Radiotherapy is a frequently used strategy to fight cancer - however, this therapy is seldom curative, and increasing evidence points to the great advantage of combining radiation therapy with immunotherapy to increase the curative potential. Immunotherapy has a great potential as cancer therapy, with the prospect of training our own immune cells to actively kill cancer cells, not only targeting primary tumors, but also metastases. Within this platform, one induces not only a treatment for current cancers, but could potentially prevent recurrence in the future by the induction of an immune memory response. In the current project (XVac, supported by the Danish innovation fund) we are elucidating various strategies for combining radiation therapy and immune stimulatory compounds. The primary goal of the XVac project is the development of new drug delivery technology platforms, which can induce a potent anti-tumor immune response. We will investigate how the combination of radiation and immunotherapy engages the immune system. We will evaluate 1) the infiltration of immune cells to the tumor lesions, 2) systemic alterations in immune reactivity and immune regulation, and 3) the ability to induce T-cell recognition of cancer specific antigens. These parameters will be evaluated as markers of tumor rejection capacity in pre-clinical tumor models. Mutation-derived antigens (neoantigens) are of prime interest as these provide a set of tumor specific targets and are truly foreign to the immune system. For the identification of the T-cell specific neoantigen landscaping pre- and post-immunization, a novel technology, based on multimerized peptide-MHC-I coupled with a specific barcode, will be performed. This technique gives the possibility of detecting >1000 antigen-specific T cells in one sample. This will allow us to gain insight to T cells that recognize targeted tumor cells and provide basis for rational design of personalized cancer vaccines. Radiotherapy is effective, but does not effectively stimulate a specific immunogenic eradication of all cancer cells. With the combination of immunotherapy this could be overcome. The end goal of this project is to find effective and safe delivery mechanisms of immune stimulatory compounds that can provide sustained clinical efficacy in cancer patients.