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Ribosome-inactivating proteins (RIPs) are potent protein toxins for cancer therapy, and they have strong ability to inhibit protein synthesis and induce cell death via inactivation of ribosomes in eukaryotic cells. However, the delivery of RIPs has been a challenging task due to their large molecular weight and lack of targeting property. Low molecular weight protamine (LMWP), a transmembrane peptide, has been proved to effectively promote transmembrane transportation, whereas the enzyme-activatable system can enhance the specificity by enhancing the tumor drug concentration through enzymatic reaction. We herein constructed a self-assembly and stimuli-responsive fusion gelonin delivery system. Gelonin, a typical RIP protein, was assembled with nickel ferrite nanoparticles by self-assembling between hexa-histidine tag (His-tagged) and nickel ions. Both in vitro and in vivo results indicated that the magnetic nanoparticle carriers and the applied linkers did not damage the pharmaceutical effect of gelonin, and the whole drug delivery system showed good biocompatibility, sensitive selectivity, and significantly enhanced cytotoxic activity. This in turn presented theranostic nanoparticles as efficient delivery vehicle for clinical use.

Advances in recombinant DNA technology have opened up new possibilities of exploiting toxic proteins for therapeutic purposes. Bringing forth these protein toxins from the bench to the bedside strongly depends on the availability of production methods that are reproducible, scalable and comply with good manufacturing practice (GMP). The type I ribosome-inhibiting protein, gelonin, has great potential as an anticancer drug, but is sequestrated in endosomes and lysosomes. This can be overcome by combination with photochemical internalization (PCI), a method for endosomal drug release. The combination of gelonin-based drugs and PCI represents a tumor-targeted therapy with high precision and efficiency. The aim of this study was to produce recombinant gelonin (rGel) at high purity and quantity using an automated liquid chromatography system. The expression and purification process was documented as highly efficient (4.4 mg gelonin per litre induced culture) and reproducible with minimal loss of target protein (~50% overall yield compared to after initial immobilized metal affinity chromatography (IMAC)). The endotoxin level of 0.05&ndash, 0.09 EU/mg was compatible with current standards for parenteral drug administration. The automated system provided a consistent output with minimal human intervention and close monitoring of each purification step enabled optimization of both yield and purity of the product. rGel was shown to have equivalent biological activity and cytotoxicity, both with and without PCI-mediated delivery, as rGelref produced without an automated system. This study presents a highly refined and automated manufacturing procedure for recombinant gelonin at a quantity and quality sufficient for preclinical evaluation. The methods established in this report are in compliance with high quality standards and compose a solid platform for preclinical development of gelonin-based drugs.

We have reported that calcitonin receptor (CTR) is widely expressed in biopsies from the lethal brain tumour glioblastoma by malignant glioma and brain tumour-initiating cells (glioma stem cells) using anti-human CTR antibodies. A monoclonal antibody against an epitope within the extracellular domain of CTR was raised (mAb2C4) and chemically conjugated to either plant ribosome-inactivating proteins (RIPs) dianthin-30 or gelonin, or the drug monomethyl auristatin E (MMAE), and purified. In the high-grade glioma cell line (HGG, representing glioma stem cells) SB2b, in the presence of the triterpene glycoside SO1861, the EC50 for mAb2C4:dianthin was 10.0 pM and for mAb2C4:MMAE [antibody drug conjugate (ADC)] 2.5 nM, 250-fold less potent. With the cell line U87MG, in the presence of SO1861, the EC50 for mAb2C4:dianthin was 20 pM, mAb2C4:gelonin, 20 pM, compared to the ADC (6.3 nM), which is >300 less potent. Several other HGG cell lines that express CTR were tested and the efficacies of mAb2C4:RIP (dianthin or gelonin) were similar. Co-administration of the enhancer SO1861 purified from plants enhances lysosomal escape. Enhancement with SO1861 increased potency of the immunotoxin (>3 log values) compared to the ADC (1 log). The uptake of antibody was demonstrated with the fluorescent conjugate mAb2C4:Alexa Fluor 568, and the release of dianthin-30:Alexa Fluor488 into the cytosol following addition of SO1861 supports our model. These data demonstrate that the immunotoxins are highly potent and that CTR is an effective target expressed by a large proportion of HGG cell lines representative of glioma stem cells and isolated from individual patients.

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.

Gelonin from the Indian plant Gelonium multiflorum belongs to the type I ribosome-inactivating proteins (RIPs). Like other members of RIPs, this toxin glycoprotein inhibits protein synthesis of eukaryotic cells, hence, it is largely used in the construction of immunotoxins composed of cell-targeted antibodies. Lysosomal degradation is one of the main issues in targeted tumor therapies, especially for type I RIP-based toxins, as they lack the translocation domains. The result is an attenuated cytosolic delivery and a decrease of the antitumor efficacy of these plant-derived toxins, therefore, strategies to permit their release from endosomal vesicles or modifications of the toxins to make them resistant to degradation are necessary to improve their efficacy. Using infrared spectroscopy, we thoroughly analyzed both the secondary structure and the thermal unfolding of gelonin. Moreover, by the combination of two-dimensional correlation spectroscopy and phase diagram method, it was possible to deduce the sequence of events during the unfolding, confirming the typical characteristic of the RIP members to denature in two steps, as a sequential loss of tertiary and secondary structure was detected at 58 &deg, C and at 65 &deg, C, respectively. Additionally, some discrepancies in the unfolding process between gelonin and saporin-S6, another type I RIP protein, were detected.

Gelonin is a plant toxin that exerts potent cytotoxic activity by inactivation of the 60S ribosomal subunit. The high-level expression of soluble gelonin still remains a great challenge and there was no detailed biophysical analysis of gelonin from Escherichia coli (E. coli) yet. In this study, the soluble and high-yield expression of recombinant gelonin (rGel) was achieved in E. coli BL21 (DE3) for the first time, with a yield of 6.03 mg/L medium. Circular dichroism (CD) analysis indicated that rGel consisted of 21.7% α-helix, 26.3% β-sheet, 18.5% β-turn, and 32.3% random coil, and it could maintain its secondary structure up to 60 °C. The antitumor activity of rGel was evaluated in two colon cancer cell lines—HCT116 and HCT-8, and it was clearly demonstrated that rGel exerted antiproliferative activity against these two cell lines by inhibiting cellular protein synthesis. These findings provide insights for researchers involved in the expression of similar biotoxins, and the biophysical characterizations of gelonin will favor its further therapeutic applications.

Delivery of toxins, such as the ricin A chain, Pseudomonas exotoxin, and gelonin, using antibodies has had some success in inducing specific toxicity in cancer treatments. However, these antibody-toxin conjugates, called immunotoxins, can be bulky, difficult to express, and may induce an immune response upon in vivo administration. We previously reported delivery of a recombinant variant of gelonin (rGel) by the full-length prostate-specific membrane antigen (PSMA) binding aptamer, A9, to potentially circumvent some of these problems. Here, we report a streamlined approach to generating aptamer-rGel conjugates utilizing a chemically synthesized minimized form of the A9 aptamer. Unlike the full-length A9 aptamer, this minimized variant can be chemically synthesized with a 5′ terminal thiol. This facilitates the large scale synthesis and generation of aptamer toxin conjugates linked by a reducible disulfide linkage. Using this approach, we generated aptamer-toxin conjugates and evaluated their binding specificity and toxicity. On PSMA(+) LNCaP prostate cancer cells, the A9.min-rGel conjugate demonstrated an IC50 of ∼60 nM. Additionally, we performed a stability analysis of this conjugate in mouse serum where the conjugate displayed a t1/2 of ∼4 h, paving the way for future in vivo experiments.

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

Owing to the extraordinary potency in inhibiting protein translation that could eventually lead to apoptosis of tumor cells, ribosome-inactivating proteins (RIPs) such as gelonin have been considered attractive drug candidates for cancer therapy. However, due to several critical obstacles (e.g., severe toxicity issues caused by a lack of selectivity in their mode of action and the low cytotoxicity via poor cellular uptake, etc.), clinical application of RIPs is yet far from being accomplished. To overcome these challenges, in the present study, we engineered gelonin fusion proteins with anti-insulin-like growth factor-1 receptor (IGF-1R) affibody (“IAFF”) via the genetic recombinant method and the SpyCatcher/SpyTag-mediated conjugation method. To this end, recombinant gelonin-anti-IGF-1R affibody (rGel-IAFF), gelonin-SpyCatcher (Gel-SpyCatcher) and SpyTag-IAFF fusion proteins were produced from the E. coli expression system, and gelonin-IAFF conjugate was synthesized by mixing Gel-SpyCatcher and SpyTag-IAFF. After preparation of both rGel-IAFF and Gel-IAFF conjugate, their components’ functionality was characterized in vitro. Our assay results confirmed that, while both Gel-IAFF and Gel-SpyCatcher retained equipotent N-glycosidase activity to that of gelonin, IAFF was able to selectively bind to IGF-1R overexpressed U87 MG brain cancer cells over low expression LNCaP cells. The results of cellular analyses showed that rGel-IAFF and Gel-IAFF conjugate both exhibited a greater cell uptake in the U87 MG cells than gelonin, but not in the LNCaP cells, yielding a significantly augmented cytotoxicity only in the U87 MG cells. Remarkably, rGel-IAFF and Gel-IAFF conjugate displayed 22- and 5.6-fold lower IC50 values (avg. IC50: 180 and 720 nM, respectively) than gelonin (avg. IC50: 4000 nM) in the U87 MG cells. Overall, the results of the present research demonstrated that fusion of gelonin with IAFF could provide an effective way to enhance the anti-tumor activity, while reducing the associated toxicity of gelonin.

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.

HER2 is overexpressed in 20-30% of breast tumors and is associated with aggressiveness and increased risk of recurrence and death. The HER2 protein is internalized as a part of its activity, and may therefore be utilized as a target for the specific intracellular delivery of drugs. Photochemical internalization (PCI) is a novel technology now undergoing clinical evaluation for its ability to improve the release into the cytosol of drugs entrapped in the endo/lysosomal compartment. PCI employs an amphiphilic photosensitizer which localizes in the membranes of endo/lysosomes. Subsequent light exposure (visible light) causes destabilization of the endo/lysosomal membranes. PCI has been proven highly effective for improving the cytosolic delivery of targeted toxins based on type I ribosome inactivating protein toxins such as gelonin. We examined the impact of the level of target antigen expression on PCI efficacy. Four human breast cancer cell lines (MDA-MB-231, BT-20, Zr-75-1 and SK-BR-3) covering a wide range of HER2 expression were included in the present study. PCI of the HER2-targeted fusion toxin MH3-B1/rGel was found to be highly effective in all four cell lines. The increase in PCI-mediated efficacy was not directly correlated with the cellular levels of HER2 as assessed by western blots, the overall uptake of MH3-B1/rGel as measured by flow cytometry, the amount of MH3-B1/rGel localized to endo/lysosomes assessed by confocal microscopy or the cell sensitivity to the photochemical treatment itself (photosensitizer and light without MH3-B1/rGel). However, correcting the PCI efficacy for the baseline cellular sensitivity to rGel revealed a linear correlation (R(2)=0.80) with HER2 expression. The present report therefore concludes the cellular sensitivity to the toxin as an important parameter for PCI efficacy and also indicates PCI of a HER2-targeted fusion toxin as an attractive treatment alternative for breast cancer patients with both HER2-low and -high expression.

A wide range of anti-cancer therapies have been shown to induce resistance upon repetitive treatment and such adapted resistance may also cause cross-resistance to other treatment modalities. We here show that MES-SA/Dx5 cells with adapted resistance to doxorubicin (DOX) are cross-resistant to photodynamic therapy (PDT). A DOX-induced increased expression of the reactive oxygen species (ROS)-scavenging proteins glutathione peroxidase (GPx) 1 and GPx4 in MES-SA/Dx5 cells was indicated as the mechanism of resistance to PDT in line with the reduction in PDT-generated ROS observed in this cell line. ROS-induced p38 activation was, in addition, shown to be reduced to one-third of the signal of the parental MES-SA cells 2h after PDT, and addition of the p38 inhibitor SB203580 confirmed p38 activation as a death signal after PDT in the MES-SA cells. The MES-SA/Dx5 cells were also cross-resistant to ionizing radiation in agreement with the increased GPx1 and GPx4 expression. Surprisingly, PDT-induced endo/lysosomal release of the ribosome-inactivating protein gelonin (photochemical internalization (PCI)) was more effective in the PDT-resistant MES-SA/Dx5 cells, as measured by synergy calculations in both cell lines. Analysis of death-inducing signaling indicated a low activation of caspase-3 and a strong PARP I cleavage after PDT and PCI in both cell lines. The PARP I activation was, however, stronger after PCI than after PDT in the MES-SA cells, but not in the MES-SA/Dx5 cells, and therefore cannot explain the strong PCI effect in the MES-SA/Dx5 cells. In conclusion PCI of recombinant gelonin circumvents ROS resistance in an apoptosis-independent manner.

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.

Aromatic-aromatic interactions play an important role in the enzyme-substrate recognition mechanism and in stabilization of proteins. Gelonin--a ribosome inactivating protein (RIP) from the plant Gelonium multiflorum--belongs to type-I RIPs and shows N-glycosylation activity which has been used as a model to explain the role of aromatic-aromatic stack pairing in RIPs. RIPs have a different substrate binding site and catalytic site. Role of tyrosine residues at the binding site has already been known but the role of tyrosine residues at catalytic site is still unclear. In this study, the role of tyrosine-adenine-tyrosine aromatic stack pairing at the catalytic site was studied by in silico mutation studies using molecular dynamic simulations. Through this study we report that, despite the fact that aromatic stack pairing aids in recognition of adenine at binding site, both the tyrosine residues of stack pairing play a crucial role in the stabilization of adenine at catalytic site. In the absence of both the tyrosine residues, adenine was unstable at catalytic site that results in the inhibition of N-glycosylation activity of gelonin protein. Hence, this study highlights the importance of π-π stack pairing in the N-glycosidic activity of gelonin by determining its role in stabilizing adenine at catalytic site.

Chemotherapy, radiation, and surgery are the conventional treatment modalities for cancer. The success achieved with these approaches has been limited due to several factors like chemoresistance to drugs, non-specificity leading to peripheral toxicity, and non-resectable tumors. To combat these problems, the concept of targeted therapy using immunotoxins was developed. Immunotoxins are chimeric proteins with a cell-selective ligand chemically linked or genetically fused to a toxin moiety and can target cancer cells overexpressing tumor-associated antigens, membrane receptors, or carbohydrate antigens. Ligands for these receptors or monoclonal antibodies or single chain variable fragments directed against these antigens are fused with bacterial or plant toxins and are made use of as immunotoxins. Pseudomonas exotoxin, anthrax toxin, and diphtheria toxin are the commonly used bacterial toxins. Ricin, saporin, gelonin, and poke weed antiviral protein are the plant toxins utilized in immunotoxin constructs. Several such fusion proteins are in clinical trials, and denileukin difitox is a FDA-approved fusion protein. In spite of the promise shown by bacterial- and plant toxin-based chimeric proteins, their clinical application is hampered by several factors like immunogenicity of the toxin moiety and non-specific toxicity leading to vascular leak syndrome. In order to overcome these problems, a novel generation of immunotoxins in which the cytotoxic moiety is an endogenous protein of human origin like proapoptotic protein or RNase has been developed. This review summarizes the advances in this new class of fusion protein and the future directions to be explored.

On the basis of the observation that in the hormonotoxin oLH-gelonin conjugation of the toxin occurs via the alpha-subunit of lutropin, an attempt was made to develop a general method for generation of similar hybrid proteins involving other glycoprotein hormones. In this approach sites suitable for conjugation would be first introduced into the alpha-subunit, hybridized with any native hormone specific beta-subunit of choice (LH-beta, FSH-beta, TSH-beta) following which the toxic component gelonin would be added on in the form of gelonin-S-S-alpha---beta complex. Thus, thiolated lutropin alpha-subunit recombined well with free lutropin beta-subunit, yielding a hybrid which was active in terms of receptor binding, immunoreactivity, and steroidogenic properties. However, subsequent conjugation with thiolated gelonin, a ribosome inactivating protein, resulted in dissociation of beta-subunit from its non-covalent union with the thiolated alpha-subunit. It is concluded that the addition of positively charged gelonin at particular sites on the alpha-subunit led to the destabilization of the lutropin quaternary structure.

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]

B lymphocyte stimulator (BLyS) is crucial for B-cell survival, and the biological effects of BLyS are mediated by three cell surface receptors designated B cell–activating factor receptor (BAFF-R), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), and B-cell maturation antibody (BCMA). Increased expression of BLyS and its receptors has been identified in numerous B-cell malignancies. We generated a fusion toxin designated rGel/BLyS for receptor-mediated delivery of the recombinant gelonin (rGel) toxin to neoplastic B cells, and we characterized its activity against various B-cell tumor lines. Three mantle cell lymphoma (MCL) cell lines (JeKo-1, Mino, and SP53) and two diffuse large B-cell lymphoma (DLBCL) cell lines (SUDHL-6 and OCI-Ly3) expressing all three distinct BLyS receptors were found to be the most sensitive to the fusion toxin (IC50 = 2–5 pmol/L and 0.001–5 nmol/L for MCL and DLBCL, respectively). The rGel/BLyS fusion toxin showed specific binding to cells expressing BLyS receptors and rapid internalization of the rGel component into target cells. The cytotoxic effects of rGel/BLyS were inhibited by pretreatment with free BLyS or with soluble BAFF-R, TACI, and BCMA decoy receptors. This suggests that the cytotoxic effects of the fusion toxin are mediated through BLyS receptors. The rGel/BLyS fusion toxin inhibited MCL cell growth through induction of apoptosis associated with caspase-3 activation and poly (ADP-ribose) polymerase cleavage. Our results suggest that BLyS has the potential to serve as an excellent targeting ligand for the specific delivery of cytotoxic molecules to neoplastic B cells expressing the BLyS receptors, and that the rGel/BLyS fusion toxin may be an excellent candidate for the treatment of B-cell malignancies especially MCL and DLBCL. [Mol Cancer Ther 2007;6(2):460–70]

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.

Summary Background Ribosome-inactivating proteins (RIPs) are expressed in many plants. Because of their anti-infectious and anti-proliferative effects, intensive research is going on for applying these toxins in therapy against viral infections or malignancies. Recently, we demonstrated that type I allergy against RIPs from elderberry can occur. Objective Stimulated by our study, a group of RIP researchers reported that some of the employees had suspected allergy to RIPs. Methods and Results We tested their sera in ELISA on natural RIPs. Specific IgE in four subjects were found against dianthin30, gelonin, momordin, PAP-S, saporin, ricin and volkensin. In contrast, asparin and lychnin did not show any IgE binding. When separating extracts of plants containing the toxins in SDS-PAGE, RIPs appeared to be the predominant constituents. Interestingly, among the other plant proteins, they were exclusively recognized by IgE in immunoblot. RIPs derived from close botanical families share high sequence homologies. Nevertheless, in IgE inhibition experiments with human sera, cross-reactivity between RIPs also derived from non-related plants could be demonstrated. Conclusion We conclude that sensitization and IgE induction to RIPs may occur upon exposure. This has to be considered when applying them in therapy against malignancies or viral infections.

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.

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 objective of this study was to determine if photochemical internalization (PCI) of gelonin can improve the treatment outcome as compared to photodynamic therapy (PDT) and gene transduction of fibroblast-like synoviocytes (FLS) in vitro. For this purpose synovial tissue was obtained under synovectomy of rheumatoid arthritis (RA) patients. Primary single cell suspensions were treated with the photosensitizer meso-tetraphenylporphine (TPPS2a) and light exposure (PDT) followed by evaluation of the cell survival by flow cytometry. PCI of gelonin was performed on FLS in passages 4 and 5 after removal from patients followed by measurements of protein synthesis 24 h after treatment. Additionally FLS were transduced with an adenovirus encoding the E.coli. lacZ gene and treated with PCI to evaluate the effect on the transduction rate. As a result all the cells in the primary cell suspension were susceptible to PDT but CD 106- (FLS) and CD14-positive (monocytes) cells were more sensitive to inactivation by PDT than CD2- (T-cells) and CD19-positive (B-cells) cells. With respect to protein synthesis FLS became up to 4-fold more sensitive to light when combining the photochemical treatment with the gelonin incubation. The fraction of virally transduced FLS was approximately doubled by means of PCI. In conclusion our experiments showed that PCI increased the cytotoxic effect of gelonin and adenoviral transduction of FLS derived from RA patients.

Here we examined the intrinsic nuclease activity of diphtheria toxin (DTx) to determine the mechanism by which it catalyzes DNA degradation. Results show that DTx degrades double-stranded DNA (dsDNA) by non-processive, endonucleolytic attack, without apparent specificity for nucleotide sequence. Moreover, divalent cation composition determines whether supercoiled dsDNA is cleaved by the introduction of single-strand nicks or double-strand breaks. Circular single-stranded DNA (ssDNA) is also a substrate for endonucleolytic attack. Pre-incubation of DTx with a 2000-fold excess of NAD, the natural substrate for the toxin's ADP-ribosyltransferase (ADPrT) activity, inhibited the transfer of radiolabeled ADP-ribose to elongation factor 2 but had no effect on the degradation of radiolabeled DNA. Based on this result and the fact that compounds known to inhibit the ADPrT activity of DTx had no effect on its nuclease activity and pre-incubation of DTx with DNA had no effect on ADPrT activity, we conclude that the ADPrT and nuclease active sites of DTx are functionally and spatially distinct. Moreover, studies with an ADPrT-inactivated form of DTx indicate that nuclease activity alone can lead to target cell lysis.

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.

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.

Gelonin, a type I ribosome-inactivating plant toxin, executes N-glycosidase activity on eukaryotic ribosomes. However, on intact cells, gelonin is relatively non-toxic, due to an incapability to penetrate cell membranes. Recently, a novel method, photochemical internalization (PCI), was invented for the translocation of membrane-impermeable molecules including gelonin to the cytosol [K. Berg et al., Cancer Res. 59 (1999) 1180–1183]. The combination of gelonin and photoactivation of endosomal and lysosomal localizing photosensitizers gives strong synergistic cytotoxic effects. In this study, we have evaluated the intracellular transport and stability of gelonin. By fluorescence microscopy, it was shown that gelonin co-localizes with the endosomal and lysosomal localizing photosensitizer, aluminum phthalocyanine with two sulfonate groups on adjacent phenyl rings, and both molecules re-localized to cytosol subsequently to light exposure. Gelonin accumulated in endosomal compartments by incubation at 18°C was released to cytosol by PCI with concomitant inhibition of protein synthesis indicating that PCI can be executed through rupture of endosomal vesicles. The cathepsin inhibitor L -trans-epoxysuccinyl-leucyl amido(4-guanido)butane increased the cytotoxic effect of gelonin after PCI when gelonin was provided as a 2 h pulse followed by 4 h chase before PCI. Thus, although gelonin can enter the cytosol from lysosomes, lysosomal degradation is a limiting factor for the outcome of PCI of gelonin.

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.

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

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.

Gelonin is a single chain ribosome inactivating protein (RIP) with potential application in the treatment of cancer and AIDS. Diffraction quality crystals grown using PEG3350, belong to the space group P2(1), with it a = 49.4 Angstrom b = 44.9 Angstrom, c = 137.4 Angstrom and beta = 98.4 degrees, and contain two molecules in the asymmetric unit. Diffraction data collected to 1.8 Angstrom resolution has a R(m) value of 7.3%. Structure of gelonin has been solved by the molecular replacement method, using ricin A chain as the search model. Crystallographic refinement using X-PLOR resulted in a model for which the r.m.s deviations from ideal bond lengths and bond angles are 0.012 Angstrom and 2.7 degrees, respectively The final R-factor is 18.4% for 39,806 reflections for which I > 1.0 sigma(I).The C-alpha atoms of the two molecules in the asymmetric unit superpose to within 0.38 Angstrom for 247 atom pairs. The overall fold of gelonin is similar to that of other RIPs such as ricin A chain and alpha-momorcharin, the r.m.s.d. for C-alpha superpositions being 1.3 and 1.4 Angstrom, respectively The-catalytic residues (Glu166, Arg169 and Tyr113) in the active site form a hydrogen bond scheme similar to that observed in other RIPs. The conformation of Tyr74 in the active site, however, is significantly different from that in alpha-momorcharin. Three well defined water molecules are located in the active site cavity and one of them, X319, superposes to within 0.2 Angstrom of a corresponding water molecule in the structure of alpha-momorcharin. Any of the three could be the substrate water molecule in the hydrolysis reaction catalysed by gelonin.Difference electron density for a N-linked sugar moiety has been observed near only one of the two potential glycosylation sites in the sequence. The amino acid at position 239 has been established as Lys by calculation of omit electron density maps.The two cysteine residues in the sequence, Cys44 and Cys50, form a disulphide bond, and are therefore not available for disulphide conjugation with antibodies. Based on the structure, the region of the molecule that is involved in intradimer interactions is suggested to be suitable for introducing a Cys residue for purposes of conjugation with an antibody to produce useful immunotoxins.

The plant toxin gelonin is an extremely potent inhibitor of protein synthesis, similar in action to ricin. The mature protein primary sequence was obtained using conventional sequencing techniques. Gelonin was found to be composed of 258 amino acids and contains 21 lysine residues. This toxin shares approximately 33% sequence homology with trichosanthin and ricin A chain. A 774 bp synthetic gene encoding gelonin was synthesized and expressed in E. coli. Recombinant gelonin (approximately 28 kD) expression was monitored and demonstrated by western analysis. Purification and functional activity studies demonstrated that this protein behaves identically to that of the natural product. Recombinant gelonin (RG) thus joins a growing list of recombinant toxins currently available for use in the construction of recombinant immunotoxins composed of gelonin fused to binding domains of antibodies, growth factors, or other cytokines.

Therapeutic agents capable of targeting tumor cells present as established tumors and micrometastases have already demonstrated their potential in clinical trials. Immunotoxins targeting hematological malignancies and solid tumors have additionally demonstrated excellent clinical activity. This review focuses on our design and characterization studies of constructs composed of recombinant gelonin toxin fused to either growth factors or single-chain antibodies targeting solid tumor cells, tumor vasculature or hematological malignancies. These agents demonstrate cytotoxicity at nanomolar or sub-nanomolar levels. All of these constructs display impressive selectivity and specificity for antigen-bearing target cells in vitro and in vivo and are excellent clinical trial candidates.

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 paper describes a method suitable for purifying immunotoxin containing type 1 ribosome-inactivating protein, gelonin. The separation of free (unreacted) 80G, a monoclonal antibody against alpha-fetoprotein (AFP), from semipurified 80G-gelonin conjugate was unsuccessful by conventional CM-Sepharose ion-exchange chromatography because the isoelectric point of the conjugate did not increase enough to reach that of gelonin alone. In contrast, Blue Sepharose affinity chromatography could efficiently separate free 80G from the semipurified conjugate because the conjugate was bound to the column by its gelonin moiety while free 80G was not in buffer containing NaCl of a particular concentration range. However, a small amount of conjugate containing gelonin modified with N-succinimidyl 3-(2-pyridyldithio)propionate, but not with 2-iminothiolane, could not bind to the column. The conjugate purified by the use of Blue Sepharose showed selective cytotoxicity against AFP-producing human hepatoma cells.

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.

Epidermal growth factor receptor (EGFR) plays an important role in the growth and metastasis of many solid tumors. Strategies that target EGFR hold promising therapeutic potential for the treatment for non-small cell lung cancer (NSCLC), as EGFR is normally overexpressed in these tumors. This study was designed to determine whether an anti-EGFR immunotoxin has anti-tumor activity against NSCLC, and if so, to further investigate the possible mechanisms of cytotoxicity. A fusion protein of anti-EGFR single-chain variable fragment (anti-EGFR scFv) and the plant toxin gelonin (rGel) was constructed, expressed in bacteria, and purified to homogeneity. Cytotoxicity of anti-EGFR scFv/rGel (E/rG) immunotoxin was assessed on A549, HCC827, and H1975 cells (EGFR-overexpressing NSCLC-derived cell lines) and A549 xenografts in nude mice. Cytotoxicity experiments using E/rG on A549, HCC827, and H1975 cells demonstrated that E/rG can specifically inhibit proliferation of these cells, whereas it did not affect the proliferation of Raji cells that do not express EGFR. Treatment for A549 xenografts in nude mice with E/rG resulted in significant suppression of tumor growth compared to controls. Immunofluorescence in frozen tissue sections confirmed that E/rG could specifically bind to tumor tissues in nude mice bearing A549 tumor xenografts, while rGel alone showed no binding activity. Furthermore, E/rG inhibited the growth of A549 cells by cytotoxic effects that blocked tumor proliferation, and the immunotoxin-induced cell death may be mediated by autophagy. These results showed that E/rG might have significant potential as a novel clinical therapeutic agent against human NSCLC.

Photochemical internalisation (PCI) is a novel technology for release of endocytosed macromolecules into the cytosol. The technology is based on the use of photosensitizers that locate in endocytic vesicles, and that upon activation by light induce a release of macromolecules from the endocytic vesicles. PCI has been shown to stimulate delivery of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane. The preclinical evaluation of PCI has been performed with aluminum phthalocyanine disulfonate (AlPcS(2a)) as photosensitizer. AlPcS(2a), due to its large number of isomers potentially with batch-to-batch ratio variations, is not an optimal photosenstizer for clinical use. Disulfonated tetraphenyl chlorin (TPCS(2a)) has therefore been developed by di-imide reduction of disulfonated tetraphenyl porphine (TPPS(2a)). The synthesized TPCS(2a) contains 3 isomers as shown by HPLC with low (4%) inter-batch variation with respect to isomer formation, less than 0.5% (w/w) of the starting material TPPS(2a) and absorbs light at 652 nm. As prerequisites for a PCI photosensitizer TPCS(2a) was found to localize in intracellular granules assumed to be endocytic vesicles. In cells in culture TPCS(2a)-PCI induced activation of gelonin as seen by enhanced cytotoxicity, increased transfection efficacy by an enhanced green fluorescence protein (EGFP)-encoding plasmid, induced gene silencing by siRNA towards EGFP and induced in a synergistic manner tumor growth delay by TPCS(2a)-mediated PCI of bleomycin in CT26.CL25 carcinomas growing subcutaneously in athymic mice. TPCS(2a)-PCI of bleomycin was found superior to meso-tetraphenyl chlorin-based photodynamic therapy (mTHPC-PDT) with respect to inhibition of tumor growth. The tumor growth delay by PCI of bleomycin was independent of the time of bleomycin administration between 3 h prior to light to immediately after light, while bleomycin administered 24 h prior to or 24 h after the light exposure induced suboptimal or only additive effects on tumor growth delay respectively. TPCS(2a)-PDT and -PCI induced indistinguishably strong edema the first 3-4 days after TPCS(2a)-administration and only weak erythema the first day after TPCS(2a) administration. In contrast, mTHPC-PDT induced moderate edema the first 7 days after mTHPC administration, but strong erythema resulting in open wounds and escar formation the first 2-3 days after mTHPC administration. The pharmacokinetic properties of TPCS(2a) were evaluated in athymic mice. The plasma pharmacokinetics was best fit to a 2-compartment model with half-lives of 0.78 and 36 hrs. TPCS(2a) was found to be a clinically suitable PCI photosensitizer for photochemical activation of molecules that do not readily penetrate the cellular plasma membrane.

Inactivation of ribosomes by gelonin, from Gelonium multiflorum, requires ATP and extraribosomal protein(s) present in the rabbit reticulocyte lysate [SPERTI, S. et al. (1991) Biochem. J. 277, 281-284]. On the anion exchanger Mono Q the activity responsible for the sensitization of ribosomes to gelonin resolves in two peaks which both display a kinase activity on ribosomal proteins. However, staurosporin, an inhibitor of several protein kinases, strongly inhibits phosphorylation of ribosomal proteins without affecting the gelonin-promoting activity of Mono Q peaks. All the evidence collected contradicts a direct link between sensitization to gelonin and phosphorylation of ribosomes.