A Molecular Approach to Develop gG1 Fc Isoforms with Enhanced Affinity to Immune Cells Surface FcgammaIIIa Receptor

摘要

The interaction between antibodies (IgG)and Immune cells surface FcgammaRIIIa receptor triggers a variety of immune responses including antibody-dependent cell mediated cytotoxicity (ADCC), antibody neutralization, phagocytosis, inflammation and even tissue injury in case of autoimmune diseases. Recent studies showed that IgG1 upper hinge region and FcgammaRs polymorphism play a major role in the interaction with Fcgamma receptors and in the stability of the immune complex hence strong inflammatory signal. To further investigate this issue, we have developed a tool box of IgG1 Fc Isoforms to illustrate the affinity/upper-hinge relation between mutated IgG1 Fc regions and extracellular domain variants (F176V) of the Fcgamma receptor CD 16a. Our strategy consist of designing different random upper-hinge mutated variants of IgG1 Fc domain and reproduce the naturally occurring two variants of CD 16 (176F AND 176V), expressing them as a recombinant fusion proteins in Pichia Pastoris and purify them. The interactions were assayed using Surface Plasmon Resonance (Biacore) method and finally an In Silico approach were used to analyze the Biacore data in order to identify the major interaction and key residues that underline the affinity between the Fc region and CD 16 variants. As a result we found that the affinity of the Fc region to the CD 16a is affected by polar interactions rather than complex energy and our molecular approach yielded an Fc mutant that has a higher affinity with CD 16a 176F variant.