Characterization of Ribosome-Inactivating Proteins Isolated from Bryonia dioica and Their Utility as Carcinoma-Reactive Immunoconjugates

Abstract

Two ribosome-inactivating proteins (RIPs) were isolated and characterized 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 reported RIP, referred to here as bryodin 1 (BD1). The amino-terminal sequence obtained for BD2 was similar, but distinct from BD1, ricin A chain, trichosanthin, and momorcharin. BD2-specific monoclonal antibodies were generated and found not to react with BD1 or ricin A chain. Purified BD1 and BD2 RIP inhibited protein synthesis in a cell-free in vitro translation assay at EC50 values of 7 and 9 pM, respectively. Intravenous administration of BD1 was less toxic to mice than BD2, with LD50 values of > 40 for BD1 and 10-12 mg/kg for BD2. Primary human endothelial cells were 5-8-fold less sensitive to BD1 and BD2 than compared to ricin A chain. BD1 and BD2 were constructed as immunoconjugates with the chimeric form of BR96 (chiBR96), a carcinoma-reactive, internalizing antibody. ChiBR96-BD1 and chiBR96-BD2 were found to bind to and kill BR96 antigen-positive carcinoma cells while not killing antigen-negative 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.

Construction, Expression, and Characterization of BD1-G28-5 sFv, a Single-chain Anti-CD40 Immunotoxin Containing the Ribosome-inactivating Protein Bryodin 1

“…3). These values are consistent with those previously reported for rBD1 and for native BD1 isolated from the plants (14,15).…”

“…The crystal structure of recombinant BD1 (rBD1) was resolved to 2.1 Å and indicated structural homology with other type I RIPs as well as the A chain of type II RIPs. BD1 was found to possess potent protein synthesis inhibitory activity in a cell-free system and was 20 -30-fold less toxic in rodents than were other plant or bacterial toxins used in immunotoxin construction (14,15).In this report, we describe the construction and in vitro characterization of a single-chain BD1-containing immunotoxin targeted to the human CD40 receptor. This immunotoxin, BD1-G28-5 sFv, was expressed in E. coli as a single polypeptide and consists of BD1 fused to the single-chain Fv (sFv) region of the anti-CD40 monoclonal antibody G28-5.…”

“…The crystal structure of recombinant BD1 (rBD1) was resolved to 2.1 Å and indicated structural homology with other type I RIPs as well as the A chain of type II RIPs. BD1 was found to possess potent protein synthesis inhibitory activity in a cell-free system and was 20 -30-fold less toxic in rodents than were other plant or bacterial toxins used in immunotoxin construction (14,15).…”

Identification of a specific tyrosine residue in Bryodin 1 distinct from the active site but required for full catalytic and cytotoxic activity

“…Keywords: bryodin; immunotoxin; membrane interaction; mutagenesis; ribosome-inactivating protein Bryodin-1 (BDI), a type 1 RIP isolated from Bryonia dioica (Stirpe et al, 1986), may have a therapeutic advantage over other RIPs due to its lower toxicity in vivo (Siegall et al, 1994). Type 1 RIPs have a common N-glycosidase activity, a basic isoelectric point, and molecular mass of 26-30 kDa (Barbieri et al, 1993).…”

“…These data indicate that Y141 plays an important role in the enzymatic activity of BDI and that Y140, although not essential for catalytic activity, is required for full BDI function. Because residues 140 and 141 are distinct from residues implicit in the active site, they may be involved in ribosomal and/or membrane interactions or in intracellular trafficking of the toxin and immunotoxin.Keywords: bryodin; immunotoxin; membrane interaction; mutagenesis; ribosome-inactivating protein Bryodin-1 (BDI), a type 1 RIP isolated from Bryonia dioica (Stirpe et al, 1986), may have a therapeutic advantage over other RIPs due to its lower toxicity in vivo (Siegall et al, 1994). Type 1 RIPs have a common N-glycosidase activity, a basic isoelectric point, and molecular mass of 26-30 kDa (Barbieri et al, 1993).…”