High-throughput profiling of antibody repertoire in ovarian cancer ascitic fluid

Background-aims: Circulating antibodies directed against self-antigens are a hallmark of several chronic diseases including autoimmune diseases and cancer. There is a growing need for the discovery of new autoantigens useful as biomarkers for the diagnosis, the prognosis and to guide therapeutic strategies. Here we present a high-throughput and unbiased approach to profile the immune responses in ovarian cancer as a model disease by identifying those tumor associated antigens (TAA) recognized by autoantibodies. Experimental design: Our technological platform combines a selection of a Phage display cDNA library followed by protein microarray and ELISA analyses of readouts and finally the correlation with clinical data. As an initial step, we characterized ascitic fluids from patients for their antibody response respectively to soluble, insoluble intracellular proteins and cell surface expressed antigens present in an ovarian cancer cell line (OVCAR3). Secondly, the most reactive ascites were used in biopanning experiments, where an ORF cDNA phage library was screened in order to isolate ORFs recognized by antibodies present in those ascitic fluids. Further, hundreds of selected ORFs were produced as GST-fusion proteins and used to construct proteins microarray. These were tested with a set of ascitic antibodies from cancer patients. Candidate antigens were validated by ELISA screening on ascitic fluids from 153 patients that includes ovarian cancer patients, other cancer and non-cancerous controls. Result and discussion: We describe here an unbiased high-throughput (HT) approach to profile the ovarian cancer antibody repertoire. With this approach, we have identified and validated a set of different ovarian cancer specific IgG interacting tumor-associated antigens, which have significantly proven to correlate with patient survival. Moreover, we demonstrated the presence of autoantibodies in ascites targeting cell surface antigens expressed by tumor cells and its role in tumor regulation by activating the complement system.