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. 2015 Jun 1;194(11):5497-508.
doi: 10.4049/jimmunol.1401218. Epub 2015 Apr 22.

Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions

Algirdas Grevys  1 , Malin Bern  1 , Stian Foss  1 , Diane Bryant Bratlie  2 , Anders Moen  3 , Kristin Støen Gunnarsen  1 , Audun Aase  2 , Terje Einar Michaelsen  4 , Inger Sandlie  1 , Jan Terje Andersen  5
Affiliations

Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions

Algirdas Grevys et al. J Immunol. .

Abstract

Engineering of the constant Fc part of monoclonal human IgG1 (hIgG1) Abs is an approach to improve effector functions and clinical efficacy of next-generation IgG1-based therapeutics. A main focus in such development is tailoring of in vivo half-life and transport properties by engineering the pH-dependent interaction between IgG and the neonatal Fc receptor (FcRn), as FcRn is the main homeostatic regulator of hIgG1 half-life. However, whether such engineering affects binding to other Fc-binding molecules, such as the classical FcγRs and complement factor C1q, has not been studied in detail. These effector molecules bind to IgG1 in the lower hinge-CH2 region, structurally distant from the binding site for FcRn at the CH2-CH3 elbow region. However, alterations of the structural composition of the Fc may have long-distance effects. Indeed, in this study we show that Fc engineering of hIgG1 for altered binding to FcRn also influences binding to both the classical FcγRs and complement factor C1q, which ultimately results in alterations of cellular mechanisms such as Ab-dependent cell-mediated cytotoxicity, Ab-dependent cellular phagocytosis, and Ab-dependent complement-mediated cell lysis. Thus, engineering of the FcRn-IgG1 interaction may greatly influence effector functions, which has implications for the therapeutic efficacy and use of Fc-engineered hIgG1 variants.

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Figures

FIGURE 1.
FIGURE 1.
Crystal structure illustrations of hIgG1 Fc variants. Amino acid residues targeted to modulate binding to hFcRn are highlighted. (A) The key Fc residues involved in binding to FcRn (I253, H310, and H435) at the CH2–CH3 interface are highlighted in green spheres. (B) M428 and N434 of the CH3 domains are highlighted in blue spheres. (C) The Fc residues M252, S254, and T256 of the CH2 domains are highlighted in orange spheres. (D) H433 and N434 of the CH3 domains are highlighted in red spheres. In all crystal structure illustrations the biantennary glycans attached to the N297 residues of the CH2 domains are shown in red. The figures were designed using PyMOL (http://www.pymol.org) with the crystallographic data of hIgG1 (Protein Data Bank accession code 1HZH) (70).
FIGURE 2.
FIGURE 2.
Production and integrity of WT hIgG1 and Fc-engineered variants. (A) Total amounts of anti-NIP hIgG1 variants produced from transient transfection of HEK293E cells. (B) Nonreducing SDS-PAGE of WT hIgG1 and Fc-engineered variants. (C) Binding of titrated amounts (1000.0–0.5 ng/ml) of anti-NIP WT hIgG1 and Fc-engineered variants to NIP-conjugated BSA. (D) A DSF thermal stability histogram showing the tm values for WT hIgG1 and Fc-engineered variants. Data are presented as mean ± SEM of experiments performed in triplicate (n = 3).
FIGURE 3.
FIGURE 3.
Measurements of pH-dependent binding to hFcRn. ELISA binding of titrated amounts (1000.0–0.5 ng/ml) of WT hIgG1 and Fc-engineered variants to hFcRn at (A) pH 6.0 and (B) pH 7.4. Data are mean ± SEM of one representative experiment out of three.
FIGURE 4.
FIGURE 4.
Binding properties of Fc-engineered hIgG1 variants toward classical hFcγRs. ELISA binding of titrated amounts (1000.0–0.5 ng/ml) of WT hIgG1 and the Fc-engineered variants to (A) hFcγRI, and calculation of relative binding of WT hIgG1 and the Fc-engineered variants to (B) hFcγRI, (C) hFcγRIIa-R131, (D) hFcγRIIa-H131, (E) hFcγRIIb, (F) hFcγRIIIa-V158, (G) hFcγRIIIa-F158, and (H) hFcγRIIIb. The experiments were performed at pH 7.4, and obtained data are shown as mean ± SEM of one experiment performed in triplicate (A), and as mean ± SEM of three independent experiments performed in triplicate (B–H). *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA test.
FIGURE 5.
FIGURE 5.
ADCP activity of hIgG1 Fc-engineered variants. (A) ADCP activity measured using PMNs and titrated amounts (5000.0–0.04 ng/ml) of WT hIgG1 and Fc-engineered variants. (B) Histogram showing the average of ADCP activity. (C) ADCP activity measured using PBMCs and titrated amounts (5000.0–0.04 ng/ml) of WT hIgG1 and Fc-engineered variants in the presence of human serum as complement source. (D) Histogram showing the average of ADCP activity in the presence of complement source. Data are shown as mean ± SEM of one experiment performed in triplicate (A and C) and as mean ± SEM of three independent experiments performed in triplicate (B and D). *p < 0.05, **p < 0.01 by one-way ANOVA test.
FIGURE 6.
FIGURE 6.
ADCC activity of hIgG1 Fc-engineered variants. (A) ADCC activity measured using PBMCs and titrated amounts (200.0–0.06 ng/ml) of WT hIgG1 and Fc-engineered variants against high-haptenated SRBCs (NIP60Fab). (B) Histogram showing the relative ADCC activity of the hIgG1 variants against high-haptenated SRBCs. (C) ADCC activity measured using PBMCs and titrated amounts (200.0–0.06 ng/ml) of WT hIgG1 and Fc-engineered variants against low-haptenated SRBCs (NIP4Fab). (D) Histogram showing the relative ADCC activity of the hIgG1 variants against low-haptenated SRBCs. (E) ADCC activity measured using pure NK cells and titrated amounts (200.0–0.06 ng/ml) of WT hIgG1 and Fc-engineered variants against high-haptenated SRBCs. (F) Histogram showing the relative ADCC activity of hIgG1 variants against high-haptenated SRBCs. (G) ADCC activity measured using pure NK cells and titrated amounts (200.0–0.06 ng/ml) of WT hIgG1 and Fc-engineered variants against against low-haptenated SRBCs. (H) Histogram showing the relative ADCC activity of hIgG1 variants against low-haptenated SRBCs. (I) ADCC activity measured using pure monocytes and titrated amounts (200.0–0.06 ng/ml) of WT hIgG1 and Fc-engineered variants against high-haptenated SRBCs. (J) Histogram showing the relative ADCC activity of hIgG1 variants against high-haptenated SRBCs. (K) ADCC activity measured using pure monocytes and titrated amounts (200.0–0.06 ng/ml) of WT hIgG1 and Fc-engineered variants against low-haptenated SRBCs. (L) Histogram showing the relative ADCC activity of hIgG1 variants against at low-haptenated SRBCs. Data are presented as mean ± SEM of one experiment performed in triplicate (A, C, E, G, I, and K), and mean ± SEM of three independent experiments performed in triplicate (B, D, F, H, J, and L). *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA test.
FIGURE 7.
FIGURE 7.
Binding properties of hIgG1 Fc-engineered variants toward C1q and complement factors. ELISA results showing binding of WT hIgG1 and Fc-engineered variants to (A) pure hC1q, (B) C1q present in human serum, and (C) pure C1q present in C1q-depleted human serum and activation of complement factors (D) C3 and (E) C5. Experiments were performed at pH 7.4 and results are shown as mean ± SEM of one representative experiment out of three.
FIGURE 8.
FIGURE 8.
ADCML activity of hIgG1 Fc-engineered variants. (A) ADCML activity measured against high-haptenated SRBCs in the presence of titrated amounts (900.0–3.7 ng/ml) of WT hIgG1 and Fc-engineered variants. (B) Histogram showing relative ADCML activity of the hIgG1 variants against high-haptenated SRBCs. (C) ADCML activity against low-haptenated SRBCs in the presence of titrated amounts (900.0–3.7 ng/ml) of WT hIgG1 and Fc-engineered variants. (D) Histogram showing relative ADCML activity of the hIgG1 variants against low-haptenated SRBCs. Experiments were performed at pH 7.4 and results are shown as mean ± SEM of one experiment performed in triplicate (A and C), and as mean ± SEM of three independent experiments performed in triplicate (B and D). ***p < 0.001 by one-way ANOVA test.
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References

    1. Chan A. C., Carter P. J. 2010. Therapeutic antibodies for autoimmunity and inflammation. Nat. Rev. Immunol. 10: 301–316. - PubMed
    1. Weiner L. M., Surana R., Wang S. 2010. Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat. Rev. Immunol. 10: 317–327. - PMC - PubMed
    1. Idusogie E. E., Presta L. G., Gazzano-Santoro H., Totpal K., Wong P. Y., Ultsch M., Meng Y. G., Mulkerrin M. G. 2000. Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc. J. Immunol. 164: 4178–4184. - PubMed
    1. Lazar G. A., Dang W., Karki S., Vafa O., Peng J. S., Hyun L., Chan C., Chung H. S., Eivazi A., Yoder S. C., et al. 2006. Engineered antibody Fc variants with enhanced effector function. Proc. Natl. Acad. Sci. USA 103: 4005–4010. - PMC - PubMed
    1. Natsume A., In M., Takamura H., Nakagawa T., Shimizu Y., Kitajima K., Wakitani M., Ohta S., Satoh M., Shitara K., Niwa R. 2008. Engineered antibodies of IgG1/IgG3 mixed isotype with enhanced cytotoxic activities. Cancer Res. 68: 3863–3872. - PubMed

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