Background: Recent clinical trials of FcRn antagonists have demonstrated that reducing immunoglobulin G (IgG) autoantibodies in blood can improve patient outcomes in a range of autoimmune disorders, including rheumatoid arthritis (clinicaltrials.gov identifier NCT04991753), myasthenia gravis [1], and chronic inflammatory demyelinating polyneuropathy [2]. However, FcRn antagonists require days to weeks to reduce circulating IgG down to 20-50% of baseline [3] and may not be suitable for alleviating flares where a rapid and substantial reduction in IgG is necessary. Likewise, plasmapheresis is a therapeutic option for several serious rheumatic autoimmune disorders but takes 2 weeks to achieve an IgG reduction to 30% of baseline [4]. As an alternative, the IgG degrading enzyme of Streptococcus pyogenes (IdeS) rapidly cleaves IgG to sever IgG effector functions from antigen-binding activity and is approved for the desensitization of crossmatched kidney transplant patients [5]. Due to its catalytic mechanism of action, IdeS reduces the levels of full-length IgG almost completely within minutes and has high potential for addressing flares and cases where a more substantial IgG reduction is required for therapeutic effect. However, wild type IdeS is highly immunogenic, cannot be safely administered multiple times, and has a short half-life, and has thus not realized its clinical potential.

Objectives: Using computational tools and rational human design, we engineered variants of IdeS with reduced immunogenicity and extended in vivo half-life, thus addressing the two major deficits of the wild type enzyme.

Methods: We identified antigenic regions in IdeS that are presented on HLA class II (HLA-II) to stimulate CD4+ T cells. IdeS was incubated with dendritic cells in culture and IdeS peptides that were processed for HLA-II presentation were isolated and analyzed by mass spectrometry. Next, mutations were made in these antigenic regions to reduce epitope affinity for HLA-II, guided by predictions from machine learning algorithms and validated by biochemical assays for peptide/HLA-II interactions. Having reduced epitopes that are presented to T cells, we approached the removal of surface epitopes that are exposed to B cells by borrowing a molecular mechanism of immune escape from viruses, namely decorating the surface of IdeS with N-linked glycans to shield underlying protein features that would be recognized as foreign.

Results: The combination of HLA-II epitope reduction and hyperglycosylation yielded a drug candidate that has markedly reduced reactivity against anti-IdeS antibodies in serum from human donors who recently recovered from strep infection. Furthermore, since the molecular weight of IdeS is close to the filtration size limit of the glomerular barrier, hyperglycosylated IdeS has significantly extended pharmacokinetics in a rabbit model and depletes full-length IgG for more than 5 days, compared to 2 days for wild type IdeS. The duration of IgG depletion was further extended to weeks by fusing an IdeS variant to human serum albumin to facilitate FcRn-mediated recycling.

Conclusion: The superior pharmacokinetic and pharmacodynamic properties of the engineered IdeS variants support their potential for the rapid and sustained degradation of IgG autoantibodies, while their reduced immunogenicity based on ex vivo assessments suggests redosing will be possible for long-term management of recurrent flares or chronic autoimmune diseases.

REFERENCES: [1] Zhu LN, et al. (2023) Neural Regen Res 18(8):1637-1644.

[2] Briani C, Visentin A. (2022) Neurotherapeutics 19(3):874-884.

[3] Peter HH, et al. (2020) J Allergy Clin Immunol 146(3):479-491.e5.

[4] Guptill JT, et al. (2016) Autoimmunity 49(7):472-479.

[5] Huang E, Maldonado AQ, Kjellman C, Jordan SC. (2021) Am J Transplant 22(3):691-697.

Acknowledgements: NIL.

Disclosure of Interests: Maximilian Sauer Cyrus Biotechnology, Cyrus Biotechnology, Benjamin Dutzar Cyrus Biotechnology, Cyrus Biotechnology, Emily Frazier Cyrus Biotechnology, Cyrus Biotechnology, Kui Chan Cyrus Biotechnology, Cyrus Biotechnology, Daniel Farrell Cyrus Biotechnology, Gavin Robertson Cyrus Biotechnology, Yifan Song Cyrus Biotechnology, Cyrus Biotechnology, Eric Tarcha Cyrus Biotechnology, Cyrus Biotechnology, Erik Procko Stock and options in Cyrus Biotechnology., Employed by Cyrus Biotechnology.