Abstract 885: Generation and characterization of NK-92®(aNK™) cells with hypoimmunogenic modifications

Abstract The next generation of NK-92® (also known as aNKTM) cells will include inducible kill switches and enhanced cytotoxicity to make them safer and more effective, without the need to irradiate them before infusion into patients. Because of their enhanced longevity in vivo and their allogeneic nature, NK-92 cells are susceptible to rejection by the patient’s own immune system and need to be equipped with mechanisms to prevent such rejection. We have genetically engineered NK-92 cells by targeting a combination of molecules that allow them to be undetected by the patient’s T-cells as well as resistant to lysis by peripheral blood NK cells (PBNKs). HLA-I-deficient NK-92 cells generated by electroporation of Cas9-sgRNA ribonucleoprotein complexes to knockout the common β2 microglobulin subunit (NK-92_B2MKO) completely inhibited CD8+ T-cell proliferation after 7 days in a mixed lymphocyte reaction with peripheral blood mononuclear cells (PBMCs) isolated from multiple healthy donors. Because downregulation of HLA-I on the cell surface can increase lysis by PBNKs via the ‘missing-self’ mechanism, we further engineered NK-92_B2MKO cells to re-express the non-classical MHC class I molecule HLA-E (NK-92_B2MKO_HLAE) to deliver an inhibitory signal through engagement with the NKG2A/CD94 heterodimer. Using flow cytometry-based cytotoxicity assays, we confirmed that the lack of HLA-I molecules rendered NK-92_B2MKO cells highly susceptible to lysis by both resting and IL-2-activated PBNKs from healthy donors, relative to wild type NK-92. We also demonstrate that re-expression of HLA-E on NK-92_B2MKO cells provides significant protection against both resting and IL-2 activated PBNKs (42.5% ± 9.0% reduction in killing at 5:1 E:T ratio, p=0.0002). HLA-E can also convey a lysis-activating signal through the NKG2C/CD94 dimer. NKG2C+ NK cells are naturally present at low numbers in blood and can be expanded in individuals who are seropositive for human cytomegalovirus. To determine whether NK-92_B2MKO_HLAE cells are sensitive to killing by NKG2C+ NK cells, we performed co-culture assays using PBNKs and our engineered NK-92 cells and examined CD107a degranulation in different NK subsets. We observed that HLA-E expression completely inhibits degranulation in NKG2A+ NK cells, while it was not increased any further in NKG2C+ cells, suggesting that the re-expression of HLA-E did not make NK-92_B2MKO_HLAE cells more susceptible to NKG2C+ PBNK lysis. We report here that abolishment of HLA class-I and re-expression of HLA-E on NK-92 cells effectively inhibited CD8+ T-cell recognition as well as provided significant protection against resting and IL-2 activated PBNKs. Our results also suggest that these modifications are likely sufficient to allow the engineered NK-92 cells to evade immune alloreactivity in a majority of patients after infusion. Citation Format: Ivan Ting Hin Fung, Laurent Boissel, Patrick Soon-Shiong, Hans Klingemann. Generation and characterization of NK-92®(aNK™) cells with hypoimmunogenic modifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 885.