Generation of recombinant antibodies against the beta-(1,6)-branched beta-(1,3)-D-glucan Schizophyllan

Abstract

Schizophyllan is a homopolysaccharide consistent of a beta-(1,3)-D-glucan main chain with single beta-(1,6)-linked glucose residues at approximate every third glucose moiety. It forms a triple helical structure in aqueous solution, which is stable at temperatures up to 135 °C as well as at pH values up to pH 12 and is responsible for the pseudo plastic flow behavior of Schizophyllan solutions. Additionally, it shows bioactivities like antitumoral and Immunomodulating effects. Due to these properties it is interesting for various applications in different industries. Therefore, an anti-Schizophyllan antibody would be of great value since it could be used for quantitative analysis and for investigation of e the glucan bioactivity. After establishment of glucan immobilization onto functionalized microwell plates as well as generation of an immune library, 4 recombinant antibodies (rAbs) were successfully generated by phage display technology and produced as bivalent scFv-Fc. The rAbs JoJ48C11, JoJ48F1 and JoJ49D10 were generated from the immune library and JoJ58B9 from naive library HAL9. Immunochemical investigations of these rAbs showed for each a specificity which is not directed solely against SCH but also against other beta-glucans with the same primary and triple helical structure as Schizophyllan, like Scleroglucan, Cinerean and Fructican. For further investigation of binding-specificity, the molecular structure of JoJ48C11 as Fab and a complex structure with Laminarihexaose were solved using X-ray crystallography. Based on the derived crystal structures, computational modeling and mutagenesis studies a structural insight into Schizophyllan binding of JoJ48C11 was gained. This proved the assumed combination of the beta-(1,6)-bound glucose residues and the triple helical structure as conformational antigen epitope for JoJ48C11. In sum, this study describes the first successful generation of monoclonal antibodies via phage display from a human naive and mouse immune antibody library, which recognize a structural epitope of beta-(1,6)-branched beta-(1,3)-D-glucans like SCH. Additionally, it shows the first structural description of such an anti-beta-D-glucan antibody complex.