The Aspergillus toxin restrictocin is a suitable cytotoxic agent for generation of immunoconjugates with monoclonal antibodies directed against human carcinoma cells

Abstract

The protein toxin restrictocin, isolated from the mould Aspergillus restrictus, inactivates protein synthesis in eukaryotic cells by blocking the ribosome elongation cycle. This protein acts as a specific nuclease that cuts off a small fragment from the 28-S rRNA. Biochemical and biological characterization of this toxin indicated that it is a non-glycosylated polypeptide of M , 16836, exhibiting in cell-free systems a protein synthesis inhibition capacity similar to that of the ricin A chain. This polypeptide seemed unable to penetrate most of the cancer cell lines tested, as measured by its low in vitro cytotoxicity. In addition in vivo studies in BALB/c mice demonstrated that restrictocin toxicity was very low and that in rabbits, after intravenous injection 15% of the toxin was still present in the blood stream 24 h later.After derivatization with N-succinimidyl 3-(2-pyridyldithio)propionate and reduction by dithiothreitol, the restrictocin maintained its protein synthesis inhibitory activity, as assayed in a cell-free system. This derivatized toxin was then coupled to monoclonal antibodies (MBrl, MLuC1, MLuC2, MOv17, MOv18, MOv19) which exhibited a restricted spectrum of reactivity against human carcinomas. The biochemical and biological characterization of the immunoconjugates indicated that (a) when restrictocin was coupled to monoclonal antibodies with an average molar ratio of about 2, the immunoconjugates maintained the binding activity of the antibody and protein synthesis inhibition activity of the toxin; (b) four immunoconjugates were tested for cytotoxicity and three of them obtained with the MBrl, MLuCl and MOvl7 monoclonal antibodies exhibited a good level of cytotoxicity for relevant target cells and low or no toxicity for the irrelevant cell lines. The MLuC2 monoclonal antibody which gave rise to a completely ineffective immunoconjugate, induced internalization of less than one tenth of the antigenic sites whereas the MBrl, MLuCl and MOv17 monoclonal antibodies exhibited about one third of the antigenic sites internalized. From these data it is concluded that, providing an appropriate target antigen and coupling procedure are selected, restrictocin can be considered a suitable toxin for immunoconjugate generation.

The Therapeutic Potential of Fungal Ribotoxins

“…However, although not so frequently employed, ribotoxins have several advantages for their use in the design of immunotoxins; namely, their small size, high thermostability, poor immunogenicity, resistance to proteases, and their highly efficient ability to inactivate ribosomes [3,4,117]. In fact, different ribotoxins have been used as components of immunotoxins [117][118][119][120][121][122][123][124][125]. The first ribotoxin-based immunotoxins were constructed by chemical conjugation with mitogillin [122], restrictocin [117][118][119], or α-sarcin [121].…”

“…In fact, different ribotoxins have been used as components of immunotoxins [117][118][119][120][121][122][123][124][125]. The first ribotoxin-based immunotoxins were constructed by chemical conjugation with mitogillin [122], restrictocin [117][118][119], or α-sarcin [121]. Some years later second-generation versions were also produced by fusing restrictocin cDNA with that encoding the scFv region of a monoclonal antibody directed against the human transferrin receptor, joined by a linear flexible peptide to promote the independent folding of the two immunotoxin moieties.…”

The Genus Aspergillus

“…Penetration of this protein synthesis inhibitor seems only possible if the integrity of the cell membrane is disturbed (Munoz er al., 1985). Intemalisation of this protein can be also achieved by the construction of immunoconjugates, prepared by the attachment of the ribotoxin to monoclonal antibodies specific of receptors present on the surface of the target cells (Conde et al, 1989;Wawrzynczak et al, 1991). The occurrence of this ribosome inactivating protein in the course of an Aspergillus infection and its presence on the conidial surface has prompted several groups to consider this molecule as a potential virulence factor in aspergillosis Arruda et al, 1992;Brandhorst and Kenealy, 1992).…”

Systemic immunotoxin therapy of cancer: advances and prospects

“…Tumour localisation is significantly enhanced for ricin A chain immunotoxins made with antibody Fab' or F(ab')2 fragments compared with analogous immunotoxins made with intact antibody (Fulton et al, 1988a;Rostaing-Capaillon & Casellas, 1990). The molecular size of immunotoxins can also be reduced by selecting smaller toxin components such as asarcin or restrictocin which form immunotoxins having comparable potency to those made with the larger plant-derived toxin A chains or RIPs (Orlandi et al, 1988;Conde et al, 1989;Wawrzynczak et al, 1991c (Stoudemire et al, 1990). In further clinical trials of the anti-melanoma immunotoxin, attempts have been made to increase the duration of therapy by suppressing the host response with cyclophosphamide, prednisone, azathioprine and cyclosporin A, used singly or in combination (Spitler et al, 1989;Oratz et al, 1990).…”