KIR/PD-1-based Inhibitory Chimeric Antigen Receptor Reduces On-target Off-tumor Toxicity Of CAR-T Therapy

Chimeric Antigen Receptor(CAR)is a synthetic chimeric molecule based on thedual signal theory of T cell activation,which contains an antigen recognition domain and a T cell activation domain.CAR gene-modified T cells(CAR-T)can effectively activate and specifically kill tumor cells after recognizing the target antigen.At present,CAR-T therapy has made breakthrough progress in the field of tumor treatment.CD19-targeted CAR-T cell treatment for relapsed and refractory B-cell leukemia has a 90%complete remission(CR).Because of the non-specificity of the target,CAR-T cells will attack normal tissue cells that express the low level of target antigens,resulting in an“on-target off-tumor”side effect.In the treatment of hematological malignancy,CAR-T cells will clear normal B cells and increase the risk of infection.In the treatment of solid tumors,CAR-T cells will target important tissues or organs,such as liver and lungs,leading to organ failure and endangering patients'lives.Therefore,“on-target off-tumor”effect is an urgent problem to be solved in the clinical application of CAR-T,especially in the treatment of solid tumors.The study has designed a novel inhibitory CAR(i CAR)that includes the extracellular domain of the killer immunoglobulin-like receptor(KIR)and intracellular domain of the programmed cell death receptor 1(PD-1).KIR domain of i KP CAR recognizes human leukocyte antigen(HLA)on the surface of normal tissue cells and activates its PD-1 signal to inhibit T cell activation.Because HLA is highly expressed in normal tissues,i KP CAR will activate PD-1 signal and inhibit CAR-T cell activation when recognizing normal tissues,and then prevents CAR-T cells from killing normal cells.While tumor cells have low or no HLA expression,the inhibitory signal of i KP CAR will not be triggered,then CAR-T cells are activated and kill tumor cells.By co-expressing i KP CAR in the clinically-used CD19-CAR-T cells and HER2-CAR-T cells,we explore whether these CAR-T cells can kill tumor cells but not normal cells in vitro and in vivo,so that i KP CAR can solve the problem of on-target off-tumor effects of CAR-T cells.First,we found that i KP CAR did not affect CAR expression,cell proliferation,activity and CD8⁺/CD4⁺subgroups of CAR-T cells.Next,we analyzed intrcellular PD-1 signaling pathway once recognizing HLA-C1 on normal cells,the results showed that the activation of CAR-T cells were inhibited by dephosphorylating the key protein P-Zap70 in CAR-T cells.At last,we demonstrated that CD19-CAR-T cells expressing i KP CAR(i KP-19-CAR-T)could kill CD19⁺HLA-C1^-Burkitt's lymphoma cell line Daudi cells in vitro effectively,while reduce the killing effect on CD19⁺HLA-C1⁺normal B cells significantly.The results of animal experiments showed that i KP-19-CAR-T cells cleared Daudi cells in immunodeficient mice while preserving healthy human B cells,but CD19-CAR-T cells cleared both Daudi cells and healthy human B cells in mice.In addition,we found that i KP-19-CAR-T cells expressed a lower level of exhaustion molecule PD-1,produced more central memory T cells(T_CM)and released less cytokines both in vitro and in vivo.In terms of solid tumor treatment,we had also demonstrated that HER2-CAR-T cells expressing i KP CAR(i KP-HER2-CAR-T)could effectively clear the gastric cancer cell line MGC803of HER2⁺HLA-C1^low,but exhibit lower cytotoxicity against HER2⁺HLA-C1^highMGC803 significantly.The related phenotypes of i KP-HER2-CAR-T cells were also detected,the results were consistent with i KP-19-CAR-T cells.In summary,we developed a KIR/PD-1-based inhibitory CAR and proved that CAR-T cells expressing an i KP CAR could selectively kill tumor cells that do not express or express low levels of HLA,while spare the normal cells that express high levels of HLA both in hematologic and solid tumor,which provides a new way to solve the problem of“on target off tumor”effects of CAR-T cells from signal regulation.