Use of specific polypeptide in the preparation of drug for treating and preventing prostate cancer

Abstract

Provided are the use of a specific polypeptide in the preparation of a drug for treating and preventing prostate cancer, and the use of the specific polypeptide in the preparation of an immune inducer. The specific polypeptide is derived from TRPM8 protein, and has a good effect on the treatment and prevention of prostate cancer in each stage, especially androgen-independent prostate carcinoma.

Description

Application of specific peptides in the preparation of drugs for treating and preventing prostate cancer Technical Field

[0001] The present invention relates to the field of medicine, and more particularly to the use of a specific polypeptide in preparing a drug for treating and preventing prostate cancer and the use of a specific polypeptide in preparing an immune inducer. Background Art

[0002] Prostate cancer is the second most common cause of cancer-related death in men worldwide and is one of the major diseases affecting men's health. The incidence of prostate cancer is associated with race, ethnicity, geographic location, and genetic factors. Its clinical manifestations include frequent urination, decreased urination force, difficulty starting or stopping urine flow, blood in semen, pelvic pain or discomfort, and bone pain. In the early stages of prostate cancer, it is generally androgen-sensitive prostate cancer, which is usually treated with surgery or radiotherapy, followed by adjuvant endocrine therapy including the anti-androgen drugs carbilutamide and enzalutamide or the luteinizing hormone-releasing hormone agonists leuprorelin and goserelin. However, it will eventually progress to androgen-independent prostate carcinoma (AIPC).

[0003] Androgen-independent prostate cancer is insensitive to androgen suppression therapy and continues to grow. Cancer cells are poorly responsive to these treatments, or even completely lose their response, and relapse as AIPC, ultimately leading to patient death. This drug resistance in prostate cancer is a serious challenge in treatment, and most hormone-dependent cancers experience varying degrees of recurrence during 1 to 3 years of treatment despite receiving hormone therapy. Therefore, advanced prostate cancer is difficult to effectively treat due to the lack of methods to block drug resistance.

[0004] Therefore, clinicians have been hoping to find more effective methods to prevent and treat prostate cancer.

[0005] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present invention, and therefore it may contain information that does not constitute the prior art known to those skilled in the art.

[0006] Summary of the Invention

[0007] In order to solve one or more of the above problems existing in the prior art, the present invention provides a use of a specific polypeptide in preparing a drug for treating or preventing prostate cancer and a use of a specific polypeptide in preparing an immune inducer.

[0008] In the use of the specific polypeptide of the present invention in the preparation of a drug for preventing or treating prostate cancer, the specific polypeptide is derived from TRPM8 protein.

[0009] According to one embodiment of the present invention, the amino acid sequence of the specific polypeptide may be as shown in SEQ ID NO: 1.

[0010] According to one embodiment of the present invention, the above-mentioned drug can be prepared by combining the above-mentioned specific polypeptide with complete Freund's adjuvant.

[0011] According to one embodiment of the present invention, the above-mentioned medicine may be an injection.

[0012] According to one embodiment of the present invention, the prostate cancer may be androgen-independent prostate cancer.

[0013] The present invention also provides a use of a specific polypeptide in preparing an immune inducer, wherein the immune inducer is used to induce prostate tissue-specific immunity, and the specific polypeptide is derived from the TRPM8 protein.

[0014] According to one embodiment of the present invention, the amino acid sequence of the specific polypeptide may be as shown in SEQ ID NO: 1.

[0015] According to one embodiment of the present invention, the above-mentioned specific polypeptide can be combined with complete Freund's adjuvant to prepare the above-mentioned immune inducer.

[0016] According to one embodiment of the present invention, the immune inducer may be an injection.

[0017] The specific polypeptide of the present invention has a good effect on the treatment and prevention of prostate cancer at all stages, especially androgen-independent prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0019] FIG1 is a diagram of Luc RM-1 Luciferase detection according to an embodiment of the present invention.

[0020] FIG2 shows a statistical graph of tumor weights of treatment groups according to an embodiment of the present invention.

[0021] FIG3 shows a statistical graph of tumor weight in the prevention group according to an embodiment of the present invention.

[0022] FIG4 is a survival analysis diagram of treatment groups according to an embodiment of the present invention.

[0023] FIG5 is a survival analysis diagram of the prevention group according to an embodiment of the present invention.

[0024] 6a and 6b show representative images of pathological sections and inflammation scores of the prostate of the blank group and the control group according to an embodiment of the present invention.

[0025] 7a and 7b are schematic diagrams showing HE staining of tumor tissue and statistics of relative tumor necrosis area in the treatment group according to an embodiment of the present invention.

[0026] 8a and 8b are schematic diagrams of HE staining of tumor tissue and statistics of relative tumor necrosis area in the prevention group according to an embodiment of the present invention.

[0027] FIG9 shows a statistical graph of IFN-γ levels in serum of the treatment group according to an embodiment of the present invention.

[0028] FIG10 shows a statistical graph of IFN-γ levels in serum of a prevention group according to an embodiment of the present invention.

[0029] 11a and 11b are graphs showing relative fluorescence quantum numbers of treatment groups 13 days apart according to an embodiment of the present invention.

[0030] 12a and 12b are graphs of the endpoint fluorescence quantum number of the prevention group according to an embodiment of the present invention.

[0031] FIG13 is a survival curve of mice according to a comparative example of the present invention.

[0032] FIG. 14 is a statistical graph showing tumor weight according to a comparative example of the present invention.

[0033] 15a and 15b are schematic diagrams showing HE staining of tumor tissue and statistics of relative necrosis area of ​​tumor according to a comparative example of the present invention. DETAILED DESCRIPTION

[0034] The present invention is described in detail below through specific embodiments so that those skilled in the art can easily implement the present invention according to the contents disclosed in this specification. The embodiments described below are only some embodiments of the present invention, not all. Based on the embodiments described in this specification, all other embodiments obtained by those skilled in the art without paying creative work are within the scope of protection of the present invention. It should be noted that the embodiments in this specification and the features in the embodiments can be combined with each other unless there is a conflict.

[0035] Prostate cancer is unique in that its growth and progression are dependent on androgens, and androgen deprivation therapy (ADT) is an effective treatment strategy in clinical practice. Early-stage PCa can achieve good therapeutic results with radical surgery or radical radiotherapy. However, as the disease progresses, it gradually transforms into androgen-independent PCa, also known as androgen-independent prostate cancer (AIPC). Metastatic AIPC is a particularly serious form of prostate cancer.

[0036] Prostate cancer primarily occurs in immunosuppressive individuals. Prostate cancer patients have a low tumor mutation burden, and in recent years, the use of ICB (intracellular chemotherapeutic drug) strategies for prostate cancer has been relatively limited. In clinical trials, IMvigor210, atezolizumab, and pembrolizumab have demonstrated promising efficacy in treating cancer. Therefore, the state of the prostate cancer tumor microenvironment plays a crucial role in drug therapy.

[0037] In late-stage disease, the use of DC vaccines in combination with systemic or local cytoreductive therapy can overcome antigen-specific tolerance and reverse the tumor's immunosuppressive environment. Therefore, the inventors believe that the enhanced local prostate inflammation and immune infiltration induced by subcutaneous injection of TRPM8 short peptides may play an important role in the management of prostate cancer. Based on these findings regarding the impact of immune cells on cancer, they investigated the therapeutic efficacy of specific peptides derived from the TRPM8 protein in an orthotopic model of prostate cancer.

[0038] Regarding the relationship between inflammation and cancer treatment, one view holds that the anti-cancer effect of inflammation is primarily manifested in the activation of immune-related inflammatory cells and inflammatory factors in the body, which inhibit tumors. This includes both innate and adaptive immunity. Currently, immunotherapy approaches for cancer focus on enhancing the recognition of cancer cell surface antigens and blocking the immune escape of tumor cells. The goal is to initiate or restart a self-sustaining cycle of cancer immunity without generating an unrestricted autoimmune inflammatory response.

[0039] Tumor immunotherapy aims to activate the human immune system, relying on its own immune system to kill cancer cells and tumor tissue, thereby causing tumor regression. Unlike previous surgeries, chemotherapy, radiotherapy, and targeted therapies, immunotherapy targets the body's own immune system, not tumor cells and tissue. It primarily works by preventing or attenuating tumor-induced immunosuppression, thereby achieving immune-mediated tumor clearance. The goal of immunotherapy is not necessarily to completely eradicate advanced cancer, but rather to restore an immune balance that inhibits the rate and progression of tumor development. Currently developed cancer vaccines leverage the immune system to enhance resistance to cancer, thereby achieving therapeutic goals.

[0040] Tumor immunotherapy is one of the most promising research directions in the current field of tumor treatment. Cancer immunotherapy can be divided into passive immunotherapy and active immunotherapy. Passive immunotherapy uses short-term innate immune enhancement or adoptive immunity to restore T helper cell (Th)-1 response by providing exogenous proinflammatory cytokines and monoclonal antibodies. Active immunotherapy can stimulate the patient's own immune response and stimulate specific immunity, thereby activating immune cells, natural killer cells or cytotoxic T cells, or producing antibodies against tumor-specific antigens, specifically targeting tumor antigens.

[0041] Prostate cancer is a slow-growing malignancy with a low mutational burden, often referred to as a "cold tumor." Due to the low number and activity of anti-tumor immune cells within the prostate cancer TME (TME), anti-tumor immunity is limited. Blocking PD-1 and PD-L1 in prostate cancer does not restore the anti-tumor response induced by tumor-infiltrating CD8+ T cells, yet this is seen in other tumor types. These two contrasting aspects illustrate the difficulty of immunotherapy for prostate cancer compared to immunotherapy for other malignancies.

[0042] To address this, the inventors explored a different approach to cancer immunity. Specifically, they sought to promote DC maturation, enhance DC-T cell activation, and reverse changes in the tumor microenvironment, thereby boosting the role of anti-tumor immune cells in the prostate cancer TME (TME), while also modulating the interaction between PD-1 and PD-L1. The tumor immune microenvironment (TME) also influences the effectiveness of immune responses.

[0043] TRPM8 (Transient receptor potential melastatin 8, a member of the transient receptor potential cation channel subfamily M) protein is widely distributed in the body, including the prostate, pancreas, testis, thymus, lung, skin, bladder, liver, brain, intestine, sperm, etc. TRPM8 protein consists of 1104 amino acids, has a complex structure and numerous antigenic epitopes. In normal prostate, TRPM8 expression in apical secretory epithelial cells remains at a moderate level. Fuessel et al. (Fuessel, S., et al., Multiple tumor marker analyses (PSA, hK2, PSCA, trp-p8) in primary prostate cancers using quantitative RT-PCR. Int J Oncol, 2003. 23(1): p. 221-8) demonstrated that TRPM8 expression is very low in normal prostate cells, but its expression increases dramatically in prostate cancer cells. Therefore, TRPM8 is considered to be a tumor-promoting factor in prostate cancer. In the early stages of prostate cancer, TRPM8 expression increases in response to androgens. However, as the tumor progresses to the advanced, invasive, and androgen-independent stages, TRPM8 expression decreases. Furthermore, the androgen-dependence of TRPM8 expression and the expression of TRPM8 channels in the cytoplasm or cell membrane are associated with the degree of differentiation of prostate epithelial cells. These findings suggest that TRPM8 plays a tumor-specific role in tumor proliferation, rather than simply being a consequence of tumor development.

[0044] TRPM8 is a protein expressed in the prostate and whose expression is increased in prostate cancer. Because its expression is increased in prostate cancer and it is associated with tumor aggressiveness, targeting TRPM8 may be important for the treatment of prostate cancer.

[0045] In prostate cancer, TRPM8 is overexpressed and is associated with the prognosis and potential treatment of prostate cancer. It can be identified as a diagnostic marker for prostate cancer and a potential prognostic marker for AIPC. The extracellular structure of the TRPM8 protein present in prostate cancer is targeted by cytotoxic T lymphocytes, enhancing the immune system's targeted attack on prostate cancer. At the same time, TRPM8 is a non-selective Ca 2+ Ion channels play an important role in maintaining cell homeostasis and tumorigenesis. They mainly regulate cell growth, migration and apoptosis, immune regulation and inflammatory response, making them a target for the treatment of prostate cancer.

[0046] Therefore, the inventors have attempted to develop a drug that can specifically modulate the local immune system of the prostate to achieve anti-tumor effects. Thus, the drug can be an immune inducer for inducing prostate tissue-specific immunity, thereby preventing and treating prostate cancer, particularly androgen-independent prostate cancer, by inducing prostate tissue-specific immune enhancement.

[0047] Previous research by the inventors has shown that specific polypeptides derived from the TRPM8 protein (sometimes also referred to as "TRPM8 short peptides") have unique prostate tissue specificity. As an antigen, upon entering the body, they activate the systemic immune system to attack the prostate, resulting in a model of chronic non-bacterial prostatitis. Furthermore, the local inflammatory response in prostate tissue induced by the TRPM8 short peptide is accompanied by enhanced systemic immunity, an increase in immune cells, and an increase in inflammatory factors. Therefore, the inventors hypothesize that activation of the systemic immune system will target prostate tissue that has already become cancerous, thereby achieving the purpose of treating androgen-independent prostate cancer.

[0048] In addition, in the preventive administration experimental scheme, the administration of TRPM8 short peptides formed a local inflammatory response accompanied by the enhancement of systemic immunity, which had a positive effect on the survival time of mice and the reduction of tumor weight. It can form an immune attack or suppression effect on the tumor in the early stage of prostate cancer, so that the early tumor development is poor or does not occur, thereby achieving a therapeutic effect. In the treatment stage, it is mainly through activating the immune system, targeting the activation of different types of immune cells, promoting the APC function of DC, the activation and proliferation of T cells, the phagocytosis of macrophages, etc., inhibiting the growth ability of PCa or enhancing its necroptosis ability, thereby achieving the therapeutic effect. TRPM8, as an immune peptide, activates the enhancement of local prostate immunity and systemic immunity, improving the microenvironment of tumor occurrence. Whether TRPM8 short peptides are administered in the prevention stage or the treatment stage, there is a difference in the initial stage of intervention, which is related to the development stage of prostate cancer. This may lead to the possibility that the same dose of TRPM8 short peptides may have different effects in prevention and treatment. The essence of the reason may still be the difference in the tumor microenvironment state of prostate cancer.

[0049] Therefore, the local prostate immune response activated by immune TRPM8 short peptide has an inhibitory effect on the progression of AIPC treatment, and also proves the bridging role of TRPM8 in the progression of inflammation and cancer.

[0050] It is speculated that there may be a TRPM8 short peptide with a stronger binding force to PD-L1, which can antagonize the binding of PD-L1 on the surface of AIPC tumor cells and the PD-1 molecule of T lymphocytes, restore the ability of T lymphocytes in the local TME of AIPC to recognize tumor cells in AIPC, inhibit the immune escape of tumor cells in AIPC caused by the immune editing process, further promote the killing of AIPC, and ultimately promote the "tumor immune editing" process from the "clearance → balance → escape" trend to the "escape → balance → clearance" trend, thereby improving the prognosis of AIPC.

[0051] Based on this, the present inventors attempted to use specific polypeptides isolated from TRPM8 protein as antigens to specifically regulate local prostate tissues, thereby enhancing the autoimmune system to achieve the purpose of preventing and treating tumors, thereby completing the present invention.

[0052] Among them, the TRPM8 protein is a non-selective cation channel and a thermoreceptor activated by cold temperatures (8-28°C) and cooling substances such as menthol. It is composed of four identical six-transmembrane structures (S1-S6). The inventors designed and synthesized six peptides based on the six extracellular loops of the TRPM8 channel protein. Among them, the specific peptide T-8 (its amino acid sequence is shown in SEQ ID NO: 1, CSEEM RHRFR QLDTK LNDLKG) is derived from the second extracellular loop of the TRPM8 protein (amino acid residues 1075-1094) and has low homology with other members of the TRP family. Immunization with the specific peptide T-8 alone can induce mild prostatitis, and inflammatory cells include CD4+ T cells, macrophages, and granulocytes. Specific peptide T-8 plus CFA (Complete Freund's adjuvant) or Al(OH)3, or a combination of the three, can aggravate epithelial degeneration, inflammatory infiltration, and congestion in the model. Therefore, the specific peptide T-8 is preferably used in combination with the inflammatory agent CFA to manufacture the medicament of the present invention. Inflammatory cells are predominantly found in the glandular stroma and peri-glandular area, and occasionally in the luminal space. Elevated levels of IL-1β in the prostate and TNF-α and CRP in plasma are observed. Furthermore, phenotypic characteristics of EAP were assessed, demonstrating increased pelvic pain and urinary frequency. The specific peptide T-8 is a useful, convenient, and economical tool for establishing EAP models. Its chemical synthesis is simple, the preparation of immune antigen solutions is straightforward, the success rate of modeling is high, and the animal production required for antigen preparation is reduced.

[0053] Therefore, in the embodiments of the present invention, the specific polypeptide T-8 derived from the TRPM8 protein is used as an antigen to specifically regulate the local tissue of the prostate, thereby enhancing the autoimmune system to achieve the purpose of preventing and treating tumors.

[0054] It should be noted that in the examples herein, experiments were conducted using C57BL / 6 mice. Compared to nude mice, C57BL / 6 mice have a normal immune system, making them more suitable for evaluating the activity of anticancer drugs or vaccines. Furthermore, the C57BL / 6 mouse prostate cancer cell line RM-1 used is an androgen-independent prostate cancer cell line.

[0055] Example

[0056] 1. Experimental Preparation

[0057] 1.1 Animals

[0058] [Corrected 02.09.2024 according to Rule 91] C57BL / 6 male mice were used. Animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health of the United States and approved by the Institutional Animal Ethics and Use Committee of China Pharmaceutical University. The selected mice weighed approximately 24-26 g and were 10-12 weeks old. The mice were kept in a closed and well-ventilated environment with a light / dark cycle of 12h / 12h, temperature control of (20-25°C) and humidity control of (40-60%), and preventive measures were taken to prevent pathogen infection. The mice had free access to food and water. The animals selected and the experimental procedures performed were in strict accordance with the Regulations on the Management of Laboratory Animals promulgated by the State Science and Technology Commission.

[0059] 1.2 Reagents

[0060] Specific peptide T-8 (amino acid sequence: CSEEM RHRFR QLDTK LNDLKG, SEQ ID NO: 1), with a molecular weight of 2.57695 kDa, is an antigenic epitope that induces autoimmune prostatitis (further synthesized and purified by Nanjing GenScript Biotechnology Co., Ltd., Catalog No. C3949G);

[0061] Complete Freund's adjuvant, product number F5881, was purchased from Sigma-Aldrich, USA;

[0062] PBS buffer;

[0063] Hematoxylin-eosin stain;

[0064] 4% formaldehyde solution;

[0065] Anhydrous ether;

[0066] C57BL / 6 mouse prostate cancer cell line (RM-1);

[0067] 10% normal fetal bovine serum;

[0068] RMPI-1640 basal medium;

[0069] 0.25% trypsin-EDTA digestion solution (containing phenol red);

[0070] Trypan blue;

[0071] Table 1: Cell culture reagents

[0072] Table 2: Preparation of 4% paraformaldehyde solution

[0073] Table 3: Hematoxylin staining solution formula

[0074] Dissolve hematoxylin in 1L of distilled water with heating, then add sodium iodate and potassium aluminum sulfate to promote its rapid dissolution, then add citric acid and chloral hydrate, stir, and it will turn purple-red.

[0075] Preparation of eosin staining solution:

[0076] Dissolve 2.50 g of eosin in 500 mL of distilled water, add 10 mL of concentrated hydrochloric acid, let it stand overnight, filter the material together with the filter paper to prevent it from drying in an oven, and dissolve the dried material in 1000 mL of 95% ethanol to prepare eosin stock solution, which can be diluted with 95% ethanol to a 1:1 solution when used.

[0077] Preparation of specific polypeptide T-8 emulsion:

[0078] Preparation of the specific polypeptide T-8 emulsion for the 22.50 mg / kg group: (1) Dissolve 43.2 mg of specific polypeptide T-8 lyophilized powder in 19.2 mL of 0.01 M PBS and shake until it is evenly dissolved to obtain a specific polypeptide T-8 solution; (2) Take out 12.0 mL of the specific polypeptide T-8 solution and transfer it to a centrifuge tube; (3) Take 12.0 mL of complete Freund's adjuvant and suspend it thoroughly. The suspension should be allowed to stand overnight without stratification and set aside;

[0079] Preparation of the specific polypeptide T-8 emulsion for the 11.25 mg / kg group: (1) Take 6.0 mL of the specific polypeptide T-8 solution obtained in (1) above for the 22.50 mg / kg group and transfer it to a centrifuge tube; (2) Add 6.0 mL of PBS to the centrifuge tube; (3) Take 12.0 mL of complete Freund's adjuvant and suspend thoroughly. The suspension should not separate after overnight standing, and then set aside;

[0080] Preparation of the specific polypeptide T-8 emulsion for the 2.250 mg / kg group: (1) Take 3.0 mL of the specific polypeptide T-8 solution obtained in (1) of the 4.500 mg / kg group in Example 1 above and place it in a centrifuge tube; (2) Add 9.0 mL of PBS to the centrifuge tube; (3) Take 12.0 mL of complete Freund's adjuvant and suspend thoroughly. The suspension should preferably remain stationary overnight without stratification and be set aside.

[0081] 1.3 Instruments

[0082] Table 4 Instrument List

[0083] 2. Experimental methods

[0084] 2.1 Cell culture and construction of RM-1-Luciferase stable transfected cell lines

[0085] 2.1.1 Lentiviral infection of target cells to construct stable strains

[0086] RM-1 cells were infected with the packaged PGMLV-CMV-Luc-PGK-Puro lentivirus to obtain the Luc RM-1 stable strain.

[0087] On the first day, take 1×10 5 RM-1 cells were seeded into 24-well plates. The next day, before infection, the viral stock solution was removed from the -80°C freezer and thawed in an ice bath. The viral stock solution was diluted with 500 μL of complete medium to an MOI > 500. The original medium from the treatment group was aspirated, and 300 μL of the medium containing the lentivirus dilution was added to the treated cells. On the third day, the culture medium was replaced. After 16 hours of infection, the entire volume of the culture medium containing the lentivirus was replaced with 500 μL of complete medium. On the fifth day, infection efficiency was assessed by observing fluorescence under an inverted fluorescence microscope to estimate the efficiency of lentivirus infection of the target cells. Appropriate eukaryotic resistance selection cells were selected (puromycin full lethal concentration 2 μg / mL, maintenance concentration 1 μg / mL). Two rounds of drug selection (one round, two days, determined by preliminary antibiotic testing) were generally performed until the cells stabilized. After stabilization, the cells were maintained in RPMI 1640 complete medium supplemented with 10% FBS, 1% Pen / Strep, and 1 μg / mL puromycin.

[0088] 2.1.2 Luc RM-1Luciferase Detection

[0089] Luciferase was detected using samples collected from the constructed Luc RM-1 cells.

[0090] (1) Remove RM-1 and Luc RM-1 cells from the incubator, collect the cell pellet by centrifugation and transfer it to a 1.5 mL EP tube. Resuspend the cells in PBS and count them. Take a tube containing 3×10 5 Transfer the suspension of 100 cells to a new 1.5 mL EP tube and collect the cell pellet (equivalent to the amount of cells in a 24-well plate) by centrifugation.

[0091] (2) Cell lysis:

[0092] Add 120 μL of cell lysis buffer and tap the tube wall with your finger to fully lyse the cells. At the same time, use a pipette to pipette several times to ensure that the lysis buffer and cells are in full contact. Usually, the cells will be lysed after 30 seconds of contact with the cells.

[0093] (3) Centrifuge the fully lysed product at 10,000 rpm for 5 min. Transfer the supernatant to a new 1.5 mL EP tube for subsequent testing.

[0094] (4) Turn on the fluorescence detection function of the Infinite / M1000 instrument and set the parameters to 10 s for the measurement time and 2 s for the measurement interval.

[0095] (5) Add 20 μL of sample to each well of the all-white ELISA plate, repeat three times, then add 20 μL of luciferase detection reagent to each well, gently pipette and mix 2-3 times (be careful to avoid bubbles, otherwise it will affect the value), and then measure the RLU (Relative light unit).

[0096] 2.1.3 Cell culture

[0097] RM-1 prostate cancer cells were stably cultured in RPMI 1640 + 10% FBS + 1% Pen / Strep complete medium for 3-4 generations. Cells were extracted during the logarithmic phase of RM-1 prostate cancer cell growth and stained with trypan blue. Cell morphology was observed under an inverted microscope and counted using a hemocytometer. The cell suspension was diluted to 4×10 6 The cell suspension was prepared by mixing the matrix gel with the cell suspension in a volume ratio of 1:1 (on ice). The final concentration of the cell suspension was 2×10 6 / mL, for future use.

[0098] 2.2 Animal Model and Immunity

[0099] 2.2.1 Establishment of an orthotopic prostate cancer model

[0100] The mouse was anesthetized with ether, fixed in the supine position, and disinfected with iodine. A 1 cm midline incision was made above the mouse's external genitalia, and the bladder was lifted up to expose the ventral prostate. A microsyringe was used to draw up the cell suspension and inject 5 μL of RM-1-Luc cell suspension into the subcapsular capsule of the dorsal lobe of the prostate, with a total of 10,000 cells. The capsule at the injection site bulged upward to form a raised bleb, which was considered a satisfactory standard. The needle was then slowly withdrawn, and then sealed with tissue glue. After confirming that there was no overflow, the normal anatomical position of the prostate and bladder was restored, and the incision was closed with interrupted 1-0 silk thread and double-layer sutures. The mouse was returned to its cage. The intraoperative operation needs to be gentle to avoid damaging normal tissue.

[0101] 2.2.2 Specific peptide T-8 treatment of prostate cancer animals (treatment group)

[0102] In addition to the blank group and the specific peptide T-8 subcutaneous injection control group, the model group and the treatment group were all RM-1 modeled on day 0. On the 21st day of the experiment, 0.01M PBS (model group) or specific peptide T-8 emulsion was injected subcutaneously at five points. The injection was repeated on the 35th day of the experiment. Natural death of the mice was the experimental endpoint. The treatment groups included a 2.250mg / kg low-dose treatment group (also referred to as the 2.250mg / kg group), an 11.25mg / kg medium-dose treatment group (also referred to as the 11.25mg / kg group), and a 22.50mg / kg high-dose treatment group (also referred to as the 22.50mg / kg group). Dosing was based on the weight of the mice before each dose.

[0103] 2.2.3 Specific peptide T-8 prevents prostate cancer in animals (prevention group)

[0104] In addition to the blank group, the model group and the prophylactic administration group were all RM-1 modeled on day 0, which was recorded as day 0. On day 0, PBS (model group) or specific peptide T-8 emulsion was injected subcutaneously at five points. The second injection was performed on day 14 of the experiment, and RM-1-Luc cells were injected orthotopically into the prostate on day 28. The mice were dosed according to their body weight before each administration. The prophylactic administration groups included a 2.250 mg / kg low-dose prophylactic group (also known as the 2.250 mg / kg group); an 11.25 mg / kg medium-dose prophylactic group (also known as the 11.25 mg / kg group); and a 22.50 mg / kg high-dose prophylactic group (also known as the 22.50 mg / kg group).

[0105] 2.3 Comparison of survival and mortality rates of mice

[0106] The death time of all mice was recorded, and survival curves of mice in all groups were prepared.

[0107] 2.4 Evaluation of tumor growth potential

[0108] After the experiment, the mice were dissected, the weight and volume of the tumor tissues were measured, and tumors were photographed. The mice's condition and weight were monitored three times a week.

[0109] The efficacy of solid tumors is expressed as the percentage of tumor growth inhibition. The calculation method is:

[0110] Tumor growth inhibition rate % = (1-T / C) × 100%

[0111] T: average tumor weight of the treatment group; C: average tumor weight of the negative control group (i.e., model group).

[0112] Evaluation criteria: Tumor growth inhibition percentage <40% is considered ineffective; ≥40% and statistical P < 0.05 is considered effective. The experiment needs to be repeated once to determine the efficacy.

[0113] 2.5 Pathological observation

[0114] Sections were sliced ​​at 5 μm thickness along the largest cross-section. Each section was dewaxed, stained with hematoxylin and eosin, and mounted with gum before observation under a microscope. Sections were scanned using a scanner and analyzed using Image J. Areas to be calculated were circled and then calculated using a dedicated software analysis system. Area data were recorded and ratios were calculated. Tumor area, necrosis area, and necrosis area / tumor area (area ratio) were calculated.

[0115] Using a randomized, blinded method, pathological sections were randomly numbered and independently scored by three researchers to assess prostate histomorphological changes and the degree of inflammatory cell infiltration. Scoring criteria were: 0, no inflammation; 1, mild inflammatory cell infiltration in the prostate stroma, perivascular, or periglandular areas; 2, moderate infiltration with epithelial cell degeneration; and 3, atrophy of the majority of the acinar epithelium, significant inflammatory cell infiltration, and severe congestion. The final score for each pathological section was the mean of the scores of the three researchers.

[0116] Tissue dehydration: Tissues fixed with 4% formaldehyde were dehydrated. The dehydration procedure is shown in Table 5.

[0117] Table 5: Tissue dehydration time and procedures

[0118] Hematoxylin-eosin staining steps:

[0119] Dewaxing: xylene I (10 min), xylene II (10 min); ethanol washing: anhydrous ethanol I (2 min), anhydrous ethanol II (2 min), 95% ethanol (1 min), 75% ethanol (1 min); rinsing with running water (2 min), hematoxylin solution (8 min), rinsing with running water (3 s), differentiation: 1% hydrochloric acid ethanol (10 s, 70% ethanol and concentrated hydrochloric acid), blueing with running water (20 min), eosin staining (2 min), rinsing with running water (3 s), dehydration: 95% ethanol (30 s), 95% ethanol II (30 s), anhydrous ethanol I (2 min), anhydrous ethanol II (2 min); transparentization: xylene I (2 min), xylene II (2 min), xylene III (2 min), neutral resin sealing.

[0120] 2.6 Enzyme-linked immunosorbent assay

[0121] Blood was collected before mice were sacrificed and allowed to rest at 4°C for 1 hour. The blood was then centrifuged at 3000 rpm for 10 minutes to obtain the supernatant, which was stored at -20°C. The concentration of the cytokine interferon-γ (IFN-γ) in the immunized animals was measured using an ELISA kit.

[0122] 2.7 Mouse imaging analysis of tumor cell infiltration and growth

[0123] Imaging is performed approximately 15 minutes after the mouse is injected intraperitoneally with 100 μL of fluorescein (30 mg / mL in PBS) prior to imaging. After conventional anesthesia (gas or needle anesthesia), the mouse is placed on the imaging darkroom platform. The mouse is anesthetized with chloral hydrate in the induction chamber. Software controls the platform's elevation to an appropriate field of view, automatically turning on the illumination (bright field) to capture the first background image. Next, the illumination is automatically turned off, and in the absence of external light (dark field), the specific photons emitted by the mouse are captured. Overlaying the bright and dark field background images visually displays the location and intensity of the specific photons within the animal, completing the imaging process.

[0124] 2.8 Statistics

[0125] SPSS v21.0 software was used for statistical analysis of differences. One-way analysis of variance (ANOVA) was used to determine differences between groups. P < 0.05, P < 0.01, and P < 0.001 indicated statistically significant differences, extremely significant differences, and very significant differences, respectively. Graphs were drawn using GraphPad Prism v7.0 software.

[0126] 3. Results

[0127] 3.1 Luc RM-1 Luciferase Assay

[0128] The results of Luc RM-1 Luciferase detection are shown in Figure 1. The detection was performed using luciferase detection reagent, and the experiment was repeated three times. The average value was taken, and the result showed that the transfection efficiency reached 99.8%.

[0129] 3.2 Tumor weight and tumor inhibition rate

[0130] Table 6 shows the tumor inhibition rate of the treatment group, and Figure 2 shows the tumor weight of the treatment group (N=9). As shown in Table 6 and Figure 2, there were significant differences in tumor weight and tumor inhibition rate in the mice treated with 11.25 mg / kg compared with the model group (P<0.05).

[0131] Table 6: Tumor inhibition rate of treatment groups

[0132] * indicates P < 0.05 compared with the model group

[0133] Table 7 shows the tumor inhibition rate of the prevention group, and Figure 3 shows the tumor weight of the prevention group (N=7). As shown in Table 7 and Figure 3, there were significant differences in tumor weight and tumor inhibition rate in the 11.25 mg / kg group compared with the model group (P<0.05).

[0134] Table 7: Tumor inhibition rate in the prevention group

[0135] * indicates P < 0.05 compared with the model group

[0136] 3.3 Analysis of mouse survival time

[0137] Figure 4 shows a survival analysis graph of the treatment group (wherein, N=9, * represents P<0.05). As shown in Figure 4, compared with the model group, there was a significant difference in the survival time of mice in the treatment group of 2.25 mg / kg and 22.50 mg / kg (P<0.05). Figure 5 shows a survival analysis graph of the prevention group (wherein, N=7, * represents P<0.05). As shown in Figure 5, compared with the model group, there was a significant difference in the survival time of mice in the prevention group of 2.25 mg / kg (P<0.05).

[0138] 3.4 Histopathology

[0139] 3.4.1 Prostate tissue pathology scoring

[0140] Figures 6a and 6b show representative images of pathological sections and inflammation scores of the prostate in the blank group and the control group, wherein Figure 6a is a representative image of pathological sections of the prostate in the blank group and the control group (i.e., the group in which specific polypeptide T-8 was subcutaneously injected to induce specific immune inflammation in prostate tissue, i.e., the inflammation model control group); Figure 6b is the inflammation score of the prostate in the blank group and the control group (** represents P < 0.0001 compared with the blank group). The specific operation is to prepare pathological sections of prostate tissue from each group of mice, and evaluate the difference in pathological scores between the control group and the blank group by prostatitis histology. As shown in Figures 6a and 6b, the glands in the prostate of the blank group are evenly distributed, with only occasional single scattered inflammatory cell infiltration in the matrix; the glands in the prostate of the control group are evenly distributed and local inflammatory cell infiltration increases. There is a significant statistical difference in pathological scores between the control group and the blank group (P < 0.0001). The results show that the control group successfully enhanced local inflammatory infiltration of the prostate.

[0141] 3.4.2 Tumor Necrosis Area / Tumor Area Ratio

[0142] Tumor tissue showed diffuse, sheet-like distribution of tumor cells, with nested structures visible in some areas. The intercellular boundaries were unclear, the cytoplasm was weakly eosinophilic, the nuclei were round or oval, with significant atypia and a high nuclear-cytoplasm ratio. Some nucleoli were visible, and mitotic figures were readily discernible. Some tumor cells were necrotic with nuclear pyknosis, and minimal inflammatory cell infiltration was observed in the interstitial tissue at the tumor margin and in the necrotic areas.

[0143] Figures 7a and 7b are schematic diagrams of the tumor necrosis area / tumor area ratio in the treatment groups, with Figure 7a showing a tumor image in the treatment group and Figure 7b showing a graph of the tumor necrosis area / tumor area ratio in the treatment group (N = 6, * indicates P < 0.05 compared to the model group). Figures 7a and 7b show that the tumor necrosis area / tumor area ratio in the 2.25 mg / kg and 11.25 mg / kg treatment groups was significantly different from that in the model group (P < 0.05).

[0144] Figures 8a and 8b are schematic diagrams of the tumor necrosis area / tumor area ratio in the prevention group, with Figure 8a showing a tumor image in the treatment group and Figure 8b showing a graph of the tumor necrosis area / tumor area ratio in the treatment group (N = 5, * indicates P < 0.05 compared to the model group). Figures 8a and 8b show that compared to the model group, the tumor necrosis area / tumor area ratio in the 2.25 mg / kg and 22.50 mg / kg groups exhibited significant differences (P < 0.05).

[0145] 3.5 Biochemical indicators

[0146] Figure 9 shows the IFN-γ content in the serum of the treatment group (N=7, * represents P<0.05, ** represents P<0.01, *** represents P<0.001). As shown in Figure 9, compared with the model group, there was a significant difference in IFN-γ in the serum of mice in the treatment group with 11.25 mg / kg administration (P<0.01); compared with the model group, there was a significant difference in IFN-γ in the serum of mice in the treatment group with 22.50 mg / kg administration (P<0.05). Figure 10 shows the IFN-γ content in the serum of the prevention group (N=7, * represents P<0.05, ** represents P<0.01, *** represents P<0.001). As shown in Figure 10, compared with the model group, there was a significant difference in IFN-γ in the serum of mice in the prevention group with 11.25 mg / kg administration (P<0.05); there was an extremely significant difference in IFN-γ in the serum of mice in the prevention group with 22.50 mg / kg administration (P<0.001).

[0147] 3.6 Mouse Imaging

[0148] Figures 11a and 11b are relative fluorescence quantum number diagrams of the treatment group at intervals of 13 days, wherein Figure 11a is a live imaging diagram of mice on day 0 and day 14 of the treatment group, and Figure 11b is a statistical diagram of the relative fluorescence quantum number of the treatment group on day 13 (N=5). As shown in Figures 11a and 11b, compared with the model group, the relative fluorescence quantum number (relative fluorescence quantum number = fluorescence quantum number on day 14 / fluorescence quantum number on day 0) of mice in the treatment administration 2.25mg / kg, 11.25mg / kg, and 22.50mg / kg groups decreased after 13 days, but there was no significant difference. Figures 12a and 12b are endpoint fluorescence quantum number diagrams of the prevention group, wherein Figure 12a is a live imaging diagram of mice at the endpoint of the prevention group, and Figure 12b is a statistical diagram of the fluorescence quantum number at the endpoint of the prevention group (N=6, * indicates P<0.05 compared with the model group). As shown in Figures 12a and 12b, compared with the model group, there were significant differences in the preventive administration groups of 2.25 mg / kg and 22.5 mg / kg (P<0.05).

[0149] From the above results, it can be seen that in the treatment group, compared with the model group, there were significant differences in the tumor weight and tumor inhibition rate of mice in the 11.25 mg / kg treatment group (P<0.05). In the prevention group, compared with the model group, there were significant differences in the tumor weight and tumor inhibition rate of mice in the 11.25 mg / kg treatment group (P<0.05). In the treatment group, compared with the model group, there were significant differences in the survival time of mice in the 2.25 mg / kg and 22.50 mg / kg treatment groups (P<0.05). In the prevention group, compared with the model group, there were significant differences in the survival time of mice in the 2.25 mg / kg treatment group (P<0.05). In the treatment group, compared with the model group, there were significant differences in the ratio of tumor necrosis area to tumor area in the 2.25 mg / kg and 11.25 mg / kg treatment groups (P<0.05). In the prevention group, the ratio of tumor necrosis area to tumor area was significantly different in the 2.25 mg / kg and 22.50 mg / kg groups compared with the model group (P < 0.05). In the treatment group, the serum IFN-γ level in the 11.25 mg / kg group was significantly different from that in the model group (P < 0.01); and the serum IFN-γ level in the 22.50 mg / kg group was significantly different from that in the model group (P < 0.05). In the prevention group, the serum IFN-γ level in the 11.25 mg / kg group was significantly different from that in the model group (P < 0.05); and the serum IFN-γ level in the 22.50 mg / kg group was extremely significantly different from that in the model group (P < 0.001). In the treatment group, the relative fluorescence quantum number of mice in the treatment groups of 2.25 mg / kg, 11.25 mg / kg, and 22.50 mg / kg decreased after 13 days compared with the model group, but there was no significant difference. In the prevention group, there was a significant difference between the 2.25 mg / kg and 22.5 mg / kg prevention groups and the model group (P<0.05). In other words, the experimental results showed that the specific peptide T-8 has a therapeutic and preventive effect on androgen-independent prostate cancer in terms of inhibiting tumor growth, prolonging mouse survival time, local immune cell infiltration in tumor tissue, necrotic foci formed by immune cells attacking tumor tissue, and serum cytokine IFN-γ levels.

[0150] It should be noted that, although the specific polypeptide T-8 was used in combination with complete Freund's adjuvant in this example, the present invention is not limited thereto, and aluminum hydroxide adjuvant or other adjuvants may also be added.

[0151] In addition, in this embodiment, an injection containing the specific polypeptide T-8 is used as a drug, but the present invention is not limited thereto. The medicament containing the specific polypeptide T-8 can also be made into an emulsion, suspension, oil, tablet, capsule or other dosage form for use. However, the effect is better when it is directly injected into the body as an injection.

[0152] Comparative Example

[0153] This study used enzalutamide, a commonly used drug in clinical practice, as a comparative example to demonstrate its efficacy in mice with prostate cancer following inoculation with RM-1 prostate cancer cells. Enzalutamide, an androgen receptor inhibitor, was approved by the FDA in August 2012 for the treatment of metastatic castration-resistant prostate cancer (mAIPC).

[0154] 1. Experimental Methods

[0155] 1.1 Group settings

[0156] Male C57BL / 6 mice weighing 22-24 g and aged 6-8 weeks were selected and randomly divided into a model group (modeling but not drug administration) and a positive drug group (modeling and drug administration).

[0157] 1.2 RM-1-Luc cell recovery

[0158] After removing the frozen RM-1-Luc cells from the -80°C freezer, heat them in a 37°C water bath, ensuring that the cryoprotectant dissolves within 1 minute. Transfer the cryovial to a clean bench and use a rubber-tipped pipette to aspirate the cryoprotectant into a 10mL centrifuge tube. Add 4mL of culture medium. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cells in 1mL of culture medium. Add 7mL of culture medium to a new cell culture flask. Add the resuspended cell suspension to the flask, tighten the cap, and place in a cell culture incubator for incubation at 37°C and 5% CO2.

[0159] 1.3 Cell passaging

[0160] Remove cells in the logarithmic growth phase, remove the culture medium from the cell culture flask using a rubber-tipped pipette, and add 2 mL of PBS to wash. Then, add 2 mL of trypsin-EDTA solution to digest the cells for 2 minutes. After digestion, observe cell morphology under a microscope. If the cells are round, the digestion is appropriate. At this point, add 4 mL of culture medium to the flask to terminate digestion, and pipette the cells to detach them from the walls.

[0161] Transfer the liquid to a 10mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and add 2mL of culture medium to the centrifuge tube to resuspend the cells. Take a new T25 cell culture flask, add 7mL of culture medium and 1mL of cell suspension to the flask, gently shake to mix, and continue cell culture.

[0162] 1.4 Cell cryopreservation

[0163] After cell digestion and centrifugation (same steps as above), resuspend the cells in freezing solution and transfer them to cryopreservation tubes. After sealing with sealing film, freeze them in a -80℃ refrigerator.

[0164] 1.5 Cell Count

[0165] Transfer RM-1-Luc cells in the logarithmic growth phase to a clean bench. Remove any remaining culture medium with a rubber-tipped pipette. Wash twice with PBS. Add 2 mL of trypsin to the flask and digest the cells for 2 minutes. After digestion, discard the trypsin, add 4 mL of complete culture medium to the flask to terminate digestion, and pipette the cells to detach.

[0166] Transfer the liquid to a 10mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend the cells in an appropriate amount of serum-free medium. Transfer 10 μL of the resuspended cell suspension to a suitable container and add 10 μL of 4% trypan blue solution. Mix thoroughly. Place a coverslip on a cell counting slide and add 10 μL of the cell suspension and 4% trypan blue mixture from the edge of the coverslip. Allow the liquid to flow naturally under the coverslip through a siphon effect to avoid the formation of bubbles.

[0167] Place the cell counting slide under a microscope and adjust the focus to clearly observe the lines and live cells on the slide. Count only the live cells in the upper left, lower left, upper right, and lower right corners, following the principle of counting the top corner, not the bottom corner, and the left corner, not the right corner.

[0168] The formula for calculating the concentration of cell suspension is:

[0169] 1.6 Preparation of cell suspension

[0170] After cell counting, add an appropriate amount of serum-free culture medium to the cell suspension according to the dilution formula to dilute the cell suspension to the target concentration (2×10 6 / mL) for later use.

[0171] 1.7 Prostate cancer in situ model

[0172] After anesthetizing the mouse, place it in the supine position on a heating pad to prevent hypothermia during and after surgery. Secure the mouse's limbs with medical tape to prevent struggling. Moisten the abdominal hair with 75% ethanol and remove the perineal hair. Use ophthalmic scissors to make an incision approximately 1 cm above the genitals to expose the abdominal cavity. Quickly locate the spermatic cord by identifying the bladder and the prostate, which is attached to it. Lift the prostate with ophthalmic forceps, positioning it in a vertical position. Use a microinjector to draw 5 μL of cell suspension and insert the needle vertically into the prostate tissue to a depth of at least 5 mm. Push the syringe plunger to allow the suspension to enter the tissue. Successful injection is indicated by visible swelling and transparency of the tissue. After injection, the needle remains in the tissue for 1–2 seconds before being withdrawn. Apply biological glue to the needle site. Reposition the internal organs to their normal physiological position and suture the incision. Exercise gentleness during the procedure to avoid excessive trauma to the mouse. The sham operation group only underwent anesthesia, abdominal opening, prostate positioning and suturing, and no cell suspension injection was performed.

[0173] 1.8 Drug configuration

[0174] Preparation of Enzalutamide: Enzalutamide was dissolved in DMSO to prepare a solution, and 50 mg / kg was administered orally by gavage to mice in the positive drug group. The drug was administered once a day starting 21 days after modeling and continued for 28 days.

[0175] 1.9 Collection of materials

[0176] Blood, prostate, tumor, spleen and other tissues were collected from each mouse and fixed with paraformaldehyde for more than 48 hours before subsequent operations.

[0177] 1.10 Tissue dehydration and staining

[0178] [Corrected 02.09.2024 according to Rule 91] Place the fixed prostate tissue in a metal dehydration box and mark each box with a marker. Place the metal dehydration boxes in a basket and arrange them neatly. Dehydrate them in the order listed in Table 8 below. After the tissue is dehydrated, it is paraffin-embedded and sliced. The slice thickness is about 5μm. The slices are glued in a constant temperature water bath at 40℃. Then, use a clean glass slide to pick out the complete and appropriate pathological slices and place them in an enamel tray to dry overnight. Place the dried slices in an oven at 68℃ for 120 minutes to prevent them from falling off. After the pathological slices cool to room temperature, store them in the slice box.

[0179] Table 8: Tissue dehydration time and procedures

[0180] Pathological sections were air-dried overnight and then oven-fixed at 68°C for 60 minutes. The procedure was performed in the following order: xylene dewaxing, gradient ethanol dehydration, staining, differentiation, staining, and xylene clearing. After staining, the sections were mounted with neutral resin and allowed to stand overnight. Before microscopic observation, excess neutral resin was wiped off with a xylene-soaked cotton ball. Microscopic evaluation and photography were then performed. The detailed procedure is shown in Table 9 below.

[0181] Table 9 HE staining process

[0182] 2 Experimental results

[0183] 2.1 Survival curve

[0184] The survival time of the model group and the positive drug group is shown in Figure 13. As shown in Figure 13, there is no significant difference in the positive drug group compared with the model group (P>0.05).

[0185] 2.2 Tumor inhibition rate

[0186] The average tumor weights of the positive drug group and the model group are shown in Figure 14. The tumor inhibition rate was calculated based on the average tumor weights of the two groups (see Table 10 below). As shown in Figure 14 and Table 10, there was no significant difference in the tumor inhibition rate in the positive drug group compared to the model group, and the downward trend in tumor weight was relatively small.

[0187] Table 10 Tumor inhibition rate of each group in the comparative example

[0188] 2.3 Tumor tissue pathology sections

[0189] The HE staining results for the model and positive drug groups are shown in Figures 15a and 15b. Figure 15a shows a tumor image for the treatment group, and Figure 15b shows a graph of the tumor necrosis area / tumor area ratio for each group. As shown in Figures 15a and 15b, the tumor sections in the positive drug group showed a relatively higher proportion of necrosis (the pinkish portion represents necrotic tissue, and the purple portion represents normal tumor tissue), but no significant difference was observed.

[0190] 3. Experimental conclusions:

[0191] The positive drug group showed no significant difference in survival time analysis, tumor inhibition rate and tumor necrosis area ratio compared to the model group, but both groups showed a trend of inhibiting disease progression. Compared with the comparative example, the therapeutic effects of each drug administration group in the embodiment were better.

[0192] In summary, subcutaneous injection of the specific polypeptide T-8 has a good specific effect in both the prevention and treatment of in situ androgen-independent prostate cancer. It should be noted that the present invention can be used alone or in combination with other drugs such as androgens in the preventive stage and at all stages of prostate cancer.

[0193] The above embodiments, particularly any "preferred" embodiments, are possible examples of implementations and are presented merely for a clear understanding of the principles of the present invention. Many changes and modifications may be made to the above embodiments without departing substantially from the spirit and principles of the technology described herein. All modifications are intended to be included within the scope of this disclosure.

[0194] All documents mentioned in this specification are incorporated herein by reference, as if each document were incorporated herein by reference in its entirety.

[0195] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope of protection of the present invention.

Claims

1. Use of a specific polypeptide in the preparation of a drug for preventing or treating prostate cancer, characterized in that, The specific polypeptide is derived from the TRPM8 protein.

2. Use of the specific polypeptide according to claim 1 in the preparation of a medicament for treating prostate cancer, characterized in that, The amino acid sequence of the specific polypeptide is as shown in SEQ ID NO:

1.

3. Use of the specific polypeptide according to claim 2 in the preparation of a medicament for treating prostate cancer, characterized in that, The drug is prepared by using the specific polypeptide in combination with complete Freund's adjuvant.

4. Use of the specific polypeptide according to claim 3 in the preparation of a medicament for treating prostate cancer, characterized in that, The drug is an injection.

5. Use of the specific polypeptide according to any one of claims 1 to 4 in the preparation of a medicament for treating prostate cancer, characterized in that, The prostate cancer is androgen-independent prostate cancer.

6. Use of a specific polypeptide in the preparation of an immune inducer, characterized in that, The immune inducer is used to induce prostate tissue-specific immunity, and the specific polypeptide is derived from the TRPM8 protein.

7. Use of the specific polypeptide according to claim 6 in the preparation of an immune inducer, characterized in that, The amino acid sequence of the specific polypeptide is as shown in SEQ ID NO:

1.

8. Use of the specific polypeptide according to claim 6 or 7 in the preparation of an immune inducer, characterized in that, The immune inducer is prepared by using the specific polypeptide in combination with complete Freund's adjuvant.

9. Use of the specific polypeptide according to claim 8 in the preparation of an immune inducer, characterized in that, The immune inducer is an injection.

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