title: Integrin binding human antibody constant domains--probing the C-terminal structural loops for grafting the RGD motif.
abstract: Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL--a disulfide-bridged cyclic heptapeptide that confers binding to human αvβ3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvβ3 integrin in ELISA or to αvβ3-expressing K562 cells in FACS analysis. Additionally, blocking of αvβ3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops.
authors.affiliation: Christian Doppler Laboratory for Antibody Engineering, Vienna Institute of BioTechnology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, Vienna, Austria.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Traxlmayr
authors.forename: Gordana
authors.initials: G
authors.lastname: Wozniak-Knopp
authors.forename: Bernhard
authors.initials: B
authors.lastname: Antes
authors.forename: Gerhard
authors.initials: G
authors.lastname: Stadlmayr
authors.forename: Florian
authors.initials: F
authors.lastname: Rüker
authors.forename: Christian
authors.initials: C
authors.lastname: Obinger
mesh_terms: D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005434:Flow Cytometry; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics / Q000378:metabolism*; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism*; D010843:Pichia; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011961:Receptors, Fc / Q000378:metabolism; D017452:Receptors, IgG / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000378:metabolism
chemical_list: D015395:Histocompatibility Antigens Class I; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D016023:Integrins; D011961:Receptors, Fc; D017452:Receptors, IgG; D013205:Staphylococcal Protein A; C100688:Fc receptor, neonatal