Modulation of the effector functions of a human IgG1 through engineering of its hinge region.
We report here the engineering of a humanized anti-human EphA2 mAb (mAb 12G3H11) in an effort to explore the relationship between the hinge of a human IgG1 and its effector functions. mAb 12G3H11, used here as a model, is directed against the human receptor tyrosine kinase EphA2, which is an actively investigated target for cancer therapy due to its up-regulation in many cancer cells. Various rational modifications were introduced into the hinge region of mAb 12G3H11. These mutations were predicted to modulate the hinge's length, flexibility, and/or biochemical properties. We show that the upper and middle hinge both play important, although functionally distinct roles. In particular, middle hinge modifications predicted to decrease its rigidity or length as well as eliminating either one of its two cysteine residues had a strong negative impact on C1q binding and complement-dependent cytotoxicity. Disruption of covalent bonds between both H chains may account in part for these effects. We also describe middle hinge mutants with a significantly decreased ability to bind FcgammaRIIIA and trigger Ab-dependent cell-mediated cytotoxicity. Conversely, we also generated upper hinge mutants exhibiting an increase in C1q binding and complement-dependent cytotoxicity activity. Therefore, this approach represents a novel strategy to fine-tune the biological activity of a given human IgG1. We also define, for the first time in such a systematic fashion, the relationship between various characteristics of the middle and upper hinge and the corresponding effector functions.
Dall'Acqua
William F
WF
Department of Antibody Discovery and Protein Engineering, MedImmune, One MedImmune Way, Gaithersburg, MD 20878, USA. dellacquaw@medimmune.com
Cook
Kimberly E
KE
Damschroder
Melissa M
MM
Woods
Robert M
RM
Wu
Herren
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000920:Antibody-Dependent Cell Cytotoxicity / Q000235:genetics; D001666:Binding Sites, Antibody / Q000235:genetics; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D015922:Complement C1q / Q000737:chemistry / Q000378:metabolism; D003168:Complement Fixation Tests; D003545:Cysteine / Q000235:genetics; D003601:Cytotoxicity Tests, Immunologic; D014644:Genetic Variation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D036104:Receptor, EphA2 / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017452:Receptors, IgG / Q000737:chemistry / Q000378:metabolism
D000911:Antibodies, Monoclonal; C496124:FCGR3A protein, human; D007074:Immunoglobulin G; D021122:Protein Subunits; D017452:Receptors, IgG; D015922:Complement C1q; D036104:Receptor, EphA2; D003545:Cysteine