Epigenetic reprogramming of allogeneic NK cells via antigen-specific “training” to generate target-specific memory-like NK cells for B-cell lymphoma without genetic engineering.

2546 Background: Clinical application of CAR-NK cells is restricted by manufacturing complexity, cost, and potential genotoxicity. Furthermore, synthetic CAR expression may disrupt native NK biology and impose steric hindrance. We hypothesized that NK cells can be epigenetically reprogrammed ex vivo to acquire antigen-specific, memory-like cytotoxicity via a novel, non-genetic “Targeted Priming Platform” (TPP), bypassing the fundamental constraints of CAR engineering. Methods: Healthy donor NK cells were co-cultured with CD19+ Raji cells in the presence of a CD16xCD19 bispecific engager (BiKE) and IL-12/15/18 cytokines. After a 7-day priming phase and subsequent rest, cells were re-challenged to assess recall responses. Deep profiling was performed using mass cytometry, ATAC-seq, and ChIP-seq. In vivo efficacy was evaluated in NSG mice engrafted with CD19+ Raji lymphoma. Comparisons were made against cytokine-only primed (Cyt-NK) and naïve NK cells. Results: TPP-NK cells demonstrated a stable, antigen-specific recall response. Upon secondary challenge with CD19+ targets, TPP-NKs exhibited superior expansion, degranulation (CD107a), and IFN-γ production compared with Cyt-NK or naïve controls. This specificity was absent in mismatched target settings. Epigenetic analysis revealed that TPP induced durable chromatin remodeling, characterized by stable hypomethylation and open chromatin at critical cytotoxicity loci (e.g., IFNG, PRF1, GZMB), distinct from the transient changes observed in Cyt-NKs. In vivo, a single infusion of TPP-NKs combined with low-dose BiKE induced sustained remission and significantly prolonged survival compared with controls (P < 0.001). Conclusions: We demonstrate a paradigm-shifting strategy to generate “target-trained” memory NK cells without genetic engineering. TPP epigenetically imprints antigen specificity, combining the precision of antibody therapy with the persistence of cellular therapy. This scalable, “off-the-shelf” platform offers a safer, cost-effective alternative to CAR-based modalities for B-cell malignancies.