Comprehensive Exploration of NK Cell Education in ADCC Triggered By a Monoclonal Antibody or a Bispecific Killer Engager Molecule (BiKE)

The responsiveness of NK cells to malignant cells that lack self-HLA class I (HLA-I) expression is controlled by NK cell education governed by the interaction between selected inhibitory NK cell receptors and their cognate HLA-I ligands. However, it remains unclear to what extent education impacts the ability of NK cells to perform antibody-dependent cellular cytotoxicity (ADCC) as previous studies show inconsistent results and are limited in resolution. Moreover, no study has yet addressed if NK cell education impacts ADCC triggered by novel CD16-binding therapeutics such as bispecific killer engagers (BiKEs). In this study we systematically characterized NK cell degranulation against a panel of tumor cell lines with defined HLA-I expression in the presence of either the monoclonal antibody rituximab or a recently developed CD34xCD16 BiKE. Using high-resolution flow cytometry, our data revealed that NK cells educated by one or two KIRs did not have significantly enhanced ADCC capacity over uneducated KIR⁺ or KIR^- NK cells, especially when increasing the antibody concentration. Contrasting co-education by KIRs, NKG2A was consistently associated with improved responsiveness and superior ADCC. Surprisingly, these results were despite that KIR-education correlated with higher CD16 expression that was markedly lower on NKG2A⁺ NK cells compared to both KIR-educated and uneducated subsets. An extended analysis uncovered that the inhibitory KIR2DL1-C245 receptor, reported to mediate weak NK cell education compared to the KIR2DL1-R245 variant, further repressed the ADCC capacity of NKG2A⁺ NK cells from donors expressing its ligand HLA C2. This highlights that also inhibitory KIRs can dampen the ADCC potential of NK cells similar to that of activating KIRs which also were discovered to restrict ADCC even when stimulated by its ligand. Finally, our study unveiled that BiKE-triggered ADCC has a similar relationship to NK cell education and its hierarchies as that triggered by a monoclonal antibody. In conclusion, the ADCC capacity of an NK cell is more regulated by restrictive mechanisms of education conferred by activating or selected inhibitory KIRs in individuals carrying the cognate KIR ligand than that of inhibitory KIRs conferring higher education potential. Hence, this study establishes that NK cell education does not enhance the ADCC capacity of an NK cell per se while providing new insights that also inhibitory KIRs can dampen the ADCC potential even when the target cell lacks its ligand. Our results add to the fundamental understanding of NK cell biology as well as to avenues of therapeutic applications for NK cells.

Uhlin:Michael Uhlin: Patents & Royalties: For the use of the BiKE molecule used in the study.