Abstract 4774: Identification and characterization of colon cancer associated antigens which would be key therapeutic targets in the prevention of disease relapse or progression

Introduction: Colon cancer (CRC) is an immunogenic tumor and adaptive immunity may play a role in inhibiting disease relapse. A vaccine which could boost cellular immunity in CRC patients would prevent disease recurrence. However, there have been few defined immunogenic proteins, implicated in driving the malignant phenotype, that have been identified as vaccine candidates. Our group has shown that overexpressed tumor associated proteins, abundant in the cancer state but expressed at basal levels in normal cells, can be immunogenic. We hypothesized that ideal candidate antigens would be ones already validated as prognostic markers in multivariate analysis. Methods: We performed a directed literature review using search words e.g. colon cancer, prognosis, biomarker to identify such proteins in CRC. We selected a panel of eight proteins based on (1) incidence of expression, (2) independent predictor of poor prognosis and (3) early disease recurrence, and (4) known biologic function in CRC pathogenesis. Using our algorithm program we identified peptides predicted to be high affinity binders across multiple HLA DR alleles; characteristics shown to be associated with native epitopes of self tumor antigens. We synthesized the 19 peptides that were predicted to be the highest binders. We evaluated the potential immunogencity of the peptides with 1. IFN-g ELISPOT assays using PBMC's from CRC patients and controls and 2. specific IgG antibody response with ELISA using serum from CRC patients and controls. We quantitated antigen gene expression using qPCR in cell lines representing microsatellite unstable (MSI), chromosome unstable (CIN) CpG island methylator phenotype (CIMP) CRC. Summary: The eight proteins are: CDC25B, COX-2, EBAG9, EGFR, Fascin-1, IGF-1R, PRL-3 and VCP. For all candidate antigens there was a greater IFN-g response in CRC patients than in controls; for three proteins, this difference reached statistical significance (CDC25B p=.013, COX-2 p=.013, PRL-3 p=.018). For ELISA positive response, the cut-off point was set at values of the mean of control serum plus two standard deviations. All six proteins were positive. All seven antigen genes were overexpressed relative to normal colon. Conclusions: 1. biologically relevant CRC associated proteins can be identified that are associated with prognosis and potentially expressed in the majority of colorectal cancers, 2. epitopes predicted to bind multiple DR alleles, derived from candidate antigens, elicit