Epitope Edited Hematopoietic Stem Cells to Enable Synergistic Immunotherapy Combinations for Acute Myeloid Leukemia

Despite treatment advances, acute myeloid leukemia (AML) is still associated with an unfavorable outcome for >50% of patients. Whereas novel immunotherapies, such as CAR-T cells, bispecific and toxin conjugated antibodies (mAb), demonstrated clinical efficacy when targeting dispensable lineage antigens (Ag), such as CD19 in B-ALL, the same approach cannot be exploited for AML, due to lack of actionable leukemia-restricted Ags. Suitable targets are shared with healthy progenitor or mature myeloid cells, leading to on-target/off-tumor toxicity and impairment of hematopoietic reconstitution. Several AML immunotherapies are currently under development, but their use is restricted to a limited time window, likely insufficient for disease eradication. To address these issues, we reasoned that precise modification of the targeted epitopes in donor HSPC used in HSCT results in loss of mAb recognition, without affecting normal protein expression, regulation, and function ( Nature, accepted). Epitope editing allows targeting genes essential for leukemia survival regardless of shared expression or functional role in normal HSPC, thus minimizing the risk of tumor immune escape by Ag loss or downregulation. Cytokine receptors such as FLT3, KIT and CD123 are found in >85% of AML cases and their mutation (e.g., FLT3-ITD) or overexpression is associated with poor prognosis. We identified amino acid substitutions in the FLT3, KIT and CD123 extracellular domains that preserve physiologic surface expression, ligand-binding, kinase phosphorylation, colony-forming capacity, proliferative response, transcriptional and phospho-proteomic profile of CD34+ HSPCs but avoid detection by a therapeutic monoclonal Ab. Cells expressing these variants were resistant to CAR-T killing and did not induce CAR activation and proliferation during in vitro co-culture. We were able to introduce these mutations into CD34+ HSPCs by adenine base editing (ABE) with high efficiencies (90%, 85% and 75% for FLT3, KIT and CD123, respectively) without the need for dsDNA breaks. After xenotransplant into NBSGW mice, FLT3, KIT or CD123 epitope-editedHSPC sustained long-term multilineage hematopoiesis indicating editing and preserved functionality of HSCs. Upon treatment with FLT3 CAR-T in vivo, we observed sparing of human CD34+38- HSPCs, granulo-mono progenitors (GMP), B-cells and B-progenitors in the bone marrow of mice engrafted with FLT3-edited HSPCs compared to AAVS1 controls. Treatment with CD123 CAR-T cells showed protection of epitope-edited myeloid lineages, including granulocytes, DCs and HSPCs. We next generated advanced in vivo models with co-engraftment of human HSPCs and patient-derived AML xenografts (PDX), to demonstrate the selective resistance of epitope edited HSPCs and their progeny, while PDX were eradicated by FLT3 or CD123 CAR-T. To further enhance therapeutic efficacy, we are now exploring the combination of epitope-edited HSPCs and pharmacological agents with potential synergistic effects with CAR-T cells. FLT3 tyrosine kinase inhibitors (e.g., Crenolanib) have the potential to enforce surface expression of FLT3 by impairing its intracellular recycling, thus enhancing CAR-T mediated killing, but their use may be limited by overlapping toxicities on healthy hematopoiesis, in particular in the post-HSCT setting. To assess if epitope-editing could prevent not only CAR- but also TKI-mediated toxicities, we treated mice co-engrafted with FLT3- or AAVS1- edited HSPCs and a FLT3-ITD+ AML PDX with combinations of FLT3-CAR and a 2-week course of Crenolanib. Leukemia burden was reduced by Crenolanib alone and eradicated by FLT3-CAR-T. Critically, we observed preservation of healthy hematopoietic lineages only in mice engrafted with epitope-edited HSPCs, including the CAR+Crenolanib group, while AAVS1 controls showed overlapping off-tumor toxicity when treated with the CAR+Crenolanib combination. In conclusion, we believe that epitope edited HSPCs may not only enable safe and effective CAR-T immunotherapies for AML, but also allow their combination with pharmacological blockade of leukemia survival/proliferative pathways to achieve synthetic lethality mechanisms, while still avoiding dose-limiting toxicities. Further exploration of immunotherapy-synergistic combinations will be fundamental to improve the outcomes of difficult-to-treat high-risk AML patients.