Improving the efficacy of oncolytic vaccinia virus-mediated breast cancer immunotherapy

• Author / Creator

  Umer, Brittany Anne

• Vaccinia virus (VACV) has shown promise as an oncolytic agent for treating a variety of tumour types, with
  preliminary results suggesting that this strategy holds promise for treating breast cancer. Our lab has previously modified VACV by deleting virally-encoded enzymes responsible for dNTP production. Specifically, the F4L gene (∆F4L), responsible for de novo dNTP synthesis, and the J2R gene (∆J2R), responsible for dTTP salvage, have been deleted from the VACV genome (DF4LDJ2R VACV). These mutations increase viral specificity for replication in cancer cells, presumably due to elevated levels of these enzymes in tumours caused by rapid growth. While this modified VACV was effective in treating orthotopic bladder cancer in rats and mice, only limited therapeutic benefits were observed in mouse models of breast cancer. This project investigated methods to improve the efficacy of vaccinia virus mediated breast cancer immunotherapy.
  In the first set of studies, I established improved virus production and quality control protocols for use in pre- clinical testing. Using purified virus, we established the maximum tolerable dose in our intratumoural murine breast cancer models to be used in subsequent studies.
  I then tested two strategies for improving the efficacy of VACV treatment of breast cancer. In the first method, VACV was combined with image-guided radiation therapy (IG-RT), a commonly used clinical treatment for breast cancer. While IG-RT causes DNA damage resulting in cell death, it is also known to stimulate the immune system to induce anti-tumour immunity. We hypothesized that VACV and IG-RT would work synergistically to improve therapeutic responses. In vitro, radiation synergized with VACV to result in improved cell killing. However, in vivo, the combination was antagonistic, and decreased survival compared to radiation alone. This discrepancy between in vitro and in vivo drug interactions suggested that the cause of this antagonism might be due to effects on the tumour microenvironment. To investigate mechanisms that might be causing antagonism, I performed experiments to investigate how IG-RT+VACV might alter the tumour immune-cell microenvironment (TiME). We observed that treating irradiated tumours with VACV increased CD8+ T cells in tumours and spleens, along with regulatory T cells (Tregs). Despite these increased Tregs, the ratio of CD8+ T cells to Tregs remained constant. Interestingly, virus treatment caused a significant increase in PD-1 expression, leading us to speculate that immune-exhaustion may contribute to antagonism. However, adding an anti-PD-1 antibody to the treatment regimen did not reverse the antagonistic effect caused by VACV oncotherapy. Although the mechanism remains to be elucidated, our results suggest caution should be taken when assessing treatment combinations in the clinic.
  In the second method to improve efficacy, I attempted to improve the immunogenicity of VACV by deleting immunomodulatory genes from the virus genome. Stimulation of the immune system is an important aspect of virotherapy, and the 200 kb VACV genome encodes an arsenal of proteins responsible for inhibiting the immune system of its host. I sought to increase anti-tumour immune responses of VACV therapy by removing immunomodulatory genes from the VACV genome and to decipher how these deletions impact anti-cancer immune responses. I performed a head-to-head comparison of six mutant oncolytic VACVs, each harbouring deletions in genes that modulate different cellular pathways such as nucleotide metabolism, apoptosis, inflammation, and chemokine and interferon signalling. I found that even minor changes to the VACV genome can impact the immune cell compartment in the tumor microenvironment. Viral genome modifications had the capacity to alter lymphocytic and myeloid cell compositions in tumors and spleens, and also impacted PD-1 expression and percentages of virus and tumor-targeted CD8+ T cells. I also observed that the most promising candidate genes for deletion were those which interfere with interferon signalling, mainly, through deletion of the viral B8R and B18R genes, which resulted in improved median survival and complete responses. Collectively, this research helps focus attention on the more critically important pathways that modulate the immune response in the context of VACV oncolytic virotherapy.

• Subjects / Keywords

  • Vaccinia virus
  • Oncolytic virus
  • Immunotherapy
  • Breast cancer

• Graduation date

  Spring 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-f5ak-jf55

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.