FC050: Nefecon® Selectively Modifies the Composition of Circulating Immune Complexes in IGA Nephropathy

Abstract

Abstract BACKGROUND AND AIMS IgA nephropathy (IgAN) is characterized by the mesangial deposition of IgA-containing immune complexes (IgA-IC). There is, however, no clear association between extent of mesangial IgA-IC deposition and disease severity in IgAN, suggesting that the composition of the IgA-IC, rather than the actual amount, may be critical in determining pathogenic consequences. The NEFIGAN trial (NCT01738035) assessed the safety and efficacy of a novel targeted-release formulation of budesonide (Nefecon®), designed to deliver the drug to the gut-associated lymphoid tissue (GALT)-rich distal ileum in patients with IgAN. The trial comprised a 6-month run-in, a 9-month treatment and a 3-month follow-up phase. A total of 48 patients received Nefecon® 16 mg/day, 51 patients received Nefecon® 8 mg/day and 50 patients received placebo. The key result of the study was that Nefecon® 16 mg/day, added to optimized renin–angiotensin system blockade, reduced proteinuria and stabilized estimated glomerular filtration rate in patients with IgAN. These findings have now been replicated in the NefIgArd study, which released data in 2021. Here, we report the composition of IgA-IC in the serum of patients treated with Nefecon® 16 mg/day or placebo in the NEFIGAN trial, assessed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). This study was supported by a grant from Calliditas Therapeutics AB. METHOD Using Jacalin Agarose and elution with 0.1 M d-galactose, IgA-ICs were affinity-purified from serum of patients who received placebo (n=18) or Nefecon® 16 mg/day (n=18) at the start of treatment (SOT) and end of treatment (EOT). All protein samples were reduced and alkylated prior to digestion with trypsin, dried using a Savant SpeedVac (Thermo Fisher Scientific, UK) and dissolved in 40 µL of 0.1% formic acid. Each sample, spiked with iRT standard peptides (Biognosys, Switzerland), was loaded onto an Evotip (Evosep Biosystems, Denmark) column. Mass spectrometry analyses were performed on the Evosep One (Evosep) platform, coupled to the TIMS TOF Pro (Bruker, US). For protein identification and quantification, raw data were processed with FragPipe (V16), based on at least two peptides per protein. The protein intensities thus generated were Log_2-transformed and internally normalized using IGHA1 protein as standard. For each patient, the normalized protein amount at SOT was subtracted from the amount at EOT. A probabilistic dropout model was applied to the data to estimate fold changes between treatment and placebo groups at EOT. The proteins were ranked using t-statistic, and a gene set enrichment analysis was performed to determine the gene sets significantly affected by Nefecon® 16 mg/day versus placebo. RESULTS Gene ontology analysis revealed that treatment with Nefecon® 16 mg/day led to a significant protein enrichment in IgA-IC associated with pathways driving biological processes involved in the negative regulation of gene expression and transcription, gene silencing and epigenetics. In parallel, treatment with Nefecon® was also associated with an enrichment of proteins associated with extracellular vesicle and exosome formation. CONCLUSION For the first time, we show that it is possible to pharmacologically modify the composition of circulating IgA-IC in IgAN. Treatment with Nefecon® 16 mg/day for 9 months selectively altered the protein composition of IgA-IC in a way that is likely to have significant consequences on the downstream mesangial response to IgA-IC accumulation; this may, in part, explain the antiproteinuric effect of this approach and the longer-term kidney function protection that has been reported in both the NEFIGAN and NefIgArd clinical trials. Studies are ongoing to precisely delineate the impact of these compositional changes on the pathogenicity of IgA-IC in IgAN.