10 research outputs found
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
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 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
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
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
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<sup>+</sup> 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
Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC-1
<p><b>Copyright information:</b></p><p>Taken from "Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC"</p><p>http://www.biomedcentral.com/1472-6750/7/84</p><p>BMC Biotechnology 2007;7():84-84.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2216013.</p><p></p> line 32-05-12 (filled squares) was cultured as a control. Viable cell density (A, solid lines), antibody concentration in the culture supernatant (A, dotted lines), and cell viability (B) were analyzed in the fed-batch culture. The oligosaccharide structures of the final products from 32-05-12 (C), FG1 (D), and FG16 (E) were analyzed using MALDI-TOF MS. The relative composition of each peak is shown as the relative amount to the total amount of oligosaccharide detected
Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC-0
<p><b>Copyright information:</b></p><p>Taken from "Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC"</p><p>http://www.biomedcentral.com/1472-6750/7/84</p><p>BMC Biotechnology 2007;7():84-84.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2216013.</p><p></p>n or hygromycin resistance gene and a short hairpin siRNA expression cassette controlled by the human tRNApromoter (B or C). The siRNA tandem expression plasmid consisted of a hygromycin resistance gene and two short hairpin siRNA expression cassettes targeting and (D). The transcribed shRNAs and tRNA-shRNA fusion product were processed into siRNAs by Dicer
Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC-2
<p><b>Copyright information:</b></p><p>Taken from "Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC"</p><p>http://www.biomedcentral.com/1472-6750/7/84</p><p>BMC Biotechnology 2007;7():84-84.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2216013.</p><p></p>A). The antigen-binding activity of the antibody was measured by ELISA (B). Antibody purified from the serum-free fed-batch cultures of cells transformed by the and tandem siRNA expression vector, FG1 (open circles), FG16 (open squares), and the parental cell line 32-05-12 (filled circles) are shown. Cytotoxicity (%) and absorbance are indicated as the mean values ± SD of triplicates