CD8+ T-cells recognise pathogens and cancer through a specific interaction between the T-cell receptor (TCR) and a 8-14 amino-acid residue peptide presented by class I major histocompatibility complex (pMHCI) molecules expressed on the target cell surface. The first structures of murine and human TCR/pMHC complexes, published in 1996, revealed a number of important features of the TCR/pMHC interface. Currently, <25 unique human TCR/pMHC complexes are reported in the literature. This is a relatively low number compared with the number of antibody or unligated pMHC structures. The lack of structural information regarding human TCR/pMHC complexes has compromised the determination of a comprehensive and accepted set of rules that govern T-cell antigen recognition. Difficulties in generating TCR/pMHC complex crystals partly explain the low number of these structures. The first part of this thesis reports the development of a new crystallization screen (TOPS) designed specifically for the generation of such protein crystals. I also had access to MART-1-specific TCRs, the MART-1 protein being expressed by virtually all fresh melanoma tumour specimens. Different human leukocyte antigen (HLA)- A*0201-restricted peptides from this protein are presented at the melanoma cell surface. As TCRs are known to bind to cancer-derived “self” peptides with weak affinity, there is considerable interest in designing enhanced affinity TCRs for the recognition of HLA-A*0201-MART-1. My work concentrated on the MART-1- specific TCR MEL5 and its affinity-enhanced variant selected by phage display, α24β17. I analysed the biophysical properties of α24β17 and determined that it bound HLA-A*0201-MART-1 with >30,000-fold enhanced affinity and distinct thermodynamics. Comparison of TCR/HLA-A*0201-MART-1 complex structures solved with TOPS and binding biophysics showed that: (i) TCR affinity can be enhanced by increasing interactions between the TCR and the MHC surface; (ii) soluble α24β17 retains the peptide specificity by a novel mechanism involving interactions with solvent molecules; and, (iii) MEL5 interaction with the physiologically relevant MART-127-35 nonameric antigen led to a peptide anchor residue switch, a TCR-induced modification that has never been observed before. I also initiated a preliminary study on the generation of genetically modified Jurkat cells and CD8+ T-cells expressing a range of affinity-enhanced TCRs directed against melanoma for adoptive cell therapy. These results suggested that melanoma specificity is retained after MEL5 transduction and that there is no need to optimize beyond a TCR affinity threshold to obtain optimal T-cell activation. Collectively, these data shed light on the complex and unpredictable nature of T-cell antigen recognition.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:569949 |
| Date | January 2013 |
| Creators | Madura, Florian |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/46103/ |
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