Immunoconjugate generation between the ribosome inactivating protein restrictocin and an anti-human breast carcinoma MAB

Abstract

In the perspective of therapeutic approaches the monoclonal antibody, MBrl, with a quite restricted spectrum of reactivity for human breast carcinoma, was coupled to restrictocin (Res), a ribosome inactivating protein produced by Aspergillus restrictus. In a cell-free system this toxin was found to have an activity comparable to that of other plant toxins, but its in vitro toxicity was shown to be low on different cell lines. Three batches of MBr1-Res conjugate were prepared and their specificity, efficiency, and maximum level of cytotoxicity were analyzed on the cell line MCF-7 expressing the relevant antigen, on several irrelevant tumor cell lines, and on normal cells. Conjugates were from 600 to 1500 times more efficient than the uncoupled derivatized Res towards MCF-7 cells and were completely ineffective on the other target cells. The antigen-driven cytotoxicity was confirmed by the nontoxicity of an irrelevant conjugate on MCF-7 cells. The cytotoxic efficiency of MBr1-Res was low when compared to the binding level of MBr1 at the same concentration and a portion of treated cells (from 10% to 30%) survived the treatment. The heterogeneity of expression of the relevant antigen, together with its only partial internalization, could account for these limitations. The lysosomotropic agent ammonium chloride and the carboxylic ionophore monensin were tested as potentiating agents but in both cases the cytotoxicity remained unmodified. A neutralization assay performed on a xenogenic model indicated that the MBr1-Res conjugate was capable of reducing the tumor take. These data indicate the possibility of using the Res to prepare a reproducible and highly selective breast cancer conjugate. However, there are still a number of problems which must first be solved before we can consider its clinical application.

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

“…These data further support our previous suggestion [18] that restrictocin, like many other toxins [3, 411, needs an adequate internalization pathway to be effective.…”

“…To this aim, several immunoconjugates were generated with it and their characterization has been reported here. A detailed study of one of these conjugates has been reported elsewhere [18].…”

“…Due to its incapacity to penetrate normal and pathological cells, restrictocin exerted a low toxicity in vivo in BALB/ c mice and in vitro o n cultured cells [18]. Finally, preliminary observations indicated that this molecule, when injected in its native form, was poorly immunogenic.…”

Biochemical, cytotoxic and pharmacokinetic properties of an immunotoxin composed of a mouse monoclonal antibody Fib75 and the ribosome‐inactivating protein α‐sarcin from Aspergillus giganteus

“…Restrictocin has previously been reported to form immunotoxins with cytotoxic activity (Orlandi et al, 1988;Conde et al, 1989). A restrictocin immunotoxin made with a mouse monoclonal IgM antibody MBrl, recognising an antigen associated with human breast carcinoma, had a cytotoxic potency similar to that reported for an MBrl -ricin-A-chain immunotoxin (Canevari et al, 1985) although the cytotoxic activities of these two immunotoxins, and of the unconjugated restrictocin and ricin A chain, were not directly compared in the same assay.…”

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).…”