Study On The Role And Mechanism Of Adoptive Tumor Neoantigen Reactive T Cells In Therapy Of Lung Cancer

BackgroundLung cancer is the leading cause of cancer death worldwide.Nearly 25%of all cancer deaths are due to lung cancer.For non-small cell lung cancer（NSCLC）,surgical removal of cancer in early stages of the disease prolongs survival of patients,however,the overall 5-year survival rate is only 15%.Patients in advanced stage of the disease are less prone to benefit from surgical interventions or systemic treatments.Cancer Statistics revealed that the 5-year survival rate of patients with advanced NSCLC is merely 6%.Targeted therapeutic drugs and immune checkpoint inhibitors（ICIs）have significantly improved the disease prognosis in clinical applications and have been incorporated to the standard management of advanced stage NSCLC in recent years.However,due to the negative driver genes or low expression of PD-1/PD-L1,some patients are unable to benefit from targeted drugs or ICIs.Additionally,the immune-related adverse events（ir AEs）and even lethal toxicity shown in the application of ICIs also limit the widespread use of such drugs.Therefore,exploring an effective and low-toxic strategy for improving the survival rate and the quality of life of patients with NSCLC remains a challenge for researchers.Cancer immunotherapy was rated as the top ten scientific breakthroughs in 2013by Science.Adoptive cell therapy（ACT）,represented by chimeric antigen receptor T cells therapy（CAR-T）combined with ICIs have become the focus of research and clinical practice,and has urgent clinical needs.The malignant transformation of cells depends on the continuous accumulation of genomic DNA damage.The mutant products induce the expression of neoantigens and activate the tumor-specific T cells which perform killing function.The higher neoantigen mutation burden and more tumor infiltrating lymphocytes（TILs）induce a stronger antitumor effect by neoantigen reactive T cells（NRT）.NSCLC possesses a higher mutation burden and more TILs infiltration,making it an ideal candidate for the adoptive transfer of NRT cells.However,there are also some challenges such as the identifying,infiltrating,killing and surviving of T cells in the suppressive tumor microenvironment（TME）in the treatment of solid tumors with ACT.To date,a variety of studies pertaining to break through these bottlenecks,including editing chemokine receptors,designing costimulatory domains and secretory cytokines CARs to T cells to promote their infiltration,activation and proliferation in tumor tissues.Moreover,combining cancer vaccines or ICIs is also a good idea to regulate the suppressive TME or revitalize the exhausted T cells and enhance the response to cancer immunotherapy.In addition,with the deepening research of immune metabolism,strategies based on immune cell metabolic reprogramming are also used to enhance the efficacy of ACT,including up-regulation of glucose transporter 1（GLUT1）to promote T cell survival,overexpression of T cell transcriptional activator PGC-1αto boost mitochondrial function,enhancement of phosphoenolpyruvate（PEP）synthesis to propel T cell proliferation and effector function.There are also reports of by conditional deleting lactate dehydrogenase A（LDHA）on myeloid derived cells and macrophages arming to regulate the immunosuppressive TME.Therefore,how to overcome the hurdles of ACT in treating solid tumors through the T cell metabolism reprogramming has now become a research hotspot of ACT therapy.Objective1、In the present study,we aimed to investigate the antitumour effect of adoptive transfusion of NRT cells induced by RNA mutanome vaccine,in order to accumulate experience for ACT of lung cancer.2、We focused on the key enzyme for glycolysis LDHA and explored the mechanisms of how the metabolic reprogramming regulating T cell function and tumor progression in order to formulate strategies for augment ACT therapy of solid tumor.Method1、NRT cells preparation and functional identification in vitro:WES and RNA-seq of cells were carried out to discover gene mutations;Tumor neoantigens were predicted based on gene mutation,MHC typing and gene expression information;NRT cells were prepared according to neoantigen sequences;Flow cytometry,ELISA,PCR and other techniques were used to assay the function of NRT cells.2、Study on the antitumor effect of NRT in vivo:NRT cells were expanded and adopted to tumor-bearing mice to detect the therapeutic effect.3、Study on the function and mechanism of LDHA in CD8+T cells:Ldha^fl/fl and Ldha^-/-mice were obtained by floxp system;Flow cytometry,Western blotting,PCR,RNA-seq,Metabolic flux and other techniques were used to explore the CD8+T cells function and mechanisms of metabolic reprogramming in vitro.4、Study on the effect of LDHA in lung cancer:Flow cytometry and pathological examination were applied to identify the different TME between Ldha^fl/fland Ldha^-/-tumor-bearing mice.Results1、NRT cells induced by neoantigen RNA vaccine have stronger cytokine secretion and killing activity than conventional T cells.2、Adoptive transfusion of NRT cells has strong antitumor effect in the tumor-bearing mice model.3、LDHA plays an important role in the activation,proliferation and executive effector function of CD8+T cells.The deletion of LDHA on T cells impaires the above functions.4、Deletion of LDHA on T cells promotes tumor progression in mice.Metabolic reprogramming based on LDHA is an important target for regulating tumor immunity.ConclusionGene sequencing technology and epitope prediction system can predict the mutation sites of tumor neoantigens.RNA vaccine based on neoantigens can induce NRT cells and adoptive transfusion of NRT cells has strong antitumor effect;LDHA is a critical enzyme in the activation,proliferation and effector function of CD8+T cells,and also plays an important role in antitumor immune response.Tumor NRT cells adoptive transfusion combined with CD8+T cell metabolic reprogramming strategy has good research potential and practical significance in immunotherapy of lung cancer.