Evaluation Of Specific Killing Effects To Bladder Cancer Cells Of Adoptive Cellular Immunotherapy

Background: The current treatment therapy for malignancy are mainly made of surgery,chemotherapy and radiotherapy,while the factors such as the fast progression,relapse of tumor and the poorly condition of patients limited the application of the conventional therapy.To deal with that,a new therapy is needed urgently.Adoptive cellular immunotherapy is one of the most promising methods of anti-tumor by using patinet's own immune system in recent years.With the extensive use of therapeutic DC vaccines based on dendritic cell(DC)induced T cell immunity,cytotoxic T lymphocytes,DC-CIK,etc.,researchers are recently paying great attention to the gene modified T cells by using T cell receptor or chimeric antigen receptor,both of which can lyse the target tumor cells specifically.DC cells have the ability of antigen presentation and co-stimulation of T cells,which can actively induce specific anti-tumor immunity and immune memory in vivo.Therefore,DC vaccines or CTLs that based on DC induced immunity have a remarkable anti-tumor ability.CAR T can specifically recognize the tumor associate antigen(TAA)only by its scFv fragment which expressed on surface of T cells instead of relying on the major histocompatibility complex(MHC),which is known as non-MHC restriction.With antigen recognition specifically,CAR can transfer the signals to immunoreceptor tyrosine-based activation motif(ITAM)of intracellular region which activate the cytotoxic effects of T cells to lyse the target cells.In order to enhance the proliferation and activation of gene modified T cells,researchers added co-stimulating factors to the intracellular region of CAR structure to modified the first,second,and third generation of CAR according to the numbers of co-stimulating factors.Up to now,there are hundreds of clinical trials of CAR T therapy for hematologic tumors and solid tumors are under way.Kymriah and Yescarta that targeting CD19 have been approved by the FDA for the treatment of hematological malignancies.Prostate steam cell antigen(PSCA)is a tumor-associated antigen which is highly expressed on the malignant tumor cell surface such as prostate cancer,bladder cancer,and pancreatic cancer.There are plenty of clinical trials of CAR T to treat prostate cancer and pancreatic cancer targeting the PSCA,and some of them had achieved effective results.Therefore,PSCAis a very promising target for cancer immunotherapy.However,there is still no reports concerning its application in the treatment of bladder cancer.In present study,we designed a third generation of chimeric antigen receptor called PSCAscFvCD28-CD137-CD3?,sinsysized into nonviral minicircle DNA(mcDNA)vector,and transduced into T cells invirtu of electroporation,and prepared PSCA-CAR-T effectors.In addition,we prepared therapeutic DC vaccine and antigen specific CTLs by loading tumor lysis onto DCs and cocultured these DCs with T cells.Specific antitumor responses of CAR-T and CTLs were estimated in following experimental studies in vitro and in vivo,and the results indicated that CAR-T and CTLs were siginificantly inhibited the tumor growth,and the antitumor efficiency was much stronger in CAR-T effectors when compared with CTLs.The results indicated that adoptive cellular immunotherapy might be effective method for treatment of bladder cancer,and CAR-T therapy seem to be more effective method.Part 1 Preparation of PSCA-CAR-T and cancer specific CTLs and their antitumor effects in vitroObjective: To prepare the PSCAscFv-CD28-CD137-CD3?CAR T and bladder cancer specific CTLs and detecte their the anti-tumor capability in vitroMethods: Human peripheral blood mononuclear cells(PBMC)were collected by density gradient centrifugation,DCs and T lymphocytes were harvested by adherent method,DC vaccines were prepared by loading tumor cell lysates,and CTLs were harvested by coculturing DC vaccine with T cells.Minicircle DNA containing the target gene was transferred into T cells by using electroporation,and PSCA-CAR-T effectors were prepared.A significant expression of GFP+ cells was observed under fluorescence microscope after the transduction,and the incidence of CAR expression on surface of T lymphocytes was detected by flow cytometry.The expression of PSCA on bladder cancer cell line RT4 and prostate cancer cell line PC-3M was simultaneously detected by flow cytometry.CCK-8 was used to determine the killing efficiency of CTLs and CAR T on RT4 and PC-3M tumor cell line respectively.ELISA was used to detect the concentration of IL-2 and IFN-? in the coculturing supernatant of CTL and CAR T with tumor cells.Results:1.The bladder cancer cell line RT4 specific DC vaccine and cytotoxic T lymphocytes were prepared successfully;2.The PSCAscFv-CD28-CD137-CD3? CAR T cells were successfully prepared by using electroporation of mcDNA into T cells.Flow cytometry analysis showed that the transduction efficiency was 47.9%.3.Both bladder cancer specific CTL and PSCA-CAR T exhibited an obvious killing effect on PSCA-positive RT4 cells,and PSCA-CAR T has better killing effect when compared with CTL and normal T cells.While there is no significant difference was found in PSCA-negetive PC-3M cell lines.4.Bladder cancer-specific CTL and PSCA-CAR T can both secrete a large amount of IL-2 and IFN-? when stimulated by PSCA-positive RT4 cells,and the secretion was much higher in PSCA-CAR T comparing with CTL or normal T cells.While no difference was found when stimulated by PSCA-negtive PC-3M cells.Part 2 Anti-tumor effects of PSCA-CAR T and bladder cancer specific CTLs in tumor bearing miceObjective: To evaluate the anti-tumor ability of PSCA-CAR T and bladder cancer specific CTLs in tumor bearing mice.Methods: NOD-SCID mouse bladder cancer model was established by inoculation of PSCA+ bladder cancer cell line RT4.After tumor formation,mice were randomly divided into 3 groups treated with CTL,CAR T cells,and normal T cells respectively to evaluate the antitumor effects in mice.The concentrations of IL-2 and IFN-? in serum of mice in each group were detected by ELISA.After the treatment,mice were sacrificed,the tumor tissues were acquired and treated with immunohistochemical staining to detect the expression of PSCA antigen and the infiltration of human CD3+T lymphocytes.Results:1.NOD/SCID tumor-bearing mice model was successfully constructed by using bladder cancer cell line RT4.2.Both CTL and CAR T cells can inhibit the growth of tumor in mice and the tumor volume of CAR T cell treated group is significantly smaller than that of CTL cell treated group,as well as control group.3.Immunohistochemical staining showed that PSCA antigen was overexpressed in tumor tissues,and CD3 positive T cells was highly expressed in both CAR T group as well as CTL group.Conclusion:1.Minicircle DNA vector containing the gene sequence that encoding CAR can be introduced into T cells by electroporation with a high efficiency of CAR expression.2.Bladder cancer specific CTL cells can be induced by DC vaccine loaded with tumor cell lysates.3.PSCA-CAR T and CTLs can exhibit antigen-dependent activation and specific killing effects in vitro,and significantly elevated secretion of IL-2 and IFN-? can be found in the co-cultureing supernatant.4.CTL and CAR T play an effective role in inhibiting tumor growth in tmor-loading mice.