Intrinsic Functional Potential of NK-Cell Subsets Constrains Retargeting Driven by Chimeric Antigen Receptors

• Published in: Cancer immunology research 2018-04, Vol.6 (4), p.467-480
• Main Authors: Oei, Vincent Yi Sheng, Siernicka, Marta, Graczyk-Jarzynka, Agnieszka, Hoel, Hanna Julie, Yang, Weiwen, Palacios, Daniel, Almåsbak, Hilde, Bajor, Malgorzata, Clement, Dennis, Brandt, Ludwig, Önfelt, Björn, Goodridge, Jodie, Winiarska, Magdalena, Zagozdzon, Radoslaw, Olweus, Johanna, Kyte, Jon-Amund, Malmberg, Karl-Johan
• Format: Article
• Language: English
• Subjects: Animals; Antigens, Neoplasm - genetics; Antigens, Neoplasm - immunology; Cell Line, Tumor; Cytotoxicity, Immunologic; Electroporation; Gene Expression; HLA Antigens - genetics; HLA Antigens - immunology; Humans; Killer Cells, Natural - immunology; Killer Cells, Natural - metabolism; Lymphocyte Activation - immunology; Lymphocyte Subsets - immunology; Lymphocyte Subsets - metabolism; Medicin och hälsovetenskap; Mice; NK Cell Lectin-Like Receptor Subfamily C - antagonists & inhibitors; Receptors, Antigen, T-Cell - genetics; Receptors, Antigen, T-Cell - metabolism; Receptors, Chimeric Antigen - genetics; Receptors, Chimeric Antigen - metabolism; Receptors, KIR - antagonists & inhibitors
• ISSN: 2326-6066; 2326-6074; 2326-6074
• DOI: 10.1158/2326-6066.CIR-17-0207

Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19 targets, and maintained their intrinsic degranulation response toward CD19 K562 cells. The response of redirected NK-cell subsets against CD19 targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA-specific KIR(s), displayed superior ability to kill CD19 , HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells. .