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Updated: Jul 4, 2026
Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells
Published on: October 22, 2012
Identification and functional validation of MHC class I epitopes in the tumor-associated antigen 5T4
William H Shingler1, Priscilla Chikoti, Susan M Kingsman
1Oxford BioMedica (UK) Ltd, Medawar Centre, Oxford Science Park, Oxford, OX4 4GA, UK. w.shingler@oxfordbiomedica.co.uk
Unlabelled:
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 interferon gamma (IFN gamma) ELISpot assay to validate one predicted A1 and two A2 epitopes.
Conclusion:
iTopia represents a rapid and high-throughput technique to identify CTL epitopes.
Insights
A new method, iTopia, rapidly identifies cytotoxic T lymphocyte (CTL) epitopes for cancer vaccines like TroVax. This technique aids in understanding immune responses and refining cancer therapies.
Area of Science:
- Immunology
- Vaccinology
- Oncology
Background:
- TroVax, a cancer vaccine targeting 5T4 antigen, has undergone clinical trials for colorectal cancer.
- Monitoring 5T4-specific immune responses is crucial for therapeutic refinement and assessing efficacy.
- Cytotoxic T lymphocytes (CTLs) are vital for anti-cancer immunity, but identifying their epitopes is challenging.
Purpose of the Study:
- To evaluate the iTopia technique for high-throughput identification of CTL epitopes against the 5T4 tumor antigen.
- To identify potential CTL epitopes that bind to common human leukocyte antigen (HLA) class I alleles.
Main Methods:
- Utilized iTopia, a high-throughput method assessing peptide binding to HLA alleles.
- Screened overlapping peptides of the 5T4 antigen against four HLA class I alleles (A*0101, A*0201, A*0301, B*0702).
- Assessed binding affinity and complex stability of identified peptides.
- Validated predicted epitopes using interferon-gamma (IFN-γ) ELISpot assays with patient peripheral blood mononuclear cells (PBMCs).
Main Results:
- Identified 46 putative CTL epitopes binding to at least one of the four tested HLA alleles.
- Characterized the physical binding properties, relative affinity, and complex stability of these epitopes.
- Validated one A1 and two A2 epitopes using IFN-γ ELISpot assays.
Conclusions:
- iTopia is a rapid and high-throughput method for identifying CTL epitopes.
- This technique facilitates the discovery of novel epitopes for cancer vaccines.
- The identified epitopes can aid in monitoring immune responses to vaccines like TroVax.
