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-Å structure 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

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

<div><p>Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from <i>N</i>-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 <i>N</i>-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.</p></div

Oligosaccharide analysis of anti-CD20 IgG1 rituximab variants.

<p>Each composition value is the relative amount of total complex-type oligosaccharides detected.</p><p>Fu(+): fucosylated complex-type sugar chains, Fu(−): nonfucosylated complex-type sugar chains, High-Man: high-mannose-type sugar chains, N.D.: not detected.</p><p>Oligosaccharide analysis of anti-CD20 IgG1 rituximab variants.</p

Data collection and refinement statistics for the IgG1-Fc-Y296W/shFcγRIIIa complex.

<p>Data collection and refinement statistics for the IgG1-Fc-Y296W/shFcγRIIIa complex.</p

Binding affinities of anti-CD20 IgG1 rituximab variants to shFcγRs.

<p>The values in parentheses indicated fold changes relative to wild-type. The mean <i>K</i>_D value (n = 3) is indicated on the Y axis; bars ± standard deviations (SDs). Fu(+): fucosylated; Fu(-): nonfucosylated.</p><p>Binding affinities of anti-CD20 IgG1 rituximab variants to shFcγRs.</p

Structure of IgG1-Fc-Y296W complexed with shFcγRIIIa.

<p>(A) Overall structures of nonfucosylated Fc fragments in a complex with the bis-<i>N</i>-glycosylated soluble form of Fcγ receptor IIIa (shFcγRIIIa): left, Y296W Fc; right, wild-type Fc (PDB code: 3AY4). Chains A and B of the Fc fragment and shFcγRIIIa are colored marine, pink, and yellow, respectively. Carbohydrate residues are represented as spheres. (B) Close-up view of the interaction interface between Fc and shFcγRIIIa: upper, Y296W Fc; lower, wild-type Fc (PDB code: 3AY4). Carbohydrate residues are represented as sticks, and Lys–128 of sFcγRIIIa and Trp/Tyr–296 of Fc are represented as transparent spheres.</p

Binding affinities of anti-CD20 IgG1 rituximab variants for shFcRIIIa (V), shFcRIIIa (F), shFcRIIIb, shFcRIIa, and shFcRI.

<p>Binding affinities of the nonfucosylated antibodies (A, C, E, G, I) and fucosylated antibodies (B, D, F, H, J) for shFcRIIIa (V), shFcRIIIa (F), shFcRIIIb, shFcRIIa, and shFcRI were determined using Surface Plasmon Resonance(SPR) measurement. The mean <i>K</i>_D value (n = 3) is indicated on the Y axis; bars ± standard deviations (SDs). Dashed lines indicate the <i>K</i>_D value of the wild-type antibodies. *, the <i>K</i>_D value of Y296P was more than 100 × 10⁻⁸ M. n.d., not detected.</p

ADCC activity of anti-CD20 IgG1 variants.

<p>ADCC activities of anti-CD20 IgG1 rituximab fucosylated (A, C) or nonfucosylated (B, D) variants (WT: closed circle, Y296W: closed square, Y296A: closed diamond shape, Y296K: closed triangle, anti DNP antibody: bar) were measured by the LDH release method using CD20⁺ B-cell lymphoma cell line Raji cells as target cells and human PBMCs from two healthy donors (donors 1 [A, B] and donor 2 [C, D]) as effector cells at an E/T ratio of 20/1.</p

Comparison among Models to Estimate the Shielding Effectiveness Applied to Conductive Textiles

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

A Nonfucosylated Variant of the anti-HIV-1 Monoclonal Antibody b12 Has Enhanced Fc?RIIIa-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