Data are represented as mean S.D. strictly dependent on specific recognition of the target antigen Polyphyllin A and could be blocked by GD2-specific antibody or anti-idiotypic antibody occupying the CARs cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD2-specific NK cells was also found against primary NB cells and GD2 expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells. Polyphyllin A values 0.05 were considered as significant. Data were analysed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Results Generation of NK cells carrying GD2-specific chimeric antigen receptors FANCC GD2-specific scFv(ch14.18) antibody fragments were derived from constructs encoding scFv(ch14.18)-Fc fusion proteins that carry heavy and light chain variable domains of the chimeric mAb ch14.18 [34, 35]. To address potential differences in the functionality of scFv(ch14.18) molecules that depend on the orientation of the variable domains, we employed scFv fragments where VH and VL of antibody ch14.18 were either assembled in the orientation VH-linker-VL, or VL-linker-VH, with the synthetic (G4S)4 sequence serving as a flexible linker. Chimeric antigen receptors were constructed by inserting the scFv fragments designated scFv(ch14.18)HL and scFv(ch14.18)LH between a sequence encoding an N-terminal immunoglobulin heavy-chain signal peptide, and sequences encoding a Myc-tag, the CD8 hinge region (amino acids 105C165) and the CD3- chain in the retroviral transfer vector pLXSN [30] (Fig. 1A). Open in a separate window Fig 1 Transduction of NK-92 cells with retroviral vectors encoding chimeric scFv(ch14.18)- antigen receptors. (A) Schematic representation of pL-scFv(ch14.18)–SN constructs. The Moloney murine leukaemia virus 5 long terminal repeat (LTR) controls Polyphyllin A the expression of chimeric scFv(ch14.18)- antigen receptors which consist of an N-terminal immunoglobulin heavy-chain leader peptide (signal peptide), a GD2-specific single-chain antibody scFv(ch14.18) with heavy (VH) and light chain (VL) variable domains in VH-linker-VL (HL) or VL-linker-VH (LH) orientation, a Myc-tag, the hinge region of CD8 and the CD3- chain. The neomycin-resistance gene for G418 selection of transduced cells is driven by the SV40 early promoter. (B) Surface expression of chimeric scFv(ch14.18)- antigen receptors. After G418 selection of transduced cells (G418), NK-92-scFv(ch14.18)HL- and NK-92-scFv(ch14.18)LH- cells expressing homogenous levels of the CARs on their surface were enriched by immunomagnetic separation with mAb 9E10 and goat antimouse IgG-coated magnetic beads (MACS). Single cell clones were derived by limiting dilution (LD). Representative clones are shown. After each selection step, surface expression of scFv(ch14.18)- receptors was determined by flow cytometry using mAb 9E10 and FITC-labelled goat antimouse secondary antibody. NK-92 cells transduced with empty pLXSN served as a control. Amphotropic retroviral vector particles were produced by stable transfection of FLYA-JET packaging cells [31], and used for transduction of human NK-92 cells. After selection with G418, expression of scFv(ch14.18)HL- and scFv(ch14.18)LH- receptor proteins on the cell surface was analysed by flow cytometry. At this step the majority of cells in the selected cell pools displayed low or undetectable expression of the CARs (Fig. 1B, left panels). To enrich NK-92 cells that express more homogenous receptor levels, cells were sorted with Myc-tag specific mAb 9E10 and immunomagnetic beads (Fig. 1B, middle panels), followed by limiting dilution to obtain single cell clones. This yielded stable NK-92 cell clones consistently expressing high levels of CARs (Fig. 1B, right panels). We did not observe a difference in expression levels between clones carrying scFv(ch14.18)HL- or scFv(ch14.18)LH- (Fig. 1B and data not shown), indicating that the orientation of VH and VL within scFv(ch14.18) had no influence on the overall expression or surface display of the receptors. Surface expression of GD2 on NB cells As a prerequisite for the analysis of CAR functionality and activity of retargeted NK-92 cells, first surface expression of GD2 by established NB cell lines and primary NB cells was investigated by flow cytometry using fluorochrome-labelled GD2-specific murine mAb 14.G2a. Control cells were treated with an irrelevant isotype-matched antibody. Established human UKF-NB3, Kelly, BE(2)C and LAN-1 NB cells displayed intermediate to high levels of GD2 on their surface, whereas only a very weak signal was determined with anti-GD2 antibody for SK-N-SH cells (Fig. 2A). Analysis of primary NB cells from the BM of 12 relapsed NB patients revealed markedly enhanced GD2 expression in these samples when.