The Aspergillus toxin restriction is a suitable cytotoxic agent for generation of immunoconjugates with monoclonal antibodies directed against human carcinoma cells.
The protein toxin restriction, 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 Mr 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 restriction 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 (MBr1, 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 MBr1, MLuC1 and MOv17 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 MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited about one third of the antigenic sites interanalized. 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.
Departamento de Investigación, Centro Ramón y Cajal, Madrid, Spain.
F P
FP
Conde
R
R
Orlandi
S
S
Canevari
D
D
Mezzanzanica
M
M
Ripamonti
S M
SM
Muñoz
P
P
Jorge
M I
MI
Colnaghi
D000485:Allergens; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000493:pharmacokinetics / Q000494:pharmacology*; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D001230:Aspergillus; D002277:Carcinoma; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D005656:Fungal Proteins / Q000032:analysis / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects*; D011817:Rabbits; D012260:Ribonucleases; D012276:Ricin / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured / Q000187:drug effects
C081947:ASPF1 protein, Aspergillus fumigatus; D000485:Allergens; D000903:Antibiotics, Antineoplastic; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D005656:Fungal Proteins; D000922:Immunotoxins; C055724:MBr1-Res immunotoxin, Aspergillus; C036196:MITF protein, Aspergillus restrictus; D012276:Ricin; D012260:Ribonucleases