Advanced uricase engineering for improved outcomes in gout patients

Ended Project Grant Awarded Last action

Award ID
R43AR084989
Recipient
Awarding agency
Obligated
$298,915

Award facts

Completion date
Place of performance
Washington, USA

Award details come from USAspending.gov.

Description

As the agency reported it.

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 development of technologies for the reduction of IMMUNOGENICITY in protein therapeutics generally. The outcome of this project has the potential to greatly expand treatment options for patients with severe gout, offering a safer and more effective therapeutic alternative.

Transactions

Action Obligated Date
Amendment $0
Original award $298,915

Quick answers

Who received award R43AR084989?

R43AR084989 was awarded to Cyrus Biotechnology, Inc. by National Insititue of Arthritis and Musculoskeletal and Skin Diseases (National Institutes of Health) on September 20, 2024.

How much is award R43AR084989 worth?

$298,915 has been obligated on R43AR084989.

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