Cancer Therapy Using Viral- and Bacterial Proteins, as Vectors for Vaccines or as Carriers of Cytostatics

New cancer therapies are urgently needed, since available treatment options today have negative side effects, and cure only about half of the patients with invasive cancer. One, relatively new, option is to vaccinate against cancer, by introducing an antigen that is present on the tumor cells into the patient to stimulate specific immunity against the tumor. For this purpose viral capsid proteins, which can self-assemble into so called virus-like particles (VLPs), can be engineered to contain tumor antigens in the form of DNA, whole protein or peptides and be used as vaccines. Here, VLPs based on murine polyomavirus (MPyV) and murine pneumotropic virus (MPtV) containing the extracellular and transmembrane part of the breast cancer antigen Her2/neu, or the whole prostate cancer antigen PSA, have been produced. As mentioned above there are side effects with cancer treatment, and the use of the common cytostatic anti-cancer drug Cisplatin has a number of side effects, including; nephrotoxicity (kidney damage); neurotoxicity (nerve damage); and ototoxicity (hearing loss). To possibly inhibit some of this toxicity we attempted to make use of the “enhanced permeability and retention” (EPR) effect that causes macromolecules to accumulate more in tumor tissue than in normal tissue, since tumor blood vessels are leaky, and tumors lack effective lymphatic drainage. The use of a macromolecule as a carrier for Cisplatin would therefore hold the potential to reduce some of its negative side effects. For this purpose it was investigated whether the macromolecule right-handed coiled coil “RHCC” protein from bacterium Staphylothermus marinus, that can incorporate heavy metals, would also incorporate cisplatin containing the metal platinum. The overall aim of the first three papers in this thesis was to develop and determine pre-clinical efficacy of MPyVand MPtV-VLPs carrying Her2/neu or PSA against tumors expressing these tumor antigens. The overall aim of paper IV was to investigate RHCC’s potential to carry cisplatin efficiently to tumors, while retaining the cytotoxic effect of the drug. In paper I we demonstrated that homologous vaccination with human Her2/neu-VLPs was more efficient against outgrowth of human Her2/neu-expressing tumors than heterologous vaccination with rat Her2/neu-VLPs, while against rat Her2/neu-tumors, rat Her2/neu-VLPs were more efficient. Furthermore, we observed that vaccination with MPtVLPs was more efficient than vaccination with MPyVLPs, and that Her2MPtVLPs could be used for therapeutic vaccination. In paper II we demonstrated that both CD4 + and CD8 + T cells are involved in the tumor protective response after Her2MPtVLP vaccination. In paper III immunization with PSA-MPyVLPs, given together with CpG and loaded onto dendritic cells, was shown to protect against outgrowth of PSA-expressing tumor cells. In paper IV RHCC was shown to incorporate cisplatin, and the complex entered human tumor cells, while retaining the cytotoxic potential of the drug both in vitro and in vivo. In conclusion, in this thesis it is shown that VLPs based on MPyV and MPtV were efficient vectors for tumor antigens in cancer vaccination, evoking both CD4 + and CD8 + cell responses. In addition, we show that RHCC can function as a carrier for cisplatin, and that it could potentially reduce some of the negative side effects with cisplatin treatment. LIST OF PUBLICATIONS I Andreasson K, Tegerstedt K, Eriksson M, Curcio C, Cavallo F, Forni G, Dalianis T, Ramqvist T. : Murine pneumotropic virus chimeric Her2/neu virus-like particles as prophylactic and therapeutic vaccines against Her2/neu expressing tumors. Int J Cancer. 2009 Jan 1;124(1):150-6. II Andreasson K, Eriksson M, Tegerstedt K, Ramqvist T, Dalianis T. : CD4 + and CD8 + T cells can act separately in tumour rejection after immunization with murine pneumotropic virus chimeric Her2/neu virus-like particles. PLoS One. 2010 Jul 19;5(7):e11580. III Eriksson M, Andreasson K, Weidmann J, Lundberg K, Tegerstedt K, Dalianis T, Ramqvist T. : Murine polyomavirus virus-like particles carrying full-length human PSA protect BALB/c mice from outgrowth of a PSA expressing tumor. PLoS One. 2011;6(8):e23828. IV Eriksson M, Hassan S, Larsson R, Linder S, Ramqvist T, Lovborg H, Vikinge T, Figgemeier E, Muller J, Stetefeld J, Dalianis T, Ozbek S. : Utilization of a right-handed coiled-coil protein from archaebacterium Staphylothermus marinus as a carrier for cisplatin. Anticancer Res. 2009 Jan; 29(1):11-8. LIST OF ABBREVIATIONS ADCC Antibody-Dependent Cellular Cytotoxicity AIDS Acquired Immune Deficiency Syndrome APC Antigen-Presenting Cell AS Adjuvant System BCR B Cell Receptor BKV BK Virus BPV Bovine PapillomaVirus BrdU BromodeoxyUridine BSA Bovine Serum Albumin CAR Chimeric Antigen Receptor CCR C-C chemokine Receptor CD Cluster of Differentiation CpG Cytosine-phosphate-Guanosine CsCl Caesium Chloride CTL Cytotoxic T Lymphocyte CTLA Cytotoxic T Lymphocyte Antigen cVLP chimeric Virus-Like Particle DC Dendritic Cell DNA DeoxyriboNucleic Acid EBV Epstein-Barr Virus EGF Epidermal Growth Factor EGFR Epidermal Growth Factor Receptor ELISA Enzyme-Linked ImmunoSorbent Assay ELISpot Enzyme-Linked Immunosorbent Spot EPR Enhanced Permeability and Retention FDA Fluorescein DiAcetate FMCA Fluorometric Microculture Cytotoxicity Assay GM-CSF Granulocyte-Macrophage Colony Stimulating Factor HaPyV Hamster PolyomaVirus HBV Hepatitis B Virus Her2 Human epidermal growth factor receptor 2 HLA Human Leukocyte Antigen HPV Human PapillomaVirus HPyV Human PolyomaVirus 131 I Iodine-131 ICCS IntraCellular Cytokine Staining IFN InterFeroN Ig Immunoglobulin IL InterLeukin i.p. intraperitoneal/ly i.v. intravenous/ly JCV JC Virus kDa kiloDalton LAL Limulus Amebocyte Lysate LCMV Lymphocytic ChorioMeningitis Virus LPS LipoPolySaccharide LPV Lymphotropic Virus LT Large T mAb monoclonal Antibody MAPK Mitogen-Activated Protein Kinase MCV Merkel Cell polyomaVirus MHC Major Histocompatibility Complex MPL MonoPhosphoryl Lipid A MPtV Murine Pneumotropic Virus MPyV Murine PolyomaVirus mRNA messenger RiboNucleic Acid MT Middle T NK Natural Killer ORF Open Reading Frame PAMP Pathogen-Associated Molecular Pattern PAP Prostatic Acid Phosphatase PET Positron Emission Tomography PI3K PhosphatidylInositol 3-Kinase PML Progressive Multifocal Leukoencephalopathy PRR Pattern Recognition Receptor PSA Prostate Specific Antigen PTEN Phosphatase and TENsin homolog RAS RAt Sarcoma Rb Retinoblastoma RHCC Right-Handed Coiled Coil RNA RiboNucleic Acid s.c. subcutaneous/ly SCID Severe Combined ImmunoDeficiency SDS-PAGE Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis ST Small T SV40 Simian Virus 40 TAA Tumor-Associated Antigen TAP Antigen Peptide Transporter TCR T Cell Receptor TGF Transforming Growth Factor Th T helper TIL Tumor Infiltrating Lymphocyte TLR Toll-Like Receptor TNF Tumor Necrosis Factor TRAIL TNF-Related Apoptosis-Inducing Ligand Treg Regulatory T cell TSA Tumor-Specific Antigen TSV Trichodysplasia Spinulosa-associated polyomaVirus VEGF Vascular Endothelial Growth Factor VLP Virus-Like Particle VP Viral Protein 90 Y Yttrium-90 A Angstrom