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Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with Fc?RIIIa and Other Fc? Receptors.

ABSTRACT: Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG) improves the binding affinity for Fc? receptor IIIa (Fc?RIIIa) and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr-296 of IgG1-Fc plays a critical role in the interaction with Fc?RIIIa, particularly in the enhanced Fc?RIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr-296 residue in the antibody in the interaction with various Fc? receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr-296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fc? receptors: shFc?RI, shFc?RIIa, shFc?RIIIa, and shFc?RIIIb. Some of the mutations affected the binding of antibody to not only shFc?RIIIa but also shFc?RIIa and shFc?RIIIb, suggesting that the Tyr-296 residue in the antibody was also involved in interactions with Fc?RIIa and Fc?RIIIb. For Fc?RIIIa binding, almost all Tyr-296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to Fc?RIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFc?RIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFc?RIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for Fc?RIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr-296 in interactions with various Fc? receptors, and have applications in the modulation of the IgG1-Fc function of therapeutic antibodies.

SUBMITTER: Isoda Y

PROVIDER: S-EPMC4596520 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors.

Isoda Yuya Y Yagi Hirokazu H Satoh Tadashi T Shibata-Koyama Mami M Masuda Kazuhiro K Satoh Mitsuo M Kato Koichi K Iida Shigeru S

PloS one 20151007 10

Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG) improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa) and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr-296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylat ...[more]

PMID: 26444434

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Project description:Chronic HIV-infection modulates the expression of Fc gamma receptors (Fc?Rs) on immune cells and their antibody-dependent effector function capability. Given the increasingly recognized importance of antibody-dependent cellular cytotoxicity (ADCC) in HIV-specific immunity, we investigated the cellular distribution of Fc?RIIIa on cytotoxic lymphocytes-natural killer cells and CD8+ T cells-and the effect of the Fc?RIIIa-F158V variant on ADCC capacity in HIV-infected individuals (n = 23) and healthy controls (n = 23). Study participants were matched for F158V genotypes, carried two copies of the FCGR3A gene and were negative for Fc?RIIb expression on NK cells. The distribution of CD56dimFc?RIIIabright and CD56negFc?RIIIabright NK cell subsets, but not Fc?RIIIa surface expression, differed significantly between HIV-1 negative and HIV-1 positive donors. NK cell-mediated ADCC responses negatively correlated with the proportion of the immunoregulatory CD56brightFc?RIIIadim/neg cells and were lower in the HIV-1 positive group. Intriguingly, the Fc?RIIIa-F158V variant differentially affected the NK-mediated ADCC responses for HIV-1 negative and HIV-1 positive donors. Healthy donors bearing at least one 158V allele had higher ADCC responses compared to those homozygous for the 158F allele (48.1 vs. 34.1%), whereas the opposite was observed for the HIV-infected group (26.4 vs. 34.6%), although not statistically significantly different. Furthermore, Fc?RIIIa+CD8bright and Fc?RIIIa+CD8dim T cell subsets were observed in both HIV-1 negative and HIV-1 positive donors, with median proportions that were significantly higher in HIV-1 positive donors compared to healthy controls (15.7 vs. 8.3%; P = 0.016 and 18.2 vs. 14.1%; P = 0.038, respectively). Using an HIV-1-specific GranToxiLux assay, we demonstrate that CD8+ T cells mediate ADCC through the delivery of granzyme B, which was overall lower compared to that of autologous NK cells. In conclusion, our findings demonstrate that in the presence of an HIV-1 infection, the cellular distribution of Fc?RIIIa is altered and that the functional consequence of Fc?RIIIa variant is affected. Importantly, it underscores the need to characterize Fc?R expression, cellular distribution and functional consequences of Fc?R genetic variants within a specific environment or disease state.

Dataset Information

Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with Fc?RIIIa and Other Fc? Receptors.

Publications

Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors.

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