Background The principal goal of this study is to generate ’off-the-shelf’ CD19-CAR-T cells utilizing umbilical cord blood (UCB) as source of T lymphocytes and characterize through multi-omics their phenotype and functional properties.

Methods T cells were isolated from UBCs (N=15) and, upon activation in vitro using CD3 and CD28 mAbs, transduced with lentiviral vectors encoding for CD19-CD28z-or CD19–4-1BBz-CARs. Engineered T cells were also produced from the peripheral blood lymphocytes (PBL; N=5) as reference. The multidimensional phenotype analysis was utilized to assess the differentiation and activation status of the T cells. CD19-CAR-T cells were co-incubated or not with either CD19⁺ or CD19^- target cells to mimic the antigen-mediated engagement of the CARs, and then, multi-omics analyses, including metabolomics, transcriptomics, and in vitro functional assays, (Elispot, Luminex) were performed.

Results CD19-CAR T cells with early stage of differentiation (CD45RA⁺) co-expressing either ICOS or BTLA were observed in UCB- vs. PBL-CAR T cells (p<0.0002-<0.05). Distinct transcriptomic and functional profiles were also observed according to the source of T cells used for engineering and the method of activation of the cells. The transcriptomic profiling of these cells showed that chemokines (e.g., CCL1, CCL2, CCL5), CD38 and CD69 molecules, involved in chemotaxis, adhesion and activation, were up-regulated in UCB- vs. PBC-CD19-CAR-T cells (p<0.001). Similar pattern was observed for IFN signaling molecules and IL-2Rγ chain. Other molecules mediating pro-inflammatory functions, such as IL-3, IL-26, Il-27, TLR2 and 5, were overexpressed in PBMC-CD19-CAR-T cells (p<0.001). Differential MicroRNA (miRNAs) patters were also identified. The multiplex analyses of the antigen-specific release of cytokines, chemokines and growth factors, highlighted superior levels of TH1-associated cytokines or cytotoxic molecules (p<0.05), and no or lower levels of pro-inflammatory molecules (e.g., CD40L, IL-1β, IL-3, IL-10, IL-15) by UCB- vs. PBMC-CAR-T cells. Differential antigen-specific metabolomic signatures were also found in association with the source of T lymphocytes expressing the CD19-CARs.

Conclusions Although the integration of the obtained multi-omics data sets is still ongoing, the deep molecular profiling of ‘off-the-shelf’ CD19-CAR-T cells allowed to assess that UCB-derived T cells are endowed with superior anti-tumor ‘fitness’ and lower risk of inducing in vivo cytokine release syndrome.

Ethics Approval The study obtained ethics approval of the Sidra Medicine institutional review board(s), ID#1500788b and all the participants gave informed consent before taking part.