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 immuno-competent 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 gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), 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

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

LOAd703 and beyond : Advancing immunostimulatory gene therapy for cancer

In the last decade, immunotherapy has come into the limelight of cancer treatments. Immunotherapy aims to stimulate the body’s own immune system to create or reinvigorate anti-tumor immune responses. Impressive results with long-term remissions have been achieved in patients with B-cell malignancies and malignant melanoma treated with chimeric-antigen receptor (CAR) T cells and immune checkpoint inhibitors, respectively. However, many patients still do not respond to these therapies or relapse. Oncolytic virotherapy represents an immunotherapy approach, in which viruses are programed to selectively replicate in tumor cells, thereby leading to direct tumor cell killing. Moreover, these viruses can be modified to introduce therapeutic transgenes into the tumor microenvironment. In this thesis, I evaluated oncolytic viruses from the Lokon Oncolytic Adenovirus (LOAd) platform. These viruses are engineered to express immunostimulatory transgenes that specifically activate cells central in the induction of anti-tumor immunity, such as dendritic cells (DCs), T cells and natural killer (NK) cells. The herein investigated viruses are LOAd703 and LOAd732, which both express CD40L and 4-1BBL, while LOAd732 additionally expresses IL-2. In paper I, we assessed the immunological status of advanced cancer patients treated with LOAd703 in combination with chemotherapy in the LOKON002 trial. We showed that intratumoral LOAd703 treatment could mediate inflammation in patients’ tumors as seen by an induction of multiple inflammatory genes, including gene profiles associated with response to checkpoint inhibition. In addition, there was a distinct profile for responding patients. In paper II, we demonstrated that LOAd703 therapy is feasible in B-cell lymphoma and that it can be combined with CAR T-cell therapy as priming lymphoma cells with LOAd703 enhanced the secretion of chemokines promoting T-cell migration and boosted the cytotoxic T-cell function leading to better lymphoma cell killing. In paper III, we showed in vivo in a melanoma model that a murine version of LOAd703 could induce anti-tumor immune responses, sensitize tumors to checkpoint inhibitors and facilitate systemic immune responses in a twin-tumor model, in particular when combined with anti-PD-L1. In paper IV, we present a novel virus, LOAd732, and its functionality was confirmed in regards to the oncolytic capability, transgene expression and immunostimulatory effect. LOAd732 activated DCs that were capable of stimulating antigen-specific T cells, as well as NK cells. Moreover, LOAd732-stimulated cells could withstand immunosuppression mediated by cytokines (TGF-β1, IL-10) commonly present in the microenvironment of many tumors

LOAd703 and beyond : Advancing immunostimulatory gene therapy for cancer

In the last decade, immunotherapy has come into the limelight of cancer treatments. Immunotherapy aims to stimulate the body’s own immune system to create or reinvigorate anti-tumor immune responses. Impressive results with long-term remissions have been achieved in patients with B-cell malignancies and malignant melanoma treated with chimeric-antigen receptor (CAR) T cells and immune checkpoint inhibitors, respectively. However, many patients still do not respond to these therapies or relapse. Oncolytic virotherapy represents an immunotherapy approach, in which viruses are programed to selectively replicate in tumor cells, thereby leading to direct tumor cell killing. Moreover, these viruses can be modified to introduce therapeutic transgenes into the tumor microenvironment. In this thesis, I evaluated oncolytic viruses from the Lokon Oncolytic Adenovirus (LOAd) platform. These viruses are engineered to express immunostimulatory transgenes that specifically activate cells central in the induction of anti-tumor immunity, such as dendritic cells (DCs), T cells and natural killer (NK) cells. The herein investigated viruses are LOAd703 and LOAd732, which both express CD40L and 4-1BBL, while LOAd732 additionally expresses IL-2. In paper I, we assessed the immunological status of advanced cancer patients treated with LOAd703 in combination with chemotherapy in the LOKON002 trial. We showed that intratumoral LOAd703 treatment could mediate inflammation in patients’ tumors as seen by an induction of multiple inflammatory genes, including gene profiles associated with response to checkpoint inhibition. In addition, there was a distinct profile for responding patients. In paper II, we demonstrated that LOAd703 therapy is feasible in B-cell lymphoma and that it can be combined with CAR T-cell therapy as priming lymphoma cells with LOAd703 enhanced the secretion of chemokines promoting T-cell migration and boosted the cytotoxic T-cell function leading to better lymphoma cell killing. In paper III, we showed in vivo in a melanoma model that a murine version of LOAd703 could induce anti-tumor immune responses, sensitize tumors to checkpoint inhibitors and facilitate systemic immune responses in a twin-tumor model, in particular when combined with anti-PD-L1. In paper IV, we present a novel virus, LOAd732, and its functionality was confirmed in regards to the oncolytic capability, transgene expression and immunostimulatory effect. LOAd732 activated DCs that were capable of stimulating antigen-specific T cells, as well as NK cells. Moreover, LOAd732-stimulated cells could withstand immunosuppression mediated by cytokines (TGF-β1, IL-10) commonly present in the microenvironment of many tumors

Gemcitabine reduces MDSCs, tregs and TGF beta-1 while restoring the teff/treg ratio in patients with pancreatic cancer

Background: Cancer immunotherapy can be potentiated by conditioning regimens such as cyclophosphamide, which reduces the level of regulatory T cells (tregs). However, myeloid suppressive cells are still remaining. Accordingly to previous reports, gemcitabine improves immune status of cancer patients. In this study, the role of gemcitabine was further explored to map its immunological target cells and molecules in patients with pancreatic cancer. Methods: Patient blood was investigated by flow cytometry and cytokine arrays at different time points during gemcitabine treatment. Results: The patients had elevated myeloid-derived suppressor cells (MDSCs), and Tregs at diagnosis. Myeloid cells were in general decreased by gemcitabine. The granulocytic MDSCs were significantly reduced while monocytic MDSCs were not affected. In vitro, monocytes responding to IL-6 by STAT3 phosphorylation were prevented to respond in gemcitabine medium. However, gemcitabine could not prevent STAT3 phosphorylation in IL-6-treated tumor cell lines. TGF beta-1 was significantly reduced after only one treatment and continued to decrease. At the same time, the effector T cell:Treg ratio was increased and the effector T cells had full proliferative capacity during the gemcitabine cycle. However, after a resting period, the level of suppressor cells and TGF beta-1 had been restored showing the importance of continuous conditioning. Conclusions: Gemcitabine regulates the immune system in patients with pancreatic cancer including MDSCs, Tregs and molecules such as TGF beta-1 but does not hamper the ability of effector lymphocytes to expand to stimuli. Hence, it may be of high interest to use gemcitabine as a conditioning strategy together with immunotherapy

Systemic immunity upon local oncolytic virotherapy armed with immunostimulatory genes may be supported by tumor-derived exosomes

Immunostimulatory gene therapy utilizing oncolytic viruses (OVs) as gene vehicles is a promising immunotherapy for cancer. Since viruses are immunogenic, systemic delivery can be troublesome due to neutralizing antibodies. Nevertheless, local delivery by intratumoral injection seems to induce systemic immune reactions. In this study, we demonstrate a novel mechanism of action of armed OV therapy suggesting that exosomes released by tumor cells infected with armed OV may participate to activate the immune system and this may also support systemic immunity. Tumor cell-derived exosomes commonly exert immunosuppressive functions. We hypothesized that exosomes derived from OV-infected tumor cells may instead be immunostimulatory. Human melanoma cells were infected by OVs armed with costimulatory molecules CD40 ligand (CD40L) and 4-1BB ligand (4-1BBL). Exosomes were purified and investigated for the presence of CD40L/4-1BBL mRNA and protein, and for their capacity to stimulate immune responses. The results show that the exosomes cargo transgenes. The exosomes from CD40L/4-1BBL-expressing tumor cells, or the viruses themselves, could stimulate robust dendritic cell (DC) activation with an enhanced level of major histocompatibility complex (MHC) and costimulatory molecules. Hence, exosomes after OV infection can locally activate immune responses at the tumor site and encounter immune cells such as DCs

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

MOESM1 of Gemcitabine reduces MDSCs, tregs and TGFβ-1 while restoring the teff/treg ratio in patients with pancreatic cancer

Additional file 1. Gating strategies for flow cytometry

Adenovirus-mediated CD40L gene transfer increases Teffector/Tregulatory cell ratio and upregulates death receptors in metastatic melanoma patients

Background and aims: Malignant melanoma is an aggressive tumor sensitive for immunotherapy such as checkpoint blockade antibodies. Still, most patients with late stage disease do not respond, and the side effects can be severe. Stimulation of the CD40 pathway to initiate anti-tumor immunity is a promising alternative. Herein, we demonstrate immune profiling data from melanoma patients treated with an adenovirus-based CD40 ligand gene therapy (AdCD40L). Methods: Peripheral blood mononuclear cells and plasma were collected from malignant melanoma patients (n = 15) enrolled in a phase I/IIa study investigating intratumoral delivery of AdCD40L with or without low dose cyclophosphamide. Cells were analyzed by flow cytometry while plasma samples were analyzed by a multi-array proteomics. Results: All patients had an increased Teffector/Tregulatory cell ratio post therapy. Simultaneously, the death receptors TNFR1 and TRAIL-R2 were significantly up-regulated post treatment. Stem cell factor (SCF), E-selectin, and CD6 correlated to enhanced overall survival while a high level of granulocytic myeloid-derived suppressor cells (gMDSCs), IL8, IL10, TGFb1, CCL4, PlGF and Fl3t ligand was highest in patients with short survival. Conclusions: AdCD40L intratumoral injection induced desirable systemic immune effects that correlated to prolonged survival. Further studies using CD40 stimulation in malignant melanoma are warranted