13 research outputs found
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
Conformational effects of N-glycan core fucosylation of immunoglobulin G Fc region on its interaction with Fcγ receptor IIIa
Abstract Antibody-dependent cellular cytotoxicity (ADCC) is promoted through interaction between the Fc region of immunoglobulin G1 (IgG1) and Fcγ receptor IIIa (FcγRIIIa), depending on N-glycosylation of these glycoproteins. In particular, core fucosylation of IgG1-Fc N-glycans negatively affects this interaction and thereby compromises ADCC activity. To address the mechanisms of this effect, we performed replica-exchange molecular dynamics simulations based on crystallographic analysis of a soluble form of FcγRIIIa (sFcγRIIIa) in complex with IgG1-Fc. Our simulation highlights increased conformational fluctuation of the N-glycan at Asn162 of sFcγRIIIa upon fucosylation of IgG1-Fc, consistent with crystallographic data giving no interpretable electron density for this N-glycan, except for the innermost part. The fucose residue disrupts optimum intermolecular carbohydrate-carbohydrate interactions, rendering this sFcγRIIIa glycan distal from the Fc glycan. Moreover, our simulation demonstrates that core fucosylation of IgG1-Fc affects conformational dynamics and rearrangements of surrounding amino acid residues, typified by Tyr296 of IgG1-Fc, which was more extensively involved in the interaction with sFcγRIIIa without Fc core fucosylation. Our findings offer a structural foundation for designing and developing therapeutic antibodies with improved ADCC activity
Conformational effects of N-glycan core fucosylation of immunoglobulin G Fc region on its interaction with Fcγ receptor IIIa
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
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
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 to shFcγRs.
<p>The values in parentheses indicated fold changes relative to wild-type. The mean <i>K</i><sub>D</sub> 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
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
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><sub>D</sub> value (n = 3) is indicated on the Y axis; bars ± standard deviations (SDs). Dashed lines indicate the <i>K</i><sub>D</sub> value of the wild-type antibodies. *, the <i>K</i><sub>D</sub> value of Y296P was more than 100 × 10<sup>−8</sup> M. n.d., not detected.</p