High Cytotoxic Efficiency of Lentivirally and Alpharetrovirally Engineered CD19-Specific Chimeric Antigen Receptor Natural Killer Cells Against Acute Lymphoblastic Leukemia

Abstract

Autologous chimeric antigen receptor-modified (CAR) T cells with specificity for CD19 showed potent antitumor efficacy in clinical trials against relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL). Contrary to T cells, natural killer (NK) cells kill their targets in a non-antigen-specific manner and do not carry the risk of inducing graft vs. host disease (GvHD), allowing application of donor-derived cells in an allogenic setting. Hence, unlike autologous CART cells, therapeutic CD19-CAR-NK cells can be generated as an off-the-shelf product from healthy donors. Nevertheless, genetic engineering of peripheral blood (PB) derived NK cells remains challenging and optimized protocols are needed. In our study, we aimed to optimize the generation of CD19-CAR-NK cells by retroviral transduction to improve the high antileukemic capacity of NK cells. We compared two different retroviral vector platforms, the lentiviral and alpharetroviral, both in combination with two different transduction enhancers (Retronectin and Vectofusin-1). We further explored different NK cell isolation techniques (NK cell enrichment and CD3/CD19 depletion) to identify the most efficacious methods for genetic engineering of NK cells. Our results demonstrated that transduction of NK cells with RD114-TR pseudotyped retroviral vectors, in combination with Vectofusin-1 was the most efficient method to generate CD19-CAR-NK cells. Retronectin was potent in enhancing lentiviral/VSV-G gene delivery to NK cells but not alpharetroviral/RD114-TR. Furthermore, the Vectofusin-based transduction of NK cells with CD19-CARs Müller et al.

Highly Efficient Generation of Transgenically Augmented CAR NK Cells Overexpressing CXCR4

“…Similar high gene transfer rates to primary NK cells were reached using Vecotfusin-1 as an enhancer, which might even be increased when using a different viral envelope. For Vectofusin-1, it was recently demonstrated that transgene delivery to primary NK cells can be improved by pseudotyping LVs with the envelope protein from endogenous feline virus (RD114) or baboon endogenous retrovirus (BaEV) instead of VSV-G ( 5 , 6 ). In the field of CAR T-cells gamma-retroviral vectors or VSV-G pseudotyped LVs are most commonly used for their generation.…”

“…Transduction efficiencies of NK cells vary tremendously depending on the cell source, the viral vector system, and the transduction enhancer used ( 4 ). Lentiviral vectors (LVs) pseudotyped with the glycoprotein of the vesicular stomatitis virus (VSV-G) are classically used to generate CAR T-cells but are less efficient for NK cells with transduction efficiencies of 20–40% ( 5 , 6 ). Therefore, optimization of gene transfer protocols for VSV-G pseudotyped LV for the generation of CAR NK cells is urgently required.…”

Immunotherapy with NK cells: recent developments in gene modification open up new avenues

“…73 The use of poly-L-lysine and DEAE-dextran has only been reported in a few NK cells transduction studies. 91 We and others showed enhanced viral transduction efficiency for CAR-NK cells with not only Retronectin, 88,92 but also Vectofusion-1, which has been reported as a promising alternative transduction enhancer. 93 Our data showed that Vectofusin-1 was as effective as Retronectin using VSV-G-pseudotyped LV, but demonstrated higher transduction efficiency for the RD114-TR-pseudotyped α-RV.…”

“…86,87 Recently, we and others demonstrated the dependency of the resulting transduction efficiency on the pseudotyping as RD114-TR-or Baboonpseudotyped lentiviruses transduced NK cells via the surface entry receptor ASCT2 that is upregulated following cytokine stimulation. 88,89 In addition, different transduction enhancers have been tested to increase the viral transduction efficiency of NK cells (e.g., polybrene, Retronectin, protamine sulfate, poly-L-lysine, DEAE-dextran, Vectofusin-1). The cationic polymers protamine sulfate and polybrene reduce the viral repulsion of NK cells, thereby enhancing the membrane fusion.…”

Humanized Mice Are Precious Tools for Preclinical Evaluation of CAR T and CAR NK Cell Therapies

“…BaEV-LVs were shown to generate functional CAR expressing NKs. Another study also showed high-level CAR delivery into NK cells employing an alpha-retroviral vector system [ 54 ]. These results will pave the way to move CAR NK cell therapy into the clinic.…”