Transfection with mRNA for CD19 Specific Chimeric Antigen Receptor Restores Natural Killer Cell Mediated Killing of CLL Cells.

Abstract

B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world and is characterized by monoclonal proliferation of functionally deficient lymphocytes, with relative decrease in T and Natural Killer (NK) cells. Available monoclonal antibody (mAb) therapies elicit a response in almost half of the patients, but durable remissions are rare. B-CLL cells are virtually impervious to T and NK cell-mediated cytotoxicity. However, a feasible approach to overcome this resistance is to redirect the specificity of T or NK cells by making them express chimeric antigen receptors (CAR). Such a method has been successfully tested in NK cells against a number of CD19-expressing cells; however the loading of cells with CD19-CAR relied on retrovirus-based transfection methods. We describe here a non-retroviral method for obtaining NK cells expressing high levels of CD19-CAR, based on electroporation of messenger RNA (mRNA). We used a CAR that is comprised of the single chain Fv portion of a mouse mAb against CD19 fused to the signaling intracellular domain of CD3ζ subunit. The corresponding cDNA was subcloned by PCR into a plasmid allowing T7-dependant mRNA synthesis. In vitro synthesis of the CD19-CAR mRNA was followed by a poly-adenylated tail elongation step in order to improve in vivo stability and translation of the messenger. Electroporation of NK-92 cells with this construct, using a Biorad GenePulser II apparatus, yields up to 78% expression efficiency and as high as 90% cell survival. Cytotoxicity of transfected NK-92 cells was tested against 3 target cell lines (NK sensitive, CD19 negative K562; CD19 expressing, NK resistant REH and SUPB15) as well as primary CLL cells from 6 untreated patients. A non-radioactive fluorochrome-based flow cytometric assay was used to determine NK-mediated cytotoxicity, at effector to target (E:T) ratios ranging from 1:1 to 10:1. Expression of the CD19-CAR on the surface of NK-92 cells do not change their cytolytic properties against K562: 70% ±5% killing compared to 74% ±2% killing for non-transfected NK-92, (E:T ratio of 10:1). Expression of CD19-CAR in NK-92 cells completely overcomes the resistance of REH and SUPB15 cell lines: 76% ±7% and 73% ±5% killing respectively, compared to 21% ±10% and 18% ±7% killing respectively with non-transfected NK-92 (E:T ratio of 10:1). Moreover, previously resistant primary CLL cells became highly sensitive to lysis by NK-92 expressing CD19-CAR: 46–82% killing, as opposed to 7–22% killing for non-transfected NK-92 (E:T ratio of 10:1). These results show that NK cells can be engineered using mRNA transfection by electroporation to target and effectively kill resistant primary CLL cells.