PROCHEM

Protein stabilization by chemistry: total synthesis of an MHC class I scaffold

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 Obiettivo del progetto (Objective)

'Technologies for T cell visualisation and T cell depletion/enrichment are steadily making their way into the clinic and developments seem dictated by availability and reliability of the peptide-Major Histocompatibility Complex (pMHC) reagents used. A bottleneck has always been the laborious process of expression, refolding and purification of individual pMHCs. Recently, conditional MHC technology, pioneered in the host laboratory, solved this problem in part by enabling high-throughput loading of one MHC allele with a wide panel of peptide ligands. However, the rapid, parallel generation of a variety of MHC alleles is still a major hurdle. Therefore, proposed research aims at the total chemical synthesis of conditional MHC platforms, to make high-throughput generation of different MHC alleles reality. Recent advances in solid phase peptide synthesis (SPPS) have now enabled the generation of long polypeptides and even small proteins. This proposal aims to use SPPS rather than biological expression systems for total protein synthesis. The major advantage of using chemical techniques for protein synthesis is (1) that it allows the facile introduction of (non-natural) amino acids with functionalities that stabilise the protein by enhanced H-bonding, intrastrand crosslinking or intramolecular cross-linking (cyclisation) and that enable immobilisation or conjugation, (2) that protein yields are higher, and (3) that it simplifies the controlled generation of GMP grade proteins for biomedical use. The outcomes of this project will benefit the development of adoptive T cell therapy and will enable epitope scans through entire genomes of emerging pathogens (e.g. pandemic H1N1 Influenza), thus contributing to vaccine development.'

Introduzione (Teaser)

Major histocompatibility complex (MHC) class I is a key receptor of the immune system. European scientists chemically synthesised a 'universal' MHC molecule to detect antigen-specific T cells.

Descrizione progetto (Article)

T lymphocytes constitute the main arm of cell-mediated immune responses. They get activated through their T-cell receptor (TCR), which recognises different peptides presented on MHC molecules by other cells.

In the past, to detect and quantify antigen-specific T cells, scientists have developed MHC tetramer assay reagents. In this technology four peptide-loaded MHC molecules are engineered to bind the protein streptavidin in a tetrameric complex. The use of fluorescently-labelled streptavidin facilitates the visualisation and quantitation of bound T cells.

Overall, the MHC tetramer assay has great prospects for use in cancer immunotherapy as a means of monitoring T cell depletion or enrichment. However, its widespread use has been limited by the laborious processes required for synthesis and purification of individual peptide-MHC reagents.

Recent technological advances in solid phase peptide synthesis (SPPS) have enabled the synthesis of MHC molecules. The EU-funded 'Protein stabilization by chemistry: total synthesis of an MHC class I scaffold' (PROCHEM) project also used chemical synthesis techniques to produce MHC molecules. The scope was to generate a truncated version of an MHC molecule based on specific human leukocyte antigen alleles.

In this context, scientists developed an empty MHC platform by preparing peptide fragments separately and connecting them through chemical ligation.

This MHC platform can in principle be loaded with desired peptide ligands, considerably increasing the applicability of MHC-based assay reagents. Apart from the field of adoptive T cell therapy, this novel approach could be used in vaccine development and the definition of novel antigens.