Identification and characterization of immunodominant T cell epitopes
- restricted by HLA-A1, A2, A3, A24
- derived from the MCV T antigens and other MCC associated novel antigens
- monitoring of responses against the identified peptides in normal donors and MCC patients
Identification of MCC associated antigens
The reverse immunology approach to identify MCV derived peptides, i.e. small and large T antigens, will be pursued. Thus, in a first step several freely available internet databases will be used for in silico prediction of peptides. Secondly, the actual binding of the peptides to the appropriate HLA molecule will be established by experimental binding analyses of peptides. This is conducted using a recently developed high throughput technique taking advantage of binding of synthetic peptides to recombinant HLA molecules and detection of stable construct in an ELISA based setup. Subsequently, proteins against which auto-antibodies can be detectedwill be analyzed for potential T cell epitopes in the same way (CCIT).
For mass spectrometry analysis of naturally presented MHC ligands fresh frozen tumor tissues from suitable patients are collected; optionally, previously established MCV+ and MCV- MCC cell lines will be used. Thousands of HLA-restricted peptides will be eluted and besides MCV T antigen encoded viral MHC ligands also MCC-restricted MHC ligands derived from over-presented or exclusively presented proteins will be identified. For the latter type of ligands lable-free relative quantification will be applied. Development of this quantification method was finished recently and has great potential to detect mutated human and viral epitopes. The method is not restricted to cell lines, but can also be applied to primary human cancer and normal tissues. (IMM)
Determination of immunogenicity of MCC associated peptides
High-throughput in vitro immunogenicity validation of natural MCC peptides will be performed to enable selection of immunogenic ligands against which T cell responses in the context of epitope spreading may occur. Knowledge about natural presentation of ligands perfectly adds to binding data and data about immunodominance as processing of the peptide from its encoding antigen is demonstrated (IMM). Blood samples from healthy donors and patients are screened using multidimensional encoding of MHC multimers in complex with MCC derived peptides. This part of WP3 takes advantage of an exchange technology in which HLA molecules stabilized by an UV sensitive peptide disintegrate upon UV exposure allowing exogenous peptide to complex with the empty HLA/B2M complex. Thus, huge numbers of HLA/peptidecomplexes can be generated in hours instead of weeks. Moreover, by a combinatorial setup in which each specific HLA/peptide complex is labeled with two different fluorochromes or quantum dots, a very high number of specificities can be studied in a single analysis, i.e. using limited volumes of donor or patient blood. T cells recognizing MCC peptide/HLA complexes can be detected directly ex vivo, or after in vitro stimulation with peptide and/or selection e.g., using CD137 antibody to sort activated cells. These experiments will present important information concerning the immunodominance of MCC peptides (CCIT).
T cell lines
MCC-specific T cell lines or clones for functional analysis of T cell/tumor cell interaction will be generated using standard cloning protocols (CCIT).
Comparison predicted and mass spectrometry characterized peptides
This part of WP3 studies and compares eluted peptides detected on the surface of MCC samples with the peptides that elicits actual responses in the patients and donors. This part of the project will be very informative on the presence of peptides available for targeting of a yet un-compromised T-cell population, and potentially also offer sequences left undetected by the in silico characterization (CCIT & IMM).