GMP-compliant manufacturing of allogeneic peripheral blood CAR-NK cells for the treatment of acute myeloid leukemia.

Abstract

Chimeric Antigen Receptor (CAR)-T cell therapy has shown promising results in hematological malignancies but faces limitations such as the need for autologous products or additional modifications of allogeneic T cell sources to prevent graft-versus-host disease (GvHD). In acute myeloid leukemia (AML), its application is particularly challenging due to antigen overlap between malignant cells and healthy hematopoietic stem cells, leading to severe and often fatal toxicities due to on-target, off-tumor effect. Natural killer (NK) cells offer a promising alternative for CAR therapy, as they are well tolerated and exert antitumor activity beyond CAR-mediated mechanisms. Peripheral blood (PB) is an accessible NK cell source with a relatively high proportion of mature NK cells; however, generating clinically relevant numbers of highly transduced PB-derived CAR-NK cells in closed systems remains challenging, and production protocols need optimization. Here, we evaluated different manufacturing strategies to generate NKG2D CAR-NK cells from PB in the CliniMACS Prodigy closed platform. We established an optimized protocol that achieved enhanced transduction efficiency, yielding high numbers of CAR+ cells. The final product met good manufacturing practice (GMP)-aligned safety and sterility requirements, displayed superior in vitro cytotoxicity against AML cell lines compared to the same product manufactured in the preclinical setting, and delayed tumor progression in an AML xenografted mouse model without cytokine support. This standardized, GMP-compliant protocol provides a robust platform for producing PB-derived CAR-NK cell therapies in a closed platform, advancing their clinical translation.