Application of CAR-T cells modified with bispecific antigen chimeric receptors in the treatment of prostate cancer

Problem to be solved: 1. Determine one or more prostate cancer specific antigens by immunohistochemical screening of pathological sections, including PSMA, ROR-1, PSMA-PDL1, CYR61, PSCA, Lewis Y, EGFR antigens, etc. 2. Construct a retroviral vector pLNCX-PMA-PDL1 CAR targeting the aforementioned specific chimeric mixed antigen receptor (such as PSMA-PDL1 CAR) and a retroviral vector pLNCX-LUC2 carrying luciferase, and package the corresponding virus using a retroviral packaging kit. 3. The pLNCX-LUC2 virus infects tumor cells such as Hela, Huh7, PC3, and LNCaP, and selects the stable expression of LUC2 cell line pLNCX-PMA-PDL1. CAR virus infects T cells, and selects the stable expression of PSMA-PDL1 cell line for subsequent experiments. 4. Luciferase bioluminescence was used to detect the killing effect of human peripheral blood lymphocytes (CAR-T) on PSMA positive tumor cells in vitro. 5. The pLNCX-LUC2 virus stable cell line was loaded into nude mice, and the inhibition of tumor growth in vivo by human peripheral blood lymphocytes (PBL-CAR), or the inhibition of tumor metastasis by PBL-CAR was tested by PC3 in nude mice's artificial lung metastasis model. The planned goal is to identify one or more prostate cancer specific antigens and construct them into specifically recognized chimeric antigen receptors (CARs), providing a foundation for the next development of CAR-T cells. 2. Clarify the targeting and killing activity of T cells modified with chimeric antigen receptor (CAR) on prostate cancer cells, and conduct animal experiments to provide favorable conditions for the use of CAR-T cells in clinical research of prostate cancer. Application of CAR-T cells modified with dual specific antigen chimeric receptors in the treatment of prostate cancer: Prostate Cancer (PCa) poses a threat to the life and health of middle-aged and elderly men. According to the data published by Siefel and others in CACancerJClin magazine in 2015, the incidence rate of PCa reached 26% in the United States in 2015, ranking first in the incidence rate of cancer, and second in the mortality rate of 9%. Although the incidence rate of prostate cancer in China is lower than that in Europe and the United States, in recent years, with the aging of the population, changes in living habits, and the gradual popularity of physical examination, the incidence rate of prostate cancer is also on the rise, and has ranked the third in male urogenital malignancies. Prostate cancer has a hidden onset and can develop into mid to late stages, which can easily lead to difficult problems such as metastasis and drug resistance, seriously affecting the quality of life and lifespan of patients. Early detection and treatment of prostate cancer is of great significance. Androgen castration or endocrine therapy that blocks androgens at the androgen receptor level is the standard treatment plan for advanced prostate cancer. However, 80% -90% of advanced prostate cancer patients undergo hormonal therapy for 12 to 18 months before transitioning from androgen dependent to more aggressive hormone independent prostate cancer, which relapses after male castration treatment and forms androgen resistant prostate cancer. Once the tumor exhibits hormone independent characteristics, it will develop high resistance to all chemotherapy drugs and will be ineffective against any other further treatment methods. Therefore, more effective targeted therapy has become the inevitable choice for the next response to prostate cancer. Chimeric antigen receptors (CARs) are formed by the fusion of antibody specific targeting regions and T cell signaling regions. When T cells express CARs, they confer T cell antigen specificity, thereby targeting and killing tumor cells. At present, CAR-T cell therapy has achieved good efficacy in B-cell derived tumors. In theory, CAR-T cells can also play a role in solid tumors, but further exploration and improvement are needed. However, CAR-T cell solid tumor treatment faces three major challenges (1) lack of appropriate specific tumor associated antigen (2) CAR-T cells are not easy to accumulate in the tumor area (3) tumor microenvironment has immunosuppressive effect on CAR-T cells. The CAR-T cell therapy strategy can achieve ideal results in hematological tumors. One important reason is that hematological tumors have specific targets, such as the expression of CD19 in B-cell hematological malignancies reaching almost 100%, while in solid tumors, it is difficult to find specific antigens like hematological tumors. Most antigens are relatively high expressed on tumor cells, and normal cells also have a small amount of expression, Therefore, it is difficult to avoid non-specific killing of normal cells. Heterogeneity is one of the more prominent characteristics of solid tumors, which means that even tumors in the same patient and site may not all express the same tumor associated antigens. Even on a tumor lesion, no single target antigen can cover all tumor cells. If only a single target CAR-T cell is used to treat solid tumors, the highly heterogeneous nature of solid tumors theoretically determines that they will inevitably recur or cannot be eradicated. Therefore, the selection of tumor specific antigens is the biggest bottleneck for CAR-T treatment of solid tumors.