Background/Purpose: The striking restriction of GCA to individuals over 50 years old suggests that age-related molecular changes may drive the pathogenesis of this disease. The specific age-related mechanisms triggering this large-vessel vasculitis remain unknown, limiting diagnostic and therapeutic approaches. Previous work investigating temporal artery biopsies revealed extensive antibody deposition in diseased tissue, suggesting autoantibody responses to unknown vascular antigens may play a role in disease pathogenesis.

Methods: To identify novel autoantibodies in patients with systemic vasculitis, a phage immunoprecipitation sequencing (PhIP-Seq) study was conducted among 956 patients with 6 different types of vasculitis and compared the results to over 60,000 samples from individuals without vasculitis. PhIP-seq is an unbiased approach to identify autoantibody targets across the entire human proteome. This method utilizes a library of 49-amino-acid peptides that covers the human genome cloned into T7 phage that are immunoprecipitated with patient antibodies and analyzed via next-generation sequencing (Fig 1).

Results: In 235 patients with GCA, PhIP-seq revealed autoantibodies targeting the amyloid-forming cleavage product of MFGE8, known as medin, in 12 (5.1%) of patients and 0% of patients without vasculitis (Fig 2). Anti-medin autoantibodies were absent in other types of vasculitis except for 1/183 (0.5%) patients with Takayasu arteritis. PhIP-seq in an orthogonal validation cohort confirmed GCA-specific autoreactivity to medin peptides. Medin is the most common human amyloid, found in the medial layer of large arteries of nearly all individuals over 50 years old. Medin amyloid has been shown to be phagocytosed by giant cells in GCA and its parent protein, MFGE8, has been associated with GCA in genome-wide association studies. Additional exploration into conformational antibody responses with recombinant expression and fibril formation of medin, followed by immunogold labeling with patient sera and TEM visualization, demonstrated autoantibodies targeting medin amyloid fibrils in patients with GCA.

Conclusion: Unbiased, proteome-wide PhIP-seq identified autoantibodies to medin in patients with GCA, connecting age-related amyloid accumulation to disease pathogenesis and implicating medin as a candidate autoimmune antigen. Despite the relatively low seroprevalence by this assay, these antibodies may be clinically significant due to their high specificity for GCA and the potential links between medin and GCA disease pathogenesis.

The human proteome phage display library is designed to represent all proteins in the human body, including all isoform variants, in a tiled design of 49 amino acid peptides with 24 amino acid overlaps. The phage library is incubated with patient serum and antibodies that bind peptides on phage are immunoprecipitated (IP) and amplified in E. coli. After 3 rounds of incubation, IP, and enrichment, the amplified phage are sequenced to identify immunogenic peptides.

GCA patient serum binds to peptides corresponding to the medin peptide sequence in PhIP-seq. Other forms of vasculitis and healthy controls (HC) do not demonstrate medin-specific enrichment. Medin peptides present in samples were normalized to reads per 100,000 (RPK), averaged and log-transformed.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/age-related-antigen-discovery-in-giant-cell-arteritis/