Conjugate vaccines targeting the tumor vasculature

Cancer cells acquire critical hallmarks which eventually facilitate the formation of malignant tumors. In this thesis, we highlighted two important hallmarks, the induction of angiogenesis and the avoidance of immune destruction. Several immunotherapies have been developed aiming to counteract these hallmarks. For example, tyrosine kinase inhibitors can inhibit signaling of VEGF. Unfortunately, such treatments have so far often lead to therapy resistance. In this thesis, we focused on a different approach to target the tumor vasculature, namely by the development of vaccines targeting the tumor vasculature-specific targets. We recently found that the intermediate filament protein Vimentin is externalized by endothelial cells of the tumor vasculature. Vimentin-targeting therapy can potentially reverse endothelial cell anergy and promote immune infiltration, supporting anti-tumor immunity. In general, extracellular Vimentin was shown to be pro-angiogenic and functionally able to mimic VEGF action. Importantly, both passive and active antibody-based immunotherapies against extracellular Vimentin were shown to specifically and safely inhibit tumor vascularization and tumor growth in vivo, including vaccination of client-owned domestic dogs presenting with spontaneous bladder carcinoma. In addition, in a mouse model B16F10 melanoma, vascular Icam1 expression was increased, while programmed cell death protein 1 (Pd-l1) expression was reduced upon vaccination against extracellular Vimentin. These data illustrate that extracellular Vimentin is a vascular immune checkpoint molecule, and antagonizing extracellular Vimentin simultaneously alleviated immune suppression and repressed tumor angiogenesis. As mentioned earlier, vaccination against extracellular Vimentin can reverse endothelial cell anergy, making the tumor microenvironment more permissive for immune cells. Vaccination of mice against extracellular Vimentin, combined with both anti-PD1 and anti-CTLA4 ICI, resulted in the most pronounced inhibition of B16F10 tumor growth, as well as the highest intratumoral T cell infiltration.These data suggest a potential synergy between vaccination against extracellular Vimentin and anti-PD1 and anti-CTLA4 therapy. In addition to testing and validating the vaccine against extracellular Vimentin, we aimed to identify novel vascular targets and test the efficacy of target-specific conjugate vaccines in several tumor models. To identify highly selective tumor endothelial markers, we performed a search for oncofetal genes that are expressed in the tumor vasculature. This screening revealed several interesting tumor endothelial targets, including Fbn2, Emilin-2, Lox, Serpine1, and Sfrp2. Upon vaccination, vaccines against all previoulsy mentioned targets resulted in the induction of a target-specific antibody response in mice. Importantly, vaccination also resulted in a significant inhibition of tumor growth, as well as a reduced tumor vessel density. Besides the general overexpression of specific genes in the tumor vasculature, it was shown that part of the specificity oncofetal gene expression in tumor endothelial cells relies on the selective expression of splice variants. Gene expression data described above was further analyzed for alternative splicing (AS) events. The analysis resulted in the identification of 9 unique genes displaying AS events, including Versican. Alternative splicing of Versican resulted in the incorporation of the oncofetal Gagß domain (exon 8) in tumor endothelial cells, known as the V1 isoform. Targeting the Gagß domain of Versican resulted in tumor growth inhibition of B16F10 melanoma and 4T1 breast carcinoma. These data show that oncofetal splice variants are valuable targets for therapeutic anticancer approaches, selectively targeting the tumor vasculature. In conclusion, in this thesis we described an innovative and novel screening approach focusing on oncofetal expression profiles to identify novel therapeutic targets in the tumor vasculature. In addition, we generated several conjugate vaccines targeting these tumor vasculature-specific proteins and observed a potent anti-tumor effect for all tested vaccines. We believe that the knowledge gained from these studies stimulates the further evaluation of the presented conjugate vaccines, as well as their potential combination with other anti-cancer therapies such as immune checkpoint blockade.