DNA- and protein vaccination in combination with immune checkpoint blockade for active immunotherapy against neuroblsatoma

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This renewal proposal describes two therapeutic strategies (DNA- and protein vaccination) for active immunotherapy against neuroblastoma (NB), the most common solid tumor in childhood. Since metastases are found in a majority of cases at diagnosis, the development of new therapeutic approaches against this aggressive malignancy is of great importance.The first therapeutic strategy we aim to use is based on vaccination with a bicistronic DNA plasmid encoding for both a tumor-associated antigen (TAA) and an immune stimulating cytokine. An ubiquitin sequence linked to the TAA ensures its proteasomal degradation and enhances presentation to cytotoxic T lymphocytes (CTLs), thus increasing a cellular anti-NB immune response. The secretion of a bioactive cytokine in microenvironment of the antigen presentation stimulates CTLs without induction of well-known cytokine-dependent side effects when administered systemically. The second therapeutic strategy implements a poor investigated concept of CTL activation by IgG-based protein vaccines based on the principle of cross-presentation. For this, we aim to investigate anti-tumor effects of vaccination with a fusion protein consisting of TAA epitopes linked to the constant heavy chain of human immunoglobulin G1. Here, the IgG structure facilitates the uptake of the fusion protein by antigen presenting cells resulting in increased presentation of TAA epitopes.The first bicistronic DNA vaccine is already generated and characterized. This vaccine encodes for the TAA tyrosine hydroxylase (TH), which is over expressed in NB, and the CTL stimulating cytokine interleukin 15 (IL-15). This project was funded by the DFG (project start: 01.07.2015; funding: 2 years; presumable overall duration: 4 years). Currently, we evaluate this vaccine in a pre-clinical model in combination with an immune checkpoint blockade (PD-1, CTLA-4 and regulatory T cells (Treg)).By analogy with the TH+IL-15-based vaccination strategy, a second bicistronic DNA vaccine against MYCN-positive tumors, that do not respond to standard therapy and are associated with poor survival, will be generated. To increase the efficacy of vaccination, an additional DNA sequence encoding for the cytokine IL-21, which stimulates CTL and inhibits Treg, will be integrated into the vaccine. Moreover, the new concept of an IgG-based MYCN fusion protein consisting of MYCN epitopes and IgG1 heavy chain will be investigated. Finally, the MYCN-based vaccines will be evaluated in combination with immuncheckpoint blockade (PD-1, CTLA-4, Treg).These multimodal strategies are important in that they combine TAA-specific anti-tumor response with immune stimulating agents (cytokines and/or inhibition of immune checkpoints), thus offering the potential for additive or synergistic anti-tumor efficacy.