with B16 2×10⁻⁶ on the right and left back near the groin. 5 One tumor cell, the subcutaneous tumor is approximately 90-100mm in size. 3 At that time, the right subcutaneous tumor was irradiated with a single dose of 8 Gy using a Rad Source RS-2000 biological X-ray irradiator, and irradiated continuously for 3 days. Starting one day before radiotherapy, the experimental group mice were treated daily by gavage with the androgen receptor antagonist enzalutamide (25 mg / kg, MCE) (e.g. Figure 5 (As shown). Tumor growth was monitored and the tumor microenvironment was analyzed by flow cytometry. Samples were collected on the fourth day after radiotherapy. The volume of both tumors was measured every two days during the experiment, with the tumor volume being the long axis × short axis. 2 ÷2.

[0060] Experimental results showed that, regardless of whether the tumor was on the irradiated or unirradiated side, the tumor volume of male mice treated with androgen receptor antagonists was significantly reduced after radiotherapy (e.g., ...). Figure 6 (As shown), the experimental group mice had tumor-infiltrating CD8+. + T cell exhaustion was low, and effector function was significantly enhanced (e.g., T cells were less exhausted, and effector function was significantly enhanced). Figure 7-8 (As shown). Experimental results indicate that radiotherapy significantly reduces CD8 tumor infiltration in male mice by lowering androgen levels. + The degree of T cell exhaustion enhances effector function.

[0061] Example 3

[0062] To investigate the effect of androgen receptor antagonists on the growth of B16 melanoma cells, this application used prostate cancer LNCaP cells with high androgen receptor expression as a positive control under in vitro conditions. The cells were treated with the androgen receptor antagonist enzalutamide (MCE, HY-70002) in addition to radiotherapy (8Gy×3). The experimental concentration of the androgen receptor antagonist was 10μM, and it was added starting from day 0 of plate formation.

[0063] Colony formation assay: B16 melanoma cells were evenly seeded into 6-well plates at densities of 500 (control group), 2500, 5000, 7500, 10000, and 20000 cells / well, respectively. After culturing in a cell culture incubator at 37°C and 5% CO2 for 2 days, a single irradiation of 8 Gy was performed. After 15 days, the culture medium was aspirated, and staining and photography were performed using Coomassie Brilliant Blue R-250 solution.

[0064] Experimental results show that radiotherapy combined with the androgen receptor antagonist enzalutamide does not inhibit the growth of B16 melanoma cells (e.g., Figure 9 As shown), the growth of LNCaP prostate cancer, used as a positive control, was significantly inhibited (e.g. Figure 10 (As shown).

[0065] Example 4

[0066] To investigate the regulatory role of the androgen / AR signaling axis in the anti-tumor function of radiotherapy combined with immunotherapy, this application constructed a bilateral B16 cell tumor-bearing model. Male mice, female mice, and castrated male mice were treated with unilateral hypofractionated radiotherapy (8 Gy × 3) combined with a PD-1 inhibitor (100 μg / mouse / dose, Junshi) (e.g., Figure 11 (As shown).

[0067] Four-week-old C57 mice were divided into three groups: a male sham-operated group, a male castration-operated group, and a female group. The male sham-operated group consisted of 6 mice, the male castration-operated group consisted of 8 mice, and the female group consisted of 6 mice.

[0068] Four-week-old male mice were randomly assigned to either castration or sham surgery. For surgical castration, male mice were anesthetized with 1% sodium pentobarbital. A midline incision was then made in the scrotum, followed by another incision in the testicular sac. The vas deferens and epididymal fat pad were carefully exposed. The blood vessels supplying the testis were clamped with hemostatic forceps, and the testis, vas deferens, and adipose tissue were dissected below the forceps. The contralateral testis was treated with the same procedure. The scrotum was closed using surgical tissue adhesive (Vetbond tissue adhesive, 3M). The sham surgery group underwent the same procedure, but without removal of the gonads. The mice recovered for four weeks prior to tumor cell inoculation. On day 0, 2 × 10⁶ B16 was subcutaneously injected into the right back near the groin of the mouse. 5Two tumor cells were subcutaneously injected with B16 (2 × 10⁶ cells) into the left back near the groin of the mouse on day 3. 5 When the right subcutaneous tumor reached approximately 90-100 mm³, it was irradiated with a single dose of 8 Gy using a Rad Source RS-2000 biological X-ray irradiator over three consecutive days. Starting from day 1 of radiotherapy, mice were administered a PD-1 inhibitor (100 μg / mouse / dose) via intraperitoneal injection every three days. Samples were collected on day four after radiotherapy. Tumor volume was measured bilaterally every two days during the experiment, with the tumor volume calculated as the long axis × short axis. 2 ÷2. Dynamically monitor tumor growth and analyze CD8. + Changes in T cell differentiation status and function.

[0069] Experimental results showed that, compared with the sham-operated group of male mice, such as Figure 12 As shown, the bilateral tumor volume of male mice in the castration group was significantly reduced, and the tumors in this group of mice infiltrated CD8. + The exhaustion level of T cells was significantly reduced (e.g. Figure 13 As shown), the effector function is significantly enhanced (e.g. Figure 14-15 (As shown).

[0070] Example 5

[0071] Androgen receptor antagonists enhance the antitumor effects of combined radiotherapy and immunotherapy by affecting a variety of immune cells.

[0072] Six- to eight-week-old male C57 mice were divided into a control group (radiotherapy + PD-1 inhibitor) and an experimental group (radiotherapy + PD-1 inhibitor + androgen receptor antagonist enzalutamide). The control group consisted of 8 mice, and the experimental group consisted of 8 mice.

[0073] On day 0, mice were subcutaneously injected with B16 2×10⁶ on the right back near the groin. 5 Two tumor cells were subcutaneously injected with B16 (2 × 10⁶ cells) into the left back near the groin of the mouse on day 3. 5 One tumor cell was observed, and the right subcutaneous tumor was approximately 90-100 mm in size. 3 At that time, the right subcutaneous tumor was irradiated with a single dose of 8 Gy using a Rad Source RS-2000 biological X-ray irradiator for 3 consecutive days. Starting from day 1 of radiotherapy, the experimental mice were injected intraperitoneally with a PD-1 inhibitor (100 μg / mouse / dose) every 3 days. Starting one day before radiotherapy, the experimental group mice were administered an androgen receptor antagonist (Enzalutamide, 25 mg / kg, MCE) by gavage daily (e.g., 1 day prior to radiotherapy). Figure 16(As shown). Tumor growth was monitored and the tumor microenvironment was analyzed. Samples were collected on the fourth day after radiotherapy, and tumor invasion CD45 was calculated using flow cytometry. + Cells, CD3 + T cells and CD8 + The total number of T cells and the number of infiltrating cells per unit mass of tumor were calculated as the number of infiltrating cells divided by the tumor mass. During the experiment, the volume of both tumors was measured every two days, and the tumor volume was calculated as the long axis × short axis. 2 ÷2.

[0074] The experimental results showed that in the experimental group (radiotherapy + PD-1 inhibitor + androgen receptor antagonist), the tumor volume on both the irradiated and unirradiated sides of male mice was significantly smaller than that in the control group, with average reductions of 58.38% and 55.80%, respectively (e.g., Figure 17 As shown), CD8 infiltrates in the tumor on the irradiated side of the experimental group mice. + T cell exhaustion was significantly lower than in the control group (e.g. Figure 18 (as shown), CD8 + T cell effector function was significantly enhanced compared to the control group (e.g., Figures 19-20 (As shown).

[0075] In both irradiated and unirradiated sides, CD45 levels were higher in tumors of male mice in the experimental group (radiotherapy + PD-1 inhibitor + androgen receptor antagonist). + Cells, CD3 + T cells and CD8 + The number of T cells per unit tumor mass was significantly higher than that in the control group (radiotherapy + PD-1 inhibitor). Figure 21 ), irradiated side CD45 + Cells, CD3 + T cells and CD8 + The number of T cells per unit tumor mass increased by 53.5%, 43.8%, and 50.9%, respectively; CD45 on the non-irradiated side + Cells, CD3 + T cells and CD8 + The number of T cells per unit tumor mass increased by 39.4%, 45.8%, and 63.7%, respectively. The experimental results indicate that the antitumor effect of androgen receptor antagonists enhanced by radiotherapy combined with immunotherapy depends on leukocytes, T cells, and CD8+. + The number of T cells increases.

[0076] Example 6

[0077] Six- to eight-week-old C57 female mice were randomly divided into a control group (radiotherapy + PD-1 inhibitor) and an experimental group (radiotherapy + PD-1 inhibitor + androgen receptor antagonist enzalutamide). There were eight mice in the control group and eight mice in the experimental group.

[0078] On day 0, mice were subcutaneously injected with B16 2×10⁶ on the right back near the groin. 5 Two tumor cells were subcutaneously injected with B16 (2 × 10⁶ cells) into the left back near the groin of the mouse on day 3. 5 One tumor cell was observed, and the right subcutaneous tumor was approximately 90-100 mm in size. 3 The right subcutaneous tumor was irradiated with a single dose of 8 Gy using a Rad Source RS-2000 biological X-ray irradiator over three consecutive days. Starting from day 1 of radiotherapy, mice were administered a PD-1 inhibitor (100 μg / mouse / dose) via intraperitoneal injection every three days. Starting one day before radiotherapy, mice in the experimental group were administered an androgen receptor antagonist (Enzalutamide, 25 mg / kg, MCE) via gavage daily. Tumor growth was monitored, and the tumor microenvironment was analyzed. Samples were collected on the fourth day after radiotherapy. During the experiment, the volume of both tumors was measured every two days, with the tumor volume calculated as the long axis × short axis. 2 ÷2.

[0079] However, in female mouse models, no significant anti-tumor effect or survival advantage was observed in the experimental group (radiotherapy + PD-1 inhibitor + androgen receptor antagonist). Figure 22 (As shown in the figure), this may be related to the lower androgen levels in female mice.

[0080] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. The present invention has been described in detail with reference to preferred embodiments. Those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Application of androgen receptor antagonists in combination with PD-1 inhibitors in the preparation of drugs for use in combination with radiotherapy agents to treat male non-gonadal tumors.

2. Application of androgen receptor antagonists in combination with PD-1 inhibitors in the preparation of drugs for use in combination with radiotherapy agents to optimize the immune microenvironment of male non-gonadal tumors.

3. Application of androgen receptor antagonists in combination with PD-1 inhibitors in the preparation of drugs for use in combination with radiotherapy agents to enhance the immune response against androgenic nongonadal tumors.

4. Application of androgen receptor antagonists in combination with PD-1 inhibitors in the preparation of drugs for use in combination with radiotherapy agents to reduce the volume of male non-gonadal tumors.

5. Androgen receptor antagonists combined with PD-1 inhibitors in the preparation of drugs for use in combination with radiotherapy to reduce CD8 infiltrating male non-gonadal tumors. + Application in drugs that address the degree of T cell depletion.

6. Androgen receptor antagonists combined with PD-1 inhibitors in the preparation of drugs for use in combination with radiotherapy to enhance CD45 in male non-gonadal tumors. + Cells, CD3 + T cells and CD8 + Application in drugs that increase the number of T cells per unit tumor mass.

7. The use according to any one of claims 1-6, characterized in that, The androgen receptor antagonist is a drug that can block the binding of androgens to their receptors and inhibit the biological effects of androgens; preferably, the androgen receptor antagonist is selected from enzalutamide, flutamide, bicalutamide, abiraterone, celenes, etc.

8. The use according to any one of claims 1-6, characterized in that, The non-gonadal tumor refers to a tumor that occurs in non-reproductive gland tissue. Preferably, the non-gonadal tumor is selected from B16 melanoma, MC38 colorectal cancer, CT26 colorectal cancer, LLC1 lung cancer, Pan02 pancreatic cancer, H22 liver cancer, etc.

9. The use according to any one of claims 1-6, characterized in that, The radiotherapy agent includes drugs that cause DNA damage, including those commonly referred to as gamma rays, X-rays, and / or the directed delivery of radioactive isotopes to tumor cells. The radiotherapy agent is administered prior to the administration of an androgen receptor antagonist, which is applied to the non-gonadal tumor or substantially adjacent to it.

10. A pharmaceutical combination for treating male non-gonadal tumors, comprising a therapeutically effective amount of an androgen receptor antagonist, a PD-1 inhibitor, and an effective amount of a radiotherapy agent administered to a subject in need, said radiotherapy agent comprising the use of a drug that causes DNA damage, the radiotherapy agent comprising those commonly referred to as gamma rays, X-rays, and / or the directed delivery of a radioactive isotope to non-gonadal tumor cells; said androgen receptor antagonist is a drug capable of blocking the binding of androgens to their receptors and inhibiting the biological effects of androgens; preferably, said androgen receptor antagonist is selected from enzalutamide, flutamide, bicalutamide, abiraterone, celecoxib, etc.

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