Investigation Of New Strategies For T Cell Targeted Therapy Of Tumor Treatment

PART ONE Novel strategy research of aptamer-T cell targeted tumor immunotherapyBackground:At present,the incidence and mortality of cancer worldwide are increasing.WHO World Cancer Research Center reported that there was 1 cancer patient in every 8 deaths.The WHO also proposed that if we cannot explore more effective treatments,there will be 26 million new cancer cases worldwide by 2030,and malignant tumors will become the number one killer of human health and become the largest in the world.A large proportion of the new cancer cases are concentrated in developing countries.Among the four malignant tumors including liver cancer,esophageal cancer,gastric cancer and lung cancer,China’s new cases and deaths rank first in the world.According to the latest statistics,malignant tumor deaths account for 23.91%of all deaths among residents,and the incidence and death of malignant tumors have continued to rise in the past decade.The annual medical expenses caused by malignant tumors exceed 220 billion,and the situation of prevention and control is severe.In addition,malignant tumors are a large class of diseases that seriously endanger human life and health and restrict social and economic development,and have become one of the important causes of death in the world’s population.The development of a tumor-targeted immunotherapy is highly required.The most advanced application is the use of CD 19 chimeric antigen receptor(CAR)-T(CAR-T)cells to B-cell malignancies.However,there are still side effects including:(1)Potential carcinogenicity of lentiviral or retroviral insertion into the host cell genome;(2)After CAR-T cells are returned to the body,the immune cells of the body release a large amount of cytokines such as IFN-γ,GM-CSF,TNF-α,IL-6 and IL-10 within a short period of time,which induces the production of serious cytokines;(3)Many tumor cells currently lack specific antigens and their scFv;(4)CAR-T cell construction technology is difficult and time-consuming and costly;(5)Immune escape and intrinsical off-target effects because of tumor cell antigen loss;(6)Poor infiltration and enrichment of T cells in solid tumor sites or tumor microenvironment tissues;(7)T cell exhaustion leading to tumor recurrence in patients.The above disadvantages have severely limited the antitumor effect of CAR-T cells and their application to the field of immunotherapy for solid tumors.Objective:To overcome the potential carcinogenic risk of lentivirus inserted into the genome of cells and to address the shortcomings of many tumor cells that lack specific antigens and their scFv,we try to explore the use of aptamers instead of scFv to couple to the surface of T cells by a non-viral manner to construct aptamer-T cells.Aptamers are used to increase the enrichment and targeting of T cells to solid tumors,thereby enhancing the anti-tumor effect of T cells.Method:Here we developed aptamer-T cell using a non-viral method for tumor therapy.Tumor cells surface-specific ssDNA aptamers were conjugated to CD3+T cells(aptamer-T cells)using N-azidomannosamine(ManNAz)sugar metabolic cell labeling and click chemistry.The effects of the aptamer-T cells on tumor were detected in vitro and in vivo.Results:We found that the aptamer-T cells could specifically target and bind to tumor cells(such as SGC-7901 gastric cancer cell and CT26 colon carcinoma cell)in vitro and in mice after adoptively transfer in.Aptamer-T cells led to significant regression in tumor volume due to being enriched at tumor microenvironment and producing strong cytotoxicity activities of CD3+T cells with enhanced perforin,granzyme B,CD107a,CD69 and FasL expression.Moreover,aptamer-T cells displayed even stronger antitumor effects than an anti-PD 1 immune-checkpoint monoclonal antibody(mAb)treatment in mice and combination with anti-PD 1 yielded synergic anti-tumor effects.Conclusion:In this study,aptamers that specifically bind to tumor cells were successfully couplled to the surface of T cells to construct aptamer-T by non-natural sugar metabolism and biological orthogonal reaction.In mouse tumor models,aptamer-T cells showed targeted and enriched to tumor cells.In addition,aptamer-T cells showed significantly anti-tumor effects and improved mouse survival.This study uncovers the strong potential of the adoptive non-viral aptamer-T cell strategy as a feasible and efficacious approach for tumor-targeted immunotherapy.PART TWO Study of novel CD19 scFv-IL10R-CAR-T and its antitumor effectBackground:The application of chimeric antigen receptor(CAR)T cell targeted therapy in the field of tumor treatment is receiving attention,is one of the most promising developments in immunotherapy of malignant tumors.The development of a tumortargeted immunotherapy is highly required.The most advanced application is the use of CD 19 chimeric antigen receptor(CAR)-T(CAR-T)cells to B-cell malignancies.However,there are still side effects including:(1)after CAR-T cells are returned to the body,the immune cells of the body release a large amount of cytokines such as IFN-y,GM-CSF,TNF-α,IL-6 and IL-10 within a short period of time,which further induce serious cytokine strom;(2)T cell exhaustion leading to tumor recurrence in patients,and so on.Objective:On the basis of the third-generation CAR(CD19scFv-CD28-4-1BB-CD3ζ),we further explored and optimized the CAR structure by incorporating IL-10R in order to reducing the cytokine storm and enhance T cells to exert antitumor function for a long time.Method:In order to achieve the above purpose,the extracellular segment of the IL-10 receptor(IL-10R)was introduced into the CAR structure.Then CAR was integrated into CD3+T cells using lentivirus.When CAR-T cells are returned to the body,T cells express IL10R and neutralize IL-10 in peripheral blood.On the one hand,it weakens the cytokine storm caused by IL-10.On the other hand,T cell exhaustion was attenuated and improved the anti-tumor function of T cells.Results:We constructed the new third-generation CAR structure expressing IL-10R and completed the lentiviral packaging.WB experiments verified the expression of CAR and the transfection of CD3+T cells with lentivirus to prepare CAR-T cells.FCM results showed that a large proportion of CD3+T cells expressed CAR.The results showed that CAR-T cells had cytotoxicity on Nalm6-a B-line acute lymphoblastic leukemia cell line in vivo and in vitro.At the same time,we found that the fusion expressed IL-10R extracellular segment significantly reduced IL-10 in the environment of T cells.Conclusions:The IL-10R was successfully constructed into the CAR to generate a new thirdgeneration CAR(CD 19scFv-Il-10R-CD28-4-1BB-CD3ζ)that simultaneously expresses IL-10R,and CD19scFv-Il-10R-CD28-4-3BB-CD3ζ-T cells were generated using the lentiviral transduction T-cell method.CAR-T cells showed significantly cytotoxicity on Nalm6 cells in vivo and in vitro.In addition,the fusion expressed of the extracellular segment of IL-10R significantly reduced IL-10 in the environment of T cells and alleviated T cell exhaustion.