MULTIPLEX SILENCING OF SIX TARGETS FOR ENGINEERING OF NEXT GENERATION, "OFF-THE-SHELF" CAR T-CELLS

Background & Aim Despite very promising clinical achievements related to treatments for haematological malignancies, challenges for chimeric antigen receptor (CAR) T-cell therapy remain, including a lack of efficacy against solid tumour cancers and limited accessibility to these treatments. Therefore, the need to multiplex engineer next generation CAR T-cells is essential for providing CAR T-cells as off-the-shelf products (allogenic) and capable of durable treatment responses. Persistence of CAR T cells is directly related to treatment durability. Rejection of CAR T cells by the host immune system, and impaired CAR T-cells fitness (continued functional activity) are the main factors hampering persistence of these cells. Therefore, to overcome these challenges and draw the future of CAR T-cell therapy, we have developed an efficient strategy to create next generation CAR T-cells using a novel bimodal gene construct (miCAR) for simultaneous CAR expression and microRNA (miRNA) mediated gene silencing. More precisely, we have used a multiplex miCAR gene construct to develop miCAR T-cells comprising of a gene responsible for the expression of anti-CD19 CAR, and miRNAs for silencing of six cell surface receptors, namely the T-cell receptor (TCR), human leukocyte antigen class I (HLA-I), CD52, PD1, TIM3 and TIGIT. Methods, Results & Conclusion Primary T-cells were modified via lentiviral vector transduction, expanded in G-Rex cell culture plates, and in final step purified by depletion of TCR-expressing cells. Produced miCAR T-cells were characterized by flow cytometric immunophenotyping, functional activity against CD19-expressing tumor cells, mixed lymphocyte reactions to demonstrate protection from allogeneic CD8 T-cells and NK cells, and assay demonstrating the loss of alloreactivity of miCAR T-cells. We demonstrate robust and reliable production of miCAR T-cells with >99% TCR negativity, efficient silencing of all targeted receptors, favorable immunophenotypic profile (52-71% being naïve memory phenotype), and specific cytotoxicity against CD19-expressing cells. Moreover, we demonstrate negligible alloreactivity of miCAR T-cells and protection from both CD8 T-cell and NK cell mediated depletion with "tuned" silencing of HLA–I (70 ± 10%). In conclusion, using a single gene construct, and from a single gene-engineering step, we successfully created next generation CAR T-cells with silencing of six target genes, which are functionally active, non-alloreactive and hypoimmunogenic. Despite very promising clinical achievements related to treatments for haematological malignancies, challenges for chimeric antigen receptor (CAR) T-cell therapy remain, including a lack of efficacy against solid tumour cancers and limited accessibility to these treatments. Therefore, the need to multiplex engineer next generation CAR T-cells is essential for providing CAR T-cells as off-the-shelf products (allogenic) and capable of durable treatment responses. Persistence of CAR T cells is directly related to treatment durability. Rejection of CAR T cells by the host immune system, and impaired CAR T-cells fitness (continued functional activity) are the main factors hampering persistence of these cells. Therefore, to overcome these challenges and draw the future of CAR T-cell therapy, we have developed an efficient strategy to create next generation CAR T-cells using a novel bimodal gene construct (miCAR) for simultaneous CAR expression and microRNA (miRNA) mediated gene silencing. More precisely, we have used a multiplex miCAR gene construct to develop miCAR T-cells comprising of a gene responsible for the expression of anti-CD19 CAR, and miRNAs for silencing of six cell surface receptors, namely the T-cell receptor (TCR), human leukocyte antigen class I (HLA-I), CD52, PD1, TIM3 and TIGIT. Primary T-cells were modified via lentiviral vector transduction, expanded in G-Rex cell culture plates, and in final step purified by depletion of TCR-expressing cells. Produced miCAR T-cells were characterized by flow cytometric immunophenotyping, functional activity against CD19-expressing tumor cells, mixed lymphocyte reactions to demonstrate protection from allogeneic CD8 T-cells and NK cells, and assay demonstrating the loss of alloreactivity of miCAR T-cells. We demonstrate robust and reliable production of miCAR T-cells with >99% TCR negativity, efficient silencing of all targeted receptors, favorable immunophenotypic profile (52-71% being naïve memory phenotype), and specific cytotoxicity against CD19-expressing cells. Moreover, we demonstrate negligible alloreactivity of miCAR T-cells and protection from both CD8 T-cell and NK cell mediated depletion with "tuned" silencing of HLA–I (70 ± 10%). In conclusion, using a single gene construct, and from a single gene-engineering step, we successfully created next generation CAR T-cells with silencing of six target genes, which are functionally active, non-alloreactive and hypoimmunogenic.