Gabriele Casirati
Postdoctoral Fellow, Dana-Farber Cancer Institute
Epitope Engineered Hematopoietic Stem and Progenitor Cells to Enable CAR-T Cell Immunotherapy for Acute Myeloid Leukemia
While novel immunotherapies demonstrated efficacy when targeting dispensable lineage antigens (Ag), the same approach cannot be exploited for AML, due to lack of actionable leukemia-restricted Ags. Targets are shared with healthy progenitor or mature myeloid cells, leading to on-target/off-tumor toxicity and impairment of hematopoietic reconstitution. We reasoned that precise modification of the targeted epitopes in donor HSPCs would result in loss of recognition, without affecting protein expression, regulation, and function. Epitope-editing allows targeting genes essential for leukemia survival regardless of expression in normal HSPC, minimizing the risk of tumor escape. As targets, we selected FLT3, KIT and CD123, which are found in >85% of AML cases. Through library screening, we identified amino-acid changes in FLT3, KIT and CD123 extracellular-domains that preserve expression, ligand-binding and intracellular activation but avoid detection by therapeutic Abs. Cells expressing these variants were resistant to CAR-T killing during co-culture. We identified gRNAs that enable introduction of these mutations by adenine base editors, which achieved up to 90%, 85% and 75% editing efficiency in CD34+ HSPCs for FLT3, KIT and CD123, respectively, either as single or multiplex editing. After xenotransplant into NBSGW mice, FLT3, KIT or CD123-edited HSPC sustained long-term multilineage hematopoiesis. Upon treatment with FLT3-CAR-T, we observed sparing of HSPCs, granulo-mono progenitors, and B-cells in mice engrafted with FLT3-edited cells. Similarly, treatment with CD123-CAR-T showed protection of CD123-epitope-edited granulocytes, DC and HSPCs. We confirmed selective resistance of edited HSPCs in mice co-engrafted with human AML xenografts, which were eradicated by FLT3- or CD123-CAR-T. In conclusion, transplantation of epitope-engineered HSPCs endowed with selective resistance to CAR-T-cells or mAbs is a novel approach to enable effective and safer immunotherapies for AML. This approach can also be exploited as non-genotoxic conditioning to allow engraftment and in vivo selection of engineered cells, without the need for chemo- or radiotherapy myeloablation.
