Sang Taek Jung1, Tae Hyun Kang2, Inger Sandlie3, Philip W. Tucker2, and George Georgiou1. (1) Department of Chemical Engineering, University of Texas at Austin, University Station C0400, Austin, TX 78712, (2) Institute for Cellular and Molecular Biology, University of Texas at Austin, University Station C0400, Austin, TX 78712, (3) Department of Molecular Biosciences and Centre for Immune Regulation, University of Oslo, Oslo, Norway
In IgG molecules removal of N-linked glycan results in a conformational change that abolishes binding to Fc gamma receptor proteins on the surface of leukocytes and clearance of abnormal target cells. We have developed a robust flow cytometric screening system for the isolation of aglycosylated Fc mutants exhibiting high affinity to desired Fcƒ× receptors despite the lack of glycosylation. A series of complex libraries (107-x109 clones) were generated by random or insertional mutagenesis of the Fc domain and screened for binding to purified Fc gamma receptors. Mutant aglycosylated Fcs that bind with high affinity and specificity were isolated and characterized in vitro. The aglycosylated Fc gamma receptor binding mutant proteins contain between 1-11 amino acid substitutions. We found that the mutations allowed highly selective binding of an aglycosylated, anti-Her2 (trastuzumab) to the Fcƒ×RI receptor, with desired affinity indistinguishable from that of CHO-derived, glycosylated trastuzumab (Herceptin). New screening using this methodology and judiciously constructed new mutant library, ultra-high affinity aglycosylated antibody Fc mutant exhibiting much higher affinity to Fcƒ×RI than commercial glycosylated trastuzumab have been also isolated. For the isolated aglycosylated antibody Fc mutants, antibody dependent cell mediated cytotoxicity (ADCC) and antibody dependent cell mediated phagocytosis (ADCP) are being investigated.