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Interaction kinetics with transcriptomic and secretory responses of CD19-CAR natural killer-cell therapy in CD20 resistant non-hodgkin lymphoma


We investigated the cytolytic and mechanistic activity of anti-CD19 chimeric antigen receptor natural killer (CD19.CAR.NK92) therapy in lymphoma cell lines (diffuse large B-cell, follicular, and Burkitt lymphoma), including rituximab- and obinutuzumab-resistant cells, patient-derived cells, and a human xenograft model. CD19.CAR.NK92 therapy significantly increased cytolytic activity at E:T ratios (1:1–10:1) via LDH release and prominent induction of apoptosis in all cell lines, including in anti-CD20 resistant lymphoma cells. The kinetics of CD19.CAR.NK92 cell death measured via droplet-based single cell microfluidics analysis showed that most lymphoma cells were killed by single contact, with anti-CD20 resistant cell lines requiring significantly longer contact duration with NK cells. In addition, systems biology transcriptomic analyses of flow-sorted lymphoma cells co-cultured with CD19.CAR.NK92 revealed conserved activation of IFNγ signaling, execution of apoptosis, ligand binding, and immunoregulatory and chemokine signaling pathways. Furthermore, a 92-plex cytokine panel analysis showed increased secretion of granzymes, increased secretion of FASL, CCL3, and IL10 in anti-CD20 resistant SUDHL4 cells with induction of genes relevant to mTOR and G2/M checkpoint activation, which were noted in all anti-CD20 resistant cells co-cultured with CD19.CAR.NK92 cells. Collectively, CD19.CAR.NK92 was associated with potent anti-lymphoma activity across a host of sensitive and resistant lymphoma cells that involved distinct immuno-biologic mechanisms of cell death.

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AME, TK, DR supported by TUFTS NIH CTSI pilot funding UL1TR002544, NIH grants 1R33CA223908-01 and R01 GM127714-01A1.

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DR and AME, designed and conducted in vitro research, analyzed results and wrote the paper. SS, TK designed and conducted microfluidics experiment, analyzed results and wrote the manuscript. SS, FP and MM performed experiments. KD analyzed data reviewed literature. AB and YC performed microarray and bioinformatic data analysis.

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Correspondence to Andrew M. Evens.

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Conflict of interest

Funding in part of this study for provided by Nantkwest to AME, other roles for AME advisory board (with honorarium): Bayer, Seattle Genetics, Affimed, Verastem, Pharmacyclics, Research to Practice, and Physician Education Resource; Research support: Takeda, Seattle Genetics, Merck, NIH/NCI, Leukemia and Lymphoma Society, and ORIEN. YC founder and consultant with financial interests in Oncomics LLC.

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Ravi, D., Sarkar, S., Purvey, S. et al. Interaction kinetics with transcriptomic and secretory responses of CD19-CAR natural killer-cell therapy in CD20 resistant non-hodgkin lymphoma. Leukemia 34, 1291–1304 (2020).

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