124 Optimizing the generation from umbilical cord blood of ‘off-the-shelf’ CD19-chimeric antigen receptor (CAR) expressing T cells

Background T lymphocytes expressing CD19-chimeric antigen receptor (CAR) showed the improvement of overall survival of patients with B-cell malignancies. Allogeneic CAR-T cells can overcome the limitation of the availability of patient‘s lymphocytes, reducing the waiting time for the treatment and decreasing the cost of manufacturing. This study is aimed at the optimizing the generation of ‘off-the-shelf’ CAR-T cells utilizing Umbilical Cord Blood (UCB) to isolate T lymphocytes. Methods UCBs have been collected at the time of childbirth from volunteer pregnant women at Sidra Medicine. Following the magnetic depletion of non-T cells, UCB-T lymphocytes were activated in vitro for 48 hr. by agonistic CD3/CD28 mAbs either conjugated to magnetic beads (Dynabeads) or to a colloidal polymeric nanomatrix (TranAct; Miltenyi Biotec). T cells generated in vitro were either i. untransduced (UT), or transduced with lentiviral encoding for ii. CD19-CD28z or iii. CD19-4-1BBz CARs. N=32 T cell cultures have been generated from fresh UCB (N=3) and, as control, from the peripheral blood lymphocytes of healthy donors (PBL; N=3) and used for deep phenotype analyses (28 markers) at different time points (Day +9 and Day+14) of the in vitro culture. Cytokines, perforin and granzyme B release (EliSpot or FluoroSpot) and cytotoxic activity (Delfia assay) have been assessed upon the co-culture with CD19+ or CD19- target cells. Results Enrichment of CD4+CAR+ T cells, besides CD8+CAR+, were observed in UCB-CAR- vs. PBL-CAR-T cells (40–59% of positive cells; as well as of CD45RA+ cells (40–60 vs. 20–30% of positive cells; p Conclusions The deep characterization of CD19-CAR-T cells contributed to validate the generation of anti-tumor ‘off-the-shelf’ CAR-T cells from UCB. Ethics Approval The study was approved by Sidra Medicine’s Ethics Board, approval number 1812044429.