Engineering TCRs for improved stability by targeting the non-CDR framework regions

McMurran, Catriona 2022. Engineering TCRs for improved stability by targeting the non-CDR framework regions. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

T-cell receptors (TCRs) form a key part of the immune system by recognising peptides presented on the cell surface by the major histocompatibility complex. Recombination events between different gene segments create a highly diverse TCR repertoire to bind diverse targets, with sequence diversity occurring not only at the binding interface but across entire TCR variable domains. Framework regions that do not make direct binding contacts are still variable in sequence and may influence function. Framework diversity is also a vital consideration for the use of TCRs in pharmaceutical contexts, where different TCRs may have different developability challenges such as stability and aggregation propensity. This thesis examines the framework regions of the TCR through multiple different approaches. A comparison of structures with different framework sequences highlighted key residues at inter-domain interfaces and identified specific amino acids that are best placed to make these inter-domain contacts. These key residues reflect those previously identified in published stabilising strategies and offers a new model for predicting and evaluating stabilising mutagenesis. A novel dataset of experimentally determined melting points for over 200 soluble TCRs was collated, revealing a broad range of intrinsic protein stabilities. Examination for sequence features that could determine overall protein stability did not support theories that certain genes or combinations of genes were inherently more stable, or that the hydrophobicity of surface-exposed residues was the determining factor; instead TCR thermal stability is highly variable even between similar sequences. Alongside these broader analyses, a detailed study of stabilising mutagenesis was carried out on two related TCRs to identify point mutations in framework regions that improve thermal stability. The most successful of these mutations were shown to improve stability in not only closely related molecules but also a panel of sequence-diverse TCRs, representing a more general approach to framework engineering for more stable molecules.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Medicine
Date of First Compliant Deposit:	22 November 2022
Last Modified:	06 Jan 2024 03:36
URI:	https://orca.cardiff.ac.uk/id/eprint/154345

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