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

Abstract

To avoid non-specific binding of intact ricin-antibody conjugates, we prepared a new blocked thioether-linked ricin-antibody IT, in which the galactose binding 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.

Recombinant ricin toxin A chain cytotoxicity against carcinoembryonic antigen expressing tumour cells mediated by a bispecific monoclonal antibody and its potentiation by ricin toxin B chain

“…These results suggest that the B chain translocation function is still active when the lectin site is blocked, and therefore that the lectin site is not involved. This is in agreement with results of studies using intrinsically blocked whole ricinantibody conjugates (Cattel et al, 1988). A blocked ricin immunotoxin had cytotoxic activity as high as that of an unblocked ricin conjugate, but was much more selective than the unblocked reagent.…”

“…RTB can be administered in free form, or conjugated to the same targeting antibody as the RTA immunotoxin or any antibody recognising the immunotoxin conjugate (McIntosh et al, 1983;Vitetta et al, 1983Vitetta et al, , 1984, and is thought to aid cellular incorporation of immunotoxin by facilitating translocation through the cell membrane (McIntosh & Thorpe, 1984). In cases where the targeted antigen is rapidly and efficiently internalised by the cell the lack of B chain may be advantageous but nonessential, but there are instances in which RTA immunotoxin is not readily incorported and the transport function of the B chain then becomes of major importance (McIntosh et al, 1983;Eccles et al, 1987 (Thorpe & Ross, 1982;Gregg et al, 1987;Cattel et al, 1988). Galactose blocking can be very effective in aiding selectivity of ricin-antibody conjugates in vitro, and a galactose-blocked ricin anti-iodiotype antibody conjugate has been tested in vivo against a guinea pig B cell leukaemia line (Gregg et al, 1987).…”

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

“…We preparaed and purified the immunotoxins as described previously [4]. The monoclonal antibody AR-3 was linked to intact ricin via a thioether bond.…”

“…The toxin was ricin, a glycoprotein extracted from Ricinus communis seeds. Proteins were obtained as described previously [4]. The monoclonal antibody used to synthesize the irrelevant immunotoxin was Movl8, an IgG1 that recognizes an antigen expressed on epithelial ovarian tumours.…”

“…Following the same strategy we prepared [4] a new thioether-linked ricin-antibody immunotoxin in which the galactose-binding site of the ricin was sterically hindered. The immunotoxin was synthesized by linking the AR-3 monoclonal antibody, previously derivatized with N-hydroxysuccinimidyl-S-acetyl thioacetate (SATA), to ricin iodoacetylated with the N-hydroxysuccinimidylester of iodoacetic acid (SIA).…”