# Structural determination of the IdeS-IgG complex to guide therapeutic development of IdeS

> **NIH NIH F32** · BOSTON CHILDREN'S HOSPITAL · 2020 · $50,584

## Abstract

IdeS, an IgG-antibody degrading enzyme produced by Streptococcus pyogenes bacteria, is a promising
therapeutic candidate for treating IgG-mediated autoimmune disorders and preventing organ transplant
rejection. The therapeutic use of IdeS in humans is limited by pre-existing immunity against S. pyogenes and
IdeS. The proposed research will structurally characterize the IdeS-IgG complex to gain mechanistic insight into
IdeS’s function and how it achieves such high specificity for IgG. Structural information gleaned from this work
will be leveraged to guide resurfacing of IdeS with polyethylene glycol (PEG) chains to reduce immunogenicity
and improve pharmacokinetic properties while maintaining IgG binding and cleavage activity. Specific Aim 1:
Determine the molecular composition of the IdeS–IgG complex. We will use size exclusion chromatography
with multi-angle static light scattering (SEC-MALS) and negative stain electron microscopy (EM) to test the
hypothesis that IdeS binds as a dimer to IgG. SEC-MALS will yield the molecular weight of the IdeS-IgG complex
from which we can determine the stoichiometric composition of the complex. We will use negative-stain EM to
visualize this complex to characterize its overall conformation and composition. The results will help to determine
whether IdeS binds IgG in a 2:1 or 1:1 ratio. Specific Aim 2: Structurally characterize the IdeS-IgG complex.
We will use hydrogen-deuterium exchange (HDX) coupled to mass spectrometry and X-ray crystallography to
elucidate the structure of the IdeS-IgG complex and test the hypothesis that IdeS binds to an exosite in IgG.
We will identify residues in IdeS and IgG that participate in their interaction and inform on how IdeS is capable
of extreme specificity for IgG. Specific Aim 3: Resurface IdeS and test in vivo. We will test the hypothesis
that resurfacing IdeS with PEG (i.g., PEGylation) will enhance its therapeutic potential by reducing
immunogenicity and increasing its circulatory half-life. We will use structural data from Aim 2 to guide mutation
of Lys residues in IdeS necessary for IgG binding or close to binding interfaces to prevent PEGylation. Mutations
will be chosen by computational analysis of the residues using Rosetta and then determining whether these
mutations are evolutionarily represented using sequence homology to related enzymes. We propose that this
rational approach will yield IdeS resurfaced with PEG that maintains its enzymatic activity. A real-time FP
enzyme assay will be developed to validate activity of non-PEGylated and PEGylated IdeS variants. Sandwich
ELISAs using rabbit polyclonal IdeS antibodies will test the ability of PEGylation to block immunogenicity in vitro.
In vivo cleavage activity and clearance of a PEGylated IdeS candidate construct will be assayed in mice by
measuring changes in IgG serum levels over time.

## Key facts

- **NIH application ID:** 10296713
- **Project number:** 7F32GM133086-03
- **Recipient organization:** BOSTON CHILDREN'S HOSPITAL
- **Principal Investigator:** Jordan Micheal Anderson
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $50,584
- **Award type:** 7
- **Project period:** 2019-09-01 → 2021-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10296713

## Citation

> US National Institutes of Health, RePORTER application 10296713, Structural determination of the IdeS-IgG complex to guide therapeutic development of IdeS (7F32GM133086-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10296713. Licensed CC0.

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