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Ribosome inactivating proteins (RIPs) as family of anti-cancer drugs recently received much attention due to their interesting anti-cancer mechanism. In spite of small drugs, RIPs use the large-size effect (LSE) to prevent the efflux process governed by drug resistance transporters (DRTs) which prevents inside of the cells against drug transfection. There are many clinical translation obstacles that severely restrict their applications especially their delivery approach to the tumor cells. As the main goal of this review, we will focus on trichosanthin (TCS) and gelonin (Gel) and other types, especially scorpion venom-derived RIPs to clarify that they are struggling with what types of bio-barriers and these challenges could be solved in cancer therapy science. Then, we will try to highlight recent state-of-the-arts in delivery of RIPs for cancer therapy.

With the high efficacy of protein-based therapeutics and plenty of intracellular drug targets, cytosolic protein delivery in a cell-specific manner has attracted considerable attention in the field of precision medicine. Herein, we present an intracellular protein delivery system based on a target-specific repebody and the translocation domain of Pseudomonas aeruginosa exotoxin A. The delivery platform was constructed by genetically fusing an EGFR-specific repebody as a targeting moiety to the translocation domain, while a protein cargo was fused to the C-terminal end of the delivery platform. The delivery platform was revealed to efficiently translocate a protein cargo to the cytosol in a target-specific manner. We demonstrate the utility and potential of the delivery platform by showing a remarkable tumor regression with negligible toxicity in a xenograft mice model when gelonin was used as the cytotoxic protein cargo. The present platform can find wide applications to the cell-selective cytosolic delivery of diverse proteins in many areas.

Therapeutically potent macromolecular drugs have shown great promise for overcoming the limitations of small-molecule anti-cancer drugs. But tumor cell-selective intracellular delivery of the macromolecules remains a major hurdle for their successful clinical application. To overcome this challenge, we engineered a novel genetic fusion protein (F3-Gel) that composed of F3 peptide, a tumor-homing peptide, and gelonin, a plant-derived ribosome-inactivating protein (RIP), and then evaluated its anti-cancer activity in vitro and in vivo. The F3-Gel-encoding gene was synthesized by genetic recombination, and F3-Gel was successfully expressed in E coli. The anti-cancer activity of the produced F3-Gel was evaluated by various in vitro assays, which revealed that F3-Gel maintained equipotent protein synthesis inhibition activity (IC50=11 pmol/L) as unmodified gelonin (IC50=10 pmol/L). Furthermore, F3-Gel displayed enhanced cellular uptake into cancer cells (U87 MG, HeLa, LnCaP and 9L) than noncancerous cells (293 HEK and SVGp12). Compared with gelonin, F3-Gel exerted significantly higher cytotoxicity against these cancer cells. F3-Gel displayed significantly greater inhibition of protein translation in U87 MG cells: F3-Gel (0.5 μmol/L) was able to reduce the protein level to less than 50%, while gelonin (1 μmol/L) did not affect the intracellular protein level. In a U87 MG xenograft tumor-bearing mouse model, F3-Gel was accumulated in the tumor site at much higher levels and maintained for a prolonged time compared with gelonin. Administration of F3-Gel (0.5, 0.75 mol/kg, iv) caused 36% and 66%, respectively, inhibition of tumor growth in U87 MG xenograft mice, suggesting that it is a promising candidate drug for cancer treatment. Furthermore, this study demonstrates that fusion of F3 peptide to a potent macromolecule could provides an effective method for targeting tumors and eventually could improve their druggability.

Toxic gene therapy (or suicidal gene therapy) is gaining enormous interest, specifically for the treatment of cancer. The success of this therapy lies in several crucial factors, including the potency of gene products to kill the transfected tumor cells and the transfection ability of the transfection vehicles. To address the potency problem, in the present study, we engineered two separate mammalian transfection plasmids (pSAP and pGEL) containing genes encoding ribosome inactivating proteins (RIPs), gelonin and saporin. After the successful preparation and amplification of the plasmids, they were tested on various cancer cell lines (HeLa, U87, 9L, and MDA-MB-435) and a noncancerous cell line (293 HEK) using polyethyleneimine (PEI) as the transfection agent. Transfection studies performed under varying gene concentration, incubation time, and gene-to-PEI ratios revealed that, compared to the treatment of pGFP (GFP expression plasmid)/PEI, both pGEL/PEI and pSAP/PEI complexes could induce significantly augmented cytotoxic effects at only 2 μg/mL gene concentration. Importantly, these cytotoxic effects were observed universally in all tested cancer cell lines. Overall, this study demonstrated the potential of pGEL and pSAP as effective gene candidates for the toxic gene-based cancer therapy.

Despite substantial advances in its treatment, brain cancer remains a life-threatening disease with a poor survival rate. The main challenges for the conventional chemotherapy include an insufficient efficacy of drugs and toxicity caused by their nonselective mode of action. Recently, great attention has been paid to highly potent macromolecules such as gelonin, a type 1 ribosome-inactivating protein that inhibits protein translation. However, gelonin is poorly internalised into tumour cells and cannot distinguish between cancer and normal cells. To overcome these challenges, we engineered in this study a recombinant gelonin fusion protein with chlorotoxin, known as a brain cancer-homing peptide. The gelonin-chlorotoxin (Gel-CLTX) fusion chimera, produced in

Immunotoxins are a novel class of cancer therapeutics that contains a cytotoxic agent fused to a targeting moiety. Various toxic agents from different sources are used in immunotoxin development, including bacterial, plant and human origin cytotoxic elements. Although bacterial and plant-derived toxins are highly toxic and commonly used in immunotoxins, their immunogenicity for human restricted their application in cancer therapy. Here, we discuss the advantages and limitations of bacterial toxins such as Pseudomonas and Diphtheria toxins, plant toxins such as ricin and gelonin, and some endogenous protein of human origin such as RNases and Granzymes. This article will also review different generations of immunotoxins with special focus on immunotoxins which are under clinical trials or approved for clinical use. Finally, current deimmunization strategies for development of new less-immunogenic recombinant immunotoxins will be discussed.mAbs: Monoclonal antibodies; EF2: elongation factor 2; ITs: Immunotoxins; DT: Diphtheria toxin; PE: Pseudomonas exotoxin; dgA: de-glycosylated A-chain of ricin; rGel: recombinant de-glycosylated form of gelonin; NKC: natural killer cells; HTR: human transferrin receptor; EGF: epidermal growth factor; GM-CSF: granulocyte-macrophage colony-stimulating factor; DAB389: truncated Diphtheria toxin; B-CCL: B-cell chronic lymphocytic leukemia; RCC: renal cell carcinoma; GVHD: Graft-versus-host disease; EGFR: epidermal growth factor receptor; AML: acute myeloid leukemia; Fab: fragment antigen-binding; dsFv: disulfide-stabilized fragment variable; scFv: single-chain fragment variable; B-ALL: B-lineage Acute Lymphoblastic Leukemia; Fv: fragment variable; HCL: hairy cell leukemia; IL-2R: Interleukin-2 receptor; CR: complete response; CLL: chronic lymphocytic leukemia; ATL: adult T-cell leukemia; DARPins: designed Ankyrin repeat proteins; pmol: picomolar; HAMA: human-anti mouse antibody.

Despite significant progress in prostate cancer treatment, yet, it remains the leading diagnosed cancer and is responsible for high incidence of cancer related deaths in the U.S. Because of the insufficient efficacy of small molecule anti-cancer drugs, significant interest has been drawn to more potent macromolecular agents such as gelonin, a plant-derived ribosome inactivating protein (RIP) that efficiently inhibits protein translation. However, in spite of the great potency to kill tumor cells, gelonin lacks ability to internalize tumor cells and furthermore, cannot distinguish between tumor and normal cells. To address this challenge, we genetically engineered gelonin fusion proteins with varied numbers of F3 peptide possessing homing ability to various cancer cells and angiogenic blood vessels. The E. coli produced F3-gelonin fusion proteins possessed equipotent activity to inhibit protein translation in cell-free protein translation systems to unmodified gelonin; however, they displayed higher cell uptake that led to significantly augmented cytotoxicity. Compared with gelonin fusion with one F3 peptide (F3-Gel), tandem-multimeric F3-gelonins showed even greater cell internalization and tumor cell killing ability. Moreover, when tested against LNCaP s.c. xenograft tumor bearing mice, more significant tumor growth inhibition was observed from the mice treated with tandem-multimeric F3-gelonins. Overall, this research demonstrated the potential of utilizing tandem multimeric F3-modified gelonin as highly effective anticancer agents to overcome the limitations of current chemotherapeutic drugs.

The TNF-like weak inducer of apoptosis (TWEAK; TNFSF12) receptor Fn14 (TNFRSF12A) is expressed at low levels in normal tissues but frequently highly expressed in a wide range of tumor types such as lung, melanoma, and breast, and therefore it is a potentially unique therapeutic target for these diverse tumor types. We have generated a recombinant protein containing a humanized, dimeric single-chain anti–fibroblast growth factor-inducible 14-kDa protein (Fn14) antibody fused to recombinant gelonin toxin as a potential therapeutic agent (designated hSGZ). The hSGZ immunotoxin is a highly potent and selective agent that kills Fn14-positive (Fn14+) tumor cells in vitro. Treatment of cells expressing the MDR protein MDR1 (ABCB1B) showed no cross-resistance to hSGZ. Induced overexpression of Fn14 levels in MCF7 cells through HER2 (ERBB2) signaling translated to an improved therapeutic index of hSGZ treatment. In combination with trastuzumab, hSGZ showed an additive or synergistic cytotoxic effect on HER2+/Fn14+ breast cancer cell lines. Also, hSGZ treatment inhibited Erb3/Akt signaling in HER2-overexpressing breast cancer cells. Pharmacokinetic studies in mice revealed that hSGZ exhibited a biexponential clearance from plasma with a rapid initial clearance (t1/2α = 1.26 hours) followed by a seven-fold longer plasma half-life (t1/2β = 7.29 hours). At 24, 48, and 72 hours after injection, uptake of the hSGZ into tumors was 5.1, 4.8, and 4.7%ID/g, with a tumor-to-muscle ratio of 5.6, 6.2, and 9.0, respectively. Therapeutic efficacy studies showed significant tumor inhibition effects using an MDA-MB-231/Luc breast cancer xenograft model. Our findings show that hSGZ is an effective anticancer agent and a potential candidate for clinical studies. Cancer Res; 73(14); 4439–50. ©2013 AACR.

Plant-derived Type I toxins are candidate anticancer therapeutics requiring cytosolic delivery into tumor cells. We tested a concept for two-stage delivery, whereby tumor cells precoated with an antibody-targeted gelonin toxin were killed by exposure to endosome-disrupting polymer nanoparticles. Co-internalization of particles and tumor cell-bound gelonin led to cytosolic delivery and >50-fold enhancement of toxin efficacy. This approach allows the extreme potency of gelonin to be focused on tumors with significantly reduced potential for off-target toxicity.

To investigate the applicability of fusion biotoxins combining pore-forming toxins (PFTs) and ribosome-inactivating proteins (RIPs) for the anti-cancer treatment.Membrane active PFTs tend to destabilize cell membranes of tumor cells, but lack a warhead inducing significant cause of cell death. Cell-impermeable RIPs possess a powerful warhead, yet not able to enter the tumor cells. To address these challenges for anti-tumor effects, we introduced a fusion strategy of conjugating melittin (a PFT) and gelonin (a type 1 RIP) via chemical and recombinant methods, followed by in vitro assays and in vivo animal studies.In vitro characterization results confirmed that the chimeric gelonin-melittin fusion proteins retained equivalent intrinsic activity to that of unmodified gelonin in inhibiting protein translation. However, chemically conjugated gelonin-melittin (cGel-Mel) and recombinant chimeric gelonin-melittin fusion (rGel-Mel) exhibited greater cell uptake, yielding a significantly enhanced cytotoxic activity over treatment of gelonin, melittin or physical mixture of gelonin and melittin. Remarkably, cGel-Mel and rGel-Mel displayed 32- and 10-fold lower IC50 than gelonin in the cell lines. The superior anti-tumor efficacy of multivalent cGel-Mel to monovalent rGel-Mel suggested that valency could be a crucial factor for the extent of melittin-mediated cell uptake. Tumoricidal effects observed from animal studies were in good accordance with our findings from the cellular assays.This study successfully demonstrated that fusion of biotoxins could provide a simple yet effective way to synergistically augment their anti-tumor activity.

This article details the development and validation of an enzyme-linked immunosorbent assay (ELISA) for the quantification of gelonin in mouse plasma. The ELISA was validated for intra- and inter-day variability and for accuracy over a standard curve range of 7.5-100 ng/mL. The assay was then applied to assess gelonin pharmacokinetics in mice. Results from the ELISA were compared to data obtained from a parallel study conducted with (125)Iodine-labeled gelonin, with quantification via gamma counting. The ELISA demonstrated good precision, as the percent coefficient of variation of quality control samples in intra-day and inter-day validation ranged from 5.4-9.3% and 2.9-7.3%, respectively. Sample recoveries ranged from 98.3-105% of nominal values. The ELISA method yielded lower plasma concentrations of gelonin than found from the less-specific gamma counting method. Consequently, pharmacokinetic analyses yielded significantly higher estimates for volume of distribution (106 ± 31 vs. 55.8 ± 13 mL/kg) and plasma clearance (34.7 ± 6.6 vs. 10.9 ± 2.1 mL/min/kg) for data determined by ELISA vs. by gamma counting.

Recombinant immunotoxins, consisting of single-chain variable fragments (scFv) genetically fused to polypeptide toxins, represent potentially effective candidates for cancer therapeutics. We evaluated the affinity of various anti-Her2/neu scFv fused to recombinant gelonin (rGel) and its effect on antitumor efficacy and off-target toxicity. A series of rGel-based immunotoxins were created from the human anti-Her2/neu scFv C6.5 and various affinity mutants (designated ML3-9, MH3-B1, and B1D3) with affinities ranging from 10−8 to 10−11 mol/L. Against Her2/neu-overexpressing tumor cells, immunotoxins with increasing affinity displayed improved internalization and enhanced autophagic cytotoxicity. Targeting indices were highest for the highest affinity B1D3/rGel construct. However, the addition of free Her2/neu extracellular domain (ECD) significantly reduced the cytotoxicity of B1D3/rGel because of immune complex formation. In contrast, ECD addition had little impact on the lower affinity constructs in vitro. In vivo studies against established BT474 M1 xenografts showed growth suppression by all immunotoxins. Surprisingly, therapy with the B1D3-rGel induced significant liver toxicity because of immune complex formation with shed Her2/neu antigen in circulation. The MH3-B1/rGel construct with intermediate affinity showed effective tumor growth inhibition without inducing hepatotoxicity or complex formation. These findings show that while high-affinity constructs can be potent antitumor agents, they may also be associated with mistargeting through the facile formation of complexes with soluble antigen leading to significant off-target toxicity. Constructs composed of intermediate-affinity antibodies are also potent agents that are more resistant to immune complex formation. Therefore, affinity is an exceptionally important consideration when evaluating the design and efficacy of targeted therapeutics. Mol Cancer Ther; 11(1); 143–53. ©2011 AACR.

Cancer is one of the leading causes of death around the world. Tumor-targeted drug delivery is one of the major areas in cancer research. Aptamers exhibit many desirable properties for tumor-targeted drug delivery, such as ease of selection and synthesis, high binding affinity and specificity, low immunogenicity, and versatile synthetic accessibility. Over the last several years, aptamers have quickly become a new class of targeting ligands for drug delivery applications. In this review, we will discuss in detail about aptamer-based delivery of chemotherapy drugs (e.g. doxorubicin, docetaxel, daunorubicin, and cisplatin), toxins (e.g. gelonin and various photodynamic therapy agents), and a variety of small interfering RNAs. Although the results are promising which warrants enthusiasm for aptamer-based drug delivery, tumor homing of aptamer-based conjugates after systemic injection has only been achieved in one report. Much remains to be done before aptamer-based drug delivery can reach clinical trials and eventually the day-to-day management of cancer patients. Therefore, future directions and challenges in aptamer-based drug delivery are also discussed.

TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor (FGF)-inducible 14 (Fn14) are a TNF superfamily ligand–receptor pair involved in many cellular processes including proliferation, migration, differentiation, inflammation, and angiogenesis. The Fn14 receptor is expressed at relatively low levels in normal tissues, but it is known to be dramatically elevated in a number of tumor types, including brain and breast tumors. Thus, it seems to be an excellent candidate for therapeutic intervention. We first analyzed Fn14 expression in human tumor cell lines. Fn14 was expressed in a variety of lines including breast, brain, bladder, skin, lung, ovarian, pancreatic, colon, prostate, and cervical cancer cell lines. We then developed an immunoconjugate containing a high-affinity anti-Fn14 monoclonal antibody (ITEM-4) conjugated to recombinant gelonin (rGel), a highly cytotoxic ribosome-inactivating N-glycosidase. Both ITEM-4 and the conjugate were found to bind to cells to an equivalent extent. Confocal microscopic analysis showed that ITEM4-rGel specifically and rapidly (within 2 hours) internalized into Fn14-positive T-24 bladder cancer cells but not into Fn14-deficient mouse embryonic fibroblasts. Cytotoxicity studies against 22 different tumor cell lines showed that ITEM4-rGel was highly cytotoxic to Fn14-expressing cells and was 8- to 8 × 104-fold more potent than free rGel. ITEM4-rGel was found to kill cells by inducing apoptosis with high-mobility group box 1 protein release. Finally, ITEM4-rGel immunoconjugate administration promoted long-term tumor growth suppression in nude mice bearing T-24 human bladder cancer cell xenografts. Our data support the use of an antibody–drug conjugate approach to selectively target and inhibit the growth of Fn14-expressing tumors. Mol Cancer Ther; 10(7); 1276–88. ©2011 AACR.

Photochemical internalisation (PCI) is a delivery technology that employs a sub-lethal form of photodynamic therapy (PDT) in which a photosensitiser is activated by light to break down intracellular membranes and release macromolecules into the cytosol where they can be biologically active. Although PCI does enhance the PDT killing of transplanted tumours in mice after local injection of the cytotoxic agent, gelonin, the redistribution of gelonin from intracellular organelles into the cytosol has only previously been demonstrated in vitro. This study is designed to understand the factors controlling the efficacy of PCI in vivo and to document the mechanism of action. Using the photosensitiser AlS(2)Pc in studies on normal rat liver, we have demonstrated in vivo that gelonin is initially taken up into lysosomes, but can be released into the cytosol using PCI. Furthermore, PCI enhances the PDT effect after systemic administration of gelonin (volume of necrosis increased x2.5 when gelonin is given one hour before light), with the remarkably low dose of 5 microg/kg (10,000 times lower than the LD50); in the absence of light, there is no effect with 500 microg/kg. These results suggest that PCI may have a useful role to play in the site specific activation of cytotoxic agents like gelonin, given at a dose level that has no effect in the absence of light.

The amino groups in the beta-subunit of ovine luteinizing hormone (oLH) were modified by thiolation using N-succinimidyl-3-(2-pyridyldithio) propionate so that it may be coupled in a disulfide linkage to similarly modified ribosome inactivating protein, gelonin. The modified beta-subunit was able to hybridize with free LH alpha-subunit and the complex retained full biological activity. However, when gelonin was coupled to the beta-subunit, the resulting conformational changes masked or eliminated the sites necessary for intersubunit recognition of the free alpha-subunit. This has important implications for the design in the synthesis of gonadotropin-toxin/drug conjugates.

The effectiveness of gelonin to arrest protein synthesis, thereby limiting the growth of cancer cells was studied by encapsulating it into liposomes. The protein was extracted from the seeds of Indian plant Gelonium multiflorum by ammonium sulfate precipitation and purified using cation-exchange and gel-filtration chromatography. Biological activity of purified gelonin was determined using a rabbit reticulocyte lysate assay in the cell-free translational experiments. Gelonin was encapsulated in conventional liposomes prepared by the dry film method in order to retain biological activity of the entrapped protein. Carcinogenesis was induced in Swiss albino mice by intravenous administration of DBN (10 mg kg(-1) body weight) at weekly intervals. Marker enzyme assays (GGT, AChE, and GST), GSH levels, cell proliferation assay, hepatocyte DNA analysis, histological examination of micro sections of liver tissues were parameters used to monitor carcinogenesis induction, and regression in mice. From the in vitro experiments conducted, it was observed that gelonin upon its encapsulation into liposome, resulted in significant destruction of the transformed liver cells by its cytotoxic effects that arrest protein synthesis. Various parameters studied to monitor regression also suggested mass cell destruction to liver upon administration of liposomal gelonin in mice exposed to DBN.

Human single-chain Fv directed against fibroblast growth factor receptor 3 (FGFR3) have been shown to block proliferation of RT112 bladder carcinoma cells in vitro. Here, we examined the ability of the recombinant gelonin toxin (rGel) to enhance this inhibitory effect in vitro and in vivo on the bladder cancer cell line RT112 and the corresponding xenografts. Immunotoxins were genetically engineered by fusing FGFR3-specific Fv fragments (3C) to the NH2 terminus of rGel and expressed as a soluble protein in Escherichia coli. The 3C/rGel fusion construct showed an IC50 of 200 nmol/L against log-phase RT112 cells compared with 1,500 nmol/L for free rGel. Immunofluorescence studies showed that the 3C/rGel construct internalized rapidly into the cytoplasm of RT112 cells within 1 h of exposure. The mechanism of immunotoxin-induced cell death was found to be mediated by apoptosis. RT112 tumor xenografts in severe combined immunodeficient mice treated with 50 mg/kg 3C/rGel exhibited considerable growth delay relative to control tumors and a significant reduction of 55% to 70% in mean tumor size. Immunohistochemical analysis showed that tumors from mice treated with 3C/rGel displayed considerable apoptotic damage compared with control groups. Subcellular location of FGFR3 in immunotoxin-treated tumors indicated a translocation of FGFR3 to the nuclear membrane in contrast to tumors from saline-treated controls. These results show that FGFR3-driven immunotoxins may be an effective therapeutic agent against human bladder and other tumor types overexpressing FGFR3. [Mol Cancer Ther 2008;7(4):862–73]

The purpose of this study is to evaluate the early response of tumors to a vascular-disrupting agent (VDA) VEGF121/recombinant toxin gelonin (rGel) using dynamic [(18)F]FPPRGD2 positron emission tomography (PET) and kinetic parameter estimation.Two tumor xenograft models: U87MG (highly vascularized) and A549 (moderately vascularized), were selected, and both were randomized into treatment and control groups. Sixty-minute dynamic PET scans with [(18)F]FPPRGD2 that targets to integrin αvβ3 were performed at days 0 (baseline), 1, and 3 since VEGF121/rGel treatment started. Dynamic PET-derived binding potential (BPND) and parametric maps were compared with tumor uptake (%ID/g) and the static PET image at 1 h after the tracer administration.The growth of U87MG tumor was obviously delayed upon VEGF121/rGel treatment. A549 tumor was not responsive to the same treatment. BPND of treated U87MG tumors decreased significantly at day 1 (p 0.05), and the difference was more significant at day 3 (p 0.01), compared with the control group. However, the tracer uptake (%ID/g) derived from static images at 1-h time point did not show significant difference between the treated and control tumors until day 3. Little difference in tracer uptake (%ID/g) or BPND was found between treated and control A549 tumors. Considering the tracer retention in tumor and the slower clearance due to damaged tumor vasculature after treatment, BPND representing the actual specific binding portion appears to be more sensitive and accurate than the semiquantitative parameters (such as %ID/g) derived from static images to assess the early response of tumor to VDA treatment.Quantitative analysis based on dynamic PET with [(18)F]FPPRGD2 shows advantages in distinguishing effective from ineffective treatment during the course of VEGF121/rGel therapy at early stage and is therefore more sensitive in assessing therapy response than static PET.

The number of epidermal growth factor receptor (EGFR)-targeting drugs in the development for cancer treatment is continuously increasing. Currently used EGFR-targeted monoclonal antibodies and tyrosine kinase inhibitors have specific limitations related to toxicity and development of resistance, and there is a need for alternative treatment strategies to maximize the clinical potential of EGFR as a molecular target. This study describes the design and production of a novel EGFR-targeted fusion protein, rGel/EGF, composed of the recombinant plant toxin gelonin and EGF. rGel/EGF was custom-made for administration by photochemical internalization (PCI), a clinically tested modality for cytosolic release of macromolecular therapeutics. rGel/EGF lacks efficient mechanisms for endosomal escape and is therefore minimally toxic as monotherapy. However, PCI induces selective and efficient cytosolic release of rGel/EGF in EGFR-expressing target cells by light-directed activation of photosensitizers accumulated selectively in tumor tissue. PCI of rGel/EGF was shown to be highly effective against EGFR-expressing cell lines, including head and neck squamous cell carcinoma (HNSCC) cell lines resistant to cetuximab (Erbitux). Apoptosis, necrosis and autophagy were identified as mechanisms of action following PCI of rGel/EGF in vitro. PCI of rGel/EGF was further shown as a highly tumor-specific and potent modality in vivo, with growth inhibitory effects demonstrated on A-431 squamous cell carcinoma (SCC) xenografts and reduction of tumor perfusion and necrosis induction in SCC-026 HNSCC tumors. Considering the small amount of rGel/EGF injected per animal (0.1 mg/kg), the presented in vivo results are highly promising and warrant optimization and production of rGel/EGF for further preclinical evaluation with PCI.

Vascular endothelial growth factor (VEGF) and its receptors (FLT-1 and KDR) are overexpressed by human bladder cancer cells and tumor endothelial cells, respectively. Strategies that target VEGF receptors hold promise as antiangiogenic therapeutic approaches to bladder cancer. A fusion protein of VEGF121 and the plant toxin gelonin (rGel) was constructed, expressed in bacteria, and purified to homogeneity. Cytotoxicity experiments of VEGF121/rGel on the highly metastatic 253J B-V human bladder cancer cell line demonstrated that the VEGF121/rGel does not specifically target these cells, whereas Western blot analysis showed no detectable expression of KDR. Treatment with VEGF121/rGel against orthotopically implanted 253J B-V xenografts in nude mice resulted in a significant suppression of bladder tumor growth (approximately 60% inhibition; P < .05) compared to controls. Immunohistochemistry studies of orthotopic 253J B-V tumors demonstrated that KDR is highly overexpressed in tumor vasculature. Immunofluorescence staining with antibodies to CD-31 (blood vessel endothelium) and rGel demonstrated a dramatic colocalization of the construct on tumor neovasculature. Treated tumors also displayed an increase in terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling staining compared to controls. Thus, VEGF121/rGel inhibits the growth of human bladder cancer by cytotoxic effects directed against the tumor vascular supply and has significant potential as a novel antiangiogenic therapeutic against human bladder cancer.

Tumors provide an extremely abnormal microenvironment that stimulates neovascularization from surrounding vessels and causes altered gene expression within vascular cells. Up-regulation of vascular endothelial growth factor (VEGF) receptors has allowed selective destruction of tumor vessels by administration of a chimeric protein consisting of VEGF121 coupled to the toxin gelonin (VEGF/rGel). We sought to determine whether there is sufficient up-regulation of VEGF receptors in endothelial cells participating in ocular neovascularization to permit a similar strategy. After intravenous injection of 45 mg/kg VEGF/rGel, but not uncoupled recombinant gelonin (rGel), there was immunofluorescent staining for rGel within choroidal neovascularization in mice and regression of the neovascularization occurred, demonstrating successful vascular targeting via the systemic circulation. Intraocular injection of 5 ng of VEGF/rGel also caused significant regression of choroidal neovascularization and regression of retinal neovascularization in two models, transgenic mice with expression of VEGF in photoreceptors and mice with ischemic retinopathy, whereas injection of 5 ng of rGel had no effect. These data suggest that the strategy of vascular targeting can be applied to nonmalignant neovascular diseases and could serve as the basis of a new treatment to reduce established ocular neovascularization.

Photochemical internalisation (PCI) is a technique for releasing biologically active macromolecules from endocytic vesicles by light activation of a photosensitiser localised in the same vesicles of targeted cells. This study investigated the PCI of the toxin gelonin as a way of enhancing the effect of photodynamic therapy (PDT) on a human malignant fibrous histiocytoma transplanted into nude mice using the photosensitiser disulphonated aluminium phthalocyanine (AlPcS(2a)). Pharmacokinetic studies after intraperitoneal administration showed that the serum level of AlPcS(2a) fitted a biexponential model (half-lives of 1.8 and 26.7 h). The tumour concentration was roughly constant up to 48 h, although fluorescence microscopy showed that the drug location was initially mainly vascular, but became intracellular by 48 h. To compare PDT with PCI, 48 h after intraperitoneal injection of 10 mg kg(-1) AlPcS(2a), and 6 h after direct intratumour injection of 50 microg gelonin (PCI) or a similar volume of phosphate-buffered saline (PDT controls), tumour-bearing animals were exposed to red light (150 J cm(-2)). Complete response was observed for more than 100 days in 50% of the PCI tumours but only 10% of the PDT tumours (P0.01). In tumours examined histologically 4 days after light delivery, the depth of necrosis was 3-4 mm after PDT, but 7 mm after PCI. The deeper effect after PCI demonstrates that the light fluence needed to kill tumour is less than with PDT. We conclude that PCI with gelonin can markedly enhance the effect of PDT on this type of tumour and may have a role clinically as an adjunct to surgery to control localised disease.

VEGF(121)/rGel, a fusion protein composed of the growth factor VEGF(121) and the recombinant toxin gelonin (rGel), targets the tumor neovasculature and exerts impressive cytotoxic effects by inhibiting protein synthesis. We evaluated the effect of VEGF(121)/rGel on the growth of metastatic MDA-MB-231 tumor cells in SCID mice. VEGF(121)/rGel treatment reduced surface lung tumor foci by 58% compared to controls (means were 22.4 and 53.3, respectively; P.05) and the mean area of lung colonies by 50% (210 +/- 37 m(2) vs 415 +/- 10 m(2) for VEGF(121)/rGel and control, respectively; P.01). In addition, the vascularity of metastatic foci was significantly reduced (198 +/- 37 vs 388 +/- 21 vessels/mm(2) for treated and control, respectively). Approximately 62% of metastatic colonies from the VEGF(121)/rGel-treated group had fewer than 10 vessels per colony compared to 23% in the control group. The VEGF receptor Flk-1 was intensely detected on the metastatic vessels in the control but not in the VEGF(121)/rGel-treated group. Metastatic foci present in lungs had a three-fold lower Ki-67 labeling index compared to control tumors. Thus, the antitumor vascular-ablative effect of VEGF(121)/rGel may be utilized not only for treating primary tumors but also for inhibiting metastatic spread and vascularization of metastases.

A number of protein toxins of bacterial and plant origin have cytosolic targets, and knowledge about these toxins have provided us with essential information about mechanisms that can be used to gain access to the cytosol as well as detailed knowledge about endocytosis and intracellular sorting. Such toxins include those that have two moieties, one (the B-moiety) that binds to cell surface receptors and another (the A-moiety) with enzymatic activity that enters the cytosol, as well as molecules that only have the enzymatically active moiety and therefore are inefficient in cell entry. The toxins discussed in the present article include bacterial toxins such as Shiga toxin and diphtheria toxin, as well as plant toxins such as ricin and ribosome-inactivating proteins without a binding moiety, such as gelonin. Toxins with a binding moiety can be used as vectors to translocate epitopes, intact proteins, and even nucleotides into the cytosol. The toxins fall into two main groups when it comes to cytosolic entry. Some toxins enter from endosomes in response to low endosomal pH, whereas others, including Shiga toxin and ricin, are transported all the way to the Golgi apparatus and the ER before they are translocated to the cytosol. Plant proteins such as gelonin that are without a binding moiety are taken up only by fluid-phase endocytosis, and normally they have a low toxicity. However, they can be used to test for disruption of endosomal membranes leading to cytosolic access of internalized molecules. Similarly to toxins with a binding moiety they are highly toxic when reaching the cytosol, thereby providing the investigator with an efficient tool to study endosomal disruption and induced transport to the cytosol. In conclusion, the protein toxins are useful tools to study transport and cytosolic translocation, and they can be used as vectors for transport to the interior of the cell.

The poly(amidoamine)s (PAAs) ISA 1 and ISA 23 display pH-dependent conformational change and pH-dependent membrane perturbation. These properties confer potential for use as endosomolytic polymers for intracytoplasmic delivery of toxins and genes. Both polymers are relatively non-toxic, and moreover ISA 23 has the beneficial property in vivo, of being non hepatotropic when administered intravenously. Although ISA 23 and ISA 1 demonstrate ability to transfect cells, ISA 1 is also able to promote intracellular delivery of non-permeant toxins. The aim of this study was to synthesise random and block copolymers of ISA 1 and ISA 23 and investigate whether these second generation hybrids would allow optimisation of PAA biological characteristics. Random and block copolymers of ISA 1 and ISA 23 were synthesised by hydrogen transfer polyaddition to generate a library of PAAs with an ISA 23:ISA 1 molar ratios of 2:1 to 4: 1. The resultant polymers have a pI slightly below 7.4 and a (M) over bar (w) of 19900-49000 g/mol and a (M) over bar (n) of 13100-24100 g/mol. Whereas none of the random or block copolymers were haemolytic at pH 7.4 all demonstrated pH-dependent membrane activity. At pH 5.5 they caused 50-60% haemoglobin (Hb) release over 1 h. This was slightly less than that seen for ISA 23 (80% Hb release). None of the copolymers were cytotoxic against B16F10 cells during a 72 h incubation (IC50 > 2 mg/ml; MTT assay). The ability of the random and block copolymer PAAs to deliver the toxin gelonin was also examined, but only ISA 1 and the block copolymer B2 (ISA 23:ISA 1 at a 2:1 molar ratio) were able to promote intracellular delivery, as measured by cytotoxic activity. It would be interesting to study the body distribution of B2 and determine whether this toxin-delivering PAA is able to escape liver capture.

Intravenous injection of gelonin and deglycosylated gelonin led to rapid clearance from the blood. Both molecules distributed similarly in liver and kidney suggesting that they followed the same pathway. Deglycosylation reduced the uptake by a third in liver, but did not affect uptake by kidney. Studies with Triton WR1339 showed a classical lysosomal pathway for both molecules. The deglycosylated molecule was degraded to a greater extent than native gelonin as seen by the presence of acid soluble radioactivity. Cell separation showed that while endothelial cells mainly took up native gelonin, Kupffer cells took up the deglycosylated molecule.

Gelonin is a type I plant toxin that has potential as an effective anti-tumor agent by virtue of its enzymatic capacity to inactivate ribosomes and arrest protein synthesis, thereby effectively limiting the growth of cancer cells. Being a hydrophilic macromolecule, however, gelonin has limited access to its target subcellular compartment, the cytosol; it is effectively plasma membrane-impermeant and subject to rapid degradation within endosomes and lysosomes upon cellular uptake as it lacks the membrane-translocating capability that is typically provided by a disulfide-linked B polypeptide found in the type II toxins (e.g. ricin). These inherent characteristics generate the need for the development of a specialized cytosolic delivery strategy for gelonin as an effective anti-tumor therapeutic agent. Here we describe an efficient means of delivering gelonin to the cytosol of B16 melanoma cells. Gelonin was co-encapsulated inside pH-sensitive liposomes with listeriolysin O, the pore-forming protein that mediates escape of the intracellular pathogen Listeria monocytogenes from the endosome into the cytosol. In in vitro experiments, co-encapsulated listeriolysin O enabled liposomal gelonin-mediated B16 cell killing with a gelonin IC50 of approximately 0.1 nM with an extreme efficiency requiring an incubation time of only 1 h. By contrast, cells treated with equivalent concentrations of unencapsulated gelonin or gelonin encapsulated alone in pH-sensitive liposomes exhibited no detectable cytotoxicity. Moreover, treatment by direct intratumor injection into subcutaneous solid tumors of B16 melanoma in a mouse model showed that pH-sensitive liposomes containing both listeriolysin O and gelonin were more effective than control formulations in curtailing tumor growth rates.

We have previously reported the identification of a unique thymocyte-specific surface molecule, JL1, which was detected using the monoclonal antibody (mAb), anti-JL1. Interestingly, JL1 was shown to be expressed in most leukemias, irrespective of their immunophenotype, and subpopulations of normal bone marrow (BM) mononuclear cells (MNCs). Here we investigated the potential usefulness of the anti-JL1 mAb as a therapeutic tool for leukemia. We demonstrated that the proliferation of cultured human leukemia cells was dramatically inhibited in vitro by anti-JL1 mAb conjugated with the polypeptide toxin, gelonin, but not by gelonin alone. We then systematically investigated the reactivity of the anti-JL1 mAb against normal human tissues to evaluate possible side effects along with various hematopoietic and nonhematopoietic tumor cell lines. All of 33 types of normal tissues except thymus and subpopulation of BM MNCs were clearly devoid of JL1 expression. Among tumor cell lines, all the nonhematopoietic cell lines tested were negative for JL1 expression, while some hematopoietic cell lines contained JL1 antigen. Collectively, the results showed the cytotoxic effects of anti-JL1-based immunotoxin against JL1-positive leukemic cells, sparing most normal tissues other than thymocytes and some BM MNCs. Therefore, we strongly suggest that gelonin-conjugated anti-JL1 mAb immunotoxin could be developed as a potential immunotherapeutic agent in the treatment of various types of JL1-positive acute leukemias.

This study describes the synthesis, characterization, and reactivity of new methoxypoly(ethylene glycol) (mPEG) derivatives containing a thioimidoester reactive group. These activated polymers are able to react with the lysyl epsilon-amino groups of suitable proteins, generating an amidinated linkage and thereby preserving the protein's positive charge. mPEG derivatives of molecular weight 2000 and 5000 Da were used, and two spacer arms were prepared, introducing chains of different lengths between the hydroxyl group of the polymer and the thioimidate group. These mPEG derivatives were used to modify gelonin, a cytotoxic single-chain glycoprotein widely used in preparation of antitumoral conjugates, whose biological activity is strongly influenced by charge modification. The reactivity of mPEG thioimidates toward lysil epsilon-amino groups of gelonin was evaluated, and the results showed an increased degree of derivatization in proportion to the molar excesses of the polymer used and to the length of the alkyl spacer. Further studies showed that the thioimidate reactive is able to maintain gelonin's significant biological activity and immunogenicity. On the contrary, modification of the protein with N-hydroxysuccinimide derivative of mPEG strongly reduces the protein's cytotoxic activity. Evaluation of the pharmacokinetic behavior of native and PEG-grafted gelonin showed a marked increase in plasma half-life after protein PEGylation; in particular, the circulating life of the conjugates increased with increased molecular weight of the polymer used. The biodistribution test showed lower organ uptake after PEGylation, in particular by the liver and spleen.

Patients with widespread cancer respond initially to combination chemotherapy, immunotherapy, and/or radiotherapy, but most relapse with chemoresistant disease. Novel methods of killing resistant neoplastic stem cells are needed. One such approach is therapy with targeted toxins composed of tumor cell selective ligands covalently linked to group I peptide toxins (group II and III peptide toxins act on the cell surface). The targeted toxin is delivered to the cell by a tumor selective ligand. Once bound, the ligand-receptor complex is internalized. The catalytic domain escapes to the cytosol. The toxin then enzymatically modifies a critical cell function (protein synthesis, p21 Rho activity, protein kinase signaling, cyclic AMP signaling or others). The irreversibly damaged cells fails to divide and, eventually, undergoes lysis or programmed cell death. Targeted peptide toxins used to date in the treatment of chemotherapy refractory cancers include ricin toxin, Pseudomonas exotoxin, pokeweed antiviral protein, saporin, gelonin and diphtheria toxin. In this review, we have focused on the applications of genetically engineered diphtheria toxin for cancer therapy.

The use of toxins for cancer therapy has great promise. Gelonin, a potent plant toxin, causes cell death by inactivating the 60S ribosomal subunit. Recently, we developed a novel gene delivery system using biodegradable cationic heparin-polyethyleneimine (HPEI) nanogels. In the current study, the antitumor activity of a recombinant plasmid expressing gelonin (pGelonin) on human ovarian cancer was assessed. The application of HPEI nanogels, was also evaluated. Gelonin-cDNA was cloned into the pVAX1 plasmid vector and transfected into SKOV3 human ovarian cancer cells using biodegradable cationic HPEI nanogels. The expression of gelonin in vitro and in vivo was confirmed using RT-PCR and western blot analysis. Cell viability and apoptosis were examined using an MTT assay and flow cytometric analysis. For the in vivo study, an SKOV3 intraperitoneal ovarian carcinomatosis model was established, and nude mice were randomly assigned into four groups receiving i.p. administration of pGelonin/HPEI complexes, pVAX/HPEI complexes, HPEI alone and 5% glucose solution. The tumor weight was monitored, and a TUNEL assay and Ki-67 immunohistochemistry were performed to evaluate apoptosis and cell proliferation in the tumor tissue sections, respectively. Gelonin was efficiently expressed in SKOV3 cancer cells in vitro and in vivo using pGelonin incorporated with HPEI nanogels. The pGelonin/HPEI complexes inhibited cell viability and induced apoptosis in the cell culture. Treatment for intraperitoneal carcinomatosis with pGelonin/HPEI complexes reduced the tumor weight by ~58.55% compared to the control groups (P

The bottlenecks of current chemotherapy in the treatment of colorectal cancer lie in the ineffectiveness of the existing anti-cancer small molecule drugs as well as the dose-limiting toxicity caused by the nonselective action on normal tissues by such drugs. To address these problems, we introduce a novel therapeutic strategy based on tumor targeting using a non-internalizing anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb) and intracellular delivery of the extremely potent yet cell-impermeable protein toxin gelonin via the aid of a cell-penetrating peptide (also termed as protein transduction domain; PTD). A chimeric TAT-gelonin fusion protein was genetically engineered, and it displayed remarkably enhanced anti-cancer activity against human colorectal cancer cells, with IC50 values being several orders of magnitude lower than the unmodified gelonin. On the other hand, a chemically synthesized conjugate of heparin and a murine anti-CEA mAb, T84.66 (termed T84.66-Hep) was found able to bind highly specifically to CEA over-expressing LS174T colorectal cancer cells. When mixing together, TAT-gelonin and T84.66-Hep could associate tightly and automatically through an electrostatic interaction between the cationic TAT and anionic heparin. In preliminary in vivo studies using LS174T s.c. xenograft tumor bearing mouse, selective and significantly augmented (58-fold) delivery of TAT-gelonin to the tumor target was observed, when compared with administration of TAT-gelonin alone. More importantly, efficacy studies also revealed that only the TAT-gelonin/T84.66-Hep complex yielded a significant inhibition of tumor growth (46%) without causing gelonin-induced systemic toxicity. Overall, this study suggested a generic strategy to effectively yet safely deliver potent PTD-modified protein toxins to the tumor.

Photochemical internalization (PCI) is a unique procedure for site-specific delivery of several types of membrane-impermeable molecules to the cytosol of target cells. The technology is based on photochemical-induced release of endocytosed macromolecules from endosomes and lysosomes into the cytosol. The purpose of this study was to evaluate the therapeutic potential of PCI of the type I ribosomal-inactivating protein gelonin in an animal model. The photosensitizer aluminum phthalocyanine disulfonate (AlPcS2a) was injected intraperitoneally (10 mg/kg) into athymic female BALB/c (nu/nu) nude mice (8–9 mice per group) with subcutaneously growing human adenocarcinoma (WiDr) tumors 48 hr before exposure to 135 J/cm2 of red light focused on the tumor. Six hours before light exposure a single dose of 50 μg gelonin was administrated intratumorally. Tumor growth was measured at least twice a week. After immunomagnetic separation of in vivo growing tumor cells the subcellular localization of the photosensitizer was evaluated by fluorescence microscopy. The photosensitizer localized in endocytic vesicles in in vivo growing WiDr cells. Furthermore, it was found that in vitro gelonin treatment of WiDr cells isolated from photosensitizer-treated mice potentiated a light-induced decrease of clonal survival. Complete remission in 6 of 9 (67%) of the treated mice were induced. Our findings indicate that photochemical treatment with the photosensitizer AlPcS2a activates the cytotoxic potential of gelonin in vivo. These results demonstrate that the synergistic effect of combining photoactivation of photosensitizer located in endocytic vesicles and gelonin is indeed a result of PCI of gelonin. © 2001 Wiley-Liss, Inc.

Immunotoxins are a class of targeted therapeutic agents under development by various research groups. The murine monoclonal antibody designated ZME-018 recognizes a high molecular weight glycoprotein present on most human melanoma cells and biopsy specimens and has been utilized for clinical imaging studies in patients with melanoma. The plant toxin gelonin is a ribosome-inactivating protein (RIP) with n-glycosidase activity similar to that of ricin A chain. In previous studies by our group, the gelonin toxin was sequenced, cloned and expressed in E. coli. The purified recombinant gelonin (RG) was found to have identical protein synthesis inhibitory activity to that of natural gelonin (NG). For comparative purposes, chemical conjugates of antibody ZME and either RG or NG were produced using the heterobifunctional crosslinking reagents SPDP and SMPT. The ZME-NG and ZME-RG immunotoxins were found to be 10(4)- to 10(5)-fold more cytotoxic to antigen-positive human melanoma cells than free toxin. NG toxin alone was cytotoxic to intact cells (IC(50) = 100 nM) while RG was nontoxic to cells at doses up to 1 microM. Both ZME-NG and ZME-RG immunoconjugates were nontoxic to antigen-negative (Me-180) cells. ZME-RG immunotoxins constructed with the more stable SMPT reagent were slightly more effective in culture than conjugates made with SPDP. Tissue distribution studies in tumor-bearing nude mice demonstrated that tumor uptake of the ZME-RG immunotoxin was similar to that of the intact ZME antibody with reduced distribution to normal organs compared to an immunoconjugate produced with NG. Pharmacokinetic studies showed that the terminal-phase plasma half-life of ZME-RG was similar to that of ZME itself (42 h vs 50 h) and almost threefold higher than that of ZME-NG (11.5 h). The area under the concentration curve (Cxt) for ZME-RG was 50% lower than that for ZME due to an increased apparent volume of distribution (Vd(a)) but was almost tenfold higher than the Cxt for ZME-NG. These studies suggest that immunoconjugates comprising RG demonstrate identical in vitro cytotoxic effects to immunoconjugates produced with NG and immunotoxins with RG display improved in vivo pharmacodynamics and tissue distribution compared to immunotoxins containing NG.

Missile therapy, which destroys cancer cells specifically, has been advocated as an effective modality for the treatment of carcinoma. We have developed an immunoconjugate consisting of the monoclonal antibody MSN-1 (IgM), which reacts strongly with endometrial adenocarcinomas, combined with a plant hemitoxin named gelonin via a disulfide bond using N-succinimidyl-3-(2-pyridyldithio) propionate and 2-iminothiolane, and examined its selective cytotoxicity in athymic mice. The reductions in resected weights of target tumor cells, at the local site of MSN-1-gelonin immunoconjugate treatment, were 96% with local administration and 75% with caudal vein administration, as compared with the untreated group. There was no weight loss in treated mice. Our results suggest that this MSN-1-gelonin immunoconjugate has potential clinical applications in the treatment of endometrial adenocarcinomas.

UNLABELLED Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide. However, there is currently no effective therapy strategy in the clinical practice. Recombinant phytotoxin gelonin fused to other factors have been used to treat different cancers. But there have been no reports of gelonin gene therapy. In this study, we have constructed a recombinant plasmid which contained a tumor-specific survivin promoter to drive phytotoxin gelonin (pSur-Gel). And the cytotoxicity effects of pSur-Gel in HCC were also validated both in vitro and in vivo. The expression level of survivin was detected in different liver cancer cell lines and normal liver cell lines by western blot analysis, and a survivin promoter-driven green fluorescent protein (GFP) expression vectors (pSur-GFP) was also tested in liver cancer cell line HepG2 and normal liver cell line LO2. Moreover, phytotoxin gelonin expression experiment and cytotoxicity experiment of pSur-Gel was performed in HepG2 cells and LO2 cells in vitro. Furthermore, anti-tumor effect of pSur-Gel against HepG2 xenografts and toxicity of this gene were evaluated in the mice model. Finally, LDH release assay, apoptosis assay and immunoblot analyse LC3 conversion (LC3-I to LC3-II) were tested. We found that the expression of survivin protein was higher in liver cancer cell lines compared with the normal liver cells. Further study showed that the pSur-GFP and pSur-Gel was expressed specially in liver cancer cell other than in normal liver cells. pSur-Gel plasmid could effectively inhibit the proliferation of liver cancer cells (*P

Targeted endocytic uptake is a first step toward tissue-specific cytoplasmic macromolecular delivery; however, inefficient escape from the endolysosomal compartment makes this generally impractical at present. We report here a targeted cytolysin approach that dramatically potentiates endosomal release of an independently targeted potent gelonin immunotoxin. Fibronectin domains engineered for affinity to EGF receptor or carcinoembryonic antigen were fused to the plant toxin gelonin or bacterial pore-forming cytolysins. These fusion proteins display synergistic activity in both antigen-specific cytotoxicity in vitro, enhancing potency by several orders of magnitude, and in tumor growth inhibition in vivo. In addition, the number of internalized gelonin molecules required to induce apoptosis is reduced from approximately 5 × 106 to less than 103. Targeted potentiation shows promise for enhancing cytoplasmic delivery of other macromolecular payloads such as DNA, siRNA, and miRNA., National Science Foundation (U.S.) (Grant U54CA119349), National Institutes of Health (U.S.) (Grant CA101830), National Institutes of Health (U.S.) (Grant AI065824), National Science Foundation (U.S.). Graduate Research Fellowship

The ineffectiveness of small molecule drugs against cancer has generated significant interest in more potent macromolecular agents. Gelonin, a plant-derived toxin that inhibits protein translation, has attracted much attention in this regard. Due to its inability to internalize into cells, however, gelonin exerts only limited tumoricidal effect. To overcome this cell membrane barrier, we modified gelonin, via both chemical conjugation and genetic recombination methods, with low molecular weight protamine (LMWP), a cell-penetrating peptide (CPP) which was shown to efficiently ferry various cargoes into cells. Results confirmed that gelonin-LMWP chemical conjugate (cG-L) and recombinant gelonin-LMWP chimera (rG-L) possessed N-glycosidase activity equivalent to that of unmodified recombinant gelonin (rGel); however, unlike rGel, both gelonin-LMWPs were able to internalize into cells. Cytotoxicity studies further demonstrated that cG-L and rG-L exhibited significantly improved tumoricidal effects, with IC50 values being 120-fold lower than that of rGel. Moreover, when tested against a CT26 s.c. xenograft tumor mouse model, significant inhibition of tumor growth was observed with rG-L doses as low as 2 μg/tumor, while no detectable therapeutic effects were seen with rGel at 10-fold higher doses. Overall, this study demonstrated the potential of utilizing CPP-modified gelonin as a highly potent anticancer drug to overcome limitations of current chemotherapeutic agents.

Antibodies and antibody domains are ideal agents for targeting the surface of cells, and fusion proteins between cell-targeting domains and cytotoxic proteins may be particularly effective therapeutic reagents. We constructed a family of immunofusion proteins linking the humanized Fab, F(ab')2, or single-chain antibody form of the H65 antibody (which recognizes the CD5 antigen on the surface of human T cells) with the plant ribosome-inactivating protein gelonin. To maximize the product yield and simplify the production process, each fusion protein was linked to a bacterial signal sequence for expression in E. coli as a secreted protein. More than 30 fusion genes were assembled with antibody domains and gelonin in various physical orientations. Each immunofusion accumulated in the bacterial culture supernatant in a properly folded, active form. Bacteria transformed with each fusion gene were then grown in a fermentor, and product was recovered from the cell-free fermentation broth by column chromatography. All of the immunofusion proteins were purified by a single process and each was tested for cytotoxicity toward antigen-positive human cells. A compact cGMP fermentation area was built to manufacture these fusion proteins. Our integrated approach to microbial protein production, including molecular genetics, bacterial fermentation, and initial isolation, is described in detail.

In this study we investigated the possibility of treating Heymann's Nephritis (HN) by destroying antibody producing cells by targetting a toxin, gelonin - conjugated to gp330, the renal brush border antigen. HN was induced in rats by immunizing them with purified gp330. The gelonin-gp330 conjugate was administered 12 days after the antigenic challenge. Serum was screened for circulating antibodies. Proteinurea was estimated. The gp330-gelonin conjugate-treated animals had a circulating antibody titre in the serum much lower than that of diseased (untreated) animals. Proteinurea seen in diseased animals was not observed in treated animals. This work suggests the possibility of using a toxin-antigen conjugate for immunomodulating antibody mediated autoimmune renal disease.

In this study, we compared the ability of different bispecific monoclonal antibodies (BsmAb) and immunotoxins to deliver the type 1 ribosome-inactivating proteins (RIP) saporin and gelonin through the CD25 or CD30 target molecules to Hodgkin's lymphoma cells. An anti-CD25/antisaporin and an anti-CD30/antisaporin BsmAb enhanced the toxicity of the relevant RIP against the CD25+CD30+ L540 Hodgkin's lymphoma cell line, although targeting by anti-CD30 BsmAb appeared eight times more efficient. Two anti-CD30/antigelonin BsmAb, reacting with different epitopes of the gelonin molecule, were able to enhance gelonin toxicity against L540 cells and had a synergistic effect when used in combination. Among CD25-CD30+ Hodgkin's lymphoma lines, which were resistant to targeting by anti-CD25/saporin BsmAb, one (L428) was sensitive to both gelonin and saporin delivered by anti-CD30 BsmAb. Another CD25-CD30+ cell line (COLE) was completely resistant to the toxic effect of gelonin targeted by the two synergistic BsmAb, as well as to an anti-CD30/gelonin immunotoxin. However, these cells were partially sensitive to saporin delivered by an anti-CD30/anti-saporin BsmAb, and they were efficiently killed by an anti-CD30/saporin immunotoxin. These results indicate that heterogeneity in the sensitivity to certain RIP, such as gelonin, exists among tumor cells of the same histotype.

Antibody ZME-018 is directed against the gp240 glycoprotein on the surface of more than 80% of human melanoma cell lines and fresh biopsy specimens. Previous studies in our laboratory described the in vitro cytotoxicity and specificity of an immunoconjugate composed of mAb ZME-018 and the plant toxin gelonin. The present study described the in vivo pharmacokinetics and therapeutic effects of ZME-gelonin in human xenograft/nude mouse models. Pharmacokinetic studies of 125I-labeled ZME-018 and ZME-gelonin demonstrated a shorter terminal-phase plasma half-life of the immunoconjugate than native ZME (20.6 h compared to 41.3 h). The initial volume of distribution of the ZME-gelonin was also higher compared to that of ZME alone (2.85 ml compared to 1.94 ml) suggesting an enhanced distribution of the conjugate outside the vasculature. The corresponding area under the concentration/time curve for the ZME-gelonin conjugate was 40% lower than that of ZME alone (80.8 compared to 139.6 microCi.ml-1 x min). In nude mice bearing well-developed human tumor A375 melanoma xenografts, administration of 125I-labeled ZME and ZME-gelonin resulted in tumor-to-blood ratios of 1.9 +/- 0.5 and 1.5 +/- 0.6 respectively by 72 h. Compared with ZME, ZME-gelonin conjugate caused an increase in the content of radiolabel in kidney, spleen and liver. Treatment of nude mice bearing well-developed (150 mm3) s.c. A375-M xenografts with divided doses of ZME-gelonin, ZME, gelonin, or saline resulted in suppression of tumor growth in the immunotoxin group but virtually no retardation of tumor growth in the control groups. Using a murine model for a rapidly growing lethal metastatic human melanoma, treatment with ZME-gelonin resulted in a mean survival of 44 days, 213% increase in mean survival time compared with the saline treatment (14.2 +/- 2 day survival). Given these encouraging results, we are proceeding with further preclinical development of this immunotoxin.

An immunotoxin specific for cells infected with human cytomegalovirus (HCMV) was constructed by attaching the ribosome-inactivating enzyme, gelonin, through a disulfide linkage to polyclonal human immunoglobulin (IgG). In uninfected cells, there was no difference between [35S]methionine incorporation in untreated cultures and those treated with immunotoxin at 100 micrograms/ml. In HCMV-infected cells, there was a significant decrease in [35S]methionine incorporation in the immunotoxin-treated cultures, suggesting a selective cytotoxic effect on the virus-infected cells. An immunotoxin specific for murine cytomegalovirus (MCMV) was prepared by linking gelonin to polyclonal anti-MCMV IgG. Using this same parameter for assay of cytotoxicity, the anti-MCMV immunotoxin had a 50% cytotoxic concentration of 35 micrograms/ml in MCMV-infected cells and greater than 200 micrograms/ml in uninfected cells. MCMV yields measured at 7 days postinoculation were reduced by 2 log10 in cultures treated with immunotoxin at 20 micrograms/ml at 1 day postinoculation. These data suggest immunotoxins may have potential for eliminating CMV-infected cells from the host.

Inactivation of ribosomes by gelonin, a ribosome-inactivating protein with RNA N-glycosidase activity on 28 S rRNA, requires macromolecular cofactors present in post-ribosomal supernatants. One of these cofactors has been purified from a rat liver extract and identified as an RNA about 70 nt long which in sequence analysis shows a high level of similarity with mammalian (bovine) tRNA(Trp). The pattern of the sequencing gel is consistent with the co-existence in the preparation of two 3′-immature tRNA(Trp) species, missing only A75, or both A75 and C74. In the presence of ATP, CTP and tRNA nucleotidyltransferase, the gelonin-stimulating RNA is a good acceptor of tryptophan. An oligodeoxynucleotide complementary to positions 55 to 72 of mammalian (bovine) tRNA(Trp) hybridizes with the gelonin-stimulating RNA as demonstrated by gel mobility shift and ribonuclease H digestion. The oligodeoxynucleotide-directed ribonuclease H treatment also abolishes the gelonin-promoting activity of crude preparations of RNA, giving strong evidence that the only active RNA is a tRNA(Trp)-like molecule.

It is well established that bispecific antibodies (BsAbs) can be used effectively in targeting the ribosome-inactivating protein (RIP), saporin, against neoplastic B cells. We have now extended this delivery system for use with gelonin. By measuring antigen-binding characteristics and epitope mapping a panel of anti-gelonin MAbs using the IAsys resonant mirror bisensor, we were able to rapidly select the most suitable for making BaAbs. The Fab' fragments from these MAbs were chemically conjugated with Fab' from either anti-CD22 or anti-CD38. Cytotoxicity assays showed that BsAbs were highly efficient at delivering gelonin to cultured Daudi cells and achieved levels of toxicity which correlated closely with the affinity of the BsAbs. Using pairs of anti-CD22 BsAbs we were able to generate bivalent BsAb-gelonin complexes which achieved IC50 values of 2 x 10(-11) M gelonin, a potency which is equivalent to that reached by saporin in this targeting system. However, because gelonin is 5-10 times less toxic than saporin, the therapeutic ratio for gelonin is superior, making it potentially a more useful agent for human treatment. Cytotoxicity assays and kinetic analysis showed that targeting gelonin via CD38 was 2-5 times less effective than delivery through CD22. However, with a pair of BsAbs designed to co-target gelonin via CD22 and CD38, the cytotoxicity achieved equalled that obtained with a pair of anti-CD22 BsAbs (IC50 = 1 x 10(-11) M). This important result suggests that the anti-CD38 helps bind the gelonin to the cell and is then 'dragged' or 'piggy-backed' into the cell by the anti-CD22 BsAb. The implication of these findings for cancer therapy is discussed.

1. 1. The actions of various compounds with antiproliferative and/or immunomodulatory activities including the ribosome-inactivating protein gelonin, the tetrapeptide tuftsin, the opioid peptide leucine-enkephalin, the antireproductive tripeptide Thr-Ser-Lys, the melatonin analog 6-methoxy-2-benzoxazolinone, and the amino acid derivatives 5-hydroxytryptamine and oxalysine on mouse embryonic development and organogenesis in vitro were investigated. 2. 2. The various compounds were tested up to a concentration of 300 μg/ml. It was found that only 6-methoxy-2-benzoxazolinone, a derivative of tryptophan, produced an increase in the number of abnormal embryos and reductions in the final somite numbers and axial lengths of embryos. 3. 3. 6-Methoxy-2-benzoxazolinone treatment also resulted in an increased incidence of abnormal morphogenesis of various organ primordia, including abnormal yolk sac circulation, twisting of body axis, absence of forelimb bud and opening of cranial neural tube.

SUMMARY Ribosomal inactivating proteins such as gelonin (Gel) and ricin A chain (RTA) conjugated to MoAbs bind to specific target cells, and upon internalization inhibit protein synthesis, ultimately resulting in cell death. We report here that Gel anti-T cell MoAb conjugates are more cytotoxic than RTA conjugates when tested against human peripheral blood mononuclear cells (PBMC). This increased cytotoxicity is observed whether Gel is conjugated to the anti-T cell MoAb or to an anti-mouse immunoglobulin Fab′ fragment which then binds to the murine anti-human T cell MoAb. Gel conjugates are not only effective at lower concentrations, but also produce a greater extent of inhibition of cellular proliferation. Moreover, a 10 min exposure to a Gel conjugate is as effective as a 90 h exposure to an RTA conjugate. When part of anti-T cell F(ab′)2 or Fab′ conjugates, Gel affects the early steps in cellular intoxication more than RTA, Gel conjugates bind more avidly and accelerate the modulation of antigen. In contrast, when part of whole IgG conjugates, Gel does not affect the binding to or modulation of surface antigen compared with RTA, while it does increase conjugate cytotoxicity. These observations suggest that Gel may be delivered more efficiently into the cytosol than RTA. A divergent intracellular pathway for Gel is also supported by the inability of chemical potentiators, which strongly enhance RTA potency, to affect Gel potency. These properties of Gel might also be advantageous for targeted immunoconjugates made with other MoAbs or receptor-binding molecules.

This study was carried out to evaluate our monoclonal antibody (MoAb) to alpha-fetoprotein (AFP), 80G, as a carrier for targeting AFP-producing hepatoma. Pharmacokinetic analysis showed that the MoAb 80G was actively incorporated into AFP-producing HuH-7N cells (xenograft of human hepatoma cell line, HuH-7) in nude mice. Four conjugates composed of MoAb 80G, and a type 1 ribosome-inactivating protein, gelonin, were prepared. They involve two disulfide-linked and two thioether-linked conjugates. The binding activity of conjugates against AFP remained as high as that of intact 80G according to enzyme-linked immunosorbent assay. The in vitro cytotoxic effects of all the conjugates were specific against AFP-producing HuH-7 cells. Of these conjugates, two containing gelonin modified with 2-iminothiolane were more potent than the others. They showed significant antitumor activity upon AFP-producing HuH-7N cells in nude mice. However, the disulfide conjugate was more toxic to mice than the thioether conjugate judging from the loss in body weight and the liver damage. These results suggest that our MoAb 80G is a suitable carrier for targeting AFP-producing hepatoma cells, and that the noncleavable thioether conjugate is promising as an AFP-producing hepatoma-targeted drug delivery system.

Conjugates of IL-2 with the ribosome-inactivating protein gelonin were prepared using heterobifunctional reagents to link the proteins via disulfide, acid-labile, and noncleavable linkers. In each case, one protein was modified using 2-iminothiolane. The sulfhydryl groups so introduced were then reacted either with 2-nitro-5-dithiobenzoate groups or with iodoacetamido groups which had been introduced into the second protein. In the case of the acid-labile linkage, a reagent which forms a labile bond upon reaction with amino groups, 4-(iodoacetamido)-1-cyclohexene-1,2-dicarboxylic acid anhydride (its synthesis is described in this paper) was used to modify the toxin. The conjugates were separated from nonconjugated proteins by gel filtration on Sephadex G100 (SF). Each was analyzed with respect to its ribosome-inactivating activity, its ability to bind to the IL-2 receptor, and its in vitro cytotoxicity. The ribosome-inactivating activity of gelonin was unaffected by modification with 2-iminothiolane and was retained in conjugates prepared using this reagent. Modification of the toxin with 4-(iodoacetamido)-1-cyclohexene-1,2-dicarboxylic acid anhydride to form the acid-labile link drastically reduced the activity of the toxin. However, the activity of the toxin was recovered following acid treatment to release the native protein. Conjugates containing each type of linkage exhibited both specific binding and selective cytotoxicity toward cells expressing the IL-2 receptor. The most potent of these toxins, that containing the disulfide linkage, exhibited a cytotoxicity which was 2 orders of magnitude greater than that of unconjugated gelonin.