GMP-compliant, automated process for generation of CAR NK cells in a closed system for clinical use

Abstract

Background & Aim Major advances have been made in harnessing natural killer (NK) cells in cancer immunotherapy in recent years. Regulated by their germ-line encoded activating and inhibitory receptors, NK cells can recognize and eliminate tumor cells rapidly without prior sensitization. Clinical evidence has shown that donor-derived NK cells have low risk in inducing graft-versus-host-disease (GvHD) as well as a reduced risk of life-threatening cytokine storms during therapy. Both properties make NK cells ideal for allogeneic application. To enhance the anti-tumor specificity and efficiency, NK cells can be further modified by chimeric antigen receptors (CARs). Promising clinical outcome of CAR-engineered NK cells has been recently reported for patients with relapsed or refractory B cell malignancies. Methods, Results & Conclusion In this study purified NK cells were cultivated with a feeder cell-free protocol including cytokines in NK MACS GMP Medium. For generation of CAR NK cells we used a Baboon endogenous virus (BaEV)-pseudotyped lentiviral vector system to efficiently transduce NK cells with various CARs and routinely obtained transduction efficiencies between 30-70%. To facilitate the clinical application of CAR NK cells, we implemented this protocol into a highly efficient, automated process to generate CAR NK cells under good manufacturing practice (GMP)-compliant conditions in a closed system by using CliniMACS Prodigy device. The process covers the complete procedure of NK cell manufacturing, including separation, activation, gene modification, and expansion/cultivation. High purity of NK cells (mean 94%) could be achieved by CD3 depletion followed by subsequent CD56 enrichment, resulting in a mean log depletion of T cells of 4.3. Inhouse evaluation runs with leukapheresis products (n=11) achieved a mean transduction efficiency of 52.0% CAR+ cells, resulting in 5.2 – 17.9 x 108 (mean 7.17 x 108) CAR+ NK cells after 14 days of culture. In summary, we developed a novel process for automated NK cell purification, transduction, and cultivation in a closed GMP-compatible system. The high level of automation enables standardized, consistent, and operator independent genetic engineering of NK cells for therapeutic applications.