Abstract A15: Rewiring the tumor microenvironment with an oncolytic virus to enhance and support tumor-infiltrating lymphocytes for ovarian cancer immunotherapy
Authors: Santos João Manuel , Hëinio Camilla , Cervera-Carrascon Victor , Siurala Mikko , Quixabeira Dafne , De Gruijl Tanja , Lassus Heini , Kanerva Anna , Hemminki Akseli 
Published: June 2021

Abstract
The natural occurrence of immune cells in the tumor microenvironment of ovarian cancer (OC) facilitates the ex vivo generation and expansion of tumor-infiltrating lymphocytes (TIL) for infusion in the context of adoptive T-cell transfer. Yet, the lack of tumor-reactive TILs and the suppressive microenvironment have hindered the translation of TIL therapy into meaningful clinical responses in OC patients. Therefore, generating a proinflammatory tumor microenvironment with oncolytic adenoviruses, in order to induce a steady and potent antitumor activity, may prove an effective add-on to TIL therapy for OC. Besides their well-established efficacy in OC, oncolytic adenoviruses genetically modified to carry the interleukin-2 (IL-2) and tumor necrosis factor alpha (TNFa) transgenes may further support and promote T-cell mediated antitumor immunity. In this study, we assessed the capability of an oncolytic virus coding for IL-2 and TNFa (Ad5/3-E2F-D24-hIL-2-IRES-TNFa; TILT-123) to enhance the reactivity of TILs towards OC in a suppressive microenvironmental context. Fresh tumor tissue from regional metastatic or primary sites was obtained from stage III-IV OC patients. Part of the samples were diced in small fragments for the generation and rapid expansion of TILs under clinically relevant in vitro conditions. Phenotypic characterization of TILs was performed by flow cytometry after rapid expansion. Following TIL production and characterization, TILs were co-cultured with T cell-depleted autologous OC single-cell suspensions in the presence or absence of oncolytic virus. TIL reactivity was determined by quantifying Interferon (IFN)-gamma in culture supernatants through ELISA. The remainder of the tumor sample was dissociated overnight and cultured in the presence or absence of oncolytic virus. Supernatants were harvested at different days post-infection for detection of relevant cytokines. Following a 14-day rapid expansion period, the final TIL product was composed mostly of CD8+ T cells (56%), CD4+ T cells (27%) and a small fraction of CD3+CD56+ NK-like T cells (2%). When TILs were cocultured with T-cell depleted OC single-cell suspensions, treatment with oncolytic virus coding for IL-2 and TNFa caused a significant increase in the release of IFNg by TILs as compared to conditions where no virus or the backbone oncolytic virus (without cytokine transgenes) was added (p&lt;0.05). Microenvironmental changes by the cytokine-encoding oncolytic adenovirus were evidenced by increased release of proinflammatory cytokines such as, TNFa, IL-2 and IFNg in 7-day cell cultures of ex vivo single-cell suspensions derived from OC patients. In summary, tailoring the tumor microenvironment with IL-2 and TNFa using oncolytic viruses sharpens the TIL reactivity in an immune-suppressive OC microenvironmental context. Hence, this approach may enable effective responses in OC patients treated with TIL therapy. This evidence supports further clinical validation.
Citation Format: João Manuel Santos, Camilla Hëinio, Victor Cervera-Carrascon, Mikko Siurala, Dafne Quixabeira, Tanja De Gruijl, Heini Lassus, Anna Kanerva, Akseli Hemminki. Rewiring the tumor microenvironment with an oncolytic virus to enhance and support tumor-infiltrating lymphocytes for ovarian cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A15.