Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 420 (1-2), 1-7

Selective Deactivation of Serum IgG: A General Strategy for the Enhancement of Monoclonal Antibody Receptor Interactions

Affiliations

Selective Deactivation of Serum IgG: A General Strategy for the Enhancement of Monoclonal Antibody Receptor Interactions

Kavitha Baruah et al. J Mol Biol.

Abstract

Serum IgG is a potent inhibitor of monoclonal antibody (mAb) binding to the cell-surface Fcγ receptors (FcγRs), which mediate cytotoxic and phagocytic effector functions. Here, we show that this competition can be eliminated, selectively, by the introduction to serum of (i) an enzyme that displaces Fc from FcγRs and (ii) a modification present in the therapeutic mAb that renders it resistant to that enzyme. Specifically, we show that (i) EndoS (endoglycosidase S) cleaves only complex-type glycans of the type found on IgG but (ii) is inactive against an engineered IgG Fc with oligomannose-type glycans. EndoS thus reduces FcγR binding of serum IgG, but not that of engineered mAb. Introduction of both the engineered mAb and endoglycosidase in serum leads to a dramatic increase in FcγR binding compared to the introduction of mAb in serum alone. Antibody receptor refocusing is a general technique for boosting the effector signal of therapeutic antibodies.

Figures

None
Fig. 1
Fig. 1
Endoglycosidase-mediated deactivation of serum IgG. (a) Binding of human IgG1 Fc to immobilized FcγRIIIa was determined, by ELISA, in the presence of PBS (phosphate-buffered saline) or increasing concentrations of human serum and detected using a secondary antibody specific for the monoclonal Fab domain (Supplementary Methods). (b) The crystal structure of FcGlcNAc (blue ribbon) overlaid with structures of glycosylated human IgG Fcs using SHP by superposition of Cα residues from one protomer while leaving the second protomer free (pink ribbons; PDB IDs 1FC1, 1H3T, 1H3U, 1H3V, 1H3W, 1H3X, 1H3Y, 2DTQ, 2DTS, 3DNK, 3D03 and 3HKF). Broken lines are drawn between equivalent Cα atoms (Tyr296) in FcGlcNAc and naturally glycosylated structures and indicate a displacement of approximately 8 Å. For crystallographic analysis, FcGlcNAc (Supplementary Methods) was concentrated to 7.0 mg/mL and was crystallized after 42 days with the use of the sitting-drop vapor diffusion method using 100 nL protein plus 100 nL precipitant equilibrated against 95 μL reservoirs. Crystals grew at room temperature in a precipitant containing 25% (w/v) polyethylene glycol 1500 and 0.100 M SPG System buffer (pH 4). Crystals were flash frozen by immersion in a cryoprotectant containing the mother liquor diluted in 25% (v/v) glycerol and then rapidly transferred to a gaseous nitrogen stream. X-ray diffraction data were recorded at beamline I03 at Diamond Light Source, Oxfordshire, England. Data were processed and scaled using DENZO and SCALEPACK, and the structure was solved using Phaser with native Fc (PDB accession number 3AVE) as a search model. Model building was performed with Coot and iteratively refined using restrained refinement with TLS in the CCP4 supported program REFMAC5. (c) Binding of EndoS-treated or mock-treated human sera to immobilized FcγRIIIa determined using an anti-human IgG secondary antibody (Supplementary Methods).
Fig. 2
Fig. 2
Resistance of oligomannose containing Fc glycoforms to EndoS-mediated hydrolysis. Matrix-assisted laser desorption/ionization mass spectrometry spectra of PNGase-F released N-glycans from IgG1 Fc expressed in GnT-I-deficient HEK (human embryo kidney) 293S cells (Supplementary Methods). These expression systems yielded, respectively, Fc with complex-type (a–c) or oligomannose-type (d–f) glycans, which were exposed to no enzyme (a and d), EndoS (b and e) or EndoH (c and f). The spectra of the oligomannose glycans reveal the presence of GnT-I-independent fucosylation. The cleavage of the core GlcNAcβ1 → 4GlcNAc bond by endoglycosidases results in the removal of a single GlcNAc (predicted Δm/z = 203.1) or Fucα1 → 6GlcNAc (predicted Δm/z = 349.1). Symbolic representation of glycan structures follows that of Harvey et al.: ◊,   Gal; ■,   GlcNAc; ○,   Man; formula image,   Fuc. The linkage position is shown by the angle of the lines linking the sugar residues (vertical line,   2-link; forward slash,   3-link; horizontal line,   4-link; back slash,   6-link). Anomericity is indicated by continuous lines for β-bonds and by broken lines for α-bonds.
Fig. 3
Fig. 3
EndoS-mediated deactivation of serum leads to enhancement of mAb binding to FcγRIIIa. (a) ELISA showing the interaction between monoclonal IgG1 containing oligomannose (Man5GlcNAc2) glycans and immobilized FcγRIIIa in the presence of PBS, serum, serum and EndoS, serum and EndoH or serum and EndoS and EndoH (Supplementary Methods). Binding was detected using a secondary antibody specific for the monoclonal Fab domain as in Fig. 1a. Data points represent the calculated mean of three independent measurements from a total of four experiments. (b) Schematic illustration of the differential binding of FcγRIIIa to oligomannose and natural Fc glycoforms in the presence of EndoS. Deactivated FcGlcNAc is shown in blue, and activated Fc in complex with FcγRIIIa (gray surface; PDB ID 1T83) is shown in pink. Glycans are shown as yellow spheres.

Similar articles

See all similar articles

Cited by 22 PubMed Central articles

See all "Cited by" articles

References

    1. Nimmerjahn F., Ravetch J.V. Fcγ receptors as regulators of immune responses. Nat. Rev., Immunol. 2008;8:34–47. - PubMed
    1. Siberil S., Dutertre C.A., Fridman W.H., Teillaud J.L. FcγR: the key to optimize therapeutic antibodies? Crit. Rev. Oncol./Hematol. 2007;62:26–33. - PubMed
    1. Stavenhagen J.B., Gorlatov S., Tuaillon N., Rankin C.T., Li H., Burke S. Fc optimization of therapeutic antibodies enhances their ability to kill tumor cells in vitro and controls tumor expansion in vivo via low-affinity activating Fcγ receptors. Cancer Res. 2007;67:8882–8890. - PubMed
    1. Bruhns P., Iannascoli B., England P., Mancardi D.A., Fernandez N., Jorieux S., Daeron M. Specificity and affinity of human Fcγ receptors and their polymorphic variants for human IgG subclasses. Blood. 2009;113:3716–3725. - PubMed
    1. Jefferis R. Antibody therapeutics: isotype and glycoform selection. Expert Opin. Biol. Ther. 2007;7:1401–1413. - PubMed

Publication types

MeSH terms

Associated data

LinkOut - more resources

Feedback