Characterization of ribosome-inactivating proteins isolated from Bryonia dioica and their utility as carcinoma-reactive immunoconjugates.
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.
Molecular Immunology Department, Bristol-Myers Squibb, Pharmaceutical Research Institute, Seattle, Washington 98121.
C B
CB
Siegall
S L
SL
Gawlak
D
D
Chace
E A
EA
Wolff
B
B
Mixan
H
H
Marquardt
D000595:Amino Acid Sequence; D000818:Animals; D000912:Antibodies, Neoplasm / Q000737:chemistry* / Q000276:immunology; D016923:Cell Death; D004730:Endothelium, Vascular / Q000166:cytology; D005260:Female; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry* / Q000276:immunology / Q000633:toxicity; D007928:Lethal Dose 50; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification / Q000633:toxicity; D010944:Plants / Q000737:chemistry*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000187:drug effects*; D014118:Toxins, Biological; D014407:Tumor Cells, Cultured
D000912:Antibodies, Neoplasm; D000922:Immunotoxins; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D054789:Ribosome Inactivating Proteins, Type 1; D014118:Toxins, Biological; C051829:bryodin