Comparison of blocked and non-blocked ricin-antibody immunotoxins against human gastric carcinoma and colorectal adenocarcinoma cell lines

Summary To avoid non-specific binding of intact ricin-antibody conjugates, we prepared a new blocked thioether-linkedricin-antibody IT, in which the galactosebinding site of ricin had lost the ability to bind to galactosidic residues of Sepharose 6B gel. As carrier agent, the monoclonal antibody AR-3, which defines the CAR-3 tumour-associated antigenic determinant expressed selectively on different human carcinoma cell lines, was used. Purification of the new conjugate was performed in three sequential steps: (1) by HPLC gel filtration on TSK G3000SW to remove the unconjugated ricin: (2) by affinity chromatography on Affi-Gel Blue to separate the free antibody from the conjugate and (3) by affinity chromatography on Sepharose 6B to separate the galactose-binding IT from the non-binding moiety. The cytotoxicity of the blocked and non-blocked thioether-linked IT was compared with that of classical ricin-antibody IT conjugated via SPDP and that of ricin A chain IT. The comparison was made on two different target cell lines (KATO III human gastric carcinoma and HT-29 human colorectal carcinoma) versus two control cell lines (HL-60 promyelocytic pre-leukaemic and COLO38 melanoma). The results showed that the blocked thioether IT displayed a more selective toxicity to target cells than the non-blocked IT and was much more potent than the ricin A chain conjugate.This work was supported by Grants from the Italian National Research Council (C.N.R. PFTBMS n. 86.01476.57 and n. 86.01748.03), the Ministry of Public Education and the Italian Association for Cancer Research (AIRC)Abbreviations used: IT, immunotoxin; PBS, phosphate-buffered saline (100 mM sodium phosphate, 150 mM NaCl); SPDP, N-succinimidyl-3-(2-pyridyldithio)-propionate; PBS-EDTA, phosphatebuffered saline containing 1 mM EDTA; DMF, dimethylformamide; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SIA, N-hydroxuccinimidyl ester of iodoacetic acid; SATA, N-succinimidyl-S-acetylthiolacetate