Development and characterization of an oncolytic adenovirus armed with the immune checkpoint stimulator SA-4-1BBL

Abstract

Background: 4-1BBL is a co-stimulatory molecule that is important for T-cell expansion and induction of robust CD8+ T-effector and memory responses, critical for tumor rejection. The natural 4-1BBL is a trimeric transmembrane protein that has no activity in soluble form. To overcome this limitation, a unique soluble form of 4-1BBL was developed by linking core streptavidin (SA) to the extracellular domain of murine 4-1BBL (SA-4-1BBL). This chimeric protein forms oligomers that have pleiotropic effects on cells of innate, adaptive, and regulatory immunity. However, production and purification of SA-4-1BBL is cost- and time-intensive. Therefore, development of an expression platform that is more efficient, translational, and augments anti-tumor efficacy of SA-4-1BBL will have a significant impact for clinical development of this immune adjuvant. Thus, we developed a novel oncolytic adenoviral system to deliver SA-4-1BBL to solid tumors. Methods: The AdenoQuick system was used to construct OAds expressing SA-4-1BBL or SA (control). Transgene expression was confirmed by western blot and SA-4-1BBL biological function was evaluated by splenocyte proliferation assay in vitro. The OAdSA-4-1BBL and Ó 2022 American Medical Association. All rights reserved. OAdSA-mediated cancer killing effect was evaluated by crystal violet staining, the activation of autophagy-mediated immunogenic cell death was assessed by detection of autophagy markers LC3-I, LC3-II and p62. The damage-associated molecular patterns molecules adenosine triphosphate, high-mobility group box 1, and calreticulin were evaluated by luminescence, ELISA, and flow cytometry, respectively. Tumor expression of SA, SA-4-1BBL, and Hexon was evaluated by immunohistochemistry. The OAdSA-4-1BBL therapeutic efficacy was evaluated in C57BL/6 mice-bearing subcutaneous TC-1 lung tumor. Results: We successfully constructed OAds expressing SA or SA-4-1BBL. We demonstrated that SA-4-1BBL can produce biologically active oligomers, selectively induces autophagy- mediated immunogenic cell death in murine and human lung cancer cell lines with little cytotoxicity to normal cell lines. The transgene expressions within the subcutaneous tumor were confirmed and OAdSA-4-1BBL shows greater anti-tumor efficacy than control viruses in a TC-1 tumor mouse model. Conclusion: Overall, our data suggests that OAd expressing the immune checkpoint stimulator SA-4-1BBL may represent alternative therapeutic strategy against lung cancer, which could be used alone or in combination with immune checkpoint inhibitors, or other approaches aimed at enhancing the anti-tumor immune response.