Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development.

To study the role of the diphthamide modification on eukaryotic elongation factor 2 (eEF2), we generated an eEF2 Gly(717)Arg mutant mouse, in which the first step of diphthamide biosynthesis is prevented. Interestingly, the Gly(717)-to-Arg mutation partially compensates the eEF2 functional loss resulting from diphthamide deficiency, possibly because the added +1 charge compensates for the loss of the +1 charge on diphthamide. Therefore, in contrast to mouse embryonic fibroblasts (MEFs) from OVCA1(-/-) mice, eEF2(G717R/G717R) MEFs retain full activity in polypeptide elongation and have normal growth rates. Furthermore, eEF2(G717R/G717R) mice showed milder phenotypes than OVCA1(-/-) mice (which are 100% embryonic lethal) and a small fraction survived to adulthood without obvious abnormalities. Moreover, eEF2(G717R/G717R)/OVCA1(-/-) double mutant mice displayed the milder phenotypes of the eEF2(G717R/G717R) mice, suggesting that the embryonic lethality of OVCA1(-/-) mice is due to diphthamide deficiency. We confirmed that the diphthamide modification is essential for eEF2 to prevent -1 frameshifting during translation and show that the Gly(717)-to-Arg mutation cannot rescue this defect.

Triterpenoid saponin augmention of saporin-based immunotoxin cytotoxicity for human leukaemia and lymphoma cells is partially immunospecific and target molecule dependent.

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.

Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis.

Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

The endocytic uptake pathways of targeted toxins are influenced by synergistically acting Gypsophila saponins.

The expression of the epidermal growth factor (EGF) receptor is upregulated in many human tumors. We developed the targeted toxin SE, consisting of the plant toxin saporin-3 and human EGF. The cytotoxic effect of SE drastically increases in a synergistic manner by a combined treatment with Saponinum album (Spn), a saponin composite from Gypsophila paniculata L. Here we analyzed which endocytic pathways are involved in the uptake of SE and which are mandatory for the Spn-mediated enhancement. We treated HER14 cells (NIH-3T3 cells transfected with human EGF receptor) with either chlorpromazine, dynasore, latrunculin A, chloroquine, bafilomycin A1 or filipin and analyzed the effect on the cytotoxicity of SE alone or in combination with Spn. We demonstrated that SE in combination with Spn enters cells via clathrin- and actin-dependent pathways and the acidification of the endosomes after endocytosis is relevant for the cytotoxicity of SE. Notably, our data suggest that SE without Spn follows a different endocytic uptake pathway. SE cytotoxicity is independent of blocking of clathrin or actin, and the decrease in endosomal pH is irrelevant for SE cytotoxicity. Furthermore, Spn has no influence on the retrograde transport. This work is important for the better understanding of the underlying mechanism of Spn-enhanced cytotoxicity and helps to describe the role of Spn better.

Epidermal growth factor receptor expression affects the efficacy of the combined application of saponin and a targeted toxin on human cervical carcinoma cells.

Cervical cancer is the second most common cancer in women worldwide. Targeting the epidermal growth factor receptor (EGFR) is a very promising approach since it is overexpressed in about 90% of cervical tumors. Here, we quantified the toxic effect of SE, a targeted toxin consisting of epidermal growth factor (EGF) as targeting moiety and the plant toxin saporin-3, on 3 common human cervical carcinoma cell lines (HeLa, CaSki and SiHa) and recently established lines (PHCC1 and PHCC2) from 2 different individuals. A human melanocytic and a mouse cell line served as negative control. Additionally, we combined SE with saponinum album, a saponin composite from Gypsophila paniculata, which exhibited synergistic properties in previous studies. The cell lines, except for SiHa cells, revealed high sensitivity to SE with 50% cell survival in the range of 5-24.5 nM. The combination with saponin resulted in a remarkable enhancement of cytotoxicity with enhancement factors ranging from 9,000-fold to 2,500,000-fold. The cytotoxicity of SE was clearly target receptor specific since free EGF blocks the effect and saporin-3 alone was considerably less toxic. For all cervical carcinoma cell lines, we evinced a clear correlation between EGFR expression and SE sensitivity. Our data indicate a potential use of targeted toxins for the treatment of cervical cancer. In particular, the combination with saponins is a promising approach since efficacy is drastically improved.

Creation and characterization of a xenograft model for human cervical cancer.

OBJECTIVE: Most of primary human cancer tissues show effective engraftment and proliferation after transplantation onto Scid mice. However xenotransplantation of vital specimens of cervical carcinoma has not been successful in the past, also the generation of cell lines from primary cervical cancer has hardly ever been possible. The lack of appropriate xenograft models impedes the search for improved specific therapeutic agents. METHODS: We explored the efficiency of different techniques for tumor transplantation and describe the first protocol to enable reliable and efficient engraftment of human cervical cancer in Scid beige mice. To demonstrate the value of this tumor model, we explored the therapeutic potency of a novel immunotoxin (SA2E). SA2E is a chimeric protein constructed by fusing the human epidermal growth factor and the plant protein toxin saporin. RESULTS: About 70% of transplanted tumors exhibited potent proliferation, and multiple retransplantation was possible in 40%. Local treatment with the immunotoxin SA2E had a dose dependent therapeutic effect and achieved a tumor volume reduction of up to 60%. CONCLUSIONS: Reliable engraftment and high reproducibility make this novel xenograft model an attractive test system to identify new therapeutic agents for cervical cancer.

Sortase-A mediated chemoenzymatic lipidation of single-domain antibodies for cell membrane engineering.

PURPOSE: Membrane engineering has versatile applications in adoptive cell therapies, immune therapy or drug delivery. Incorporation of lipidated antibody-derived ligands into cells may enforce supraphysiological cell interactions that offer new therapeutic approaches. A challenge is the defined synthesis of lipidated ligands that effectively interact with such membranes. METHODS: Sortase-A was used to attach a PEGylated, dimyristyl lipid-anchor on single-domain antibodies (VHH). The membrane insertion was investigated on liposomal bilayers, myeloid-derived suppressor cells (MDSC) and T cells. RESULTS: The lipidated VHHs remodeled liposomal as well as cellular membranes. The VHH carrying liposomes were successfully targeted towards antigen-positive cells. MDSC and T cells were both modified with lipidated VHHs as detected with an FITC-anti-llama antibody. T cells that carried an anti-CD11b VHH showed cellular association in vitro with CD11b+Gr-1+ MDSC in a two-dimensional magnetic activated cell sorting / flow-cytometry assay. CONCLUSION: The applied combination of chemoenzymatic ligation, PEGylated lipid anchors and single-domain antibodies delivers water-soluble and chemically defined lipidated ligands, which readily associate with liposomal and cellular membranes. This enables liposomal drug targeting and artificial cell-cell interactions. Hence, the presented concept for lipidation of single-domain antibodies is promising for further application in the field of drug delivery or cell-based therapies.

A simple method for isolation of Gypsophila saponins for the combined application of targeted toxins and saponins in tumor therapy.

Saponinum album (SAP) is a complex mixture of triterpene saponins from Gypsophila paniculata L. Although most of the saponins from SAP are characterized, the separation of pure saponins remains time consuming and costly, involving different chromatographic techniques. Recently it was shown that SAP drastically enhanced the cytotoxicity of a chimeric toxin consisting of the N-glycosidase saporin and human epidermal growth factor (Sap-EGF) in cell culture experiments. In view of a potential therapeutic use of the coadministration of SAP and Sap-EGF in tumor therapy, an economic and time-saving method for the isolation of pure saponins from the crude SAP mixture in high amounts is required. In this study we isolated a single saponin by a simple chromatographic method. The isolated saponin was characterized by mass spectrometry and was shown to enhance the cytotoxicity of Sap-EGF on HER14 cells.

Sortagging of liposomes with a murine CD11b-specific VHH increases in vitro and in vivo targeting specificity of myeloid cells.

The therapeutic index of drugs can be increased via drug encapsulation in actively targeted, meaning ligand modified drug delivery systems. The manufacturing of such targeted drug delivery systems, in particular the conjugation between drug carrier and ligand, can be done by enzymatic conjugation methods, exploiting the site-specific, bioorthogonal nature of these reactions. The use of such enzymes like Sortase-A transpeptidase requires efficient purification methods, as residuals of the enzyme may be responsible for immunogenic potential and drug product instabilities. These instabilities may be based on the enzymatic reverse reaction, meaning here a cleavage between ligand and drug carrier. In the presented work, two differently PEGylated formulations were modified with variable fragments of camelid heavy chain-only antibodies (VHH) via Sortase-A, purified by different methodologies and tested for ligand cleavage upon storage. Strongly PEGylated liposomes (PEGhigh-LS) were found to retain higher amounts of Sortase-A than lowly PEGylated ones (PEGlow-LS) after dialysis purification. Surprisingly, this did not correlate with ligand stability during storage. PEGhigh-LS were less prone for degradation, compared to PEGlow-LS, which showed a ligand cleavage of 20% after an 8 weeks storage at 2-8 °C. Nonetheless, overall degradation could be minimized by an additional affinity bead purification procedure. Liposomes modified with a CD11b-specific VHH were tested for their in vitro and in vivo targeting ability towards CD11b+ cells. Specific targeting of CD11b was achieved in vitro and in vivo on various cell types. PEGylation decreased the targeting effect in vitro, however no differences between PEGhigh or PEGlow formulations were observed in vivo. The obtained results underline the need for a thorough characterization of novel conjugation strategies as well as an early in vivo characterization of such targeted drug delivery systems.

Biodistribution and efficacy of [131I]A33scFv::CDy, a recombinant antibody-enzyme protein for colon cancer.

In antibody-directed enzyme-prodrug therapy (ADEPT), an antibody-bound enzyme localizes to tumor tissue, where it selectively converts a subsequently administered non-toxic prodrug into a cytotoxic drug. A33scFv::CDy is a bifunctional fusion construct comprising a single chain antibody against the gpA33 antigen and the prodrug-converting enzyme cytosine deaminase. gpA33 is highly and homogeneously expressed in >95% of all colorectal cancers. Here we describe the biodistribution and tumor-targeting capacity of 131I labeled A33scFv::CDy. 131I labeling of A33scFv::CDy was performed by the chloramine-T method, and the properties of the resulting [131I]A33scFv::CDy conjugate were determined in vivo and in vitro, including biodistribution studies in nude mice bearing human LIM1215 colon carcinoma xenografts. The [131I]A33scFv::CDy conjugate bound specifically to colorectal cancer cells in vitro with KD = 15.8 nM as determined by a saturation assay. in vivo, the tumor uptake of [131I]A33scFv::CDy peaked at 87% injected dose/g 47 h post injection. Normal tissue uptake was low, and activity in blood was lower than in tumor at all time-points studied (6-92 h). The tumor-to-blood ratio increased over time with a maximum of 8.1 at 67 h post injection. [131I]A33scFv::CDy thus shows a biodistribution that makes it attractive for both radioimmunotherapy (RIT) and ADEPT. Preliminary therapeutic experiments showed a significant reduction of tumor size in mice treated with the A33scFv::CDy-5-fluorocytosine/5-fluorouracil ADEPT system. This work demonstrates the feasibility of ADEPT and RIT based on the A33scFv::CDy recombinant construct.

Saponins in tumor therapy.

Saponins are plant glycosides with favorable anti-tumorigenic properties. Several saponins inhibit tumor cell growth by cell cycle arrest and apoptosis with IC50 values of up to 0.2 microM. We discuss diverse groups of saponins (dioscins, saikosaponins, julibrosides, soy saponins, ginseng saponins and avicins) investigated in relation to tumor therapy and focus on cellular and systemic mechanisms of tumor cell growth inhibition both in vitro and in vivo. The review also describes saponins in combination with conventional tumor treatment strategies, which result in improved therapeutic success. Some combinations of saponins and anti-tumorigenic drugs induce synergistic effects with potentiated growth inhibition.

Pentaglycine lipid derivates - rp-HPLC analytics for bioorthogonal anchor molecules in targeted, multiple-composite liposomal drug delivery systems.

The quantification of lipids and assessment of lipid composition is an indispensable step during the pharmaceutical development of novel lipid based drug delivery systems such as liposomes. Broad excipient screenings of such formulations raise the need for versatile analytical methods. Even more demanding complexity is generated by introduction of targeted systems requiring functionalized lipids. We addressed this demand by developing an rp-HPLC based analytical method with evaporative light scattering detection (ELSD) for the simultaneous analysis of commonly used phosphatidylcholines, cholesterol and bilayer surface-modifying cationic, anionic or PEGylated lipids, which can be analyzed in combination with novel pentaglycine lipids suitable as targeting ligand anchor. The method was validated for specificity, precision, accuracy and sample stability. We monitor the continuous and scalable manufacturing of two pentaglycine-modified liposomal formulations and track the modification of these drug delivery systems with a single-domain antibody utilizing bioorthogonal Sortase-A technology. Both the presented analytical and preparative techniques can help to improve the quality control and to accelerate the pharmaceutical development of such targeted drug delivery systems.

Sortaggable liposomes: Evaluation of reaction conditions for single-domain antibody conjugation by Sortase-A and targeting of CD11b+ myeloid cells.

Active targeting with ligand coated liposomal drug delivery systems is a means to increase the therapeutic index of drugs. Stable ligand coating requires bilayer anchorage of the commonly proteinaceous ligands and hence a conjugation of lipid structures towards amino acids. This often leads to heterogeneous reaction products especially when chemical coupling methods are employed. Chemoenzymatic Sortase-A mediated transpeptidation (sortagging) is a useful tool to avoid this protein heterogeneity through its site-specific, bioorthogonal ligation mechanism. Manufacturing of such sortaggable, pentaglycine modified liposomes was developed by adaption of a scalable solvent injection technique. The pentaglycine liposomes were prepared with different degrees of PEGylation and steric accessibility of the pentaglycine motif. Comparable hydrodynamic diameters (146-188 nm) of the different formulations were obtained after a flow rate screening. The sortagging reactivity of a single-domain antibody (VHH) towards the pentaglycine liposomes was strongly dependent on the steric accessibility of the pentaglycine nucleophile. Adjusting the pentaglycine to ligand ratio improved conversion rates up to 80%. The liposome bound VHH was accessible for its soluble antigen as shown by a chromatography-based binding assay. Mono- and granulocytes could be selectively targeted in vitro by conjugation of BMX1, a VHH directed towards human myeloid cell surface marker CD11b. Confocal microscopy revealed intracellular localization of the targeted liposomes. The developability of those pentaglycine liposomes as well as their proof of principle for targeted drug delivery shows their potential for further investigation, for example as delivery platform for diagnostics or drugs into the tumor microenvironment.

Genetic screen in myeloid cells identifies TNF-α autocrine secretion as a factor increasing MDSC suppressive activity via Nos2 up-regulation.

The suppressive microenvironment of tumors remains one of the limiting factors for immunotherapies. In tumors, the function of effector T cells can be inhibited by cancer cells as well as myeloid cells including tumor associated macrophages and myeloid-derived suppressor cells (MDSC). A better understanding of how myeloid cells inhibit T cell function will guide the design of therapeutic strategies to increase anti-tumor responses. We have previously reported the in vitro differentiation of MDSC from immortalized mouse hematopoietic progenitors and characterized the impact of retinoic acid and 3-deazaneplanocin A on MDSC development and function. We describe here the effect of these compounds on MDSC transcriptome and identify genes and pathway affected by the treatment. In order to accelerate the investigation of gene function in MDSC suppressive activity, we developed protocols for CRISPR/Cas9-mediated gene editing in MDSC. Through screening of 217 genes, we found that autocrine secretion of TNF-α contributes to MDSC immunosuppressive activity through up-regulation of Nos2. The approach described here affords the investigation of gene function in myeloid cells such as MDSC with unprecedented ease and throughput.

A cleavable molecular adapter reduces side effects and concomitantly enhances efficacy in tumor treatment by targeted toxins in mice.

Two of the main problems associated with administration of receptor-targeted toxins in tumor therapy are severe systemic side effects and low transfer of the toxins into the cytosol after binding to the tumor cell surface. To improve chimeric toxins in this respect we have developed a molecular adapter that links the toxic moiety and ligand. The adapter is designed to improve cytosolic uptake, retain the toxin inside the cytosol and detoxify the drug after cell death. The plant toxin saporin linked either directly or via the adapter to epidermal growth factor (EGF) served to evaluate efficacy to inhibit tumor growth and reduce side effects in vivo. The lethal dose for BALB/c mice was three times less for the adapter-containing toxin (SA2E) than for the adapter-free construct (SE). Furthermore, SE only reduced the average weight of induced tumors by 33% whereas SA2E-treated mice exhibited 71% reduction with an almost complete suppression in 60% of the cases. Additionally, severe side effects like hyperalgesia, alopecia and death were drastically reduced in SA2E-treated animals. Tumors without target receptor were only slightly affected by SA2E and the reduction in side effects less pronounced indicating specific depletion from the blood by target receptor expressing cells.

Identification of inhibitors of myeloid-derived suppressor cells activity through phenotypic chemical screening.

Tumors are infiltrated by cells of the immune system that interact through complex regulatory networks. Although tumor-specific CD8+ T cells can be found in peripheral blood and tumor samples from cancer patients, their function is inhibited by immunosuppressive cells such as regulatory T cells, tumor-associated macrophages, and myeloid-derived suppressor cells (MDSC). Recent clinical successes have demonstrated that alleviating immunosuppression and T cell exhaustion translates into long-term clinical benefits. Although tremendous progress has been achieved, tools that afford unbiased approaches and screenings to uncover new potential inhibitors or gene targets are lacking. In this study, we describe a system based on immortalized progenitors that allows straightforward investigation of myeloid cells. We show that bone marrow progenitors immortalized through the transduction of NUP98-HOXB4 transgene can be differentiated into CD11b+Gr-1+ MDSC that express Arginase-1 and PD-L1, produce reactive oxygen and nitrogen species, and suppress T cell function in vitro. To uncover chemical probes that interfere with MDSC biology, we performed a chemical phenotypic screening and identified 3-deazaneplanocin A as a novel modulator of MDSC functions. We characterized and compared the effect of 3-deazaneplanocin-A and all-trans retinoic acid, a well-known modulator of MDSC activity, on the expression of effector molecules and immunosuppressive functions of MDSC. Altogether, this proof-of-principle opens new possibilities for the identification of drugs targeting myeloid cells with immunosuppressive activities.

The activity of myeloid cell-specific VHH immunotoxins is target-, epitope-, subset- and organ dependent.

The central role of myeloid cells in driving autoimmune diseases and cancer has raised interest in manipulating their function or depleting them for therapeutic benefits. To achieve this, antibodies are used to antagonize differentiation, survival and polarization signals or to kill target cells, for example in the form of antibody-drug conjugates (ADC). The action of ADC in vivo can be hard to predict based on target expression pattern alone. The biology of the targeted receptor as well as its interplay with the ADC can have drastic effects on cell apoptosis versus survival. Here we investigated the efficacy of CD11b or Ly-6C/Ly-6G-specific variable fragments of camelid heavy chain-only antibodies (VHH) conjugated to Pseudomonas exotoxin A to deplete myeloid cells in vitro and in vivo. Our data highlight striking differences in cell killing in vivo, depending on the cell subset and organs targeted, but not antigen expression level or VHH affinity. We observed striking differences in depletion efficiency of monocytes versus granulocytes in mice. Despite similar binding of Ly-6C/Ly-6G-specific VHH immunotoxin to granulocytes and monocytes, granulocytes were significantly more sensitive than monocytes to immunotoxins treatment. Our results illustrate the need of early, thorough in vivo characterization of ADC candidates.

The saponin-mediated enhanced uptake of targeted saporin-based drugs is strongly dependent on the saponin structure.

Saponins are a group of plant glycosides consisting of a steroid or triterpenoid aglycone to which one or more sugar chains are attached. They exhibit cell membrane-permeabilizing properties and, thus, have been investigated for their therapeutic potential. Recently, at a non-permeabilizing concentration saponinum album from Gypsophila paniculata L. has been described to enhance the cytotoxicity of a chimeric toxin in a cell culture model. To elucidate whether this enhancing effect is also mediated by other saponins, we analyzed the ability of seven different saponins to enhance the cytotoxicity of a targeted chimeric toxin. The chimeric toxin is composed of saporin, a plant ribosome-inactivating toxin, a cleavable adapter, and human epidermal growth factor (EGF). Cytotoxicity on EGF receptor (EGFR)-bearing cells was analyzed both alone and after combined application of saponin and chimeric toxin. Only two of the tested saponins, quillajasaponin and saponinum album, enhanced cytotoxicity by more than 1,000-fold, whereas the enhancement factors of the other saponins were only approximately 10-fold. In contrast to saponinum album, quillajasaponin enhanced the cytotoxicity both on control cells lacking EGFR and on target cells, indicating that, in this case, the enhancement is not target cell receptor specific. This is also the case for some of the saponins with low enhancement factors. Saponinum album resulted in a more than 13,600-fold receptor-specific enhancement, decreasing the 50% inhibitory concentration (IC(50)) from 2.4 nM to 0.18 pM, which renders it the best option to promote saporin-3-based drug uptake while retaining specificity for the EGFR.

Tumor Targeting and Drug Delivery by Anthrax Toxin.

Anthrax toxin is a potent tripartite protein toxin from Bacillus anthracis. It is one of the two virulence factors and causes the disease anthrax. The receptor-binding component of the toxin, protective antigen, needs to be cleaved by furin-like proteases to be activated and to deliver the enzymatic moieties lethal factor and edema factor to the cytosol of cells. Alteration of the protease cleavage site allows the activation of the toxin selectively in response to the presence of tumor-associated proteases. This initial idea of re-targeting anthrax toxin to tumor cells was further elaborated in recent years and resulted in the design of many modifications of anthrax toxin, which resulted in successful tumor therapy in animal models. These modifications include the combination of different toxin variants that require activation by two different tumor-associated proteases for increased specificity of toxin activation. The anthrax toxin system has proved to be a versatile system for drug delivery of several enzymatic moieties into cells. This highly efficient delivery system has recently been further modified by introducing ubiquitin as a cytosolic cleavage site into lethal factor fusion proteins. This review article describes the latest developments in this field of tumor targeting and drug delivery.

Combined application of saponin and chimeric toxins drastically enhances the targeted cytotoxicity on tumor cells.

Immunotoxins have to be administered in high doses due to low cytosolic uptake with the consequence of severe side effects. Recently we found that the cytotoxic activity from Agrostemma githago seeds can be attributed to a synergistic toxicity of a triterpenoid saponin and a ribosome-inactivating protein. Here we investigated whether saponins are able to enhance the efficacy of a receptor-specific chimeric toxin consisting of saporin-3, epidermal growth factor and a molecular adapter previously shown to reduce side effects on non-target cells. Pre-applied saponin enhances the target cell-specific cytotoxic effect, dependent on the cell line, between 3560- and 385,000-fold with an IC50 up to 0.67 pM. Non-target cells are not affected at the same concentration. At the optimal concentrations of the chimeric toxin and saponin application of either one of the components shows no cytotoxicity at all proving a synergistic effect. In the presence of saponin ligand-free saporin-3 does not exhibit any cytotoxic effect up to 0.1 nM providing further evidence for an increased specificity. This synergistic effect is in the same order of magnitude as in a mouse model. Our investigations clearly demonstrate that a combined administration of saponin and chimeric toxins opens up a promising perspective for tumor therapy.