Abstract B64: Coexpression of a CD19-OX40-CD3ζ CAR with membrane-bound IL-15 enhances natural killer cell function
Authors: Buren Luxuan G. , Guo Chao , Fan Yanying , Aronov Alexander , Wu Xiumin , Liu Daofeng , Xie Ming-Hong , Lazetic Sasha , Chan Ivan H. , Trager James B. 
Published: March 2020

Abstract
Introduction: Chimeric antigen receptors (CARs) have been used successfully to retarget T cells in patients with hematologic malignancies. Natural killer (NK) cells offer an alternative to T cells for cellular immunotherapy and are suitable for allogeneic use as they are not HLA-restricted and patients who receive NK cell treatment do not develop graft-versus-host disease (GVHD). Therefore, NK cells can provide an attractive alternative for “off-the-shelf” cellular therapy. Here, we investigated multiple approaches to modify and enhance CD19 CAR expression on NK cells. It has been previously reported that costimulatory domains play an important role in proliferation, efficacy, and persistence of CAR-T cells both in vitro and in vivo. To understand how CAR structure effects the functional behavior of NK cells, we assessed the capability of various costimulatory signaling domains, including CD28, OX40, CD28-41BB, etc., all coupled to CD3ζ, to enhance CD19 CAR signaling and cytotoxicity in NK cells. We demonstrated that NK activity and persistence can be elevated by simultaneous expression of chimeric constructs directing the expression of a CD19 CAR and a membrane-bound form of IL-15 (mbIL-15).
Methods: NK cells were generated by coculture of peripheral blood mononuclear cells (PBMC) with genetically modified irradiated K562 feeder cells. NK cells were transduced at a MOI of 1-2 with a gamma-retrovirus encoding a CD19 CAR and mbIL-15. NK expansion and CAR expression were evaluated by flow cytometry. In vitro cytotoxicity of transduced NK cells was measured using both flow cytometry and the IncuCyte S3 live cell analysis system. The in vivo activity of engineered NK cells was further assessed in a xenograft tumor model in NSG mice, using a Nalm6 leukemia cell line.
Results: CD19 CAR constructs containing various costimulatory domains were all expressed in expanded and transduced NK cells. CAR expression in multiple donor NK cells was typically between 60-90%. In cytotoxicity assays, CD19 CAR constructs containing the costimulatory domains of OX40, CD28, or CD27 generally exhibited the greatest cytotoxic activity against Nalm6 and Raji tumor cell lines in vitro. High expression of these three CD19 CAR constructs was also maintained over the course of 4 weeks in addition to sustained NK proliferation and cell numbers, indicating an increase in NK cell persistence. In comparing these constructs in an in vivo Nalm6 tumor model, all three constructs demonstrated greater antitumor efficacy relative to unmodified NK cells. The CD19 CAR construct containing the OX40 costimulatory domain showed moderately enhanced efficacy than the CD28 or CD27 variants in vivo.
Conclusion: Transduction of NK cells with CD19-OX40-CD3ζ CAR and mbIL15 increases their cytotoxic activity and persistence. Based on these data, further development of NK CD19 CAR for clinical use will be pursued.
Citation Format: Luxuan G. Buren, Chao Guo, Yanying Fan, Alexander Aronov, Xiumin Wu, Daofeng Liu, Ming-Hong Xie, Sasha Lazetic, Ivan H. Chan, James B. Trager. Coexpression of a CD19-OX40-CD3ζ CAR with membrane-bound IL-15 enhances natural killer cell function [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B64.