T cell antigen receptors (TCRs) on CD8+ T cells recognise endogenously processed peptides bound to major histocompatibility complex (pMHC) antigens presented on the cell surface on almost all types of cells in the body, including tumour cells. The majority of tumour-associated peptide antigens (TAPAs) are derived from non-mutated self-proteins and are therefore subject to immunological tolerance, mainly through negative selection of high avidity T cells in the thymus. In addition, there is low presentation of pMHC on the surface of cancer cells. As a result, T cell responses tend to be weak and ineffective at killing tumour cells. ImmTACs (Immune mobilising monoclonal T cell receptors Against Cancer) are bispecific soluble biologics comprising a soluble TCR with an enhanced affinity for tumour-associated pMHCI fused to a humanised anti-CD3 single-chain antibody fragment (scFv) which redirect and activate T cells to lyse tumour cells. In this study, the potency, sensitivity, and specificity of ImmTACs was investigated for pMHCI epitopes derived from four tumour associated antigens (TAAs): (1) gp100, (2) MAGE-A3, (3) Melan-A/MART-1, and (4) NY-ESO-1/LAGE-1. A comprehensive range of assays and methodologies have been established to characterise the ImmTAC reagents. Cytokine release assays such as IFN-γ and Granzyme B ELISpot were used to evaluate the specificity and biological activity of ImmTACs. In concentration-response experiments, all four ImmTACs produced EC50 values in the range of 100 picomolar or lower demonstrating a high degree of sensitivity despite low epitope numbers. Killing assays, including LDH-release for assessing short-term lysis and IncuCyte technology to visualise long-term killing kinetics in real time, show that redirected T cells potently kill their targets. Furthermore, in vitro screening against a panel of antigen negative, primary human cell lines have shown that ImmTACs are highly specific and only activate T cells against target cells presenting their cognate pMHC. The potency of ImmTACs was also investigated using tumour infiltrating lymphocytes (TILs) extracted from tumour specimens and with tumour-derived cancer cells as targets. An HLA-A2, gp100 specific ImmTAC has received phase I clinical trial regulatory approval in the UK and in the US on the basis of this in vitro data, which has been used to determine minimal anticipated biological effect level (MABEL). The clinical trial is currently in progress.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:571761 |
| Date | January 2013 |
| Creators | Adams, Katherine Jane |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/47578/ |
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