| Purpose:The immunosuppressive tumor microenvironment(TIME)has been identified as a major problem impinging upon the efficacy of chimeric antigen receptor(CAR)T cells,especially in solid tumors[1].Programmed death-1(PD-1;CD279),a type I transmembrane receptor localized at the cell surface,is the most extensively expressed T-cell inhibitory receptor(IR)on T cells,B cells,NK cells,DCs,monocytes,macrophages,and even activated vascular endothelial cells[2].PD-1 pathway seems to be a“master switch”in TIME since in some cases a single dose of anti-PD therapy can change from an initially highly suppressive TIME to a highly active inflammatory site[3].A number of cancer types overexpress PD-L1 to impair the T cell antitumor response and evade the immune system via PD-1/PD-L1 interactions[4].However,the PD1-PDL1/2 pathway is also very important for maintaining normal peripheral immune tolerance,and direct targeting of PDL1 may cause serious on target off tumor effects.How to tune the affinity of CAR to different targets through redesign CAR structure to avoid the immunosuppressive signal of PD1 and on target off tumor effect is a valuable research.Methods:The PD1 fragment was inserted into the structure of second-generation CAR targeting CD19,thus new bi-affinity CARs targeting CD19 and PDL1 was constructed,named as PD1-BAT1,2,3 respectively.First,we verified CAR were stably expressed on the surface of T cells transfected by lentivirus.Second,we detected the killing efficiency targeting CD19 and/or PDL1,cytokine secretion,in vitro culture and specific proliferation of the bi-affinity CAR T cells.Thirdly,we further verified the safety and effectiveness of the new bi-specific CAR T cells in NSG mouse bearing NALM-6-PDL1 model.Fourthly,in order to explore the reasons behind the different CAR T cells showing different biological effect,we further analyzed the transcriptome and metabolism of CAR T cells incubating with NALM-6-PDL1.At last,we explored the congruent relationship between the different biological effects of CAR T cells and different structures of CAR.Results:1.The novel bi-affinity CAR were stably expressed on the surface of T cells,but the expression efficiency of the PD1-BAT2 was lower than the other CAR structures.2.Compared with the conventional second-generation CAR T cells,PD1-BAT1/2 cells augment the immune response towards CD19~+PDL1~+tumor cells while PD1-BAT1 cells show PD1-related-off-target toxicity towards CD19~-PDL1~+cells.3.PD1-BAT2/2 cells show weaker antigen-specific cytokine release,compared with the conventional second-generation CAR T cells.4.PD1-BAT2 cells maintain improved antitumor activity in the NSG mouse model bearing NALM-6-PDL1 cells without inducing on target off tumor effect,whereas the group administered PD1-BAT1 cells presented severe side effects,depilation,and irritability.5.PD1-BAT2/3 cells exhibit ameliorative differentiation,exhaustion,and apoptosis against tumor cells 6.Compared with the conventional second-generation CAR T cells,PD1-BAT3 cells had a higher oxygen consumption rate of mitochondria after incubation with NALM-6-PDL1.7.PD1-BAT2/3 cells had a weaker affinity for CD19 than 2G-T cells.Conclusion:Based on the conventional second-generation CAR structure targeting CD19,we constructed the novel bi-affinity CAR reversing the immunosuppressive signal of PD1.The novel bi-affinity CAR T cells enhanced anti-tumor effects towards tumors with high expression of CD19 and PDL1 but didn’t kill PDL1 positive cells.At the same time,the novel bi-affinity CAR T cells not only reversed the immunosuppressive signal of PD1 but also reduced cytokines release against tumors,so the novel bi-affinity CAR T cells may avoid the CRS in the future clinical application.The novel dual-target CAR T cells may provide a more effective and safe treatment for tumors.Hence,the novel bi-affinity CAR T cells may serve to address the critical challenges currently facing CAR T cell therapy by increasing its efficacy and safety... |
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