Description:
<jats:title>Abstract</jats:title>
<jats:p>Introduction</jats:p>
<jats:p>Neuroblastoma (NB) is a neuroectodermal tumor of childhood characterized by a poor prognosis. Therefore, the establishment of new adjuvant therapeutic strategies is a prerequisite, especially for advanced stage 4 patients. The T cell independent antigen ganglioside GD2 is highly expressed on most NB which makes it an interesting target for immunotherapeutic strategies. In general, NK cells show a major histocompatibility complex (MHC) class I-independent target cell recognition and are able to actively lyse tumors with down regulated MHC class I expression; therefore they represent a suitable tool for immunotherapeutic applications. In order to specifically direct the cytotoxic abilities of NK cells towards NB cells, the human NK cell line NK-92 was genetically engineered to express a chimeric receptor, consisting of a GD2-specific ch14.18scFv-antibody fragment and the signal transducing zeta-chain of the CD3 complex (NK-92-scFv(ch14.18)-zeta).</jats:p>
<jats:p>Methods</jats:p>
<jats:p>In order to determine specificity of NK-92-scFv(ch14.18)-zeta, FACS analysis was performed. For this purpose, we employed an anti-idiotypic-antibody (anti-Id-Ab), which mimics the GD2 epitope and is directed against the binding domain of ch14.18. In vitro cytotoxicity assays measuring chromium (Cr) 51 release with GD2+ and GD2- NB and melanoma cell lines were performed, in order to prove functionality of transduced NK-92 cells. Furthermore, cytotoxic activity of NK-92-scFv(ch14.18)-zeta was blocked using the α-Id-Ab in Cr51 release assays.</jats:p>
<jats:p>Results</jats:p>
<jats:p>FACS analysis using an α-Id-Ab revealed high expression of the chimeric receptor on NK-92-scFv(ch14.18)-zeta, indicating specificity for GD2. Furthermore, genetically modified NK-92 specifically lysed GD2+ tumor cells at an E:T ratio of 6.3:1, ranging from 70% (M21 cells, GD2+ melanoma) to 79% (LAN-1, GD2+ human NB) cytotoxicity. In contrast to that, cytotoxicity against GD2- NB cell line SK-N-SH was decreased (16% specific cytotoxicity). Importantly, parental NK-92 cells showed only diminished cytotoxicity towards GD2 expressing tumor cells compared to NK-92-scFv(ch14.18)-zeta. Additionally, we could show that blocking the chimeric receptor on NK-92-scFv(ch14.18)-zeta with the α-Id-Ab leads to a significant decrease of cytotoxicity towards GD2+ LAN-1 cells (20% specific cytotoxicity).</jats:p>
<jats:p>Conclusion</jats:p>
<jats:p>The results achieved so far clearly demonstrate specificity and functionality of the chimeric receptor molecule on NK-92-scFv(ch14.18)-zeta and provide an important base line for additional experiments to further illuminate the cytotoxic potential and anti-tumor efficacy of NK-92-scFv(ch14.18)-zeta in NB xenograft mouse models.</jats:p>
<jats:p>Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5601.</jats:p>