Antiphosphatidylserine Antibody Combined with Irradiation Damages Tumor Blood Vessels and Induces Tumor Immunity in a Rat Model of Glioblastoma

Purpose: The vascular targeting antibody bavituximab is being combined with chemotherapy in clinical trials in cancer patients. Bavituximab targets the membrane phospholipid, phosphatidylserine, complexed with 2-glycoprotein I. Phosphatidylserine is normally intracellular but becomes exposed on the luminal surface of vascular endothelium in tumors. Phosphatidylserine exposure on tumor vessels is increased by chemotherapy and irradiation. Here, we determined whether treatment with the murine equivalent of bavituximab, 2aG4, combined with irradiation can suppress tumor growth in a rat model of glioblastoma. Experimental Design: F98 glioma cells were injected into the brains of syngeneic rats where they grow initially as a solid tumor and then infiltrate throughout the brain. Rats with established tumors were treated with 10 Gy whole brain irradiation and 2aG4. Results: Combination treatment doubled the median survival time of the rats, and 13 of animals were rendered disease free. Neither treatment given individually was as effective. We identified two mechanisms. First, irradiation induced phosphatidylserine exposure on tumor blood vessels and enhanced antibody-mediated destruction of tumor vasculature by monocytes/macrophages. Second, the antibody treatment induced immunity to F98 tumor cells, which are normally weakly immunogenic. Surviving rats were immune to rechallenge with F98 tumor cells. In vitro, 2aG4 enhanced the ability of dendritic cells (DCs) to generate F98-specific cytotoxic T cells. Phosphatidylserine exposure, which is induced on tumor cells by irradiation, likely suppresses tumor antigen presentation, and 2aG4 blocks this tolerogenic effect. Conclusion: Bavituximab combined with radiotherapy holds promise as a vascular targeting and immune enhancement strategy for the treatment of human glioblastoma. (Clin Cancer Res 2009;15(22):687180)

[1]  R. Mirimanoff,et al.  Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. , 2005, The New England journal of medicine.

[2]  S. Riddell,et al.  Efficient cross-priming of tumor antigen-specific T cells by dendritic cells sensitized with diverse anti-MICA opsonized tumor cells. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[3]  J. Yodoi,et al.  Persistent oxidative stress in cancer , 1995, FEBS letters.

[4]  V. Fadok,et al.  Regulation of macrophage cytokine production by phagocytosis of apoptotic and post-apoptotic cells. , 1998, Biochemical Society transactions.

[5]  Rolf F. Barth,et al.  Rat brain tumor models in experimental neuro-oncology:The 9L, C6, T9, F98, RG2 (D74), RT-2 and CNS-1 Gliomas , 2004, Journal of Neuro-Oncology.

[6]  V. Fadok,et al.  Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-beta1 secretion and the resolution of inflammation. , 2002, The Journal of clinical investigation.

[7]  N. Kawashima,et al.  Immune-mediated inhibition of metastases after treatment with local radiation and CTLA-4 blockade in a mouse model of breast cancer. , 2005, Clinical cancer research : an official journal of the American Association for Cancer Research.

[8]  M. Westphal,et al.  Anti-VEGF antibody treatment of glioblastoma prolongs survival but results in increased vascular cooption. , 2000, Neoplasia.

[9]  D. Zagzag,et al.  Angiogenesis in Gliomas: Biology and Molecular Pathophysiology , 2005, Brain pathology.

[10]  S. Bhangoo,et al.  Adjuvant Gamma Knife Stereotactic Radiosurgery at the Time of Tumor Progression Potentially Improves Survival for Patients with Glioblastoma Multiforme , 2005, Neurosurgery.

[11]  S. Ran,et al.  Antitumor Effects of a Monoclonal Antibody that Binds Anionic Phospholipids on the Surface of Tumor Blood Vessels in Mice , 2005, Clinical Cancer Research.

[12]  M. Dhodapkar,et al.  Antitumor Monoclonal Antibodies Enhance Cross-Presentation of Cellular Antigens and the Generation of Myeloma-specific Killer T Cells by Dendritic Cells , 2002, The Journal of experimental medicine.

[13]  P. Williamson,et al.  Back and forth: the regulation and function of transbilayer phospholipid movement in eukaryotic cells. , 1994, Molecular membrane biology.

[14]  R. Brekken,et al.  Combination of a monoclonal anti‐phosphatidylserine antibody with gemcitabine strongly inhibits the growth and metastasis of orthotopic pancreatic tumors in mice , 2006, International journal of cancer.

[15]  Naoto Tsuchiya,et al.  Clinical evaluation of dendritic cell vaccination for patients with recurrent glioma: results of a clinical phase I/II trial. , 2005, Clinical cancer research : an official journal of the American Association for Cancer Research.

[16]  P. Devaux,et al.  Ion regulation of phosphatidylserine and phosphatidylethanolamine outside-inside translocation in human erythrocytes. , 1987, Biochimica et biophysica acta.

[17]  A. Clavreul,et al.  Effects of syngeneic cellular vaccinations alone or in combination with GM-CSF on the weakly immunogenic F98 glioma model , 2006, Journal of Neuro-Oncology.

[18]  Andrea Vondracek,et al.  Interaction between Phosphatidylserine and the Phosphatidylserine Receptor Inhibits Immune Responses In Vivo1 , 2005, The Journal of Immunology.

[19]  C. Orosz,et al.  Phenotype and functional activity of tumor-infiltrating lymphocytes isolated from immunogenic and nonimmunogenic rat brain tumors. , 1991, Cancer research.

[20]  J. Kim,et al.  B7.1 expression by the weakly immunogenic F98 rat glioma does not enhance immunogenicity , 2000, Gene Therapy.

[21]  R. Steinman,et al.  Enhanced T-Cell Responses to Glioma Cells Coated With the Anti-EGF Receptor Antibody and Targeted to Activating FcγRs on Human Dendritic Cells , 2008, Journal of immunotherapy.

[22]  G. Schuler,et al.  An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. , 1999, Journal of immunological methods.

[23]  Z. Fuks,et al.  Radiation and ceramide-induced apoptosis , 2003, Oncogene.

[24]  S. Ran,et al.  Increased exposure of anionic phospholipids on the surface of tumor blood vessels. , 2002, Cancer research.

[25]  Gerold Schuler,et al.  Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity? , 2002, Trends in immunology.

[26]  J. Turnay,et al.  Inhibition of Phosphatidylserine Recognition Heightens the Immunogenicity of Irradiated Lymphoma Cells In Vivo , 2004, The Journal of experimental medicine.

[27]  B. Ross,et al.  Development of a syngeneic rat brain tumor model expressing EGFRvIII and its use for molecular targeting studies with monoclonal antibody L8A4. , 2005, Clinical cancer research : an official journal of the American Association for Cancer Research.

[28]  P. Thorpe.  Vascular Targeting Agents as Cancer Therapeutics , 2004, Clinical Cancer Research.

[29]  S. Ran,et al.  Phosphatidylserine is a marker of tumor vasculature and a potential target for cancer imaging and therapy. , 2002, International journal of radiation oncology, biology, physics.

[30]  P. Thorpe,et al.  Radiation-Enhanced Vascular Targeting of Human Lung Cancers in Mice with a Monoclonal Antibody That Binds Anionic Phospholipids , 2007, Clinical Cancer Research.

[31]  S. Ran,et al.  Infarction of solid Hodgkin's tumors in mice by antibody-directed targeting of tissue factor to tumor vasculature. , 1998, Cancer research.

[32]  S. Demaria,et al.  Combining radiotherapy and immunotherapy: a revived partnership. , 2005, International journal of radiation oncology, biology, physics.

[33]  G. Finocchiaro,et al.  Dendritic cells pulsed with glioma lysates induce immunity against syngeneic intracranial gliomas and increase survival of tumor-bearing mice , 2006, Neurological research.

[34]  P. Thorpe,et al.  A monoclonal antibody that binds anionic phospholipids on tumor blood vessels enhances the antitumor effect of docetaxel on human breast tumors in mice. , 2005, Cancer research.

[35]  M. Berger,et al.  Centralized databases available for describing primary brain tumor incidence, survival, and treatment: Central Brain Tumor Registry of the United States; Surveillance, Epidemiology, and End Results; and National Cancer Data Base. , 1999, Neuro-oncology.

[36]  Zvi Fuks,et al.  Tumor Response to Radiotherapy Regulated by Endothelial Cell Apoptosis , 2003, Science.

[37]  F. Goñi,et al.  Sphingomyelinase Activity Causes Transbilayer Lipid Translocation in Model and Cell Membranes* , 2003, Journal of Biological Chemistry.

[38]  Jin He,et al.  Vascular Imaging of Solid Tumors in Rats with a Radioactive Arsenic-Labeled Antibody that Binds Exposed Phosphatidylserine , 2008, Clinical Cancer Research.

[39]  A. Schroit,et al.  Aminophospholipid asymmetry: A matter of life and death. , 2003, Annual review of physiology.

[40]  R. Schmidt-Ullrich,et al.  Ionizing radiation-induced, mitochondria-dependent generation of reactive oxygen/nitrogen. , 2001, Cancer research.

[41]  R. Barth,et al.  Rat Brain Tumor Models to Assess the Efficacy of Boron Neutron Capture Therapy: A Critical Evaluation , 2004, Journal of Neuro-Oncology.

[42]  N. Davoust,et al.  How to drain without lymphatics? Dendritic cells migrate from the cerebrospinal fluid to the B-cell follicles of cervical lymph nodes. , 2006, Blood.

[43]  P. D. de Groot,et al.  Plasma Protein β-2-Glycoprotein 1 Mediates Interaction between the Anti-tumor Monoclonal Antibody 3G4 and Anionic Phospholipids on Endothelial Cells* , 2006, Journal of Biological Chemistry.

[44]  M. Westphal,et al.  Inhibition of glioma angiogenesis and growth in vivo by systemic treatment with a monoclonal antibody against vascular endothelial growth factor receptor-2. , 2001, Cancer research.

[45]  Keith L. Black,et al.  Vaccination with Tumor Lysate-Pulsed Dendritic Cells Elicits Antigen-Specific, Cytotoxic T-Cells in Patients with Malignant Glioma , 2004, Cancer Research.