Engineering safe anti-CD19-CD28ζ CAR T cells with CD8a hinge domain in serum-free media for adoptive immunotherapy.

Background: Despite the curative potential, high cost of manufacturing and the toxicities limits the wider access of Chimeric Antigen Receptor (CAR) T cell therapy in global medicine. CARs are modular synthetic antigen receptors integrating the single-chain variable fragment (scFv) of an immunoglobulin molecule to the TCR signaling. CARs allow HLA independent, T cell mediated destruction of tumor cells independent of tumor associated-HLA downregulation and survive within the patient as 'living drug.' Here we report a safer approach for engineering alpha beta T cells with anti- CD19-CD28ζ CAR using self-inactivating (SIN) lentiviral vectors for adoptive immunotherapy. Method: αβ T cells from the peripheral blood (PB) were lentivirally transduced with CAR construct containing hinge domain from CD8α, transmembrane and co-stimulatory domain from CD28 along with signaling domain from CD3ζ and driven by human UBC promoter. The cells were pre-stimulated through CD3/CD28 beads before lentiviral transduction. Transduction efficiency, fold expansion and phenotype were monitored for the CAR T cells expanded for 10-12 days. The antigen-specific tumor-killing capacity of CD19 CAR T cells was assessed against a standard CD19 expressing NALM6 cell lines with a flow cytometry-based assay optimized in the lab. Results and conclusion: We have generated high titer lentiviral vectors of CAR with a titer of 9.85 ± 2.2×10 Conclusion: We have generated, validated, and characterized a reproducible indigenous workflow for generating anti-CD19 CAR T cells