Structural characterization of a mutated, ADCC-enhanced human Fc fragment.
We report here the three-dimensional structure of a human Fc fragment engineered for enhanced antibody dependent cell mediated cytotoxicity (ADCC). The triple mutation S239D/A330L/I332E ('3M') was introduced into the C(H)2 portion of a human immunoglobulin G1 (IgG1) Fc. These three substitutions typically result in an about 10-100-fold increase in human IgG1 binding to human Fc gamma RIIIA (CD16). The recombinantly produced Fc/3M fragment was crystallized and its structure solved at a resolution of 2.5A using molecular replacement. No dramatic structural changes were observed in Fc/3M when compared with unmutated human Fc fragments. However, we found that the relative positions of its C(H)2 domains allowed for an unusually 'open' conformation of the entire fragment. Although this particular structural feature could be due to crystallization artifacts or intrinsic variability, we propose that molecular mechanisms at the basis of the enhanced interaction between Fc/3M and CD16 could include enhanced Fc openness as well as the introduction of additional hydrophobic contacts, hydrogen bonds and/or electrostatic interactions at the corresponding interface. The existence of a more pronounced cleft between the two Fc chains as well as of repulsive, electrostatic intra-chain interactions may also account in part for the decreased thermostability of both Fc/3M and a 3M-modified humanized anti-human EphA2 IgG1 when compared with their respective unmutated counterparts.
Oganesyan
Vaheh
V
Department of Antibody Discovery and Protein Engineering, MedImmune, Inc., One MedImmune Way, Gaithersburg, MD 20878, USA.
Damschroder
Melissa M
MM
Leach
William
W
Wu
Herren
H
Dall'Acqua
William F
WF
D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology*; D002152:Calorimetry, Differential Scanning; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D018360:Crystallography, X-Ray; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D008024:Ligands; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017452:Receptors, IgG / Q000737:chemistry; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D044366:Transition Temperature
D002241:Carbohydrates; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D008024:Ligands; D008670:Metals; D050505:Mutant Proteins; D017452:Receptors, IgG