Oncolytic viroimmunotherapy combines the inflammatory nature of viral vectors with the delivery of virally-encoded tumor associated antigens (TAAs) to induce robust anti-tumor T cell responses. We have previously reported that vesicular stomatitis virus (VSV) vectors expressing truncated forms of the TAAs NRAS, Cytochrome-C1, and TYRP1 provide superior therapeutic benefit to mice bearing B16 melanoma tumors compared to treatment with VSV expressing the full-length form of these proteins. Based on structural modeling, we determined that each carboxyl-(C)-terminal truncation disrupted domains important for antigen stability. Others have demonstrated a direct association between the stability of an antigen, its degradation rate, and the generation of immunogenic epitopes that can be utilized to activate cognate T cell responses. Thus, we hypothesized that modulation of tumor antigen stability and degradation would increase the availability of immunogenic epitopes for presentation on MHC complexes, thereby improving cognate T cell responses in the context of VSV immunotherapy. In this work, we first demonstrated that the 19 amino acid C-terminal truncation of the melanoma antigen, TYRP1, identified in the cDNA library significantly reduced the protein’s half-life. We found that truncated TYRP1 underwent autophagy-mediated degradation, consistent with the CD4+ T cell-dependent responses observed in vivo. To extend these findings, we used the crystal-structure of the model antigen ovalbumin (OVA) to identify putative mutations which would reduce protein stability and promote antigen degradation. We found that one mutant exhibited a significantly reduced half-life compared to the wild-type antigen and was degraded in a 26S-proteasomal dependent manner. Infection of murine antigen presenting cells with VSV vectors expressing the destabilized OVA antigen resulted in enhanced presentation of the immunogenic OVA epitope, SIINFEKL, and activation of cognate T cells in vitro and in vivo compared to expression of the wild-type antigen. Correspondingly, SIINFEKL-specific T cell activation by the virally delivered destabilized OVA resulted in improved long term survival in a murine B16OVA melanoma model. In a final approach, we aimed to utilize pharmacological activation of the unfolded protein response (UPR), a cellular mechanism to promote degradation of misfolded proteins, to promote antigen presentation. We found that UPR activation using the small molecule thapsigarigin (Tg) induced proteasomal-mediated degradation of endogenously encoded OVA in our B16OVA murine melanoma cell line. This was associated with enhanced presentation of the SIINFEKL epitope and improved recognition and activation of cognate CD8+ T cells in vitro. Intratumoral administration of Tg in combination with systemic VSV-OVA immunotherapy significantly reduced early tumor growth and had modest improvements in overall survival. Overall, the mechanisms investigated here provide a framework for improving therapeutic outcomes with viral immunotherapeutic agents by manipulating the stability and degradation of virally-encoded TAAs to promote anti-tumor T cell responses.
|Commitee:||Galanis, Eva, Cattaneo, Roberto, Pavelko, Kevin, Barry, Michael, Ramirez-Alvarado, Marina|
|School:||College of Medicine - Mayo Clinic|
|Department:||Virology and Gene Therapy|
|School Location:||United States -- Minnesota|
|Source:||DAI-B 82/4(E), Dissertation Abstracts International|
|Subjects:||Virology, Immunology, Oncology|
|Keywords:||Antigen presentation, Oncolytic virus, Protein degradation, Viral immunotherapy|
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