The galactose-binding sites of the cytotoxic lectin ricin can be chemically blocked in high yield with reactive ligands prepared by chemical modification of glycopeptides containing triantennary N-linked oligosaccharides

Abstract

A glycopeptide containing a triantennary N-linked oligosaccharide from fetuin was modified by a series of chemical and enzymic reactions to afford a reagent that contained a terminal residue of 6-(N-methylamino)-6-deoxy-D-galactose on one branch of the triantennary structure and terminal galactose residues on the other two branches. Binding assays and gel filtration experiments showed that this modified glycopeptide could bind to the sugar-binding sites of ricin. The ligand was activated at the 6-(N-methylamino)-6-deoxy-D-galactose residue by reaction with cyanuric chloride. The resulting dichlorotriazine derivative of the ligand reacts with ricin, forming a stable covalent linkage. The reaction was confined to the B-chain and was inhibited by lactose. Bovine serum albumin and ovalbumin were not modified by the activated ligand under similar conditions, and we conclude, therefore, that the reaction of the ligand with ricin B-chain was dependent upon specific binding to sugar-binding sites. Ricin that had its galactose-binding sites blocked by the covalent reaction with the activated ligand was purified by affinity chromatography. The major species in this fraction was found to contain 2 covalently linked ligands per ricin B-chain, while a minor species contained 3 ligands per B-chain. The cytotoxicity of blocked ricin was at least 1000-fold less than that of native ricin for cultured cells in vitro, even though the activity of the A-chain in a cell-free system was equal to that from native ricin. Modified ricin that contained only 1 covalently linked ligand was also purified. This fraction retained an ability to bind to galactose affinity columns, although with a lower affinity than ricin, and was only 5- to 20-fold less cytotoxic than native ricin.

Antibody-Based Immunotoxins for the Treatment of Cancer

“…As research progressed, toxin autonomic cell-binding domains were recognized and removed. The resulting toxin fragment, that could no longer bind normal cells, was coupled to an antibody [12][13][14][15]. Although full length immunoglobulin G (IgG) has good in vivo half-life and effector functions, its large size limits antibody tissue penetration, especially in solid tumors, and complicates the manufacturing process [6].…”

“…Since the B chain is known to reduce the cytotoxicity of the A chain [91], another strategy, named blocked-ricin (bR), was developed. In this strategy, the whole ricin toxin is used and its oligosaccharide-binding sites are blocked [13,14]. Unfortunately, RIP-derived immunotoxins were found to have some clinical disadvantages.…”

“…N901 recognizes the natural killer cell-associated antigen, a neural cell adhesion molecule isoform that is expressed on SCLC cell lines and fresh human tumors [14]. In a phase I study in 21 patients with relapsed or refractory SCLC, only one patient showed PR out of the 20 patients that have been evaluated.…”

Lineage specific treatment of adult patients with acute lymphoblastic leukemia in first remission with anti‐B4‐blocked ricin or high‐dose cytarabine

“…In blocked ricin, the highaffinity galactose binding sites of the B-chain are blocked by the covalent attachment of ligands to those sites. 7 Thus, blocked ricin retains the cytotoxicity of native ricin but has minimal, nonspecific toxicity because of the reduction in nonspecific binding.…”

Systemic immunotoxin therapy of cancer: advances and prospects

“…A blocked ricin having a non-target cell toxicity about 1,000-fold lower than that of the native toxin has been generated by chemically attaching oligosaccharide ligands with a high affinity for the galactosebinding sites (Lambert et al, 1991). Trials of immunotoxins made with this blocked ricin are in progress or planned to take place in patients with B-cell tumours and small cell lung carcinoma.…”