Abstract 3286: Development and characterization of an oncolytic adenovirus armed with the immune checkpoint stimulator SA-4-1BBL
Authors: Gómez-Gutiérrez Jorge G. , Ramos-Gonzalez Martin , Garza-Morales Rodolfo , Tarique Mohammad , Arguc Feyza , Batra Lalit , Shirwan Haval , Yolcu Esma 
Published: June 2022

Abstract
Introduction: 4-1BBL is a co-stimulatory molecule that is particularly important for the expansion of T cells 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, and in soluble form, has 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 prototype oncolytic adenoviral system to deliver SA-4-1BBL (OAdSA-4-1BBL) to the solid tumors.
Methods: The AdenoQuick 2.0 system was used to construct two OAds, one expressing SA-4-1BBL and the other expressing SA (as control virus). The expression of transgenes was confirmed by western blot and the SA-4-1BBL biological function was evaluated by splenocyte proliferation assay in vitro. The OAdSA-4-1BBL and OAdSA-mediated cancer killing effect was evaluated by crystal violet staining, the activation of autophagy-mediate immunogenic cell death was assessed by detection of autophagy markers (e.g., LC3-I and II as well as p62), the damage-associated molecular patterns (DAMPs) molecules such as adenosine triphosphate (ATP), high mobility group box 1 protein (HMGB1), and calreticulin were evaluated by luminescence, ELISA, and flow cytometry, respectively. Expression of SA, SA-4-1BBL, and Hexon within the tumors were evaluated by immunohistochemistry. The OAdSA-4-1BBL therapeutic efficacy was evaluated in a C57BL/6 mice bearing subcutaneous TC-1 lung tumor.
Results: We successfully constructed two 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 in 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.
Conclusions: 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.
Citation Format: Jorge G. Gómez-Gutiérrez, Martin Ramos-Gonzalez, Rodolfo Garza-Morales, Mohammad Tarique, Feyza Arguc, Lalit Batra, Haval Shirwan, Esma Yolcu. Development and characterization of an oncolytic adenovirus armed with the immune checkpoint stimulator SA-4-1BBL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3286.