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. 2024 Jan-Dec;16(1):2402701.
doi: 10.1080/19420862.2024.2402701. Epub 2024 Sep 15.

Systematic analysis of Fc mutations designed to reduce binding to Fc-gamma receptors

Geoff Hale  1 , Jelle De Vos  2 , Alastair Douglas Davy  3 , Koen Sandra  2 , Ian Wilkinson  1
Affiliations

Systematic analysis of Fc mutations designed to reduce binding to Fc-gamma receptors

Geoff Hale et al. MAbs. 2024 Jan-Dec.

Erratum in

  • Correction.
    [No authors listed] [No authors listed] MAbs. 2024 Jan-Dec;16(1):2416272. doi: 10.1080/19420862.2024.2416272. Epub 2024 Oct 20. MAbs. 2024. PMID: 39428558 Free PMC article. No abstract available.

Abstract

Elimination of the binding of immunoglobulin Fc to Fc gamma receptors is highly desirable for the avoidance of unwanted inflammatory responses to therapeutic antibodies and fusion proteins. Many different approaches have been used in the clinic, but they have not been systematically compared. We have now produced a matched set of anti-CD20 antibodies with different Fc subclasses and variants and compared their activity for binding to C1q, Fc-gamma receptors and in cell-based assays. Most of the variants still have significant levels of activity in one or more of these assays and many of them have impaired temperature stability compared with the corresponding wild-type antibody.

Keywords: C1q; CD16; CD32; CD64; DSF; Fc receptor; Fc region; Fc silent; FcγRI; FcγRII; FcγRIII; antibody effector function; antibody engineering; therapeutic antibody.

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Conflict of interest statement

This research was sponsored by mAbsolve Limited. mAbsolve has a proprietary interest in STR mutations and licenses them to the biotech and pharmaceutical industry. Geoff Hale and Ian Wilkinson have financial interests in mAbsolve.

Figures

Three scatter graphs each showing a good linear correlation between the two sets of samples.
Figure 1.
Comparison of two sets of CD20 antibodies binding to FcγRI from different species. (a) human, (b) cynomolgus monkey, (c) mouse. Binding was measured by surface plasmon resonance and expressed as a percentage of the binding of wild-type IgG1. Sample set 1 was prepared in HEK cells and sample set 2 in CHO cells.
Four overlaid chromatograms. Wild-type IgG1 shows three peaks at the longest elution time, variant LALA shows three peaks at a shorter elution time, variant LALAPG shows three peaks at an even shorter elution time and variant STR shows a single peak at the shortest elution time, indicating no interaction with the column.
Figure 2.
Affinity chromatography of CD20 antibodies on a column containing immobilized FcγRIIIa (158 V). Elution profiles of wild-type IgG1, variants L234A/L235A (LALA), L234A/L235A/P329G (LALAPG) and L234S/L235T/G236R (STR) are shown.
A scatter graph showing that the affinity HPLC method tends to give much higher responses than the SPR method.
Figure 3.
Comparison of the binding of CD20 antibodies to FcγRIIIa (158 V) measured by surface plasmon resonance compared with affinity HPLC. The SPR results are expressed as percentage of the binding of wild-type IgG1 and the HPLC results are expressed as a percentage of the retention time of wild-type IgG1. IgG1 samples filled red circles ●, IgG2 samples filled blue circles ●, IgG3 samples open blue squares □, IgG4 samples filled orange triangles ▲.
Five scatter graphs each showing reasonable but non-linear correlations between the two methods. Responses in the cell-based assay are generally higher than in the SPR assay but there are exceptions, particularly for the different versions of FcγRII and for IgG3, which consistently gives high responses by SPR and low responses in the cell-based assays.
Figure 4.
Comparison of the binding of CD20 antibodies to human fc receptors and their activity in a cell-based luminescence assay. (a) FcγRI, (b) FcγRIIa (131 H), (c) FcγRIIa (131 R), (d) FcγRIIb, (e) FcγRIIIa (158F), (f) FcγRIIIa (158 V). Binding and activity are expressed as a percentage of wild-type IgG1. Wild-type IgG3 is indicated with an open square □, other samples with a closed circle ●.
Scatter graphs to compare the two assay methods. The Promega assay gives slightly higher activity than the SVAR assay for FcγRIIa, but the Svar assay gives much higher responses for FcγRIIIa.
Figure 5.
Comparison of Promega and Svar cell-based assay systems. (a) FcγRIIaγ (131 H), (b) FcγRIIIa (158 V). Activity is expressed as a percentage of wild-type IgG1.
Plots showing the degree of unfolding as a function of temperature. The most thermally stable samples are wild-type IgG1 and the variant L234S/L235T/G236R (STR).
Figure 6.
Thermal stability of CD20 antibodies measured by differential scanning fluorimetry. Representative melt curves (normalized first differential) are shown for: (a) wild-type IgG1, IgG2, IgG3 and IgG4-P and (b) wild-type IgG1, L234S/L235S/G236R (STR), L234A/L235A (LALA), and N297A (aglycosyl).
See this image and copyright information in PMC

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