Optimization, Pharmacokinetics, and Efficacy of an IgG Protease for the Treatment of Immune Thrombocytopenia and Other IgG-Mediated Autoimmune Diseases

ABSTRACT Immune thrombocytopenia (ITP) is an autoimmune disorder caused by antiplatelet IgG autoantibodies that cause a marked reduction in platelet counts, increasing the risk of severe bleeding and bruising, and lowering patient quality of life. Current treatments, including corticosteroids and intravenous immunoglobulin, have partial efficacy and side effects. Efgartigimod alfa, an antagonist of the neonatal Fc receptor that lowers circulating IgG levels, has demonstrated limited efficacy that supports reduction of IgG as an effective therapeutic mechanism but also underscores the need for enhanced depth of IgG depletion. We are developing the bacterial IgG protease IdeS (Immunoglobulin Degrading Enzyme from Streptococcus pyogenes), engineered for reduced immunogenicity and an extended in vivo half-life, to treat ITP by the rapid and durable depletion of antiplatelet autoantibodies. Engineering of the IdeS sequence leverages computational AI protein design tools with extensive experimental validation to identify and remove T cell epitopes that are displayed on HLA class II (HLA-II). By incorporating glycosylation sites and fusing IdeS with human serum albumin (HSA), surface epitopes exposed to B cells are shielded and the enzyme's half-life is extended. As a result, we have created short-acting (CYR241) and long- acting (CYR212) drug development candidates for assessment in a chronic ITP mouse model. Additionally, we have computationally designed de novo albumin-binding domains (ABD) with potential for low immunogenicity and intermediate pharmacokinetics (PK), allowing for control over the therapeutic duration of IgG depletion and minimizing infection risks associated with hypogammaglobulinemia. (Aim 1.1) In this proposal, we will experimentally evaluate the immunogenic risk of the ABD sequence by displaying ABD-derived peptides on yeast and measuring binding to common HLA-II alleles following a HLA-DM catalyzed antigen loading reaction. (Aim 1.2) As necessary, we will remove potential immunogenic epitopes by mutagenesis and finalize an IdeS-ABD fusion protein sequence. (Aim 2) We will evaluate the PK of the short (CYR241), putative medium (IdeS-ABD), and long-acting (CYR212) IdeS proteins in a double-humanized hSA/hFcRN mouse model to provide human- like PK. (Aim 3) Utilizing a non-lethal chronic ITP mouse model, we will assess the therapeutic efficacy of CYR241, CYR212, and our final IdeS-ABD candidate. The model, driven by continuous passive transfer of polyclonal rabbit anti-mouse thrombocyte antibodies that mimics sustained autoantibody synthesis, will allow for evaluation of the IdeS variants' ability to mitigate disease through sustained platelet count improvement and antibody degradation activity over time. By the program's completion, we expect to have a set of three development candidates with different durations of action that are ready for IND-enabling studies. Clinical considerations will eventually inform which candidate/s proceed, balancing efficacy and safety. This innovation not only addresses a clinical need for improved ITP management but also opens possibilities for treating a range of IgG-mediated autoimmune diseases.