Immunotoxins: An Update

Thrush, Gerald R.; Lark, Laura R.; Clinchy, Birgitta; Vitetta, Ellen S.

doi:10.1146/annurev.immunol.14.1.49

Cited by 158 publications

(111 citation statements)

References 115 publications

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“…The response rates observed in phase I/II trials have often been higher than those reported for some of the conventional antiblastic drugs. 11,16,17 In this research, the cytotoxic effect of Rituximab conjugated to the RIP saporin-S6 has been studied for the first time. Moreover, we explored the possibility of combining immunotoxin and the chemotherapic drug Fludarabine, to augment the efficiency of killing target cells.…”

Section: Introductionmentioning

confidence: 99%

The conjugate Rituximab/saporin-S6 completely inhibits clonogenic growth of CD20-expressing cells and produces a synergistic toxic effect with Fludarabine

et al. 2004

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Immunotoxins are chimeric proteins consisting of a toxin coupled to an antibody. To date, several clinical trials have been conducted, and some are still ongoing, to evaluate their anti-tumor efficacy. In this view, we chemically constructed an anti-CD20 immunotoxin with the mAb Rituximab and the type 1 ribosome-inactivating protein (RIP) saporin-S6, designed for B cells non-Hodgkin's lymphoma (NHL) therapy. This immunotoxin showed a specific cytotoxicity for the CD20 þ cell lines Raji and D430B, evidenced by inhibition of protein synthesis, evaluation of apoptosis and clonogenic assay. Upon conjugation, saporin-S6 increased its toxicity on target cells by at least 2 logs, with IC 50 values of 0.1-0.3 nM. The percentage of AnnexinV þ cells was over 95% in both cell lines treated with 10 nM immunotoxin. A complete elimination of Raji clones was reached with the 10 nM immunotoxin, whereas a mixture of free RIP and mAb gave about 90% of clonogenic growth. Rituximab/ saporin-S6, at 10 nM concentration, also induced apoptosis in 80% of lymphoma cells from NHL patients. Moreover, sensitivity of Raji to Rituximab/saporin-S6 was augmented when cells were coincubated with Fludarabine. The synergistic toxic effect of the two drugs led to a total elimination of the neoplastic population.

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Section: Introductionmentioning

confidence: 99%

The conjugate Rituximab/saporin-S6 completely inhibits clonogenic growth of CD20-expressing cells and produces a synergistic toxic effect with Fludarabine

et al. 2004

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“…The separation process is an important step since it is necessary to avoid steric hindrance and allows an effective translocation of the toxin into the cytoplasm 25 . This can be accomplished by disulfide bonding 26 for the coupling of the toxin to a carrier molecule 9 .…”

Section: Introductionmentioning

confidence: 99%

Expression and purification of cysteine introduced recombinant saporin

Günhan

Swe

Palazoglu

et al. 2008

Protein Expression and Purification

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Saporin, a ribosome inactivating protein is widely used for immunotoxin construction. Here we describe a mutation of saporin (sap)-3 DNA by introducing a cysteine residue, followed by protein expression and purification by ion exchange chromatography. The purified Cys255sap-3, sap-3 isomer and commercially purchased saporin, were tested for toxicity using assays measuring inhibition for protein synthesis. The IC 50 values showed that the toxicity of the Cys255sap-3 is equivalent to the sap-3 isomer and commercial saporin. Reactivity of Cys255sap-3 was confirmed by labeling with a thio-specific fluorescent probe as well as conjugation with a nonspecific mouse IgG. We have found that a single cysteine within saporin provides a method for antibody conjugation that ensures a uniform and reproducible modification of a saporin variant retaining high activity.

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“…IT therapy seems an excellent additional treatment in view of the potent and specific killing of malignant cells in vitro as well as in vivo. 1,2 ITs are constructed by linking chemically or genetically a cell binding moiety to a toxin. The commonly used toxins belong to the ribosome-inactivating proteins 3,4 and monoclonal antibodies (MoAbs) are usually used as targeting moiety.…”

Section: Introductionmentioning

confidence: 99%

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1999

Leukemia

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Despite the strong in vitro activity of some immunotoxins (ITs), clinical application did not result in complete cure. The outcome of therapy may be improved by combining ITs with ITcytotoxicity enhancing agents. We studied the effect of various agents that influence the intracellular routing of ITs on the activity of the anti-B cell IT CD22-recombinant (rec) ricin A. In protein synthesis inhibition assays the carboxylic ionophores monensin and nigericin enhanced the activity of the IT 117-and 382-fold, respectively, against the cell line Daudi, and 81-and 318-fold, respectively, against the cell line Ramos. IT activity to Daudi and Ramos was enhanced to a lesser extent by the lysosomotropic amines chloroquine (14-and 11-fold, respectively) and NH 4 Cl (nine-and 10-fold, respectively). However, the combination of NH 4 Cl and chloroquine induced more than an additive effect (145-and 107-fold, respectively). Cytotoxicity was not influenced by brefeldin A, all-trans retinoic acid (ATRA), verapamil and perhexiline maleate. Bacitracin enhanced the IT cytotoxicity in contrast to the other protease inhibitors aprotinin, leupeptin and soybean trypsin inhibitor, albeit enhancement was weak (two-fold). The enhancers exerted only a negligible effect on bone marrow progenitor cells. We recently developed a flow cytometric cytotoxicity assay in which cell elimination can be assessed. In order to detect enhancement in this assay, we used 5 × 10 2.06 log). To determine the applicability of the IT in combination with enhancers in vivo we investigated the effect of human serum. Human serum inhibited IT activity which could not be restored by monensin and nigericin because of complete inhibition of these enhancers by serum. In contrast, chloroquine partially restored the activity of CD22-rec ricin A in the presence of human serum. We conclude that monensin, nigericin and the combination of NH 4 Cl and chloroquine can be used instead of NH 4 Cl to potentiate CD22-rec ricin A activity in purging autologous bone marrow transplants contaminated with malignant B cells. Chloroquine might be a promising enhancer of CD22-rec ricin A for treating patients in vivo.

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Immunotoxins: An Update

Cited by 158 publications

References 115 publications

The conjugate Rituximab/saporin-S6 completely inhibits clonogenic growth of CD20-expressing cells and produces a synergistic toxic effect with Fludarabine

The conjugate Rituximab/saporin-S6 completely inhibits clonogenic growth of CD20-expressing cells and produces a synergistic toxic effect with Fludarabine

Expression and purification of cysteine introduced recombinant saporin

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