Immuno-mass Spectrometric Identification of Predictive Biomarkers of Response and/or Toxicity to Immune Checkpoint Inhibitors

Immune checkpoint inhibitors (ICIs) are humanized antibodies that target specific inhibitory molecules, such as programmed cell death protein 1 (PD-1) and its ligand, PD-L1. The limitations of ICIs are high cost, limited response rate and development of immune-related adverse events (irAEs). Predictive and monitoring biomarkers of ICIs are an unmet need. We hypothesized that patients who develop irAEs will produce specific autoantibodies indicative of an autoimmune-like response. We developed a novel methodology that involves immunoaffinity purification of serum autoantibodies, incubation with target autoantigens and mass spectrometric identification of autoantibody targets. Using our methodology and electrochemiluminescence immunoassay for validation, we found that patients treated with pembrolizumab have anti-thyroglobulin antibody titers that are significantly associated with irAE development. Furthermore, we identified several potential autoantibody targets in a patient with ICI-induced myocarditis. Predictive biomarkers of ICIs will save significant resources, ensure proper patient selection for treatment, and spare certain patients from irAEs.M.Sc.2020-11-21 00:00:0

A proteome-wide immuno-mass spectrometric identification of serum autoantibodies

Abstract Background Autoantibodies are produced when tolerance to self-antigens is broken and they can be mediators of tissue injury and systemic inflammation. They are excellent biomarkers because they are minimally invasive to screen and are highly abundant in serum due to limited proteolysis and slow clearance. Conventionally used methods of identifying autoantibodies in patient sera include indirect immunofluorescence, enzyme-linked immunoabsorbent assays (ELISAs) and protein microarrays. Here we present a novel proteome-wide immuno-mass spectrometric method to identify serum autoantibody targets. Methods Serum samples from patients with inflammatory bowel disease (IBD) were analyzed by ELISA for the presence of autoantibodies to CUB and zona pellucida-like domain-containing protein 1 (CUZD1). Protein was extracted from the human pancreas as well as 16 other human tissues to make a complex tissue lysate protein mixture. Antibodies in patient sera were immobilized and purified on protein G magnetic beads and subsequently incubated with pancreatic lysate containing CUZD1 or the aforementioned complex tissue lysate. After extensive washing, antibody-bound protein antigens were trypsin-digested and identified using shotgun mass spectrometry. Results The protocol was optimized for the immunoaffinity purification of autoantibody targets from tissue lysate, using CUZD1 from pancreatic lysate and anti-CUZD1 autoantibodies present in IBD patient serum as a proof-of-concept. Pancreatic secretory granule membrane major glycoprotein 2, whose autoantibodies are a known biomarker of Crohn’s disease, was also immunoprecipitated from IBD patient serum, as an additional internal positive control. Conclusions This study demonstrates the effectiveness of a proteomic approach to identify serum autoantibody targets, using immunoaffinity purification followed by tandem mass spectrometry. Our methodology is applicable for proteome-wide analysis of autoantibody targets in a wide variety of clinical settings

Correction to: A proteome-wide immuno-mass spectrometric identification of serum autoantibodies

In the original version of the article [1], an error was noticed under the heading “Immunoprecipitation on protein G magnetic beads” in “Methods” section