CAR T-cell therapies are efficient in treating B- and plasma-cell malignancies by targeting cell-of-origin antigens, eradicating both tumor cells as well as healthy counterparts. Their loss can be compensated by immunoglobulin substitution until they regenerate from hematopoietic stem and progenitor cells (HSPCs). However, similar on-target, off-tumor cell-of-origin elimination would be detrimental in HSPC-derived malignancies, such as acute myeloid leukemia (AML) as CAR T-cell activity might lead to terminal ablation of hematopoiesis. To address this challenge, we developed adaptor-mediated CAR T-cells displaying the scFv E2, AdFITC(E2), that binds to fluorescein conjugated to antigen-binding adaptors in diabody format (Db-FM).

We generated second-generation AdFITC(E2)-CAR T-cells and multiple Db-based adaptors against selected AML antigens. We then tested the Dbs targeting CD33 and CD117 with respect to their ability to mediate CAR T-cell biocidal activity as single adaptors or in combination in vitro and in vivo.

In vitro cytotoxicity assays of healthy-donor-derived AdFITC(E2)-CAR T-cells, both against cell lines (MOLM14-CD117+GFP+Luc+) as well as patient-derived AML blasts, revealed that dual adaptor use significantly improved tumor cell lysis compared to the equimolar concentration of single adaptors. In therapeutic xenogeneic mouse models engrafted with MOLM14 cells where AdFITC(E2)-CAR T-cells in combination with CD117 or CD33 Db-FM administered as single adaptors were as efficient as direct CAR T-cells against the same antigens. Injection of AdFITC(E2)-CAR T-cells and both CD117 and CD33 Db-FM effectively inhibited tumor growth, outperforming monotherapies, and leading in some cases to full AML elimination in bone marrow and blood.

We tested an adaptor CAR T-cell approach using multiple adaptors to modulate and enhance CAR T-cell activity, focusing on AML as a relevant disease model. The high heterogeneity in antigen expression on AML cells supports a combinatorial targeting strategy individualized based on the respective AML immunophenotype. Due to their relatively small molecular weight, Db-based adaptors are rapidly cleared from the body, allowing for rapid control over AdFITC-CAR T-cell on-off activity. We envision that this approach has the clinical potential to improve CAR T-cell safety profiles by enhancing the specificity toward target cells.