Abstract 6802: CXCR4 transgene improves in vivo migration and efficacy of engineered iPSC-derived natural killer cells
Authors: Millar Hillary J. , Walker David , Campion Liam R. , Budd Ciara , Kolluri Aarti , Sharma Harsh , Gurung Buddha , Carton Jill M. , Perry Daniel J. , Alexander Nicholas , Levitsky Hyam , Naso Michael , Morse Barry A. 
Published: March 2024

Abstract
Chimeric antigen receptor (CAR)-engineered induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells have shown a favorable safety profile and early evidence of clinical efficacy in the treatment of patients with certain hematologic malignancies. CAR-iNK cells retain phenotypic and functional similarities to primary natural killer cells, with additional capabilities for recognition of tumors. For the treatment of cancers that are located in the bone marrow, it is critical that iNK cells are able to migrate efficiently to relevant sites of disease. CXCR4 (C-X-C chemokine receptor type 4) is a chemokine receptor specific for stromal-derived-factor-1 (SDF-1), and mediates the migration of hematopoietic cells to the bone marrow upon binding SDF-1. Here we describe novel engineering of iPSCs resulting in efficient migration and efficacy of iNK cells. iPSC were sequentially engineered at defined loci with Crispr/MAD7/HDR to incorporate Allo-EvasionTM edits, a CAR targeting antigens expressed on different hematological malignancies, constitutive interleukin-15 (IL-15) cytokine support, with and without wild-type CXCR4. iPSCs were then differentiated into natural killer cells using a proprietary differentiation process. Cytolysis of a panel of target cells by iNK was evaluated using an Incucyte-based killing assay. Anti-tumor efficacy was tested in disseminated xenograft models of hematological malignancies following intravenous injection of engineered iNK cells. Additionally, in vivo migration was evaluated using immunohistochemistry (IHC) and flow cytometry (FACS) of blood, bone marrow, and other organs. Engineering with CXCR4 resulted in a bulk population of iNK with a heterogenous expression of CXCR4. No significant impact of CXCR4 engineering was detected on iPSC differentiation, phenotype, or iNK yield. iNKCXCR4+ and iNKCXCR4− cells had equivalent target tumor cell cytolysis in vitro, indicating the CXCR4 transgene does not affect killing potential. In vivo, iNKCXCR4+ cells migrated to the bone marrow of both tumor-bearing and non-tumor-bearing animals, and the presence of these cells was enhanced in tumor-bearing mice, indicating CXCR4-mediated migration and possibly CAR-mediated expansion and/or retention of iNK. Migration to the bone marrow was not observed with iNKCXCR4− cells. As a result, iNKCXCR4+ cells significantly impacted anti-tumor efficacy, effectively eliminating bone disease. We have demonstrated the CAR-specific killing and enhanced iNK migration and subsequent tumor killing in the bone marrow, with the addition of a CXCR4 transgene in iPSC-derived NK cells. The addition of the CXCR4 transgene in iPSC-derived effector cells may allow for a higher therapeutic response in axial-skeletal located tumors.
Citation Format: Hillary J. Millar, David Walker, Liam R. Campion, Ciara Budd, Aarti Kolluri, Harsh Sharma, Buddha Gurung, Jill M. Carton, Daniel J. Perry, Nicholas Alexander, Hyam Levitsky, Michael Naso, Barry A. Morse. CXCR4 transgene improves in vivo migration and efficacy of engineered iPSC-derived natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6802.