iPSC-Derived CAR Neutrophils Possess Potent Activity Against Solid Tumors In Vivo

While chimeric antigen receptor (CAR)-T cells demonstrate high efficacy in eliminating liquid cancers (lymphoma, leukemia, multiple myeloma), and promise exists for CAR NK cells for lymphoma, many challenges remain in applying CAR lymphoid therapies for solid tumors including ineffective trafficking to the tumor microenvironment. Neutrophils provide a logical alternative to cellular immunotherapy since they can migrate to, and into, solid tumors better than any other cell type. However, neutrophils can have pro- or anti-tumor properties. Thus, enforcing anti-tumor properties through genetic engineering would be essential before successfully translating adoptive neutrophil immunotherapies into the clinic. Here, we established induced pluripotent stem cell (iPSC) technologies to generate anti-GD2 CAR neutrophils (iNeuts) using ETV2 modified mRNA and tested their anti-tumor potential. The disialganglioside (GD2) antigen is highly expressed in a variety of pediatric and adult tumors, including melanoma, and neuroblastoma, while its expression in normal post-natal tissues is low and limited to peripheral nerve pain fibers. The 3^rd generation anti-GD2 CAR (14g2A-CD28-OX40-CD3z) construct was integrated into AAVS1 locus using CRISPR/Cas9. iNeuts generated from CAR iPSCs displayed typical neutrophil morphology and phenotype similar to unmodified wild-type (WT) WT-iNeuts, and retained CAR expression. Measurement of cytokine production by iNeuts in cultures without or with melanoma revealed that CAR-iNeuts produce higher levels of IFNg, TNFa, IL13, IL4, and IL10 after stimulation with tumor as compared with WT-iNeuts, while no differences were observed in IL12 and IL6 levels. GD2 CAR-iNeuts displayed cytotoxic activity against GD2-expressing neuroblastoma and melanoma in vitro,while cytotoxicity of GD2-negative cancers was minimal. Serial infusions of GD2 CAR-iNeuts exhibited strong antitumor activity against subcutaneous and metastatic melanoma in vivoleading to marked reduction in tumor burden and increased survival. Administration of iNeuts was safe and didn't produce toxicity as determined by clinical evaluation or histopathologic analysis. Overall, our data support the feasibility of clinical translation of GD2 CAR-iNeuts for solid tumors including melanoma.

Jung:CRISPR Therapeutics: Current Employment. Zhang:Universal Cells: Current Employment. Huttenlocher:Genentech: Research Funding. Capitini:WiCell: Honoraria; Novartis: Honoraria; Elephas Bio: Honoraria; Nektar Therapeutics: Honoraria; Bayer: Honoraria. Slukvin:Umoja Biopharma: Consultancy, Current equity holder in private company; Cynata Therapeutics: Consultancy, Current equity holder in publicly-traded company.