Amino Acid Sequence Analysis, Gene Construction, Cloning, and Expression of Gelonin, a Toxin Derived from<i>Gelonium multiflorum</i>

Rosenblum, Michael G.; Kohr, W A; Beattie, Kenneth L.; Beattie, Wanda G.; Marks, W; Toman, P. David; Cheung, Lawrence H.

doi:10.1089/jir.1995.15.547

Cited by 44 publications

(37 citation statements)

References 25 publications

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“…We have used immunotoxins based on rGel, which have shown improved stability and reduced toxicity compared with other toxins (37). The rGel was fused to an FGFR3-specific scFv for studies of in vitro and in vivo inhibition of bladder carcinoma cell proliferation.…”

Section: Discussionmentioning

confidence: 99%

Antitumor activity of fibroblast growth factor receptor 3–specific immunotoxins in a xenograft mouse model of bladder carcinoma is mediated by apoptosis

Martı́nez-Torrecuadrada

Cheung

López-Serra

et al. 2008

Molecular Cancer Therapeutics

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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 NH 2 terminus of rGel and expressed as a soluble protein in Escherichia coli. The 3C/rGel fusion construct showed an IC 50 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 immunotoxininduced 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]

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Section: Discussionmentioning

confidence: 99%

Antitumor activity of fibroblast growth factor receptor 3–specific immunotoxins in a xenograft mouse model of bladder carcinoma is mediated by apoptosis

Martı́nez-Torrecuadrada

Cheung

López-Serra

et al. 2008

Molecular Cancer Therapeutics

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“…Recombinant gelonin (rGel) 2 is an ϳ30-kDa N-glycosidase with activity similar to the ricin A chain but exhibiting better stability and lower immunogenicity (13,14). The use of rGel in tumor-targeted cytotoxic agents has been well studied (15,16).…”

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confidence: 99%

Convergent Potency of Internalized Gelonin Immunotoxins across Varied Cell Lines, Antigens, and Targeting Moieties

Pirie¹,

Hackel²,

Rosenblum

et al. 2011

Journal of Biological Chemistry

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Gelonin-based immunotoxins vary widely in their cytotoxic. Results were matched with cytotoxicity measurements made at equivalent concentration and exposures. Unexpectedly, when matched internalization and cytotoxicity data were combined, a conserved internalized cytotoxicity curve was generated that was common across experimental conditions. Considerable variations in antigen expression, trafficking kinetics, extracellular immunotoxin concentration, and exposure time were all found to collapse to a single potency curve on the basis of internalized immunotoxin. Fifty percent cytotoxicity occurred when ϳ5 ؋ 10 6 toxin molecules were internalized regardless of the mechanism of uptake. Cytotoxicity observed at a threshold internalization was consistent with the hypothesis that endosomal escape is a common, highly inefficient, rate-limiting step following internalization by any means tested. Methods designed to enhance endosomal escape might be utilized to improve the potency of gelonin-based immunotoxins.Immunotoxins are a promising approach to the targeted delivery of highly potent, cancer-specific, cytotoxic agents. Immunotoxins are frequently composed of a targeting moiety (derived from antibodies or other cell-binding proteins) either chemically conjugated or genetically fused to highly cytotoxic plant or bacterial protein toxins. Clinical success for immunotoxins has been mostly limited to hematological malignancies due to transport limitations in solid tumors (1). Such limitations have been extensively studied experimentally (2) and with several computational models (3, 4).The potency of a particular immunotoxin is dependent on the ability to deliver the toxin to the cytoplasm, which is commonly considered to be the rate-limiting step. For some native toxins such as ricin, intracellular delivery is achieved through lectin binding, followed by internalization and toxin release with membrane fusion or retrograde trafficking (5). Immunotoxins attempt to recreate this scenario by replacing the indiscriminate lectin binding with cancer-specific antigen binding as a means of targeting and internalization (6). Subsequent intracellular trafficking, release, and endosomal escape are often achieved using existing toxin characteristics, translocation domains, protease cleavage sites, disulfide bonds, and/or signaling peptides (7-10). However, the inclusion of toxins with domains facilitating cytoplasmic access can also lead to increased nonspecific toxicity in vivo (11,12).Gelonin is a plant toxin and classified as a type I ribosomeinactivating protein because it lacks any cell-binding or cytoplasmic delivery domains. Recombinant gelonin (rGel) 2 is an ϳ30-kDa N-glycosidase with activity similar to the ricin A chain but exhibiting better stability and lower immunogenicity (13,14). The use of rGel in tumor-targeted cytotoxic agents has been well studied (15, 16). Furthermore, rGel has been shown to be active without cleavage from the binding domain and without negative impact on the targeting agent's pharmacokinetics (...

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“…17,18 An analog of rGel was used which contained an engineered cysteine in the C-terminus, allowing site-specific conjugation of the toxin to the antibody component. The HUM-195/rGel conjugate has been examined in numerous in vitro models demonstrating impressive, specific cytotoxic effects.…”

confidence: 99%

“…It is a single chain protein that inactivates the ribosomal 60S subunit by cleaving rRNA adenine N-glycoside bonds in a sequence-specific fashion and is a potent inhibitor of protein synthesis. 17 Unlike dual-chain toxins such as ricin, gelonin lacks a carbohydrate-binding domain and, by itself, cannot bind to or enter mammalian cells without a carrier such as an antibody or growth factor.…”

Section: Introductionmentioning

confidence: 99%

Phase 1 study of an anti-CD33 immunotoxin, humanized monoclonal antibody M195 conjugated to recombinant gelonin (HUM-195/rGEL), in patients with advanced myeloid malignancies

Borthakur¹,

Rosenblum

Talpaz

et al. 2012

Haematologica

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© F e r r a t a S t o r t i F o u n d a t i o nwith doses up to 10 mg/m 2 administered every 76-98 h for six doses. 16 Recombinant gelonin (rGel) is an engineered, bacterially-expressed recombinant version of gelonin toxin originally isolated from the seeds of Gelonium multiflorum. It is a single chain protein that inactivates the ribosomal 60S subunit by cleaving rRNA adenine N-glycoside bonds in a sequence-specific fashion and is a potent inhibitor of protein synthesis.17 Unlike dual-chain toxins such as ricin, gelonin lacks a carbohydrate-binding domain and, by itself, cannot bind to or enter mammalian cells without a carrier such as an antibody or growth factor.HUM-195/rGel is an immunotoxin created by conjugating rGel with via an N-succinimidyl-3-(2-pyridyl-dithio)-propionate linkage. 17,18 An analog of rGel was used which contained an engineered cysteine in the C-terminus, allowing site-specific conjugation of the toxin to the antibody component. The HUM-195/rGel conjugate has been examined in numerous in vitro models demonstrating impressive, specific cytotoxic effects. [18][19][20] In a bone marrow purging model using HL60 cells mixed with mobilized peripheral blood progenitor cells, incubation with HUM-195/rGel followed by freeze-thawing, simulating marrow purging, led to a 2-log reduction of leukemic cells from the normal progenitor cells. 19Additional in-vivo models of leukemia also confirmed the conjugate's activity. 20 Here we report the results of the first in-human phase I study of HUM-195/rGel in patients with CD33-expressing myeloid malignancies. Design and Methods ObjectiveThe primary objective of the study was to determine the safety and toxicity of the HUM-195/rGel immunotoxin in patients with relapsed or refractory myeloid malignancies and define a recommended dose for phase 2 studies. Secondary objectives were to examine the biological effects of HUM-195/rGel, including the ability to elicit antileukemic responses and human anti-gelonin antibody (HAGA) responses. EligibilityPatients aged ≥18 years with any of the following diagnoses in whom prior therapies had failed were eligible: relapsed or refractory AML, refractory anemia with excess blasts, chronic myelomonocytic leukemia, accelerated or myeloid blastic phase of chronic myelogenous leukemia or myeloproliferative disorders and >5% blasts in the blood or bone marrow. In addition, as assessed by flow cytometry, CD33 expression was required in ≥80% of blasts. Because of concerns associated with proliferative diseases, eligibility was restricted to patients with a white blood cell count <10x10 9 /L in those with AML, myelodysplastic syndrome, chronic myeloid leukemia in myeloid blastic phase and myeloproliferative disorders and ≤30x10 9 /L in those with chronic myeloid leukemia in accelerated phase. Other eligibility criteria included: (i) being off any cytotoxic chemotherapy for 2 weeks prior to enrollment and free from residual toxicity of grade ≥2 from prior therapies; (ii) serum creatinine ≤ 1.5 times the upper limit of normal laborat...

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Amino Acid Sequence Analysis, Gene Construction, Cloning, and Expression of Gelonin, a Toxin Derived fromGelonium multiflorum

Cited by 44 publications

References 25 publications

Antitumor activity of fibroblast growth factor receptor 3–specific immunotoxins in a xenograft mouse model of bladder carcinoma is mediated by apoptosis

Antitumor activity of fibroblast growth factor receptor 3–specific immunotoxins in a xenograft mouse model of bladder carcinoma is mediated by apoptosis

Convergent Potency of Internalized Gelonin Immunotoxins across Varied Cell Lines, Antigens, and Targeting Moieties

Phase 1 study of an anti-CD33 immunotoxin, humanized monoclonal antibody M195 conjugated to recombinant gelonin (HUM-195/rGEL), in patients with advanced myeloid malignancies

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