WO2025137979A1 - Use of specific polypeptide in the preparation of drug for treating and preventing prostate cancer - Google Patents

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

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

Background Art

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

Androgen-independent prostate cancer is insensitive to androgen suppression therapy and will continue to grow. Cancer cells have low responsiveness to the above treatment, or even completely lose their response, and relapse in the form of AIPC, eventually leading to the patient's death. This drug resistance of prostate cancer is a serious challenge in the treatment process, and most hormone-dependent cancers still relapse to varying degrees during the 1-3 years of treatment despite hormone therapy. Therefore, advanced prostate cancer is difficult to treat effectively due to the lack of methods to block drug resistance.

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

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.

Summary of the invention

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 and preventing prostate cancer and a use of a specific polypeptide in preparing an immune inducer.

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.

According to one embodiment of the present invention, the amino acid sequence of the specific polypeptide can be shown as SEQ ID NO: 1.

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

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

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

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

According to one embodiment of the present invention, the amino acid sequence of the above-mentioned specific polypeptide can be shown as SEQ ID NO: 1.

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.

According to one embodiment of the present invention, the immune inducing agent may be an injection.

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

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:

Figure 1 is a Luc RM-1 Luciferase detection diagram according to an embodiment of the present invention.

FIG. 2 shows a statistical graph of tumor weights of treatment groups according to an embodiment of the present invention.

FIG. 3 shows a statistical graph of tumor weight of the prevention group according to an embodiment of the present invention.

FIG. 4 is a survival analysis graph of treatment groups according to an embodiment of the present invention.

FIG. 5 is a survival analysis diagram of the prevention group according to an embodiment of the present invention.

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.

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.

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.

FIG. 9 shows a statistical graph of IFN-γ content in serum of a treatment group according to an embodiment of the present invention.

FIG. 10 shows a statistical graph of IFN-γ content in serum of a prevention group according to an embodiment of the present invention.

11a and 11b are graphs showing relative fluorescence quantum numbers of treatment groups at 13-day intervals according to an embodiment of the present invention.

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

FIG. 13 is a survival curve of mice according to a comparative example of the present invention.

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

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

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 creative work belong to 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 without conflict.

The uniqueness of prostate cancer lies in its dependence on androgen for growth and progression, and androgen deprivation therapy (ADT) is an effective treatment strategy in clinical practice. Early PCa can achieve good treatment effects through radical surgery or radical radiotherapy, and as the disease progresses, it slowly transforms into androgen-independent PCa, namely androgen-independent prostate cancer (AIPC). Among them, metastatic androgen-independent prostate cancer (AIPC) is a more serious type of prostate cancer.

Prostate cancer mainly occurs in immunosuppressive populations. Prostate cancer has a low tumor mutation burden and the application of ICB treatment strategies for prostate cancer has been relatively limited in recent years. In clinical trials, IMvigor210, atezolizumab, pembrolizumab, etc. have shown certain efficacy in the treatment of cancer, so the state of the prostate cancer tumor microenvironment is of great significance for drug treatment.

The use of DC vaccines in combination with systemic or local cytoreductive therapy in the late stage can overcome antigen-specific tolerance and reverse the tumor immunosuppressive environment. Therefore, the inventors believe that the enhanced local inflammation and increased immune infiltration of the prostate caused by subcutaneous injection of TRPM8 short peptides may play an important role in the control of prostate cancer. Based on the above effects of immune cells on cancer, an attempt was made to study the therapeutic effect of specific peptides derived from TRPM8 protein on the in situ model of prostate cancer.

Regarding the relationship between inflammation and cancer treatment, there is a view that the anti-cancer effect of inflammation is mainly reflected in the activation of immune-related inflammatory cells and inflammatory factors in the body to inhibit tumors, which includes innate immunity and adaptive immunity. At present, the main idea of immunotherapy for cancer is to enhance the recognition of cancer cell surface antigens and block the immune escape of tumor cells. The goal is to start or restart the self-sustaining cycle of cancer immunity, but it will not produce an unrestricted autoimmune inflammatory response.

Tumor immunotherapy aims to activate the human immune system, relying on the body's own immune function to activate and kill cancer cells and tumor tissues, causing tumor regression. Unlike previous surgery, chemotherapy, radiotherapy and targeted therapy, immunotherapy targets not tumor cells and tissues, but the body's own immune system. It mainly works by preventing or weakening tumor-induced immunosuppression, thereby achieving immune-mediated tumor clearance. The goal of immunotherapy is not necessarily to completely eradicate advanced cancer, but to restore a state of immune balance and inhibit the speed and progress of tumor development. The cancer vaccines currently being developed use the effects of the body's own immune system to enhance resistance to cancer, thereby achieving the purpose of treatment.

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 is to provide exogenous proinflammatory cytokines and monoclonal antibodies, using short-term innate immune enhancement or adoptive immune restoration of T helper cell (Th)-1 response. 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.

Prostate cancer is a slow-growing, low-mutation-load malignant tumor known as a "cold tumor." Due to the small number and low activity of immune cells with anti-tumor activity in the prostate cancer TME, the anti-tumor immune effect is limited. Blocking the effects of PD-1 and PD-L1 in prostate cancer does not restore the anti-tumor response induced by tumor-infiltrating CD8+T cells, but this can be seen in other types of tumors. This comparison from two aspects illustrates the difficulty of immunotherapy for prostate cancer compared with immunotherapy for other malignant tumors.

In response to this, the inventor tried another approach to cancer immunity. That is, by promoting the maturation of DC cells, strengthening the activation of DC-T cells, and reversing the changes in the tumor microenvironment, the role of anti-tumor immune cells in prostate cancer TME was promoted, while regulating the interaction between PD-1 and PD-L1. The tumor immune microenvironment (TME) also affects the immune effect.

TRPM8 (Transient receptor potential melastatin 8, member of transient receptor potential cation channel subfamily M) protein is widely distributed in the body, including prostate, pancreas, testis, thymus, lung, skin, bladder, liver, brain, intestine, sperm, etc. TRPM8 protein is composed of 1104 amino acids, with a complex structure and numerous antigenic epitopes. In normal prostate, the expression of TRPM8 in apical secretory epithelial cells remains at a moderate level. Fuessel (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) et al. demonstrated that TRPM8 is expressed at a very low level in normal prostate cells, but its expression increases sharply in prostate cancer cells. Therefore, TRPM8 is considered to be a carcinogenic factor in prostate cancer. In the early stage of prostate cancer, the expression of TRPM8 is increased by androgen regulation. However, as the tumor develops, the expression of TRPM8 decreases in the late, invasive and androgen-independent stages. In addition, the androgen dependence of TRPM8 expression and the expression of TRPM8 channels in the cytoplasm or cell membrane are related to the degree of differentiation of prostate epithelial cells. Based on the above, it can be inferred that TRPM8 has a tumor-specific role in tumor proliferation, not just the result of tumor development.

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.

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 channel that plays an important role in maintaining cell homeostasis and tumorigenesis. It mainly regulates cell growth, migration and apoptosis, immune regulation and inflammatory response, making it a target for the treatment of prostate cancer.

Therefore, the inventor attempts to develop a drug that can specifically regulate the enhancement of the local autoimmune system of the prostate tissue to achieve the anti-tumor purpose. Thus, the drug can be an immune inducer for inducing prostate tissue-specific immunity, and prevents and treats prostate cancer, especially androgen-independent prostate cancer, by inducing prostate tissue-specific immune enhancement.

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

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. In the early stage of prostate cancer, an immune attack or suppression effect can be formed on the tumor, so that the early tumor is poorly developed 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 necrosis and apoptosis ability, so as to achieve the therapeutic effect. TRPM8, as an immune peptide, activates the enhancement of local prostate immunity and systemic immunity, and improves the microenvironment of tumor occurrence. Whether TRPM8 short peptides are given in the prevention stage or the treatment stage, there are only differences in the initial stage of intervention, which is related to the development stage of prostate cancer. This may cause the same dose of TRPM8 short peptides in prevention and treatment to have different effects. The essence of the reason may still be different from the tumor microenvironment state of prostate cancer.

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

It is speculated that there may be TRPM8 short peptides with stronger binding force to PD-L1, which can antagonize the binding of PD-L1 on the surface of AIPC tumor cells and PD-1 molecules on 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.

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

Among them, TRPM8 protein is a non-selective cation channel and a temperature receptor activated by cold temperature (8-28°C) and cooling substances such as menthol. It consists 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 polypeptide 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 a low homology with other members of the TRP family. Immunization with specific polypeptide T-8 alone can induce mild prostatitis, and inflammatory cells include CD4+T cells, macrophages, and granulocytes. Specific polypeptide 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. Therefore, it is preferred that the specific polypeptide T-8 is used in combination with the inflammatory agent CFA to manufacture the drug of the present invention. Inflammatory cells are mostly found in the glandular stroma and peri-glandular area, and occasionally in the lumen. The levels of IL-1β in the prostate and TNF-α and CRP in the plasma are elevated. In addition, the phenotypic characteristics of EAP were also evaluated, and the degree of pelvic pain and frequent urination were increased. The specific polypeptide T-8 is a useful, convenient and economical tool for establishing an EAP model. It has low chemical synthesis difficulty, simple preparation of immune antigen solution, high modeling success rate and saves animals required for antigen preparation.

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.

It should be noted that in the examples of this specification, C57BL/6 mice were used for the experiments. Compared with nude mice, C57BL/6 mice have a normal immune system, which is beneficial for evaluating the activity of anticancer drugs or vaccines. In addition, the C57BL/6 mouse prostate cancer cell line RM-1 used is an androgen-independent prostate cancer cell.

Example

1. Experimental Preparation

1.1 Animals

[Corrected 02.09.2024 in accordance with Article 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 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 12h/12h of light/darkness, temperature control of (20-25°C) and humidity control of (40-60%), and preventive measures were taken to prevent pathogen infection. Mice were free to eat and drink. The animals selected and the experimental operations performed were strictly in accordance with the Regulations on the Management of Laboratory Animals promulgated by the State Science and Technology Commission.

1.2 Reagents

Specific peptide T-8 (amino acid sequence is CSEEM RHRFR QLDTK LNDLKG, i.e., 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 Biotech Co., Ltd., catalog number C3949G);

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

PBS buffer;

Hematoxylin-eosin stain;

4% formaldehyde solution;

Anhydrous ether;

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

10% normal fetal bovine serum;

RMPI-1640 basal medium;

0.25% trypsin protein-EDTA digestion solution (containing phenol red);

Trypan blue;

Table 1: Cell culture reagents

Table 2: Preparation of 4% paraformaldehyde solution

Table 3: Hematoxylin staining solution formula

Hematoxylin is dissolved in 1L of distilled water with heating, and then sodium iodate and potassium aluminum sulfate are added to promote its rapid dissolution, and then citric acid and chloral hydrate are added and stirred until it turns purple-red.

Preparation of eosin stain:

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

Preparation of specific polypeptide T-8 emulsion:

Preparation of the specific polypeptide T-8 emulsion used in the 22.50 mg/kg group: (1) Take 43.2 mg of specific polypeptide T-8 lyophilized powder, dissolve it in 19.2 mL 0.01 M PBS, and shake it to make it dissolve evenly to obtain a specific polypeptide T-8 solution; (2) Take out 12.0 mL of the specific polypeptide T-8 solution and put it into 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;

Preparation of the specific polypeptide T-8 emulsion used in the 11.25 mg/kg group: (1) Take 6.0 mL of the specific polypeptide T-8 solution obtained in (1) of the 22.50 mg/kg group and put it into 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 it thoroughly. The suspension should not stratify after being left to stand overnight and is ready for use;

Preparation of the specific polypeptide T-8 emulsion used in 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 the above Example 1 and put it into 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 it thoroughly. The suspension should be allowed to stand overnight without stratification and is ready for use.

1.3 Instruments

Table 4 Instrument List

2. Experimental methods

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

2.1.1 Lentivirus infection of target cells to construct stable strains

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

On the first day, RM-1 cells were inoculated in 24-well plates at 1×10 ⁵ cells. On the second day, before infection, the virus stock solution was taken out of the -80℃ refrigerator and melted in an ice bath. The virus stock solution was diluted with 500μL complete medium at MOI>500, the original medium of the treatment group was removed, and 300μL medium containing the lentivirus dilution solution was added to the cells of the treatment group. On the third day, the culture medium was replaced. After 16h of infection, the culture medium containing the lentivirus was replaced with 500μL complete culture medium. On the fifth day, the infection efficiency was detected, and the fluorescence was observed under an inverted fluorescence microscope to estimate the efficiency of lentivirus infection of the target cells. Appropriate eukaryotic resistance screening cells (Puromycin full lethal concentration 2μg/mL, maintenance concentration 1μg/mL) were selected. Generally, two rounds of drug screening (1 round for 2 days, determined by the antibiotic pre-experiment) were performed, and the cells were stable. After stabilization, RPMI 1640+10% FBS+1% Pen/Strep+1μg/mL Puromycin complete medium was used to maintain the cells.

2.1.2 Luc RM-1Luciferase Detection

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

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

(2) Cell lysis:

Add 120 μL of cell lysis buffer, tap the tube wall with your fingers to fully lyse the cells, and use a pipette to blow several times to ensure that the lysis buffer and cells are in full contact. Usually, the cells will be lysed after the lysis buffer contacts the cells for 30 seconds.

(3) Centrifuge the fully lysed product at 10,000 rpm for 5 min. After centrifugation, transfer the supernatant into a new 1.5 mL EP tube for subsequent testing.

(4) Turn on the fluorescence detection of the Infinite/M1000 instrument and set the parameters: the measurement time is 10 s and the measurement interval is 2 s.

(5) Add 20 μL of the sample to each well of the all-white ELISA plate, repeat three times, and then add 20 μL of luciferase detection reagent to each well. Use a pipette to 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).

2.1.3 Cell culture

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 the cells were counted using a hemocytometer. The cell suspension was diluted to 4×10 ⁶ cells/ml with serum-free medium, and then the matrix gel was mixed with the cell suspension at a volume ratio of 1:1 (on ice), and the final concentration of the cell suspension was 2×10 ⁶ cells/mL for later use.

2.2 Animal models and immunization

2.2.1 Establishment of orthotopic prostate cancer model

The mouse was anesthetized with ether, fixed in the supine position, disinfected with iodine, and a 1 cm midline incision was made above the mouse's external genitalia. The bladder was lifted up to expose the ventral prostate. A microsyringe was used to draw the cell suspension and inject 5 μL of RM-1-Luc cell suspension into the 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 vesicle as a satisfactory standard. Then slowly withdraw the needle, and then use tissue glue to seal it. After confirming that there is no overflow, restore the normal anatomical position of the prostate and bladder, and close the incision with 1-0 silk thread interrupted and double-layer sutures. Put the mouse back in the cage. The operation needs to be gentle during the operation to avoid damaging normal tissue.

2.2.2 Specific peptide T-8 for the treatment of prostate cancer animals (treatment group)

Except for the blank group and the control group of subcutaneous injection of specific peptide T-8, the model group and the treatment group were all modeled with RM-1 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, and the natural death of mice was the experimental endpoint. The treatment groups included a 2.250mg/kg low-dose treatment group (also called a 2.250mg/kg group), a 11.25mg/kg medium-dose treatment group (also called a 11.25mg/kg group), and a 22.50mg/kg high-dose treatment group (also called a 22.50mg/kg group). The mice were dosed according to their body weight before each dose.

2.2.3 Specific peptide T-8 to prevent prostate cancer in animals (prevention group)

In addition to the blank group, the model group and the preventive administration group were all modeled with RM-1 on day 0, and the day of RM-1 modeling 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 administered according to their body weight before each administration. The preventive administration groups included a 2.250 mg/kg low-dose preventive group (also called a 2.250 mg/kg group); a 11.25 mg/kg medium-dose preventive group (also called a 11.25 mg/kg group); and a 22.50 mg/kg high-dose preventive group (also called a 22.50 mg/kg group).

2.3 Comparison of survival and mortality rates of mice

The death time of all mice was recorded, and the survival curves of all groups of mice were prepared.

2.4 Evaluation of tumor growth potential

After the experiment, the mice were dissected, the weight and volume of the tumor tissue were measured, and the tumor was photographed. The status and weight of the mice were tracked three times a week.

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

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

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

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

2.5 Pathological observation

Slices were cut along the maximum cross section at a thickness of 5 μm. Each slice was dewaxed, stained with hematoxylin and eosin, and observed under a microscope after being sealed with gum. The slices were scanned with a scanner, and image analysis was performed using Image J. After the area to be calculated was circled, the area was calculated using a specific software analysis system. The area data was recorded and the ratio was calculated. The values of tumor area and necrosis area and the values of necrosis area/tumor area (area ratio) were calculated.

The different pathological sections were randomly numbered in a random and blinded manner, and then three researchers independently scored to evaluate the morphological changes of prostate tissue and the degree of inflammatory cell infiltration in different pathological sections. The scoring criteria were: 0 points, no inflammation; 1 point, mild inflammatory cell infiltration in the prostate stroma, perivascular or periglandular areas; 2 points, moderate infiltration with epithelial cell degeneration; 3 points, atrophy of most alveolar epithelial cells, significant inflammatory cell infiltration and severe congestion. The final score of each pathological section was the average of the scores of the three researchers.

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

Table 5: Tissue dehydration time and procedures

Hematoxylin-eosin staining steps:

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.

2.6 Enzyme-linked immunosorbent assay

Blood was collected before killing the mice and allowed to stand at 4°C for 1 hour. The blood was then centrifuged at 3000r/10min to obtain the supernatant, which was stored at -20°C. The concentration of cytokine interferon-γ (IFN-γ) in the immunized animals was detected by ELISA detection kit.

2.7 Mouse imaging analysis of tumor cell infiltration and growth status

Before imaging, 100 μL of fluorescein (30 mg/mL in PBS) was injected intraperitoneally into the mouse and imaging was performed about 15 minutes later. After conventional anesthesia (gas anesthesia or needle anesthesia), the mouse was placed on the imaging dark box platform, and the mouse was anesthetized with chloral hydrate in the induction chamber. The software controlled the platform to rise and fall to a suitable field of view, and the lighting was automatically turned on (bright field) to take the first background image. In the next step, the lighting was automatically turned off, and the specific photons emitted by the mouse were photographed in the absence of external light sources (dark field). After the background images of the bright field and the dark field are superimposed, the location and intensity of the specific photons in the animal can be intuitively displayed to complete the imaging operation.

2.8 Statistics

SPSS v 21.0 software was used for statistical analysis of differences, and one-way analysis of variance (ANOVA) was used to determine the differences between groups. P<0.05, P<0.01, and P<0.001 indicated statistically significant differences, extremely significant differences, and extremely significant differences, respectively. GraphPad Prism v 7.0 software was used for drawing.

3. Results

3.1 Luc RM-1 Luciferase Detection

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

3.2 Tumor weight and tumor inhibition rate

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, compared with the model group, there were significant differences in tumor weight and tumor inhibition rate in the treatment group of 11.25 mg/kg (P<0.05).

Table 6: Tumor inhibition rate of treatment groups

*P<0.05 compared with the model group

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, compared with the model group, there were significant differences in tumor weight and tumor inhibition rate in the 11.25 mg/kg prevention group (P<0.05).

Table 7: Tumor inhibition rate in the prevention group

*P<0.05 compared with the model group

3.3 Analysis of mouse survival time

Figure 4 shows the 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 administration 2.25 mg/kg and 22.50 mg/kg groups (P < 0.05). Figure 5 shows the 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 administration 2.25 mg/kg group (P < 0.05).

3.4 Histopathology

3.4.1 Prostate tissue pathology scoring

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 the comparison of the control group with the blank group, P<0.0001). The specific operation is to prepare pathological sections from the prostate tissue of 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.

3.4.2 Tumor necrosis area/tumor area ratio

In the tumor tissue, the tumor cells were diffusely distributed in sheets, and nest-like structures were visible in some areas. The boundaries between cells were unclear, the cytoplasm was weakly eosinophilic, the nuclei were round or oval, with large atypia, high nuclear-cytoplasmic ratio, some nucleoli were visible, and nuclear division images were easy to see. Some tumor cells were necrotic with nuclear pyknosis, and very few inflammatory cells were infiltrated in the tumor edge stroma and necrotic areas.

Figures 7a and 7b are schematic diagrams of tumor necrosis area/tumor area in the treatment group, wherein Figure 7a is a tumor picture of the treatment group, and Figure 7b is a graph of the ratio of tumor necrosis area/tumor area in the treatment group (where N=6, * represents P<0.05 compared with the model group). As shown in Figures 7a and 7b, compared with the model group, there was a significant difference in the ratio of tumor necrosis area/tumor area in the mice in the treatment and administration groups of 2.25 mg/kg and 11.25 mg/kg (P<0.05).

Figures 8a and 8b are schematic diagrams of tumor necrosis area/tumor area in the prevention group, wherein Figure 8a is a tumor picture of the treatment group, and Figure 8b is a graph of the ratio of tumor necrosis area/tumor area in the treatment group (where N=5, * represents P<0.05 compared with the model group). As shown in Figures 8a and 8b, compared with the model group, there was a significant difference in the ratio of tumor necrosis area/tumor area in the mice in the prevention administration 2.25 mg/kg and 22.50 mg/kg groups (P<0.05).

3.5 Biochemical indicators

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 of 11.25 mg/kg (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 of 22.50 mg/kg (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 of 11.25 mg/kg (P<0.05); there was a very significant difference in IFN-γ in the serum of mice in the prevention group of 22.50 mg/kg (P<0.001).

3.6 Mouse imaging

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 days 0 and 14 of the treatment group, and Figure 11b is a statistical diagram of the relative fluorescence quantum number of the treatment group on days 13 (N=5). As shown in Figures 11a and 11b, compared with the model group, the relative fluorescence quantum (relative fluorescence quantum number=fluorescence quantum number on day 14/fluorescence quantum number on day 0) of mice in the treatment and administration groups of 2.25 mg/kg, 11.25 mg/kg, and 22.50 mg/kg was reduced at intervals of 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 endpoint fluorescence quantum number 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).

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 treatment administration of 11.25 mg/kg 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 prevention administration of 11.25 mg/kg 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 treatment administration of 2.25 mg/kg and 22.50 mg/kg 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 prevention administration of 2.25 mg/kg group (P<0.05). In the treatment group, compared with the model group, there were significant differences in the ratio of tumor necrosis area/tumor area in the treatment administration of 2.25 mg/kg and 11.25 mg/kg groups (P<0.05). In the prevention group, compared with the model group, there was a significant difference in the ratio of tumor necrosis area/tumor area in the 2.25mg/kg and 22.50mg/kg groups (P<0.05). In the treatment group, compared with the model group, there was a significant difference in IFN-γ in the serum of the mice in the 11.25mg/kg group (P<0.01); compared with the model group, there was a significant difference in IFN-γ in the serum of the mice in the 22.50mg/kg group (P<0.05). In the prevention group, compared with the model group, there was a significant difference in IFN-γ in the serum of the mice in the 11.25mg/kg group (P<0.05); there was a very significant difference in IFN-γ in the serum of the mice in the 22.50mg/kg group (P<0.001). In the treatment group, compared with the model group, the relative fluorescence quantum number of mice in the treatment administration groups of 2.25mg/kg, 11.25mg/kg, and 22.50mg/kg decreased at intervals of 13 days, but there was no significant difference. In the prevention group, compared with the model group, there were significant differences in the prevention administration groups of 2.25mg/kg and 22.5mg/kg (P<0.05). That is, the experimental results showed that the specific peptide T-8 had a therapeutic and preventive effect on androgen-independent prostate cancer in terms of inhibiting tumor growth, prolonging the survival time of mice, local immune cell infiltration in tumor tissue, necrotic foci formed by immune cells attacking tumor tissue, and the content of cytokine IFN-γ in serum.

It should be noted that, although the specific polypeptide T-8 was used together 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.

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 to this. The drug containing the specific polypeptide T-8 can also be made into an emulsion, suspension, oil, tablet, capsule and other dosage forms for use. However, the effect is better when it is directly injected into the body as an injection.

Comparative Example

The present invention uses enzalutamide, which is commonly used in clinical practice, as a comparative example to verify its efficacy in mice with prostate cancer after 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).

1. Experimental Methods

1.1 Group settings

Male C57BL/6 mice aged 22-24 g and 6-8 weeks were selected and randomly divided into a model group (modeling but no drug administration) and a positive drug group (modeling and drug administration).

1.2 RM-1-Luc cell recovery

After taking out the frozen RM-1-Luc cells from the -80℃ refrigerator, heat them in a 37℃ water bath, and try to ensure that the cryopreservation solution dissolves within 1 minute. Then transfer the cell cryopreservation tube to the clean bench, use a rubber-tipped dropper to aspirate the cryopreservation solution and transfer it to a 10mL centrifuge tube and add 4mL of culture medium. After centrifugation at 1000rmp for 5min, discard the supernatant, add 1mL of culture medium to resuspend the cells. Take a new cell culture bottle, add 7mL of culture medium, add the resuspended cell suspension to the cell culture bottle, tighten the bottle cap, and place it in a cell culture incubator for culture at 37℃ and 5% CO ₂ .

1.3 Cell passaging

Take cells in the logarithmic growth phase, use a rubber-tipped dropper to absorb the culture medium in the cell culture bottle and add 2mL PBS to wash. Then add 2mL trypsin-EDTA solution to digest the cells for 2min. After digestion, observe the cell morphology under a microscope. If the cells are round, it means that the digestion degree is appropriate. At this time, add 4mL of culture medium to the culture bottle to stop digestion, and blow the cells to make them fall off the wall.

Transfer the liquid to a 10mL centrifuge tube, centrifuge at 1000rmp for 5min, discard the supernatant, 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.

1.4 Cell cryopreservation

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°C refrigerator.

1.5 Cell counting

Take RM-1-Luc cells in the logarithmic growth phase, transfer the cell culture flask to the clean bench, remove the remaining culture medium with a rubber-tipped dropper, wash twice with PBS, add 2mL of trypsin to the culture flask, and digest the cells for 2 minutes. After digestion, discard the trypsin, add 4mL of complete culture medium to the culture flask again to terminate digestion, and blow the cells to make them fall off.

Transfer the liquid to a 10mL centrifuge tube, centrifuge at 1000rmp for 5min, discard the supernatant, and add an appropriate amount of serum-free medium to the centrifuge tube to resuspend the cells. Take 10μL of the resuspended cell suspension to a suitable container, add 10μL of 4% trypan blue solution, and mix well. Cover the cover glass on the cell counting plate, add 10μL of the mixture of cell suspension and 4% trypan blue from the edge of the cover glass, and let the liquid naturally enter under the cover glass through the siphon effect to avoid the generation of bubbles.

Place the cell counting plate under a microscope and adjust the focus to clearly observe the lines and live cells on the cell counting plate. Only record the number of live cells in the upper left, lower left, upper right, and lower right corners, and follow the counting principle of counting the upper part but not the lower part, and counting the left part but not the right part.

The formula for calculating the concentration of cell suspension is:

1.6 Preparation of cell suspension

After the cell counting is completed, 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 cells/mL) for later use.

1.7 Prostate cancer in situ model

After anesthetizing the mice, let them lie on the heating plate in the supine position to prevent hypothermia during and after the operation. Fix the limbs of the mice with medical tape to prevent them from struggling. After wetting the abdominal hair with 75% ethanol, remove the hair in the perineum. Make an incision about 1 cm above the genitals with ophthalmic scissors to expose the abdominal cavity. Quickly locate the spermatic cord by finding the bladder and find the prostate attached to the spermatic cord. Lift the prostate with ophthalmic forceps so that the prostate is in a vertical position. Use a microinjector to absorb 5μL of cell suspension, and the needle is inserted vertically into the prostate tissue with a depth of not less than 5mm. Push the syringe piston to allow the suspension to enter the tissue, and observe that the tissue becomes swollen and transparent as a sign of successful injection. After the injection is completed, the needle stays in the tissue for 1 to 2 seconds before being withdrawn, and the needle eye is coated with biological glue. Reposition the internal organs to their normal physiological position and suture the incision. Pay attention to gentle movements during the operation to prevent excessive damage to the mouse. The sham operation group only underwent anesthesia, abdominal opening, prostate positioning and suturing, but no cell suspension injection.

1.8 Drug configuration

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

1.9 Collection of materials

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

1.10 Tissue dehydration and staining

[Corrected 02.09.2024 in accordance with Article 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, and perform dehydration treatment in the order 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 40℃ constant temperature water bath, and then the complete and appropriate pathological slices are fished out with a clean glass slide and placed in an enamel tray to dry overnight. The dried slices are placed in an oven at 68℃ for 120 minutes to prevent detachment. After the pathological slices have cooled to room temperature, they are stored in the slice box.

Table 8: Tissue dehydration time and procedures

The pathological sections were dried overnight and then placed in an oven (68°C) for baking and fixation for 60 minutes. The following steps were followed: xylene dewaxing, ethanol gradient dehydration, staining, differentiation, staining, xylene transparentization, etc. After staining, the sections were sealed with neutral resin and left to stand overnight. Before microscopic observation, the excess neutral resin on the surface could be wiped off with a cotton ball soaked in xylene, and then microscopic observation and photography were performed. For the specific process, see Table 9 below.

Table 9 HE staining process

2 Experimental results

2.1 Survival curve

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).

2.2 Tumor inhibition rate

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 is no significant difference in the tumor inhibition rate of the positive drug group compared with the model group, and the downward trend of tumor weight is relatively small.

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

2.3 Tumor tissue pathology sections

The HE staining results of the model group and the positive drug group are shown in Figures 15a and 15b. Figure 15a is a tumor picture of the treatment group, and Figure 15b is a graph of the ratio of tumor necrosis area to tumor area of each group. As shown in Figures 15a and 15b, the proportion of necrosis area in the tumor section of the positive drug group is relatively higher (the pink part is the necrotic part, and the purple part is the normal tumor tissue), but there is still no significant difference.

3. Experimental conclusion:

The positive drug group had no significant difference in survival time analysis, tumor inhibition rate and tumor necrosis area ratio compared with the model group, but both showed a trend of inhibiting the disease process. Compared with the comparative example, the therapeutic effect of each drug administration group in the embodiment was better.

In summary, subcutaneous injection of specific polypeptide T-8 has good specific effects in both 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.

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

All documents mentioned in this specification are cited in this application as references, just as if each document was fully cited in this specification as references.

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 protection scope of the present invention.

Claims (9)

A 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 TRPM8 protein. The use of the specific polypeptide according to claim 1 in the preparation of a drug for treating prostate cancer is characterized in that the amino acid sequence of the specific polypeptide is as shown in SEQ ID NO: 1. The use of the specific polypeptide according to claim 2 in the preparation of a drug for treating prostate cancer is characterized in that the drug is prepared by combining the specific polypeptide with complete Freund's adjuvant. The use of the specific polypeptide according to claim 3 in the preparation of a drug for treating prostate cancer, characterized in that the drug is an injection. The use of the specific polypeptide according to any one of claims 1 to 4 in the preparation of a drug for treating prostate cancer, characterized in that the prostate cancer is androgen-independent prostate cancer. The invention discloses an application of a specific polypeptide in the preparation of an immune inducer, wherein the immune inducer is used to induce prostate tissue-specific immunity, and the specific polypeptide is derived from TRPM8 protein. The use of the specific polypeptide according to claim 6 in the preparation of an immune inducer is characterized in that the amino acid sequence of the specific polypeptide is as shown in SEQ ID NO: 1. The use of the specific polypeptide according to claim 6 or 7 in the preparation of an immune inducer is characterized in that the immune inducer is prepared by mixing the specific polypeptide with complete Freund's adjuvant. The use of the specific polypeptide according to claim 8 in the preparation of an immune inducer is characterized in that the immune inducer is an injection.