Oral Presentation Melbourne Protein Group Student Symposium 2013

Polyclonal T-cell Receptors Recognise an Unusually Long Tumour Antigenic Peptide Complexed to HLA-B*0702 (#3)

Kok-Fei Chan 1 2 3 , Stephanie Gras 3 , Lars Kjer-Nielsen 2 , James McCluskey 2 , Jamie Rossjohn 3 , Weisan Chen 1 4
  1. Ludwig Institute for Cancer Research, Austin-Melbourne Branch, The University of Melbourne, Heidelberg, VIC, Australia
  2. Department of Microbiology and Immunology, University of Melbourne, Melbourne, VIC, Australia
  3. Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, Australia
  4. La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia

We recently reported a novel immunodominant 13 mer epitope derived from cancer testis antigen NY-ESO-1. This peptide is restricted to HLA-B*0702 and is naturally presented by melanoma cells. Although it adopts an extensive bulge in the centre when complexed to HLA-B7, unlike other reported long peptides1,2,3,4, the TCD8+ cell response to this 13 mer is not only immunodominant but also involves broad TCR repertoire, at least 10 different Vβ families5. We hypothesise that the polyclonal recognition of NY-ESO-160-72/HLA-B7 is probably a result of flexible arrangement of the bulged peptide upon being recognised by different TCRs. To date, four NY-ESO-160-72-specific TCD8+ cell clones with different TCRs have been generated and functionally characterised in various in vitro assays. These clones recognise naturally presented NY-ESO-160-72 presented by melanoma cell line. They show different fine specificities when probed by a series of single amino acid substituted NY-ESO-160-72 peptides. They also display different peptide-MHC I (pMHC I) tetramer binding characteristics. In addition, the blocking of CD8 co-receptor has been demonstrated to impair the recognition of pMHC I complex by these clones. All individual full-length TCR alpha and beta chain sequences have been identified and cloned. DNA sequence analysis revealed that the TCRs bear different complementarity-determining region (CDR3) sequences. These TCRs have been shown to form functional surface receptor in TCR-negative human thymoma cell line after retroviral transduction. High purity soluble recombinant TCRs and NY-ESO-160-72/HLA-B7 complex have been successfully produced. Gel filtration assay and Surface Plasmon Resonance analysis confirmed the interaction between purified recombinant TCRs and NY-ESO-160-72/HLA-B7 complex. Recombinant protein crystals have been successfully grown in CrystalMation screenings. High resolution X-ray diffraction dataset of 2.0 Å, 2.9 Å and 3.2 Å have been collected for three TCR-NY-ESO-160-72/HLA-B7 crystals. The knowledge acquired from this study will aid to the current understanding of the TCR-pMHC I interaction and may have important implications for future vaccine design.

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