CHARACTERIZATION OF RIBOSOME-INACTIVATING PROTEINS ISOLATED FROM BRYONIA-DIOICA AND THEIR UTILITY AS CARCINOMA-REACTIVE IMMUNOCONJUGATES

Two ribosome-inactivating proteins (RIPs) were isolated and characteri zed from the roots of Bryonia dioica. One of these was a novel 27-kDa protein termed bryodin 2 (BD2), while the second was a previously repo rted RIP, referred to here as bryodin 1 (BD1). The amino-terminal sequ ence obtained for BD2 was similar, but distinct from BD1, ricin A chai n, trichosanthin, and momorcharin. BD2-specific monoclonal antibodies were generated and found not to react with BD1 or ricin A chain. Purif ied BD1 and BD2 RIP inhibited protein synthesis in a cell-free in vitr o translation assay at EC50 values of 7 and 9 pM, respectively. Intrav enous administration of BD1 was less toxic to mice than BD2, with LD(5 0) values of >40 for BD1 and 10-12 mg/kg for BD2. Primary human endoth elial cells were 5-8-fold less sensitive to BD1 and BD2 than compared to ricin A chain. BD1 and BD2 were constructed as immunoconjugates wit h the chimeric form of BR96 (chiBR96), a carcinoma-reactive, internali zing antibody. ChiBR96-BD1 and chiBR96-BD2 were found to bind to and k ill BR96 antigen-positive carcinoma cells while not killing antigen-ne gative carcinoma cells. Bryodins represent RIPs that may be useful in constructing immunotoxin conjugates with reduced toxicity and vascular sensitivity, as compared to ricin A chain immunotoxins.