* To identify novel human tumour antigens and to synthesize peptides maximizing their recognition by T cells.
* To optimize and standardize peptide sensitization protocols for MHC class II as well as class restricted T cell responses.
* To demonstrate recognition of native tumour cells by peptide-sensitized T cells and to control tumour cell growth in vitro and eventually in vivo.
It is clear that MHC; class l-restrictea cytotoxic lymphocytes can recognise and destroy tumour cells in vitro and in vivo, but CD4+ MHC class II-restricted help is commonly required for optimal responses. The CD4+ cells themselves may also exert direct cytotoxic activity. Despite this, most of our knowledge on tumour-specific responses and peptideepitopes suitable for adoptive (or active) immunotherapy is restricted to class I-directed responses. Many potential class I-presented tumour antigens have been identified over the last 5 years; therefore EUCAPS will notrepetitively seek further novel class I-restricted tumour antigens. Rather, we propose to study particularly MHC class II-restricted anti-tumour responses and to limit ourselves to the definition of novel MHC class II-presented tumour epitopes, of which only a very small number is known.
We will employ two main strategies
1) elution of endogenous peptides from class II molecules of tumour cells (concentrating on melanoma and leukemia), and sensitisation of T cells in vitro against peptides corresponding to these;
2) sensitisation of T cells from tumour-infiltrating lymphocytes (TIL) or peripheral blood cells against tumour cells, generation of T cell clones and identification of the antigen recognized by them.
T cells obtained by either approach will be characterized in terms of anti-tumour specificity and function and will be propagated in long-term culture for use in adoptive immunotherapy. Although the feasibility of adoptive immunotherapy with antigen-specific T cells has been demonstrated using CMV or EBV-specific lines and clones as well as TIL-derived lines, experience is very limited and tumour-specific clones have not been utilised. One reason for this is the technical difficulty of generating sufficient cells. Moreover, like other somatic tissues, T cells age in culture, changing function, developing variants and becoming more easily energized and apoptotic. Culture techniques which prevent or delay this could therefore be applied beneficially for adoptive immunotherapy and will be explored in this project by culture in low oxygen tension with supplemental anti-oxidants in continuous flow bioreactors.
The measurable objectives in this concerted action proposal are therefore, first, to identify novel MHC class II-restricted tumour antigens and to synthesise peptides maximising their recognition by T cells; second, to establish a "virtual" (computerized) and a more limited (physical) tumour cell line bank of material characterized for expression of HLA and the appropriate tumour antigens, as a tool for the participants. Information will be available to all participants when searching for a particular combination of HLA restriction element and tumour antigen, fusion protein or mutant on coprotein target. This information could also be made available publicly as a European service. Third, to optimise and standardize sensitisation protocols both for class I and class II-restricted responses. Because there are still very few examples where T cells have been shown to recognize native tumour after sensitisation to synthetic peptides, it is important to use such peptides as controls and to search for others that can be employed in the same way.
Standard protocols will be improved upon in attempts by participants to examine variables in a concerted effort to identify factors of critical importance in the generation of tumour-specific T cells. 1) Optimisation of antigen presentation: what type of antigen presenting cell? 2) Optimisation of cytokine requirements for sensitisation; 3) optimisation of peptide structure required for optimal sensitisation. Fourth, to demonstrate recognition of native tumour bypeptide-sensitised T cells in the absence of exogenous peptide. Fifth, to control tumour cell growth in vitro, or directly to Iyse tumour, by such sensitised cells. Sixth, to perform adoptive immunotherapy using these cultured cells at those centers with the appropriate infrastructure to carryout such clinical trials (the majority).
Therefore, the prime objectives of the proposed concerted action are 1) to facilitate the identification of novel MHC class II-restricted tumour antigens; 2) to establish reliable standardized and optimised culture protocols for generation of peptide-sensitised class I- and II-restricted T cell lines that recognize tumour cells and can be used in adoptive immunotherapy; 3) to generate information concerning which peptides to use for which tumours, which antigen presenting cells and cytokines to employ in which culture sequence. These results, which are also directly relevant to the identification of synthetic peptide vaccines representing defined T cell epitopes, will be disseminated as widely as possible by electronic and conventional publishing means.
The project adheres to the priority research tasks in Area 4.1.3 of "Cancer Research": "Cell selective targeting to explore, and to implement in clinical practise, techniques which cause preferential destruction of tumour cells", and, in particular, the research task "Identification of tumour-specific antigens and the production of antigen-specific tumour vaccines".