Two-Pronged Cell Therapy for B-Cell Malignancies: Engineering NK Cells to Target CD22 and Redirect Bystander T Cells to CD19

Background: T-cell therapy with CD19-CAR T cells has been very successful for the treatment of B-cell derived malignancies in humans. However, cytokine release syndrome, neurotoxicity, and development of CD19- escape variants have emerged as potential limitations. Developing CAR NK-cell based therapies might overcome some of these side effects since NK cells do not rapidly expand or persist long-term after adoptive transfer. However, CAR NK-cell therapies are less effective than CAR T-cell therapies in preclinical models. To overcome these limitations we have devised a strategy to genetically modify NK cells with CD22-CARs and CD19/CD3-bispecific T-cell engager (CD19-ENG) molecules. These NK cells should not only kill CD22+ B cells directly, but also redirect bystander T cells to malignant CD19+ B cells, enhancing antitumor effects and preventing immune escape.

Methods: NK cells were generated using K562s expressing 41BBL and membrane bound IL15, and genetically modified with a retroviral vector encoding a CD22-CAR with a 41BB.ζ endodomain and/or a retroviral vector encoding CD19-ENG and mOrange separated by an IRES. To mimic immune escape, CD19 or CD22 knockout (ko) Ph+ leukemia cells (BV173) were generated by CRISPR/cas9. The effector function of genetically modified NK cells was evaluated using standard immunological assays.

Results: After transduction 70-80% of NK cells expressed CD22-CARs, and ~50% expressed CD22-CARs and CD19-ENGs as judged by FACS analysis. We performed coculture and cytotoxicity assays using non-transduced (NT), CD22-CAR, CD19-ENG, and CD22-CAR/CD19-ENG NK cells as effectors and BV173 (CD19+/CD22+), BV173.koCD19, BV173.koCD22, Daudi (CD19+/CD22+), and KG1a (CD19-,CD22-) as targets. Cocultures were preformed +/- T cells and after 24 hours IFNγ and IL2 was determined by ELISA. In the absence of T cells, CD22-CAR and CD22-CAR/CD19-ENG NK cells only recognized CD22+ targets as judged by IFNγ production. Moreover, CD22-CAR/CD19-ENG and CD19-ENG NK cells efficiently redirected T cells to secrete IFNγ in the presence of CD19+/CD22- targets. No NK-cell population produced IL2, however CD22-CAR/CD19-ENG and CD19-ENG NK cells induced IL2 production of T cells in the presence of CD19+ targets. No significant cytokine production was observed in the absence of antigen (media, KG1a). Specificity of generated NK cells was confirmed in cytotoxicity assays. In vivo studies to confirm our in vitro findings are in progress.

Conclusions: We have generated for the first time NK cells that kill B-cell malignancies through a CAR (CD22) and simultaneously redirect bystander T cells to a 2^nd B-cell antigen (CD19) to enhance antitumor effects and prevent immune escape. Genetic modification of NK cells to enhance their antitumor activity and redirect bystander T cells may present a promising addition to current cell therapies for B-cell malignancies.