657. A CD46-Targeting Recombinant Protein that Increases the Therapeutic Efficacy of Monoclonal Antibodies for Cancer

CANCER - IMMUNOTHERAPY II 656. An A20-Silenced, Stroma-Targeted DC Vaccine Reverses the Immunosuppressive Tumor Microenviroment Resulting in Potent Antitumor Activity Xiao-Tong Song,1,2 Xiaoou Zhou,1,2 Lisa Rollins,1,2 Cliona M. Rooney,1,2 Stephen Gottschalk.1,2 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital, The Methodist Hospital, Houston, TX; 2 Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX.

Dendritic cell (DC) vaccines have produced disappointing benets in most clinical studies of cancer. This is most likely due to the presence of immunosuppressive cells within the tumor. Inhibition may come from regulatory T cells (Tregs) and also from the tumor supporting stroma, since cancer associated broblasts (CAFs; the central component of the tumor stroma) secrete inhibitory cytokines. Moreover, the extracellular matrix itself may protect malignant cells from effective immunity. We have previously shown that an A20-silenced DC vaccine renders DC resistant to Treg mediated immunosuppression and the aim of this project was to evaluate the effects of an A20-silenced DC vaccine targeting broblast activating protein (FAP) expressed on CAFs in the murine B16 tumor model. Bone marrow derived DCs were genetically modied to express an A20-specic shRNA and full-length murine FAP (DC-shA20FAP). DC-shA20-FAP vaccination induced potent CD4+ and CD8+ FAP-specic T-cell responses in C57BL/6 mice as judged by IFN-g Elispot assays in contrast to a DC vaccine expressing FAP only. Vaccinating B16 melanoma bearing C57BL/6 mice with DC-shA20FAP induced a signicant decrease of tumor-inltrating macrophages (CD11b+; 5.75% vs 28.92%) and myeloid-derived suppressor cells (CD11b +Gr1 hi; 4.37% vs 2.48%) in comparison to DC-shA20 vaccinated mice. Conversely, DC-shA20-FAP vaccination induced a 2-fold upregulation of CD80 expression on tumor-inltrating DCs as well as an increase of inltrating CD8+ T cells (19.31% vs 7.24%). DC-shA20-FAP vaccination resulted in potent antitumor effects in subcutaneous and metastatic B16 melanoma models. We show here that an A20-silenced, FAP-targeted DC vaccine induces potent FAPspecic immune response, reverses the immunosuppressive tumor microenvironment and has potent antitumor activity. Thus, targeting the tumor stroma has the potential to improve current DC vaccine approaches for cancer.

657. A CD46-Targeting Recombinant Protein that Increases the Therapeutic Efcacy of Monoclonal Antibodies for Cancer

Hongjie Wang,1 Ying Liu,1 Zong Li,1 Akseli Hemminki,2 Andre Lieber.1 1 Medical Genetics, University of Washington, Seattle, WA; 2 University of Helsinki, Helsinki, Finland. Monoclonal antibodies (mAbs) have emerged as a class of novel oncology therapeutics. Despite their commercial successes, each of these mAbs is only effective in a fraction of patients. It is now recognized that one of the major impediments to their therapeutic efcacy is the overexpression of complement inhibitory proteins, e.g. CD46, on the cell surface of solid tumors and hematologic malignancies. To address the problem we capitalized on our earlier nding that adenovirus serotype 35 uses the CD46 as a high-afnity attachment receptor. From an E.coli expression library of Ad35 ber knob mutants, we selected a variant (Ad35K++) that had a higher afnity to CD46 than the natural Ad35 ber knob. Ad35K++ is produced as a soluble protein in E.coli and can be easily puried. Ad35K++ binding results in the transient removal of CD46 from the surface of tumor cells for approximately 72 hours. During this time period, tumor cells that are normally resistant to mAb therapy become S256

susceptible and can be killed by complement dependent cytotoxicity. In in vitro studies, we demonstrated an enhancing effect of Ad35K++ for the following mAbs: rituximab/Rituxan (used for the treatment of B-cell non-Hodgkin’s lymphoma), alemtuzumab/Campath (for chronic lymphocytic leukemia), trastuzumab/Herceptin (for breast cancer), cetuximab/Erbitux (for colon cancer), gemtuzumab/ Myelotarg (for acute myelogenous leukemia), and ofatumumab/ Arzerra (for lymphoma and CLL). Thus far, in vivo studies have been completed for the combination of Ad35K++ and the CD20targeting mAb rituximab. We demonstrated in two murine lymphoma models that rituximab plus Ad35K++ achieved superior anti-tumor effects and animal survival when compared to animals treated with rituximab alone. For example, sixty percent of mice injected with human lymphoma Raji cells were cured with Ad35K++/rituximab treatment, while all mice treated with only rituximab died within 19 days after Raji cell transplantation. In vivo studies with ofatumumab and trastuzumab are ongoing and results will be presented. Our long-term goal is to test Ad35K++ in combination with rituximab in treatment-refractory B-cell lymphoma patients. These patients are unable to mount efcient antibody responses, which facilitates repeat treatment with Ad35K++. In preparation for this clinical trial, we are currently performing toxicity and efcacy studies in double transgenic huCD46/huCD20 mice with syngeneic lymphomas and in non-human primates. Notably, we have shown that baboon (but not macaque) CD46 is recognized by Ad35K++ and that treatment of baboon CD20+ B-cells with Ad35K++ enhances rituximab-mediated complement-dependent killing in vitro.

658. Treatment of Cancer Patients with IntegrinTargeted Oncolytic Adenoviruses Ad5-D24-RGD and Ad5-RGD-D24-GMCSF

Sari Pesonen,1,2 Vincenzo Cerullo,1,2 Sophie Escutenaire,1,2 Mari Raki,1,2 Lotta Kangasniemi,3 Eerika Karli,1,2 Elina Haavisto,3 Minna Oksanen,1,2 Timo Joensuu,4 Anna Kanerva,1,5 Akseli Hemminki.1,2 1 Cancer Gene Therapy Group, Transplantation Laboratory, Haartman Institute and Finnish Institute of Molecular Medicine, University of Helsinki, Helsinki, Finland; 2HUSLAB, Helsinki University Central Hospital, Helsinki, Finland; 3 Oncos Therapeutics Inc., Helsinki, Finland; 4International Comprehensive Cancer Center Docrates, Helsinki, Finland; 5 Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Helsinki, Finland. Advanced tumors are often immunosuppressive meaning that the host immune defence mechanism does not effectively attack malignant cells. Tumor inltrating dendritic cells (DCs) play a key role in the immune defence against cancer but may not be optimally activated in the tumor microenvironment due to lack of co-stimulatory molecules. Granulocyte-macrophage colony stimulating factor (GMCSF) is a potent activator of DCs and a local production of GM-CSF at the tumor site can result in the differentiation and activation of DCs. Oncolytic viruses are an experimental strategy against advanced solid tumors. Arming the virus with an immune stimulatory molecule is an appealing strategy and may increase overall antitumor potency. Therefore, we generated an oncolytic adenovirus 5 coding for human GM-CSF. Furthermore, RGD modication in the HI-loop of the ber knob and a 24bp constant region 2 deletion in E1A were introduced to increase tumor selectivity. 9 patients were treated with Ad5-D24RGD (the same virus without GMCSF) and 7 patients with Ad-RGDD24-GMCSF. All patients had progressing advanced solid tumors refractory to available therapies. In general, both viruses were well tolerated. Typical side effects were grade 1-2 dyspnea, fatigue, and fever. 77% (10/13) of evaluable patients showed virus in circulation for at least 2 weeks after treatment. Objective clinical benet (by RECIST) was seen in 2 out of 5 evaluable patients treated with Ad5RGD-D24-GMCSF whereas no patients treated with Ad5-D24-RGD Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy