A clinically adaptable method to enhance the cytotoxicity of natural killer cells against B-cell malignancies

Abstract

Background aims. Retroviral transduction of anti-CD19 chimeric antigen receptors significantly enhances the cytotoxicity of natural killer (NK) cells against B-cell malignancies. We aimed to validate a more practical, affordable and safe method for this purpose. Methods. We tested the expression of a receptor containing CD3ζ and 4-1BB signaling molecules (anti-CD19-BB-ζ) in human NK cells after electroporation with the corresponding mRNA using a clinical-grade electroporator. The cytotoxic capacity of the transfected NK cells was tested in vitro and in a mouse model of leukemia. Results. Median anti-CD19-BB-ζ expression 24 h after electroporation was 40.3% in freshly purified (n =18) and 61.3% in expanded (n = 31) NK cells; median cell viability was 90%. NK cells expressing anti-CD19-BB-ζ secreted interferon (IFN)-γ in response to CD19-positive target cells and had increased cytotoxicity. Receptor expression was detectable 6 h after electroporation, reaching maximum levels at 24–48 h; specific anti-CD19 cytotoxicity was observed at 96 h. Levels of expression and cytotoxicities were comparable with those achieved by retroviral transduction. A large-scale protocol was developed and applied to expanded NK cells (median NK cell number 2.5 × 10⁸, n = 12). Median receptor expression after 24 h was 82.0%; NK cells transfected under these conditions exerted considerable cytotoxicity in xenograft models of B-cell leukemia. Conclusions. The method described here represents a practical way to augment the cytotoxicity of NK cells against B-cell malignancies. It has the potential to be extended to other targets beyond CD19 and should facilitate the clinical use of redirected NK cells for cancer therapy.

Key Words::

• acute lymphoblastic leukemia
• cell therapy
• natural killer cells
• non-Hodgkin lymphoma

Acknowledgments

We thank Drs Linhong Li and Madhusudan V. Peshwa (MaxCyte Inc.) for guidance and technical support with the electroporation procedure. This work was supported by grants CA113482, CA70089 and CA21765 from the National Cancer Institute, a grant from the Fondation des Gouverneurs de l'espoir, a Singapore Translational Research Award from the National Medical Research Council of Singapore, and by the American Lebanese Syrian Associated Charities (ALSAC).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.