Comparison Of Anti-tumor Ability Of CAR-T Cells Based On Affinity Of ScFv

ObjectiveChimeric antigen receptor T cells(CAR-T)targeting tumor-associated antigens can cause off-target toxicity during the treatment of solid tumors.The single-chain variable fragment(scFv)of CAR-T cells plays a key role in the specific recognition of target cells.At present,decreasing the affinity of CAR-T cells by changing the scFv has become an effective strategy,but the decreasing of CAR affinity may also impair the anti-tumor functions of CAR-T cells.Human epidermal growth factor receptor 2(HER2),a member of the transmembrane epidermal growth factor receptor family,expressing at high levels on the surface of various tumor cells,can be used as a target for CAR-T cell therapy.In this study,we chose HER2 as the target antigen of CAR-T cells,and HER2 targeting scFv as the extracellular recognition domain of CAR.This study aims to investigate the correlation between CAR-T cell anti-tumor ability and scFv affinity,when the latter is below the threshold for off-target toxicity.MethodsWe constructed CAR transfection lentiviral plasmids with different affinity by genetic engineering.Then used the plasmid to transfect 293T cells to obtain lentivirus.CD8~+T cells were isolated from the peripheral blood of healthy donors.And we used lentivirus to transfect CD8~+T cells to product HER2-specific FRP5-CAR-T cells and ch A21-CAR-T cells.Then flow cytometry was used to detect the transfection efficiency.CAR-T cells proliferation and differentiation were detected by cell counting and flow cytometry at different time points.Then HER2 expression of MCF-7 and MRC-5 cell lines were detected by flow cytometry.Incubate the MCF-7and MRC-5 cell lines with different affinities CAR-T cells respectively to test the cytotoxicity.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression levels of IL-2 and IFN-?in the co-incubation supernatant of CAR-T cells and the target cells.Imaging flow cytometry was used to detect the immune synapse formation ability of CAR-T cells after incubation with target cells.Western blot was used to detect changes in intracellular activation-related signaling proteins of activated CAR-T cells.Mouse models were used to verify the effect of scFv affinity on the anti-tumor ability of CAR-T cells in vivo.Results1.We constructed CAR transfection lentiviral plasmids with different affinity.2.We constructed FRP5-CAR-T cells and ch A21-CAR-T cells with different affinity.scFv affinities had no significant effect on CAR-T cells proliferation and differentiation in vitro.3.Flow cytometry results showed that MCF-7 highly expresses HER2 and MRC-5lowly expresses HER2.The results of cytotoxicity experiments showed that increasing the affinity of scFv of CAR-T cells enhanced the CAR-T cells cytotoxicity,and no off-target toxicity was detected.ELISA results showed that higher affinity ch A21-CAR-T cells could secrete higher levels of IL-2 and IFN-?.Imaging flow cytometry results showed that higher affinity scFv could increase the ability of CAR-T cells to form immune synapses.Western blot results showed that the higher affinity of ch A21-CAR-T cells had higher expression levels of intracellular activation-related proteins,compaired with the FRP5-CAR-T cells.Mouse model results showed that ch A21-CAR-T cells could better inhibit tumor growth,compaired with the FRP5-CAR-T cells,indicating that increasing the affinity of scFv could enhance the anti-tumor ability of CAR-T cells in vivo.ConclusionThis study compared the effects of two low-affinity scFv on the anti-tumor ability of CAR-T cells.Our results showed that the higher affinity scFv can increase CAR-T cell cytokine secretion,immune synapse formation ability and CAR-T cell activation signal,thereby enhancing CAR-T cell anti-tumor ability in vivo and in vitro.It is suggested that decreasing the affinity of scFv could prevent on-target off-tumor,while finding a balance between anti-tumor efficacy and off-target toxicity.And according to the different clinical situations,the intensity of tumor antigen expression of patients should be taken into account to achieve the purpose of precise treatment.