Th177 - Crispr-mediated Deletion of PD1 to Enhance the Efficacy of Human CAR Tregs

Sonya Mangat – Department of Microbiology & Immunology, The University of British Columbia, Vancouver BC, Canada – BC Children’s Hospital Research Institute, Vancouver BC, Canada; Jana Gillies – Department of Surgery, The University of British Columbia, Vancouver BC, Canada – BC Children’s Hospital Research Institute, Vancouver BC, Canada; Vivian Fung – Department of Surgery, The University of British Columbia, Vancouver BC, Canada – BC Children’s Hospital Research Institute, Vancouver BC, Canada; Manjurul Haque – Department of Surgery, The University of British Columbia, Vancouver BC, Canada – BC Children’s Hospital Research Institute, Vancouver BC, Canada; Majid Mojibian – Department of Surgery, The University of British Columbia, Vancouver BC, Canada – BC Children’s Hospital Research Institute, Vancouver BC, Canada; Andrew Brown – Department of Surgery, The University of British Columbia, Vancouver BC, Canada – BC Children’s Hospital Research Institute, Vancouver BC, Canada; Avery Lam – Department of Pathology, Stanford University, Stanford CA, USA; Megan Levings – Department of Surgery, The University of British Columbia, Vancouver BC, Canada – BC Children’s Hospital Research Institute, Vancouver BC, Canada

Abstract Text: Regulatory T cells (Tregs) are being tested clinically as therapeutic tools for treating transplant rejection and autoimmunity. Pre-clinical studies have shown that the efficacy of Tregs can be enhanced by using chimeric antigen receptors (CARs) to confer specificity for disease-relevant antigens, such as allogeneic HLA molecules in a transplant setting. However, recent data has suggested that PD1 signalling limits the activation potential of Tregs and may contribute to Treg dysfunction. We hypothesised that CRISPR-mediated removal of PDCD1 (encoding PD1) would improve the sensitivity of CAR Tregs to antigen stimulation, thereby enhancing their therapeutic efficacy. Human CAR Tregs were generated using a previously characterised HLA-A2-specific CAR, PD1 was removed by CRISPR and successfully edited cells were identified by incorporation of a homology directed repair DNA template that encoded a truncated CD19 (ΔCD19) reporter. A high degree of gene editing was achieved with >50% of the CAR+ Tregs expressing ΔCD19. When stimulated with HLA-A2+PDL1+ K562 cells, PD1-deficient CAR Tregs expressed higher levels of activation markers than PD1-sufficient CAR Tregs, demonstrating a greater sensitivity to antigen stimulation. However, these cells maintained their characteristic expression of FOXP3 and Helios following transfer into HLA-A2-transgenic NSG mice, suggesting PD1-ablation did not compromise Treg stability. Upon co-culture with mature HLA-A2+ dendritic cells, PD1-deficient CAR Tregs suppressed the expression of MHC class II and co-stimulatory molecules more effectively than PD1-sufficient CAR Tregs, demonstrating PD1-ablation enhanced the suppressive potency of CAR Tregs. Future work will test the therapeutic efficacy of PD1-deficient CAR Tregs in a humanised mouse model.