Immunotoxin construction with a ribosome-inactivating protein from barley.
ABSTRACT The aim of this study was to determine the suitability of a ribosome-inactivating protein (RIP) from barley endosperm for use as an immunotoxin. This barley RIP is identical with the 30-kDa protein first reported by Coleman and Roberts [(1982) Biochim. Biophys. Acta 696, 239] and sequenced by Asano and co-workers [(1986) Carlsberg Res. Commun. 51, 75]. Use of the terms barley toxin I, II, and III is proposed to describe the three isoforms resolved by cation-exchange chromatography. An improved procedure for isolating the protein involving the steps of aqueous extraction, ammonium sulfate precipitation, and cation-exchange HPLC is described. Barley toxin II retained activity after exposure to ca. 40% acetonitrile and 0.1% trifluoroacetic acid or lyophilization. In a comparative study using the rabbit reticulocyte lysate assay, the protein was about 68% and 30% as potent as gelonin and ricin A-chain (RTA), respectively. Introduction of SH groups with 2-iminothiolane resulted in a substantial loss of activity as the number of thiol groups approached four. Therefore, it was necessary to limit thiolation to an average of one to two SH groups per toxin molecule. Anti-transferrin receptor-based immunotoxins constructed with RTA, gelonin, and barley toxin II exhibited comparable cytotoxicity against a human colon tumor cell line. We conclude that the availability of raw material, ease of purification, and stability of barley toxin II to lyophilization and denaturing conditions render it a suitable protein for the construction of immunotoxins.
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ABSTRACT: The term ribosome-inactivating protein (RIP) is used to denominate proteins mostly of plant origin, which have N-glycosidase enzymatic activity leading to a complete destruction of the ribosomal function. The discovery of the RIPs was almost a century ago, but their usage has seen transition only in the last four decades. With the advent of antibody therapy, the RIPs have been a subject of extensive research especially in targeted tumor therapies, which is the primary focus of this review. In the present work we enumerate 250 RIPs, which have been identified so far. An attempt has been made to identify all the RIPs that have been used for the construction of immunotoxins, which are conjugates or fusion proteins of an antibody or ligand with a toxin. The data from 1960 onwards is reviewed in this paper and an extensive list of more than 450 immunotoxins is reported. The clinical reach of tumor-targeted toxins has been identified and detailed in the work as well. While there is a lot of potential that RIPs embrace for targeted tumor therapies, the success in preclinical and clinical evaluations has been limited mainly because of their inability to escape the endo/lysosomal degradation. Various strategies that can increase the efficacy and lower the required dose for targeted toxins have been compiled in this article. It is plausible that with the advancements in platform technologies or improved endosomal escape the usage of tumor targeted RIPs would see the daylight of clinical success.Current Pharmaceutical Design 10/2014; 20(42):6584-643. · 3.29 Impact Factor
Article: Immunotoxins[Show abstract] [Hide abstract]
ABSTRACT: Many types of tumour cell express high levels of surface antigens which are largely absent from normal cells. Monoclonal antibodies against these surface antigens can be coupled to potent toxins to form conjugates (immunotoxins) which selectively kill the antigen-bearing cells. This article describes the components of immunotoxins, their modes of action and their clinical applications.Trends in Biotechnology. 01/1986; 3(7):175-179.
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ABSTRACT: 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 cargos 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 2μg/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.Journal of Controlled Release 08/2013; · 7.63 Impact Factor