Abstract 6292: Novel seroprofiling assay reveals previously undescribed autoantibody responses in immunotherapy treated cancer patients
Authors: Wang Eric Y. , Rosen Connor E. , Dai Yile , Sanmamed Miguel F. , Ring Aaron M. 
Published: September 2020

In contrast to cell-mediated immunity, the potential role of antibody-mediated “humoral” immunity in the context of immunotherapy is greatly under-appreciated. This is in spite of clues that patient autoantibodies can dramatically affect cancer progression. For instance, patients who fortuitously produce autoantibodies that target their tumor (e.g., anti-HER2 and anti-MUC1) have enhanced survival. Autoantibodies also underlie numerous cancer-associated paraneoplastic syndromes and immune-related adverse events. Thus, the humoral immune system represents a rich source of natural products (antibodies) that can exert either anti-neoplastic activity or harmful toxicities. A major barrier to uncovering functional humoral responses in immunotherapy is the paucity of methods to comprehensively determine autoantibody targets, particularly for autoantibodies targeting extracellular proteins that could plausibly mediate disease-modifying effects. To address this, we developed a yeast-surface display-based method that enables rapid discovery of seroreactivities. In short, we created a library of ~3,500 extracellular proteins, with each protein displayed on genetically-barcoded yeast clones. We incubate a small sample (~50µL) of patient serum or plasma with the library, use magnetic selection to isolate antibody-coated yeast clones, and use next-generation sequencing of genetic barcodes to identify bound proteins. Hundreds of samples can be screened against thousands of proteins simultaneously. Compared to platforms such as peptide arrays and phage-display libraries, yeast-surface display enables sampling of conformational epitopes that benefit from enhanced folding in a eukaryotic secretory environment. Using this assay, we screened a longitudinal cohort of 63 NSCLC patients treated primarily with anti-PD-L1 and anti-PD-1 checkpoint inhibition along with a variety of other antibody immunotherapies (anti-OX40, anti-VEGFA, anti-TNF, etc.). We successfully detected the therapeutic antibodies administered to these patients, which served as internal positive controls. We also identified novel autoantibody reactivities against proteins that have not yet been described in the context of cancer and that could potentially have disease-modifying effects. These include autoantibodies targeting chemokines (e.g., CXCL1/2/3), type 1 interferons, growth factors (e.g., VEGFB), and adhesion receptors (e.g., MADCAM1). We validated many of these reactivities using recombinant protein-based enzyme-linked immunosorbent assays and luciferase immunoprecipitation systems. Ultimately, these data indicate that humoral immunity could have wide-ranging functional effects in the context of immunotherapy and may provide not only insights into potential therapeutic targets but also novel patient-derived immunotherapeutic antibody agents.

Citation Format: Eric Y. Wang, Connor E. Rosen, Yile Dai, Miguel F. Sanmamed, Aaron M. Ring. Novel seroprofiling assay reveals previously undescribed autoantibody responses in immunotherapy treated cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6292.