Functional evaluation of HMGB1 as immune therapeutic effector molecule for cell-based vaccination strategies

Abstract

Cancer is a leading cause of death worldwide although extensive research in human cancer medicine is carried out. To develop and improve molecular anticancer therapies, the characterisation of the structure and function of cancer-related genes and proteins is essential. The dog is one of the companion animals being considered as an invaluable model system. The spontaneous development of tumours in the context of an intact immune system in dogs and the striking similarities to human neoplasias offers new prospects for cancer immunotherapy in contrast to immunocompromised rodent models. Within this thesis, the molecular structure and function of the cancer related canine HMGB1, RAGE, IL-1alpha, IL-1beta, and TNF-alpha genes and proteins was analysed. The results revealed highly conserved molecular homologies and similar expression patterns in dogs and humans. Further parts of this thesis were conducted to support the development of a cell-based cancer immunotherapy vaccination strategy with genetically engineered recombinant HMGB1 secreting canine DCs in dogs. For transfection, a novel femtosecond laser based opto-perforation method and a protocol based on the application of a new type of laser generated positively charged gold nanoparticles was established. The functionality of different HMGB1 vectors was analysed. Subsequently, the time dependent secretion of recombinant HMGB1 from mammary epithelial cells was induced by TNF-alpha stimulation. In another cell stimulating strategy an increased proliferative response of canine B cell lymphoma cell was induced by CpG ODN application in combination with IL-2. To monitor the fate of transplanted cells such as DC-vaccines non invasively in vivo, more safely methods in comparison to the commonly used standard techniques are needed. Thus, within the last part of this thesis the generation of antibody fragments targeting specifically canine DCs and improved cell tracking of SPIO labelled cells via MRI was carried out. Concluding, the results of this thesis allowed establishing innovative methods contributing to the development of therapeutic approaches targeting at neoplasias in dogs as patients, but also providing benefit for human medicine in terms of the dog as model organism.