Identification and functional validation of MHC class I epitopes in the tumor-associated antigen 5T4

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Abstract
The cancer vaccine TroVax, modified vaccinia Ankara encoding the tumor-associated antigen 5T4, has been tested in phase I and II studies in colorectal cancer patients. Monitoring of 5T4-specific immune responses in patients receiving TroVax is critical since it could inform future refinements to the therapeutic or provide a surrogate marker of clinical efficacy. Tumor-specific cytotoxic T lymphocyte (CTL) are considered to be a key component of an effective anti-cancer immune response. Though numerous techniques have been employed to identify CTL epitopes, many are labor intensive, of variable reliability or biased toward common alleles such as human leukocyte antigen (HLA)-A2. A new high-throughput technique, iTopia, enables peptides to be evaluated on the basis of their physical binding properties for HLA alleles. This technique has been utilized to rapidly screen a panel of overlapping peptides, spanning the length of 5T4. Initially, peptides which bound to four class I alleles (A*0101, A*0201, A*0301 and B*0702) were identified and their physical binding characteristics assessed further by analysis of relative affinity and complex stability. 46 putative CTL epitopes have been identified which bind to at least one of the four HLA alleles. Using PBMCs from patients vaccinated with TroVax, we have used the interferonγ (IFNγ) ELISpot assay to validate one predicted A1 and two A2 epitopes. Conclusion: iTopia represents a rapid and high-throughput technique to identify CTL epitopes.