# Advanced Uricase Engineering for Improved Outcomes in Gout Patients

> **NIH NIH R43** · CYRUS BIOTECHNOLOGY, INC. · 2024 · $298,915

## Abstract

ABSTRACT
 Gout, a common form of arthritis triggered by high uric acid levels, affects a considerable patient
population and the market for gout therapeutics is projected to grow significantly. A recombinant and pegylated
uricase, pegloticase (trade name Krystexxa), is administered by intravenous infusion to patients with chronic
refractory gout, but its usage is limited due to high immunogenicity and severe adverse effects. Our proposal
addresses these challenges by engineering uricase variants that are long-acting with reduced immunogenicity.
In preliminary experiments, we screened uricases from different organisms and chose a bacterial uricase for its
high expression, catalytic activity, and the presence of just a small number of antigenic epitopes presented on
human leukocyte antigen class II (HLA-II) based on computational prediction algorithms and experimental
MAPPs analysis. Using high throughput activity screens at elevated temperatures, we identified mutations that
dramatically increase the enzyme's thermostability and improve activity in vivo. Computational modeling
assisted in the removal of free cysteines and their replacement by an intersubunit disulfide that further stabilizes
the enzyme for a final melting temperature of 75°C. In separate experiments, mutations predicted to decrease
affinity of uricase epitopes for HLA-II allotypes were validated by two methods that measure peptide binding to
HLA-II under acidic conditions in the presence of an HLA-II-specific chaperone, thus mimicking the antigen
presentation pathway within antigen-presenting cells. Finally, an AI-based protein design tool was used to create
de novo albumin-binding domains (ABDs) that were fused to the uricase for the purposes of improving
pharmacokinetics and shielding the protein surface from anti-drug antibodies (ADAs). (Aim 1.1) We propose to
combine each of these features into uricase variants, thus optimizing for stability, reduced immunogenicity, and
extended half-life in a single uricase sequence. (Aim 1.2) A set of uricase variants will then be administered
intravenously and subcutaneously to C57BL/6J inbred mice and serum exposures measured over time. Next,
the binding of serum albumin to ABDs localized on the outside of the uricase D2 symmetric tetramer is
hypothesized to shield underlying surface epitopes from ADA recognition. (Aim 1.3) We will determine whether
the binding of serum albumin to a uricase-ABD fusion protein reduces reactivity of polyclonal anti-uricase
antibodies in vitro and (Aim 1.4) reduces the generation of ADAs in vivo in mice. (Aim 2) Finally, taking the
mutations that were shown to reduce the affinity of peptide epitopes for HLA-II allotypes, we will assess the
impact of the mutations on peptide-mediated stimulation of T cells from healthy human donors. This research is
significant as it not only addresses the major liability (immunogenicity) of recombinant uricases as therapeutics
for gout, but also contributes to the broader dev...

## Key facts

- **NIH application ID:** 11008432
- **Project number:** 1R43AR084989-01
- **Recipient organization:** CYRUS BIOTECHNOLOGY, INC.
- **Principal Investigator:** Kui Kiu Chan
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $298,915
- **Award type:** 1
- **Project period:** 2024-09-20 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11008432

## Citation

> US National Institutes of Health, RePORTER application 11008432, Advanced Uricase Engineering for Improved Outcomes in Gout Patients (1R43AR084989-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/11008432. Licensed CC0.

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