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Kidney Week

Abstract: SA-PO830

Potency Assessment of a Recombinant IgA Protease: Toward the Treatment of IgA Nephropathy

Session Information

Category: Glomerular Diseases

  • 1401 Glomerular Diseases: From Inflammation to Fibrosis

Authors

  • Shu, Chutian, Alebund Biotech, Inc., Shanghai, China
  • Jin, Jing, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
  • Zhu, Yuan, Alebund Biotech, Inc., Shanghai, China
  • Liang, Chencai, Alebund Biotech, Inc., Shanghai, China
  • Mareczko, Andrew, Alebund Biotech, Inc., Shanghai, China
Background

Microbial IgA proteases represent a structurally heterogenous group of proteins. Despite their structural diversity, IgA proteases are highly specific to cleave the hinge region of human IgA1 heavy chain. Here we explore the potential use of an IgA protease derived from Streptococcus strain TIGR4 for the treatment of IgA nephropathy by the clearance of IgA deposits in the glomerular mesangial area.

Methods

We designed a TIGR4 with a truncation at its N-terminus in a fusion configuration with Fc for extending its plasma half-life. We then expressed this TIGR4-Fc fusion in E. coli strain BL21 under a Tac promoter, purified through MabSelect PrismATM columns, and assessed its purity, stability, and potency.

Results

Recombinant TIGR4-Fc was expressed as a ~350 kDa dimer attributable to dimeric Fc in its native fold. The purity is estimated to be >90% on an SDS PAGE. At 10 mg/mL, it remains stable in pbs at 20°C for over a week without visible precipitation. When subjecting TIGR4-Fc to pbs of human IgA1, enzymatic cleavage of the heavy chain occurs. With TIGR4-Fc at 2 and 20 µg/mL, and the substrate at 1,000 µg/mL, all IgA was cleaved by the enzyme. When TIGR4-Fc is at 0.2 µg/mL, a partial cleavage of IgA was observed with an incremental of cleavage over a period of 24 hours.

Conclusion

These results demonstrated the feasibility of constructing a recombinant IgA protease in fusion with Fc. The resulting biologic exhibited stability in solution and showed an exceptional enzymatic potency for human IgA1. Based on the calculated EC90 of 0.2 µg/mL, a therapeutic dose of TIGR4-Fc of as little as 1 mg is sufficient to clear 90% of IgA contents in an adult patient. The Fc fusion may further extend the in vivo efficacy to 10-15 days in clearing IgA in both circulation and existing kidney deposits.

Fab and Fc of IgAH are cleavage fragments.