Background
Fc&x025B;R1-deficient Natural Killer (g-NK) cells are a naturally occurring subset of NK cells that occur in a portion of individuals latently infected with cytomegalovirus. The lack of Fc&x025B;R1-adapter protein results in exclusive signaling through CD3 and consequently, significantly enhanced CD16-mediated antibody-dependent cellular cytotoxicity (ADCC) compared to conventional NK (cNK) cells in bulk coculture assays with tumor cells. However, the resulting cellular mechanisms underlying this enhanced ADCC activity of g-NK cells are not fully understood.
Methods
Artificial intelligence-powered Time-lapse Imaging Microscopy In Nanowell Grids (TIMING™) was applied to compare cNK and g-NK cell interaction dynamics and cytotoxicity kinetics at a single-cell level with the human multiple myeloma cell line, LP-1, with or without daratumumab, an anti-CD38 monoclonal antibody (mAb). Nanowell grids were seeded with target and NK cells at varying E:T ratios. Cell identity, migration, interaction dynamics, and cytotoxicity were quantified and statistically tested for the effect of mAb on cNK or g-NK cells.
Results
Consistent with results of bulk ADCC assays, g-NK cells exhibited significantly increased LP-1 cell killing (1.8X) compared to cNK at a single-cell resolution. Increased ADCC activity of g-NK cells was primarily driven by increased motility and synapse formation with target cells. Individual g-NK cells demonstrated a ~1.5X faster rate of single-cell motility and were ~1.7X more likely to form a synapse with a target cell compared to cNK (1:1 E:T). Once a NK/target cell synapse had formed, single-cell cytotoxicity was similar between cNK and g-NK in the absence of mAb; however, consistent with the Fc&x025B;R1-deficient phenotype, mAb drove higher cytotoxicity with g-NK cells (1:1 E:T) and increased serial killing (at 1:3 E:T). Interestingly, addition of mAb did not increase synapse formation suggesting that mAb does not contribute significantly to this process.
Conclusions
In addition to stronger signal transduction through CD3 after CD16 engagement, enhanced ADCC of g-NK cells is due to increased motility and synapse formation with target cells which occurs independent of CD16/mAb/target interaction. The resulting greater frequency of synapse formation leads to overall greater single-target and serial killing by g-NK cells compared to cNK. Higher ADDC potency through increased g-NK cell motility is consistent with findings that motility correlates with higher potency in CAR-T cell products. These results provide new mechanistic insights into how g-NK cells improve therapeutic efficacy of mAbs for the treatment of cancer or autoimmune disease. IDP-023 is currently in clinical trials for the treatment of advanced hematologic cancers (NCT06119685).
Acknowledgements
Research reported in this abstract was supported, in part, by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number R44GM149106.
Ethics Approval
All human materials utilized in these studies were obtained through commercial sources which adhere to all relevant ethical and legal requirements.