Abstract A119: Combating primary and secondary checkpoint blockade resistance using immunostimulatory CD40L/4-1BBL-encoding oncolytic virotherapy for melanoma
Authors: Wenthe Jessica , Šilanskas Mantas , Eriksson Emma , Loskog Angelica 
Published: February 2019

Abstract
In recent years, cancer immunotherapies significantly advanced the clinical management of metastatic melanoma. In particular, treatment with immune checkpoint inhibitors increased survival rates compared with standard therapy. However, not all patients benefit from checkpoint blockade. Primary treatment resistance is connected to poor T-cell infiltration in the tumors. This can be due to limited activation of antigen-presentation cells such as dendritic cells (DCs) and the high threshold of activating low-affinity T-cells that may respond to the tumor antigens. CD27 signaling in T-cells during activation lowers the T-cell receptor signaling threshold, which may be important to activate tumor-targeting T-cells. Secondary resistance to checkpoint blockade therapy includes loss of MHC-I on the tumor cells. Hence, inducing natural killer (NK) cell activation in parallel to antitumor T-cells may be crucial. LOAd703 was designed to optimize antitumor immune activation. LOAd703 is an oncolytic adenovirus (serotype Ad5/35) carrying two immunostimulatory transgenes; a trimerized membrane-bound CD40 ligand (TMZ-CD40L) and the full-length 4-1BB ligand (4-1BBL) (patent filed: PCT/EP2015/057489). The viral replication is restricted to tumor cells with a hyperphosphorylated retinoblastoma pathway due to a deletion in E1A, but transgenes are expressed under the control of a cytomegalovirus (CMV) promoter, which enables transgene expression even in the surrounding tumor microenvironment. Herein, we investigated LOAd703 in a melanoma model and its immunostimulatory effect on DC maturation to induce antigen-specific T-cell responses. LOAd703 infected the human melanoma cell line 526-mel and efficiently induced tumor cell death as evaluated by MTS viability assay. The viability of infected cells was reduced to 15% at 72 hours post infection compared to uninfected cells. Transgene expression of both TMZ-CD40L and 4-1BBL was confirmed by flow cytometry post infection. To evaluate the immunostimulatory capacity of LOAd703, immature DCs were differentiated from CD14+ monocytes using granulocyte-macrophage colony-stimulating factor and interleukin-4 and infected with virus. LOAd703-infected DCs upregulated the expression of maturation markers, such as CD83, CD86 and MHC molecules as analyzed by flow cytometry. Interestingly, CD70 that is required for CD27 stimuli of T-cells was highly upregulated on the DCs using LOAd703. Furthermore, the chemokine receptor CCR7 and the adhesion molecule ICAM-1 were increased upon LOAd703 infection, which are crucial for lymph node homing and the initiation of a systemic response. Next, the functional capacity of LOAd703-matured DCs to induce antigen-specific T-cell responses was assessed in a CMV model, in which CMV-peptide pulsed DCs are utilized to induce expansion of CMV-specific T-cells. LOAd703-matured DCs from CMV+ donors were pulsed with CMV-peptide and co-cultured with autologous peripheral blood mononuclear cells for 11 days. LOAd703-matured DCs were able to expand CMV-specific T-cells, but to a lower degree than the positive control Poly I:C matured DCs. However, the CMV-specific T-cells expanded from the positive control had higher expression of PD-1 compared to the LOAd703 group, indicating that LOAd703 leads to the expansion of less exhausted and potentially more functional T-cells. Moreover, LOAd703 also induced a massive expansion of NK cells, which is probably driven by 4-1BBL. In conclusion, LOAd703 killed human melanoma cells by oncolysis and induced the expression of TMZ-CD40L and 4-1BBL in infected cells. Furthermore, LOAd703 infection activated DCs to express costimulatory molecules including high levels of CD70 and CCR7, which in turn could promote the expansion of antigen-specific T-cells with low PD-1 expression, as well as the potent expansion of NK cells.
Citation Format: Jessica Wenthe, Mantas Šilanskas, Emma Eriksson, Angelica Loskog. Combating primary and secondary checkpoint blockade resistance using immunostimulatory CD40L/4-1BBL-encoding oncolytic virotherapy for melanoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A119.