PROCHEM · Protein stabilization by chemistry: total synthesis of an MHC class I scaffold
FP7 — People (Marie Curie Actions)
- Duration
- 2011-01-01 → 2013-04-22
- EU contribution
- €161,749
- Participants
- 1
- Scheme
- MC-IEF
Lines connect the coordinator with its partners.
Results in brief
Protein stabilization by chemistry: total synthesis of an MHC class I scaffold
Summary report: Cancer immunotherapy benefits from technology for T cell visualization and T cell depletion/enrichment. Techniques that currently allow visualization of antigen-specific T cell responses involve biotinylation of soluble MHC monomers loaded with a peptide of choice and subsequent tetramerization by binding to a streptavidin. Currently, availability and reliability of the peptide-Major Histocompatibility Complex (pMHC) reagents dictates their development and subsequent applications. Recently, the development of MHC tetramers with exchangeable peptide ligand has partly solved the problem of the tedious and laborious preparation of individual MHCs. However, the development of an improved technology suitable for high throughput screening and ultimately of empty stable MHCs is highly desirable. PROCHEM’s main goal is to design and prepare, by chemical total synthesis, a truncated version of the MHC class 1 protein based on the human allele HLA::A2.1 that aims to furnish a practical tool to detect antigen specific T cells. Ultimately, we want to generate a stable empty MHC (α1,α2) platform by using chemical modification along the peptide sequence and by circularization of the platform. We use solid phase peptide synthesis to prepare peptide fragments that, after being properly functionalized, are connected to each other by mean of native chemical ligation and click reaction to furnish the desired MHC (α1,α2) platform. The sequence of the synthesized platform has been confirmed by SDS PAGE analysis and MSMS.
Data: CORDIS, © European Union
Project 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.
Original text from CORDIS.
Participants
- STICHTING HET NEDERLANDS KANKER INSTITUUT-ANTONI VAN LEEUWENHOEK ZIEKENHUIS · AmsterdamCoordinatorNetherlands
Links
Data: CORDIS, © European Union