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

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 nonfucosylated IgG1. However, the importance of the Tyr-296 residue in the antibody in the interaction with various Fcγ 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 Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr-296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa 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 FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa 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 shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr-296 in interactions with various Fcγ receptors, and have applications in the modulation of the IgG1-Fc function of therapeutic antibodies.

A nonfucosylated variant of the anti-HIV-1 monoclonal antibody b12 has enhanced FcγRIIIa-mediated antiviral activity in vitro but does not improve protection against mucosal SHIV challenge in macaques.

Eliciting neutralizing antibodies is thought to be a key activity of a vaccine against human immunodeficiency virus (HIV). However, a number of studies have suggested that in addition to neutralization, interaction of IgG with Fc gamma receptors (FcγR) may play an important role in antibody-mediated protection. We have previously obtained evidence that the protective activity of the broadly neutralizing human IgG1 anti-HIV monoclonal antibody (MAb) b12 in macaques is diminished in the absence of FcγR binding capacity. To investigate antibody-dependent cellular cytotoxicity (ADCC) as a contributor to FcγR-associated protection, we developed a nonfucosylated variant of b12 (NFb12). We showed that, compared to fully fucosylated (referred to as wild-type in the text) b12, NFb12 had higher affinity for human and rhesus macaque FcγRIIIa and was more efficient in inhibiting viral replication and more effective in killing HIV-infected cells in an ADCC assay. Despite these more potent in vitro antiviral activities, NFb12 did not enhance protection in vivo against repeated low-dose vaginal challenge in the simian-human immunodeficiency virus (SHIV)/macaque model compared to wild-type b12. No difference in protection, viral load, or infection susceptibility was observed between animals given NFb12 and those given fully fucosylated b12, indicating that FcγR-mediated activities distinct from FcγRIIIa-mediated ADCC may be important in the observed protection against SHIV challenge.

Structural basis for improved efficacy of therapeutic antibodies on defucosylation of their Fc glycans.

Removal of the fucose residue from the N-glycans of the Fc portion of immunoglobulin G (IgG) results in a dramatic enhancement of antibody-dependent cellular cytotoxicity (ADCC) through improved affinity for Fcγ receptor IIIa (FcγRIIIa). Here, we present the 2.2-Šstructure of the complex formed between nonfucosylated IgG1-Fc and a soluble form of FcγRIIIa (sFcγRIIIa) with two N-glycosylation sites. The crystal structure shows that one of the two N-glycans of sFcγRIIIa mediates the interaction with nonfucosylated Fc, thereby stabilizing the complex. However, fucosylation of the Fc N-glycans inhibits this interaction, because of steric hindrance, and furthermore, negatively affects the dynamics of the receptor binding site. Our results offer a structural basis for improvement in ADCC of therapeutic antibodies by defucosylation.

Nonfucosylated rituximab potentiates human neutrophil phagocytosis through its high binding for FcgammaRIIIb and MHC class II expression on the phagocytotic neutrophils.

OBJECTIVE: Antibody-dependent cellular cytotoxicity mediated by natural killer cells via leukocyte receptor IIIa (FcgammaRIIIa) is greatly enhanced by the absence of the core fucose of Fc oligosaccharides, and is closely related to the clinical efficacy of anticancer processes in humans in vivo. Here, we focused on the physiological functions of nonfucosylated anti-CD20 IgG1 rituximab, in particular those functions mediated by human neutrophils, which highly express FcgammaRIIIb, a highly homologous FcgammaR to FcgammaRIIIa. MATERIALS AND METHODS: After treatment with anti-CD20, the response of neutrophils to fluorescently labeled CD20(+) B-cell lymphoma in human whole blood was quantitatively analyzed by measuring their activities of antibody-dependent phagocytosis and major histocompatibility complex (MHC) class II expression on the phagocytotic neutrophils using flow cytometry. RESULTS: In human whole blood, most of the added CD20(+) B-cell lymphoma died shortly, within 4 hours, irrespective of the presence or absence of anti-CD20. Neutrophils were not directly concerned in the death because depletion of neutrophils from human whole blood did not affect the phenomenon. However, neutrophils aggressively phagocytosed newly dead lymphoma cells, and the nonfucosylated anti-CD20 effectively enhanced neutrophil phagocytosis solely by enhancing binding for the phagocytosis coreceptor FcgammaRIIIb. Noteworthy, more increased expression of MHC class II was also observed on the phagocytotic neutrophils than those observed on spontaneous and fucosylated anti-CD20 stimulated phagocytotic neutrophils. CONCLUSIONS: Our data showed that antibody therapy composed of nonfucosylated rituximab can activate human neutrophil functions involving phagocytosis and MHC class II expression, which may favorably potentiate the adaptive immune response in cancer patients.

The N-linked oligosaccharide at Fc gamma RIIIa Asn-45: an inhibitory element for high Fc gamma RIIIa binding affinity to IgG glycoforms lacking core fucosylation.

Human leukocyte receptor IIIa (Fc gamma RIIIa) plays an important role in mediating therapeutic antibodies' antibody-dependent cellular cytotoxicity (ADCC), which is closely related to the clinical efficacy of anticancer processes in humans in vivo. The removal of the core fucose from oligosaccharides attached to the Fc region of antibodies improves Fc gamma RIIIa binding, allowing the antibodies to enhance dramatically the antibody effector functions of ADCC. In this study, the contribution of Fc gamma RIIIa oligosaccharides to the strength of the Fc gamma RIIIa/antibody complex was analyzed using a serial set of soluble human recombinant Fc gamma RIIIa lacking the oligosaccharides. A nonfucosylated antibody IgG1 appeared to have a significantly higher affinity to the wild-type Fc gamma RIIIa fully glycosylated at its five N-linked oligosaccharide sites than did the fucosylated IgG1, and this increased binding was almost abolished once all of the Fc gamma RIIIa glycosylation was removed. Our gain-of-function analysis in the Fc gamma RIIIa oligosaccharide at Asn-162 (N-162) confirmed that N-162 is the element required for the high binding affinity to nonfucosylated antibodies, as previously revealed by loss-of-function analyses. Interestingly, beyond our expectation, the Fc gamma RIIIa modified by N-162 alone showed a significantly higher binding affinity to nonfucosylated IgG1 than did the wild-type Fc gamma RIIIa. Attachment of the other four oligosaccharides, especially the Fc gamma RIIIa oligosaccharide at Asn-45 (N-45), hindered the high binding affinity of Fc gamma RIIIa to nonfucosylated IgG1. Our data clearly demonstrated that N-45 is an inhibitory element for the high Fc gamma RIIIa binding affinity mediated by N-162 to nonfucosylated antibodies. This information can be exploited for the structural-based functional study of Fc gamma RIIIa.