Abstract 767: Gypsophila saponins significantly augment the cytotoxicity of saporin-based anti-CD19, -CD22, -CD38 and -CD71 immunotoxins in human leukemia and lymphoma

Abstract

Abstract Expanding the therapeutic window of immunotoxins (IT) is an important goal that would open up greater therapeutic possibilities for this class of drug. Saponins from Gypsophila paniculata have been shown to augment the cytotoxicity of the ribosome-inactivating protein (RIP) saporin and saporin containing cytotoxins that are directed against EGFR in cell culture and mouse models of human cancer. The mechanism through which saponins exert this effect is not clearly understood but is probably mediated through membrane disruption of endosomes that contain internalised IT thus allowing more efficient escape of IT to the cytosol and thence direct access of the RIP to target ribosomes. Here we show that Gypsophila saponins (SA) dramatically and significantly augment the cytotoxicity of saporin-based ITs directed against CD19, CD22, CD38 and CD71 expressed on the membrane surface of five different human leukaemia and lymphoma cell lines. We observed a high degree of variability in the augmentive effect exerted by SA, not only between different cell lines but also between different target molecules expressed on the same or on different cell lines. For instance SA augmented the anti-CD19 IT BU12-Saporin by only 10-fold in NALM-6 cells (representing a reduction in IT IC50 from 3 × 10−10M to 3 × 10−11M by SA) but by a massive 9.43 million-fold in Daudi cells (representing a reduction in IT IC50 from 5 × 10−9M to 5.3 × 10−16M by SA), despite there being a similar expression level of CD19 by each cell line. Through antibody blocking experiments and the use of cells not expressing the antigen targeted by the IT under study we revealed that SA does bring about a modest reduction in the immunospecificity of IT activity. However, by scheduling pulse exposure of target cells to IT first followed by washout and then exposure to SA it was possible to retain full immunospecificity. We speculate that the differences in SA augmentive effect on IT cytotoxicity between different cell lines and different target molecules on the same cell line probably represents individual variations in lipid/cholesterol membrane composition, particularly within lipid microdomains (lipid rafts) to which target molecules such as CD19 and CD38 migrate prior to receptor-mediated endocytosis following cross linking by the antibody component of IT. The variations in augmentive effect of SA on IT cytotoxicity observed for the different cell lines in the present study makes understanding the mechanism behind this phenomenon very important if saponins are to be sucessfully developed as a treatment modality to be used in conjunction with saporin-based ITs or cytotoxins for individual patients with haematological and other types of malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 767. doi:10.1158/1538-7445.AM2011-767