Blocked and not blocked whole-ricin-antibody immunotoxins: intraperitoneal therapy of human tumour xenografted in nude mice

Abstract

A blocked immunotoxin, consisting of ricin and AR-3 monoclonal antibody joined by a short thioether bond, was previously synthesized. This conjugate had lost the ability to bind the galactosidic residues of Sepharose 6B, probably because of the steric restraint of the antibody molecule on the ricin B chain. In in vitro assays immunotoxin was active only on cells expressing the corresponding AR-3 epitope. The in vivo activity of our blocked immunotoxin was assessed by injecting it directly into the peritoneal cavity of tumour-bearing nude mice. The animals were i.p. grafted with the HT-29 cell line, which was derived from a human colorectal adenocarcinoma expressing the antigen CAR-3, against which the AR-3 monoclonal antibody is directed. The best protocol tested, to arrive at the optimal regimen for the i.p. blocked immunotoxin therapy, required the administration of the immunotoxin (2 micrograms) on days 4 and 6 after the graft. The mice were killed on different subsequent days to determine the therapeutic effects. Histological sections of the different organs were prepared and stained with haematoxylin/eosin and were also examined by an immunocytochemical method with AR-3 monoclonal antibody to confirm the presence of the relating antigen on the tumour cell surface. The blocked immunotoxin substantially suppressed tumour growth of the grafted HT-29 cells, without showing any undesirable ricin toxicity. Most importantly, established transplanted HT-29 tumour cells treated with blocked immunotoxin almost completely regressed, while under the same conditions the not blocked immunotoxin, an irrelevant immunotoxin, ricin, and the AR-3 alone failed to inhibit tumour growth.