Identification of IgG1 Fc mutations affecting enzymatic inactivation by IdeS via deep mutational scanning Restricted; Files Only
Yoza, Rea (Spring 2024)
Abstract
IdeS (Immunoglobulin-degrading enzyme from Streptococcus pyogenes) is a cystine protease produced by Streptococcus pyogenes, a gram-positive bacterium, used to circumvent host immune responses. This is primarily achieved through the mechanism of IdeS in cleaving the Fab region of IgG1 from Fc, leading to the destruction of comprehensive effector function by IgG. In this, IdeS is observed to hold high specificity for IgG and binds to its lower hinge region. This specificity allows it to have unique potential for use in immunotherapy and in research. However, to fully take advantage of IdeS as a tool or in combatting its use by pathogens to evade host immune systems, it is vital to elucidate structures and residues on its substrate, specifically the Fc and hinge regions of IgG, that are significant to the activity and binding of IdeS. Thus, modulation of IdeS activity on Fc is an area of active research. In this investigation, the effect of all possible point mutations in the hinge and Fc region of IgG1 on the binding affinity and activity of IdeS was tested via deep mutational scanning in combination with a cell surface display platform. Cell sorting was utilized to collect and identify Fc mutants with the highest/ lowest binding affinities and the best/worst cleavability by IdeS. The initial results of the mutational scanning provides insight into which residues are potentially significant for IdeS binding and activity which should be beneficial for the development of IdeS or antibody related treatments.
Table of Contents
Table of contents
1. Introduction………………………………………………………………...1
2. Materials and Methods
a. Expression and purification of IdeS protease……………………………………..6
b. Inactive IdeS biotinylation………………………………………………………...6
c. Expression of wild type Fc in mammalian cells…………………………………..7
d. Flow cytometry characterization of IdeS binding and activity……………………7
e. Fluorescence activated cell sorting of Fc mutant library to select mutants affecting IdeS binding and activity………………………………………………………….8
f. Processing of sorted cell populations for High-throughput sequencing…………..9
g. Site directed mutagenesis of wild type Fc………………………………………...9
h. Expression of Fc hinge mutants from mammalian cells…………………………10
i. IdeS activity assay………………………………………………………………..11
j. Mass spectrometry analysis of IdeS activity……………………………………..11
3. Results
a. Determination of IdeS binding affinity and activity towards Fc wild type via mammalian cell surface display………………………………………………….12
b. Deep mutational scanning of IgG Fc and hinge region library to identify IgG Fc mutants with changes in binding affinity and activity of IdeS…………………..13
c. Deep mutational scanning validation by IdeS activity analyzed by SDS-PAGE..14
d. Deep mutational scanning validation by assessment of IdeS activity with mass spectrometry…………………………………………………………………...…15
e. Fc mutant thermostability analysis………………………………………………15
4. Discussion ……………………………………………………………..…..17
5. Figures…………………………………………………………………..…23
6. References ……………………………………………………………..…38
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File download under embargo until 24 May 2026 | 2024-04-29 23:48:06 -0400 | File download under embargo until 24 May 2026 |
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