Epitope Editing of Hematopoietic Stem Cells Enables Adoptive Immunotherapies for Acute Myeloid Leukemia

Chimeric antigen receptor T cells (CAR-T), bispecific and toxin-conjugated antibodies are promising adoptive immunotherapies that can overcome the limitations of conventional cancer treatment and have demonstrated striking efficacy when targeting dispensable lineage antigens (Ag), such as CD19 or CD22 in B-cell acute lymphoblastic leukemia (B-ALL) and B-phenotype lymphomas​​. Although these clinical successes are poised to revolutionize the oncology field, the absence of actionable tumor-restricted markers currently hampers their safe application for other common malignancies, such as acute myeloid leukemia (AML), a life-threatening hematological cancer arising from hematopoietic stem and progenitor cells (HSPCs)​​. Since AML shares most of its surface markers with normal HSPCs or differentiated myeloid cells, on-target toxicities of antibody (Ab)-based immunotherapies would result in treatment-related myeloid aplasia and impairment of hematopoietic reconstitution​. Furthermore, due to its intra-tumoral heterogeneity and plasticity, targeting more than one surface antigen might be required to eradicate AML, thus exacerbating the risk of overlapping toxicity​​. Nonetheless, a range of anti-AML immunotherapies targeting myeloid or HSPCs antigens are currently under development​​, but their role will likely be time-restricted to remission induction or bridge treatment before allogeneic HSPC transplantation (HSCT), thus reducing the chances of AML eradication. Removal of the targeted antigen through CRISPR-Cas knock-out (KO) or exon-skipping from donor HSPCs used in allogeneic transplantation has recently been proposed​​ as a possible solution to reduce on-target adverse effects associated with anti-CD33 treatments and this approach is currently under evaluation in early-phase clinical testing (NCT04849910). However, while these studies provided evidence for the dispensable role of CD33 for HSPCs engraftment and myeloid differentiation, long-term studies on the functionality of CD33-KO myeloid cells in humans are lacking​. Furthermore, targeting a non-essential gene for AML survival would facilitate the occurrence of tumor escape mechanisms through antigen loss or downregulation, as observed in CD19-negative relapses after CD19 CAR-T therapies​​ or mismatched HLA loss after haploidentical HSCT​. Here, we show that precise editing of the targeted epitope on FLT3 (CD135), KIT (CD117) and IL3RA (CD123) in HSPCs results in near complete loss of Ab binding without the need for gene KO, thus allowing preservation of physiologic protein expression, regulation, and intracellular signaling. Critically, this strategy enables targeting genes fundamental for leukemia survival regardless of shared expression and functional role in normal HSPCs, which retain in vivo repopulating and multilineage differentiation capacities. Furthermore, we show that multiplex epitope-engineering of HSPCs is feasible and allows more effective immunotherapies against multiple targets without incurring overlapping off-tumor toxicities. We envision that this approach will provide novel opportunities to treat relapsed/refractory AML and allow safer non-genotoxic conditioning.