Improved binding of a bivalent single-chain immunotoxin results in increased efficacy for in vivo T-cell depletion

Thompson, Jeffrey P.; Stavrou, Scott; Weetall, Marla; Hexham, J M; Digan, Mary Ellen; Wang, Zhuri; Woo, Jeong Soo; Yu, Yongjun; Mathias, Askale; Liu, Yuan Yi; Ma, Shenglin; Gordienko, Irina; Lake, Phil; Neville, David M.

doi:10.1093/protein/14.12.1035

Cited by 56 publications

(56 citation statements)

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“…We examined the ability of these binding scaffolds to retain affinity within the fusion construct and found that although affinity was retained to a considerable degree, there was some loss of affinity compared with the parent molecule, likely attributable to partial misfolding and instability in the fusion construct. This is consistent with work by others designing direct fusion immunotoxins (42,43). We believe that this misfolding and instability also influence the synthetic yields of each of the immunotoxins.…”

Section: Discussionsupporting

confidence: 91%

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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Section: Discussionsupporting

confidence: 91%

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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“…7 CD3ε is a component of the T-cell receptor. 8 The CD3 subunits are expressed on the vast majority of mature T-cell neoplastic cells.…”

Section: Introductionmentioning

confidence: 99%

Resimmune, an anti-CD3 recombinant immunotoxin, induces durable remissions in patients with cutaneous T-cell lymphoma

et al. 2015

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Resimmune is a second-generation recombinant immunotoxin composed of the catalytic and translocation domains of diphtheria toxin fused to two single chain antibody fragments reactive with the extracellular domain of CD3ε. We gave intravenous infusions of Resimmune 2.5 -11.25 μg/kg over 15 minutes to 30 patients (25 with cutaneous T-cell lymphoma, 3 with peripheral T-cell lymphoma, 1 with T-cell large granular lymphocytic leukemia and 1 with T-cell prolymphocytic leukemia) in an inter-patient dose escalation trial. The most common adverse events were fever, chills, hypotension, edema, hypoalbuminemia, hypophosphatemia, and transaminasemia. Among the 25 patients with cutaneous T-cell lymphoma, there were nine responses for a response rate of 36% (95% CI, 18%-57%) including four complete remissions (16%, 95% CI, 5%-36%). The durations of the complete remissions were 72+, 72+, 60+ and 38+ months. There were five partial remissions lasting 3, 3, 3+, 6+ and 14 months. Of 17 patients with a modified skin weighted assessment tool score <50, 17 patients with stage IB/IIB, and 11 patients with both a score <50 and stage IB/IIB, nine (53%), eight (47%), and eight (73%) had responses, respectively. Further studies of Resimmune in patients with low tumor burden, stage IB-IIB cutaneous T-cell lymphoma are warranted. This trial is registered at clinicaltrials.gov as #NCT00611208. Resimmune, an anti-CD3ε recombinant immunotoxin, induces durable remissions in patients with cutaneous T-cell lymphoma ABSTRACT MethodsThe Resimmune study was a single-arm, multicenter interpatient dose escalation phase 1 trial in patients with advanced CD3 + T-cell malignancies. The study was performed under the sponsorship of Angimmune, LLC, registered at clinical trials.gov as NCT00611208, and approved by Institutional Review Boards at the participating institutions. Thirty patients were treated with a single course of Resimmune at doses ranging from 2.5 to 11.25 μg/kg intravenously twice daily for 4 days. Eligibility and diagnosisPatients with CD3 + T-cell malignancies, diagnosed by morphological, histochemical, and cell surface criteria, in whom systemic therapy had failed were eligible for the study.

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“…Furthermore, the bivalent immunotoxin production in an EF-2 mutant CHO cell expression system was limited to 5 mg/liter and could not be increased by selection for multiple gene insertions (Neville, unpublished data). Due to this limitation, except for three reports from our laboratory (12,20,25), all recombinant immunotoxin production for therapeutic uses has been limited to Escherichia coli production, which has required denaturation and refolding from inclusion bodies (6). However, refolding of the multidomain structure of the bivalent immunotoxin from E. coli was inefficient, and complete bioactivity was not recovered (25).…”

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confidence: 99%

“…This immunotoxin selectively kills human T cells and is the most efficacious of a variety of anti-T-cell immunotoxins that have been developed (20). The bivalent immunotoxin contains the first 390 amino acid residues of diphtheria toxin (DT) and two tandem sFv molecules that are responsible for binding the immunotoxin to the CD3ε␥ subunit of the T-cell receptor complex on human T cells.…”

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confidence: 99%

Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

Woo

Liu

Stavrou

et al. 2004

Appl Environ Microbiol

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The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G 4 S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut ؉ ) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15°C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P. pastoris.The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G 4 S) was developed for treatment of T-cell leukemia and autoimmune diseases and tolerance induction for transplantation. This immunotoxin selectively kills human T cells and is the most efficacious of a variety of anti-T-cell immunotoxins that have been developed (20). The bivalent immunotoxin contains the first 390 amino acid residues of diphtheria toxin (DT) and two tandem sFv molecules that are responsible for binding the immunotoxin to the CD3ε␥ subunit of the T-cell receptor complex on human T cells. The first 390 amino acid residues of DT (DT390) contain the catalytic domain or A chain of DT that inhibits protein synthesis by ADP ribosylation of EF-2 and the translocation domain that translocates the catalytic domain to the cytosol by interactions with cytosolic Hsp90 and thioredoxin reductase (17).The inhibition of protein synthesis by the catalytic domain makes it difficult to produce the toxin-related proteins in most eukaryotic cells (e.g., wild-type CHO cells, insect cells, and yeasts). The use of toxin-resistant eukaryotic cells can overcome the immunotoxin toxicity. However, selection and characterization of toxin-resistant eukaryotic cells are tedious, labor-intensive, ...

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Improved binding of a bivalent single-chain immunotoxin results in increased efficacy for in vivo T-cell depletion

Cited by 56 publications

References 24 publications

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

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

Resimmune, an anti-CD3 recombinant immunotoxin, induces durable remissions in patients with cutaneous T-cell lymphoma

Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

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