Abstract 559: Targeting hematological malignancies and solid tumors with switchable chimeric antigen receptor-engineered iPSC-derived natural killer cells
Authors: Li Xiao-Hua , Goldenson Benjamin , Thangaraj Jaya Lakshmi , Gynn Matthew , Gumber Diana , Do Myan , Willert Karl , Kaufman Dan S. 
Published: June 2022

Abstract
Switchable chimeric antigen receptors (sCAR) provide an important new strategy to precisely regulate CAR-mediated anti-tumor activity. The sCAR system combines a CAR that recognizes peptide neoepitope (PNE) with an injectable “switch” molecule that consists of an anti-tumor Fab linked to PNE. Configurating PNE at different location or chain of the Fab enables us to identify best switches optimal for tumor targeting. Previous studies demonstrate that this sCAR system expressed in T cells provides tight control over anti-tumor activity. Here, we translated this approach to engineer natural killer (NK) cells with the sCAR to provide a universal, targeted cell-therapy approach in a patient-nonspecific manner. First, we engineered human induced pluripotent stem cells (iPSCs) with the sCAR combining the PNE-specific CAR scFv with our previously described NK cell optimized CAR4 signaling motifs consisting of the NKG2D transmembrane domain, 2B4 co-stimulatory domain and the CD3ζ chain. We selected clones that maintained highest level of pluripotency and most stable expression of the sCAR4 on the surface. Next, we generated mature sCAR4-expressing iPSC-derived NK cells that expressed common surface receptors similar to that of donor peripheral blood mononuclear cell-derived NK cells. We then used three panels of switches with specificity to CD19 (consisting of 9 different configurations), to CD33, CD123 and CLL1 (1 configuration each), and to FRIZZLED7 (FZD7; 6 configurations) to target CD19+ B cell lymphoma, acute myeloid leukemia (AML), and ovarian cancer, respectively. All three targets demonstrated switch-specific dose response in killing tumor cell lines. Different configurations conferred variable efficiencies in switch-mediating tumor cell killings and the optimal switch concentrations were found to be different from what was observed previously with sCAR-expressing T cells. In a comparison with the “conventional” (non-switchable) CARs (cCARs), iPSC-NK cells expressing either sCAR4 and treated with an anti-CD19 switch or iPSC-NK cells with an anti-CD19-cCAR4 demonstrated similar level of cytotoxicity against B cell leukemia cells. Finally, we have built a mouse model and are currently testing the iPSC-NK-sCAR system in vivo. Together, this iPSC-NK-sCAR strategy enables close control over CAR-mediated activity with flexibility to target multiple tumor types and a potential to become a novel off-the-shelf therapy.
Citation Format: Xiao-Hua Li, Benjamin Goldenson, Jaya Lakshmi Thangaraj, Matthew Gynn, Diana Gumber, Myan Do, Karl Willert, Dan S. Kaufman. Targeting hematological malignancies and solid tumors with switchable chimeric antigen receptor-engineered iPSC-derived natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 559.