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. 2009 Sep;33(9):1255-9.
doi: 10.1016/j.leukres.2008.11.024. Epub 2009 Jan 14.

Transfection with mRNA for CD19 specific chimeric antigen receptor restores NK cell mediated killing of CLL cells

Laurent Boissel  1 , Monica Betancur, Winfried S Wels, Hande Tuncer, Hans Klingemann
Affiliations

Transfection with mRNA for CD19 specific chimeric antigen receptor restores NK cell mediated killing of CLL cells

Laurent Boissel et al. Leuk Res. 2009 Sep.

Abstract

An emerging treatment option for chronic lymphocytic leukemia (CLL) is to make cytotoxic immune cells express a chimeric antigen receptor (CAR) that recognizes specific surface molecules on CLL cells. Here an mRNA coding for an anti-CD19 CAR was transfected into the NK-92 cell line by electroporation. In contrast to cDNA, mRNA resulted in high transfection efficiency (47.2 +/- 8% versus <5% for cDNA) with minimal effect on cell viability. NK-92 cells expressing anti-CD19 CAR killed previously resistant CD19+ B-ALL cell lines, as well as primary CLL cells and therefore may present a safe, cell-based, targeted treatment for patients with CLL.

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Conflict of interest statement

Conflict of interest statement

H. Klingemann is co-founder and shareholder in ZelleRx Corp.

Figures

Fig. 1
Fig. 1
Schematic representation of the CD19-CAR mRNA. VH and VL: extracellular single strand antibody domains. 5′ and 3′ UTR are from human globin and enhance mRNA stability as well as provide a poly-adenylation sequence.
Fig. 2
Fig. 2
Viability of cells after electroporation. NK-92 cells were stained with PI 24 h after electroporation with GFP (■) or αCD19-CAR (formula image), either in the form of DNA or mRNA, or without any nucleic acid (□, control).
Fig. 3
Fig. 3
Expression and stability of the exogenous proteins. (A) Right: FACS plots of a representative experiment showing GFP expression (FITC), αCD19-CAR expression (APC), and PI staining (PE) following mRNA electroporation. Left: Expression levels of GFP (■) or αCD19-CAR (formula image) proteins at 24 h post-electroporation in NK-92 cells electroporated with DNA or mRNA, shown in percentage of the whole cell population. (B) Expression of GFP (●) or αCD19-CAR (formula image) proteins over time in hours in NK-92 cells electroporated with mRNA.
Fig. 4
Fig. 4
Cytolytic properties of electroporated NK-92 cells against CD19+ cell lines. Cytolytic efficiency, expressed in percentage of killed target cells, of NK-92 non-electroporated (formula image), and electroporated without nucleic acid (□), with GFP mRNA (■), or with αCD19-CAR mRNA (formula image), against cell lines K562, REH, SUP-B15, and SR-91. The data correspond to an effector to target ratio of 5:1.
Fig. 5
Fig. 5
Cytolytic properties of electroporated NK-92 cells against CLL cells. Cytotoxicity, expressed in percentage of killed target cells, of NK-92 electroporated without nucleic acid (□), or with αCD19-CAR mRNA (formula image), against five primary CLL MNC samples. The data are for an effector to target ratio of 5:1.
Fig. 6
Fig. 6
Effects of irradiation on cytolytic properties of electroporated NK-92 cells. Cytotoxicity, expressed in percentage of killed target cells, of NK-92 against cell lines K562, REH, and SUP-B15 for an effector to target ratio of 5:1. NK-92 cells were electroporated without nucleic acid (control, full box color) or with αCD19-CAR mRNA (stripes), and irradiated either 4 h prior to electroporation (■) or 20 h after electroporation (formula image).
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