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<0.05). The preferential selection of early stage of differentiation (CCR7+CD28+CD27+CD137+CD62L+) for CAR-T cells isolated from both source of lymphocytes occurred. LAG3 and TIM-3 expressing T cells were found with higher frequency in UCB- vs. PBL-CAR-T cells, with superior association with CD4+ UCB-derived cells. CD19-CAR-T cells secreted IFN-g(300–400 N. spot/10 × 104 T cells), regardless the co-stimulatory molecules (CD28z vs 4-1BBz), upon the engagement of CAR by CD19. A minority of IL-4 releasing T cells was found for few CAR-T cells activated with TransAct. IFN-gamma secreting CAR-T cells simultaneously released IL-2, Granzyme B and Perforin but not IL-5 and IL-17, thus belonging to TH-1/effector subset. The cytotoxic activity of these T cells against CD19+ target cells was also determined by europium release assay. Differential gene expression profile was determined in UCB-CAR-T vs. PBL-CAR-T cells bearing the different CARs following the co-culture with either CD19+ or CD19- target cells.

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.