Abstract 5018: Activation of dendritic cells by immunostimulatory CD40L/4-1BBL-encoding oncolytic virotherapy in melanoma
Authors: Wenthe Jessica , Lövgren Tanja , Šilanskas Mantas , Eriksson Emma , Loskog Angelica 
Published: August 2019

Abstract
Checkpoint blockade antibody therapy has revolutionized the treatment of metastatic melanoma. However, up to 50% of patients establish treatment resistance, which is frequently connected to a non-T cell inflamed phenotype. Poor T cell activation and infiltration is suggested to be caused by a lack of functional dendritic cells (DCs) that are crucial for T cell priming. Indeed, in progressing melanoma patients, immature DCs are often increased in the tumor lesion and draining lymph node. Herein, we investigated the effect of an immunostimulatory oncolytic adenovirus LOAd703 in a humanized melanoma-DC model. LOAd703 (serotype Ad5/35) carries two immunostimulatory transgenes; trimerized membrane-bound (TMZ)-CD40 ligand and 4-1BBL. Viral replication is restricted to tumor cells but transgene expression is controlled by a CMV promoter, which allows expression also in healthy cells of the surrounding tumor microenvironment (TME).
LOAd703 efficiently infected and killed the human melanoma cell line 526-mel as evaluated by MTS viability assay. Infected 526-mel cells expressed both TMZ-CD40L and 4-1BBL as verified by flow cytometry. To investigate LOAd703’s effect on DC maturation, immature DCs were differentiated from CD14+ monocytes using GM-CSF and IL-4 and infected with LOAd703 or stimulated with MEGACD40L®, which is a highly active CD40L oligomer. Flow cytometry analysis revealed that LOAd703-infected DCs upregulated the expression of maturation markers and co-stimulatory molecules in a similar manner as high doses of MEGACD40L®. These included CD83, CD80, CD86, CCR7, ICAM-1 and MHC molecules. Interestingly, CD70 that is required for CD27 stimuli of T cells and lowers T cell receptor signaling threshold, was highly upregulated on the DCs using LOAd703, but not MEGACD40L®. Tumor cells are often immunosuppressive which may reduce the capacity of LOAd703 to activate DCs. To model this, immature DCs were co-cultured with LOAd703-infected 526-mel cells, which is also better mimicking the events in the TME. Co-cultured DCs upregulated the same markers as before, but this upregulation was twofold higher for CD83 and CD80. In agreement with the previous observation that CD40L signaling alone did not induce CD70 expression, CD70 expression on DCs was lower in the co-culture, indicating that a direct infection of the virus and potentially signaling through TLRs together with CD40L signaling is required to induce CD70 on DCs or that it may be inhibited by immunosuppressive factors released by the tumor. LOAd703-matured DCs also upregulated PD-L1 and thus it would be of interest to combine LOAd703 therapy with PD-L1/PD-1 blockade.
In conclusion, LOAd703 killed human melanoma cells and induced the expression of TMZ-CD40L and 4-1BBL in infected cells. Moreover, LOAd703 infection activated DCs to express costimulatory molecules, as well as CCR7, which is essential for a systemic immune response.
Citation Format: Jessica Wenthe, Tanja Lövgren, Mantas Šilanskas, Emma Eriksson, Angelica Loskog. Activation of dendritic cells by immunostimulatory CD40L/4-1BBL-encoding oncolytic virotherapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5018.