Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Oct 7;10(10):e0140120.
doi: 10.1371/journal.pone.0140120. eCollection 2015.

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

Yuya Isoda  1 , Hirokazu Yagi  2 , Tadashi Satoh  3 , Mami Shibata-Koyama  4 , Kazuhiro Masuda  1 , Mitsuo Satoh  4 , Koichi Kato  5 , Shigeru Iida  1
Affiliations

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

Yuya Isoda et al. PLoS One. .

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 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.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: Yuya Isoda, Mami Shibata-Koyama, Kazuhiro Masuda, Mitsuo Satoh, and Shigeru Iida are from Kyowa Hakko Kirin Co., Ltd. Koichi Kato is a scientific advisor of Medical & Biological Laboratories Co. Ltd. These do not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. SDS-PAGE of the purified anti-CD20 IgG1 rituximab variants.
The purified fucosylated (A) and nonfucosylated (B) anti-CD20 IgG1 variants were subjected to nonreducing and reducing 5–20% SDS-PAGE analyses. Lane M: molecular mass marker, lane 1: Y296G, lane 2: Y296E, lane 3: Y296L, lane 4: Y296F, lane 5: Y296D, lane 6: Y296P, lane 7: Y296H, lane 8: Y296W, lane 9: Y296S, lane 10: Y296R, lane 11: Y296N, lane12: Y296V, lane 13: Y296 A, lane 14: Y296I, lane 15: Y296C, lane 16: Y296T, lane 17: Y296K, lane 18: Y296M, and lane 19: Y296Q.
Fig 2
Fig 2. Binding affinities of anti-CD20 IgG1 rituximab variants for shFcRIIIa (V), shFcRIIIa (F), shFcRIIIb, shFcRIIa, and shFcRI.
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 K D value (n = 3) is indicated on the Y axis; bars ± standard deviations (SDs). Dashed lines indicate the K D value of the wild-type antibodies. *, the K D value of Y296P was more than 100 × 10−8 M. n.d., not detected.
Fig 3
Fig 3. ADCC activity of anti-CD20 IgG1 variants.
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.
Fig 4
Fig 4. Structure of IgG1-Fc-Y296W complexed with shFcγRIIIa.
(A) Overall structures of nonfucosylated Fc fragments in a complex with the bis-N-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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sheridan C. First integrin inhibitor since Tysabri nears approval for IBD. Nat Biotechnol. 2014;32(3):205–7. 10.1038/nbt0314-205 - DOI - PubMed
    1. Webster RM. The immune checkpoint inhibitors: where are we now? Nat Rev Drug Discov. 2014;13(12):883–4. 10.1038/nrd4476 - DOI - PubMed
    1. Weiner GJ. Rituximab: mechanism of action. Semin Hematol. 2010;47(2):115–23. 10.1053/j.seminhematol.2010.01.011 - DOI - PMC - PubMed
    1. Hatjiharissi E, Xu L, Santos DD, Hunter ZR, Ciccarelli BT, Verselis S, et al. Increased natural killer cell expression of CD16, augmented binding and ADCC activity to rituximab among individuals expressing the Fc{gamma}RIIIa–158 V/V and V/F polymorphism. Blood. 2007;110(7):2561–4. - PMC - PubMed
    1. Weng WK, Levy R. Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J Clin Oncol. 2003;21(21):3940–7. - PubMed

Publication types

MeSH terms

Associated data

Grants and funding

This work was supported in part by JSPS KAKENHI (Grant Numbers 25102008, 24249002 to K.K.), the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO) (to K.K.), and the Okazaki ORION project. Yuya Isoda, Mami Shibata-Koyama, Kazuhiro Masuda, Mitsuo Satoh, and Shigeru Iida are from Kyowa Hakko Kirin Co., Ltd. Koichi Kato is a scientific advisor of Medical & Biological Laboratories Co. Ltd. These funders provided support in the form of salaries for authors (Y.I., M.S., K.M., M.S., S.I., K.K.), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of the authors are articulated in the ‘author contributions’ section.