Prime-Boost Immunovirotherapy Targeting High-Frequency Neoantigens in KRAS-mutant cancer types

• Applicant: Dr. Mathias Leber
• Subject Area: Hematology, Oncology ; Immunology ; Virology
• Term: from 2019 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 432906602

a. Background: Cancer remains to be a key medical and socioeconomic challenge in our society. In many tumor entities, such as pancreatic or colorectal cancer, specific mutations in the KRAS proto-oncogene have been found to be critically involved in tumor development. Adoptive transfer of KRASmut-reactive, autologous T cells has resulted in complete responses in patients with KRAS-mutant cancer, demonstrating the clinical potential of neoantigen targeting. However, relapse of individual lesions has been described and attributed to immune evasion. b. Research Aims: Here, we aim to develop a heterologous prime-boost vaccination therapy using tumor-selective, oncolytic viruses (OVs) which encode high-frequency KRAS neoantigens and target immune-resistant tumor cells as well. This approach seeks to (a) develop a novel, universal neoepitope expression cassette for OVs which ensures the correct neoantigen processing and presentation, (b) generate and characterize OVs encoding the neoantigen expression cassette, and (c) assess neoantigen-specific immune responses and therapeutic efficacy of heterologous prime-boost oncolytic vaccination therapy in a humanized mouse model of cancer. c. Methods & Approaches: The selected neoantigens correspond to the three most frequent KRAS mutations displayed on HLA-A*02:01 and will be encoded in a polyepitope expression cassette (PEC) designed to facilitate correct neoantigen processing. This is achieved by an endoplasmic reticulum (ER)-targeting sequence and ER-specific protease cleavage sites separating the individual KRAS neoantigens. Selected KRAS neoepitopes will be cloned into a tagged PEC vector, transfected into HLA-A*02:01+, KRASwt tumor cells, and the expression and ER-translocation of encoded KRAS neoantigens will be validated (by RT-PCR & immunofluorescence). Then, we will vaccinate humanized, HLA-A*02:01-transgenic mice with the PEC vector and assess KRAS neoantigen-specific immune responses (by tetramer staining/IFNγ-ELISpot). In the second part, we will generate and characterize replication-competent, tumor-specific OVs (measles virus, vesicular stomatitis virus) encoding the same KRAS neoantigen PEC. We will assess neoantigen-specific immune responses and overall efficacy after heterologous prime-boost oncolytic vaccination therapy in a humanized, HLA-A*02:01-transgenic murine model of KRAS-mutant cancer. Selected tumors will be harvested and stained for signs of inflammation, tumor infiltrating lymphocytes, and OV proteins. Residual tumor material will be subjected to total RNA sequencing to determine changes in the host cell transcriptome upon prime-boost oncolytic vaccination.d. Impact: Currently, no promising treatment options are available for patients with advanced-stage KRAS-mutant cancer. This oncolytic immunotherapy is tailored specifically towards HLA-A*02:01+ patients with high-frequency KRAS mutations and has the potential to be translated quickly and cost-effectively into the clinic.

• DFG Programme: Research Fellowships
• International Connection: Canada
• Host: Professor John Bell, Ph.D.