Targeted ‘Off-the-Shelf’ Tumor Immunotherapy Using Allogeneic T-Cell Precursors.
Authors: Zakrzewski Johannes L. , Suh David , Smith Odette M. , King Christopher , Goldberg Gabrielle L. , Jenq Robert , Holland Amanda M. , Grubin Jeremy , Lu Sydney X. , Cabrera-Perez Javier , Rizzuto Gabrielle , Sant’Angelo Derek B. , van den Brink Marcel R.M. , Markley John C. , Riviere Isabelle , Sadelain Michel , Zuniga-Pflucker Juan Carlos 
Published: October 2019

Abstract
T cell deficiencies can occur in many settings, including aging, autoimmune and genetic disorders, hematological malignancies, infectious diseases, and exposure to cytotoxic/cytostatics agents. Notch-based culture systems can be utilized for ex vivo generation of large numbers of T lineage committed lymphoid precursor cells, and we recently reported that the adoptive transfer of T cell precursors significantly enhances T cell reconstitution and function after allogeneic T cell depleted HSCT. The aim of this study was to evaluate if allogeneic T cell precursors are safe and effective when used for adoptive transfer across MHC barriers in the absence of allogeneic HSCs to overcome radiation injury, enhance T cell function and improve anti-tumor activity in immunosuppressed recipients. We found that positive selection of adoptively transferred allogeneic (C57BL/6) precursor cells ± syngeneic (BALB/c) HSCs in irradiated hosts (BALB/c) is dependent on MHC molecules on non-hematopoietic host cells, resulting in the in vivo development of host-MHC restricted allogeneic T cells. We demonstrate that negative selection of these cells is mediated by donor-derived antigen presenting cells (APCs), resulting in host-tolerance. Adoptively transferred allogeneic T cell precursors significantly improved survival of BALB/c mice after irradiation (675 cGy) and enhanced anti-tumor activity against liquid (A20 lymphoma) and solid (renal cell carcinoma) tumors in syngeneic HSCT recipients. Furthermore, we demonstrate the feasibility of genetic engineering of antigen-specific T cell precursors, by transducing them to express a chimeric antigen receptor (CAR) targeting human CD19. Transduction efficiencies were routinely in the range of 50%–70%. Adoptive transfer of CAR-expressing T cell precursors resulted in the in vivo generation of high numbers of appropriately selected CAR-expressing T cells with significantly enhanced anti-tumor activity (compared with CAR-negative T cell precursors) against a CAR-sensitive tumor, but without any undesirable auto/alloreactivity. We conclude that adoptively transferred allogeneic T cell precursors develop into host-MHC restricted and host-tolerant T cells characterized by selection of a functional TCR repertoire even in a fully mismatched thymic epithelial MHC environment. This strategy overcomes important limitations of conventional adoptive T cell therapies: rejection, alloreactivity and impaired antigen recognition due to restriction to MHC disparate from the one expressed on APCs. The use of allogeneic instead of autologous cells eliminates the risk of contamination with residual malignant patient cells and allows the generation and storage of virtually unlimited quantities of precursor cells for ‘off-the-shelf’ immunotherapy. This procedure has not only substantial logistic advantages, but it facilitates ex vivo manipulation, in particular genetic engineering, to generate antigen-specific or otherwise enhanced designer cells. Adoptive transfer of MHC mismatched and genetically enhanced T cell precursors therefore represents a promising novel strategy for targeted ‘off-the-shelf’ immunotherapy in immunosuppressed patients.