Lymphoid Tissue-Activated Donor T cells (LADTs) for Improved Donor Lymphocyte Infusion (DLI) against Blood Cancers 2250463

Abstract Introduction Donor lymphocyte infusion (DLI) is a key strategy for treating relapses after HLA-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT) for blood cancers. In DLI, T cell alloreactivity drives both the highly beneficial graft vs leukemia (GvL) effect and the potentially fatal graft vs host diseases (GvHDs), which severely limits DLI’s application and efficacy. Here, we propose to separate GvL and GvHD by engineering Lymphoid Tissue-Activated Donor T cells (LADTs) that display alloreactivity only in lymphoid tissues where cancer cells accumulate but not in organs involved in GvHD including the skin, liver and guts. Methods To generate LADTs, PBMCs were first used to expand alloreactive T cells from an HLA-mismatched donor. Then, T cell alloreactivity was abrogated by knocking out CD3ε using CRISPR/Cas9, which blocks cell surface expression of the TCR/CD3 complex. Finally, the T cells were engrafted with a B cell-sensing mechanism that includes a CD19-specific synthetic Notch receptor (synNotch) and an expression cassette that drives the expression of an exogenous copy of CD3ε in response to synNotch engagement. This mechanism enables LADTs to restore alloreactivity only in lymphoid organs where B cells are abundant but not in other organs where B cells are scarce. Results We have established a process of generating LADTs with consistent yields. LADTs bearing CD19-specific synNotch receptors restored surface expression of TCR/CD3 in response to CD19+ cells and showed strong cytotoxicity against HLA+ U266 myeloma cells but not against HLA- ones. After removing CD19+ cells, LADTs gradually lost TCR/CD3 cell surface expression. Conclusion Our study demonstrated the feasibility of LADT generation and the proof-of-concept of using LADTs for improved DLI. This cellular immunotherapy approach does not depend on cancer-specific biomarkers thus may be applied to a wide range of hematopoietic malignancies. Funding Source Nemours Foundation Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)