Correlation between CD16a binding and immuno effector functionality of an antigen specific immunoglobulin Fc fragment (Fcab).
Antigen binding immunoglobulin Fc fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions.
f-star, Gastgebgasse 5-13, 1230 Vienna, Austria.
Manuela
M
Kainer
Bernhard
B
Antes
Susanne
S
Wiederkum
Gordana
G
Wozniak-Knopp
Anton
A
Bauer
Florian
F
Rüker
Max
M
Woisetschläger
D000920:Antibody-Dependent Cell Cytotoxicity; D002460:Cell Line; D002478:Cells, Cultured; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008958:Models, Molecular; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D017452:Receptors, IgG / Q000276:immunology*
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D018719:Erb-b2 Receptor Tyrosine Kinases; D017452:Receptors, IgG; C496124:FCGR3A protein, human