Moderate Affinity Of Chimeric Antigen Receptor Engineered T Cells Targeting EGFR Family Exhibit Extensive Antitumor Activity

Adoptive immunotherapy with chimeric antigen receptor(CAR)or T cell receptor(TCR)modified T cells is a new immunotherapy strategies with rapid development.Based on non-major histocompatibility complex(MHC)manner,compared with non-specific T cells,CART cells with the characteristics of specificity recognition,activation and proliferation.Currently,CARs have considerably evolved to the third generation,containing two co-stimulatory molecules,such as CD28+CD134(OX40)or CD28+CD137(4-1BB),which have usually been demonstrated with enhanced cytokine production and tumor lytic activity and reduced activation-induced cell death(AICD)than the second or first generation CARs.The study on lyse specificity and efficiency of dual vector system after tuning the affinity,published by Kloss CC team on the?Nature Biotechnology?in 2012,the study improve the specificity of dual CAR system,also provides a new optimization direction for optimizing CAR vector.Two study papers on adjusting the affinity of CAR system for reducing on-target/off-tumor toxicity,studies have shown that CART cells with high affinity showed strong killing effects against tumor cells expressed EGFR antigen at low or high levers,while CAR system with low affinity only showed killing effects against tumor cells expressed EGFR at high lever,the killing effects was positively correlated with the expression of EGFR.Tuning the affinity of CARs specifically killing tumor cells,reducing the on-target/off-tumor toxicity by exempt normal cells expressed target antigen at low lever.But tumor cells expressed antigen will also be different due to tumor heterogeneity characteristic,cause the tumor cells to escape that expressed targeting antigen at low levels,reducing therapeutic efficiency while exempt normal cells.So tuning affinity of CART cells targeting a single antigen molecules cannot completely eliminate all of the tumor cells.EGFR family EGFR(HER1/erbB-1),HER2(ErbB-2 or neu),HER-3(erbB-3)and HER-4(erbB-4)overexpressed on breast cancer,lung,colon,prostate,head and neck and various tumor cells,the total positive rate of the four members in the EGFR family is nearly 100% in the variety of malignant tumor tissues.So we generated the G3-Herin CART cell that targeting HER1 ? HER2? HER-3and HER-4,and the G3-Herin withmoderate affinity can reduce the on-target/off-tumor toxicity against normal tissues.Part One: Generation of chimeric antigen receptor targeting EGFR familyThe moderate affinity HERIN fragment which targeting EGFR family and generate the third generation the vector of G3Herin-CAR,through enzyme identification and gene sequencing to prove that we have build G3Herin-CARvector successfully.Then the plasmid to transfection of 293 T cells,further evidence that the generated vector can efficiently packaging carry expressed the gene of G3Herin-CAR.Part Two: The chimeric antigen receptor targeting EGFR to modified T cellsThe Generated plasmid were electroporated into peripheral blood mononuclear cells(PBMC)from healthy donors,T cells were cultured in 2%AIM and IL-2.Through western blot and detection of CART cells phenotype for successfully genetic modification of T cells.Part Three: Functional experiment of CART cell which targeting EGFR in vitroThrough western blot and filtered flow experiment detection of tumor cells,then test the production of the cytokines IFN-r,TNF-a,and the killing effects to tumor cells,for evidence that the generated pNBS328-G3Herin-CAR T cells exist a broad spectrum and effective lytic activity target tumor cells which expressing the members of EGRR family.Successfully prove that the experiment original design: G3HERIN-CART cells can identified and killed a variety of tumor cells,and the moderate affinity target can ensure the curative effect while improved therapeutic safety at the same time.