Heterologous immune vaccination induces specific therapeutic CD8 T-cell immune responses against tumor-associated antigens expressed in HCC

CD8 T cells are a major component of the adaptive immune system and responsible for the recognition and elimination of malignant cells by major histocompatibility complex class I (MHCI)-restricted cytotoxicity. Immunotherapies are capable of inducing potent immune responses against tumour-associated antigens (TAA). TAAs alpha-fetoprotein (AFP) and glypican-3 (GPC3) are highly upregulated in hepatocellular carcinoma (HCC) and serve as a diagnostic marker and as potential target for immunotherapies. The aim of this project is the establishment of a therapeutic TAA-specific heterologous immune therapeutic vaccination protocol targeting HCC.

An orthotopic HCC mouse model was established in vivo using the “Sleeping Beauty” transposon system. Hydrodynamic injection was performed to transfect murine hepatocytes with plasmids containing oncogenic NRas G12V linked to murine AFP (mAFP) or GPC3 (mGPC3) in combination with myristoylated Akt1 and short hairpin against p53 (shRp53). High-affinity binding mAFP epitopes were optimized to further improve binding affinity to MHCI molecules. A heterologous vaccination protocol consisting of primary dendritic cell immunization followed by a boost with soluble heteroclitic mAFP peptide, Poly I:C and an agonistic CD40 antibody will be performed in tumour-bearing mice. Resected tumours from MHC haplotype HLA-A*02:01 patients will be collected and mass spectrometry as well as proteomics analysis performed to analyze MHC-bound peptides to assess the entirety of expressed tumour proteins, respectively. Potential neoantigens will be validated by whole exome sequencing. Additionally, HCC cells will be isolated from tumours and established in culture; the HCC cells will undergo the aforementioned methods and verify preceding results.

Flow cytometric data demonstrated a massive expansion of specific CD8 T cell immune responses against mAFP. Immunization with the optimized peptide resulted in a frequency of up to 30% mAFP-specific CD8 T cells of total CD8 T cells compared to < 1% after immunization with the wildtype peptide. T cells were detected by intracellular cytokine staining and flow cytometry.

TAAs mAFP and mGPC3 are promising targets to establish effective immunotherapeutic treatments. In contrast to conventional vaccination approaches, the combination of primary dendritic cell immunization with subsequent injection of agonistic, co-stimulatory antibodies is able to massively expand tumour-specific CD8 T cells, capable of targeting TAA-expressing HCC.