Development of an arenavirus-based immunotherapy for treatment of KRAS mutant cancer.
Authors: Raguz Josipa , Ahmadi-Erber Sarah , Bischl Romana , Scharf Irene , Deutschmann Daniela , Schmidt Sarah , Lauterbach Henning , Orlinger Klaus Karl 
Published: June 2024

e14672 Background: Oncogenic mutations in KRAS are prevalent in more than 80% of pancreatic ductal adenocarcinomas (PDAC), approximately 30% of colorectal cancer (CRC) and 15-20% of lung adenocarcinoma (LUAD). It has been demonstrated previously that CD8+ T cell responses specific for KRAS mutations can lead to tumor control and regression of metastasis. Replicating arenavirus vectors are currently evaluated in multiple clinical trials in infectious diseases and oncology and have demonstrated potent induction of target antigen specific CD8+ T cell responses of up to almost 50% of the total CD8+ T cell compartment in peripheral blood. To augment KRAS mutation specific CD8+ T cell responses in patients, we are using replicating arenavirus vectors targeting five prevalent KRAS mutations which are found in approximately 95% (PDAC), 74% (CRC), and 83% (LUAD) of the KRAS mutated tumors, representing a promising off-the-shelf immunotherapy for these indications. Here, we systematically investigate immune responses induced by mutant KRAS targeted arenavirus-vector-based cancer vaccines in HLA transgenic mice and characterize the efficiency of target cell killing of activated KRAS mutation specific CD8+ T cells. Methods: A transgene cassette consisting of peptide stretches including KRAS mutations G12D, G12V, G12C, G12R and G13D was generated by in silico aided antigen design. Vectors encoding peptide stretches of single KRAS mutations as well as the corresponding wildtype KRAS sequence served as controls in preclinical experiments. KRAS mutation specific CD8+ T cell expansion was evaluated in HLA transgenic mice and functionality of induced CD8+ T cells was evaluated by assessing CD8+ T cell mediated killing of mutant KRAS peptide loaded target cells in vivo. Results: All treatment regimens were well tolerated, and no mortalities or major adverse events were observed. Following vector administration, HLA I restricted antigen-specific CD8+ T cell responses were detected against 4 encoded mutant KRAS epitopes in HLA-A*11:01 (KRAS G12D and G12V), or HLA-B*07:02 (KRAS G12C and G12R) transgenic mice. These cells did not cross-react with wildtype KRAS epitopes presented in the same HLA I molecules. Specific cytotoxicity against KRAS G12D/V or KRAS G12C/R pulsed target cells was observed in vivo in HLA transgenic animals, indicating functionality of the induced KRAS mutation specific T cells. No specific cytotoxicity towards target cells pulsed with KRAS WT peptides was observed in any of the groups. Conclusions: This study indicates efficient induction of KRAS mutation specific T cell responses in HLA transgenic mice using an arenavirus vector based vaccine. Expanded CD8+ T cells were capable of killing cells loaded with KRAS mutation specific epitopes in vivo indicating functionality of the induced T cell responses. Based on these results, initiation of a Phase I study for the treatment of KRAS mutated cancers is planned.