Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 755

Development of a Cancer Targeting Tumor Blood Vessels

ELISABETH JM HUIJBERS

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-554-8317-3 UPPSALA urn:nbn:se:uu:diva-170887 2012 Dissertation presented at Uppsala University to be publicly examined in B42, Biomedicinsk Centrum (BMC), Husargatan 3, Uppsala, Friday, May 11, 2012 at 09:00 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English.

Abstract Huijbers, E. J. M. 2012. Development of a Targeting Tumor Blood Vessels. Acta Universitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 755. 85 pp. Uppsala. ISBN 978-91-554-8317-3.

A treatment strategy for cancer is the suppression of tumor growth by directing an to the tumor vessels, which will destroy the tissue. In this thesis we describe the development of a vaccine that targets expressed around angiogenic vasculature in most solid tumors. These antigens are alternative spliced extra domains of glycoproteins present in the extracellular matrix; e.g. the extra domain-B (ED-B) and extra domain-A (ED-A) of fibronectin and the C-domain of tenascin-C (TNCC). We show that it is possible to break self-tolerance and induce a strong antibody response against ED-B by . Furthermore, tumor growth was inhibited and the changes observed in the tumor tissue were consistent with an attack of the tumor vasculature by the . For clinical development of therapeutic , targeting self-molecules like ED-B, a potent but non-toxic biodegradable adjuvant is required. The -based Montanide ISA 720 (M720) in combination with CpG DNA fulfilled these requirements and induced an equally strong anti-self immune response as the preclinical golden standard Freund’s adjuvant. We have further characterized the immune response against ED-B generated with the adjuvant M720/ GpG. The ED-B vaccine also inhibited tumor growth in a therapeutic setting in a transgenic mouse model of pancreatic insulinoma in which tumorigenesis was already initiated. Furthermore, antibodies against ED-A and TNCC could be induced in mice and rabbits. We analyzed the expression of ED-A in breast tumors of transgenic MMTV-PyMT mice, a metastatic breast cancer model, with the aim to use this model to study the effect of an ED-A vaccine on metastasis. We also detected ED-B in canine mammary tumor tissue. Therefore vascular antigens might also represent potential therapeutic targets in dogs. All together our preclinical data demonstrate that a vaccine targeting tumor blood vessels is a promising new approach for cancer treatment.

Keywords: Vaccine, Therapeutic, Cancer, Tumor, Angiogenesis, , Vascular, Extracellular matrix

Elisabeth JM Huijbers, Uppsala University, Department of Medical Biochemistry and Microbiology, Box 582, SE-751 23 Uppsala, Sweden.

© Elisabeth JM Huijbers 2012

ISSN 1651-6206 ISBN 978-91-554-8317-3 urn:nbn:se:uu:diva-170887 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-170887)

Life is what you make of it!

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Huijbers, E., Ringvall, M., Femel, J., Kalamajski, S., Lukinius, A., Åbrink, M., Hellman, L. and Olsson, AK. (2010) Vaccination against the extra domain-B of fibronectin as a novel tumor therapy. FASEB J, 11:4535-4544

II Ringvall, M., Huijbers, E., Ahooghalandari, P., Alekseeva, L., Andronova, T., Olsson, AK. and Hellman, L. (2009) Identification of potent, biodegradable and non-toxic adjuvants for the use in therapeutic vaccination against self- antigens. Vaccine, 28:48-52

III Huijbers, E., Femel, J., Andersson, K., Björkelund, H., Hellman, L. and Olsson, AK. (2012) The non-toxic and biodegradable adjuvant Montanide ISA720/CpG can replace Freund’s in a cancer vaccine targeting ED-B - a prerequisite for clinical development. Vaccine, 30:225-230

IV Huijbers, E.§, Femel, J.§, Cedervall, J., Hein, T., Hellman, L. and Olsson, AK. Development of a therapeutic vaccine targeting blood vessels in primary tumors and metastases. Manuscript.

§ equal contribution

Reprints were made with permission from the respective publishers.

Contents

Introduction ...... 13 CANCER ...... 13 THE VASCULAR SYSTEM ...... 15 TREATMENT OF CANCER ...... 19 VASCULAR TARGETS ...... 28 SELF-TOLERANCE ...... 39 ADJUVANTS ...... 40 TUMOR MODELS ...... 41 Present investigations ...... 44 VACCINATION MECHANISM ...... 44 DEVELOPMENT OF A POTENT ADJUVANT SAFE FOR CLINICAL USE ...... 46 Paper I ...... 47 Paper II ...... 49 Paper III ...... 50 Paper IV ...... 51 Discussion ...... 52 Future perspectives and concluding remarks ...... 54 Populär vetenskaplig sammanfattning ...... 57 Nederlandse samenvatting ...... 59 Deutsche Zusammenfassung ...... 61 Acknowledgments ...... 63 References ...... 66

Abbreviations

Ab Antibody ADCC Antibody dependent--mediated cytotoxicity ALL Acute lymphoblastic leukemia APC -presenting cell AS Adjuvant system B-cell Bone marrow-; B-lymphocyte BAFF B-cell activating factor BCG Bacillus Calmette-Guérin BCR B-cell receptor C Constant Ig domain CD Cluster of differentiation CDC Complement-dependent cytotoxicity CEP Carboxyethylpyrrole CH Constant Ig domain of the heavy chain CIS Carcinoma in situ CLL Chronic lymphocytic leukemia CML Chronic myelogenous leukemia CSF-1 Colony stimulating factor 1 CTL Cytotoxic T-lymphocyte CTLA4 Cytotoxic T-lymphocyte antigen 4 DC DLL4 Delta-like ligand 4 E.coli Escherichia coli EBRT External beam radiotherapy EC Endothelial cell ECM Extracellular matrix ED-A Extra domain-A; EIIIA ED-B Extra domain-B; EIIIB EDA+ ED-A containing EDB+ ED-B containing EGF Epidermal growth factor EGFR Epidermal growth factor receptor ELISA Enzyme linked immunosorbent assay EMT Epithelial mesenchymal transition FDA American Food and Drug Administration FGF growth factor

FLK1 murine VEGFR-2 FLT-3 Fms-related tyrosine kinase 3 FN Fibronectin GEMM Genetically engineered mouse model GI Gastrointestinal tract GM-CSF Granulocyte -colony stimulating factor GMDP N-acetylglucoseamine-1-4-N-acetylmuramyl- alanyl-D-isoglutamine GMDP-A N-acetylglucoseamine-1-4-N-acetylmuramyl- alanyl-D-glutamic acid GMP Good manufacturing practice GST Glutathione-S-transferase GU Genitourinary tract HAV Hepatitis A virus HBV Hepatitis B virus HER Human epidermal growth factor receptor HLA HMGB1 High-mobility group B1 HPV Human papillomavirus I131 Radioactive iodine ICAM-1 Intercellular adhesion molecule 1 IFN Interferon Ig Immunoglobulin IL Interleukin ISA Incomplete Seppic adjuvant KHL Keyhole limpet hemocyanin KIT Stem cell factor receptor M720 Montanide ISA 720 MAb Monoclonal antibody MAC Membrane attack complex MART Melanoma antigen recognized by T-cells MDP Muramyl dipeptide MDSC Myeloid-derived suppressor cell MHC-I Major histocompatibility complex class I MHC-II Major histocompatibility complex class II MLP Monophosphoryl MMP Matrix metalloproteinase MMTV Mouse mammary tumor virus M720 Montanide ISA 720 MTD Maximum tolerated dose NCI National Cancer Institute, USA NF-κB Nuclear factor κB NK cell

NOD Nucleotide-binding oligomerization domain NSCLC Non-small cell lung carcinoma O/W oil-in-water ORO Opossum-rat-opossum PAP Prostatic acid phosphatase PBMC Peripheral-blood mononuclear cell PCR Polymerase chain reaction PDGF Platelet derived growth factor PDGFR Platelet derived growth factor receptor pIII Minor bacteriophage coat protein PKB/Akt Protein kinase B PlGF Placental growth factor PRR Pattern recognition receptor PSA Prostate specific antigen pVIII Major bacteriophage coat protein PyMT Polyoma virus middle T antigen RA Rac1 Ras-related C3 botulinum toxin substrate 1 RAGE Receptor for advanced glycation end products RCC Renal cell carcinoma RGD Arginine-Glycine-Aspartic acid, Arg–Gly–Asp RIP Rat insulin promoter RIT Radio- scFV Single-chain FV fragment SCID Severe combined immunodeficiency SEM Scanning electron microscope SIP Small immunoprotein SMC Smooth muscle cells SR protein Serine- and arginine-rich protein SV Simian virus T-cell Thymus-lymphocyte; T-lymphocyte; CD8+ T-cell/lymphocyte T241 T241 fibrosarcoma Tag T-antigen TAM Tumor-associated macrophage TCR T-cell receptor TGF-β Transforming growth factor-beta Th-cell T-helper cell/lymphocyte; CD4+ T-cells/lymphocyte TIMP Tissue inhibitor of matrix metalloproteinase TKI Tyrosine kinase inhibitor TLR Toll-like receptor TNC Tenascin-C

TNCC C-domain of tenascin-C TNFα Tumor necrosis factor α Treg Regulatory T-lymphocyte TSP Thrombospondin tTF Extracellular soluble domain of tissue factor TUR Transurethral resection V Antibody variable region VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor VLA-4 Very late antigen 4 VPF Vascular permeability factor W/O water-in-oil WBRT Whole brain radiation treatment ΤRX Thioredoxin

Introduction

CANCER Cancer is after ischemic heart the most common cause of death in the Western world and it is the third leading cause of death in the developing countries1-3. The most common types of cancer diagnosed in 2009 in Sweden were prostate cancer and breast cancer1. Over the last two decades the recorded cancer has slightly increased. Explanations for this are better screening programs and diagnostics that detect more cases as well as increasing age of the population. However, due to better treatment alternatives it is now possible to cure the disease or prolong survival drastically depicted for example by the fact that the relative 5-year survival has increased from 30% to 70% over the last 40 years in Sweden1. In North America, Sweden and Japan 80% of women diagnosed with breast cancer survive the disease due to optimized treatment compared to 60% in middle- income and below 40% in low-income countries4,5. However, once the disease has disseminated (metastasized) treatment is difficult and often can only prolong survival but not cure the disease. The formation of a tumor is characterized by uncontrolled cell growth. A tumor can be noninvasive (benign) or invasive (malign). Noninvasive tumors do not grow into the surrounding tissue and usually do not interfere with normal tissue and organ function, whereas malignant tumors invade the surrounding tissue and disturb it. Tumor cells become more malignant by successively gaining new capabilities in the multistep tumor growth pathogenesis. These capabilities defined as the hallmarks of cancer are suggested to include: sustained proliferative signaling, evasion of growth suppression, resistance to cell death, enabling of replicative immortality, induction of angiogenesis, reprogramming of energy metabolism, escape of immune destruction and activation of tumor invasion and metastasis. Genome instability generates genetic diversity, which is the cause of malignancy and inflammation6,7. Malignant tumors can eventually develop metastases, when the cancer cells are spread to distant organs enabling development of numerous new tumors within the body. Metastatic disease is more difficult to treat than a localized primary tumor and therefore accounts for 90% of all cancer-associated mortality8. Metastatic cancer cells need to acquire several new traits in order to be able to invade and settle into a new tissue environment. Obstacles that need

13 to be tackled by metastatic cancer cells are invasion of local tissue environment, intravasation (leaving the tissue and entering into the circulation), to survive shear stress and immune attacks, transport through the body, extravasation (entering new tissue via penetration of the blood vessel wall), micrometastases formation and colonization of the new milieu9,10.

Causes of cancer/risk factors Factors contributing to the development of cancer are heredity, increasing age and living conditions (physical environment and lifestyle)11. Increasing age is a risk factor for cancer since with the accumulating number of cell divisions during a human lifetime there is a higher chance for mistakes (mutations) to occur. Environmental factors have been found to act tumor promoting by induction of mutagenesis or proliferation. Some environmental factors that have been connected to cancer are virus (hepatitis B and C or human papilloma virus (HPV)), exposure to carcinogens or agents that cause chronic or infections. Chronic inflammation is believed to be the cause of 15-20% of all malignancies worldwide. Lifestyle factors like use of tobacco, unhealthy diet and physical inactivity were also found to be risk factors for cancer development11-14.

Tumor composition The majority (80%) of cancers and the most well studied tumor type are carcinomas, which are derived from epithelial cells11,15. In general a tumor can be seen as a separate ‘organ’ that in addition to tumor cells also consists of stroma. The tumor stroma contains immune cells such as , neutrophils and T-cells; mesenchymal cells e.g. matrix synthesizing , extracellular matrix (ECM) and blood vessels16. Tumor growth can also be described as a process of continues inflammation as compared to sterile self-limiting inflammation observed in wound healing. Therefore a tumor is said to resemble a ‘wound that does not heal’17. Keratinocytes (epithelial cells) in a wound undergo partial epithelial mesenchymal transition (EMT) and gain a mitogenic and motile phenotype to mediate wound closure. Tumor cells however undergo full EMT and adapt an invasive phenotype by losing their cell-cell contacts, obtaining fibroblast- like morphology and expressing mesenchymal proteins9,18-21. Tumors also contain a matrix of fibrin and fibronectin similar to a clot in wounds17. However, in tumors the clot persists due to constant vessel leakiness, whereas vascular hyperpermeability in wounds is transient. The presence of inflammatory cells in a tumor promotes further tumor growth and progression. Since these cells release mediators that maintain inflammation18,22,23. Similar growth factors are found in tumors as are

14 involved in the process of wound healing such as vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-β)18,24-26. A tumor requires blood vessels to sustain growth and therefore turns on angiogenesis, the formation of new blood vessels from preexisting ones27. In a healing wound, angiogenesis is tightly regulated and transient, instead of continuous as in a tumor. Carcinomas contain fibroblasts and myofibroblasts due to release of PDGF, a fibroblast mitogen, by activated platelets and tumor cells26. In a wound these mesenchymal cells produce ECM, growth factors and proteinases for fibrin matrix degradation18,28. Similar functions have been found for these cells in cancer18.

THE VASCULAR SYSTEM As pointed out previously tumors do not only consist of tumor cells but they also contain blood vessels and stroma cells. All cells, normal cells as well as cancer cells, need oxygen and nutrients to survive and proliferate. Cancer cells, however, are extremely fast growing compared to normal cells. This will lead to an increasing consumption of oxygen and nutrients and in order to satisfy this demand tumors recruit blood vessels6,7. The vascular system is also called the circulatory system and was first described in detail by William Harvey in the year 162829. It consists of the cardiovascular system that distributes blood and the lymphatic system that transports lymph in the body. Its function is to provide the body with oxygen and other gases, nutrients, hormones, to control homeostasis, pH and body temperature as wells as to fight diseases. The cardiovascular system includes the pulmonary circulation and the systemic circulation, which are connected by the heart. In the lungs the blood is oxygenated and then it is pumped through the arteries by the heart to be distributed within the body. The exchange of oxygen, nutrients and waste products between blood and tissue occurs in the capillaries via diffusion through the interstitial fluid. The transport of de-oxygenated (carbon dioxide rich) blood, back to the heart, occurs via venules and veins. In the heart it enters the pulmonary circulation where it will be re-oxygenated30. The lymphatic system consists of a network of lymph vessels carrying the lymph, lymphoid tissues (e.g. lymph nodes) and structures involved in the production and circulation of (e.g. bone marrow, spleen and thymus). It collects excessive interstitial fluid from the tissues and drains it into the vascular system. Additionally it absorbs and transports fatty acids and fat as chyle to the circulatory system and moves immune cells to and from the lymph nodes30,31. Arteries, arterioles, veins and venules are composed of endothelial cells and smooth muscle cells (SMCs). The SMCs provide vessel stability and regulate the blood pressure via arteriolar contraction. The endothelial cells

15 form the vessel lumen and are in contact with the blood. The capillaries are responsible for the exchange with the surrounding tissue and therefore their composition is different from other blood vessels. Capillaries lack SMC coverage and are instead stabilized by pericytes (Figure 1). The pericytes are attached to the endothelial cells and the basement membrane at the abluminal side of the vessel32. All blood vessels are surrounded by ECM, to which the cells in the tissue adhere.

pericyte EC

Vascular Blood Basement vessel Membrane lumen Figure 1. Capillary. Endothelial cells (EC) (gray) form the vessel lumen, which is surrounded by basement membrane and pericytes (black)32.

Angiogenesis The vascular system develops de novo during embryogenesis and this process is called vasculogenesis. Vasculogenesis is genetically programed, and vessels arise from endothelial cells that originate from progenitors derived from the mesoderm33-35. Blood vessels can also develop from preexisting vessels, a process called angiogenesis27. The most well studied types of angiogenesis are sprouting and intussusception (the splitting of a mother vessel)36,37. Other proposed mechanisms for angiogenesis are involving circulating endothelial progenitor cells and looping38-40. Sprouting angiogenesis is initiated by a VEGF-A gradient, which triggers the formation of a tip cell. Local release of proteases such as matrix metalloproteinases (MMPs) by the endothelial cells and other cells causes ECM degradation and creates space for the cells to migrate41-46. The tip cell then develops filopodia to sense its surroundings and to be able to navigate and migrate. Stalk cells, which form a firm cord, will follow the tip cell and proliferate to elongate the sprout. Subsequently, the tip cell will fuse with other tip cells and a vessel will be formed that will be perfused. The vessel will mature and pericytes will be attracted to stabilize it. The process of sprouting is terminated by quiescence of the vessel47. During intussusception, a process believed to be of importance during vessel remodeling, transluminal pillars (holes) appear that split the vessel. In contrast to sprouting angiogenesis, blood flow is maintained throughout the

16 whole splitting event. Contractile periendothelial cells, macrophages and blood flow are all believed to be involved in this process48. Angiogenesis is a rare event in the healthy adult but takes place under physiological conditions like wound healing and the female menstrual cycle, in which it is tightly regulated by a balance between pro- and anti- angiogenic factors35,49. However, under pathological conditions excessive or insufficient angiogenesis can occur. Conditions characterized by excessive angiogenesis are inflammatory diseases (e.g. rheumatoid arthritis), tumor growth and diabetic retinopathy. Insufficient angiogenesis occurs in ischemia and might also be connected to ulcers in the digestive tract as well as infertility35. A large number of endogenous factors regulating angiogenesis both in a positive and negative manner have been described. Some examples of positive regulators (pro-angiogenic factors) are VEGF, PDGF, fibroblast growth factor (FGF), placental growth factor (PlGF), angiopoietin and toll- like receptors (TLRs)49-55. Negative endogenous regulators (anti-angiogenic factors) are amongst others thrombospondin-1 and -2 (TSP-1, TSP-2), tumstatin, endostatin, angiostatin and histidine-rich glycoprotein50,56-60.

Vascular endothelial growth factor The most extensively studied pro-angiogenic factor is VEGF, which was first discovered under the name vascular permeability factor (VPF)61,62. The mammalian VEGF family consists of five different isoforms (VEGF- A, -B, -C, -D and PlGF), which exert their effect through binding to different VEGF receptors. The VEGF receptor-family of tyrosine kinase receptors includes three different receptors (VEGFR-1, VEGFR-2 and VEGFR-3)51. Upon binding of VEGF these receptors form hetero- or homodimers, which leads to autophosphorylation of their intracellular domain inducing a signaling cascade. VEGF-A exerts its pro-angiogenic effect through the VEGFR-2 and is involved in endothelial cell survival, mitosis and motility as well as vascular permeability51,63-65. There are five different splice variants of the human VEGF-A monomer, of which VEGF-A165 is most abundantly expressed63. VEGF-A is essential for development of the vascular system since mice lacking only a single Vegf-a allele die of vascular defects around embryonic day 1162,66. VEGFR-2-/- mice have a phenotype analogous to VEGF-A-/- mice and die around embryonic day 8.5 due to vascular defects67. The VEGFR-1-/- phenotype is characterized by excessive endothelial proliferation and occlusion of the vessel lumen and mice die around embryonic day 8.5-9. These observations indicate that VEGFR-1 is a negative regulator of angiogenesis, at least during development63,68. VEGFR- 3 is present on lymphatic vessels and stimulation of the receptor by its ligands VEGF-C and VEGF-D promotes development of lymph vessels from preexisting ones a process defined as lymphangiogenesis31.

17 Angiogenesis mediated via endogenous toll-like receptor ligands It is now clear that angiogenesis also can be induced by mechanisms that are independent of VEGF and some of these pathways are regulated by TLRs, pattern-recognition receptors (PRRs) involved in innate immunity. One example is oxidative stress promoted by myeloid cells in wound healing, inflammation and tumors that causes an accumulation of oxidative products derived from phospholipids in the , such as carboxyethylpyrrole (CEP) and other related pyrroles. CEP was found to bind to TLR2 on endothelial cells69. CEP-induced signaling through TLR2 in endothelial cells and activated ras-related C3 botulinum toxin substrate 1 (Rac1), a protein that mediates cell signaling. Activation of Rac1 promotes endothelial cell migration via integrins and hence angiogenesis69-71. Another example is the release of the protein high-mobility group B1 (HMGB1) from necrotic cells, which was shown to signal through the receptor for advanced glycation end products (RAGE), TLR2 and TLR4. Activation of these receptors induced upregulation of angiogenic factors such as VEGF in hematopoietic and endothelial cells via activation of nuclear factor κB (NF-κB)72.

The angiogenic switch Most tumors remain dormant (1-2 mm3 in size) and do not develop into an aggressive phenotype73. In 1971 Judah Folkman postulated that this might be due to the fact that angiogenesis is lacking to provide the tumor with sufficient nutrients and oxygen in order to grow larger74. One critical step in the progression of tumor development is therefore called the ‘angiogenic switch’. Hypoxia, hypoglycemia and genetic as well as inflammatory alterations are believed to trigger the angiogenic switch75-79. These triggers might induce the expression of oncogenes in tumor cells that can downregulate anti-angiogenic factors such as thrombospondin-1. But the triggers might also activate the overexpression of pro-angiogenic factors in tumor cells or in the by the tumor recruited inflammatory cells, such as macrophages and mast cells50,51,53,55-57,80,81. Eventually there will be a misbalance between pro- and anti-angiogenic factors in favor of the pro- angiogenic factors, which tilts the balance towards the development of tumor blood vessels resulting in a vascular and eventually metastatic tumor type82,83.

Tumor vessels The blood vessels in a tumor are leaky/highly permeable due to an excess of VEGF-A produced by the tumor cells or inflammatory cells recruited by the tumor. The activated tumor also overexpresses several proteins and/or receptors such as for example VEGFR-2 or survivin, an anti-apoptotic protein77,84,85, promoting an angiogenic phenotype and enhanced vessel

18 growth. Tumor vessels also show reduced pericyte coverage and partial loss of endothelial cells52,86-88. All these features lead to formation of a malfunctioning disorganized and chaotic tumor vasculature in which hierarchy is lost and perfusion is impaired. This can be seen in Figure 2 in which tumor vasculature and normal vasculature are depicted.

Figure 2. Normal vasculature and tumor vasculature. The left panel depicts normal organized microvasculature and the right panel disorganized tumor microvasculature, in which vessel hierarchy was lost. Images of polymer casts were taken in a scanning electron microscope (SEM). Reprinted with permission from the Nature Publishing Group: Nature Medicine89, copyright 2003.

TREATMENT OF CANCER In addition to the actual tumors cells, cancer consists of different types of cells, extracellular matrix and blood vessels, which all represent potential targets for tumor treatment. Classical cancer treatment is surgery (tumorectomy) to remove the tumor if possible combined with chemotherapy and/or radiotherapy as additive therapy to prevent recurrent tumor growth. In case the cancer cannot be removed chemo- and radiotherapy is used as curative therapy or for palliative care to relieve suffering. Surgery, chemotherapy and radiotherapy are until today the most widely used and most successful methods for cancer treatment. A side effect of chemo- and radiotherapy is the damage to healthy tissue that these agents cause, because they will also affect normal tissue. During recent years, new treatment options have become available and are also under development. One group of drugs used in the clinic are tyrosine kinase inhibitors (TKIs) that inhibit growth factor receptors on tumor blood vessels and tumor cells90-92. Monoclonal antibodies (MAbs) have been developed for the treatment of cancer as well. These monoclonal antibodies target either VEGF93,94, which is overexpressed in tumors to

19 promote tumor angiogenesis, or tumor antigens (HER2/Neu; ErbB2 receptor) or growth factor receptors (EGF-R, HER1)95-97. Another way of targeting tumor tissue is by immunotherapy. Different strategies are used and there are a number of therapies in preclinical development. Priming of dendritic cells with tumor-associated antigens or proteins/receptors overexpressed on the tumor endothelium, and adoptive T- cell transfer are examples of approaches used. These different strategies will be described in more detail below.

Chemotherapy The chemical warfare mustard gas was found to suppress hematopoiesis and thus cell growth. In 1942 the related agent ‘nitrogen mustard’ was used as the first chemotherapeutic drug to treat a cancer patient with non-Hodgkin’s lymphoma98. Over 20 years later the first combination of different chemotherapeutic drugs was administered to patients with acute lymphoblastic leukemia (ALL)99. However, until 1975 the genetic and biochemical mechanisms of cancer pathogenesis were still unidentified100, which made target-specific treatment impossible. Since the introduction of the first chemotherapeutic drug many new drugs have been developed. The general mechanism of these agents is the inhibition of cell proliferation. Tumor cells are highly proliferative and are therefore more sensitive to chemotherapeutics than normal cells. In Table 1 the different classes of chemotherapeutic drugs used in the clinic are listed.

20 Table 1. Chemotherapeutic drugs used as conventional chemotherapy in the clinic101

Agent group Mechanism of action Drug substance Alkylating agents Crosslinking of DNA strands by CLASSICAL: alkylation of purine bases, which cyclophosphamide, melphalan leads to apoptosis. PLATINATING: cisplatin, carboplatin Antimetabolites Interfere with DNA and/or RNA ANTIFOLATES: methotrexate synthesis. PURINE ANALOGS: fludarabine, gemcitabine THYMIDYLATE SYNTHASE INHIBITORS: fluorouracil, capecitabine Topoisomerase Topoisomerase I inhibitors interfere TOPOISOMERASE I: inhibitors with DNA unwinding and strand topotecan, irinotecan passage. TOPOISOMERASE II: Topoisomerase II inhibitors etoposide, doxorubicin interfere with DNA replication, and repair. Taxanes Promote microtubule assembly; Paclitaxel, docetaxel antimitotic Vinca alkaloids Inhibit microtubule polymerization; Vincristine, vinorelbine antimitotic Mixed bag DNA intercalating agents; ANTIBIOTICS: streptozotocin, L-asparaginase catalyzes the Anthracycline antibiotics: hydrolysis of asparagine to aspartic doxorubicin, daunomycin acid (ALL leukemic cells are not ENZYMES: L-Asparaginase able to produce asparagine).

All these therapeutic agents are given as high-dose chemotherapy (at the maximum tolerated dose (MTD)) to avoid drug resistance101,102. This is often accompanied by severe side effects caused by immunosuppression such as anemia, neutropenia or thrombocytopenia, which have to be treated concurrently103. Therefore, low-dose metronomic chemotherapy was developed, which is the continuous oral administration of conventional chemotherapy at relatively low, minimally toxic doses104-106. Metronomic treatment has shown promising results in phase II clinical trials and is now evaluated for use in phase III clinical studies107,108. Low-dose chemotherapy has been shown to activate the immune system rather then to suppress it as well as to inhibit tumor angiogenesis13,103,108,109.

Radiotherapy Radiotherapy has been used for more than a century to treat cancer. Its mechanism of action is to damage cellular DNA by ionizing radiation. This type of radiation harms the cells directly by photons or electrical charged particles or by formation of free hydroxyl radicals due to ionization of water in the body110. It is either used as curative, additive (together with

21 chemotherapy) or palliative cancer treatment. Radiotherapy is used for local treatment of tumors to avoid damage of healthy tissues.

Tyrosine kinase inhibitors Targeted therapy The mechanism of action of these drugs is the inhibition of the tyrosine kinase activity of receptor tyrosine kinases. In tumors the tyrosine kinase signaling pathways are often overactive and promote tumor growth and angiogenesis. Small molecule tyrosine kinase inhibitors used in the clinic that target multiple kinases are sorafenib (Nexavar ®), sunitinib (Sutent ®) and imatinib (Gleevec ®). Sorafenib blocks preferentially VEGFR-1, VEGFR-2, VEGFR-3, PDGF receptor β (PDGFRβ), and Raf kinases90,111,112. Sunitinib mainly inhibits VEGFR-2, PDGFRα and PDGFRβ, the stem cell factor receptor (KIT), fms-related tyrosine kinase 3 (FLT-3), colony- stimulating factor 1 (CSF-1) and rearranged during transfection kinase (RET)113-117. Imatinib preferentially targets the BCR-ABL kinase involved in the pathogenesis of chronic myelogenous leukemia (CML), KIT and PDGFRβ118,119. Since tyrosine kinase activity is required for tissue homeostasis treatment with TKIs is often accompanied by side effects. Hypertension is for example frequently seen in patients treated with sorafenib or sunitinib. Occurrence of hypertension is used as a measure of treatment effectiveness and related to prolonged disease survival120-122. Recently it was shown that sunitinib related hypertension is caused by concurrent inhibition of the PDGFR on pericytes in the heart vasculature123.

Monoclonal antibodies Over the past decade several MAbs have been developed for treatment of cancer and other diseases. Examples of monoclonal antibodies used as anti- cancer drugs in the clinic are bevacizumab (Avastin ®), a VEGF-A binding antibody93,94,124, trastuzumab (Herceptin ®), an anti-HER2 antibody125, cetuximab (Erbitux ®), an epidermal growth factor receptor (EGFR; HER1) blocking antibody126,127 and rituximab (Rituxan ®), an anti-CD20 (cluster differentiation 20) antibody128-131. Trastuzumab binds to the HER2/Neu (ErbB2) receptor, a tyrosine kinase receptor frequently overexpressed on solid tumors96. HER2 heterodimerizes with EGFR and stimulation of this receptor by its ligand epidermal growth factor promotes cell survival, differentiation, invasion and angiogenesis97,132. HER2 can also act as an oncoprotein, which does not need activation by its ligand but instead has constitutive kinase activity. Tumors overexpressing HER2 have a more aggressive phenotype and are more difficult to treat133,134. Cetuximab directly binds to the EGFR and blocks the binding site of the EGF-ligand thereby

22 preventing receptor signaling126. Rituximab for example is used to treat chronic lymphocytic leukemia (CLL). CLL is characterized by accumulation of monoclonal B-lymphocytes (B-cells) in the blood, secondary lymphoid tissues and bone marrow. Binding of rituximab to CD20, a tetraspan phosphoprotein expressed on the surface of B-cells, leads to elimination of all B-cells via antibody dependent-cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and direct induction of apoptosis128,129. Monotherapy with the anti-VEGF-A antibody (Avastin ®) has shown to be ineffective for cancer treatment in humans and only in combination with conventional chemotherapy an overall survival benefit in colorectal and lung cancer patients could be achieved35,135-139. One explanation for this observation is that anti-VEGF-A therapy does not strangulate the tumor but instead normalizes the leaky tumor vasculature. This leads to a better delivery of chemotherapeutic drugs into the tumor tissue and a greater inhibitory effect on tumor growth83,140. A monoclonal antibody in clinical trials is the anti-CTLA4 (cytotoxic T- lymphocyte antigen 4) antibody. CTLA4 is a molecule expressed on the surface of activated T-cells by which the extent of the T-cell (T-lymphocyte) response can be regulated. It competes with CD28 for B7 (CD80/CD86) binding (a co-stimulus needed for T-cell activation; see paragraph self- tolerance) and transmits inhibitory signals to shut down T-cell activation141,142. Blocking of CTLA4 shuts down this negative-feedback control loop and was found to enhance antitumor T-cell responses in cancer97,143,144. The anti-CTLA4 antibody has been shown to give significant treatment responses in patients with cancer although at the expense of severe autoimmune side effects, such as colitis or hepatitis13,145-148.

Immunotherapies Three general aspects, which complicates development of a tumor vaccine are firstly the frequently occurring downregulation of major histocompatibility complex I (MHC-I) by tumor cells, which makes them less prone to a cytotoxic T-lymphocyte (CTL) attack149,150. Secondly, tumors + + commonly cause upregulation of regulatory CD4 CD25 T-cells (Tregs) suppressing the host’s immune system151,152. Thirdly cancer patients are often immunocompromised because of chemotherapy, rendering therapeutic vaccination ineffective103. One way to circumvent some of the above problems is to induce a humoral immune response, involving CD4+ lymphocytes, in which endogenous antibodies are produced that directly bind to their target. This would create a more specifically directed immune response independent of MHC-I expression on tumor cells.

23 Preventive tumor vaccines The only example of a prophylactic tumor vaccine in clinical use is the recombinant for prevention of cervical cancer caused by HPV. Today there are two HPV vaccines, ® and ®, available on the market, which both protect against the most common HPV types (HPV-16 and HPV-18) causing cervical cancer153,154. HPV is a foreign antigen, which makes vaccination against it relatively simple, since it will be recognized by the body’s immune system as foreign. However, it is more difficult to vaccinate against tumor-associated antigens because these are self-proteins and the body has developed several mechanisms to avoid an immune responses against self-molecules. These mechanisms include that autoreactive T-cells are removed or inactivated during development and thus prevent endogenous tissue in the body from being attacked by the immune system, a phenomenon referred to as self-tolerance (see paragraph self- tolerance)155.

Therapeutic tumor vaccines Therapeutic cancer vaccines can be divided into cell-based vaccines and antigen-based vaccines. Cell-based vaccines make use of cell transfer to induce specific tumor immunity. These types of vaccines include whole cells for example allogeneic (genetically similar; from the same species) cancer cells, peripheral-blood mononuclear cells (PBMCs) activated with tumor- associated antigens or adoptive T-cell transfer. Antigen-based vaccines aim at novel presentation of peptide/protein antigens and methods to enhance the endogenous immune response. These vaccines make use of fusion proteins, whole bacteria or DNA. Proteins or DNA can either be injected directly to evoke an immune response or delivered within a vector to avoid breakdown and give additional immune stimulation. Vectors used are modified viruses (adenovirus or poxvirus of which vaccinia virus is the most widely used), bacteria both alive and attenuated (Salmonella, Mycobacterium, Listeria or Shigella)152 or yeast (nonpathogenic Saccharomyces cerevisae). The bacteria here listed are all intracellular bacteria, which have the ability to target antigen-presenting cells (APCs) directly and depending on their location in the APC they interfere with distinct antigen-presenting pathways (MHC class I or MHC class II)156. The therapeutic vaccines and their mechanism of action are listed in Table 2.

24 Table 2. Different therapeutic vaccines/ used in the clinic* and in preclinical development. Antibody (Ab), neutral killer (NK) cell, dendritic cell (DC)

Vaccine type Antigen Mechanism Name ANTIGEN-BASED VACCINES Protein Tumor-associated Ab formation, FUSION PROTEIN: carbohydrates T-cell response KHL- carbohydrate157, 158 FLK1-AP (pulse DCs by intradermal )159 Whole bacteria Bacterial antigens Immunostimulation TheraCys ®*160 by , CTL and Ab response DNA Tumor-associated CTL response and pDERMATT161 antigens, antigens or Ab response pVAX-DLL4162 on tumor depending on the pcDNA3.1-FLK1163 vasculature delivery vector used CELL-BASED VACCINES Allogeneic whole cancer All tumor cell T-cell response, NK 164, 165 cells antigens cells, macrophages, eosinophils APC (DC) vaccine Tumor-associated Activation and Sipuleucel-T*166 antigens maturation of DCs to evoke an CTL response Adoptive T-cell transfer Tumor-associated CTLs response Selection, antigens generation and activation of anti- tumor T-cells167-169

Cell-based vaccines Recently (in the year 2010) the American Food and Drug Administration (FDA) approved the first therapeutic cancer vaccine Sipuleucel-T (Provenge ®) for clinical use170,171. The FDA approval was based on the results of a randomized placebo controlled phase III clinical171. In this study treatment

25 with Sipuleucel-T gave an antigen-specific cellular and a humoral immune response and showed a 4.1-month median survival benefit and an 8.7% extended 3-year survival in patients with asymptomatic or minimal symptomatic disease compared to placebo172-174. The vaccine is an autologous active cellular immunotherapy used for treatment of metastatic castration resistant prostate cancer. In this method autologous peripheral- blood mononuclear cells, including APCs, which are dendritic cells (DCs) and macrophages, are removed from the patient and stimulated in vitro with a prostatic acid phosphatase-granulocyte macrophage-colony stimulating factor (PAP-GM-CSF) recombinant fusion protein (PA2024). In this fusion protein a prostate antigen (prostatic acid phosphatase) is coupled to GM- CSF, an immunostimulatory agent. The activated APCs are then placed back into the patient and will promote a CTL immune response against the prostate tumor cells. Sipuleucel-T is given as a 60-minute infusion of minimal 50 million activated autologous CD54+ cells (CD54 is a surrogate marker for activated APCs) every two weeks for three doses166,175-178. Another strategy under development used for the induction of an antitumor CTL response is adoptive T-cell transfer. By this method large numbers of antitumor T-cells, which specifically recognize tumor antigens, can be selected, generated and activated in vitro and transferred back into the host167,168. Allogeneic whole cell vaccines have also been tested in clinical trials with encouraging results164,165. Here irradiated whole cancer cells from another host with the same tumor type are injected into the patient with the aim to evoke an immune response against the multiple antigens expressed by these cells. Whole tumor cells are poorly immunogenic and therefore additional immunostimulating agents have to be added to the vaccine.

Antigen-based vaccines TheraCys ® consists of Bacillus Calmette-Guérin (BCG), which is a live attenuated strain of . The whole bacterial vaccine is approved by the FDA for treatment and prophylaxis of carcinoma in situ (CIS) in the urinary bladder and prophylaxis of primary or recurrent stage Ta (noninvasive papillary carcinoma) and/or T1 (invasion of subepithelial connective tissue) papillary tumors following transurethral resection (TUR). The bacteria are administered into the bladder via a catheter (intravesical), which leads to immune stimulation caused by the bacterial infection160,179-182. A recombinant DNA vaccine for treatment of melanoma named pDERMATT (plasmid DNA Encoding Recombinant MART-1 and Tetanus toxin fragment-c), consisting of a DNA plasmid encoding melanoma associated antigen ‘melanoma antigen recognized by T-cells’ (MART-1) specific to the melanocyte lineage and an immunostimulatory tetanus toxin fragment-c, was successfully used for induction of a cytotoxic T-cell response against MART-1 in mice161. In this study only the

26 of the DNA vaccine was tested and no data are available on the effect of the vaccine on tumor growth in a mouse model. Another strategy used is carbohydrate-based vaccines, which consist of a protein carrier (e.g. keyhole limpet hemocyanin (KHL)) coupled to tumor- associated carbohydrate antigens157,158,183. A foreign protein carrier is used in this strategy to make the tumor-associated carbohydrate antigens, which are self-antigens, visible to the immune system184. In a phase I the safety of a tumor-associated carbohydrate antigen-KHL vaccine injected with a immunologic adjuvant was tested in patients with relapsed prostate cancer. Antibodies against the tumor antigen could be measured and a decrease in prostate specific antigen (PSA) could be observed185. Further vaccine approaches to target carbohydrates on tumor cells are under investigation186. Despite the numerous different therapeutic vaccines in preclinical development only two are clinically approved for cancer treatment. Rosenberg et al. evaluated the overall objective response rate to different types of antigen-based cancer vaccines, in patients with metastatic cancer, as 3.3%. Data in this study were obtained from trials performed at the Surgery Branch of the National Cancer Institute (NCI) and 35 other reports of clinical vaccine trials. The majority of therapeutic antigen-based cancer vaccines activate CD8+ lymphocytes (CTLs) initiating a cellular immune response against the tumor. A vaccine-induced cellular immune response is often insufficient since the achieved circulating levels of high avidity immune cells are too low. Furthermore, these cells have to reach their tumor target and must be activated correctly to be able to destroy it187,188.

Combination of chemotherapy or radiotherapy with immunotherapy Chemotherapy and radiotherapy are ways to activate the immune system and it might therefore be an advantage to combine these treatments with immunotherapy. In the beginning of the era of chemotherapy it was believed that treatment with chemotherapeutic agents was immunosuppressive due to its anti-proliferative and cytotoxic properties103. However, high-dose chemotherapy has also been found to have immunostimulating properties, which might be of advantage for combination treatment with immunotherapy. Many types of chemotherapeutic drugs cause lymphocytopenia, which can create a positive environment for expansion of tumor-specific CTLs189,190. Therefore chemotherapy in combination with a therapeutic CTL inducing vaccine might promote expansion of effector T- cells and enhance antitumor T-cell responses13. Since cancer suppresses the immune system of the host149 dendritic cells in the tumor tissue and lymph nodes are often immature. Mature DCs,

27 however are needed for proper CD4+ and CD8+ T-cell activation. This can be a hurdle for therapeutic vaccines that aim at induction of a CTL or antibody response for which proper DC and T-cell activation is essential. Furthermore, DC function is suppressed by myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), which are recruited by the tumor. Mechanisms of actions suggested for anthracycline and cyclophosphamide are the promotion of cancer cell death and thereby release of uric acid. The release of uric acid (crystalline uric acid) was proposed to activate DCs and to promote tumor rejection191. Proteins secreted by dying tumor cells are also believed to promote immunogenic cross-presentation due to interaction with the pattern-recognition receptor TLR4 on DCs13,192. Toll-like receptors alert the immune system and bind to -associated molecular patterns, which can be of microbial or endogenous origin. Activation of TLRs initiates both innate and adaptive immune responses193. Cross-presentation is the ability of one cell (the DC) to present antigens from another cell (e.g. a virus-infected or tumor cell) to activate antigen-specific T-cells194. It has also been shown that tumor apoptosis and/or necrosis induced by chemotherapy promotes the release of tumor-associated proteins and cross-presentation of these proteins by DCs195,196. Another effect attributed to anthracyclines is the stimulation of calrecticulin translocation to the cell surface. Calrecticulin is involved in the MHC-I processing pathway and its translocation enhances of cancer cells by dendritic cells and promotes their activation13,197. Cylcophosphamide was also found to promote a systemic release of type I interferon (IFN-α, IFN-β), which results in DC, T-cell and B-cell activation leading to an antitumor response13,198-200. Furthermore, chemotherapy is believed to delete regulatory T-cells, which are upregulated by the tumor, TAMs and MDSCs, alleviating tumor induced immune suppression13,195,196,201,202. Low-dose radiation as well as certain chemotherapeutic drugs may additionally alter the phenotype of tumor cells by upregulation of the expression of Fas (death receptor), MHC-I, ICAM-1 (intercellular adhesion molecule 1) and tumor-associated antigens. This will render the cancer cells more sensitive to a T-cell mediated attack by the immune system203-206. Finally, there are also studies that support that tumor cell death caused by radiotherapy can promote cross-presentation13,192,207.

VASCULAR TARGETS Tumor endothelial cells as targets The previously described vaccines all aim at destroying tumor cells directly, but tumor endothelial cells can also be used as targets. Tumor vessels are believed to be more genetically stable than tumor cells and are therefore

28 more prone to immune recognition35,150,208-210. This is in contrast to the transformed tumor cells that have developed mechanisms to avoid recognition by the immune system such as downregulation of MHC-I149. However, one should bear in mind that chromosomal abnormalities have also been discovered in endothelial cells of solid tumors211,212 and in glioblastomas endothelial cells have even been found to originate from cancer stem-like cells213,214. An advantage of targeting endothelial cells is that they line the vessel lumen and are therefore easily accessible for the immune system compared to tumor cells, which are located at distance from the vessels77. Furthermore, targeting of a single endothelial cell will have an amplified effect due to the inhibition of as many as 100 tumor cells215,216. The reason for this is that oxygen and nutrient supply is guaranteed by blood vessels and by this means one endothelial cell supports several tissue cells.

Anti-angiogenic vaccines targeting tumor endothelial cells A bacterial DNA vaccine targeting VEGFR-2 has been evaluated163. The vaccine contains attenuated Salmonella typhimurium transformed with murine VEGFR-2 (FLK1) encoding DNA (pcDNA3.1-FLK1). Immunization of mice prior to tumor challenge promoted a CTL response against VEGFR-2 and tumor growth was reduced in a murine melanoma, colon carcinoma and lung carcinoma model. Furthermore, a bacterial vector vaccine has been designed for targeting VEGFR-2 in a HER2/neu+ breast tumor mouse model. The vaccine consisted of Listeria monocytogenes containing a plasmid encoding for polypeptides from mouse VEGFR-2 fused to the microbial adjuvant listeriolysin-O. A strong antitumor CTL response was observed. However, tumor eradication in this model was not solely due to targeting of the tumor vasculature but was also dependent on epitope spreading to HER2/neu217. Li et al. engineered a FLK1- alkaline phosphatase (FLK1-AP) fusion protein to pulse dendritic cells. With this method antitumor activity was observed in a preventive vaccine approach, which was mediated by antibodies and CTLs159. Another anti-angiogenic DNA vaccine in preclinical development targets the endothelial tip cells in the tumor vasculature. The vaccine consists of an expression plasmid pVAX1 into which the human Notch ligand delta-like ligand 4 (DLL4) is inserted (pVAX1-DLL4)162. DLL4 regulates vessel sprouting and is present in the activated endothelium of malignant tissues218,219. The vaccine induced a humoral immune response against DLL4 and inhibited tumor growth in two different mouse mammary carcinoma models. A phase I clinical trial of combination therapy of gemcitabine and a human VEGFR-2 peptide vaccine in patients with metastatic non-resectable pancreatic cancer showed a CTL response in 61% of treated patients and a disease control rate of 67%. Patients in this trial developed immunological reactions at the injection site but no severe adverse effects could be observed220. A potential problem of targeting VEGFR-2 is that this receptor

29 is also expressed on normal vasculature and is needed for endothelial cell survival and vascular homeostasis221. Therefore it is surprising that no severe side effects were observed in a phase I clinical study when targeting VEGFR-2. However, no long-term side effects were investigated in this study.

Vascular antigens One group of molecules with a highly restricted expression pattern is the extra domains of splice variants of extracellular matrix molecules present in the neovasculature of tumors or tumor stroma.

Extracellular matrix All cells in the body are surrounded by extracellular matrix, a three dimensional network/meshwork of proteins, consisting of glycoproteins, proteoglycans and collagens222. Examples of glycoproteins within the ECM are fibronectin and tenascin-C. The ECM provides organs and tissues with stability and it is important for tissue and cell adhesion, cell growth, migration, embryogenesis, hemostasis and wound healing223,224. Cell adhesion to the matrix is mediated by integrin receptors, cell surface proteoglycans (such as syndecans) and glycoproteins (e.g. discoidin domain receptors)222,225,226. In the ECM many different growth factors are sequestered, which can be released and activated by protease cleavages under certain physiological or pathophysiological conditions leading to cell proliferation and migration, cell communication, tissue homeostasis or matrix remodeling223,227,228. Endothelial cells forming the lumen of blood vessels and capillaries are surrounded by a basement membrane (see Figure 1), which is a specialized type of ECM and mainly composed of laminin, collagen type IV, perlecan and nidogen proteins224,227,228. Three molecules with a highly restricted tumor vascular expression are the extra domain-B (ED-B) and extra domain-A (ED-A) of the extracellular matrix protein fibronectin and the C-domain of tenascin-C (TNCC). These antigens are highly expressed during embryonic development229-234 and around angiogenic vasculature and in the stroma of most solid tumors235-238 but are essentially undetectable in normal adult tissue. Exceptions are situations of physiological angiogenesis such as wound healing and the female menstrual cycle during which these extra domains also reoccur235,239,240. Thus the specific expression of these molecules in tumor tissue renders them excellent for tumor-specific targeting. The expression of vascular antigens is more restricted compared to VEGFR-2, which is present on all endothelial cells in the body and essential for blood vessel homeostasis221. Therefore targeting of these extra domains is likely to give fewer side effects since only the tumor vasculature is targeted and not all blood vessels.

30 Fibronectin Fibronectin (FN) is a plasma and ECM glycoprotein consisting of two identical 220-250 kDa subunits connected by two disulphide bridges forming a dimer241,242. Furthermore, fibronectin is located in the basement membrane of blood vessels, incorporated between endothelial cells and perivascular cells in vessel morphogenesis, where it provides vessel stability243. In humans there are 20 different isoforms and in rats and mice there are 12 fibronectin variants, which are formed by alternative splicing of the fibronectin mRNA244,245. Each monomer consists of three different types of homologous repeating domains, the type I, II and III repeat (see Figure 3A)242,244,245. Plasma fibronectin is essentially devoid of the type III repeats termed extra domain-A (EIIIA or ED-A) and extra domain-B (EIIIB or ED- B) (only < 1% of plasma fibronectin contains these repeats)246-248. Both ED- A and ED-B containing fibronectin are highly expressed during embryogenesis but undetectable in normal adult tissue249-251. However, the extra domains are re-expressed during physiological and pathophysiological angiogenesis235,239,240,249,250. Fibronectin including one or both of the extra domains is also called ‘oncofetal fibronectin’, which refers to its expression during tumor angiogenesis and embryonic development235,249,250,252. To date the function of these extra repeats is unknown but it is hypothesized that incorporation of ED-A and/or ED-B into the fibronectin molecule leads to a conformational change, which makes the RGD (Arg-Gly-Asp) cell-binding site accessible for integrin binding242,253,254. Except for alternative splicing cryptic sequences in the fibronectin can also be exposed by simple stretching or proteolysis of the fibronectin molecule255,256. Splicing of fibronectin mRNA is regulated by SR proteins (proteins rich in serine- and arginine), whose activity is controlled by SR protein kinase and PKB/Akt (protein kinase B)242,257-259.

31

Figure 3. Structure of a fibronectin and tenascin-C monomer. (A) Fibronectin monomer. The different types of homologies (12 Type I, two Type II and 15 Type III) are represented. The ED-A and ED-B fragment are excluded in the numbering of the Type III repeats. The splicing pattern for ED-A and ED-B is similar in all species, whereas the splicing of the IIICS region is species-specific (five variants in humans, three in rodents and two in chickens)242,260. (B) Tenascin-C monomer. The structure depicted contains an assembly domain, 14.5 epidermal growth factor like (EGF- like) repeats, 16 type III homology repeats, and a fibrinogen globe232,260-262.

Extra domain-B The extra domain-B of fibronectin is a 91 amino acid domain, which is highly conserved between species and identical in mouse, rat, rabbit, dog, monkey, human and several other species260,263,264. Due to its high conservation it was believed that in vivo generation of antibodies targeting ED-B would be difficult or even impossible. ED-B is incorporated into fibronectin by alternative splicing during embryogenesis, tumorigenesis and angiogenesis229,235,240,242,250,251,265. Despite the high conservation of the extra domain-B, its regulation and function is still unknown. Both ED-A-/- and ED-B-/- mice are viable266 but double knock out mice (ED-A-/- and ED-B-/-) are 80% lethal on a C57BL6 background243,267. ED-B-/- mice have a similar phenotype as wild type mice. However, fibroblasts derived from these mice showed diminished proliferation and less fibronectin deposition in the pericellular matrix in vitro251,266. Transforming growth factor beta (TGF-β) has been found to regulate the splicing of the extra domain-B240,268-271. Other regulators suggested are high glucose levels268 and reduced intracellular pH240, one possible effect of hypoxia. Fibronectin containing the extra domain-B is expressed around the vasculature of most solid tumors or in the tumor

32 stroma241. Examples of ED-B expressing tumors are lung squamous carcinoma, lung alveolar adenocarcinoma, glioblastoma multiforme and invasive ductal breast carcinoma235,240,272. The expression of ED-B containing (EDB+) fibronectin is tumor type dependent and several different cell types such as endothelial cells, tumor cells or tumor-associated myofibroblasts have been found to produce this isoform273-275. The increased permeability of tumor vessels is believed to enhance the accessibility of ED- B from the blood stream276,277. Wagner et al. suggest an immunological role for ED-B and found ED-B containing fibronectin to be synthesized by activated T-cells and to be associated to their surface in vitro. They propose that EDB+ fibronectin binds to very late antigen-4 (VLA-4) on adjacent T- cells and thereby provides a co-stimulus for T-cell proliferation278. In early studies the monoclonal antibody BC-1, which recognizes human ED-B+ fibronectin was used to analyze ED-B expression in tissue. This antibody does not recognize ED-B directly but instead a cryptic epitope of 253 the FN type III7 repeat, which is unmasked by insertion of ED-B . An additional cryptic sequence on the FN type III8 was discovered recently and a high-affinity murine monoclonal antibody C6, specific for ED-B+ fibronectin is available279. Bencharit et al. solved the crystal structure of the interface between the ED-B domain and the adjacent FN type III8 repeat in 2007. The crystal structure revealed an acidic groove between the two repeats rendering it a potential binding site for fibronectin interacting molecules280. Thus the insertion of ED-B into fibronectin leads to conformational changes in the fibronectin molecule, which unmasks hindered sequences but in turn masks others253,279. Peters et al. determined in 1996 the expression pattern of total fibronectin, ED-B+ fibronectin and ED-A containing (ED-A+) fibronectin in the adult mouse246. In this study they found ED-B+ fibronectin expression in the walls of smaller blood vessels, in the smooth muscle of the gastrointestinal tract (GI), genitourinary (GU), and respiratory tracts, as well as in cartilage and the eye. Tissue staining was performed with anti-ED-B antibodies raised against an EDB-GST (glutathione-S-transferase) fusion protein and the tissue was treated with N-glycanase (Peptide: N-glycosidase F, PNGase F) to remove N-linked oligosaccharides on Asn1359 from ED-B231,248. Expression of ED-A+ fibronectin was detected in vessel walls, the lung interstitium, and smooth muscle associated with the GI, GU and respiratory tracts of mouse when staining with a human anti-ED-A antibody reactive to mouse tissue 246. However, several others could not confirm the expression of these segments in normal tissue241,250,281,282. To date one monoclonal anti-ED-B antibody, termed L19, has been developed for clinical use by the phage display technique in which binding to the ED-B fragment was screened.

33 Antibody phage display technique The antibody phage display technique, which was first described by McCafferty in 1990, is based on the expression of antibody variable (V) region fragments on the surface of bacteriophages283. For antibody development the immunoglobulin variable genes are amplified from hybridoma- or B-cells with the help of the polymerase chain reaction (PCR) and cloned into a phage expression vector containing bacteriophage virus genes. Bacterial cells are then infected with the bacteriophages, which will replicate in the host but are unable to assemble their coat since they are devoid of the genes necessary for assembly. However, insertion of the phage expression vector into the bacterial host will enable packaging of the phage DNA and the assembly of their coat containing the antibody fragment of interest as part of either the minor (pIII) or major coat protein (pVIII). By this means a phage display library can be generated. The specific clones are then selected upon the ability of the expressed surface antibody fragment to bind to antigen-coated wells in an enzyme-linked immunosorbent assay (ELISA) or to an antigen-coated column. The desired bacteriophage clone can then be amplified in E.coli. This process of selection is called ‘panning’ and usually 2-4 rounds are needed to select for a high affinity antibody. The antibody fragments derived from the phage display will be secreted into the bacterial periplasm and culture medium from which they can be purified further264,284. The diversity of antibodies generated in a phage display is only around 1012, whereas the human body can generate a much higher variety of 1015-1016 by rearrangement and somatic mutation285. Generation of a specific high affinity antibody by the phage display technique can therefore be hard to achieve. For production of monoclonal antibodies the genes encoding the specific antigen-binding site can be isolated from the phage DNA and used for the construction of a complete immunoglobulin (Ig) gene by fusing them with the antibody’s invariant parts. The constructed antibody gene can then be inserted into hybridoma cells, which then will secrete monoclonal antibodies286-288.

Monoclonal anti-ED-B antibodies The monoclonal phage antibody L19 is a 150 kDa IgG1 antibody, which recognizes ED-B. In addition derivatives of this antibody have been produced, which are a dimeric single-chain FV(scFV) 50 kDa fragment (two antibody variable (V) regions of an IgG fused) and a small immunoprotein (L19-SIP) (80 kDa). The L19 small immunoprotein consists of two scFV fragments each fused to a CH4 domain derived from human IgE that mediates their homodimerization (Figure 4)264,282,289.

34

Figure 4. L19 antibody derivatives (adapted from289). (A) The L19-IL2 construct consists of an scFV fragment fused to the cytokine IL-2. (B) Single-chain FV dimer. (C) L19-SIP is composed of two scFV fragments fused each fused to a CH4 domain derived from human IgE that mediates their homodimerization264,282,289.

To date there are many publications available, in which targeting of ED-B with the help of the L19 antibody coupled to different effector molecules has been proven to be a successful anti-cancer strategy in preclinical models and in clinical studies. In a study performed by Borsi et al. L19 coupled to tumor necrosis factor (TNF) α induced tumor necrosis290. The L19-TNFα (monoclonal antibody cytokine fusion protein) is currently evaluated in a phase Ib/II study in combination with doxorubicin for treatment of advanced solid tumors. In a phase I study for dose finding L19-TNFα in combination with melphalan is evaluated for treatment of III/IV melanoma291. Another approach in which the L19 antibody was coupled to the soluble extracellular domain of tissue factor (tTF), to induce specific thrombosis of tumor blood vessels, showed infarction of solid tumors and complete tumor eradication in 30% of mice treated with the highest dose292. The L19 antibody coupled to interleukin-2 (IL-2); a cytokine involved in neutral killer cell and macrophage recruitment, has been tested in combination with rituximab, in a mouse model of human B-cell lymphoma xenografts. Combination treatment caused complete remission of localized lymphomas and showed increased infiltration of immune cells into the tumor tissue293. In several different subcutaneous and orthotopic mouse models treatment with L19-IL2 had a significant effect on tumor growth281,294. In the preclinical models tested no side effects were reported. In a phase I/II clinical trial to evaluate the safety, tolerability and recommended dose, for further use in a phase II study with the L19-IL2, manageable and reversible toxicity could be found. Furthermore, the L19-IL2 antibody showed activity in humans and a stabilized disease level could be observed in 51% of the patients with solid tumors and in 83% of the patients with advanced renal cell carcinoma (RCC) after two treatment cycles. With the recommend dose a progression free survival of eight month could be seen in patients with RCC. Upregulation of

35 natural killer (NK) cells and CD8+ cells in the blood could also be detected295. Currently the L19-IL2 antibody is tested in a phase II clinical trial in patients with grade III/IV melanoma and in combination with dacrabazine for metastatic melanoma. A combined treatment of the L19-IL2 antibody with gemcitabine is tested in a phase I/II clinical trial for treatment of advanced pancreatic cancer291. A clinical study with L19-SIP coupled to radioactive iodine (I131) induced a sustained partial response in two out of three relapsed Hodgkin lymphoma patients tested273. According to the Philogen website recently the efficacy and dose of L19-SIP-I131 were evaluated in patients with cancer (phase I/II clinical trail)291. No data on the type of cancer patients treated in this study are available on the website. In a prospective non-randomized study in patients with multiple brain-metastasis from solid tumors L19-SIP-I131 radio-immunotherapy (RIT) is combined with Whole Brain Radiation Treatment (WBRT). Another study performed is the combination of L19-SIP-I131 radio-immunotherapy, External Beam Radiotherapy (EBRT) and chemotherapy for treatment of inoperable, locally advanced (stage III) non-small cell lung carcinoma (NSCLC)291.

Extra domain-A The extra domain-A of fibronectin is a 90 amino acid domain, which is highly conserved between species. However, unlike ED-B, which is 100% identical in mouse and human, mouse and human ED-A are only 98% identical, which includes a two amino acid difference between the species242,296. The expression of the ED-A domain is also regulated by alternative splicing of the fibronectin molecule. The inclusion of ED-A into fibronectin of fibroblasts is regulated by TGF-β1269,270,297. Furthermore, TGF-β1 was found to promote inclusion of ED-A in bovine granulose cells4 298,299 and tubular epithelial cells . The ED-A segment binds to α4β1 and α9β1 integrins (cell-surface adhesion receptors that bind to ECM) promoting cell adhesion and migration239,242,296. ED-A+ fibronectin is re-expressed during wound healing and under pathological conditions, such as occurrence of solid tumors and rheumatoid arthritis (RA)236,300. ED-A-/- mice have defective skin wound healing301. ED- A was found to stimulate keratinocytes to progress into the cell cycle302. In individuals with psoriasis hyperproliferating keratinocytes are suggested to be the source of ED-A+ fibronectin302,303. Muro and colleagues showed that activation of TGF-β is impaired in ED-A-/- mice304. Furthermore, ED-A+ fibronectin in combination with TGF-β and mechanical tension was proposed to promote in vitro differentiation of fibroblasts into myofibroblasts305-307. ED-A containing fibronectin promotes cell adhesion and spreading better than fibronectin lacking this domain254. Another function, which has been suggested for ED-A is the signaling through TLR4243,308. It has also been proposed that ED-A can activate dendritic cells via direct binding to TLR4 and promote a T-helper 1 (Th1) cell directed

36 immune response309. In vitro activated Th1 cells express mainly fibronectin containing the ED-A domain, which promotes expression of the pro- inflammatory cytokine IL-6 in monocytes310. ED-A+ fibronectin is also elevated in plasma of RA, rheumatoid vasculitis, psoriasis, diabetes and cancer patients as well as in acute vascular injury236,242,300,303,311-313. In the pathological condition of rheumatoid arthritis ED-A+ fibronectin can additionally be detected in synovial membrane, synovial fluid and cartilage extracts. Therefore Przybysz et al. suggest it as a potential diagnostic marker in the synovial fluid of RA patients300. Kriegsman et al. found both ED-A and ED-B to be expressed in the synovium of rheumatoid arthritis and osteoarthritis patients, which was determined with the murine monoclonal antibody IST-9 (ED-A) and BC-1 (ED-B) antibody314. Pro-inflammatory roles suggested for ED-A in rheumatoid arthritis, mediated via TLR4 interaction, are the stimulation of mast cells315 and priming of leukotriene synthesis in neutrophils and monocytes316. In idiopathic pulmonary fibrosis ED-A+ fibronectin was found to deposit prior to collagens in regions of active fibrosis and to correlate with increased α-smooth muscle actin expression in activated fibroblasts242,317. ED-A+ fibronectin could also be found in intimal thickening and atherosclerotic plaques of human arteries, in an experimental model of intimal thickening of the rat aorta and in cultured vascular smooth muscle cells in vitro230,318.

Monoclonal anti-ED-A antibodies Recombinant antibody fragments directed against human ED-A have been developed by the phage display technique249. Based on the anti-ED-A antibody fragment (F8) the F8 small immunoprotein was designed250 (see Figure 4C). The F8-SIP antibody stained both murine F9 teratocarcinoma and Ramos lymphoma xenografts but not normal human tissue250. With the same antibody fragment ED-A+ fibronectin was found in different primary tumors and metastases236. The F8 monoclonal antibody coupled to IL-10 (Dekavil) in combination with methotrexate is tested in a phase I clinical trial for treatment of rheumatoid arthritis291.

Expression of ED-B and ED-A in wound healing and cartilage Clark et al. detected an increase of total fibronectin levels in the basement membrane of blood vessels adjacent to cutaneous wounds between day 3 and 7 post-injury319,320. Singh P et al. determined the expression pattern of the ED-A and ED-B containing isoforms in cutaneous wound healing239 and revealed an increase in expression of both isoforms in granulation tissue from day 4 to day 7 after wounding. Expression of ED-B remained elevated through day 14 whereas ED-A expression was reduced. ED-A+ fibronectin was more abundant in the wound area than ED-B+ fibronectin229. Low amounts of ED-A+ and ED-B+ fibronectin might even be present immediately after injury in the wounded area due to release of these

37 isoforms by activated platelets, which contain a mixture of plasma and cellular fibronectin in their α-granules242,321. Both ED-A and ED-B containing fibronectin are expressed in human articular cartilage322-324 where it is probably produced by chondrocytes325.

Tenascin-C Tenascin-C (TNC) is an extracellular matrix glycoprotein, which consists of six similar subunits joined together by disulphide bonds at their NH2 terminus. In the literature different suggestions for the structural composition of a TN-C subunit can be found. One monomer is suggested to contain either 14.5 or 15 epidermal growth factor-like repeats, 15-17 type III homology repeats, one assembly domain and one fibrinogen globe (see Figure 3B)232,233,261,262,326-328. A single gene encodes the protein and its expression is regulated by a single promoter329. Tenascin-C exists as several different isoforms, depending on the alternative splicing pattern of the nine fibronectin-like type III repeats (A1, A2, A3, A4, B, AD2, AD1, C, D)232,330,331. In humans 32 different splice variants have been detected to date331-334. In mouse brain however, only six alternatively spliced fibronectin-like type III repeats could be detected232. Splicing is cell cycle- dependent and regulated by environmental conditions such as extracellular pH335-337. Pearson et al. showed that expression of TN-C in cultured fibroblasts is induced by TGF-β338. Further, it was found that tenascin-C expression can be induced by, FGF-2, EGF, PDGF-BB, mechanical stress exerted by fibroblasts, pro- and anti-inflammatory cytokines, hypoxia or reactive oxygen species 237,262,339-341. TNC-/- mice are viable but have several defects such as abnormal behavior, abnormalities in brain chemistry and low fibronectin expression in wounds342-345. The tenascin-C isoforms containing fibronectin-like type III repeats are also referred to as large tenascin-C, whereas the isoform devoid of these repeats is called small or little tenascin-C232,233,346,347. The large isoform is mainly expressed in processes as cell migration, proliferation or tissue remodeling, which occur in embryonic development, wound healing or neoplasia262,348,349. Throughout the course of wound healing the expression of tenascin-C is highest 48h after injury350. Tenascin-C is synthesized by osteoblasts in bone formation but not present in mature bone and cartilage351. The molecule is often co-localized with fibronectin in the ECM and it has been shown in vitro that small tenascin-C binds to fibronectin, whereas large tenascin-C does not352. In rheumatoid arthritis small tenascin-C is highly expressed in the synovium and maintains inflammation by activation of TLR4353. Tenascin-C has been shown to upregulate the expression of MMP- 13 and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) in breast cancer cells and fibroblasts in vitro, which could play a role in tumor growth and invasion354.

38 C-domain of tenascin-C Similar to the above-mentioned domains of fibronectin the C-domain (type III repeat C)232,330,331,355 of tenascin-C is a 91 amino acid domain inserted into tenascin-C by alternative splicing. The identity between the mouse and human variant is 95%232. The oncofetal tenascin-C including the C-domain is expressed during embryogenesis but essentially undetectable in normal adult tissue326,356,357. However, it is upregulated in solid tumors such as high-grade astrocytoma (grade III), glioblastoma and the majority of lung cancers238,326,355. Its expression pattern is mainly around angiogenic vasculature, proliferating cells and in the tumor stroma238,326,355. Accumulation of the large tenascin-C isoform in malignant cells is due to pH-insensitivity, which prevents that splicing of the mRNA occurs336. No data are at present available in the literature about the expression of the C- domain in wound healing.

SELF-TOLERANCE The immune system is only supposed to be activated by foreign molecules and not by self-molecules. This is to prevent harmful attacks on the body’s own tissues. A number of mechanisms has therefore been developed to avoid recognition of self-molecules and this is termed immunological self- tolerance358. These mechanisms include central (in the thymus and bone marrow) and peripheral (in the lymph nodes and spleen) T-lymphocyte and B- lymphocyte tolerance. Central T-cell tolerance is the negative selection of autoreactive T-cells in the thymus during embryonic development. After birth there is the peripheral tolerance characterized by that T-cell activation requires several different simultaneous stimuli. The sole presentation of the antigen on MHC by APCs, e.g. dendritic cells and macrophages, and recognition by the T-cell receptor (TCR) is insufficient for proper T-cell activation. In order to get correctly activated T-cells additional co-stimuli such as the surface receptors B7-1 (CD80) and B7-2 (CD86) on the APC and CD28 on the T-cell, as well as cytokine (IL-6, IL-12, IL-7 and TGF-β) secretion by the APC are needed142,358-360. Without presence of these co-stimulatory molecules, the T- cell will be inactivated, a process supported by regulatory T-cells358,361,362. Downregulation of these co-stimulatory molecules is an important feature of peripheral T-cell tolerance. Central B-cell tolerance is mediated when autoreactive B-cells undergo apoptosis in the bone marrow. This negative selection is triggered when the immature B-cell recognizes and binds to a self-antigen in the bone marrow; through an interaction with its receptor (BCR) that is a surface anchored IgM molecule358,363. Peripheral B-cell tolerance is the consequence when a B-cell

39 encounters its antigen in the body but does not receive any stimuli from T- helper cells. The lack of the co-stimulatory signal leads to downregulation of the BCR and to B-cell anergy358. New B-cells are produced throughout the whole time course of life, which continuously generates new B-cell specificities363.

ADJUVANTS An adjuvant is a component added to a vaccine to accelerate, prolong or enhance the immune response but that has no immunogenic effect by itself. The word adjuvant is derived from the Latin adjuvare, which means to help or aid364. The classification of adjuvants is difficult since their application and mechanism of action is so diverse. For example cytokines such as granulocyte macrophage colony stimulating factor, interleukins and interferons are sometimes also named adjuvants. The attempt made by O´Hagan and De Gregorio to divide the adjuvants into different generations however is in my opinion the clearest one365. The first generation adjuvants are alum (aluminum salts) and Freund’s adjuvant366,367. Both adjuvants are composed of particulate dispersions (alum forms aggregates and Freund’s emulsion droplets) to which the antigen adheres. In case of Freund’s adjuvant the oil base forms a water-in-oil (W/O) emulsion when mixed with the water phase (the antigen). In an emulsion the water phase is dispersed as globules in the continuous phase, which functions as a depot to promote a slow release of the antigen. This leads to a gradual exposure of the antigen to the immune system, which is believed to give a more profound immune response368,369. Another aspect is that first-generation adjuvants induce local inflammation, which enhances APC recruitment to the injection site. Polymeric particles and liposomes370,371 can also be classified as first generation adjuvants since their particle size is equal to alum and appropriate for uptake into immune cells. These newer adjuvants also adsorb or encapsulate the antigen to enhance slow release and delivery365. Furthermore, the first-generation adjuvants have been optimized and to increase tolerability in humans oil-in-water emulsions (O/W) have been developed, of which the squalene-based MF59 is an example. The second- generation adjuvants consist of the first generation adjuvants to which additional components have been added. Examples of agents added to the first-generation base are muramyl dipeptide (MDP), derived from the bacterial cell wall372, (monophosphoryl lipid A (MPL)) and oligonucleotides365. Adjuvants approved for clinical use today in preventive vaccines are alum, MF59, AS03, AS04 and (Table 3)365,373,374. Squalene, a natural derivative, composes the oil phase of MF59 and of the by GlaxoSmithKline developed Adjuvant System (AS) AS03 adjuvant.

40 Table 3. Adjuvants approved for clinical use (adapted from365,373). Hepatitis B virus (HBV), Hepatitis A virus (HAV)

Name Class Indications Alum Mineral salts Various (e.g. Gardasil®) MF59 O/W emulsion Influenza (Fluad ®)/pandemic flu 374 AS03 O/W emulsion + α-tocopherol Pandemic flu ( ®) AS04 MLP + alum HBV (Fendrix ®)375 HPV (Cervarix ®)376 Liposomes O/W emulsion HAV, Flu

All clinically approved adjuvants are weak immunostimulators and therefore not potent enough to break immune tolerance against self-antigens, which is required for development of a therapeutic cancer vaccine. An adjuvant potent enough to evoke an immune response against a self-antigen is Freund’s adjuvant, which is widely used in animal studies. However, due to its mineral oil base and addition of heat-killed Mycobacterium tuberculosis (Freund’s complete adjuvant only), it is toxic for humans and cannot be used in clinical studies377-380. Therefore the identification of new adjuvants, which can be used in therapeutic vaccines to enhance the immunogenicity against self-antigens, is an important aspect to address.

TUMOR MODELS In vivo tumor models in mice have been used extensively to study the effect of drugs for cancer treatment. A model that can be used is the subcutaneous injection of tumor cells into mice. The tumor cells have to be syngeneic, which means that they are genetically similar to the host and therefore accepted, when injected into immunocompetent mice381,382. A disadvantage of these types of models is that the injected tumor cells are fast growing and hardly resemble slow growing multi-step tumor progression in humans, which often proceeds over decades. For studies of therapeutic effects of tumor vaccines, fast growing tumor models are difficult to use since vaccines need at least two weeks to induce an antibody response and therefore it is difficult to study the effect of a vaccine in a therapeutic setting in subcutaneous disease models. Tumor cells can also be injected into athymic nude mice or severe combined immunodeficiency (SICD) mice, which accept cells from another species (xenografts)381,382. Xenograft models are commonly used in cancer . However, these models cannot be used for vaccine studies since

41 they lack B-and/or T-cells and therefore an immune response will not be activated. Tumor cells can be injected into their site of natural origin (orthotopic) or grown at a different site (heterotopic). Heterotopic subcutaneous tumor models have made an important contribution to the development of cancer treatments but to answer more complex biological questions new physiological models may be required. Studies performed in orthotopic tumor models may be preferred for investigating new treatment strategies since the microenvironment into which the cells are placed determines the tumor phenotype and therefore tumors might behave differently in different sites in the body. Models that probably better mimic the stepwise tumor progression in humans are believed to be spontaneously arising tumors381,382. However, the genetically engineered mouse models (GEMMs) of spontaneous cancer have not yet been shown to be superior to human xenograft models in predicting treatment effects in humans, which have been used for development of the most important drugs in the clinic today (chemotherapy)382-385. As stated before patients often die from metastatic disease, and therefore models that mimic the spread of a tumor to distant sites in the body can be useful tools for treating more progressed cancers.

RIP1-Tag2 transgenic mouse model of insulinoma A mouse model that is believed to reflect multi-step human tumorigenesis is the transgenic RIP1-Tag2 model of pancreatic insulinoma386. These mice express the Simian Virus-40 (SV40) large Tumor antigen (Tag) oncoprotein under the control of the Rat Insulin Promoter (RIP) in the insulin producing β-cells of the pancreatic Langerhans islets. About 50% of the pancreatic islets will become hyperplastic with the histological characteristics of carcinoma in situ. However, in order to proceed from hyperplasia to neoplasia the islets need to turn on angiogenesis and only 10% of the hyperplastic islets succeed to do so at 7-8 weeks of age. Eventually only approximately 3% of the in total 400 islets develop into solid tumors, adenomas and/or invasive carcinomas, by 12-15 weeks. The mice die around 15 weeks of age from hypoglycemia due to overproduction of insulin386-389.

MMTV-PyMT transgenic mouse model of metastatic breast cancer Another mouse model, which is believed to reflect multi-step human tumorigenesis, is the mouse mammary tumor virus-polyoma virus middle T antigen (MMTV-PyMT) transgenic mouse390. These mice carry the mouse polyoma middle T antigen (PyMT) under the control of the mouse mammary

42 tumor virus (MMTV), which only is expressed in the breast tissue. By 8-10 weeks of age the animals develop adenocarcinomas in the mammary and around week 12-13 a high incidence of pulmonary metastasis can be detected. Tumor development is characterized by four different stages: hyperplasia, adenoma/mammary intra-epithelial neoplasia, and early and late carcinoma391. The model resembles human breast cancer in that the mice gradually lose steroid hormone receptors and β1-intergin, which is associated with overexpression of ErbB2 and cyclin D1392. Cyclin D1 is important for the stimulation of the cell cycle (mitosis) and the ErbB2 receptor is required for EGF growth factor signaling393-395.

43 Present investigations

The present investigation has focused on development of a therapeutic cancer vaccine targeting tumor angiogenesis, with the aim to find a novel and efficient treatment for cancer and prevent spreading. The technique that has been used for immunization described in all four papers of this thesis is based on the injection of a fusion protein together with a potent adjuvant. Both the fusion protein and the adjuvant are required to be able to circumvent the body’s self-tolerance396,397.

VACCINATION MECHANISM How to break self-tolerance A flow chart of the vaccination mechanism we used is depicted in Figure 5. The non-self part of the fusion protein can be of bacterial origin or any other origin as long as it is recognized by the immune system as foreign. The self- part is the host protein, which is aimed to be targeted. After injection of the fusion protein, so called antigen-presenting cells (APCs), macrophages and dendritic cells, will take up the protein and digest it into peptides. These peptides will be presented via MHC-II to T-helper 2 lymphocytes (Th2- cells).

44

Figure 5. Vaccination mechanism. Antigen presenting cell (APC), T-helper-2 cell (TH), B-cell (B), Major histocompatibility complex class II (MHC-II), antigen (Ag) T-cell receptor (TCR), B-cell receptor (BCR) an IgM molecule, anti-self antibody (anti-self AB) an IgG molecule.

As previously, described autoreactive T-cells are removed in the thymus during embryonic development or anergized (inactivated) in the periphery363,398,399. The T-cell receptors (TCRs) on the surface of the T-cells present in the circulation will thus only recognize non-self peptides presented by APCs on the MHC-II molecules. Recognition of the antigen (Ag) by the TCR then leads to activation and clonal expansion (proliferation) of the specific-T-cell. Simultaneously, the self-part of the fusion-protein is recognized by the B-cell receptor (BCR) on autoreactive B-cells, which are present in the circulation. Normally these autoreactive B-cells do not become activated since they do not receive any help from autoreactive T-cells. The autoreactive B-cells will also present the foreign peptides of the fusion protein on their surface via MHC-II. In this case the previously activated T- cells will recognize the presented non-self peptides in conjunction with the MHC-II. These T-cells will stimulate the autoreactive B-cells to clonally expand and mature into antibody producing plasma cells. By this means the T-cells get ‘tricked’, into providing help to autoreactive B-cells, and antibodies against the self-protein will be produced. The antibodies produced in the spleen will enter the circulation and bind to the self-antigen and make it visible for the immune system. The formed antigen-antibody complexes, are mainly composed of immunoglobulin M (IgM) or (IgG) bound to the self-antigen287,400. The immunoglobulins will activate the classical complement pathway, which is the part of the complement system initiated by antigen-antibody complexes (immune complexes). The complement system is part of the innate immune

45 response and consists of three different pathways: the classical, lectin and alternative pathway. In total more than 20 serum glycoproteins, synthesized by hepatocytes in the liver are involved this system, which is important for the recruitment of inflammatory cells and the killing or opsonization of pathogens401,402. The antigen-bound Igs will initiate the classical pathway via their CH2 (IgG) or CH3 (IgM) domains in their Fc portion403-405. C1, a complex consisting of one C1q and two C1s and C1r molecules, will be recruited and bind to the immunoglobulin via the C1q component. This will trigger the classical complement cascade leading to formation of C3 convertase (the C4b/C2a complex)406,407. The C3 convertase acts as a major amplification step in the complement pathway because of its ability to produce large quantities of C3b408. C3b will bind directly to the antigen and to C3b receptors409 on phagocytes (macrophages and neutrophils), a process called opsonization. By this means the C3b coated antigen can be phagocytosed. Additionally C3a and C5a, formed by C3 convertase, act as chemoattractants for neutrophils and macrophages to the site of the antigen401. In a consecutive cascade the C5b6789 complex, also named the membrane attack complex (MAC), will be formed. The MAC forms ion- permeable pores in the cell membrane of the target cell resulting in osmotic cell lysis401. If the antigen is soluble the attracted macrophages and neutrophils will engulf the protein and destroy it, a process called phagocytosis410,411. In the case of a tissue-bound antigen, macrophages and neutrophils will not be able to fully engulf the protein. In this situation a process called frustrated phagocytosis is initiated, where these cells release their granule content instead. The granules contain proteolytic enzymes for lysosomal degradation and oxidases for production of reactive oxygen species promoting tissue damage and necrosis410,412.

DEVELOPMENT OF A POTENT ADJUVANT SAFE FOR CLINICAL USE As stated before the second requirement for development of a therapeutic vaccine for human use is a potent adjuvant able to break self-tolerance. The non-toxic and biodegradable squalene-based Montanide incomplete Seppic adjuvant (ISA) 720 (M720)379 can be used to induce an immune response against a non-self antigen413. However, to break self-tolerance additional stimulators of the immune system are needed. To accomplish this, additional substances can be added to the Montanide base. One such component is repetitive unmethylated CpG DNA (CpG oligonucleotides) that is present in genomes of bacteria and certain DNA viruses. CpG oligonucleotides can stimulate the innate immune response that in turn

46 enhances the adaptive immune response and thereby promotes antibody production414,415. Other immunopotentiating components, which can be added to the Montanide base, are double stranded RNA, a component of viral genomes, or muramyl dipeptides derived from bacterial peptidoglycan that is present in the bacterial cell wall416,417. The effects of these different immunostimulators are believed to be mediated via stimulation of antigen presenting cells, mainly dendritic cells414,418. CpG oligonucleotides exert their effect through TLR9415,419-422; double stranded RNA is known to trigger TLR3 and the muramyl dipeptides activate Nucleotide-binding oligomerization domain 2 (NOD2). TLR9, TLR3 and NOD2 are all receptors present on dendritic cells416,423. In paper II we have identified an adjuvant that is potent enough to break self-tolerance and safe to use in humans. In paper III we have further characterized the immune response induced with the newly developed adjuvant against the self-antigen ED-B.

Paper I Vaccination against the extra domain-B of fibronectin as a novel tumor therapy

Aim In this study we addressed if immunization against ED-B was possible and could inhibit tumor growth.

Results In our approach we have made a recombinant fusion protein consisting of a bacterial thioredoxin (TRX) part fused with the extra domain-B (ED-B), termed TRX-EDB. This fusion protein was injected together with Freund’s adjuvant into eight-weeks old female wild type C57BL6 mice. Mice were boostered twice in a period of five weeks before they were inoculated subcutaneously with T241 fibrosarcoma cells, a tumor type known to express ED-B. After a tumor growth period of three weeks animals were sacrificed and blood and tumors were removed. Nineteen out of 20 vaccinated mice responded with production of anti-ED-B antibodies and showed a 70% reduction in tumor size compared to the animals in the control group vaccinated with Freund’s adjuvant and vehicle and lacking anti-ED-B antibodies. Staining of murine grade III glioma tissue was done to examine whether the serum from TRX-EDB mice could detect native ED-B. It showed an extensive vascular staining pattern compared to normal brain tissue, which is

47 devoid of ED-B. This proves that the anti-ED-B antibodies are able to detect native tissue ED-B. To determine the type of immune response induced by the ED-B vaccine we analyzed the anti-ED-B IgG subclasses present in the serum from control and ED-B vaccinated mice. We found that the main immunoglobulin G subclass induced was IgG1, which is characteristic of a Th2-reponse, thus an antibody-mediated immune response. Stereological analysis did not reveal any difference in vessel number, vessel density or area between tumors from control and ED-B vaccinated mice. Quantification of tumor necrotic area revealed a greater necrotic area in TRX-EDB vaccinated compared to control animals. Further investigation of the tumors with electron microscopy revealed morphological changes in the tumor vasculature of ED-B vaccinated animals, which was consistent with an immune response towards the tumor vasculature expressing ED-B. Immunohistochemical analysis of tumors from ED-B vaccinated animals showed an increased number of infiltrating neutrophils compared to controls, in agreement with an immune response in the tumor. An increased amount of extravasated fibrinogen, indicative of vascular leakage, was also detected in tumors of ED-B vaccinated animals compared to controls. Since ED-B is also expressed during physiological angiogenesis we addressed the effect of the presence of anti-ED-B antibodies on wound healing, but could not detect any alterations in the healing process between ED-B vaccinated mice and controls. Fibronectin containing ED-B is also present in cartilage and we scored control and TRX-EDB vaccinated animals for arthritis development but could not detect any differences between the treatment groups.

48 Paper II Identification of potent biodegradable adjuvants that efficiently break self-tolerance – a key issue in development of therapeutic vaccines

Aim The aim of this study was to identify an adjuvant safe for clinical use that is potent enough to break self-tolerance.

Results In this investigation we screened a number of adjuvants for their ability to induce an immune response against a self-protein. We immunized 40 female Wistar Furth rats, 8-10 weeks old, with a recombinant opossum-rat-opossum (ORO) IgE protein together with Freund’s adjuvant or Montanide ISA 720 (M720) with or without additional immunostimulators. The ORO-protein contains a self-part (C3 domain of rat ε-heavy chain) flanked with two non- self parts (C2 and C4 domain of opossum ε-heavy chain). We used either M720 alone or with addition of different immunostimulatory compounds: two different CpG oligonucleotides, double stranded RNA, GMDP (N-acetylglucoseamine-1-4-N-acetylmuramyl-alanyl- D-isoglutamine) or GMDP-A (N-acetylglucosamine-1-4-N-acetylmuramyl- alanyl-D-glutamic acid). Freund’s adjuvant was used as reference in this study. M720 is a squalene-based biodegradable mineral oil, which functions as a depot to facilitate a slow release of the protein component of the vaccine, which enhances the immune response. Bacteria and certain DNA viruses contain repetitive unmethylated CpG DNA sequences that stimulate the immune response. We found that the combination of Montanide ISA 720 and CpG oligonucleotide is as effective as Freund’s adjuvant in inducing an immune response against self-antigens. Even without additions, Montanide ISA 720 was as potent as Freund’s adjuvant with respect to induction of an immune response against a non-self protein.

49 Paper III The non-toxic and biodegradable adjuvant Montanide ISA 720/CpG can replace Freund’s in a cancer vaccine targeting ED- B – a prerequisite for clinical development

Aim The objective of this paper was to analyze if the adjuvant M720/CpG identified in paper II could be used for induction of an immune response against the self-antigen ED-B. In addition the aim was to compare the immune response induced with M720/CpG with the response generated with Freund’s adjuvant, which was used as adjuvant in paper I.

Results The TRX-EDB fusion protein was injected together with M720/CpG or Freund’s adjuvant into four- to eight-weeks old female wild type C57BL6 mice. The immunization period was five weeks and booster injections were given at day 14 and 28 of the experiment. Control animals were injected with vehicle or TRX mixed with M720/CpG. Comparable anti-ED-B antibody levels could be measured one week after the second boost in mice vaccinated with Freund’s adjuvant and M720/CpG. However, the variation in individual antibody levels measured was less with M720/CpG compared to Freund’s. Surface plasmon resonance was used to determine the avidity of the antibodies generated with the different adjuvants. We found that the anti- ED-B antibodies induced with M720/CpG were of higher avidity than antibodies generated with Freund’s adjuvant. Anti-ED-B IgG subclass analysis revealed IgG1 as the major subclass induced by both adjuvants. To compare the duration of the immune response induced by the different adjuvants, anti-ED-B antibody levels were measured three months and seven months after the second boost. At the three-month time point antibody levels were comparable in both groups. However, at the seven-month time point anti-ED-B antibody levels had decreased to baseline in the Freund’s group whereas in the M720/CpG group antibodies were still clearly detectable. No difference in induction of a response against ED-B between the two adjuvants could be detected. We also investigated the kinetics of induction of anti-ED-B antibodies in naïve mice with M720/CpG and Freund’s as adjuvants. Six-week old female C57BL6 mice were immunized with the TRX-EDB fusion protein mixed with Freund’s adjuvant or M720/CpG. The mice received booster injections at day 14 and 28 of the experiment and blood samples were drawn prior to immunization and at day three, six, nine, 17, 21, 31, and 35 after the first injection. At day nine after the first

50 immunization anti-ED-B antibodies were detectable in both groups but the levels in the M720/CpG group were significantly higher than those detected in the Freund’s group. In the Freund’s group a booster injection was needed to reach comparable levels. We conclude that the production of anti-ED-B antibodies in naïve mice was faster when M720/CpG, was used as adjuvant compared to using Freund’s.

Paper IV Development of a therapeutic vaccine targeting blood vessels in primary tumors and metastases

Aim In the present studies we sought to investigate the effect on the immune response by immunization against the three target molecules ED-B, ED-A, TNCC simultaneously in one vaccine. We also aimed to determine the expression pattern of the tumor vascular antigens in different tumor models. Furthermore, we wanted to address the therapeutic effect of the ED-B vaccine on already established tumors in the orthotopic RIP1-Tag2 insulinoma model.

Results We show that self-tolerance against ED-A and TNCC can be broken in mouse and rabbits. Anti-ED-B antibody levels generated were significantly reduced by addition of one (TRX-EDA) or two (TRX-EDA and TRX- TNCC) vaccine protein components to the ED-B vaccine. The TRX part alone also significantly suppressed the antibody production against ED-B. When all three vaccine-proteins (TRX-EDB, TRX-EDA and TRX-TNCC) were combined into a single fusion protein (TRX-EDB-EDA-TNCC) the suppressive effect of the foreign fusion part TRX on the antibody production was reduced. Using affinity-purified anti-mouse antibodies from rabbit serum we found that ED-B, ED-A and TNCC was expressed in T241 fibrosarcoma but not in normal mouse tissue. ED-B was also detected in insulinomas of RIP-Tag2 mice. Immunization against ED-B induced a significant reduction in the number of tumors in the pancreas compared to control mice. This indicates that the ED-B vaccine can be used in a therapeutic setting when tumorigenesis already has been initiated. We identified ED-A in tumor tissue of transgenic MMTV-PyMT mice, a model for metastatic breast cancer, but not in normal breast tissue. The aim

51 is to use this model to study the effect of an ED-A vaccine on metastasis. We also found that ED-B is expressed in canine breast tumors and this domain may represent a potential therapeutic target in dogs. Dog-specific anti-ED-A and anti-TNCC antibodies could also be generated in rabbits and are important tools to establish the expression pattern of these molecules in dog tissue.

Discussion Monoclonal antibodies against ED-A and ED-B alone or in combination with chemotherapy or radiation are tested in phase I/II clinical trials, to deliver coupled agents specifically to the tumor tissue273,293. This supports our choice of target molecules and their disease specificity. Since the clinically tested antibodies are monoclonal no immune complexes will be formed and thus no effective immune response can be induced with these antibodies. Our strategy however is aiming to induce an endogenous production of antibodies against the vascular antigens. This will give a polyclonal immune response and consequently more binding possibilities to the target antigen. A polyclonal serum will induce immune complex formation leading to a more effective immune response. This explains why monoclonal antibodies alone are insufficient to evoke an immune response against the tumor vasculature and have to be coupled to other agents to obtain an effect. Mice with anti-ED-B antibodies present in their circulation have normal life span (we kept mice for a time period of almost two years), normal physical appearance and they show no signs of pathological changes in their tissues such as in cartilage. Furthermore, we could not observe any impaired wound healing in full-thickness wounds, a model of physiological angiogenesis, in these mice. An explanation for the outcome that the cartilage did not seem to be attacked by the immune system in TRX-EDB immunized mice could be the lack of blood vessels in this tissue424. This reduces the accessibility of the anti-ED-B antibodies for their target molecule ED-B. The reoccurrence of ED-B during wound healing could cause a problem in vaccinated animals with anti-ED-B antibodies in their blood and promote an immune reaction directed against the provisional matrix present in wounds. We found that wound healing was not impaired in mice with antibodies against ED-B present in their circulation. An explanation for this could be that ED-B+ fibronectin expression is transient in a wound compared to the permanent expression observed in tumors. Furthermore, the blood vessel quality might be different in physiological angiogenesis (wound healing) compared to pathophysiological angiogenesis (tumorigenesis). Tumor vessels are constantly leaky whereas the blood vessels in a wound are

52 only leaky within a transient time-interval18, which might not be long enough to sustain an immune response leading to tissue damage. The antibodies also need to cross the endothelium to reach their target in the ECM and therefore targeting of ED-B in a tumor might be easier. Another possibility could be that ED-B+ fibronectin is less abundantly expressed in a healing wound than ED-A+ fibronectin229,239. Therefore an immune attack directed towards ED-B might not have any impact on wound healing because ED-A+ fibronectin might be the crucial isoform required for this process. CpG oligonucleotide binds to TLR9. TLR9 is expressed by B-cells and plasmacytoid DCs and stimulation of this pattern recognition receptor leads to production of IFNα414,425,426. IFNα has been shown to induce the secretion of B-cell activating factor (BAFF) by myeloid dendritic cells and the differentiation of B-cells into antibody producing plasma cells427. BAFF was also found to promote survival and antibody secretion by autoreactive B- cells427,428. This mechanism might explain why CpG oligonucleotides can be used to break immune tolerance and induce autoantibodies. TLR9 stimulation by CpG oligonucleotides has been shown to promote B-cell proliferation, inhibit B-cell apoptosis and induce IgM production415,429. Simultaneous triggering of TLR9 and the B-cell receptor was shown to stimulate proliferation of autoreactive B-cells422,427, which might be attributed to the production of IFNα induced by TLR9 stimulation. When systemically administered type I interferons (IFNα and IFNβ) can induce autoimmune diseases such as Systemic lupus erythematosus427. This observation might therefore provide an explanation for the potent effect of TLR9 stimulation on breaking self-tolerance. CpG oligonucleotide was also found to directly activate NK cells414 and therefore the addition of CpG oligonucleotide to the vaccine maybe also promotes NK cell activation in our vaccination approach. The anti-ED-B antibodies induced with the help of the adjuvant M720/CpG had a 10-fold higher avidity for ED-B than those induced with Freund’s adjuvant. An explanation for this observation could be the presence of CpG. In a study by Siegrist et al. the addition of human specific CpG oligonucleotides (CpG 7909) to a prophylactic was shown to enhance affinity maturation of the human anti-hepatitis B antibodies induced by the vaccine430. The CpG 7909 significantly increased both antibody levels and antibody avidity. The adjuvant used in this study was alum and human-specific CpG oligonucleotide with a sequence distinct from the mouse-specific CpG 1826 used in our studies. However, the results of Siegrist et al. are in agreement with ours. The difference with the monoclonal antibodies targeting VEGF used in the clinic, which have to be administered frequently and at high doses, is that our vaccination approach triggers the endogenous production of antibodies. By this means the immune system can be directed against the tumor blood vessels, which will lead to vessel destruction and inhibition of tumor growth.

53 Additionally, the production of monoclonal antibodies is costly and therefore treatment with these agents is expensive. A vaccine in comparison requires lower doses because the body will produce the antibodies against the administered target antigen. Monoclonal antibodies however need frequent administration (every 2-3 weeks) due to their short half-life (approximately 20 days)119. The immune response induced by our vaccine is reversible, which is of advantage. Reversibility is desirable in case the endogenously produced antibodies give side effects. After vaccination against ED-B the induced anti-ED-B antibodies stayed present in the circulation for seven to 12 month, depending on which adjuvant was used. The reason for the difference in duration of the immune response is unknown. One explanation might be that the higher avidity of the antibodies induced with M720/CpG aids to keep the antigen on the surface of follicular dendritic cells (fDCs) promoting a more long-lived response. Long-lived fDCs help to present the injected fusion protein TRX-EDB to immune cells by Ig, which is bound to Fc-receptors. Furthermore, greater antibody avidity might require a lower number of cross-linked Ig molecules on the surface of the B-cell to trigger its activation. Both Montanide ISA 720 and CpG oligonucleotide 7909 have been tested separately in clinical studies and single use of these agents seems to be safe since only mild or moderate side effects were observed430-435. We observed a significant decrease in the number of tumors in RIP-Tag2 mice. The total tumor volume was also reduced although this was not statistically significant in the present study group. Nevertheless the observation indicates that the ED-B vaccine also is effective in a therapeutic setting. The other target molecules ED-A and TNCC have an expression pattern similar to ED-B and are therefore interesting to target with the same approach as used for ED-B. Our data show that it is possible to break self- tolerance against ED-B, ED-A and TNCC in mice and rabbits and therefore our vaccination technology might be an interesting approach for production of antibodies against antigens with low immunogenicity.

Future perspectives and concluding remarks The overall aim of our studies is to develop a new treatment strategy for patients with solid tumors. To achieve this goal the strategy has to be validated in additional preclinical as well as clinical studies. The high degree of conservation of ED-A, ED-B and TNCC should facilitate clinical translation of the approach. The results described in this thesis show that it is possible to break self- tolerance against vascular antigens, which are present in angiogenic vasculature of most solid tumors. When ED-B was used as a target molecule tumor growth could be inhibited in a preventive (subcutaneous T241 fibrosarcoma) and therapeutic setting (orthotopic RIP-Tag2 tumor model).

54 Furthermore, we found a non-toxic and biodegradable (M720/CpG) adjuvant potent enough to break the immune tolerance against self-antigens. This adjuvant was also effective in breaking self-tolerance against the vascular antigens ED-A, ED-B and TNCC. This demonstrates that there are less toxic alternatives to Freund’s adjuvant in experimental animal work. In addition this finding makes M720/CpG an interesting adjuvant for development of therapeutic cancer vaccines. ED-A was found to be expressed in breast tumors of MMTV-PyMT mice. This model will be used to determine the effect of an ED-A vaccine on tumor growth and metastasis. We also found that addition of TRX suppressed the formation of anti-ED-B antibodies in mice immunized with TRX-EDB and that this was also the case when mice were immunized with a vaccine consisting of TRX-EDB, TRX-EDA and TRX-TNCC. The immune response to ED-B however could be partly restored when only one molecule TRX was fused to all three antigens in one fusion protein. Further optimization is needed to obtain equal levels of specific antibodies directed to each of the three antigens when administering them simultaneously as obtained after immunization with ED-B only. We would like to perform a tumor study to address the effect of the vaccine on tumor growth after a combined immunization with all three antigens. The T241 fibrosarcoma model expresses all three vascular antigens. Simultaneous targeting of these antigens might therefore have a more profound effect on tumor growth compared to immunization against a single antigen. During the past era of cancer treatment it has become clear that combination therapy is essential for tumor eradication and to avoid drug resistance101. One situation when a tumor vaccine could potentially be useful is after resection of the primary tumor to prevent tumor recurrence and metastatic disease. However, this topic requires further investigation. In combination with chemotherapy or radiotherapy the vaccine might be used to treat non-resectable established tumors, since chemotherapy and radiation have been shown to stimulate the immune system13,108,189,190. The vaccine might also be applied to treat metastatic disease in combination with chemotherapy. We could not observe any changes in wound healing, an example of physiological angiogenesis, between TRX-EDB and control-vaccinated animals. A clinical situation in which application of a therapeutic vaccine cancer would be highly interesting is for prevention of metastases formation in patients diagnosed with a primary tumor. It has been reported previously that ED-A is expressed in primary human breast tumors and metastases236. We found that ED-A is expressed in a similar pattern in primary tumors and metastases of the transgenic MMTV-PyMT breast cancer model. This makes the MMTV-PyMT mice a suitable model to address the potential of an ED-A vaccine in prevention of breast cancer metastases.

55 To confirm the therapeutic effect of our vaccine a clinical trial has to be conducted. Dogs with tumors represent a potentially interesting group of patients for a clinical study, since they are to a large extent affected by the same type of tumors as humans. We found ED-B also to be expressed in mammary tumors of dogs and thus ED-B is also a potential target in this common dog tumor type.

56 Populär vetenskaplig sammanfattning

Utveckling av ett cancervaccin som angriper tumörblodkärl Cancer är efter kardiovaskulära sjukdomar den näst vanligaste dödsorsaken i västvärlden och den tredje i utvecklingsländerna. Det är en sjukdom som karakteriseras av elakartade svulster (tumörer) som beror på att tumörceller växer okontrollerat och kan tränga sig in i vävnader. Under det senaste seklet har man till stor del kartlagt orsakerna till cancer vilket gjort behandling möjlig. De flesta patienter som dör av sjukdomen, gör det emellertid inte av själva ursprungstumören utan pga. att tumören spridit sig genom s.k. metastasering. Med dagens behandlingsmetoder är det tyvärr mycket svårt att behandla cancer som spridit sig. Det finns därför ett stort behov av att få fram fler behandlingsmetoder framförallt för metastaserad cancer men även för primära tumörer. I denna avhandling beskrivs utvecklingen av ett vaccin som angriper nybildningen av blodkärl i tumörer (tumörangiogenes). Det är en metod som kan hjälpa till att hämma tumörtillväxten genom att man kan stoppa blodflödet och därmed svälta ut tumören genom att den inte får tillgång till viktiga näringsämnen och syre. Vaccinet riktar sig specifikt mot molekyler som uttrycks vid blodkärlsnybildning kring blodkärl i de flesta solida tumörer. Dessa molekyler utgör bland annat delar (extra domäner) av proteiner som bygger upp den vävnad som alla celler i kroppen sitter på (extracellulära matrixen). Dessa molekyler är bl.a. extra domän-B (ED-B) och extra domänen-A (ED-A) i fibronektin och C-domänen i tenascin-C (TNCC). Vi visar att det går att bryta självtolerans genom vaccinering och stimulera produktion av antikroppar mot kroppseget ED-B i det första delarbetet. Immunförsvaret är normalt inställt på att inte reagera mot kroppsegna molekyler (antigener) utan bara mot det som är främmande. Vaccinet hämmade även tumörtillväxten i möss och de observerade förändringarna i tumörvävnaden stämmer väl överens med att immunförsvaret angripit blodkärl i tumören. För att kunna använda vaccineringsmetoden kliniskt behöver man förstärka vaccinets förmåga att sätta igång ett immunsvar. Det gör man genom att tillsätta ett s.k. adjuvans och det måste i det här fallet vara tillräckligt kraftigt för att man skall kunna bryta självtoleransen mot kroppsegna antigener som ED-B men det måste även vara ofarligt att

57 användas på människor. I det andra delarbetet visar vi att det skvalen- baserade adjuvanset Montanide ISA 720 (M720) i kombination med CpG DNA (ometylerade CG rika DNA molekyler isolerade från bakterier eller virus; mänskligt DNA är metylerat) uppfyller dessa krav och skulle kunna användas kliniskt som adjuvans. Vi demonstrerar även att M720/CpG är lika effektivt som den prekliniska ‘gyllene standarden’ Freund’s adjuvans när det gäller att stimulera ett immunsvar mot ett kroppseget antigen. I den tredje artikeln (III) har vi karakteriserat det immunsvar mot ED-B som man framkallar med hjälp av M720/CpG. ED-B vaccinet visade sig dessutom ha effekt i en terapeutisk tillämpning genom att det hämmade tumörtillväxten i en transgen musmodell med insulin producerande tumörer (insulinom) i bukspottkörteln efter att tumörtillväxten redan hade kommit igång. Vi har även lyckats få ett immunsvar med vår metod med antikroppar mot ED-A och TNCC i möss och kaniner. Dessutom har vi kartlagt uttrycket av ED-A molekylen i tumörvävnad hos möss med bröstcancer. I denna musmodell vill vi i framtiden kunna undersöka vilken effekt ett ED-A vaccin kan ha på metastaser. Vi kunde även hitta ED-B i tumörvävnader hos hundar. Eftersom hundar lider av liknande sjukdomar som människor skulle de kunna inkluderas i en klinisk studie för att analysera effekterna av cancervaccinet. Sammanfattningsvis visar våra prekliniska studier att ett vaccin som angriper blodkärlsbildningen via en eller flera vägar är en ny mycket lovande strategi för behandling av cancer i olika former.

58 Nederlandse samenvatting

De ontwikkeling van een kankervaccin dat aangrijpt op de tumorbloedvaten Kanker is na ischemische hartziekten de meest voorkomende doodsoorzaak in de Westerse wereld en staat op een derde plaats in de ontwikkelingslanden. De ziekte wordt gekenmerkt door kwaadaardige gezwellen, die ontstaan door dat tumorcellen ongecontroleerd groeien en gezond weefsel kunnen binnendringen. Gedurende de vorige eeuw zijn voor een groot deel de oorzaken van kanker in kaart gebracht en werd behandeling mogelijk. Het merendeel van de kanker patiënten sterft echter niet aan het oorspronkelijke gezwel (tumor) maar aan ongeneeslijke uitzaaiingen (metastasen) die met de ziekte gepaard gaan. Met de huidige beschikbare behandelmethoden is het moeilijk om kanker die is uitgezaaid te behandelen. Daarom is er behoefte aan nieuwe mogelijkheden ter behandeling van zowel metastasen als primaire tumoren. In dit proefschrift wordt de ontwikkeling van een vaccin beschreven dat aangrijpt op de bloedvatvorming in tumoren (de tumorangiogenese). Dit is een methode die kan bijdragen aan het remmen van de tumorgroei en de bloedtoevoer naar de tumor kan stoppen, waarmee deze van voedingsstoffen en zuurstof kan worden onttrokken. Het vaccin richt zich op moleculen die aanwezig zijn tijdens de aanmaak van nieuwe bloedvaten in de meeste solide tumoren. Deze moleculen zijn o.a. extra domeinen van eiwitten die voorkomen in de extracellulaire matrix (het weefsel waarop cellen hechten); voorbeelden zijn het extra domein-B (ED-B) en extra domein-A (ED-A) van fibronectine en het C-domein van tenascine-C (TNCC). In het eerste artikel laten we zien dat het mogelijk is om doormiddel van vaccinatie de immuuntolerantie te doorbreken en om de productie van antilichamen tegen het lichaamseigen ED-B te stimuleren. Het immuunsysteem reageert normaalgesproken namelijk niet op lichaamseigen moleculen (antigenen) maar alleen op lichaamsvreemde. Bovendien kon met het vaccin de tumorgroei in muizen worden geremd en de veranderingen, waargenomen in het tumorweefsel, waren overeenkomstig met het aangrijpen van het immuunsysteem op de tumorbloedvaten. Om de vaccinatietechnologie in de kliniek toe te kunnen passen moet het vermogen van het vaccin om een immuunreactie op gang te brengen worden

59 versterkt. Dit kan worden bereikt met behulp van een zogenaamd adjuvans, dat krachtig genoeg is om de immuuntolerantie tegen lichaamseigen antigenen zoals ED-B te doorbreken. Tevens moet het adjuvans geschikt zijn voor humaan gebruik. In paper II laten we zien dat het squaleen afgeleide Montanide ISA 720 (M720) in combinatie met CpG DNA (ongemethyleerd DNA rijk aan CG van bacteriën en virussen; humaan DNA is gemethyleerd) voldoet aan deze voorwaarden en mogelijk als adjuvans in de kliniek kan worden toegepast. Bovendien laten we zien dat M720/CpG even krachtig is als de preklinische ‘gouden standaard’ Freund’s adjuvans in het opwekken van een immuunreactie tegen een lichaamseigen antigeen. In paper III hebben we de met behulp van M720/CpG opgewekte immuunreactie tegen ED-B onderzocht. Het ED-B vaccin bleek tevens effectief in een therapeutische toepassing en remde de tumorgroei in een transgeen muismodel met insulinomen (insuline producerende tumoren) in de pancreas waarin de tumorgroei al op gang was gekomen. Tevens konden we met onze methode antilichamen tegen ED-A en TNCC opwekken in muizen en konijnen. We hebben ook de expressie van ED-A bepaald in tumorweefsel van muizen met borstkanker. In dit muismodel willen we in de toekomst het effect van een ED-A vaccin op metastasen bestuderen. ED-B kon tevens worden aangetoond in tumorweefsel van honden. Honden met kanker zijn daarom potentieel geschikt voor inclusie in een klinische studie ter analyse van het effect van het kankervaccin. Samenvattend laten onze preklinische studieresultaten zien dat een vaccin dat aangrijpt op de bloedvatvorming een veelbelovende nieuwe strategie voor de behandeling van kanker is.

60 Deutsche Zusammenfassung

Die Entwicklung eines Krebsimpfstoffes der die Blutgefäße im Tumor angreift Krebs ist nach Herz-Kreislauferkrankungen die zweite Todesursache in der westlichen Welt und die dritte in den Entwicklungsländern. Die Krankheit kennzeichnet sich durch bösartige Geschwülste (Tumore), die entstehen durch das die Tumorzellen unkontrolliert wachsen und in gesundes Gewebe eindringen können. Während des vorherigen Jahrhunderts wurden zum größten Teil die Ursachen der Krankheit entdeckt und Behandlungsmethoden wurden entwickelt. Allerdings sterben die meisten Krebspatienten letztendlich nicht am ursprünglichen Tumor sondern an unheilbaren Tochtergeschwülsten (Metastasen). Mit den heutigen Behandlungsmethoden ist es schwierig die verbreiteten Tochtergeschwülste zu behandeln. Deshalb werden neue Behandlungsmöglichkeiten gebrauch, um sowohl die Metastasen als auch den Ursprungstumor anzugreifen. In dieser Doktorarbeit wird die Entwicklung eines Impfstoffes, welcher die Bildung von neuen Blutgefäßen durch den Tumor (Tumor-Angiognese) verhindert, beschrieben. Die Methode kann bei der Hemmung des Tumorwachstums behilflich sein indem sie die Blutzufuhr zum Tumor stoppt und damit dessen Nährstoff- und Sauerstoffzufuhr einschränkt. Der Impfstoff richtet sich gegen Moleküle, welche nur in unmittelbarer Nähe zu neugebildeten Gefäßen gefunden werden können und die während der Tumorangiogenese von den meisten Krebsarten gebildet werden. Zu diesen Molekülen gehören unteranderem die Extra-Domänen von Eiweißen in der extrazellulären Matrix (das Gewebe worauf Zellen haften); wie zum Beispiel die Extra-Domäne-B (ED-B) und Extra-Domäne-A (ED-A) von Fibronektin und die C-Domäne von Tenascin-C (TNCC). Im ersten Artikel zeigen wir, dass es möglich ist die Immuntoleranz mit einem Impfstoff zu durchbrechen und die Produktion von Antikörpern gegen das körpereigene ED-B zu stimulieren. Das Immunsystem reagiert normalerweise nur auf körperfremde Moleküle (Antigene) aber nicht auf körpereigene. Außerdem konnte das Tumorwachstum in Mäusen gehemmt werden und die beobachteten Veränderungen im Tumorgewebe erschienen übereinstimmend mit einer Reaktion des Immunsystems auf die Tumorblutgefäße.

61 Um die Impfung als Therapie im Menschen anwenden zu können muss die Wirkung des Impfstoffes verstärkt werden. Dies erreicht man indem man ein Adjuvans hinzufügt. Das Adjuvans muss in diesem Fall ausreichend strak sein um die Immuntoleranz gegen körpereigene Antigene wie ED-B zu durchbrechen. Außerdem benötigt man ein für den Menschen ungefährliches Adjuvans. Das von Squalen abgeleitete Montanide ISA (M720) in Kombination mit CpG DNA (unmethylierte CG reiche DNA von Bakterien und Viren; menschliche DNA ist methyliert) entspricht diesen Anforderungen und kann möglicherweise in de Klinik als Adjuvans angewendet werden. M720/CpG erzeugte eine Immunantwort gegen ein körpereigenes Antigen ebenso effektiv wie der ,,goldene Standard’’ Freund’s Adjuvans. Im dritten Artikel dieser Arbeit haben wir die Immunantwort gegen ED-B, welche unter Anwendung von M720/CpG erzeugt wurde, charakterisiert. Der ED-B- Impfstoff war eine wirksame Tumortherapie und hemmte das Tumorwachstum in einem Mausmodel mit Insulinomen (Tumore der Langerhans-Zellen) in der Bauchspeicheldrüse in welchem das Tumorwachstum schon eingesetzt hatte. Außerdem konnte eine Immunantwort mit Antikörper gegen ED-A und TNCC in Mäusen und Kaninchen erzeugt werden. Wir haben auch das Vorhandensein von ED-A in Tumoren von Mäusen, welche an Brustkrebs erkranken, gezeigt. In diesem Mausmodel wollen wir in der nahen Zukunft den Effekt eines ED-A- Impfstoffes auf Metastasen testen. Außerdem konnte ED-B im Tumorgewebe von Hunden feststellt werden. Wir planen, unsere Impfstoffe in einer klinischen Studie an Hunden, welche an Krebs erkrankt sind, zu testen. Zusammenfassend zeigen, unsere Studien, dass ein Impfstoff der die Tumorblutgefäße angreift eine vielversprechende neuartige Strategie zur Krebsbehandlung darstellt.

62 Acknowledgments

The work presented in this thesis has been carried out at the Department of Medical Biochemistry and Microbiology, Uppsala University. Funding was provided by grants from the Swedish Cancer Society, the Swedish Research Council, the Swedish Society of Medicine, Jeansson’s Foundation, Åke Wiberg Foundation, the Department of Medical Biochemistry and Microbiology and the Medical Faculty at Uppsala University.

I would like to thank everyone who has contributed to this work and supported me during my graduate studies. In particular I would like to thank:

Anna-Karin Olsson, min handledare: Du är toppen! Jag gillar att jobba med dig. Du är en jättebra handledare som låter mig prova på saker och är där när jag behöver hjälp. Hoppas vi kan fortsätta ett tag till att forska tillsammans och ha det roligt.

Min andra handledare Staffan Johansson för interesset i mitt arbete och bra hjälp, råd och kommentarer.

Lars Hellman – tack för ett bra samarbete och de extra immunologi- lektionerna.

Julia, danke für die vielen schönen Stunden im Labor und hoffenltich noch ein paar mehr… Auf viele Publikationen, gute Freundschaft und Spaß zusammen! Viel Erfolg beim Licentiate!

Jessica, tack för allt skoj på jobbet och en bra USA resa! Kanske vi kan shoppa lite till i New York någon gang!

Yanyu, good luck with your PhD studies!

Maria, för bra stunder på labbet. Det är jätteskoj att jobba ihop med dig även om vaccinet ibland hamnar på väggen. Lycka till som gruppledare.

63

Åsa, kollega och vän, för all hjälp, råd, diskussioner, fika och nattvakande i Boston. Lycka till med familjen och din framtida karriär.

Magnus Åbrink för vetenskaplig diskussion, kaffe, fika och samarbete.

Nanna Forsberg, Lene Uhrbom, Marianne Kastemar och Maria Boije för ett bra sammarbete. Nanna – lycka till i New York!

Vielen Dank auch an Wilhelm Jahnen-Dechent und Daniela Dreymüller für die gute Zusammenarbeit und eine nette Zeit in Aachen. Ihr seid herzlich Willkommen in Uppsala!

Nona ‘joon’ for being such a nice and good friend. It is great to have you close by in the lab. I promise not to say any ‘you are welcome’ in Persian without you. J Good luck in finding your way!

Ananya, for a great friendship, scary movie nights and good food. I hope there are more such nights to come! Good luck with your defense!

Familjen Danielsson för många bra timmar i Falun, svensk tradition och skidåkning. Anna, vad skönt att ha dig tillbaka in Uppsala!

Vahid ‘joon’ for having fun in the lab and in spare time. Good luck with your graduate studies!

All past and present IMBIM PhD student association members for your efforts, pub and fun times at BMC.

All people in B9:3, including the ones that have moved to D9:4 and the rest of IMBIM for lunches, fika and collaboration. Especially great thanks to the ladies at the administration.

The members of the vascular biology group at the Rudbeck Laboratory, where I started my work as a project student.

All my co-authors for their excellent contributions to this work.

64 All my friends and everyone else I have forgotten to name in this acknowledgements. It was not on purpose!

Antonia, Lothar, Ute, Kathrin, Martin, Stefan, Anne, Pavel och hela ’fredagsklubben’. For the many nice conversations, beers, burgers, barbecues and other activities.

Hamid ‘joon’ och Juan för en jättetrevlig resa till Göteborg! Katja for core, BT, fika and nights out. Also Vlad, Abhi and Majid for fun parties and nights out.

Laurens, Marjolein en Elise dankjewel voor alle Nederlandse uurtjes!

Thirza, ‘meissie’: ik mis je nog steeds na al die jaren in Uppsala. Heel veel succes met je nieuw baan. Tot ziens in NL! Dikke kus

De farmaciemeiden Caro, Suus en Mo – wat mis ik jullie! Het is altijd weer gezellig wanneer we samen zijn. Milaan was erg gezellig samen met chips in bed en limoncello en heel veel shopping. Misschien over een paar jaar ‘sex and the city’ in New York? Caro, sterkte met je zwangerschap. Suus, zonder jou was ik nooit doctor geworden want jij hebt mij ooit pipetteren geleerd hebt! Dr. Mo, heel veel succes met je opleiding to ziekenhuisapotheker! Tot snel! Jammer dat we geen hotelkamer delen in Uppsala J

Ann-Cathrin, min vän och rådgivare i stan. Du visade mig forskningen! Tack för det! Jag ser fram emot manga fler bra timmar med dig och Bosse.

Huize Griftstraat - George, Sanne en Jean-Louis – ik mis jullie, alle feestjes aan de bar, huisweekenden en de gezelligheid samen. Hopelijk kunnen we het feesten samen nog een keer over doen op mijn promotiefeest!

En natuurlijk niet te vergeten de hele ‘fam’. Pappa en ‘mutti’ op de Bünnert in Uedem, Sjors in Winterthur, Marije ergens in verwegistan en Dirk ‘im Osten’. Ik mis jullie! De reis door Zweden, nu bijna vijf jaar geleden, was fantastisch. Zelfs het schaap op de achterbank heeft genoten! Ik kijk er naar uit om jullie binnenkort weer in Uppsala te zien. Liefs

65 References

1. Prigorowsky, M. Cancerfondsrapporten 2011. (ed. Jaresand, M.) (Cancerfonden, Stockholm, 2011). 2. WHO. Cancer. http://www.who.int/mediacentre/factsheets/fs297/en/index.html access date: march 25th 2012 3. WHO. The top 10 causes of death. http://www.who.int/mediacentre/factsheets/fs310/en/index.html access date: march 25th 2012 4. Coleman, M.P., et al. Cancer survival in five continents: a worldwide population-based study (CONCORD). Lancet Oncol 9, 730-756 (2008). 5. WHO. Breast cancer: prevention and control. http://www.who.int/cancer/ detection/breastcancer/en/index1.html access date: march 25th 2012 6. Hanahan, D. & Weinberg, R.A. The hallmarks of cancer. Cell 100, 57-70 (2000). 7. Hanahan, D. & Weinberg, R.A. Hallmarks of cancer: the next generation. Cell 144, 646-674 (2011). 8. Weinberg, R.A. The biology of cancer, 588 (Garland Science, Taylor & Francis Group, LLC, New York, 2007). 9. Weinberg, R.A. Moving out: invasion and metastasis. in The biology of cancer (Garland Science, Taylor & Francis Group, LLC, New York, 2007). 10. Chambers, A.F., Groom, A.C. & MacDonald, I.C. Dissemination and growth of cancer cells in metastatic sites. Nature reviews. Cancer 2, 563-572 (2002). 11. Weinberg, R.A. The nature of cancer. in The biology of cancer, 25-56(Garland Science, Taylor & Francis Group, LLC, New York,NY, 2007). 12. Mantovani, A. & Pierotti, M.A. Cancer and inflammation: a complex relationship. Cancer Lett 267, 180-181 (2008). 13. Melief, C.J. by dendritic cells. Immunity 29, 372-383 (2008). 14. WHO. Human papillomavirus (HPV). http://www.who.int/immunization/topics/hpv/en/ access date: march 25th 2012 15. Berman, J.J. Tumor classification: molecular analysis meets Aristotle. BMC Cancer 4, 10 (2004). 16. Liotta, L.A. & Kohn, E.C. The microenvironment of the tumour-host interface. Nature 411, 375- 379 (2001). 17. Dvorak, H.F. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. The New England journal of medicine 315, 1650-1659 (1986). 18. Schafer, M. & Werner, S. Cancer as an overhealing wound: an old hypothesis revisited. Nat Rev Mol Cell Biol 9, 628-638 (2008). 19. Tse, J.C. & Kalluri, R. Mechanisms of metastasis: epithelial-to-mesenchymal transition and contribution of tumor microenvironment. Journal of cellular biochemistry 101, 816-829 (2007). 20. Nakamura, M. & Tokura, Y. Epithelial-mesenchymal transition in the skin. J Dermatol Sci 61, 7- 13 (2011). 21. Kalluri, R. EMT: when epithelial cells decide to become mesenchymal-like cells. J Clin Invest 119, 1417-1419 (2009). 22. Balkwill, F., Charles, K.A. & Mantovani, A. Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer cell 7, 211-217 (2005).

66 23. de Visser, K.E., Eichten, A. & Coussens, L.M. Paradoxical roles of the immune system during cancer development. Nature reviews. Cancer 6, 24-37 (2006). 24. Wakefield, L.M. & Roberts, A.B. TGF-beta signaling: positive and negative effects on tumorigenesis. Curr Opin Genet Dev 12, 22-29 (2002). 25. Rossiter, H., et al. Loss of vascular endothelial growth factor a activity in murine epidermal keratinocytes delays wound healing and inhibits tumor formation. Cancer research 64, 3508-3516 (2004). 26. Ostman, A. PDGF receptors-mediators of autocrine tumor growth and regulators of tumor vasculature and stroma. Cytokine Growth Factor Rev 15, 275-286 (2004). 27. Folkman, J. & Kalluri, R. Tumor angiogenesis. in Cancer Medicine (eds. Kufe, D.W., et al.) 161- 194 (B.C. Decker, Hamilton, 2003). 28. Werner, S. & Smola, H. Paracrine regulation of keratinocyte proliferation and differentiation. Trends Cell Biol 11, 143-146 (2001). 29. Harvey, W. Exercitatio anatomica de motu cordis et sanguinis in animalibus, (1628). 30. Vander, A., Sherman, J. & Luciano, D. Circulation. in Human physiology - the mechanisms of body function (ed. Kane, K.T.) 372-426 (McGraw-Hill Companies, Inc. , Boston, 1998). 31. Alitalo, K., Tammela, T. & Petrova, T.V. Lymphangiogenesis in development and human disease. Nature 438, 946-953 (2005). 32. Bergers, G. & Song, S. The role of pericytes in blood-vessel formation and maintenance. Neuro- oncology 7, 452-464 (2005). 33. Jin, S.W. & Patterson, C. The opening act: vasculogenesis and the origins of circulation. Arteriosclerosis, thrombosis, and vascular biology 29, 623-629 (2009). 34. Eichmann, A. Vasculogenesis and angiogenesis in development. in Tumor angiogenesis (eds. Marmé, D. & Fusenig, N.) 31-45 (Springer-Verlag, Berlin Heidelberg, 2008). 35. Carmeliet, P. Angiogenesis in life, disease and medicine. Nature 438, 932-936 (2005). 36. Risau, W. Mechanisms of angiogenesis. Nature 386, 671-674 (1997). 37. Burri, P.H. & Djonov, V. Intussusceptive angiogenesis--the alternative to capillary sprouting. Mol Aspects Med 23, S1-27 (2002). 38. Urbich, C. & Dimmeler, S. Endothelial progenitor cells: characterization and role in vascular biology. Circ Res 95, 343-353 (2004). 39. Gao, D., et al. Endothelial progenitor cells control the angiogenic switch in mouse lung metastasis. Science (New York, N.Y 319, 195-198 (2008). 40. Kilarski, W.W., Samolov, B., Petersson, L., Kvanta, A. & Gerwins, P. Biomechanical regulation of blood vessel growth during tissue vascularization. Nature medicine 15, 657-664 (2009). 41. Genersch, E., Hayess, K., Neuenfeld, Y. & Haller, H. Sustained ERK phosphorylation is necessary but not sufficient for MMP-9 regulation in endothelial cells: involvement of Ras-dependent and - independent pathways. J Cell Sci 113 Pt 23, 4319-4330 (2000). 42. Ben-Yosef, Y., Miller, A., Shapiro, S. & Lahat, N. Hypoxia of endothelial cells leads to MMP-2- dependent survival and death. Am J Physiol Cell Physiol 289, C1321-1331 (2005). 43. Werb, Z., Vu, T.H., Rinkenberger, J.L. & Coussens, L.M. Matrix-degrading proteases and angiogenesis during development and tumor formation. Apmis 107, 11-18 (1999). 44. Pepper, M.S., Montesano, R., Mandriota, S.J., Orci, L. & Vassalli, J.D. Angiogenesis: a paradigm for balanced extracellular proteolysis during cell migration and morphogenesis. Enzyme Protein 49, 138-162 (1996). 45. van Hinsbergh, V.W., Koolwijk, P. & Hanemaaijer, R. Role of fibrin and plasminogen activators in repair-associated angiogenesis: in vitro studies with human endothelial cells. Exs 79, 391-411 (1997). 46. Aimes, R.T., et al. Endothelial cell serine proteases expressed during vascular morphogenesis and angiogenesis. Thrombosis and haemostasis 89, 561-572 (2003). 47. Carmeliet, P., De Smet, F., Loges, S. & Mazzone, M. Branching morphogenesis and antiangiogenesis candidates: tip cells lead the way. Nat Rev Clin Oncol 6, 315-326 (2009).

67 48. Djonov, V.G., Kurz, H. & Burri, P.H. Optimality in the developing vascular system: branching remodeling by means of intussusception as an efficient adaptation mechanism. Dev Dyn 224, 391- 402 (2002). 49. Carmeliet, P. Angiogenesis in health and disease. Nature medicine 9, 653-660 (2003). 50. Nyberg, P., Xie, L. & Kalluri, R. Endogenous inhibitors of angiogenesis. Cancer research 65, 3967-3979 (2005). 51. Ferrara, N., Gerber, H.P. & LeCouter, J. The biology of VEGF and its receptors. Nature medicine 9, 669-676 (2003). 52. Hellberg, C., Ostman, A. & Heldin, C.H. PDGF and vessel maturation. Recent Results Cancer Res 180, 103-114 (2010). 53. Cao, Y., Cao, R. & Hedlund, E.M. R Regulation of tumor angiogenesis and metastasis by FGF and PDGF signaling pathways. J Mol Med (Berl) 86, 785-789 (2008). 54. Grote, K., et al. Toll-like receptor 2/6 stimulation promotes angiogenesis via GM-CSF as a potential strategy for immune defense and tissue regeneration. Blood 115, 2543-2552 (2010). 55. Nissen, L.J., et al. Angiogenic factors FGF2 and PDGF-BB synergistically promote murine tumor neovascularization and metastasis. J Clin Invest 117, 2766-2777 (2007). 56. O'Reilly, M.S., et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277-285 (1997). 57. Sund, M., et al. Function of endogenous inhibitors of angiogenesis as endothelium-specific tumor suppressors. Proceedings of the National Academy of Sciences of the United States of America 102, 2934-2939 (2005). 58. Olsson, A.K., et al. A fragment of histidine-rich glycoprotein is a potent inhibitor of tumor vascularization. Cancer research 64, 599-605 (2004). 59. Thulin, A., et al. Activated platelets provide a functional microenvironment for the antiangiogenic fragment of histidine-rich glycoprotein. Mol Cancer Res 7, 1792-1802 (2009). 60. Ringvall, M., et al. Enhanced platelet activation mediates the accelerated angiogenic switch in mice lacking histidine-rich glycoprotein. PloS one 6, e14526 (2011). 61. Senger, D.R., et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science (New York, N.Y 219, 983-985 (1983). 62. Ferrara, N. & Henzel, W.J. Pituitary follicular cells secrete a novel -binding growth factor specific for vascular endothelial cells. Biochemical and biophysical research communications 161, 851-858 (1989). 63. Olsson, A.K., Dimberg, A., Kreuger, J. & Claesson-Welsh, L. VEGF receptor signalling - in control of vascular function. Nat Rev Mol Cell Biol 7, 359-371 (2006). 64. Cohen, M.M., Jr. Vascular update: morphogenesis, tumors, malformations, and molecular dimensions. American journal of medical genetics 140, 2013-2038 (2006). 65. Ferrara, N. Vascular endothelial growth factor: basic science and clinical progress. Endocrine reviews 25, 581-611 (2004). 66. Carmeliet, P., et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435-439 (1996). 67. Shalaby, F., et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376, 62-66 (1995). 68. Fong, G.H., Rossant, J., Gertsenstein, M. & Breitman, M.L. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 376, 66-70 (1995). 69. West, X.Z., et al. Oxidative stress induces angiogenesis by activating TLR2 with novel endogenous ligands. Nature 467, 972-976 (2010). 70. Tan, W., et al. An essential role for Rac1 in endothelial cell function and vascular development. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 22, 1829-1838 (2008). 71. Arbibe, L., et al. Toll-like receptor 2-mediated NF-kappa B activation requires a Rac1-dependent pathway. Nature 1, 533-540 (2000).

68 72. van Beijnum, J.R., Buurman, W.A. & Griffioen, A.W. Convergence and amplification of toll-like receptor (TLR) and receptor for advanced glycation end products (RAGE) signaling pathways via high mobility group B1 (HMGB1). Angiogenesis 11, 91-99 (2008). 73. Almog, N. Molecular mechanisms underlying tumor dormancy. Cancer Lett 294, 139-146 (2010). 74. Folkman, J. Tumor angiogenesis: therapeutic implications. The New England journal of medicine 285, 1182-1186 (1971). 75. Dvorak, H.F. Angiogenesis: update 2005. Journal of thrombosis and haemostasis : JTH 3, 1835- 1842 (2005). 76. Rosen, L.S. Clinical experience with angiogenesis signaling inhibitors: focus on vascular endothelial growth factor (VEGF) blockers. Cancer Control 9, 36-44 (2002). 77. Matejuk, A., Leng, Q., Chou, S.T. & Mixson, A.J. Vaccines targeting the neovasculature of tumors. Vasc Cell 3, 7 (2011). 78. Gordan, J.D. & Simon, M.C. Hypoxia-inducible factors: central regulators of the tumor phenotype. Curr Opin Genet Dev 17, 71-77 (2007). 79. Gruber, M. & Simon, M.C. Hypoxia-inducible factors, hypoxia, and tumor angiogenesis. Curr Opin Hematol 13, 169-174 (2006). 80. Zhou, L., et al. Silencing of thrombospondin-1 is critical for myc-induced metastatic phenotypes in medulloblastoma. Cancer research 70, 8199-8210 (2010). 81. Kalas, W., et al. Oncogenes and Angiogenesis: down-regulation of thrombospondin-1 in normal fibroblasts exposed to factors from cancer cells harboring mutant ras. Cancer research 65, 8878- 8886 (2005). 82. Folkman, J. & Shing, Y. Angiogenesis. The Journal of biological chemistry 267, 10931-10934 (1992). 83. Jain, R.K. Taming vessels to treat cancer. Scientific American 298, 56-63 (2008). 84. Nanda, A. & St Croix, B. Tumor endothelial markers: new targets for cancer therapy. Curr Opin Oncol 16, 44-49 (2004). 85. Miettinen, M., Rikala, M.S., Rys, J., Lasota, J. & Wang, Z.F. Vascular Endothelial Growth Factor Receptor 2 as a Marker for Malignant Vascular Tumors and Mesothelioma: An Immunohistochemical Study of 262 Vascular Endothelial and 1640 Nonvascular Tumors. Am J Surg Pathol (2012). 86. Munn, L.L. Aberrant vascular architecture in tumors and its importance in drug-based therapies. Drug Discov Today 8, 396-403 (2003). 87. Jain, R.K. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science (New York, N.Y 307, 58-62 (2005). 88. Mazzone, M., et al. Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization. Cell 136, 839-851 (2009). 89. McDonald, D.M. & Choyke, P.L. Imaging of angiogenesis: from microscope to clinic. Nature medicine 9, 713-725 (2003). 90. Wilhelm, S.M., et al. Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling. Mol Cancer Ther 7, 3129-3140 (2008). 91. FDA Approval for Sorafenib Tosylate. http://www.cancer.gov/cancertopics/ druginfo/fda-sorafenib-tosylate access date: march 25th 2012 92. FDA Approval for Sunitinib Malate. http://www.cancer.gov/cancertopics/ druginfo/fda-sunitinib- malate access date: march 25th 2012 93. Ferrara, N. & Kerbel, R.S. Angiogenesis as a therapeutic target. Nature 438, 967-974 (2005). 94. FDA Approval for Bevacizumab. http://www.cancer.gov/cancertopics/druginfo/fda-bevacizumab access date: march 25th 2012 95. Li, S., et al. Structural basis for inhibition of the epidermal growth factor receptor by cetuximab. Cancer cell 7, 301-311 (2005). 96. Hudis, C.A. Trastuzumab--mechanism of action and use in clinical practice. The New England journal of medicine 357, 39-51 (2007).

69 97. Weinberg, R.A. Crowd control: tumor immunology and immunotherapy. in The biology of cancer, 655-724 (Garland Science, Taylor & Francis Group, LLC, New York, 2007). 98. Gilman, A. The initial clinical trial of nitrogen mustard. American journal of surgery 105, 574-578 (1963). 99. Frei, E., 3rd, et al. The effectiveness of combinations of antileukemic agents in inducing and maintaining remission in children with acute leukemia. Blood 26, 642-656 (1965). 100. Weinberg, R.A. The rational treatment of cancer. in The biology of cancer, 725-796 (Garland Science, Taylor & Francis Group, LLC, New york, 2007). 101. Chabner, B.A. & Roberts, T.G., Jr. Timeline: Chemotherapy and the war on cancer. Nature reviews. Cancer 5, 65-72 (2005). 102. Skipper, H.E. & Griswold, D.P. Frank M. Schabel 1918-1983. Cancer research 44, 871-872 (1984). 103. Mihich, E. Historical overview of biologic response modifiers. Cancer Invest 18, 456-466 (2000). 104. Mihich, E. On the immunomodulating effects of anti-cancer drugs and their therapeutic exploitation. Jpn J Clin Oncol 30, 469-471 (2000). 105. Browder, T., et al. Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer. Cancer research 60, 1878-1886 (2000). 106. Klement, G., et al. Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity. J Clin Invest 105, R15-24 (2000). 107. Fedele, P., et al. Efficacy and safety of low-dose metronomic chemotherapy with capecitabine in heavily pretreated patients with metastatic breast cancer. Eur J Cancer 48, 24-29 (2012). 108. Francia, G., et al. Low-Dose Metronomic Oral Dosing of a Prodrug of Gemcitabine (LY2334737) Causes Antitumor Effects in the Absence of Inhibition of Systemic Vasculogenesis. Mol Cancer Ther (2012). 109. Pasquier, E., Kavallaris, M. & Andre, N. Metronomic chemotherapy: new rationale for new directions. Nat Rev Clin Oncol 7, 455-465 (2010). 110. Magnander, K. & Elmroth, K. Biological consequences of formation and repair of complex DNA damage. Cancer Lett (2012). 111. Wilhelm, S.M., et al. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer research 64, 7099-7109 (2004). 112. Wan, P.T., et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116, 855-867 (2004). 113. Gan, H.K., Seruga, B. & Knox, J.J. Sunitinib in solid tumors. Expert Opin Investig Drugs 18, 821- 834 (2009). 114. Sun, L., et al. Discovery of 5-[5-fluoro-2-oxo-1,2- dihydroindol-(3Z)-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide, a novel tyrosine kinase inhibitor targeting vascular endothelial and platelet-derived growth factor receptor tyrosine kinase. J Med Chem 46, 1116-1119 (2003). 115. Mendel, D.B., et al. In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship. Clinical cancer research : an official journal of the American Association for Cancer Research 9, 327-337 (2003). 116. Chow, L.Q. & Eckhardt, S.G. Sunitinib: from rational design to clinical efficacy. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 25, 884-896 (2007). 117. O'Farrell, A.M., et al. SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo. Blood 101, 3597-3605 (2003). 118. An, X., et al. BCR-ABL tyrosine kinase inhibitors in the treatment of Philadelphia chromosome positive chronic myeloid leukemia: a review. Leuk Res 34, 1255-1268 (2010). 119. College voor zorgverzekeringen, Farmacotherapeutisch Kompas. www.fk.cvz.nl access date: march 25th 2012

70 120. Osterlund, P., et al. Hypertension and overall survival in metastatic colorectal cancer patients treated with bevacizumab-containing chemotherapy. British journal of cancer 104, 599-604 (2011). 121. Dahlberg, S.E., Sandler, A.B., Brahmer, J.R., Schiller, J.H. & Johnson, D.H. Clinical course of advanced non-small-cell lung cancer patients experiencing hypertension during treatment with bevacizumab in combination with carboplatin and paclitaxel on ECOG 4599. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 28, 949-954 (2010). 122. Maitland, M.L., et al. Ambulatory monitoring detects sorafenib-induced blood pressure elevations on the first day of treatment. Clinical cancer research : an official journal of the American Association for Cancer Research 15, 6250-6257 (2009). 123. Rees, M.L. & Khakoo, A.Y. Molecular mechanisms of hypertension and heart failure due to antiangiogenic cancer therapies. Heart Fail Clin 7, 299-311 (2011). 124. Genentech. Learn about Avastin today. http://www.avastin.com/avastin/patient/ access date: march 25th 2012 125. FDA approval for trastuzumab. http://www.cancer.gov/cancertopics/druginfo/fda-trastuzumab access date: march 25th 2012 126. Hubbard, S.R. EGF receptor inhibition: attacks on multiple fronts. Cancer cell 7, 287-288 (2005). 127. FDA approval cetuximab. http://www.cancer.gov/cancertopics/druginfo/fda-cetuximab access date: march 25th 2012 128. James, D.F. & Kipps, T.J. Rituximab in chronic lymphocytic leukemia. Adv Ther 28, 534-554 (2011). 129. Maloney, D.G., Smith, B. & Rose, A. Rituximab: mechanism of action and resistance. Semin Oncol 29, 2-9 (2002). 130. Smith, M.R. Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance. Oncogene 22, 7359-7368 (2003). 131. FDA approval for rituximab. http://www.cancer.gov/cancertopics/druginfo/fda-rituximab access date: march 25th 2012 132. Holbro, T., Civenni, G. & Hynes, N.E. The ErbB receptors and their role in cancer progression. Experimental cell research 284, 99-110 (2003). 133. Herbst, R.S. Review of epidermal growth factor receptor biology. International journal of radiation oncology, biology, physics 59, 21-26 (2004). 134. Nahta, R., Yu, D., Hung, M.C., Hortobagyi, G.N. & Esteva, F.J. Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer. Nature clinical practice 3, 269-280 (2006). 135. Ferrara, N. The role of VEGF in the regulation of physiological and pathological angiogenesis. Exs, 209-231 (2005). 136. Jonasch, E., et al. Treatment of metastatic renal carcinoma patients with the combination of gemcitabine, capecitabine and bevacizumab at a tertiary cancer centre. BJU Int 107, 741-747 (2011). 137. Planchard, D. Bevacizumab in non-small-cell lung cancer: a review. Expert Rev Anticancer Ther 11, 1163-1179 (2011). 138. Los, M., Roodhart, J.M. & Voest, E.E. Target practice: lessons from phase III trials with bevacizumab and vatalanib in the treatment of advanced colorectal cancer. Oncologist 12, 443-450 (2007). 139. Sandler, A., et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. The New England journal of medicine 355, 2542-2550 (2006). 140. Jain, R.K., et al. Angiogenesis in brain tumours. Nat Rev Neurosci 8, 610-622 (2007). 141. Abbas, A.K., Lichtman, A.H. & Pillai, S. Antigen receptors and accessory molecules of T lymphocytes. in Cellular and Molecular Immunology 137-149 (Saunders-Elsevier, Philadelphia, 2010). 142. Sharpe, A.H. & Freeman, G.J. The B7-CD28 superfamily. Nat Rev Immunol 2, 116-126 (2002).

71 143. Murphy, K., Travers, P. & Walport, M. Janeway's immunobiology, (Garland Science, Taylor & Francis Group, LLC, New York, NY, 2008). 144. Ribas, A., et al. Role of dendritic cell phenotype, determinant spreading, and negative costimulatory blockade in dendritic cell-based melanoma immunotherapy. Journal of immunotherapy 27, 354-367 (2004). 145. Sutmuller, R.P., et al. Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25(+) regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses. The Journal of experimental medicine 194, 823-832 (2001). 146. Phan, G.Q., et al. Cancer regression and autoimmunity induced by cytotoxic T lymphocyte- associated antigen 4 blockade in patients with metastatic melanoma. Proceedings of the National Academy of Sciences of the United States of America 100, 8372-8377 (2003). 147. Weber, J. Review: anti-CTLA-4 antibody ipilimumab: case studies of clinical response and immune-related adverse events. Oncologist 12, 864-872 (2007). 148. Kahler, K.C. & Hauschild, A. Treatment and side effect management of CTLA-4 antibody therapy in metastatic melanoma. J Dtsch Dermatol Ges 9, 277-286 (2011). 149. Ridolfi, L., Petrini, M., Fiammenghi, L., Riccobon, A. & Ridolfi, R. Human embryo immune escape mechanisms rediscovered by the tumor. Immunobiology 214, 61-76 (2009). 150. Lollini, P.L., Cavallo, F., Nanni, P. & Forni, G. Vaccines for tumour prevention. Nature reviews 6, 204-216 (2006). 151. Curiel, T.J., et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nature medicine 10, 942-949 (2004). 152. Orentas, R., Hodge, J.W. & Johnson, B.D. Cancer vaccines and tumor immunity, (John Wiley & Sons, Inc., Hoboken, NJ, 2008). 153. Ghazal-Aswad, S. Cervical cancer prevention in the human papilloma virus vaccine era. Annals of the New York Academy of Sciences 1138, 253-256 (2008). 154. Harper, D.M. Currently approved prophylactic HPV vaccines. Expert review of vaccines 8, 1663- 1679 (2009). 155. Abbas, A.K., Lichtman, A.H. & Pillai, S. Immunological tolerance. in Cellular and Molecular Immunology 243-263 (Saunders-Elsevier, Philadelphia, PA, 2010). 156. Kaufmann, S.H. & Hess, J. Impact of intracellular location of and antigen display by intracellular bacteria: implications for vaccine development. Immunology letters 65, 81-84 (1999). 157. Helling, F., et al. GD3 vaccines for melanoma: superior immunogenicity of keyhole limpet hemocyanin conjugate vaccines. Cancer research 54, 197-203 (1994). 158. Helling, F., et al. GM2-KLH : increased immunogenicity in melanoma patients after administration with immunological adjuvant QS-21. Cancer research 55, 2783-2788 (1995). 159. Li, Y., et al. against the vascular endothelial growth factor receptor flk1 inhibits tumor angiogenesis and metastasis. The Journal of experimental medicine 195, 1575-1584 (2002). 160. Food and Drug Administration. BCG live (intravesical), TheraCys. http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Compli anceActivities/Enforcement/UntitledLetters/ucm092731.htm access date: march 25th 2012 161. Quaak, S.G., et al. GMP production of pDERMATT for vaccination against melanoma in a phase I clinical trial. Eur J Pharm Biopharm 70, 429-438 (2008). 162. Haller, B.K., et al. Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma. Oncogene 29, 4276-4286 (2010). 163. Niethammer, A.G., et al. A DNA vaccine against VEGF receptor 2 prevents effective angiogenesis and inhibits tumor growth. Nature medicine 8, 1369-1375 (2002). 164. Michael, A., et al. Delayed disease progression after allogeneic cell vaccination in hormone- resistant prostate cancer and correlation with immunologic variables. Clinical cancer research : an official journal of the American Association for Cancer Research 11, 4469-4478 (2005).

72 165. Vilella, R., et al. Treatment of patients with progressive unresectable metastatic melanoma with a heterologous polyvalent melanoma whole cell vaccine. International journal of cancer. Journal international du cancer 106, 626-631 (2003). 166. Small, E.J., et al. Placebo-controlled phase III trial of immunologic therapy with sipuleucel-T (APC8015) in patients with metastatic, asymptomatic hormone refractory prostate cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 24, 3089-3094 (2006). 167. Forsberg, O., et al. High frequency of prostate antigen-directed T cells in cancer patients compared to healthy age-matched individuals. The Prostate 69, 70-81 (2009). 168. Mackensen, A., et al. Phase I study of adoptive T-cell therapy using antigen-specific CD8+ T cells for the treatment of patients with metastatic melanoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 24, 5060-5069 (2006). 169. Yee, C., et al. Adoptive therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. Proceedings of the National Academy of Sciences of the United States of America 99, 16168-16173 (2002). 170. FDA approval for Sipuleucel-T. http://www.cancer.gov/cancertopics/druginfo/fda-sipuleucel-T access date: march 25th 2012 171. Dendreon Corporation, Provenge (Sipuleucel-T) Active cellular immunotherapy of metastatic prostate cancer after failing hormone therapy. (U.S. National Institutes of Health, 2010). http://clinicaltrials.gov/ show/NCT00065442 access date: 25th march 2012 172. Department of health and human services, Statistical review and evaluation bla. (U.S. Food and Drug Administration, 2010). http://www.fda.gov/downloads/BiologicalsBloodVaccines/ CellularGeneTherapyProducts/ApprovedProducts(UCM214543.pdf access date: march 25th 2012 173. Wesley, J., Whitmore, J., Trager, J. & Sheikh, N. An overview of sipuleucel-T: Autologous cellular immunotherapy for prostate cancer. Hum Vaccin Immunother 8(2012). 174. Joniau, S., et al. Current vaccination strategies for prostate cancer. Eur Urol 61, 290-306 (2012). 175. Kantoff, P.W., et al. Sipuleucel-T immunotherapy for castration-resistant prostate cancer. The New England journal of medicine 363, 411-422 (2010). 176. Fong, M.K., Hare, R. & Jarkowski, A. A new era for castrate resistant prostate cancer: A treatment review and update. J Oncol Pharm Pract (2012). 177. Goldman, B. & DeFrancesco, L. The cancer vaccine roller coaster. Nature biotechnology 27, 129- 139 (2009). 178. Sheikh, N.A. & Jones, L.A. CD54 is a surrogate marker of antigen presenting cell activation. Cancer immunology, immunotherapy : CII 57, 1381-1390 (2008). 179. Mydlo, J.H., Usher, S.M., Camacho, F. & Freed, S. Retrospective study of efficacy of intravesical BCG alone in treatment of superficial bladder cancer. Urology 28, 173-175 (1986). 180. Lamm, D.L., et al. A randomized trial of intravesical doxorubicin and immunotherapy with bacille Calmette-Guerin for transitional-cell carcinoma of the bladder. The New England journal of medicine 325, 1205-1209 (1991). 181. Lamm, D.L., et al. Maintenance bacillus Calmette-Guerin immunotherapy for recurrent TA, T1 and carcinoma in situ transitional cell carcinoma of the bladder: a randomized Southwest Oncology Group Study. J Urol 163, 1124-1129 (2000). 182. National Cancer Institute. Stage information for bladder cancer. (2012). http://www.cancer.gov/cancertopics/pdq/treatment/bladder/HealthProfessional/page3 access date: march 25th 2012 183. Wilson, R.M. & Danishefsky, S.J. Fully synthetic carbohydrate-based antitumor vaccines. in Cancer vaccines and tumor immunity (eds. Orentas, R.J., Hodge, J.W. & Johnson, B.D.) (John Wiley & Sons Inc., Hoboken, NJ, 2008). 184. Le Poole, I.C., Gerberi, M.A. & Kast, W.M. Emerging strategies in tumor vaccines. Curr Opin Oncol 14, 641-648 (2002).

73 185. Slovin, S.F., et al. Fully synthetic carbohydrate-based vaccines in biochemically relapsed prostate cancer: clinical trial results with alpha-N-acetylgalactosamine-O-serine/threonine conjugate vaccine. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 21, 4292-4298 (2003). 186. Heimburg-Molinaro, J., et al. Cancer vaccines and carbohydrate epitopes. Vaccine 29, 8802-8826 (2011). 187. Rosenberg, S.A., Yang, J.C. & Restifo, N.P. Cancer immunotherapy: moving beyond current vaccines. Nature medicine 10, 909-915 (2004). 188. Speiser, D.E., et al. Self antigens expressed by solid tumors Do not efficiently stimulate naive or activated T cells: implications for immunotherapy. The Journal of experimental medicine 186, 645- 653 (1997). 189. Muranski, P., et al. Increased intensity lymphodepletion and adoptive immunotherapy--how far can we go? Nature clinical practice. Oncology 3, 668-681 (2006). 190. Rosenberg, S.A., Restifo, N.P., Yang, J.C., Morgan, R.A. & Dudley, M.E. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nature reviews. Cancer 8, 299-308 (2008). 191. Hu, D.E., Moore, A.M., Thomsen, L.L. & Brindle, K.M. Uric acid promotes tumor immune rejection. Cancer research 64, 5059-5062 (2004). 192. Apetoh, L., et al. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nature medicine 13, 1050-1059 (2007). 193. Kaisho, T. & Akira, S. Regulation of dendritic cell function through toll-like receptors. Current molecular medicine 3, 759-771 (2003). 194. Abbas, A.K., Lichtman, A.H. & Pillai, S. Activation of T lymphocytes. in Cellular and Molecular Immunology, 189-214 (Saunders-Elsevier, Philadelphia, 2010). 195. van der Most, R.G., Currie, A.J., Robinson, B.W. & Lake, R.A. Decoding dangerous death: how cytotoxic chemotherapy invokes inflammation, immunity or nothing at all. Cell Death Differ 15, 13-20 (2008). 196. Zitvogel, L., Apetoh, L., Ghiringhelli, F. & Kroemer, G. Immunological aspects of cancer chemotherapy. Nat Rev Immunol 8, 59-73 (2008). 197. Obeid, M., et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nature medicine 13, 54-61 (2007). 198. Schiavoni, G., et al. Cyclophosphamide induces type I interferon and augments the number of CD44(hi) T lymphocytes in mice: implications for strategies of chemoimmunotherapy of cancer. Blood 95, 2024-2030 (2000). 199. Mokyr, M.B., Place, A.T., Artwohl, J.E. & Valli, V.E. Importance of signaling via the IFN- alpha/beta receptor on host cells for the realization of the therapeutic benefits of cyclophosphamide for mice bearing a large MOPC-315 tumor. Cancer immunology, immunotherapy : CII 55, 459-468 (2006). 200. Giordani, L., et al. IFN-alpha amplifies human naive TLR-9-mediated activation and Ig production. Journal of leukocyte biology 86, 261-271 (2009). 201. Zou, W. Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol 6, 295-307 (2006). 202. Brode, S. & Cooke, A. Immune-potentiating effects of the chemotherapeutic drug cyclophosphamide. Crit Rev Immunol 28, 109-126 (2008). 203. Schlom, J. Cancer vaccines and cancer immunotherapy: a new paradigm. in Cancer vaccines and tumor immunity, foreword XV(eds. Orentas, R., Hodge, J.W. & Johnson, B.D.) (John Wiley & Sons, Inc., Hoboken, NJ, 2008). 204. Chakraborty, M., et al. Irradiation of tumor cells up-regulates Fas and enhances CTL lytic activity and CTL adoptive immunotherapy. Journal of immunology 170, 6338-6347 (2003). 205. Chakraborty, M., et al. External beam radiation of tumors alters phenotype of tumor cells to render them susceptible to vaccine-mediated T-cell killing. Cancer research 64, 4328-4337 (2004). 206. Garnett, C.T., et al. Sublethal irradiation of human tumor cells modulates phenotype resulting in enhanced killing by cytotoxic T lymphocytes. Cancer research 64, 7985-7994 (2004).

74 207. Chen, Z., et al. Combined radiation therapy and dendritic cell vaccine for treating solid tumors with liver micro-metastasis. J Gene Med 7, 506-517 (2005). 208. Hida, K., Hida, Y. & Shindoh, M. Understanding tumor endothelial cell abnormalities to develop ideal anti-angiogenic therapies. Cancer science 99, 459-466 (2008). 209. Boehm, T., Folkman, J., Browder, T. & O'Reilly, M.S. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390, 404-407 (1997). 210. Huang, J., et al. Vascular remodeling marks tumors that recur during chronic suppression of angiogenesis. Mol Cancer Res 2, 36-42 (2004). 211. Hida, K., et al. Tumor-associated endothelial cells with cytogenetic abnormalities. Cancer research 64, 8249-8255 (2004). 212. Hida, K. & Klagsbrun, M. A new perspective on tumor endothelial cells: unexpected chromosome and centrosome abnormalities. Cancer research 65, 2507-2510 (2005). 213. Ricci-Vitiani, L., et al. Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature 468, 824-828 (2010). 214. Wang, R., et al. Glioblastoma stem-like cells give rise to tumour endothelium. Nature 468, 829- 833 (2010). 215. Folkman, J. Fighting cancer by attacking its blood supply. Scientific American 275, 150-154 (1996). 216. Folkman, J. Angiogenic therapy. in Prinicples and practice of oncology (eds. DeVita, V.T., Hellman, S. & Rosenberg, S.A.) 3075-3085 (Lippincott-Raven, Philidelphia, 1997). 217. Seavey, M.M., Maciag, P.C., Al-Rawi, N., Sewell, D. & Paterson, Y. An anti-vascular endothelial growth factor receptor 2/fetal liver kinase-1 Listeria monocytogenes anti-angiogenesis cancer vaccine for the treatment of primary and metastatic Her-2/neu+ breast tumors in a mouse model. J Immunol 182, 5537-5546 (2009). 218. Jubb, A.M., et al. Expression of vascular notch ligand delta-like 4 and inflammatory markers in breast cancer. The American journal of pathology 176, 2019-2028 (2010). 219. Jubb, A.M., et al. Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer. British journal of cancer 101, 1749-1757 (2009). 220. Miyazawa, M., et al. Phase I clinical trial using peptide vaccine for human vascular endothelial growth factor receptor 2 in combination with gemcitabine for patients with advanced pancreatic cancer. Cancer science (2009). 221. Lee, S., et al. Autocrine VEGF signaling is required for vascular homeostasis. Cell 130, 691-703 (2007). 222. Engel, J. & Chiquet, M. An overview of extracellular matrix structure and function. in The extracellular matrix: an overview (ed. Mecham, R.P.) (Springer-Verlag, Berlin-Heidelberg, 2011). 223. White, E.S. & Muro, A.F. Fibronectin splice variants: understanding their multiple roles in health and disease using engineered mouse models. IUBMB Life 63, 538-546 (2011). 224. Alberts, B., et al. in Mol Biol Cell (Garland Science, New York, 2002). 225. Klass, C.M., Couchman, J.R. & Woods, A. Control of extracellular matrix assembly by syndecan-2 proteoglycan. J Cell Sci 113 ( Pt 3), 493-506 (2000). 226. Leitinger, B. & Hohenester, E. Mammalian collagen receptors. Matrix biology : journal of the International Society for Matrix Biology 26, 146-155 (2007). 227. Kalluri, R. Basement membranes: structure, assembly and role in tumour angiogenesis. Nature reviews. Cancer 3, 422-433 (2003). 228. Weinberg, R.A. Dialogue replaces monologue: heterotypic interactions and the biology of angiogenesis. in The biology of cancer, 527-586 (Garland Science, Taylor & Francis Group, LLC, New York, 2007). 229. Ffrench-Constant, C., Van de Water, L., Dvorak, H.F. & Hynes, R.O. Reappearance of an embryonic pattern of fibronectin splicing during wound healing in the adult rat. The Journal of cell biology 109, 903-914 (1989).

75 230. Glukhova, M.A., Frid, M.G., Shekhonin, B.V., Balabanov, Y.V. & Koteliansky, V.E. Expression of fibronectin variants in vascular and visceral smooth muscle cells in development. Developmental biology 141, 193-202 (1990). 231. Peters, J.H. & Hynes, R.O. Fibronectin isoform distribution in the mouse. I. The alternatively spliced EIIIB, EIIIA, and V segments show widespread codistribution in the developing mouse embryo. Cell adhesion and communication 4, 103-125 (1996). 232. Joester, A. & Faissner, A. Evidence for combinatorial variability of tenascin-C isoforms and developmental regulation in the mouse central nervous system. The Journal of biological chemistry 274, 17144-17151 (1999). 233. Erickson, H.P. & Bourdon, M.A. Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors. Annual review of cell biology 5, 71-92 (1989). 234. Faissner, A. The tenascin gene family in axon growth and guidance. Cell Tissue Res 290, 331-341 (1997). 235. Castellani, P., et al. The fibronectin isoform containing the ED-B oncofetal domain: a marker of angiogenesis. International journal of cancer 59, 612-618 (1994). 236. Rybak, J.N., Roesli, C., Kaspar, M., Villa, A. & Neri, D. The extra-domain A of fibronectin is a vascular marker of solid tumors and metastases. Cancer research 67, 10948-10957 (2007). 237. Chiquet-Ehrismann, R., Hagios, C. & Schenk, S. The complexity in regulating the expression of tenascins. Bioessays 17, 873-878 (1995). 238. Silacci, M., et al. Human monoclonal antibodies to domain C of tenascin-C selectively target solid tumors in vivo. Protein engineering, design & selection : PEDS 19, 471-478 (2006). 239. Singh, P., et al. The spatial and temporal expression patterns of integrin alpha9beta1 and one of its ligands, the EIIIA segment of fibronectin, in cutaneous wound healing. The Journal of investigative dermatology 123, 1176-1181 (2004). 240. Kosmehl, H., Berndt, A. & Katenkamp, D. Molecular variants of fibronectin and laminin: structure, physiological occurrence and histopathological aspects. Virchows Arch 429, 311-322 (1996). 241. Carnemolla, B., et al. A tumor-associated fibronectin isoform generated by alternative splicing of messenger RNA precursors. The Journal of cell biology 108, 1139-1148 (1989). 242. White, E.S., Baralle, F.E. & Muro, A.F. New insights into form and function of fibronectin splice variants. The Journal of pathology 216, 1-14 (2008). 243. Astrof, S. & Hynes, R.O. Fibronectins in vascular morphogenesis. Angiogenesis 12, 165-175 (2009). 244. ffrench-Constant, C. Alternative splicing of fibronectin--many different proteins but few different functions. Experimental cell research 221, 261-271 (1995). 245. Kornblihtt, A.R., et al. The fibronectin gene as a model for splicing and transcription studies. Faseb J 10, 248-257 (1996). 246. Peters, J.H., Chen, G.E. & Hynes, R.O. Fibronectin isoform distribution in the mouse. II. Differential distribution of the alternatively spliced EIIIB, EIIIA, and V segments in the adult mouse. Cell adhesion and communication 4, 127-148 (1996). 247. Vartio, T., et al. Differential expression of the ED sequence-containing form of cellular fibronectin in embryonic and adult human tissues. J Cell Sci 88 ( Pt 4), 419-430 (1987). 248. Peters, J.H., Trevithick, J.E., Johnson, P. & Hynes, R.O. Expression of the alternatively spliced EIIIB segment of fibronectin. Cell adhesion and communication 3, 67-89 (1995). 249. Borsi, L., Castellani, P., Allemanni, G., Neri, D. & Zardi, L. Preparation of phage antibodies to the ED-A domain of human fibronectin. Experimental cell research 240, 244-251 (1998). 250. Villa, A., et al. A high-affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo-vasculature in vivo. International journal of cancer 122, 2405-2413 (2008). 251. Astrof, S., et al. Direct test of potential roles of EIIIA and EIIIB alternatively spliced segments of fibronectin in physiological and tumor angiogenesis. Molecular and cellular biology 24, 8662- 8670 (2004).

76 252. Ruoslahti, E. Specialization of tumour vasculature. Nature reviews 2, 83-90 (2002). 253. Carnemolla, B., Leprini, A., Allemanni, G., Saginati, M. & Zardi, L. The inclusion of the type III repeat ED-B in the fibronectin molecule generates conformational modifications that unmask a cryptic sequence. The Journal of biological chemistry 267, 24689-24692 (1992). 254. Manabe, R., Ohe, N., Maeda, T., Fukuda, T. & Sekiguchi, K. Modulation of cell-adhesive activity of fibronectin by the alternatively spliced EDA segment. The Journal of cell biology 139, 295-307 (1997). 255. Baneyx, G., Baugh, L. & Vogel, V. Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension. Proceedings of the National Academy of Sciences of the United States of America 99, 5139-5143 (2002). 256. Krammer, A., Lu, H., Isralewitz, B., Schulten, K. & Vogel, V. Forced unfolding of the fibronectin type III module reveals a tensile molecular recognition switch. Proceedings of the National Academy of Sciences of the United States of America 96, 1351-1356 (1999). 257. Shi, Y. & Manley, J.L. A complex signaling pathway regulates SRp38 phosphorylation and pre- mRNA splicing in response to heat shock. Mol Cell 28, 79-90 (2007). 258. Prasad, J. & Manley, J.L. Regulation and substrate specificity of the SR protein kinase Clk/Sty. Molecular and cellular biology 23, 4139-4149 (2003). 259. Blaustein, M., et al. Concerted regulation of nuclear and cytoplasmic activities of SR proteins by AKT. Nat Struct Mol Biol 12, 1037-1044 (2005). 260. Neri, D. & Bicknell, R. Tumour vascular targeting. Nature reviews 5, 436-446 (2005). 261. Erickson, H.P. Tenascin-C, tenascin-R and tenascin-X: a family of talented proteins in search of functions. Current opinion in cell biology 5, 869-876 (1993). 262. Midwood, K.S. & Orend, G. The role of tenascin-C in tissue injury and tumorigenesis. Journal of cell communication and signaling (2009). 263. Hynes, R.O. Fibronectins, (Springer-Verlag New York Inc., New York, 1990). 264. Scheuermann, J. ETH Zürich. PhD thesis (2002). 265. Zardi, L., et al. Transformed human cells produce a new fibronectin isoform by preferential alternative splicing of a previously unobserved exon. The EMBO journal 6, 2337-2342 (1987). 266. Fukuda, T., et al. Mice lacking the EDB segment of fibronectin develop normally but exhibit reduced cell growth and fibronectin matrix assembly in vitro. Cancer research 62, 5603-5610 (2002). 267. Astrof, S., Crowley, D. & Hynes, R.O. Multiple cardiovascular defects caused by the absence of alternatively spliced segments of fibronectin. Developmental biology 311, 11-24 (2007). 268. Khan, Z.A., et al. EDB fibronectin and angiogenesis -- a novel mechanistic pathway. Angiogenesis 8, 183-196 (2005). 269. Borsi, L., Castellani, P., Risso, A.M., Leprini, A. & Zardi, L. Transforming growth factor-beta regulates the splicing pattern of fibronectin messenger RNA precursor. FEBS letters 261, 175-178 (1990). 270. Balza, E., Borsi, L., Allemanni, G. & Zardi, L. Transforming growth factor beta regulates the levels of different fibronectin isoforms in normal human cultured fibroblasts. FEBS letters 228, 42- 44 (1988). 271. Borsi, L., Balza, E., Allemanni, G. & Zardi, L. Differential expression of the fibronectin isoform containing the ED-B oncofetal domain in normal human fibroblast cell lines originating from different tissues. Experimental cell research 199, 98-105 (1992). 272. Nicolo, G., et al. Expression of tenascin and of the ED-B containing oncofetal fibronectin isoform in human cancer. Cell Differ Dev 32, 401-408 (1990). 273. Sauer, S., et al. Expression of the oncofetal ED-B-containing fibronectin isoform in hematologic tumors enables ED-B-targeted 131I-L19SIP radioimmunotherapy in Hodgkin lymphoma patients. Blood 113, 2265-2274 (2009). 274. Midulla, M., et al. Source of oncofetal ED-B-containing fibronectin: implications of production by both tumor and endothelial cells. Cancer research 60, 164-169 (2000).

77 275. Pujuguet, P., et al. Expression of fibronectin ED-A+ and ED-B+ isoforms by human and experimental colorectal cancer. Contribution of cancer cells and tumor-associated myofibroblasts. The American journal of pathology 148, 579-592 (1996). 276. Nagy, J.A., Benjamin, L., Zeng, H., Dvorak, A.M. & Dvorak, H.F. Vascular permeability, vascular hyperpermeability and angiogenesis. Angiogenesis 11, 109-119 (2008). 277. Nagy, J.A., Chang, S.H., Dvorak, A.M. & Dvorak, H.F. Why are tumour blood vessels abnormal and why is it important to know? British journal of cancer 100, 865-869 (2009). 278. Wagner, C., et al. Fibronectin synthesis by activated T lymphocytes: up-regulation of a surface- associated isoform with signalling function. Immunology 99, 532-539 (2000). 279. Balza, E., et al. A novel human fibronectin cryptic sequence unmasked by the insertion of the angiogenesis-associated extra type III domain B. International journal of cancer 125, 751-758 (2009). 280. Bencharit, S., et al. Structural insights into fibronectin type III domain-mediated signaling. Journal of molecular biology 367, 303-309 (2007). 281. Wagner, K., Schulz, P., Scholz, A., Wiedenmann, B. & Menrad, A. The targeted immunocytokine L19-IL2 efficiently inhibits the growth of orthotopic pancreatic cancer. Clin Cancer Res 14, 4951- 4960 (2008). 282. Borsi, L., et al. Selective targeting of tumoral vasculature: comparison of different formats of an antibody (L19) to the ED-B domain of fibronectin. International journal of cancer 102, 75-85 (2002). 283. McCafferty, J., Griffiths, A.D., Winter, G. & Chiswell, D.J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552-554 (1990). 284. Murphy, K., Travers, P. & Walport, M. Appendix I: Immunologists’ Toolbox. in Janeway's immunobiology (ed. Lawrence, E.) 750-751 (Garland Science, Taylor & Francis Group, LLC, New York, NY, 2008). 285. Kindt TJ, Goldsby RA, Osborne BA & J, K. Kuby Immunology, (W.H. Freeman and Company, New York, NY, 2007). 286. Murphy, K., Travers, P. & Walport, M. Appendix I: Immunologists’ Toolbox in Janeway's immunobiology (ed. Lawrence, E.) 749-750 (Garland Science, Taylor & Francis Group, LLC, New York, NJ, 2008). 287. Abbas, A.K., Lichtman, A.H. & Pillai, S. Antibodies and antigens. in Cellular and Molecular Immunology 75-96 (Saunders-Elsevier, Philadelphia, PA, 2010). 288. Nakazawa, M., Mukumoto, M. & Miyatake, K. Production and purification of monoclonal antibodies. Methods in molecular biology 657, 75-91 (2010). 289. Fowler, N. & Younes, A. There will be blood: targeting tumor vasculature. Blood 113, 2121-2122 (2009). 290. Borsi, L., et al. Selective targeted delivery of TNFalpha to tumor blood vessels. Blood 102, 4384- 4392 (2003). 291. Philogen S.p.A.. Clinical trials. http://www.philogen.com/clinical-trials.html access date: march 25th 2012 292. Nilsson, F., Kosmehl, H., Zardi, L. & Neri, D. Targeted delivery of tissue factor to the ED-B domain of fibronectin, a marker of angiogenesis, mediates the infarction of solid tumors in mice. Cancer research 61, 711-716 (2001). 293. Schliemann, C., et al. Complete eradication of human B-cell lymphoma xenografts using rituximab in combination with the immunocytokine L19-IL2. Blood 113, 2275-2283 (2009). 294. Carnemolla, B., et al. Enhancement of the antitumor properties of interleukin-2 by its targeted delivery to the tumor blood vessel extracellular matrix. Blood 99, 1659-1665 (2002). 295. Johannsen, M., et al. The tumour-targeting human L19-IL2 immunocytokine: preclinical safety studies, phase I clinical trial in patients with solid tumours and expansion into patients with advanced renal cell carcinoma. Eur J Cancer 46, 2926-2935 (2010).

78 296. Liao, Y.F., Wieder, K.G., Classen, J.M. & Van De Water, L. Identification of two amino acids within the EIIIA (ED-A) segment of fibronectin constituting the epitope for two function-blocking monoclonal antibodies. The Journal of biological chemistry 274, 17876-17884 (1999). 297. Inoue, T., Nabeshima, K., Shimao, Y., Kataoka, H. & Koono, M. Cell density-dependent regulation of fibronectin splicing at the EDA region in fibroblasts: cell density also modulates the responses of fibroblasts to TGF-beta and cancer cell-conditioned medium. Cancer Lett 129, 45-54 (1998). 298. Viedt, C., Burger, A. & Hansch, G.M. Fibronectin synthesis in tubular epithelial cells: up- regulation of the EDA splice variant by transforming growth factor beta. Kidney Int 48, 1810-1817 (1995). 299. Burger, A., et al. Fibronectin synthesis by human tubular epithelial cells in culture: effects of PDGF and TGF-beta on synthesis and splicing. Kidney Int 54, 407-415 (1998). 300. Przybysz, M., Borysewicz, K. & Katnik-Prastowska, I. Differences between the early and advanced stages of rheumatoid arthritis in the expression of EDA-containing fibronectin. Rheumatology international 29, 1397-1401 (2009). 301. Muro, A.F., et al. Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan. The Journal of cell biology 162, 149-160 (2003). 302. Szell, M., et al. Proliferating keratinocytes are putative sources of the psoriasis susceptibility- related EDA+ (extra domain A of fibronectin) oncofetal fibronectin. The Journal of investigative dermatology 123, 537-546 (2004). 303. Ting, K.M., et al. Overexpression of the oncofetal Fn variant containing the EDA splice-in segment in the dermal-epidermal junction of psoriatic uninvolved skin. The Journal of investigative dermatology 114, 706-711 (2000). 304. Muro, A.F., et al. An essential role for fibronectin extra type III domain A in pulmonary fibrosis. American journal of respiratory and critical care medicine 177, 638-645 (2008). 305. Jarnagin, W.R., Rockey, D.C., Koteliansky, V.E., Wang, S.S. & Bissell, D.M. Expression of variant fibronectins in wound healing: cellular source and biological activity of the EIIIA segment in rat hepatic fibrogenesis. The Journal of cell biology 127, 2037-2048 (1994). 306. Serini, G., et al. The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1. The Journal of cell biology 142, 873-881 (1998). 307. Tomasek, J.J., Gabbiani, G., Hinz, B., Chaponnier, C. & Brown, R.A. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 3, 349-363 (2002). 308. Okamura, Y., et al. The extra domain A of fibronectin activates Toll-like receptor 4. The Journal of biological chemistry 276, 10229-10233 (2001). 309. Lasarte, J.J., et al. The extra domain A from fibronectin targets antigens to TLR4-expressing cells and induces cytotoxic T cell responses in vivo. Journal of immunology 178, 748-756 (2007). 310. Sandig, H., et al. Fibronectin is a TH1-specific molecule in human subjects. J Allergy Clin Immunol 124, 528-535, 535 e521-525 (2009). 311. Kanters, S.D., et al. Plasma levels of cellular fibronectin in diabetes. Diabetes Care 24, 323-327 (2001). 312. Peters, J.H., Maunder, R.J., Woolf, A.D., Cochrane, C.G. & Ginsberg, M.H. Elevated plasma levels of ED1+ ("cellular") fibronectin in patients with vascular injury. J Lab Clin Med 113, 586- 597 (1989). 313. Voskuyl, A.E., et al. Levels of circulating cellular fibronectin are increased in patients with rheumatoid vasculitis. Clin Exp Rheumatol 16, 429-434 (1998). 314. Kriegsmann, J., et al. Expression of fibronectin splice variants and oncofetal glycosylated fibronectin in the synovial membranes of patients with rheumatoid arthritis and osteoarthritis. Rheumatology international 24, 25-33 (2004). 315. Gondokaryono, S.P., et al. The extra domain A of fibronectin stimulates murine mast cells via toll- like receptor 4. Journal of leukocyte biology 82, 657-665 (2007).

79 316. Lefebvre, J.S., et al. Extra domain A of fibronectin primes leukotriene biosynthesis and stimulates neutrophil migration through activation of Toll-like receptor 4. Arthritis Rheum 63, 1527-1533 (2011). 317. Kuhn, C. & McDonald, J.A. The roles of the myofibroblast in idiopathic pulmonary fibrosis. Ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis. The American journal of pathology 138, 1257-1265 (1991). 318. Magnusson, M.K. & Mosher, D.F. Fibronectin: structure, assembly, and cardiovascular implications. Arteriosclerosis, thrombosis, and vascular biology 18, 1363-1370 (1998). 319. Clark, R.A., et al. Blood vessel fibronectin increases in conjunction with endothelial cell proliferation and capillary ingrowth during wound healing. The Journal of investigative dermatology 79, 269-276 (1982). 320. Clark, R.A., et al. Fibronectin is produced by blood vessels in response to injury. The Journal of experimental medicine 156, 646-651 (1982). 321. Paul, J.I., Schwarzbauer, J.E., Tamkun, J.W. & Hynes, R.O. Cell-type-specific fibronectin subunits generated by alternative splicing. The Journal of biological chemistry 261, 12258-12265 (1986). 322. Rencic, A., Gehris, A.L., Lewis, S.D., Hume, E.L. & Bennett, V.D. Splicing patterns of fibronectin mRNA from normal and osteoarthritic human articular cartilage. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society 3, 187-196 (1995). 323. Burton-Wurster, N. & Lust, G. Fibronectin in cartilage. in Fibronectin in health and disease (ed. Carsons, S.) 243-255 (CRC Press, Boca Raton, FL, 1989). 324. Han, F., et al. Transforming growth factor-beta1 (TGF-beta1) regulates ATDC5 chondrogenic differentiation and fibronectin isoform expression. Journal of cellular biochemistry 95, 750-762 (2005). 325. Burton-Wurster, N., Lust, G. & Macleod, J.N. Cartilage fibronectin isoforms: in search of functions for a special population of matrix glycoproteins. Matrix Biol 15, 441-454 (1997). 326. Carnemolla, B., et al. Identification of a glioblastoma-associated tenascin-C isoform by a high affinity recombinant antibody. The American journal of pathology 154, 1345-1352 (1999). 327. Chiquet-Ehrismann, R. Tenascin and other adhesion-modulating proteins in cancer. Seminars in cancer biology 4, 301-310 (1993). 328. Pas, J., et al. Analysis of structure and function of tenascin-C. The international journal of biochemistry & cell biology 38, 1594-1602 (2006). 329. Gherzi, R., et al. Human tenascin gene. Structure of the 5'-region, identification, and characterization of the transcription regulatory sequences. The Journal of biological chemistry 270, 3429-3434 (1995). 330. Siri, A., et al. Human tenascin: primary structure, pre-mRNA splicing patterns and localization of the epitopes recognized by two monoclonal antibodies. Nucleic acids research 19, 525-531 (1991). 331. Ljubimov, A.V., et al. Expression of tenascin-C splice variants in normal and bullous keratopathy human corneas. Investigative ophthalmology & visual science 39, 1135-1142 (1998). 332. Guttery, D.S., Shaw, J.A., Lloyd, K., Pringle, J.H. & Walker, R.A. Expression of tenascin-C and its isoforms in the breast. Cancer Metastasis Rev 29, 595-606 (2010). 333. Joester, A. & Faissner, A. The structure and function of tenascins in the nervous system. Matrix biology : journal of the International Society for Matrix Biology 20, 13-22 (2001). 334. Berndt, A., et al. Differential expression of tenascin-C splicing domains in urothelial carcinomas of the urinary bladder. Journal of cancer research and clinical oncology 132, 537-546 (2006). 335. Borsi, L., et al. Cell-cycle dependent alternative splicing of the tenascin primary transcript. Cell adhesion and communication 1, 307-317 (1994). 336. Borsi, L., Allemanni, G., Gaggero, B. & Zardi, L. Extracellular pH controls pre-mRNA alternative splicing of tenascin-C in normal, but not in malignantly transformed, cells. International journal of cancer 66, 632-635 (1996). 337. Borsi, L., Balza, E., Gaggero, B., Allemanni, G. & Zardi, L. The alternative splicing pattern of the tenascin-C pre-mRNA is controlled by the extracellular pH. The Journal of biological chemistry 270, 6243-6245 (1995).

80 338. Pearson, C.A., Pearson, D., Shibahara, S., Hofsteenge, J. & Chiquet-Ehrismann, R. Tenascin: cDNA cloning and induction by TGF-beta. The EMBO journal 7, 2977-2982 (1988). 339. Tucker, R.P., Hammarback, J.A., Jenrath, D.A., Mackie, E.J. & Xu, Y. Tenascin expression in the mouse: in situ localization and induction in vitro by bFGF. J Cell Sci 104 ( Pt 1), 69-76 (1993). 340. Sakai, T., Kawakatsu, H., Ohta, M. & Saito, M. Tenascin induction in tenascin nonproducing carcinoma cell lines in vivo and by TGF-beta 1 in vitro. J Cell Physiol 159, 561-572 (1994). 341. LaFleur, D.W., Fagin, J.A., Forrester, J.S., Rubin, S.A. & Sharifi, B.G. Cloning and characterization of alternatively spliced isoforms of rat tenascin. Platelet-derived growth factor-BB markedly stimulates expression of spliced variants of tenascin mRNA in arterial smooth muscle cells. The Journal of biological chemistry 269, 20757-20763 (1994). 342. Mackie, E.J. & Tucker, R.P. The tenascin-C knockout revisited. J Cell Sci 112 ( Pt 22), 3847-3853 (1999). 343. Evers, M.R., et al. Impairment of L-type Ca2+ channel-dependent forms of hippocampal synaptic plasticity in mice deficient in the extracellular matrix glycoprotein tenascin-C. J Neurosci 22, 7177-7194 (2002). 344. Forsberg, E., et al. Skin wounds and severed nerves heal normally in mice lacking tenascin-C. Proceedings of the National Academy of Sciences of the United States of America 93, 6594-6599 (1996). 345. Saga, Y., Yagi, T., Ikawa, Y., Sakakura, T. & Aizawa, S. Mice develop normally without tenascin. Genes Dev 6, 1821-1831 (1992). 346. Jones, P.L. & Jones, F.S. Tenascin-C in development and disease: gene regulation and cell function. Matrix biology : journal of the International Society for Matrix Biology 19, 581-596 (2000). 347. Takeda, A., et al. Clinical significance of large tenascin-C spliced variant as a potential biomarker for colorectal cancer. World J Surg 31, 388-394 (2007). 348. Chiquet-Ehrismann, R., Mackie, E.J., Pearson, C.A. & Sakakura, T. Tenascin: an extracellular matrix protein involved in tissue interactions during fetal development and oncogenesis. Cell 47, 131-139 (1986). 349. Brack, S.S., Silacci, M., Birchler, M. & Neri, D. Tumor-targeting properties of novel antibodies specific to the large isoform of tenascin-C. Clinical cancer research : an official journal of the American Association for Cancer Research 12, 3200-3208 (2006). 350. Midwood, K.S., Williams, L.V. & Schwarzbauer, J.E. Tissue repair and the dynamics of the extracellular matrix. The international journal of biochemistry & cell biology 36, 1031-1037 (2004). 351. Mackie, E.J. & Tucker, R.P. Tenascin in bone morphogenesis: expression by osteoblasts and cell type-specific expression of splice variants. J Cell Sci 103 ( Pt 3), 765-771 (1992). 352. Ghert, M.A., Qi, W.N., Erickson, H.P., Block, J.A. & Scully, S.P. Tenascin-C splice variant adhesive/anti-adhesive effects on chondrosarcoma cell attachment to fibronectin. Cell structure and function 26, 179-187 (2001). 353. Midwood, K., et al. Tenascin-C is an endogenous activator of Toll-like receptor 4 that is essential for maintaining inflammation in arthritic joint disease. Nature medicine 15, 774-780 (2009). 354. Hancox, R.A., et al. Tumour-associated tenascin-C isoforms promote breast cancer cell invasion and growth by matrix metalloproteinase-dependent and independent mechanisms. Breast Cancer Res 11, R24 (2009). 355. Viale, G.L., et al. Occurrence of a glioblastoma-associated tenascin-C isoform in cerebral cavernomas and neighboring vessels. Neurosurgery 50, 838-842; discussion 842 (2002). 356. Dorries, U. & Schachner, M. Tenascin mRNA isoforms in the developing mouse brain. Journal of neuroscience research 37, 336-347 (1994). 357. Tucker, R.P., et al. Novel tenascin variants with a distinctive pattern of expression in the avian embryo. Development (Cambridge, England) 120, 637-647 (1994). 358. Goodnow, C.C., Sprent, J., Fazekas de St Groth, B. & Vinuesa, C.G. Cellular and genetic mechanisms of self tolerance and autoimmunity. Nature 435, 590-597 (2005).

81 359. Marrack, P. & Kappler, J. Control of T cell viability. Annu Rev Immunol 22, 765-787 (2004). 360. Abbas, A.K., Lichtman, A.H. & Pillai, S. Cytokines. in Cellular and Molecular Immunology 267- 301 (Saunders-Elsevier, Philadelphia, PA, 2010). 361. Sioud, M. An overview of the immune system and technical advances in tumor antigen discovery and validation. Methods in molecular biology (Clifton, N.J 360, 277-318 (2007). 362. Kronenberg, M. & Rudensky, A. Regulation of immunity by self-reactive T cells. Nature 435, 598- 604 (2005). 363. Abbas, A.K., Lichtman, A.H. & Pillai, S. Lymphocyte development and the rearrangement and expression of antigen receptor genes. in Cellular and Molecular Immunology 153-187 (Saunders- Elsevier, Philadelphia, PA, 2010). 364. Walker, B. & Feavers, I. Adjuvant effects on antibody titre. Methods in molecular biology 626, 187-198 (2010). 365. O'Hagan, D.T. & De Gregorio, E. The path to a successful vaccine adjuvant--'the long and winding road'. Drug Discov Today 14, 541-551 (2009). 366. Ramon, G. Procédés pour accroitre la prodcution de antitoxines. Ann Inst Pasteur 40, 1-10 (1926). 367. Freund, J. Sensitization and antibody formation after injection of tubercle bacilli and paraffin oil. Proc Soc Exp Biol Med 37, 509-513 (1937). 368. Herbert, W.J. The mode of action of mineral-oil emulsion adjuvants on antibody production in mice. Immunology 14, 301-318 (1968). 369. Miles, A.P., et al. Montanide ISA 720 vaccines: quality control of emulsions, stability of formulated antigens, and comparative immunogenicity of vaccine formulations. Vaccine 23, 2530- 2539 (2005). 370. Kreuter, J., Mauler, R., Gruschkau, H. & Speiser, P.P. The use of new polymethylmethacrylate adjuvants for split influenza vaccines. Exp Cell Biol 44, 12-19 (1976). 371. Allison, A.G. & Gregoriadis, G. Liposomes as immunological adjuvants. Nature 252, 252 (1974). 372. Ellouz, F., Adam, A., Ciorbaru, R. & Lederer, E. Minimal structural requirements for adjuvant activity of bacterial peptidoglycan derivatives. Biochemical and biophysical research communications 59, 1317-1325 (1974). 373. Mbow, M.L., De Gregorio, E., Valiante, N.M. & Rappuoli, R. New adjuvants for human vaccines. Current opinion in immunology 22, 411-416 (2010). 374. CHMP. Summary of product characteristics Pandemrix H1N1 vaccine. http://www.scribd.com/doc/20313100/Contents-and-Usage-of-GSK-s-Pandemrix-H1N1-Vaccine access date: march 25th 2012 375. Boland, G., et al. Safety and immunogenicity profile of an experimental hepatitis B vaccine adjuvanted with AS04. Vaccine 23, 316-320 (2004). 376. Harper, D.M., et al. Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomised control trial. Lancet 367, 1247-1255 (2006). 377. Opie, E.L. & Freund, J. An Experimental Study of Protective with Heat Killed Tubercle Bacilli. The Journal of experimental medicine 66, 761-788 (1937). 378. Freund, J. Some aspects of active immunization. Annu Rev Microbiol 1, 291-308 (1947). 379. Lindblad, E.B. Methods in Molecular Medicine; Vaccine Adjuvants: Preparation Methods and Research Protocols, (Humana Press, Inc., Totowa, NJ, 2000). 380. Davenport, F.M. Seventeen years' experience with mineral oil adjuvant influenza virus vaccines. Ann Allergy 26, 288-292 (1968). 381. Teicher, B.A. Tumor models for efficacy determination. Mol Cancer Ther 5, 2435-2443 (2006). 382. Francia, G. & Kerbel, R.S. Raising the bar for cancer therapy models. Nature biotechnology 28, 561-562 (2010). 383. Kerbel, R.S. Human tumor xenografts as predictive preclinical models for anticancer drug activity in humans: better than commonly perceived-but they can be improved. Cancer Biol Ther 2, S134- 139 (2003).

82 384. Talmadge, J.E., Singh, R.K., Fidler, I.J. & Raz, A. Murine models to evaluate novel and conventional therapeutic strategies for cancer. The American journal of pathology 170, 793-804 (2007). 385. Peterson, J.K. & Houghton, P.J. Integrating pharmacology and in vivo cancer models in preclinical and clinical drug development. Eur J Cancer 40, 837-844 (2004). 386. Hanahan, D. Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature 315, 115-122 (1985). 387. Bergers, G., Javaherian, K., Lo, K.M., Folkman, J. & Hanahan, D. Effects of angiogenesis inhibitors on multistage carcinogenesis in mice. Science (New York, N.Y 284, 808-812 (1999). 388. Bergers, G., Hanahan, D. & Coussens, L.M. Angiogenesis and apoptosis are cellular parameters of neoplastic progression in transgenic mouse models of tumorigenesis. Int J Dev Biol 42, 995-1002 (1998). 389. Hanahan, D. & Folkman, J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86, 353-364 (1996). 390. Guy, C.T., Cardiff, R.D. & Muller, W.J. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Molecular and cellular biology 12, 954-961 (1992). 391. Lin, E.Y., et al. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. The American journal of pathology 163, 2113-2126 (2003). 392. Fantozzi, A. & Christofori, G. Mouse models of breast cancer metastasis. Breast Cancer Res 8, 212 (2006). 393. Hunter, T. & Pines, J. Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell 79, 573-582 (1994). 394. Massague, J. G1 cell-cycle control and cancer. Nature 432, 298-306 (2004). 395. Yarden, Y. The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities. Eur J Cancer 37 Suppl 4, S3-8 (2001). 396. Vernersson, M., Ledin, A., Johansson, J. & Hellman, L. Generation of therapeutic antibody responses against IgE through vaccination. Faseb J 16, 875-877 (2002). 397. Hellman, L. Profound reduction in allergen sensitivity following treatment with a novel allergy vaccine. European journal of immunology 24, 415-420 (1994). 398. Hellman, L. Therapeutic vaccines against IgE-mediated allergies. Expert review of vaccines 7, 193- 208 (2008). 399. Kyewski, B. & Klein, L. A central role for central tolerance. Annu Rev Immunol 24, 571-606 (2006). 400. Murphy, K., Travers, P. & Walport, M. The humoral immune response. in Janeway's immunobiology (ed. Lawrence, E.) 379-420 (Garland Science, Taylor & Francis Group, LLC, New York, NY, 2008). 401. Abbas, A.K., Lichtman, A.H. & Pillai, S. Effector mechanisms of humoral immunity. in Cellular and Molecular Immunology 321-348 (Saunders-Elsevier, Philadelphia, PA, 2010). 402. Carroll, M.C. The complement system in regulation of adaptive immunity. Nature immunology 5, 981-986 (2004). 403. Daha, N.A., et al. Complement activation by (auto-) antibodies. Mol Immunol 48, 1656-1665 (2011). 404. Yasmeen, D., Ellerson, J.R., Dorrington, K.J. & Painter, R.H. The structure and function of immunoglobulin domains. IV. The distribution of some effector functions among the Cgamma2 and Cgamma3 homology regions of human immunoglobulin G1. Journal of immunology 116, 518- 526 (1976). 405. Wright, J.F., Shulman, M.J., Isenman, D.E. & Painter, R.H. C1 binding by murine IgM. The effect of a Pro-to-Ser exchange at residue 436 of the mu-chain. The Journal of biological chemistry 263, 11221-11226 (1988).

83 406. Walport, M.J. Complement. Second of two parts. The New England journal of medicine 344, 1140- 1144 (2001). 407. Walport, M.J. Complement. First of two parts. The New England journal of medicine 344, 1058- 1066 (2001). 408. Volanakis, J.E. Participation of C3 and its ligands in complement activation. Curr Top Microbiol Immunol 153, 1-21 (1990). 409. Law, S.K., Lichtenberg, N.A. & Levine, R.P. Covalent binding and hemolytic activity of complement proteins. Proceedings of the National Academy of Sciences of the United States of America 77, 7194-7198 (1980). 410. Henson, P.M. Interaction of cells with immune complexes: adherence, release of constituents, and tissue injury. The Journal of experimental medicine 134, 114-135 (1971). 411. Murphy, K., Travers, P. & Walport, M. Innate immunity. in Janeway's immunobiology (ed. Lawrence, E.) 39-108 (Garland Science, Taylor & Francis Group, LLC, New York, NY, 2008). 412. Abbas, A.K., Lichtman, A.H. & Pillai, S. Innate immunity. in Cellular and Molecular Immunology 19-46 (Saunders-Elsevier, Philadelphia, PA, 2010). 413. Johansson, J., Ledin, A., Vernersson, M., Lovgren-Bengtsson, K. & Hellman, L. Identification of adjuvants that enhance the therapeutic antibody response to host IgE. Vaccine 22, 2873-2880 (2004). 414. Krieg, A.M. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 20, 709- 760 (2002). 415. Krieg, A.M., et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374, 546- 549 (1995). 416. Kumar, H., Kawai, T. & Akira, S. Toll-like receptors and innate immunity. Biochemical and biophysical research communications 388, 621-625 (2009). 417. Murphy, K., Travers, P. & Walport, M. Allergy and hypersensitivity. in Janeway's immunobiology (ed. Lawrence, E.) 555-598 (Garland Science, Taylor & Francis Group, LLC, New York, NY, 2008). 418. Tada, H., Aiba, S., Shibata, K., Ohteki, T. & Takada, H. Synergistic effect of Nod1 and Nod2 agonists with toll-like receptor agonists on human dendritic cells to generate interleukin-12 and T helper type 1 cells. Infect Immun 73, 7967-7976 (2005). 419. Davis, H.L., et al. CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen. J Immunol 160, 870-876 (1998). 420. Speiser, D.E. & Krieg, A.M. Stimulation of toll-like receptor 9 for enhancing vaccination. in Cancer vaccines and tumor immunity (eds. Orentas, R., Hodge, J.W. & Johnson, B.D.) 43-66 (John Wiley & Sons, Inc., Hoboken, NJ, 2008). 421. Rankin, R., et al. CpG motif identification for veterinary and laboratory species demonstrates that sequence recognition is highly conserved. Antisense & nucleic acid drug development 11, 333-340 (2001). 422. Krieg, A.M. Therapeutic potential of Toll-like receptor 9 activation. Nat Rev Drug Discov 5, 471- 484 (2006). 423. Kanneganti, T.D., Lamkanfi, M. & Nunez, G. Intracellular NOD-like receptors in host defense and disease. Immunity 27, 549-559 (2007). 424. Miura, S., et al. Impairment of VEGF-A-stimulated lamellipodial extensions and motility of vascular endothelial cells by chondromodulin-I, a cartilage-derived angiogenesis inhibitor. Experimental cell research (2009). 425. Krug, A., et al. Identification of CpG oligonucleotide sequences with high induction of IFN- alpha/beta in plasmacytoid dendritic cells. European journal of immunology 31, 2154-2163 (2001). 426. Krug, A., et al. Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12. European journal of immunology 31, 3026-3037 (2001). 427. Gottenberg, J.E. & Chiocchia, G. Dendritic cells and interferon-mediated autoimmunity. Biochimie 89, 856-871 (2007).

84 428. Mackay, F. & Schneider, P. Cracking the BAFF code. Nat Rev Immunol 9, 491-502 (2009). 429. Vollmer, J. & Krieg, A.M. Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists. Advanced drug delivery reviews 61, 195-204 (2009). 430. Siegrist, C.A., et al. Co-administration of CpG oligonucleotides enhances the late affinity maturation process of human anti-hepatitis B vaccine response. Vaccine 23, 615-622 (2004). 431. Lawrence, G.W., Saul, A., Giddy, A.J., Kemp, R. & Pye, D. Phase I trial in humans of an oil-based adjuvant SEPPIC MONTANIDE ISA 720. Vaccine 15, 176-178 (1997). 432. Saul, A., et al. Human phase I vaccine trials of 3 recombinant asexual stage malaria antigens with Montanide ISA720 adjuvant. Vaccine 17, 3145-3159 (1999). 433. Cooper, C.L., et al. CPG 7909, an immunostimulatory TLR9 agonist oligodeoxynucleotide, as adjuvant to Engerix-B HBV vaccine in healthy adults: a double-blind phase I/II study. J Clin Immunol 24, 693-701 (2004). 434. Cooper, C.L., et al. Safety and immunogenicity of CPG 7909 injection as an adjuvant to Fluarix . Vaccine 22, 3136-3143 (2004). 435. Speiser, D.E., et al. Rapid and strong human CD8+ T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909. J Clin Invest 115, 739-746 (2005).

85 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 755 Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine.

ACTA UNIVERSITATIS UPSALIENSIS Distribution: publications.uu.se UPPSALA urn:nbn:se:uu:diva-170887 2012