Preclinical development and molecular characterization of an engineered Type-1 regulatory T cell product suitable for immunotherapy

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapeutic approach for many hematological disorders. However, allo-HSCT is frequently accompanied by a serious side-effect: graft-versus-host disease (GvHD). The clinical use of allo-HSCT is limited by the inability of the current immunosuppressive regimens to adequately control GvHD without impairing the graft-vs-leukemia effect (GvL) conferred by transplanted healthy immune cells. To address this, we have developed an engineered type 1 regulatory T (Tr1) cell product called CD4 IL-10 . CD4 IL−10 are obtained through lentiviral transduction that delivers the human IL10 gene into purified polyclonal CD4 + T cells. CD4 IL−10 cells may provide an advantage over standard of care immunosuppressants due to the ability to suppress GvHD through continuous secretion of IL-10 and enhance graft-versus-leukemia (GvL) effect in myeloid malignancies through targeted killing of malignant myeloid cells. Here, we established a production process aimed at current Good Manufacturing Practice (cGMP) production for CD4 IL-10 . We demonstrated that the CD4 IL−10 cell product maintains suppressive and cytotoxic functions of previously described CD4 IL−10 cells. In addition, RNA sequencing analysis of CD4 IL−10 identified novel transcriptomic changes indicating that CD4 IL−10 cells primarily upregulate cytotoxicity-related genes. These include four molecules with described roles in CD8 + T and NK cell-mediated cytotoxicity: CD244, KLRD1, KLRC1, and FASLG. Finally, it is shown that CD4 IL−10 upregulate IL-22, which mediates wound healing and tissue repair, particularly in the gut. Collectively, these results pave the way towards clinical translation of the cGMP-optimized CD4 IL−10 cell product and uncover new molecules that have a role in the clinical application of CD4 IL-10 .