Ioanna Milenova

Activation of myeloid and endothelial cells by CD40L gene therapy supports T-cell expansion and migration into the tumor microenvironment

CD40 is an interesting target in cancer immunotherapy due to its ability to stimulate T-helper 1 immunity via maturation of dendritic cells and to drive M2 to M1 macrophage differentiation. Pancreatic cancer has a high M2 content that has shown responsive to anti-CD40 agonist therapy and CD40 may thus be a suitable target for immune activation in these patients. In this study, a novel oncolytic adenovirus armed with a trimerized membrane-bound extracellular CD40L (TMZ-CD40L) was evaluated as a treatment of pancreatic cancer. Further, the CD40L mechanisms of action were elucidated in cancer models. The results demonstrated that the virus transferring TMZ-CD40L had oncolytic capacity in pancreatic cancer cells and could control tumor progression. TMZ-CD40L was a potent stimulator of human myeloid cells and T-cell responses. Further, CD40L-mediated stimulation increased tumor-infiltrating T cells in vivo, which may be due to a direct activation of endothelial cells to upregulate receptors for lymphocyte attachment and transmigration. In conclusion, CD40L-mediated gene therapy is an interesting concept for the treatment of tumors with high levels of M2 macrophages, such as pancreatic cancer, and an oncolytic virus as carrier of CD40L may further boost tumor killing and immune activation.

Shaping the tumor stroma and sparking immune activation by CD40 and 4-1BB signaling induced by an armed oncolytic virus.

Purpose: Pancreatic cancer is a severe indication with short expected survival despite surgery and/or combination chemotherapeutics. Checkpoint blockade antibodies are approved for several cancer indications, but pancreatic cancer has remained refractory. However, there are clinical data suggesting that stimulation of the CD40 pathway may be of interest for these patients. Oncolytic viruses armed with immunostimulatory genes represent an interesting approach. Herein, we present LOAd703, a designed adenovirus armed with trimerized CD40L and 4-1BBL that activates the CD40 and 4-1BB pathways, respectively. As many cells in the tumor stroma, including stellate cells and the infiltrating immune cells, express CD40 and some 4-1BB, we hypothesize that LOAd703 activates immunity and simultaneously modulates the biology of the tumor stroma. Experimental Design: Tumor, stellate, endothelial, and immune cells were infected by LOAd703 and investigated by flow cytometry, proteomics, and functional analyses. Results: LOAd703-infected pancreatic cell lines were killed by oncolysis, and the virus was more effective than standard-of-care gemcitabine. In in vivo xenograft models, LOAd703 efficiently reduced established tumors and could be combined with gemcitabine for additional effect. Infected stellate and tumor cells reduced factors that promote tumor growth (Spp-1, Gal-3, HGF, TGFβ and collagen type I), while chemokines were increased. Molecules involved in lymphocyte migration were upregulated on infected endothelial cells. Dendritic cells were robustly stimulated by LOAd703 to produce costimulators, cytokines and chemokines, and such DCs potently expanded both antigen-specific T cells and NK cells. Conclusions: LOAd703 is a potent immune activator that modulates the stroma to support antitumor responses. Clin Cancer Res; 23(19); 5846–57. ©2017 AACR.

516. A Novel Oncolytic Adenovirus Expressing Tumor Microenvironment Stimulators to Evoke and Facilitate Anti-Tumor Immune Responses

A Novel Oncolytic Adenovirus Expressing Tumor Microenvironment Stimulators to Evoke and Facilitate Anti-Tumor Immune Responses

Abstract 3662: Activation of CD40 while inhibiting IL6/STAT3 using oncolytic viruses induces mature DCs with high cytokine production but blocks PDL1 expression

The tumor microenvironment (TME) consists of tumor cells and stroma, including fibroblasts, blood vessels, immune cells and extracellular matrix. The TME supports tumor progression, metastasis as well as resistance to cancer therapeutics. In pancreatic cancer, the TME is dense due to overproduction of collagen and the tumor is infiltrated with suppressive myeloid cells such as M2 macrophages and myeloid suppressor cells. One key regulator of myeloid cells is CD40, a receptor expressed on a variety of cell types. CD40/CD40L signaling results in production of cytokines and chemokines by myeloid cells but also endothelial and epithelial cells to alert the immune system of immediate danger. 4-1BB is expressed by lymphocytes and dendritic cells (DCs). Stimulation via 4-1BBL drives lymphocyte expansion and regulates memory formation. IL6 signaling leads to STAT3 phosphorylation in myeloid cells and tumor cells leading to suppressive phenotypes, tumor proliferation, and angiogenesis. Further, STAT3 signaling enhances production of TGFb, which in turn leads to overexpression of collagen. We have constructed a family of oncolytic adenoviruses (LOAd) activating the CD40, 4-1BB and/or inhibiting IL6 signaling. The LOAd viruses (-, 700, 703, 713) were investigated for their capacity to activate human monocyte-derived DCs as well as their effect on pancreatic tumor cells and stroma (fibroblastic stellate cells, endothelial cells) using flow cytometry, MTS assay and ProSeek Proteomics. The LOAd viruses expressing a trimerized CD40L, 4-1BBL and/or a scFv IL6R showed efficient oncolysis of tumor cells but primary stellate cells were unaffected. However, stellate cells reduced tumor-promoting factors such as FGF5, PlGF, amphiregulin, Gal3, TGFb and collagen type I. Dendritic cells increased costimulators, cytokines as well as chemokines but PDL1 was not expressed when IL6/STAT3 was blocked. Infected endothelial cells upregulated receptors important for lymphocyte transmigration (ICAM, VCAM and E-Selectin). Taken together, our data demonstrates that it is possible to utilize oncolytic adenoviruses to spark immune activation at the same time changing biological processes via STAT3 blockade and/or CD40/4-1BB pathway activation to reduce factors that promotes tumor progression. Citation Format: Emma Eriksson, Ioanna Milenova, Jessica Wenthe, Anna Dimberg, Rafael Moreno, Gustav Ullenhag, Ramon Alemany, Angelica Loskog. Activation of CD40 while inhibiting IL6/STAT3 using oncolytic viruses induces mature DCs with high cytokine production but blocks PDL1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3662. doi:10.1158/1538-7445.AM2017-3662

662. An Oncolytic Adenovirus Gene Therapy Targeting Both Tumor Cell Survival and Desmoplasia in Pancreatic Cancer

An Oncolytic Adenovirus Gene Therapy Targeting Both Tumor Cell Survival and Desmoplasia in Pancreatic Cancer

Abstract 1474: A novel oncolytic adenovirus expressing tumor microenvironment modulators that activates myeloid cells, lymphocytes and endothelial cells

Immunotherapy is becoming a cornerstone in cancer treatment of many indications. So far, pancreatic cancer has shown little response to so called checkpoint blockade antibodies. However, animal data suggests that activating immunotherapies releases the effect of checkpoint blockade also in pancreatic cancer. The tumor microenvironment (TEM) supports the growth of the tumor cells and consists of stroma cells, fibroblasts, blood vessels and immune cells. In some tumor lesions, such as those in pancreatic cancer, the TEM is dense and comprises most of the lesion. The TEM regulates immune activity via its high content of M2 macrophages, myeloid derived suppressor cells and T regulatory cells. Further, the dysfunctional blood vessels in lesions are not optimal for recruiting lymphocytes. With these aspects in mind, LOAd703 was developed. LOAd703 is an oncolytic adenovirus carrying TEM modulators. LOAd703 was constructed from the ICOVIR system of oncolytic adenoviruses in which replication depends on a dysfunctional, hyperphosphorylated retinoblastoma pathway. The genome was further altered by removing E3-6.7K and gp19K, changing the serotype 5 fiber to a serotype 35 fiber to target CD46 expressed by most tumors, as well as by adding a CMV-driven transgene cassette with the human transgenes for TMZ-CD40L and 4-1BBL. Hence, the transgenes will be expressed in both tumor and stroma while oncolysis is initiated in the tumor cells. We demonstrate herein that LOAd703 infection of a panel of pancreatic cancer cell lines efficiently induced tumor cell death within 48-72 hrs post infection while LOAd703 infection of dendritic cells demonstrated an increased maturation of myeloid cells including dendritic cells (DCs). These DCs could in turn potently activate and promote expansion of both T- and NK cells. Further, LOAd703 infection of endothelial cells (HUVEC) induced upregulation of molecules involved in lymphocyte attachment, rolling and transmigration. In conclusion, LOAd703 is a novel oncolytic virus that targets both the tumor and its TME and a clinical trial is underway to elucidate its effect in pancreatic cancer. Citation Format: Angelica Loskog, Emma Eriksson, Ioanna Milenova, Rafael Moreno, Ramon Alemany. A novel oncolytic adenovirus expressing tumor microenvironment modulators that activates myeloid cells, lymphocytes and endothelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1474.

Abstract A25: Immunotherapy using LOAd700 armed with CD40 ligand controls experimental pancreatic cancer and activates immune responses

The aim of this study was to evaluate the efficacy of LOAd immunotherapy to treat pancreatic cancer in a xenograft model as well as to determine the capacity of LOAds to stimulate the immune system in in vitro models. LOAd viruses are oncolytic adenoviruses (5/35) armed with immunostimulatory genes in order to shift the tumor milieu towards immune activation at the same time providing release and spread of tumor antigens due to oncolysis. In this manner, LOAd virus is an antigen-independent immunotherapy for various solid tumors. LOAd armed with CD40 ligand (LOAd700) was constructed from the ICOVIR system by changing the virus shaft and knob to that of serotype 35 to broaden virus binding and entry into cells (pending patent EP14163704). Oncolysis in OCOVIRs is restricted to cells with a disrupted Rb pathway. Viruses were produced using A549 cells. The pancreatic cancer cell lines BxPC3, Panc01, MiaPaCa2 and PaCa3 were used for in vitro evaluation and Panc01 was used in the xenograft model in Nu/Nu mice. Human dendritic cells (DCs) were obtained by differentiation of CD14+ monocytes with GM-CSF and IL4 for 7 days. LOAd viruses infected both the panel of pancreatic tumor cell lines and human DCs with high efficacy and both the cell lines and the DCs expressed the CMV driven transgene/s post transduction. The oncolytic LOAd virus did not kill DCs nor healthy pancreatic cells present in donor islet cell isolation surplus material. In contrast, oncolysis was restricted to the tumor cell lines. The LOAd-transduced DCs increased markers of maturation such as MHC II, CD86, CD70 and CD83 and produced high levels of IL12. LOAd stimulated DCs were able to activate and expand antigen-specific T cells and NK cells as demonstrated in a CMV in vitro system. LOAd viruses could control tumor growth in a xenograft immunodeficient model due to oncolysis alone. In conclusion, LOAd700 can kill pancreatic tumor cells in vitro and control growing tumor in mice due to oncolysis alone. Further, upon DC transduction, LOAd viruses mature DCs to efficient antigen presenters and stimulators of Th1 effector cells such as T cells and NK cells. LOAd is an interesting new immunotherapy for solid malignancies including pancreatic cancer. Citation Format: Emma Svensson, Rafael Moreno, Ioanna Milenova, Lisa Christiansson, Ramon Alemany, Angelica Loskog. Immunotherapy using LOAd700 armed with CD40 ligand controls experimental pancreatic cancer and activates immune responses. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A25.

Abstract 297: Immunotherapy with a CD40L/4-1BBL double-armed oncolytic adenovirus drives Th1 immunity and control tumor progression in a pancreas cancer model

We hypothesized that an immunostimulatory oncolytic virus (LOAd703) is a potent inducer of anti-tumor immunity and tested its biological functions in models of pancreas cancer. LOAd703 is a double-armed oncolytic adenovirus that introduces the simultaneous expression of a trimerized membrane-bound CD40L and 4-1BBL locally in the tumor aiming to induce potent Th1-mediated anti-tumor immunity. By utilizing an oncolytic adenovirus serotype 5/35 for gene transfer, virally induced tumor cell oncolysis will provide a broad release of antigens at the site of immune activation. Transgene expression is driven by a separate promoter to allow for efficient expression in both tumor cells and tumor stroma while virus replication is restricted to tumor cells by E1A delta24 deletion. CD40 ligand is a potent stimulator of myeloid cells including dendritic cells (DCs) that in turn induce robust T cell responses. CD40L can also reduce the levels of myeloid suppressor cells and M2 macrophages as well as enhancing T cell infiltration into tumors. Further, CD40-mediated signaling combined with TLR stimuli (such as adenovirus) induces intense Th1 immunity. 4-1BBL is known to provide expansion and survival signaling to pre-activated T- and NK cells. Pancreatic cancer cell lines and healthy exocrine pancreas cells were transduced with LOAd703 and it efficiently killed tumor cells while normal cells remained intact as evaluated by an MTS assay. CD40L and 4-1BBL were highly expressed by the transduced tumor cells as detected by flow cytometry. Cell lysis by LOAd703 was equally efficient as a similar oncolytic virus without transgenes demonstrating that the double transgene expression did not interfere with viral replication. Repeated (6x) peritumoral injections of LOAd703 in a xenograft Nu/Nu/Panc01 model showed efficient tumor cell growth control and sustained complete responses. The effect could be further enhanced by gemcitabine in both xenograft mice and in a syngeneic immunocompetent C57BL/6/Panc02 model. Transduction of human monocyte-derived immature DCs resulted in a strong CD40L and 4-1BBL expression without lysis of the cells. Instead, the transduced DCs matured as shown by high CD83 and IL12 expression. The addition of 4-1BBL significantly enhanced DC maturation compared to a virus containing only CD40L, or no transgenes, by expressing higher levels of CD70, IL12, TNFa, IFNg and IL21. Further, LOAd703-transduced DCs pulsed with pp65 CMV peptides potently expanded antigen-specific T cells as well as NK cells. In conclusion, LOAd703 is a novel, double-armed immunostimulatory oncolytic gene therapy that initiates robust Th1 immunity, and eradicates pancreatic cancer in experimental models. A clinical trial using LOAd703 for pancreatic cancer is underway. Citation Format: Emma Svensson, Ioanna Milenova, Rafael Moreno, Ramon Alemany, Angelica Loskog. Immunotherapy with a CD40L/4-1BBL double-armed oncolytic adenovirus drives Th1 immunity and control tumor progression in a pancreas cancer model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 297. doi:10.1158/1538-7445.AM2015-297