Background
Rhesus macaques (RM), Macaca mulatta, are one of the main nonhuman primate models for the testing of vaccines and antibody-based therapeutics. Macaques, like humans, have high and a low affinity alleles of FcγRIIA (CD32A) which is expressed on macrophages, dendritic cells and other antigen presenting cells. The polymorphism in FcγRIIA potentially influences the ‘vaccinal effect’ via FcγRIIA-Ab mediated phagocytosis (ADCP) and the functionality of the IgG2 subclass in humans. The high affinity allele has H131 in both species, while the low affinity allele has R131 in humans and P131 in macaques. We determined the structures of the receptor variants in complex with the IgG1 Fc for both species, as well as the complex of the RM high-affinity allele with RM IgG2 Fc. These studies have enabled us to elucidate the molecular mechanisms underlying the affinity differences and specificity of the FcγRIIA receptor for the IgG subclasses.
Methods
Human receptors were expressed in HEK 293 cells and purified on IgG sepharose and macaque receptors expressed in CHO cells and purified using nickel magnetic beads. IgG1 Fcs were generated by papain digest of IgG1 (human) or as Fc constructs (both IgG1 and IgG2, macaque) expressed in HEK 293 cells. IgG subclass binding to FcγRIIA was evaluated by surface plasmon resonance. Complexes were made with an excess of Fc, which was removed by purification by gel filtration, and were then used to grow protein crystals for x-ray structure determination.
Results
We determined the structures of the human hFcγRIIA-H131 and hFcγRIIA-R131 in complex with hIgG1Fc to 2.4 Å and 2.8 Å, the structures of the macaque mFcγRIIA-H131 and mFcγRIIA-P131 with mIgG1Fc to 3.5 Å and 2.7 Å, and the structure of the mFcγRIIA-H131 with mIgG2Fc to 3.2 Å. In both species H131 forms an H-bond to Fc monomer A and packs against G237 stabilizing the N-terminus similar to the analogous His in FcγRIIIA. P131 and R131 instead pack against G236; R131 also forms an H-bond to the carbonyl of G236. These differences can explain the low affinity allele’s reduced affinity for IgG2 in both species.
Conclusions
We show how a single amino acid polymorphism changes the FcγRIIA-Fc complex by modulating how the N-terminus of monomer A interacts with FcγR to not only affect affinity but also specificity. Our findings suggest that these structural changes can be leveraged to engineer novel variants of human and RM antibodies to modulate ADCP and thereby influence therapeutic strategies and vaccine efficacy.
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