Immunostimulatory gene therapy targeting CD40, 4-1BB and IL-2R activates DCs and stimulates antigen-specific T-cell and NK-cell responses in melanoma models.

BACKGROUND: The activation of dendritic cells (DCs) is pivotal for generating antigen-specific T-cell responses to eradicate tumor cells. Hence, immunotherapies targeting this interplay are especially intriguing. Moreover, it is of interest to modulate the tumor microenvironment (TME), as this harsh milieu often impairs adaptive immune responses. Oncolytic viral therapy presents an opportunity to overcome the immunosuppression in tumors by destroying tumor cells and thereby releasing antigens and immunostimulatory factors. These effects can be further amplified by the introduction of transgenes expressed by the virus. METHODS: Lokon oncolytic adenoviruses (LOAd) belong to a platform of chimeric serotype Ad5/35 viruses that have their replication restricted to tumor cells, but the expression of transgenes is permitted in all infected cells. LOAd732 is a novel oncolytic adenovirus that expresses three essential immunostimulatory transgenes: trimerized membrane-bound CD40L, 4-1BBL and IL-2. Transgene expression was determined with flow cytometry and ELISA and the oncolytic function was evaluated with viability assays and xenograft models. The activation profiles of DCs were investigated in co-cultures with tumor cells or in an autologous antigen-specific T cell model by flow cytometry and multiplex proteomic analysis. Statistical differences were analyzed with Kruskal-Wallis test followed by Dunn's multiple comparison test. RESULTS: All three transgenes were expressed in infected melanoma cells and DCs and transgene expression did not impair the oncolytic activity in tumor cells. DCs were matured post LOAd732 infection and expressed a multitude of co-stimulatory molecules and pro-inflammatory cytokines crucial for T-cell responses. Furthermore, these DCs were capable of expanding and stimulating antigen-specific T cells in addition to natural killer (NK) cells. Strikingly, the addition of immunosuppressive cytokines TGF-ß1 and IL-10 did not affect the ability of LOAd732-matured DCs to expand antigen-specific T cells and these cells retained an enhanced activation profile. CONCLUSIONS: LOAd732 is a novel immunostimulatory gene therapy based on an oncolytic adenovirus that expresses three transgenes, which are essential for mediating an anti-tumor immune response by activating DCs and stimulating T and NK cells even under imunosuppressive conditions commonly present in the TME. These qualities make LOAd732 an appealing new immunotherapy approach.

Immune priming using DC- and T cell-targeting gene therapy sensitizes both treated and distant B16 tumors to checkpoint inhibition.

Immune checkpoint inhibitors have revolutionized the treatment of metastatic melanoma, but most tumors show resistance. Resistance is connected to a non-T cell inflamed phenotype partially caused by a lack of functional dendritic cells (DCs) that are crucial for T cell priming. Herein, we investigated whether the adenoviral gene vehicle mLOAd703 carrying both DC- and T cell-activating genes can lead to inflammation in a B16-CD46 model and thereby overcome resistance to checkpoint inhibition therapy. B16-CD46 cells were injected subcutaneously in one or both flanks of immunocompetent C57BL/6J mice. mLOAd703 treatments were given intratumorally alone or in combination with intraperitoneal checkpoint inhibition therapy (anti-PD-1, anti-PD-L1, or anti-TIM-3). Tumor, lymph node, spleen, and serum samples were analyzed for the presence of immune cells and cytokines/chemokines. B16-CD46 tumors were non-inflamed and resistant to checkpoint blockade. In contrast, mLOAd703 treatment led to infiltration of the tumor by CD8+ T cells, natural killer (NK) cells, and CD103+ DCs, accompanied by a systemic increase of pro-inflammatory cytokines interferon γ (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-27 (IL-27). This response was even more pronounced after combining the virus with checkpoint therapy, in particular with anti-PD-L1 and anti-TIM-3, leading to further reduced tumor growth in injected lesions. Moreover, anti-PD-L1 combination also facilitated abscopal responses in non-injected lesions.

Immunostimulatory oncolytic virotherapy for multiple myeloma targeting 4-1BB and/or CD40.

Multiple myeloma (MM) is a plasma cell malignancy that is characterized by immune dysregulation. MM is commonly treated with immunomodulating agents, but still remains incurable. Herein, we proposed and evaluated immunostimulatory Lokon oncolytic adenoviruses (LOAd) for MM treatment. LOAd viruses are serotype 5/35 chimera, which enables infection of hematopoietic cells. Oncolysis is restricted to cells with a dysregulated retinoblastoma protein pathway, which is frequently observed in MM. Further, LOAd viruses are armed with human immunostimulatory transgenes: trimerized membrane-bound CD40L (LOAd700, LOAd703) and 4-1BBL (LOAd703). LOAd viruses were assessed in a panel of MM cell lines (ANBL-6, L363, LP-1, OPM-2, RPMI-8226, and U266-84). All cells were sensitive to infection, leading to viral replication and cell killing as analyzed by quantitative PCR and viability assay. Transgene expression was verified post infection with flow cytometry. Cell phenotypes were further altered with a downregulation of markers connected to MM progression (ICAM-1, CD70, CXCL10, CCL2, and sIL-2Rα) and an upregulation of the death receptor Fas. In a co-culture of immune and MM cells, LOAd viruses promoted activation of cytotoxic T cells as seen by higher CD69, CD107a, and IFNγ expression. This was most prominent with LOAd703. In conclusion, LOAd viruses are of interest for MM therapy.