Synthesis of a BSA-Le(x) glycoconjugate and recognition of Le(x) analogues by the anti-Le(x) monoclonal antibody SH1: the identification of a non-cross reactive analogue.
A Le(x) trisaccharide functionalized with a cysteamine arm was prepared and this synthesis provided additional information on the reactivity of N-acetylglucosamine O-4 acceptors when they are glycosylated with trichloroacetimidate donors activated with excess BF(3)·OEt(2). In turn, this trisaccharide was conjugated to BSA lysine side chains through a squarate-mediated coupling. This BSA-Le(x) glycoconjugate displayed 35 Le(x) haptens per BSA molecule. The relative affinity of the anti-Le(x) monoclonal antibody SH1 for the Le(x) antigen and analogues of Le(x) in which the D-glucosamine, L-fucose or D-galactose residues were replaced with D-glucose, L-rhamnose and D-glucose, respectively, was measured by competitive ELISA experiments. While all analogues were weaker inhibitors than the Le(x) antigen, only the analogue of Le(x) in which the galactose residue was replaced by a glucose unit showed no binding to the SH1 mAb. To confirm that the reduced or loss of recognition of the Le(x) analogues by the anti-Le(x) mAb SH1 did not result from different conformations adopted by the analogues when compared to the native Le(x) antigen, we assessed the conformational behavior of all trisaccharides by a combination of stochastic searches and NMR experiments. Our results showed that, indeed, the analogues adopted the same stacked conformation as that identified for the Le(x) antigen. The identification of a trisaccharide analogue that does not cross-react with Le(x) but still retains the same conformation as Le(x) constitutes the first step to the design of a safe anti-cancer vaccine based on the dimeric Le(x) tumor associated carbohydrate antigen.
Wang
Jo-Wen
JW
Department of Chemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Asnani
Ari
A
Auzanneau
France-Isabelle
FI
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000970:Antineoplastic Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D001667:Binding, Competitive; D002240:Carbohydrate Sequence; D002417:Cattle; D003429:Cross Reactions; D006031:Glycosylation; D016256:Lewis X Antigen / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D014312:Trisaccharides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D016256:Lewis X Antigen; D014312:Trisaccharides; D012710:Serum Albumin, Bovine