P032 | DEVELOPMENT OF AUTOLOGOUS NATURAL KILLER CELL-BASED IMMUNOTHERAPYIN COMBINATION WITH MONOCLONAL ANTIBODIES FOR AGGRESSIVE B-CELL LYMPHOMAS

Aggressive B-cell lymphomas remain a major therapeutic challenge, due to frequent relapses and limited efficacy of current treatments, including chimeric antigen-receptor (CAR)-T cell therapies. Natural Killer (NK) cell-based immunotherapies may represent a promising and safe alternative to CAR-T cell therapies. However, optimizing NK cell expansion and functional enhancement remains a crucial step that directly impacts their clinical applicability. This project aimed to generate preclinical data to support the clinical translation of allogeneic and autologous NK cell therapies in aggressive B-cell lymphoproliferative disorders. We developed an adoptive cell therapy approach combining NK cell infusion with monoclonal antibodies and NK-cell engagers. To this end, we established a feeder-free, GMP-compatible protocol for the ex vivo expansion of peripheral blood-derived NK cells. The process includes CD3-CD56+ selection followed by 20-day culture in IL-2/IL-15-enriched medium yielding highly pure NK cells (95% CD56+) with robust expansion (500-800-fold). Expanded NK cells exhibit strong activation (80% CD16+, 95% NKG2D+, 93% NKp46+), enhanced cytokine secretion (IFN-γ, TNF-α), and potent cytotoxicity in vitro (>40% tumor cell lysis after 4h at 1:1 E:T ratio) confirmed by FACS-based killing assays and live-cell imaging. Notably, this protocol enables the generation of functionally equivalent NK cells from both healthy donors and immunocompromised patients (n=20), regardless of starting material. To further boost cytotoxicity, NK cells were combined with monoclonal antibodies or NK-cell engagers to enhance antibody-dependent cell-mediated Cytotoxicity (ADCC), and with immunomodulatory agents. In a patient-derived xenograft (PDX) model of diffuse large B-cell lymphoma (DLBCL), the addition of ex vivo expanded allogeneic NK cells to Rituximab/Lenalidomide combination significantly reduced tumor growth as compared to either treatment alone. Additional autologous PDX models are being generated to compare the persistence and in vivo efficacy of autologous versus allogeneic NK cells. Further studies with Tafasitamab and novel NK-cell engagers are ongoing, and the most effective strategies will be selected for clinical translation. These findings lay the groundwork for clinical application of next-generation NK cell therapies in combination with monoclonal antibodies or NK-cell engagers in refractory B-cell malignancies.