Cholesterol seems to play a central role in the augmentation of saporin-based immunotoxin (IT) cytotoxicity by triterpenoid saponins. Endolysosomal escape has been proposed as one mechanism for the saponin-mediated enhancement of targeted toxins. We investigated the effects of lipid depletion followed by repletion on Saponinum album (SA)-induced endolysosomal escape of Alexa Fluor labelled saporin and the saporin-based immunotoxin OKT10-SAP, directed against CD38, in Daudi lymphoma cells. Lipid deprived cells showed reduced SA-induced endolysosomal escape at two concentrations of SA, as determined by a flow cytometric method. The repletion of membrane cholesterol by low density lipoprotein (LDL) restored SA-induced endolysosomal escape at a concentration of 5 µg/mL SA but not at 1 µg/mL SA. When LDL was used to restore the cholesterol levels in lipid deprived cells, the SA augmentation of OKT10-SAP cytotoxicity was partially restored at 1 µg/mL SA and fully restored at 5 µg/mL SA. These results suggest that different mechanisms of action might be involved for the two different concentrations of SA and that endosomal escape may not be the main mechanism for the augmentation of saporin IT cytotoxicity by SA at the sub-lytic concentration of 1 µg/mL SA.

Purpose The aim of this work was to develop a quantitative, flow cytometric method for tracking the endolysosomal escape of a fluorescently labelled saporin toxin. Methods Flow cytometric measurements of fluorescent pulse width and height were used to track the endocytic uptake into Daudi cells of a fluorescently labelled saporin toxin and the saporin based immunotoxin, OKT10-SAP. Subsequently, measurement of changes in pulse width were used to investigate the effect of a triterpenoid saponin on the endolysosomal escape of internalised toxin into the cytosol. Live cell confocal microscopy was used to validate the flow cytometry data. Results Increased endolysosomal escape of saporin and OKT10-SAP was observed by confocal microscopy in cells treated with saponin. Fluorescent pulse width measurements were also able to detect and quantify escape more sensitively than confocal microscopy. Saponin induced endolysosomal escape could be abrogated by treatment with chloroquine, an inhibitor of endolysosomal acidification. Chloroquine abrogation of escape was also mirrored by a concomitant abrogation of cytotoxicity. Conclusions Poor endolysosomal escape is often a rate limiting step for the cytosolic delivery of protein toxins and other macromolecules. Pulse width analysis offers a simple method to semi-quantify the endolysosomal escape of this and similar molecules into the cytosol.

We have previously shown that antibody-dependent cellular cytotoxicity (ADCC) cooperates with immunotoxin (IT)-mediated killing of human leukaemia cells in an severe combined immunodeficient (SCID) mouse model of human T-cell acute lymphoblastic leukaemia (SCID-HSB-2 mice), but not in an equivalent non-obese diabetic (NOD)/SCID mouse model. In these earlier studies, we reasoned that diminished ADCC due to the functional deficit in natural killer (NK) cell activity in NOD/SCID mice resulted in a failure of effective perforin/granzyme-mediated cytotoxicity necessary for the delivery of the augmentative effect. Poly-inosinic-cytidylic acid [poly (I:C)] is a synthetic dsRNA toll-like receptor 3 (TLR3) agonist that possesses a number of biological properties that includes the in vivo activation of NK cells. We show here that intravenous (i.v.) injection of SCID mice with [poly (I:C)] results in characteristic time-related changes in serum interleukin 2 (IL-2), IL-12, and interferon γ (INFγ) cytokine levels that are consistent with TLR3 driven activation of SCID mouse NK cells. Concomitantly, there are changes in the expression levels of CD2, CD16/32 (FcγRII/RIII), CD161 (NK1.1), and F4/80 in the bulk splenocyte population. These observed changes correlate with an increase in the in vitro lytic capabilities of putative NK cells from within the splenocyte population of [poly (I:C)] treated SCID mice. We demonstrate that the in vivo activation of NK cells with [poly (I:C)] in SCID mice bearing disseminated human T-cell leukaemia xenografts resulted in a significant improvement in the therapeutic activity exerted by an intact murine monoclonal antibody against human CD7. This was also seen for a saporin-based immunotoxin constructed with the same intact antibody (HB2-SAPORIN), but not with an F(ab’)2 derivative of the same antibody or of an IT constructed with the same F(ab’)2 HB2 antibody derivative. This study further demonstrates the previously reported reinforcing role of ADCC for the therapeutic activity of IT in an SCID mouse model of human T-ALL and the potential to significantly boost this further with [poly (I:C)]. Our study provides the rationale to justify the exploration of the clinical utility of IT based therapeutics in combination with TLR3 agonists, such as [poly (I:C)], for the treatment of haematological, and possibly other, malignancies.

Triterpenoid saponins from Saponinum album (SA) significantly augment the cytotoxicity of saporin-based immunotoxins but the mechanism of augmentation is not fully understood. We investigated the effects of six small molecule pharmacological agents, which interfere with endocytic and other processes, on SA-mediated augmentation of saporin and saporin-based immunotoxins (ITs) directed against CD7, CD19, CD22 and CD38 on human lymphoma and leukaemia cell lines. Inhibition of clathrin-mediated endocytosis or endosomal acidification abolished the SA augmentation of saporin and of all four immunotoxins tested but the cytotoxicity of each IT or saporin alone was largely unaffected. The data support the hypothesis that endocytic processes are involved in the augmentative action of SA for saporin ITs targeted against a range of antigens expressed by leukaemia and lymphoma cells. In addition, the reactive oxygen species (ROS) scavenger tiron reduced the cytotoxicity of BU12-SAP and OKT10-SAP but had no effect on 4KB128-SAP or saporin cytotoxicity. Tiron also had no effect on SA-mediated augmentation of the saporin-based ITs or unconjugated saporin. These results suggest that ROS are not involved in the augmentation of saporin ITs and that ROS induction is target antigen-dependent and not directly due to the cytotoxic action of the toxin moiety.

Triterpenoid saponins from Saponinum Album (SA) exert potent lytic effects on eukaryotic cell plasma membranes and, when used at sub-lytic concentrations, significantly augment the cytotoxicity of saporin-based immunotoxins (IT). To help elucidate the mechanism(s) behind these two phenomena we investigated the role of cholesterol to both. Human Daudi lymphoma cells were lipid deprived using a combination of three different approaches. Following treatment, the total cellular lipid content was analyzed by electrospray ionization mass spectrometry (ESI-MS) and plasma membrane (PM) cholesterol content measured using the lipophilic fluorescent probe NR12S. Maximal lipid deprivation of cells resulted in a complete loss of sensitivity to lysis by SA. Similarly augmentation of the anti-CD19 immunotoxin (IT) BU12-SAPORIN by SA was lost but without a concomitant loss of intrinsic IT cytotoxicity. The lytic activity of SA was restored following incubation of lipid deprived Daudi cells with Synthecol or LDL. The augmentative effect of SA on IT cytotoxicity for Daudi cells was restored following repletion of PM cholesterol levels with LDL. NR12S fluorescence and ESI-MS analysis of cellular lipids demonstrated that restoration of SA lytic activity by Synthecol was entirely due to increased PM cholesterol levels. Restoration of cellular and PM cholesterol levels by LDL also restored the augmentative effect of SA for IT, an effect associated with repletion of PM cholesterol with minor changes in some phospholipid species. These results indicate that the lytic and IT augmentative properties of SA are cholesterol-dependent in contrast to intrinsic IT cytotoxicity that is at least partially cholesterol independent.

CONTEXT:Saponinum album (SA) is a complex mixture of triterpenoid saponins previously shown to augment the cytotoxicity of the type I ribosome-inactivating protein saporin and an EGF-saporin target toxin that could potentially be used to improve the therapeutic window of targeted toxins.OBJECTIVE:To investigate the augmentative property of SA on saporin and saporin-based immunotoxins (IT) directed against five different cell surface target molecules on human leukemia and lymphoma cells.MATERIALS AND METHODS:After determining the optimum dose of SA for each cell line, the extent of SA-mediated augmentation was established for saporin and five saporin-based ITs using XTT and an annexin V apoptosis assay. Immunospecificity was investigated using three different blocking assays. Dose-scheduling was also investigated using the XTT assay.RESULTS:Uncorrected SA-mediated augmentation ranged at best from 31.5 million-fold to, at worse, 174-fold. However, when the calculated fold-increases were adjusted for the non-immunospecific effects of SA on an off-target IT, the true augmentative effects of SA were found to be largely non-immunospecific. Antibody blocking studies demonstrated that the augmentative effect of SA was only partially immunospecific. Separate exposure of target cells to IT and SA at different times demonstrated that immunospecific augmentation of IT by SA could be achieved but only if cells were exposed to IT first and SA second.CONCLUSIONS:SA significantly, although variably, augments the cytotoxicity of saporin and saporin-based immunotoxins. Concomitant exposure to both IT and SA can result in non-immunospecific cytotoxicity that can be overcome by temporally separating exposure to each.

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

Abstract Saponins are a highly diverse group of glycosides of plant origin containing either a steroidal or triterpenoid aglycone to which one or more sugar chains are attached. They have been variously described as enhancing the cytotoxic activity of ribosome-inactivating proteins (RIPs) and ligand-directing conjugates constructed with these for eukaryotic cells. Saponins are thought to facilitate the entry of RIPs into the cytosol across the membranes of intracellular vesicular structures. This characteristic of saponins therefore makes them of interest as molecules that could be useful in improving the therapeutic index of immunotoxins (IT) in a clinical setting. In the current study we used a protein synthesis inhibition assay based on leucine incorporation together with cell proliferation studies in culture to demonstrate that saponins from Gypsophila paniculata L. potentiate the cytotoxicity of the anti-CD19 and CD38 saporin-based immunotoxins (BU12-SAPORIN and OKT10-SAPORIN, respectively) by several thousand-fold for the human lymphoma cell line Daudi. Various experiments revealed that the enhancing effect of saponins on immunotoxin activity did not appear to be dependent on extra cellular pH in the assay system we used. Kinetic studies further revealed that saponin at a predetermined optimal concentration of 2 μg/ml reduced the t10 (time taken to reduce protein synthesis to 10% of the level seen in control Daudi cells) for OKT10-SAPORIN at 0.1μg/ml from 50 hours without saponin to 12.5 hours with saponin. In blocking experiments using OKT10-SAPORIN IT at the IC50 concentration in combination with saponin together with a gross excess of native OKT10 antibody we established that the immunospecific cytotoxic activity of OKT10-SAPORIN was fully retained when leucine incorporation was used as the surrogate measure of cytotoxicity but only partially so when thymidine incorporation was used. This observation of a partial uncoupling of protein and DNA synthesis as a measure of cytotoxicity was further supported by cell proliferation experiments in culture which were undertaken in parallel. If saponins are to be of any clinical value as candidate molecules for improving the therapeutic index of immunotoxins it is imperative that the immunospecificity of IT activity is fully retained. Whilst this study clearly demonstrates that saponins from G. paniculata L. do indeed significantly potentiate IT activity it seems that this effect may only be partially immunospecific. Nevertheless, the dramatic improvements that saponins confer on IT activity argue in favor of in vivo experiments in transgenic animal models of human lymphoma combined with additional studies to explore other strategies that might be employed to improve the immunospecific effect achievable. 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 5624.