Chimeric Antigen Receptors (CARs) are used to direct and activate cytotoxic lymphocytes against virally infected or malignant cells. They have been heavily investigated in cytotoxic T cells, and, to a much lesser extent, in natural killer (NK) cells with promising results, especially for anti-CD19 CARs to mediate killing of B cell leukemias. To date, CARs that have been utilized in NK cells have primarily employed the same intracellular signaling components as T cell CARs. However, this may not be the optimal approach for CAR-expressing NK cells due to differences in key signaling pathways. To improve the activity of NK cell-CARs targeted against solid tumors, the intracellular signaling domains of an anti-mesothelin CAR were modified to express NK cell specific signaling molecules and evaluated for activity in NK-92 cells against ovarian cancer cell lines. CARs were assembled using gBlocks encoding various transmembrane, co-activating, and signaling domains. NK cell specific CARs were then transfected into NK-92 cells, a human NK cell tumor line, using Sleeping Beauty. Positive cells were selected for using a drug resistance marker and CAR surface expression was evaluated using flow cytometry. CAR expressing NK-92 cells were then evaluated for degranulation, as indicated by CD107a production, and interferon (INF)-γ release when challenged with mesothelin negative, MA148, and mesothelin positive, A1847, ovarian cell lines. In addition, beads conjugated to mesothelin were used to measure NK cell activation via the CAR alone.
In total 10 novel CARs were assembled and transfected into NK-92 cells, 6 of which were properly expressed on the cell surface. All 6 properly expressed CARs enhanced both CD107a and INF-γ production. When CAR activity alone was evaluated using mesothelin conjugated protein A beads, non-transfected NK-92 cells or cells that did not properly express the CAR all had <3% positive cells for CD107a and INF-γ. In contrast, NK-92 cells expressing CARs had between 8 and 16% positive cells for CD107a, with 5-12% positive for INF-γ. Since CAR4, expressing an NKG2D transmembrane, 2B4 co-activation, and CD3ζ signaling domain had the greatest activity, individual domains were mutated to assess their function. Mutation of the key binding residue in NKG2D for DAP10 resulted in a drastic decrease in the percentage of INF-γ producing cells, while mutation of the tyrosine residues in the CD3ζ signaling domain greatly abrogated CD107a production. As expected, mutation of all domains resulted in an inactive CAR. These studies demonstrate that CARs containing NK cell receptor domains function properly and may lead to better NK-92 cell activation. In addition, mutating CAR domains allows for dissecting the different pathways leading to either degranulation or cytokine release in NK cells. To better assess CAR activation potential in non-transformed NK cells, we are currently expressing the most promising CAR constructs into induced pluripotent stem cells (iPSCs) followed by differentiation into mature NK cells that will be tested for activity against ovarian cancer cells both in vitro and in vivo. These iPSC-NK cells will have the potential to create an “off-the-shelf” immunotherapy with the capability of targeting multiple malignancies.
Citation Format: David L. Hermanson, Branden Moriarity, Trevor Argall, Melissa A. Geller, David Largaespada, Dan S. Kaufman. Design and evaluation of novel natural killer cell chimeric antigen receptors. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A175.
- ©2016 American Association for Cancer Research.