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

Abstract

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 (...

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

“…This conclusion is supported by SPR experiments and previous binding studies with PEGylated Ec1 (9). Recently, from studies with the plant toxin gelonin, Pirie and colleagues (47) reported the existence of a near-universal threshold for the amount of internalized toxin that is required for induction of cell death. It is thus conceivable that cell death induction by PEG 20kDa -Ec1-ETA 00 obeys the same rules and internalization is significantly more effective after unveiling of PEG once the fusion toxin is localized in the tumor.…”

Improved Synthesis andIn VitroEvaluation of an Aptamer Ribosomal Toxin Conjugate

“…For ribosomal plant toxins such as gelonin, ricin, or saporin, and bacterial toxins such as PE-38, this is an innate property of the toxin itself (reviewed in [22]) and not the targeting molecule. In the case of gelonin, cytosolic access, not cell uptake, has previously been demonstrated to be the limiting step in cell killing [18]. As aptamers are unlikely to gain access to the cytosol, ligand release within the endosomal pathway may be an important role in freeing the toxin.…”

“…Efficacy of gelonin as a cargo has been shown to be dependent upon both uptake and time [18]. To ensure the toxin had enough time to effectively inhibit cell growth, cell viability was measured after 5 days of treatment.…”

Suppression of hepatoma tumor growth by systemic administration of the phytotoxin gelonin driven by the survivin promoter

“…Gelonin is a plant toxin that can make the ribosome inactive and mostly is used for recombinant protein to tumor therapy. An immunotoxin of anti-EGF receptor single-chain antibody fusion gelonin can inhibit EGF receptor-positive cancer cells proliferation in vitro [33]. We once constructed similar immunotoxin that can effectively inhibited tumor growth in mouse non-small cell lung cancer models and reflected the anti-tumor activity [18].…”