Manufacturing And Tumor Killing Function Research Of CAR-T Cells Targeting KRAS G12V/HLA-A*02:01 In Pancreatic Cancer

Background and Purpose : The incidence of pancreatic cancer continues to rise,mortality rates remain high,conventional treatments are of limited use,as a result,new and more effective treatments need to be found.Immunotherapy,especially immune cell therapy,is regarded as one of the promising treatments to overcome tumors.In recent years,besides drugs targeting immune checkpoints such as PD-1 and PD-L1 have achieved certain efficacy in tumor treatment,immune cell therapy represented by CAR-T cells have also made significant breakthroughs in hematologic malignancy.However,there are more bottlenecks in solid tumors,such as lack of specific targets and the inhibition of tumor microenvironment.Among all the limitations,finding highly specific targets is the primary issue that needs to be addressed.Traditional tumorassociated antigens(TAA)widely express in normal tissues,and targeting TAA is often associated with more severe on-target/off-tumor toxicities.Therefore,targeting tumor-specific antigens(TSA),which express only in malignant tumor tissues,is a potential and future direction for malignant tumor treatments.As a result,we selected KRAS G12 V,an intracellular TSA widely expressing in pancreatic cancer and predicting poor prognosis in a variety of malignant solid tumors,as the target of our study.Given the risk of low affinity and mismatch of TCR-T cells,we selected the human leukocyte antigen(HLA)type I allele HLA-A*02:01,which is high in the population,and used bioinformatics algorithms to predict the affinity of the major histocompatibility complex(MHC)to the antigen.The antibodies screened by phage display that can specifically recognize KRAS G12V/HLA-A*02:01 were designed and manufactured for CAR-T cells to remove the restriction that CAR-T cells can only recognize cell surface antigens.Besides,in order to break the immunosuppression from PD-1 pathway in the tumor microenvironment,we added truncated PD-1(t PD-1),which removes the intracellular structural domain,to the above-mentioned CAR-,in an attempt to enhance the tumor-killing function of CAR-T cells by competitively binding PD-1 ligands such as PD-L1 to block the PD-1 inhibitory signaling pathway.Objective :(1)The targets of CAR-T cell therapy in pancreatic cancer,KRAS G12 V and HLA-A*02:01,were identified through bioinformatics analysis,PUE#4 CAR-and PUE#5 CAR-that specifically recognize the target antigens were produced,and CAR-T cells were manufactured by electroporation with PB transposons.(2)Functional experiments in vitro:In-vitro killing and cytokine release assays were performed on two types of CAR-T cells using constructed cells that exogenously express/don’t express the target antigens,and pancreatic cancer cell lines that endogenously express/don’t express the target antigens.And then,CAR-T cells attached with truncated PD-1 were manufactured,and the immune efficacy of the combination therapy was verified by in vitro killing assays of two target cells expressing the target antigens.(3)Functional experiments in vivo:The immunodeficient mice subcutaneous transplantation tumor models were constructed using pancreatic cancer cell lines that endogenously expressed the target antigens,and PUE#4 CAR-T cells,which performed better in in-vitro functional experiments,and PUE#4 CAR-T cells with truncated PD-1attached,and PBS buffer were infused to compare the tumor growth and survival of each group of tumor-bearing mice,and tumor tissue samples were taken for immunohistochemical staining.Outcome :(1)Two types of CAR-T cells were successfully manufactured.(2)In-vitro functional experiments showed that both CART cells were able to specifically recognize and kill cells expressing target antigens,while only exhibiting intrinsic killing properties for cells not expressing target antigens,and the CAR-conjugated truncated PD-1 was able to enhance the killing efficacy of T cells.(3)In-vivo functional experiments showed that CAR-T cells significantly suppressed tumor growth and prolonged survival in tumor-bearing mice,and CAR-T cells attached with truncated PD-1 had higher activity,suggesting that their immune function was superior to that of CAR-T cells alone.Conclusion:We successfully constructed two CAR-targeting the HLA-A*02:01-restricted KRAS G12 V and validated their specificity and efficacy by experiments both in vitro and in vivo,which showed that both CAR-T cells were able to effectively kill cells expressing the target antigens and the addition of truncated PD-1 could strengthen the immune function and promote immune infiltration.This project validated the function of CAR-T cells targeting HLA-A*02:01-restricted KRAS G12 V.The results mark the feasibility of combining high-frequency KRAS point mutations with HLA alleles designed as chimeric antigen receptors and combined CAR-T cell therapy with immune checkpoint inhibition,which is expected to be a new approach for pancreatic cancer and other solid tumors expressing KRAS G12V/HLA-A*02:01,laying the foundation for exploring more and more effective combination therapeutic strategies.