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Title: Molecular engineering of high affinity T-cell receptors for bispecific therapeutics
Author: Liddy, Nathaniel
ISNI:       0000 0004 2733 6145
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2013
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Cytotoxic T lymphocytes are able to identify malignant cells by scanning for aberrant peptides presented on cell surface human leukocyte antigen (HLA) Class I molecules by virtue of an antigen binding receptor called the T-cell receptor (TCR). Peptides presented by HLA Class I complexes represent the largest array of tumour associated antigens (TAAs) and are therefore ideal targets for immunotherapeutic reagents. Cancer patients frequently mount T-cell responses to tumour-specific antigens, but these are in most cases ineffective at clearing the tumour. This is in part due to the low affinity of TCRs for self-antigens coupled with low-level expression of target peptides on the surface of cancer cells. To harness the exquisite antigen recognition property of TCRs for use as potential therapeutic proteins, the principal goal of this thesis was to generate ultra-high affinity TCRs against three clinically relevant HLA Class I melanoma-specific epitopes, including peptides derived from Melan-A/MART-1(26-35), gp100(280-288) and MAGE-A3(168-176). TCRs are membrane-bound disulphide (ds)-linked heterodimers consisting of an alpha and a beta chain. Each chain comprises three hypervariable or complementarity-determining region (CDR) loops, which assemble to form the antigen binding domains. As a general rule the CDR3 loops, and to a lesser extent the CDR1 loops, contact the peptide bound in the HLA groove and as such specificity is largely attributable to the CDR3 loops. The remaining CDR loops interact with the HLA surface and not the bound peptide. Each CDR loop was mutagenised using degenerative NNK oligonucleotides and expressed on the surface of bacteriophage as fusions to the phage coat protein pIII. Through a Darwinian process of in vitro evolution using pHLA ligand as the target molecule, mutated TCRs with improved affinity for pHLA were identified. TCRs engineered by phage display were produced as soluble ds-linked proteins and the contribution to affinity of each mutated CDR was measured by surface plasmon resonance (SPR). Using a combinatorial strategy, individual mutated CDRs were spliced into the same TCR molecule in a stepwise manner to further increase binding affinity. The final combination of mutated CDRs was shown to bind their cognate pHLA antigen with substantially improved KD values of 18 pM (Melan-A/MART-1(26-35)), 11 pM (gp100(280-288)) and 58 pM (MAGE-A3(168-176)), representing an increase over the wild-type TCR of approximately 1.8 million-fold, 1.7 million-fold and 3.7 million-fold respectively. In addition, having discovered an off-target binding profile for the high affinity MAGE-A3 TCR, the phage display methodologies were explored to 5 reestablish the specificity of this molecule. These results are significant because this has provided a platform on which, for the first time, to make TCR-based therapeutics. For example, the affinity enhanced gp100 TCR is currently undergoing clinical evaluation in a Phase I/II trial.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: RM Therapeutics. Pharmacology