WO2015143221A1 - Methods and materials for treating cancer - Google Patents

Methods and materials for treating cancer Download PDF

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Publication number
WO2015143221A1
WO2015143221A1 PCT/US2015/021574 US2015021574W WO2015143221A1 WO 2015143221 A1 WO2015143221 A1 WO 2015143221A1 US 2015021574 W US2015021574 W US 2015021574W WO 2015143221 A1 WO2015143221 A1 WO 2015143221A1
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antigen
nucleic acid
vsv
composition
composition comprises
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PCT/US2015/021574
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French (fr)
Inventor
Jose S. Pulido
Richard G. Vile
Timothy J. Kottke
Jill M. Thompson
Rosa Maria DIAZ
Christine Marie PULIDO
Alan A. MELCHER
Peter Selby
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Mayo Foundation For Medical Education And Research
University Of Leeds
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Priority to EP15765220.7A priority Critical patent/EP3119426A4/en
Priority to US15/126,333 priority patent/US10441643B2/en
Publication of WO2015143221A1 publication Critical patent/WO2015143221A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/766—Rhabdovirus, e.g. vesicular stomatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001128—CD44 not IgG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00113—Growth factors
    • A61K39/001134—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A61K39/001162—Kinases, e.g. Raf or Src
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A61K39/001164—GTPases, e.g. Ras or Rho
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20241—Use of virus, viral particle or viral elements as a vector
    • C12N2760/20243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This document relates to methods and materials for treating cancer.
  • this document relates to methods and materials for using combinations of antigens to treat cancer (e.g., melanoma such as skin melanoma or uveal melanoma, non-Hodgkin lymphoma, colorectal cancer, brain tumors, papillary thyroid carcinoma, non-small-cell lung carcinoma, or adenocarcinoma of the lung).
  • Cancer is a serious illness that affects many people every year. In general, there are several common methods for treating cancer: surgery, chemotherapy, radiation therapy, immunotherapy, and biologic therapy.
  • a cancer specialist such as an oncologist can provide a patient with various cancer treatment options.
  • an oncologist will recommend the best treatment plan based on the type of cancer, how far it has spread, and other important factors like the age and general health of the patient.
  • this document provides methods and materials for treating cancer.
  • this document provides combinations of antigens having the ability to reduce the presence of cancer (e.g., reduce established tumors) within a mammal (e.g., a human).
  • combinations of antigens e.g., a combination of a GNAQ antigen, a TYRP 1 antigen, and an N-RAS antigen, a combination of a BRAF antigen, a ⁇ - ⁇ antigen, and a YB-1 antigen, a combination of a TGF- ⁇ antigen, a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen, a combination of a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen, a combination of a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a
  • VSV vectors designed to express a GNAQ antigen, a TYRP1 antigen, and an N-RAS antigen can be used to reduce the number of cancer cells (e.g., uveal melanoma cells) within a mammal (e.g., a human).
  • VSV vectors designed to express a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen can be used to reduce the number of cancer cells (e.g., skin melanoma cells) within a mammal (e.g., a human).
  • the combinations of antigens provided herein can be used to treat cancer a cancer that overexpresses TOPO-IIa, YB-1, TYRP-1, or BRAF.
  • one aspect of this document features a composition comprising, or consisting essentially of, nucleic acid encoding a GNAQ antigen, a TYRP 1 antigen, and an N-RAS antigen, wherein the composition comprises less than 100 separate nucleic acid molecules.
  • the composition can comprise a nucleic acid molecule encoding the GNAQ antigen, a nucleic acid molecule encoding the TYRP 1 antigen, and a nucleic acid molecule encoding the N-RAS antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the GNAQ antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the TYRP1 antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the N-RAS antigen.
  • the composition can comprise less than 50 separate nucleic acid molecules.
  • the composition can comprise less than 10 separate nucleic acid molecules.
  • the composition can comprise less than 5 separate nucleic acid molecules.
  • this document features a method of treating cancer within a mammal.
  • the method comprises, or consists essentially of, administering to the mammal a composition comprising, or consisting essentially of, nucleic acid encoding a GNAQ antigen, a TYRP1 antigen, and an N-RAS antigen, wherein the composition comprises less than 100 separate nucleic acid molecules.
  • the cancer can be a melanoma.
  • the mammal can be a human.
  • the composition can comprise a nucleic acid molecule encoding the GNAQ antigen, a nucleic acid molecule encoding the TYRP 1 antigen, and a nucleic acid molecule encoding the N-RAS antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the GNAQ antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the TYRP 1 antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the N-RAS antigen.
  • the composition can comprise less than 50 separate nucleic acid molecules.
  • the composition can comprise less than 10 separate nucleic acid molecules.
  • the composition can comprise less than 5 separate nucleic acid molecules.
  • this document features a composition comprising, or consisting essentially of, nucleic acid encoding a BRAF antigen, a ⁇ - ⁇ antigen, and a YB-1 antigen, wherein the composition comprises less than 100 separate nucleic acid molecules.
  • the composition can comprise a nucleic acid molecule encoding the BRAF antigen, a nucleic acid molecule encoding the TOPO-IIa antigen, and a nucleic acid molecule encoding the YB- 1 antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the BRAF antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the TOPO- IIa antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the YB-1 antigen.
  • the composition can comprise less than 50 separate nucleic acid molecules.
  • the composition can comprise less than 10 separate nucleic acid molecules.
  • the composition can comprise less than 5 separate nucleic acid molecules.
  • this document features a method of treating cancer within a mammal.
  • the method comprises, or consists essentially of, administering to the mammal a composition comprising, or consisting essentially of, nucleic acid encoding a BRAF antigen, a TOPO-IIa antigen, and a YB- 1 antigen, wherein the composition comprises less than 100 separate nucleic acid molecules.
  • the cancer can be a melanoma.
  • the mammal can be a human.
  • the composition can comprise a nucleic acid molecule encoding the BRAF antigen, a nucleic acid molecule encoding the TOPO-IIa antigen, and a nucleic acid molecule encoding the YB-1 antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the BRAF antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the TOPO-IIa antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the YB-1 antigen.
  • the composition can comprise less than 50 separate nucleic acid molecules.
  • the composition can comprise less than 10 separate nucleic acid molecules.
  • the composition can comprise less than 5 separate nucleic acid molecules.
  • this document features a composition
  • a composition comprising, or consisting essentially of, nucleic acid encoding: (a) a TGF- ⁇ antigen, a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen, (b) a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen, (c) a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen, (d) a TOPOIIa antigen and an ABCB5a antigen, or (e) an ABCB5a antigen, a CYT-C antigen, a N-RAS antigen, and a TYRP-1 antigen, wherein the composition comprises less than 100 separate nucleic acid molecules.
  • the composition can comprise: (a) a nucleic acid molecule encoding a TGF- ⁇ antigen, a nucleic acid molecule encoding a MDR1 antigen, a nucleic acid molecule encoding a TYRP- 1 antigen, and a nucleic acid molecule encoding a KDR2 antigen, (b) a nucleic acid molecule encoding a TOPOIIa antigen, a nucleic acid molecule encoding a YB- 1 antigen, a nucleic acid molecule encoding a CDC7 kinase antigen, and a nucleic acid molecule encoding a BRAF antigen, (c) a nucleic acid molecule encoding a TOPOIIa antigen, a nucleic acid molecule encoding a YB-1 antigen, a nucleic acid molecule encoding a CDC7 kinase antigen, and a nucleic acid molecule
  • the composition can comprise a VSV vector comprising nucleic acid encoding the TGF- ⁇ antigen, the MDR1 antigen, the TYRP-1 antigen, the KDR2 antigen, the TOPOIIa antigen, the YB-1 antigen, the CDC7 kinase antigen, the BRAF antigen, the CD44 antigen, the ABCB5a antigen, the CYT-C antigen, or the N-RAS antigen.
  • the composition can comprise less than 50 separate nucleic acid molecules.
  • the composition can comprise less than 10 separate nucleic acid molecules.
  • the composition can comprise less than 6 separate nucleic acid molecules.
  • this document features a method of treating cancer within a mammal.
  • the method comprises, or consists essentially of, administering to the mammal a composition of the preceding paragraph.
  • the cancer can be a melanoma.
  • the mammal can be a human.
  • this document features a composition comprising, or consisting essentially of, nucleic acid encoding a TGF- ⁇ antigen, a KDR2 antigen, a P Glyc antigen, and a TYRP-1 antigen, wherein the composition comprises less than 100 separate nucleic acid molecules.
  • the composition can comprise a nucleic acid molecule encoding the TGF- ⁇ antigen, a nucleic acid molecule encoding the KDR2 antigen, a nucleic acid molecule encoding the P Glyc antigen, and a nucleic acid molecule encoding the TYRP-1 antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the TGF- ⁇ antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the KDR2 antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the P Glyc antigen.
  • the composition can comprise a VSV vector comprising nucleic acid encoding the TYRP1 antigen.
  • the composition can comprise less than 50 separate nucleic acid molecules.
  • the composition can comprise less than 10 separate nucleic acid molecules.
  • the composition can comprise less than 5 separate nucleic acid molecules.
  • this document features a method of treating cancer within a mammal, wherein the method comprises administering to the mammal a composition comprising nucleic acid encoding a TGF- ⁇ antigen, a KDR2 antigen, a P Glyc antigen, and a TYRP-1 antigen, wherein the composition comprises less than 100 separate nucleic acid molecules.
  • the cancer can be a melanoma.
  • the mammal can be a human.
  • this document features a composition of any one of the above recited paragraphs, wherein the composition comprises an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor can be an anti-PD-1 antibody or an anti- CTLA4 antibody.
  • this document features a method of any one of the above recited paragraphs, wherein the method comprises administering an immune checkpoint inhibitor to the mammal.
  • the immune checkpoint inhibitor can be an anti- PD-1 antibody or an anti-CTLA4 antibody.
  • Figure 1 is a graph plotting the percent survival of mice from B 16 recurrences that escaped frontline ganciclovir (GCV) treatment. The mice were treated with the indicated combinations of VSV vectors.
  • Intracranial tumors of different histology express a HIF-2aHi phenotype.
  • Tumors established in the brains of C57BL/6 (B16, GL261 or TC2 cells) or C3H (K1735) mice were dissected upon sacrifice (tumor explants), and tumor cells were seeded at lxlO 5 per well. lxlO 5 cells of each cell line cultured in vitro (cult.) were also plated.
  • HIF-2a was measured by ELISA after 24 hours. Error bars are expressed as standard deviation (SD).
  • FIG. 3 Brain derived CD1 lb + cells impose a HIF-2aHi phenotype on in vitro cultured GL261, in part through TGF- ⁇ . HIF-2a expression was measured by ELISA from: lxlO 5 GL261 cells cultured in vitro for 24 hours (lane 1); GL261 i.e.
  • Human brain tumor explants express a HIF-2aHi phenotype which diminishes with time. Human brain tumor explants were recovered from surgery and depleted of CD1 lb + cells. Tumor cells were plated at lxlO 4 per well either alone (24 hours CD1 lb ) or with 5xl0 3 CD1 lb + cells (24 hours CD1 lb + ). HIF-2a expression was measured at 24 hours. In cultures from which tumor cells survived more than a week, HIF-2a was measured from lxlO 4 tumor cells after 2 weeks, by which time CD1 lb + cells had been washed away/died (2 week CD1 lb " ). HIF-2a also was measured from lxlO 3 separated CD1 lb + cells 24 hours after explant. Results are representative of three separate measurements. Error bars are expressed as standard deviation (SD).
  • VSV-TAA therapy of intracranial GL261 tumors C57BL/6 mice bearing 5 day established i.e. GL261 tumors were treated intravenously with a total of 5xl0 6 pfu of (VSV-HIF-2a, VSV-SOX-10, and VSV-c-MYC); (VSV-HIF-2a, VSV- SOX-10, and VSV-GFP); (VSV-N-RAS, VSV-CYT-C, and VSV-TYRP-1), or (VSV- GFP) on days 6, 8, 10, 13, 15, 17, 20, 22, 24, 27, 29, and 31. Survival with time is shown.
  • FIG. 6 Checkpoint inhibition uncovers a repressed anti-tumor Thl IFN- ⁇ response.
  • VSV-GFP VSV-GFP
  • VSV-HIF-2a VSV- SOX-10
  • VSV-c-MYC VSV-c-MYC
  • B-D Splenocytes and lymph nodes were pooled from 3 C57BL/6 mice per group bearing 5 day established i.e. GL261 tumors treated with either (PBS/PBS); (VSV-GFP + anti-PDl antibody); (VSV-HIF-2a, VSV-SOX-10, and VSV-C-MYC+ IgG), or (VSV-HIF-2a, VSV-SOX-10, and VSV-C-MYC+ anti- PDl antibody).
  • FIG. 7 Anti-PDl checkpoint inhibition uncovers a Thl IFN- ⁇ anti-tumor response.
  • Double checkpoint inhibition therapy enhances treatment with VSV-antigens.
  • A C57BL/6 mice bearing 5 day established i.e. GL261 tumors were treated intravenously with a total dose of 5xl0 6 pfu of (VSV-GFP); (VSV-HIF-2a, VSV-SOX-10, and VSV-c-MYC) or PBS on days 6, 8, 10, 13, 15, and 17. On days 13, 15, and 17, these groups also were treated with either anti-PDl antibody, anti- CTLA4 antibody, anti-PDl antibody plus anti-CTLA4 antibody, or PBS as shown. Survival with time is shown.
  • B-D Splenocytes and lymph nodes were pooled from 3 C57BL/6 mice per group bearing 5 day established i.e.
  • VSV-GFP + anti-PDl + anti-CTLA4 VSV-GFP + anti-PDl + anti-CTLA4
  • VSV-HIF-2a VSV-SOX-10, and VSV-c-MYC + anti-PDl antibody + anti-CTLA4 antibody
  • VSV-HIF-2a VSV- SOX-10, and VSV-c-MYC + PBS
  • PBS + PBS PBS
  • VSV-HIF-2a, VSV-SOX-10, and VSV-c-MYC + anti-PDl antibody or
  • VSV-HIF-2a, VSV-SOX-10, and VSV-c- MYC + anti-CTLA4 antibody VSV-GFP + anti-PDl + anti-CTLA4 antibody
  • Figure 9 is a graph plotting the percent survival of mice having s.c. B 16 tumors and treated with PBS or the indicated combinations of VSV vectors.
  • Figure 10 is a schematic of an in vitro assay designed to determine which specific antigens are presented by which antigen presenting cell subtypes in order to reconstitute a Thl7 response.
  • Figure 1 1 is a bar graph plotting IL-17 levels (pg/mL) for the indicated conditions. None/none indicates no depletion of any cells; Ly6G/Ly-V-CYT-C indicates depletion of neutrophils and the reinfection with VSV-cytochrome C;
  • Ly6G/Ly-V-N-RAS indicates depletion of neutrophils and reinfection with VSV-N- RAS; Ly6G/Ly-V-TYRP- 1 indicates depletion of neutrophils and reinfection with VSV-TYRP-1 ; Ly6G/Ly-VSVGFP indicates depletion of neutrophils and the reinfection with VSV-GFP; pDC/pDC-V-CYT-C indicates depletion of plasmacytoid dendritic cells and the reinfection with VSV-cytochrome C; pDC/pDC-V-N-RAS indicates depletion of plasmacytoid dendritic cells and the reinfection with VSV-N- RAS; pDC/pDC-V-TYRP-1 indicates depletion of plasmacytoid dendritic cells and reinfection with VSV-TYRP-1 ; pDC/pDC-V-GFP indicates depletion of plasmacytoid dendritic cells and reinfection with VSV-GFP; 1 IB/1 1
  • Figure 12 is a bar graph plotting IL-17 levels (pg/mL) for the indicated conditions. None/none indicates no depletion of any cells; Ly6G/Ly-V-TGF indicates depletion of neutrophils and the reinfection with VSV-TGF ; Ly6G/Ly-V- KDR2 indicates depletion of neutrophils and reinfection with VSV-KDR2; Ly6G/Ly- V-Pglyco indicates depletion of neutrophils and reinfection with VSV-P Glyc;
  • Ly6G/Ly-VTYRP-l indicates depletion of neutrophils and the reinfection with VSV- TYRP-1;
  • pDC/pDC-V-TGF indicates depletion of plasmacytoid dendritic cells and the reinfection with VSV-TGF ;
  • pDC/pDC-V-KDR2 indicates depletion of plasmacytoid dendritic cells and the reinfection with VSV-KDR2;
  • Glyc indicates depletion of plasmacytoid dendritic cells and reinfection with VSV- P Glyc; pDC/pDC-V-TYRP-1 indicates depletion of plasmacytoid dendritic cells and reinfection with VSV-TYRP-1 ; l lB/l lB-V-TGF indicates depletion of CDl lb + cells and reinfection with VSV-TGF ; 1 IB/11B-V-KDR2 indicates depletion of CD 1 lb + cells and reinfection with VSV-KDR2; 1 IB/1 1B-V-Pglyc indicates depletion of CD1 lb + cells and reinfection with VSV-P Glyc; and 1 IB/1 lB-V-TYRP-1 indicates depletion of CD1 lb + and reinfection with VSV-TYRP-1.
  • Figure 13 is a proposed model of antigen presentation.
  • Figure 14 is contains sequence information for a truncated VSV-N-RAS virus recovered from an ASMEL.
  • Figure 15 is contains sequence information for a truncated VSV-CYT-C virus recovered from an ASMEL.
  • Figure 16 is contains sequence information for a truncated VSV-TYRP- 1 virus recovered from an ASMEL.
  • Figure 17 is a graph plotting the percent survival of mice having s.c. B16 tumors and treated with the indicated VSV vectors.
  • Figure 18 is a schematic of the indicated VSV vectors.
  • Figure 19 is a bar graph plotting IFN- ⁇ levels (pg/mL) for cells obtained from mice treated as indicated and stimulated with the indicated polypeptides.
  • Figure 20 is a schematic of an in vivo assay for assessing VSV vectors expressing IFN- ⁇ polypeptides.
  • Figure 21 is a graph plotting the percent survival of mice having B 16 tumors and treated with the indicated VSV vectors.
  • this document provides methods and materials for treating cancer.
  • this document provides combinations of antigens having the ability to reduce the number of cancer cells within a mammal (e.g., a human).
  • combinations of antigens that include a GNAQ antigen, a TYRP 1 antigen, and an N-RAS antigen, that include a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen, that include a TGF- ⁇ antigen, a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen, that include a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen, that include a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen, that include a TOPOIIa antigen and an ABCB5a antigen
  • combinations of antigens that include a GNAQ antigen, a TYRPl antigen, and an N-RAS antigen that include a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen, that include a TGF- ⁇ antigen, a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen, that include a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen, that include a TOPOIIa antigen, a YB- 1 antigen, a CDC7 kinase antigen, and a CD44 antigen, that include a TOPOIIa antigen and an ABCB5a antigen, that include an ABCB5a antigen, a CYT-C antigen, a N- RAS antigen, and a TYRP-1 antigen, or that include a TGF
  • the methods and materials provided herein can be used to treat cancer or to reduce the number of cancer cells present within any appropriate mammal such as humans, monkeys, horses, cows, sheep, dogs, cats, mice, or rats.
  • the methods and materials provided herein can be used to treat any appropriate cancer or to reduce the number of appropriate type of cancer cells present within a mammal.
  • the methods and materials provided herein can be used to treat melanoma (e.g., skin melanoma or uveal melanoma), non-Hodgkin lymphoma, colorectal cancer, brain tumors, papillary thyroid carcinoma, non-small-cell lung carcinoma, or adenocarcinoma of the lung or can be used to reduce the number of melanoma (e.g., skin melanoma or uveal melanoma), non-Hodgkin lymphoma, colorectal cancer, brain tumor, papillary thyroid carcinoma, non-small-cell lung carcinoma, or adenocarcinoma of the lung cancer cells present within a mammal.
  • melanoma e.g., skin melanoma or uveal melanoma
  • non-Hodgkin lymphoma e.g., colorectal cancer
  • brain tumor e.g., papillary thyroid carcinoma, non-small-cell lung
  • a combination of a GNAQ antigen, a TYRP1 antigen, and an N-RAS antigen can be used to treat cancer (e.g., melanoma such as uveal melanoma).
  • cancer e.g., melanoma such as uveal melanoma
  • one or more viral vectors e.g., vesicular stomatitis virus (VSV) vectors
  • VSV vesicular stomatitis virus
  • VSV vectors designed to express a GNAQ antigen, a TYRPl antigen, and an N-RAS antigen can be administered to a mammal (e.g., a human) with uveal melanoma to reduce the size or to prevent the additional growth of that melanoma.
  • YB-1 antigen can be used to treat cancer (e.g., melanoma such as skin melanoma).
  • one or more viral vectors e.g., VSV vectors
  • VSV vectors designed to express a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen can be used to treat cancer (e.g., melanoma such as skin melanoma).
  • VSV vectors designed to express a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen can be used to treat cancer (e.g., melanoma such as skin melanoma).
  • a mammal e.g., a human
  • skin melanoma to reduce the size or to prevent the additional growth of that melanoma.
  • a combination of a TGF- ⁇ antigen, a MDRl antigen, a TYRP-1 antigen, and a KDR2 antigen can be used to treat cancer (e.g., melanoma).
  • one or more viral vectors e.g., VSV vectors
  • a TGF- ⁇ antigen, a MDRl antigen, a TYRP-1 antigen, and a KDR2 antigen can be used to treat cancer (e.g., melanoma).
  • a combination of a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen can be used to treat cancer (e.g., melanoma, colorectal cancer, prostate cancer, ovarian cancer, or breast cancer).
  • cancer e.g., melanoma, colorectal cancer, prostate cancer, ovarian cancer, or breast cancer.
  • one or more viral vectors e.g., VSV vectors designed to express a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen can be used to treat cancer (e.g., melanoma, colorectal cancer, prostate cancer, ovarian cancer, or breast cancer).
  • a combination of a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen can be used to treat cancer (e.g., melanoma or prostate cancer).
  • one or more viral vectors e.g., VSV vectors
  • a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen can be used to treat cancer (e.g., melanoma or prostate cancer).
  • a combination of a TOPOIIa antigen and an ABCB5a antigen can be used to treat cancer (e.g., melanoma).
  • one or more viral vectors e.g., VSV vectors
  • a TOPOIIa antigen and an ABCB5a antigen can be used to treat cancer (e.g., melanoma).
  • RAS antigen, and a TYRP-1 antigen can be used to treat cancer (e.g., melanoma).
  • one or more viral vectors e.g., VSV vectors
  • designed to express an ABCB5a antigen, a CYT-C antigen, a NRAS antigen, and a TYRP-1 antigen can be used to treat cancer (e.g., melanoma).
  • a combination of a TGF- ⁇ antigen, a KDR2 antigen, a P glycoprotein (P Glyc) antigen, and a TYRP-1 antigen can be used to treat cancer (e.g., melanoma).
  • one or more viral vectors e.g., VSV vectors
  • a TGF- ⁇ antigen, a KDR2 antigen, a P glycoprotein (P Glyc) antigen, and a TYRP-1 antigen can be used to treat cancer (e.g., melanoma).
  • a GNAQ (guanine nucleotide binding protein, q polypeptide) antigen can have the amino acid sequence set forth in GenBank ® Accession No. AF493896.1 (GI No. 20147684) or U40038.1 (GI No. 1181670), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • GenBank ® Accession No. AF493896.1 GI No. 20147684
  • U40038.1 GI No. 1181670
  • a TYRP1 (tyrosinase-related protein 1) antigen can have the amino acid sequence set forth in GenBank ® Accession No. CAG2861 1 (GI No. 471 15303), NM_000550.2 (GI No. 169881242), CR407683.1 (GI No. 47115302),
  • XM_005251574.1 (GI No. 530390132), or X51420.1 (GI No. 37512), or a fragment of such an amino acid sequence that is between about 7 and 527 amino acid residues (e.g., between about 10 and 527 amino acid residues, between about 15 and 527 amino acid residues, between about 20 and 527 amino acid residues, between about 25 and 527 amino acid residues, between about 30 and 527 amino acid residues, or between about 30 and 200 amino acid residues) in length.
  • 7 and 527 amino acid residues e.g., between about 10 and 527 amino acid residues, between about 15 and 527 amino acid residues, between about 20 and 527 amino acid residues, between about 25 and 527 amino acid residues, between about 30 and 527 amino acid residues, or between about 30 and 200 amino acid residues
  • N-RAS neuroroblastoma RAS viral oncogene homolog
  • GenBank ® Accession No. AAB29640 GI No. 544859
  • an N-RAS antigen can have the amino acid sequence set forth in GenBank ® Accession No. AAB29640 (GI No. 544859), or a fragment of such an amino acid sequence that is between about 7 and 400 amino acid residues (e.g., between about 10 and 400 amino acid residues, between about 15 and 400 amino acid residues, between about 20 and 400 amino acid residues, between about 25 and 400 amino acid residues, between about 30 and 400 amino acid residues, or between about 30 and 200 amino acid residues) in length.
  • an N-RAS antigen can have the amino acid sequence set forth in
  • amino acid residues e.g., between about 10 and 400 amino acid residues, between about 15 and 400 amino acid residues, between about 20 and 400 amino acid residues, between about 25 and 400 amino acid residues, between about 30 and 400 amino acid residues, or between about 30 and 200 amino acid residues
  • a BRAF (v-raf murine sarcoma viral oncogene homolog B) antigen can have the amino acid sequence set forth in GenBank ® Accession No. NM_004333.4 (GI No. 187608632), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • a GNAQ antigen can have the amino acid sequence set forth in
  • GenBank ® Accession No. XM_005250045.1 (GI No. 530387105), XM 005250046.1 (GI No. 530387107), or XM_005250047.1 (GI No. 530387109), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • amino acid residues e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues
  • a TOPO-IIa (DNA topoisomerase 2-alpha) antigen can have the amino acid sequence set forth in GenBank ® Accession No. NM_001067.3 (GI No. 300193028), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • GenBank ® Accession No. NM_001067.3 GI No. 300193028
  • a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residue
  • a YB-1 (Y box binding protein 1) antigen can have the amino acid sequence set forth in GenBank ® Accession No. NM_004559.3 (GI No. 109134359), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • a GNAQ antigen can have the amino acid sequence set forth in GenBank ® Accession No. BC071708.1 (GI No.
  • XM_005270904.1 GI No. 530362706
  • a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • a TGF- ⁇ antigen can have the amino acid sequence set forth in GenBank ® Accession No. X02812 or J051 14 (GI No. 37092), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • GenBank ® Accession No. X02812 or J051 14 GI No. 37092
  • a MDR1 antigen can have the amino acid sequence set forth in GenBank ® Accession No. X58723 or X59732 (GI No. 34522), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • a KDR2 antigen can have the amino acid sequence set forth in GenBank ® Accession No. AF063658 (GI o. 3132832), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • GenBank ® Accession No. AF063658 GI o. 3132832
  • a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • a CYT-C antigen can have the amino acid sequence set forth in GenBank ® Accession No. NP_061820 (GI No. 1 1128019), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • GenBank ® Accession No. NP_061820 GI No. 1 1128019
  • amino acid residues e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues
  • An ABCB5a antigen can have the amino acid sequence set forth in GenBank ® Accession Nos. NM 029961, XM_001002680, or XM_906632 (GI No. 255708374), NM 001 163941.1 (GI No. 255708476), NM_178559.5 (GI No. 255708475),
  • NM_001 163942.1 (GI No. 255708370), or NM_001163993.2 (GI No. 574957217), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • amino acid residues e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues
  • a CDC7 kinase antigen can have the amino acid sequence set forth in GenBank ® Accession No. NM_ 009863 (GI No. 409168309), NM_001 134420.1 (GI No. 197313666), NM_003503.3 (GI No. 197313663), or NM_001 134419.1 (GI No. 197313664), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • a CD44 antigen can have the amino acid sequence set forth in GenBank ® Accession No. NM_001 177787 (GI No. 295293147), AY101 193.1 (GI No.
  • 21429240 21429240
  • AY101192.1 GI No. 21429238
  • a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • a P Glyc (P glycoprotein) antigen can have the amino acid sequence set forth in GenBank ® Accession No. M23234.1 (GI No. 187501), AY234788.1 (GI No.
  • AY425006.1 GI No. 40795902
  • AF399931.1 GI No. 3330771 1
  • EU854148.1 GI No. 194740429
  • amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
  • a GNAQ, TYRPl, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF- ⁇ antigen can have the amino acid sequence set forth in one of the GenBank ® Accession numbers indicated above or a fragment of such an amino acid sequence that is immunogenic and induces a robust IL-17 response.
  • such an antigen can include one or more mutations within the sequence provided in GenBank ® provided that the mutant antigen induces a robust IL-17 response.
  • a GNAQ, TYRPl, N- RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF- ⁇ antigen can have the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form.
  • a GNAQ antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have one or more of the following mutations: GNAQ(209) or GNAQ(R183).
  • a TYRPlantigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have one or more of the following mutations: 1-BP DEL of 368A (condition: albinism, oculocutaneous, type III), SER166TER (dbSNP: rsl04894130), ARG373TER, ARG356GLU, 1-BP DEL of 106T, 4-BP DEL of 1057AACA, or ARG93CYS (condition: albinism, oculocutaneous, type III).
  • N-RAS antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have one or more of the following mutations: Q61 (dbSNP: rsl 1554290), GLY13ASP (dbSNP: rsl21434596), GLY13ARG (dbSNP: rs l21434595),
  • THR50ILE GLY60GLU (condition: Noonen syndrome 6), PR034LEU, or
  • GLY12ASP condition: epidermal nevus, somatic.
  • a BRAF antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have the following mutation: V600 (dbSNP: rsl 13488022).
  • a TOPO-IIa antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have the following mutation: ARG486LYS (condition: resistance to inhibition of or by amsacrine).
  • a YB-1 antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have the following mutation: YB-1(S 102).
  • a MDR1 antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have one or more of the following mutations: G2677T, C3435T (dbSNP: rsl045642), GLY185VAL (dbSNP: rs l 128501; condition: colchicine resistance), or ALA893SER/THR (condition: inflammatory bowel disease).
  • a KDR2 antigen having the amino acid sequence (or a fragment thereof) as found in a naturally- occurring mutated form can have one or more of the following mutations: D717V, T771R, PROl 147SER (condition: hemangioma, capillary infantile, somatic), or CYS482ARG (dbSNP: rs34231037; condition susceptibility to hemangioma or capillary infantile).
  • An ABCB5a antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have one or more of the following mutations: G347R, M521L, P580S, or A687S.
  • a CDC7 kinase antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have the following mutation: L2101.
  • a CD44 antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have the following mutation: ARG46PRO (dbSNP: rs l21909545; condition: Indian blood group system polymorphism).
  • a TGF- ⁇ antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have one or more of the following mutations: CYS225ARG (dbSNP: rs 104894719), ARG218HIS (dbSNP: rs 104894720), ARG218CYS (dbSNP: rs l04894721), TYR81HIS (dbSNP: rsl 1 1033611),
  • CYS223ARG (dbSNP: rsl04894722), CYS223GLY (dbSNP: rs l04894722; condition: camurati-engelmann disease), or LEU10PRO (conditions: cystic fibrosis lung disease, modifier of invasive breast cancer, or susceptibilities thereto).
  • a GNAQ, TYRP1, N-RAS, BRAF, TOPO-IIa, YB-1, MDRl, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF- ⁇ antigen can have an amino acid sequence that is truncated at the C terminus.
  • a GNAQ antigen can include the N-terminal sequence of a full length GNAQ polypeptide, while lacking a portion of the C-terminal sequence of a full length GNAQ
  • the length of the missing C-terminal sequence of a truncated antigen can be from 1 to about 300 (e.g., 1 to 275, 1 to 250, 1 to 225, 1 to 200, 1 to 175, 1 to 150, 1 to 125, 1 to 100, 1 to 75, 1 to 50, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 5 to 275, 5 to 250, 5 to 225, 5 to 200, 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 275, 10 to 250, 10 to 225, 10 to 200, 10 to to 300 (e.g., 1 to 275, 1 to 250, 1 to 225, 1 to 200, 1 to 175, 1 to 150, 1 to 125, 1 to 100, 1 to 75, 1 to 50, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 5 to 275, 5 to 250, 5 to 225, 5 to 200, 5 to 175, 5 to 150, 5 to
  • the length of the missing C-terminal sequence of a truncated antigen can be between about 0.01 percent to about 85 percent (e.g., about 0.01 percent to about 85 percent, about 0.01 percent to about 75 percent, about 0.01 percent to about 65 percent, about 0.01 percent to about 55 percent, about 0.01 percent to about 45 percent, about 0.01 percent to about 35 percent, about 0.01 percent to about 25 percent, about 0.01 percent to about 15 percent, about 0.01 percent to about 10 percent, about 0.01 percent to about 5 percent, about 0.1 percent to about 85 percent, about 1 percent to about 85 percent, about 5 percent to about 85 percent, about 5 percent to about 85 percent, about 5 percent to about 75 percent
  • the combination of antigens used to treat cancer or reduce the number of cancer cells within a mammal can be antigens of another species (e.g., mouse, rat, pig, monkey, sheep, cow, dog, or cat).
  • a combination of mouse, rat, or monkey antigens can be used to treat cancer or reduce the number of cancer cells within a human.
  • GNAQ sequences from mouse are set forth in GenBank ® Accession Nos. NM_008139.5 (GI No. 145966786) and BC057583.1 (GI No. 34785834).
  • Examples of TYRP-1 sequences from mouse are set forth in GenBank ® Accession Nos.
  • NM_001282014.1 (GI No. 530537243), NM_031202.3 (GI No. 530537240), and NM_001282015.1 (GI No. 530537245).
  • Examples of N-RAS sequences from mouse are set forth in GenBank ® Accession Nos. NM_010937.2, NC_000069.6 (GI No. 372099107), and AC_000025.1 (GI No. 83280973).
  • An example of a BRAF sequence from mouse is set forth in GenBank ® Accession No. NM_139294.5 (GI No. 153791903).
  • An example of a TOPO-IIa sequence from mouse is set forth in GenBank ® Accession No. NM011623.
  • GenBank ® Accession No. M62867 An example of a TGF- ⁇ sequence from mouse is set forth in GenBank ® Accession No. M13177.1 (GI No. 201952).
  • GenBank ® Accession No. M13177.1 An example of a MDRl sequence from mouse is set forth in GenBank ® Accession No. NM_011075 (GI No. 161 169006).
  • GenBank ® Accession No. EU884114.1 GI No. 215400615.
  • GenBank ® Accession No. X57621.1 GI No. 55450
  • C061634.1 GI No.
  • NM_01 1732.2 (GI No. 1 13205058).
  • GenBank ® Accession No. M33581.1 (GI No. 199104), JQ655148.1 (GI No. 406817019), M24417.1 (GI No. 2000329), or AY864315.1 (GI No. 57791235).
  • Any appropriate vector e.g. a viral vector
  • a viral vector can be used to deliver nucleic acid encoding a GNAQ, TYRP1, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT- C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF- ⁇ antigen (or combination thereof) to cells of a mammal to treat cancer as described herein.
  • viral vectors for administering nucleic acids e.g., a nucleic acid encoding a GNAQ, TYRP1, N- RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF- ⁇ antigen (or combination thereof)
  • nucleic acids e.g., a nucleic acid encoding a GNAQ, TYRP1, N- RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF- ⁇ antigen (or combination thereof)
  • standard materials e.g., packaging cell lines, helper viruses, and vector constructs. See, for example, Gene Therapy Protocols (Methods in Molecular Medicine), edited by Jeffrey R.
  • a viral vector for delivering nucleic acid encoding a GNAQ, TYRP1, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT- C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF- ⁇ antigen (or combination thereof) can be derived from, for example, animal viruses such as adenoviruses, adeno- associated viruses, retroviruses, lentiviruses, vaccinia viruses, vesicular stomatitis virus, herpes viruses, maraba virus, or papilloma viruses.
  • lentiviral vectors can be used to deliver nucleic acid encoding a GNAQ, TYRP1, N-RAS, BRAF, ⁇ - ⁇ , YB-1, MDR1, KDR2, CYT-C,
  • VSV-IFN e.g., human interferon viral vectors such as those described elsewhere (Obuchi et al, J. Virol, 77(16):8843-56 (2003) and Jenks et al, Hum.
  • Gene Ther., 21(4):451-62 (2010) can be used to deliver nucleic acid encoding a GNAQ, TYRPl, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF- ⁇ antigen (or combination thereof) to cells of a mammal to treat cancer.
  • Any appropriate method can be used to insert nucleic acid encoding a GNAQ, TYRPl, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF- ⁇ antigen into a viral vector (e.g., a VSV vector).
  • a viral vector e.g., a VSV vector.
  • Biotechnol, 30:337-43 (2012) can be used to insert nucleic acid encoding a GNAQ, TYRP l, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF- ⁇ antigen into a VSV vector such that the antigen (e.g., the GNAQ, TYRPl, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF- ⁇ antigen) is expressed in mammalian cells.
  • the antigen e.g., the GNAQ, TYRPl, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a,
  • VSV vectors having the ability to express a GNAQ antigen, a TYRP 1 antigen, and an N-RAS antigen, a BRAF antigen, a TOPO-IIa antigen, and a YB- 1 antigen, a TGF- ⁇ antigen, a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen, a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen, a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen, a TOPOIIa antigen and an ABCB5a antigen, a TGF- ⁇ antigen, a KDR2 antigen, a P Glyc antigen, and a TYRP-1 antigen, or an ABCB5a antigen, a CYT-
  • a combination of VSV vectors having the ability to express a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen can be administered to a mammal to treat cancer (e.g., melanoma such as skin melanoma) or to reduce the number of cancer cells (e.g., melanoma cells such as skin melanoma cells) present within a mammal.
  • cancer e.g., melanoma such as skin melanoma
  • cancer cells e.g., melanoma cells such as skin melanoma cells
  • viral vectors e.g., VSV vectors
  • VSV vectors designed to express a GNAQ antigen, a TYRPl antigen, and an N-RAS antigen
  • a BRAF antigen a TOPO-IIa antigen, and a YB-1 antigen
  • TGF- ⁇ antigen a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen
  • TOPOIIa antigen a YB- 1 antigen
  • CDC7 kinase antigen a BRAF antigen
  • TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen a CD44 antigen
  • a TOPOIIa antigen and an ABCB5a antigen a TGF- ⁇ antigen, a KDR2 antigen, a P Glyc antigen, and a TYRP-1 antigen, or an ABCB5a
  • intratumoral, subcutaneous, intravenous, intraperitoneal, and intradermal administrations can be used to administer viral vectors (e.g., VSV vectors) designed to express a GNAQ antigen, a TYRPl antigen, and an N-RAS antigen to a mammal having cancer (e.g., uveal melanoma) or viral vectors (e.g., VSV vectors) designed to express a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen to a mammal having cancer (e.g., skin melanoma).
  • viral vectors e.g., VSV vectors
  • GNAQ antigen e.g., GNAQ antigen, a TYRPl antigen, and an N-RAS antigen
  • a mammal having cancer e.g., uveal melanoma
  • viral vectors e.g., VSV vectors
  • the mammal can be monitored to confirm a reduction in the number of cancer cells present within the mammal.
  • imaging techniques such as MRI and CT scans can be used to confirm that the number of cancer cells present within the mammal is reduced following administration of the viral vectors.
  • the following examination criteria can be used.
  • a GNAQ antigen, a TYRP 1 antigen, and an N-RAS antigen can be formulated with an adjuvant such as alum, monophosphoryl lipid A, liposomes, QS21, MF-59, or immunostimulating complexes (ISCOMS) and administered to a mammal having cancer (e.g., uveal melanoma). Following this administration, the number of cancer cells present within the mammal can be reduced.
  • an adjuvant such as alum, monophosphoryl lipid A, liposomes, QS21, MF-59, or immunostimulating complexes (ISCOMS)
  • a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen can be administered as a combination in the form of polypeptides to a mammal having cancer (e.g., skin melanoma). Following this administration, the number of cancer cells present within the mammal can be reduced.
  • cancer e.g., skin melanoma
  • therapy with a combination of antigens provided herein can include the use of radiation.
  • a patient when treating cutaneous melanoma, a patient can be treated with both radiation and a combination of antigens provided herein.
  • therapy with a combination of antigens provided herein can include the administration of one or more immune checkpoint inhibitors.
  • a combination of viral vectors e.g., VSV vectors designed to express a GNAQ antigen, a TYRP1 antigen, and an N-RAS antigen, a BRAF antigen, a TOPO- ⁇ antigen, and a YB-1 antigen, a TGF- ⁇ antigen, a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen, a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen, a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen, a TOPOIIa antigen and an ABCB5a antigen, a TGF- ⁇ antigen, a KDR2 antigen
  • Example 1 Treating melanoma using VSV vectors designed to express BRAF
  • mice C57BL/6 mice seeded with B 16tk tumors 5 days previously were treated with GCV i.p. at 50 mg/mL for 5 consecutive days, followed by 2 days' rest, followed by 5 further consecutive injections of GCV (days 6-10 and days 13-17).
  • VSV-BRAF+VSV-YB-l+VSV-GFP injections of VSV-BRAF+VSV-YB-l+VSV-GFP; VSV-TOPOIIa+VSV-BRAF+VSV-GFP; VSV- TOPOIIa+VSV-YB-l+VSV-GFP; or VSV-BRAF+VSV-TOPOIIa+VSV-YB-1 (3xl0 6 pfu/virus/injection).
  • VSV-IFN- ⁇ with -RAS, GNAQ, or TYRP1 is administered in one single tumor location using a 21- or 22-gauge needle, whose length may range between 15 to 20 cm under CT or ultrasound guidance.
  • VSV-hIFN- ⁇ i.e., VSV-IFN-N-RAS, VSV-IFN-GNAQ, and VVS-IFN-TYRPl
  • VSV-hIFN- ⁇ i.e., VSV-IFN-N-RAS, VSV-IFN-GNAQ, and VVS-IFN-TYRPl
  • the combination of three VSV vectors is administered as a mixture via a single intratumoral injection. The injection occurs slowly. If the tumor size is over 2 cm, this may require multiple injections. These injection sites are at least 2 cm apart from one another. Depending on the size and location of the tumor, it is estimated that the procedure will take anywhere from 30 to 60 minutes in duration.
  • the concentration (pfus) for each of the three VSV vectors in the mixture is between 10 5 and 10 9 .
  • the injection is given on day 1, and the length of the study is 28 days.
  • Tissue specimens are collected and submitted on days 1, 2, and 8 on patients that allow for another biopsy.
  • Example 3 Treating skin melanoma patients using VSV vectors designed to express
  • VSV vectors Human skin melanoma patients with stage II and III melanoma are administered adjuvantly and stage IV melanoma patients with oligometastatic melanoma (1-5 metastatic deposits) are administered a combination of three VSV vectors: (a) a VSV vector designed to express a BRAF antigen, (b) a VSV vector designed to express a TOPO-IIa antigen, and (c) a VSV vector designed to express a YB-1 antigen.
  • Patients with stage IV melanoma receive VSV (i.e., VSV-BRAF, VSV YB-1, and VSV- ⁇ - ⁇ ) in combination with ablative radiation.
  • VSV i.e., VSV-BRAF, VSV YB-1, and VSV- ⁇ - ⁇
  • VSV-BRAF, VSV-YB-1, and VSV- ⁇ - ⁇ are administered intratumorly or intravenously or subcutaneously using a 21- or 22-gauge needle, whose length may range between 15 to 20 cm under CT or ultrasound guidance.
  • the three forms of VSV i.e., VSV-BRAF, VSV-YB-1, and VSV- ⁇ - ⁇
  • the combination of three VSV vectors is administered as a mixture via a single injection.
  • Murine B16 cells (American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS; Life technologies) and L-glutamine (Life technologies).
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • Murine GL261 cells American Type Culture Collection, Manassas, VA
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • L-glutamine Life technologies
  • TRAMP-C2 (TC2) cells derived from a prostate tumor that arose in a TRAMP mouse, were characterized as described elsewhere (Kottke et ah, Cancer Res., 67: 11970-9 (2007)) and were routinely grown as tumors in C57BL/6 mice in an androgen- independent manner.
  • the K1735 melanoma cell line (Chong et ah, Hum. Gene Ther., 7: 1771-9 (1996)) was derived from H-2k C3H/He mice.
  • C57BL/6 and C3H mice were purchased from The Jackson Laboratory (B; Harbor, ME) at 6-8 weeks of age.
  • the ASMEL VSV-cDNA library was generated as described elsewhere (Kottke et ah, Nature Med., 201 1 :854-9 (2011); Pulido et ah, Nat. Biotechnoh, 30:337-43 (2012); and Alonso-Camino et al., Mol. Ther., 22: 1936-48 (2014)).
  • VSV expressing N-RAS, CYT-C, TYRP-1, HIF-2a, SOX-10, or c-MYC were isolated by limiting dilution as described elsewhere (Pulido et ah, Nat. Biotechnoh, 30:337-43 (2012); and Alonso-Camino et ah, Mol. Ther., 22: 1936- 48 (2014)). These were expanded in BHK cells and purified by sucrose gradient centrifugation. VSV-GFP was manufactured by cloning the cDNA for GFP into the plasmid pVSV-XN2 as described elsewhere (Fernandez et ah, J. Virol. , 76:895-904 (2002)). Monoclonal VSV-GFP was obtained by plaque purification on BHK-21 cells and concentrated by sucrose-gradient centrifugation.
  • mice were stereotactically injected into the brain (1 mm anterior, and 2 mm lateral to the bregma) of C57B1/6 (B16, GL261, or TC2 cells) or C3H (K1735 cells) mice. Mice were sacrificed upon sign of distress, and single-cell suspensions of brain tumor explants or in vitro cultured cells (B 16, GL261, TC2 or K1735) were plated at lxlO 5 per well in DMEM + 10% FCS and 1% penicillin-streptomycin.
  • HIF-2a polypeptide expression was measured by ELISA according to the manufacturer's instructions (USCN Life Sciences, Houston TX). lxlO 5 cells of each cell line (B16, GL261, TC2, K1735) from in vitro cultures also were plated and measured for HIF-2a polypeptide expression.
  • CD 1 lb + cells were purified from brain-cell suspensions of multiple brains, or from the spleens of C57B1/6 mice (re-suspended in Iscove's modified Dulbecco's medium (IMDM; Gibco, Grand Island, NY) + 5% FCS + 1% penicillin-streptomycin + 40 ⁇ / ⁇ 2 -ME) using CDl lb microbeads according to the manufacturer's instructions (Miltenyi Biotech, Auburn, CA). lxlO 6 CDl lb + cells were co-cultured in DMEM + 10% FCS and 1% penicillin-streptomycin with (lxlO 5 ) GL261 cells.
  • IMDM Iscove's modified Dulbecco's medium
  • FCS 1% penicillin-streptomycin + 40 ⁇ / ⁇ 2 -ME
  • HIF-2a polypeptide levels were measured by ELISA. HIF-2a polypeptide also was evaluated following co-culture of GL261 cells with brain- or spleen-derived CDl lb + cells, in the presence of 10 ng/mL recombinant TGF- ⁇ RII Fc Chimera 341-BR (R&D systems, MN).
  • Human primary glioblastoma brain tumor tissue was obtained following surgery. Within three hours of surgical resection, explants were depleted of CD l lb + cells using CDl lb microbeads. Tumor cells were then seeded at lxlO 4 cells per well in DMEM + 10% FCS + 1% penicillin-streptomycin ⁇ isolated autologous CD 1 lb + cells (5xl0 3 per well). HIF-2a polypeptide levels in cell-free supernatants were evaluated at 24 hours and again following 2 week's culture. HIF-2a polypeptide also was evaluated in cell-free supernatants from lxlO 3 isolated CD1 lb + cells, 24 hours after explant.
  • lxlO 4 GL261 cells in 2 ⁇ L PBS were stereotactically injected using a syringe bearing a 26G needle into the brain (1 mm anterior, and 2 mm lateral to the bregma) of C57BL/6 mice (7-9 mice per treatment group unless otherwise stated).
  • Virus, drug, or PBS control 100 ⁇ was administered intravenously following 5 days tumor establishment and occurred as dictated by each specific study. Mice were examined daily for overall health and, survival with time was documented.
  • control ChromPure rat IgG antibody Jackson Immunochemicals, West Grove, PA
  • anti-PD 1 antibody were injected
  • intravenous anti- PD1 was administered at 225 ⁇ g/mouse/injection and anti-CTLA4 at 0.1
  • Spleens and lymph nodes were harvested from euthanized mice and dissociated into single-cell suspensions by crushing through a 100 ⁇ filter.
  • Red blood cells were lysed with ACK lysis buffer (sterile distilled H2O containing 0.15 M NH4CI, 1.0 mM KHCO3 and 0.1 mM EDTA adjusted to pH 7.2 - 7.4) for 2 minutes.
  • Cells were re-suspended at lxlO 6 cells/mL in IMDM + 5% FCS + 1% penicillin- streptomycin + 40 ⁇ / ⁇ 2-ME.
  • VSV-N protein derived epitope peptide VSV-N52-59:RGYVYQG at 5 ⁇ g/mL
  • supernatants were evaluated for IFN- ⁇ and IL-17 response by ELISA.
  • Intra-cranial tumors of different histologies express a similar HIF-2aHi phenotype. It was hypothesized that the intra-cranial microenvironment imposes a HIF-2aHi phenotype upon different types of tumors, which is distinct from that expressed by the same tumor cells growing in culture. Consistent with this hypothesis, freshly resected i.e. tumors of different histological types, including K1735 melanoma (in C3H mice), as well as B16 melanoma, GL261 glioma, and TC2 prostate cancer (C57B1/6 mice), all expressed a HIF-2aHi phenotype. In contrast, the same cell lines grown in culture, from which the tumors were initially derived by i.e. implantation, expressed low or undetectable levels of HIF-2a ( Figure 2).
  • CD1 lb + cells in intact brain homogenate impose a HIF-2aHi phenotype on GL261 cells in vitro in part through TGF- ⁇ .
  • the HIF-2aHi phenotype of i.e. B16-ova tumors was imposed by brain-associated, but not spleen-derived, CD1 lb + cells.
  • Human tumor explants express a HIF-2aHi phenotype, which is reduced over time.
  • HIF-2aHi phenotype of primary human brain tumor samples was studied.
  • Freshly resected tumors cultured with their own autologous CD1 lb + cells exhibited a HIF- 2aHi phenotype, although levels of HIF-2a were consistently lower than in resected murine tumors ( Figure 4).
  • the CD1 lb + cells themselves did not express significant levels of HIF-2a ( Figure 4).
  • human brain tumors also express a HIF-2aHi phenotype, which is maintained, at least in part, by immune cells within the brain microenvironment.
  • Intracranial GL261 can be treated with VSV-tumor-associated antigen therapy and enhanced by addition of checkpoint inhibitors
  • mice bearing s.c. B16 tumors were treated successfully with a combination of VSV-expressed N-RAS, CYT-C, and TYRP-1, i.e. B16 tumors were only successfully treated with a combination of VSV expressed HIF-2a, SOX- 10, c- MYC, and TYRP-1.
  • Anti-PD- 1 antibody uncovers a Thl response against intra-cranial GL261.
  • the therapeutic anti-tumor response to self antigens induced by VSV-cDNA library treatment is Thl 7 CD4 + T cell mediated and no Thl IFN- ⁇ T cell responses could be detected.
  • Mixed splenocytes and lymph node cultures from mice bearing i.c. GL261 tumors following treatment with VSV-HIF-2a, VSV-SOX-10, and VSV-c-MYC did not secrete IFN- ⁇ in response to challenge with freeze/thaw lysates of explanted i.c. GL261 tumors ( Figure 6B).
  • Anti-PD 1 antibody therapy does not enhance the Thl 7 response against intracranial GL261.
  • GL261 responses were only induced when VSV expressed tumor antigens VSV-HIF-2a/SOX-10/c-MYC, as opposed to VSV-GFP, ( Figures 6B and 6D, respectively), indicating that virally- mediated expression of tumor antigens was required for an effective immune response.
  • Anti-PDl antibody enhances the Thl response against VSV.
  • 2a/SOX-10/c-MYC viroimmunotherapy with anti-PDl checkpoint inhibition de- represses an anti -tumor Thl IFN- ⁇ T cell response against both self antigens and against foreign viral antigens, but has no significant effect on the anti-tumor Thl 7 response.
  • VSV-antigen e.g., VSV-HIF-2a/SOX-10/c- MYC
  • VSV-antigen e.g., VSV-HIF-2a/SOX-10/c- MYC
  • a combination of anti-PD 1 and anti-CTLA-4 checkpoint inhibition to target separate stages of the T cell activation/repression pathway was tested in combination with VSV-antigen (e.g., VSV-HIF-2a/SOX-10/c-MYC) therapy.
  • VSV-HIF-2a/SOX-10/c-MYC therapy in combination with anti-CTLA4 exhibited a strong trend to enhancing the Thl 7 response to i.e. GL261 responses ( Figure 8D) compared to VSV-HIF-2a/SOX- 10/c-MYC therapy alone, or in combination with anti-PDl.
  • splenocyte/LN cultures from mice treated with VSV-HIF-2a/SOX-10/c-MYC therapy combined with both anti-CTLA4 and anti-PDl therapy generated the strongest Thl 7 recall responses against i.c GL261 ( Figure 8D).
  • VSV- antigen e.g., VSV-HIF-2a/SOX-10/c-MYC
  • these increases in therapy are associated with the de-repression of an anti-tumor Thl IFN- ⁇ T cell response (anti-PDl, anti-CTLA4, or both) and of the anti-tumor Thl 7 response (anti- PD1 plus anti-CTLA4).
  • VSV-TGF- ⁇ + VSV-KDR2 + VSV-P Glyc + VSV TYRP-1) cured 6/6 mice which did not experience toxicities associated with i.v. VSV treatment ( Figure 9). Two of the 8 mice treated died of hind limb paralysis, associated with a high i.v. dose (2xl0 7 pfu/injection; 9 injections) of the VSV combination.
  • mice were cured of K 1735 tumors with a lower dose of VSV-combination (VSV-TGF- ⁇ + VSV-KDR2 + VSV-P Glyc + VSV TYRP- 1) (5xl0 6 pfu/injection; 9 injections). 5/5 mice were cured with the same dose of the total library (ASMEL).
  • ASMEL total library
  • an in vitro assay ( Figure 10) was performed where splenocytes were depleted of different types of antigen presenting cells by Miltenyi beads.
  • VSV-TGF- ⁇ + VSV-KDR2 + VSV-P Glyc + VSV TYRP-1 VSV-combination
  • VSV-TGF- ⁇ + VSV-KDR2 + VSV-P Glyc + VSV TYRP-1 VSV-combination
  • depletion of certain subsets of APC abrogated IL-17 secretion.
  • Those APC were then infected with a single VSV-TAA (VSV-TGF- ⁇ , VSV-KDR2, VSV-P Glyc, or VSV TYRP-1) and re-introduced into the antigen presentation assay.
  • Example 6 Treating melanoma using VSV vectors designed to express truncated antigens
  • VSV vectors having nucleic acid that encodes truncated versions of antigens were recovered from the ASMEL cDNA library. The nucleic acids were sequenced to identify the location of the 3' truncations.
  • the VSV vector contained an N-RAS cDNA that encodes an N-RAS polypeptide lacking the following C-terminus: YRMKKLNSSDDGTQGCMGLPCVVM (SEQ ID NO: 1). See, also, Figure 14.
  • the VSV vector contained a CYT-C cDNA that encodes a CYT-C polypeptide lacking the following C-terminus: YTIKRHKWSVLKSRKLAYRPPK (SEQ ID NO:2). See, also, Figure 15.
  • the VSV vector contained a TYRP- 1 cDNA that encodes a TYRP- 1 polypeptide lacking the following C-terminus: YQCYAEEYEKLQNPNQSVV (SEQ ID NO:3). See, also, Figure 16.
  • the VSV vector contained a TGF- ⁇ cDNA that encodes a TGF- ⁇ polypeptide lacking the following C-terminus: YYV- GRKPKVEQLSNMTVRSCKCS (SEQ ID NO:4).
  • the VSV vector contained a KDR2 cDNA that encodes a KDR2 polypeptide lacking the following C-terminus: YSSEEAELLKLIEIGVQTGSTAQILQPD- SGTTLSSPPV (SEQ ID NO:5).
  • the VSV vector contained a P-glycoprotein cDNA that encodes a P-glycoprotein polypeptide lacking the following C-terminus: YFSMVSVQAGTKRQ (SEQ ID NO:6).
  • VSV encoding library derived, truncated VSV- N-RAS + VSV-CYT-C + VSV TYRP-1 (5xl0 6 pfu/100 ⁇ ), (2) VSV encoding full length polypeptides: VSV-NRAS + VSV-TYRP-1 + VSV-CYT-C, or (3) VSV-GFP. Survival of tumor- bearing C57BL/6 (n 8 mice per group) was determined. The results were representative of two separate experiments.
  • truncated antigens e.g., antigens lacking a portion of their C terminus
  • Dogs e.g., 5-10 dogs with a solitary intracranial mass consistent with a gliomas based on MRI that is surgically accessible are recruited. The diagnosis is confirmed as a high-grade (III-IV) glioma by histopathology. The dogs are otherwise in good health and able to undergo anesthesia for surgical excision and virus delivery.
  • the dogs are treated by surgical removal of the tumor and administration of either single VSV vectors (e.g., VSV-HIF-2a only) or a combination of different VSV vectors (e.g., VSV-HIF-2a + VSV-SOX-10 + VSV-cMYC).
  • single VSV vectors e.g., VSV-HIF-2a only
  • a combination of different VSV vectors e.g., VSV-HIF-2a + VSV-SOX-10 + VSV-cMYC
  • any particular combination of VSV vectors provided herein is administered to a dog having cancer.
  • a VSV-cDNA library such as an ASMEL is administered to a dog having cancer.
  • Toxicities are assessed using a standard veterinary scale of grade I-V events based on owner diaries, serial blood tests, and neurological examinations.
  • Surgical resection of the tumor is performed using the appropriate approach based on MRI.
  • Each dog is administered a standard drug regimen before craniotomy to minimize cerebral edema.
  • each dog is administered 5xl0 8 pfu of Reolysin (reovirus) injected in 5- ⁇ aliquots around the resection cavity.
  • a postoperative MRI is performed to assess the extent of resection, and then each dog is allowed to recover from anesthesia and is monitored in an intensive care unit.
  • each dog is administered prednisone (1 mg/kg body weight) PO every 12 hours for 2 days, and then the dose is tapered and discontinued over 7 days.
  • Adjustments are made to the dose of steroids depending on the clinical signs, such as changes in mentation or neurological function (i.e., hemiparesis), of each individual dog.
  • the dogs are examined by MRI of the brain performed immediately after surgery and then 4, 8, and 12 months after therapy.
  • the scans are evaluated, and the surgical resection of the tumor is defined as gross total resection (GTR) if there is complete resection of the preoperative fluid-attenuated inversion recovery signal abnormality, near total resection (NTR) if a thin ( ⁇ 3 mm) residual fluid-attenuated inversion recovery signal abnormality remains around the rim of the resection cavity, or subtotal resection (STR) if there is residual nodular fluid-attenuated inversion recovery signal abnormality.
  • GTR gross total resection
  • NMR near total resection
  • STR subtotal resection
  • Clinical response is considered as complete response (CR) if there is no evidence of the target lesion, partial response (PR) if the tumor is ⁇ 25% of the original longest diameter of the tumor, progressive disease if there is > 25% increase in the original longest diameter of the tumor, or stable disease (SD) if there are small changes that do not meet the previously defined criteria. If a dog develops recurrent or worsening neurologic signs before a scheduled MRI, an unscheduled MRI is performed at that time.
  • each dog is treated with intravenous injections of 5xl0 6 pfu of VSV-TAA (e.g., a single VSV vector such as VSV-HIF-2a only or a combination of different VSV vectors such as VSV-HIF-2a + VSV-SOX-10 + VSV- cMYC) on days 10, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, and 360 after surgery, or until tumor recurrence.
  • VSV-TAA e.g., a single VSV vector such as VSV-HIF-2a only or a combination of different VSV vectors such as VSV-HIF-2a + VSV-SOX-10 + VSV- cMYC
  • any particular combination of VSV vectors provided herein is administered to a dog having cancer as an immunological boost.
  • a VSV-cDNA library such as an ASMEL is administered to a dog having cancer as an immunological boost.
  • Dogs are monitored for 30 minutes following each injection for any immediate adverse reactions, such as severe wheals, dyspnea, or other signs of anaphylaxis. Dogs suffering from an acute severe reaction are given dexamethasone (0.01 mg/kg SC) and diphenhydramine (0.5 mg/kg IM). Dogs are followed over a 12-month period by imaging or until euthanasia. Dogs are assessed with complete physical and neurological examinations and quality of life assessments at suture removal and each VSV-TAA injection.
  • any immediate adverse reactions such as severe wheals, dyspnea, or other signs of anaphylaxis. Dogs suffering from an acute severe reaction are given dexamethasone (0.01 mg/kg SC) and diphenhydramine (0.5 mg/kg IM). Dogs are followed over a 12-month period by imaging or until euthanasia. Dogs are assessed with complete physical and neurological examinations and quality of life assessments at suture removal and each VSV-TAA injection.
  • PBMC Peripheral blood mononuclear cells
  • Example 8 Treating cancer using VSV designed to express IFN- ⁇
  • VSV encoding TYRP-1 (full length) and IFN- ⁇ was generated by inserting mTYRP-1 in the vector backbone containing IFN- ⁇ (IFN- ⁇ ) located between the M and G genes of VSV ( Figure 18).
  • PCR amplification of niTYRP-1 cDNA was prepared from B 16 cells using forward (5 ' -CTCGAGATG- AAATCTTACAACGTCC-3 ' ; SEQ ID NO: 7) and reverse (5'- CTAGCTAGCTCA- GACCATGGAGTGGTTA-3 ' ; SEQ ID NO: 8) primers.
  • VSV-mTYRP-1 was generated by inserting TYRP-1 between the G and L genes.
  • Viruses were generated from BHK cells by co-transfection of pVSV-XN2-cDNA library DNA along with plasmids encoding viral genes as described elsewhere (Fernandez et ah, J. Virol. , 76:895-904 (2002)). Virus was expanded by a single round of infection of BHK cells and purified by sucrose gradient centrifugation.
  • Splenocytes/LN from C57BL/6 mice bearing s.c. B16 tumors and treated with PBS alone or with either VSV-GFP, VSV-mTYRP-1, VSV-mIFN-p-TYRP-l, or VSV-mIFN- ⁇ were harvested.
  • Splenocytes were re-stimulated in vitro with PBS, VSV N peptide VSV-N52-59 (RGYVYQGL; SEQ ID NO:9) or with synthetic H- 2Kb-restricted melanoma peptides: murine TRP- 1222-229 (TAYRYHLL, SEQ ID NO: 10; or TWYRYHLL SEQ ID NO: l 1; TAY, TWY, respectively), TRP-2180-188 (SVYDFFVWL, SEQ ID NO: 12; TRP2), murine gplOO (EGSRNQDWL, SEQ ID NO: 13; mgplOO), or human gpl0025-33 (KVPRNQDWL, SEQ ID NO:14; hgplOO). Forty eight hours later, supernatants were assayed for IFN- ⁇ by ELISA ( Figure 19).
  • mice 5xl0 5 B16-ova tumor cells in 100 ⁇ L of PBS were injected into the flanks of C57BL/6 mice (7 mice per treatment group). Seven days later, mice were treated intra-tumorally (IT) with PBS, VSV encoding antigens, or VSV-GFP at 7xl0 8 /50 for three days every other day ( Figure 20). Survival times were determined ( Figure 21).

Abstract

This document provides methods and materials for treating cancer. For example, methods and materials for treating cancer using combinations of antigens are provided. For example, VSV vectors designed to express a GNAQ antigen, a TYRP1 antigen, and an N-RAS antigen can be used to reduce the number of cancer cells (e.g., uveal melanoma cells) within a mammal (e.g., a human). In some cases, VSV vectors designed to express a BRAF antigen, a TOPO-lla antigen, and a YB-I antigen can be used to reduce the number of cancer cells (e.g., skin melanoma cells) within a mammal (e.g., a human). The composition can comprise less than 50 separate nucleic acid molecules.

Description

METHODS AND MATERIALS FOR TREATING CANCER
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Serial No. 61/955,648 filed March 19, 2014. This disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
BACKGROUND
1. Technical Field
This document relates to methods and materials for treating cancer. For example, this document relates to methods and materials for using combinations of antigens to treat cancer (e.g., melanoma such as skin melanoma or uveal melanoma, non-Hodgkin lymphoma, colorectal cancer, brain tumors, papillary thyroid carcinoma, non-small-cell lung carcinoma, or adenocarcinoma of the lung). 2. Background Information
Cancer is a serious illness that affects many people every year. In general, there are several common methods for treating cancer: surgery, chemotherapy, radiation therapy, immunotherapy, and biologic therapy. When initially diagnosed with cancer, a cancer specialist such as an oncologist can provide a patient with various cancer treatment options. Typically, an oncologist will recommend the best treatment plan based on the type of cancer, how far it has spread, and other important factors like the age and general health of the patient.
SUMMARY
This document provides methods and materials for treating cancer. For example, this document provides combinations of antigens having the ability to reduce the presence of cancer (e.g., reduce established tumors) within a mammal (e.g., a human). As described herein, combinations of antigens (e.g., a combination of a GNAQ antigen, a TYRP 1 antigen, and an N-RAS antigen, a combination of a BRAF antigen, a ΤΟΡΟ-Πα antigen, and a YB-1 antigen, a combination of a TGF-β antigen, a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen, a combination of a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen, a combination of a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen, a combination of a TOPOIIa antigen and an ABCB5a antigen, or a combination of an ABCB5a antigen, a CYT-C antigen, a N-RAS antigen, and a TYRP-1 antigen) can be used to treat cancer (e.g., melanoma such as skin melanoma or uveal melanoma, non-Hodgkin lymphoma, colorectal cancer, brain tumors, papillary thyroid carcinoma, non-small-cell lung carcinoma, or adenocarcinoma of the lung). For example, VSV vectors designed to express a GNAQ antigen, a TYRP1 antigen, and an N-RAS antigen can be used to reduce the number of cancer cells (e.g., uveal melanoma cells) within a mammal (e.g., a human). In some cases, VSV vectors designed to express a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen can be used to reduce the number of cancer cells (e.g., skin melanoma cells) within a mammal (e.g., a human). In some cases, the combinations of antigens provided herein (e.g., a combination of a GNAQ antigen, a TYRP 1 antigen, and an N-RAS antigen or a combination of a BRAF antigen, a TOPO-IIa antigen, and a YB- 1 antigen) can be used to treat cancer a cancer that overexpresses TOPO-IIa, YB-1, TYRP-1, or BRAF.
In general, one aspect of this document features a composition comprising, or consisting essentially of, nucleic acid encoding a GNAQ antigen, a TYRP 1 antigen, and an N-RAS antigen, wherein the composition comprises less than 100 separate nucleic acid molecules. The composition can comprise a nucleic acid molecule encoding the GNAQ antigen, a nucleic acid molecule encoding the TYRP 1 antigen, and a nucleic acid molecule encoding the N-RAS antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the GNAQ antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the TYRP1 antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the N-RAS antigen. The composition can comprise less than 50 separate nucleic acid molecules. The composition can comprise less than 10 separate nucleic acid molecules. The composition can comprise less than 5 separate nucleic acid molecules.
In another aspect, this document features a method of treating cancer within a mammal. The method comprises, or consists essentially of, administering to the mammal a composition comprising, or consisting essentially of, nucleic acid encoding a GNAQ antigen, a TYRP1 antigen, and an N-RAS antigen, wherein the composition comprises less than 100 separate nucleic acid molecules. The cancer can be a melanoma. The mammal can be a human. The composition can comprise a nucleic acid molecule encoding the GNAQ antigen, a nucleic acid molecule encoding the TYRP 1 antigen, and a nucleic acid molecule encoding the N-RAS antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the GNAQ antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the TYRP 1 antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the N-RAS antigen. The composition can comprise less than 50 separate nucleic acid molecules. The composition can comprise less than 10 separate nucleic acid molecules. The composition can comprise less than 5 separate nucleic acid molecules.
In another aspect, this document features a composition comprising, or consisting essentially of, nucleic acid encoding a BRAF antigen, a ΤΟΡΟ-Πα antigen, and a YB-1 antigen, wherein the composition comprises less than 100 separate nucleic acid molecules. The composition can comprise a nucleic acid molecule encoding the BRAF antigen, a nucleic acid molecule encoding the TOPO-IIa antigen, and a nucleic acid molecule encoding the YB- 1 antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the BRAF antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the TOPO- IIa antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the YB-1 antigen. The composition can comprise less than 50 separate nucleic acid molecules. The composition can comprise less than 10 separate nucleic acid molecules. The composition can comprise less than 5 separate nucleic acid molecules.
In another aspect, this document features a method of treating cancer within a mammal. The method comprises, or consists essentially of, administering to the mammal a composition comprising, or consisting essentially of, nucleic acid encoding a BRAF antigen, a TOPO-IIa antigen, and a YB- 1 antigen, wherein the composition comprises less than 100 separate nucleic acid molecules. The cancer can be a melanoma. The mammal can be a human. The composition can comprise a nucleic acid molecule encoding the BRAF antigen, a nucleic acid molecule encoding the TOPO-IIa antigen, and a nucleic acid molecule encoding the YB-1 antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the BRAF antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the TOPO-IIa antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the YB-1 antigen. The composition can comprise less than 50 separate nucleic acid molecules. The composition can comprise less than 10 separate nucleic acid molecules. The composition can comprise less than 5 separate nucleic acid molecules.
In another aspect, this document features a composition comprising, or consisting essentially of, nucleic acid encoding: (a) a TGF-β antigen, a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen, (b) a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen, (c) a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen, (d) a TOPOIIa antigen and an ABCB5a antigen, or (e) an ABCB5a antigen, a CYT-C antigen, a N-RAS antigen, and a TYRP-1 antigen, wherein the composition comprises less than 100 separate nucleic acid molecules. The composition can comprise: (a) a nucleic acid molecule encoding a TGF-β antigen, a nucleic acid molecule encoding a MDR1 antigen, a nucleic acid molecule encoding a TYRP- 1 antigen, and a nucleic acid molecule encoding a KDR2 antigen, (b) a nucleic acid molecule encoding a TOPOIIa antigen, a nucleic acid molecule encoding a YB- 1 antigen, a nucleic acid molecule encoding a CDC7 kinase antigen, and a nucleic acid molecule encoding a BRAF antigen, (c) a nucleic acid molecule encoding a TOPOIIa antigen, a nucleic acid molecule encoding a YB-1 antigen, a nucleic acid molecule encoding a CDC7 kinase antigen, and a nucleic acid molecule encoding a CD44 antigen, (d) a nucleic acid molecule encoding a TOPOIIa antigen and a nucleic acid molecule encoding an ABCB5a antigen, or (e) a nucleic acid molecule encoding an ABCB5a antigen, a nucleic acid molecule encoding a CYT-C antigen, a nucleic acid molecule encoding a N-RAS antigen, and a nucleic acid molecule encoding a TYRP-1 antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the TGF-β antigen, the MDR1 antigen, the TYRP-1 antigen, the KDR2 antigen, the TOPOIIa antigen, the YB-1 antigen, the CDC7 kinase antigen, the BRAF antigen, the CD44 antigen, the ABCB5a antigen, the CYT-C antigen, or the N-RAS antigen. The composition can comprise less than 50 separate nucleic acid molecules. The composition can comprise less than 10 separate nucleic acid molecules. The composition can comprise less than 6 separate nucleic acid molecules.
In another aspect, this document features a method of treating cancer within a mammal. The method comprises, or consists essentially of, administering to the mammal a composition of the preceding paragraph. The cancer can be a melanoma. The mammal can be a human. In another aspect, this document features a composition comprising, or consisting essentially of, nucleic acid encoding a TGF-β antigen, a KDR2 antigen, a P Glyc antigen, and a TYRP-1 antigen, wherein the composition comprises less than 100 separate nucleic acid molecules. The composition can comprise a nucleic acid molecule encoding the TGF-β antigen, a nucleic acid molecule encoding the KDR2 antigen, a nucleic acid molecule encoding the P Glyc antigen, and a nucleic acid molecule encoding the TYRP-1 antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the TGF-β antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the KDR2 antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the P Glyc antigen. The composition can comprise a VSV vector comprising nucleic acid encoding the TYRP1 antigen. The composition can comprise less than 50 separate nucleic acid molecules. The composition can comprise less than 10 separate nucleic acid molecules. The composition can comprise less than 5 separate nucleic acid molecules.
In another aspect, this document features a method of treating cancer within a mammal, wherein the method comprises administering to the mammal a composition comprising nucleic acid encoding a TGF-β antigen, a KDR2 antigen, a P Glyc antigen, and a TYRP-1 antigen, wherein the composition comprises less than 100 separate nucleic acid molecules. The cancer can be a melanoma. The mammal can be a human.
In another aspect, this document features a composition of any one of the above recited paragraphs, wherein the composition comprises an immune checkpoint inhibitor. The immune checkpoint inhibitor can be an anti-PD-1 antibody or an anti- CTLA4 antibody.
In another aspect, this document features a method of any one of the above recited paragraphs, wherein the method comprises administering an immune checkpoint inhibitor to the mammal. The immune checkpoint inhibitor can be an anti- PD-1 antibody or an anti-CTLA4 antibody.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
DESCRIPTION OF DRAWINGS
Figure 1 is a graph plotting the percent survival of mice from B 16 recurrences that escaped frontline ganciclovir (GCV) treatment. The mice were treated with the indicated combinations of VSV vectors.
Figure 2. Intracranial tumors of different histology express a HIF-2aHi phenotype. Tumors established in the brains of C57BL/6 (B16, GL261 or TC2 cells) or C3H (K1735) mice were dissected upon sacrifice (tumor explants), and tumor cells were seeded at lxlO5 per well. lxlO5 cells of each cell line cultured in vitro (cult.) were also plated. HIF-2a was measured by ELISA after 24 hours. Error bars are expressed as standard deviation (SD).
Figure 3. Brain derived CD1 lb+ cells impose a HIF-2aHi phenotype on in vitro cultured GL261, in part through TGF-β. HIF-2a expression was measured by ELISA from: lxlO5 GL261 cells cultured in vitro for 24 hours (lane 1); GL261 i.e. tumors, dissected from the brain upon sacrifice, and plated at lxlO5 cells per well for 24 hours (lane 2); lxlO5 GL261 cells co-cultured for 24 hours with lxlO6 CD1 lb+ cells purified from normal splenocytes of C57B1/6 mice (lane 3); lxlO5 GL261 cells co-cultured for 24 hours with lxlO6 CD1 lb+ cells purified from normal brains of C57B1/6 mice (lane 4). Cultures of lanes 3 and 4 were repeated in the presence of recombinant TGF-β RII Fc chimera at 10 ng/mL (lane 5 and 6). Results are representative of three separate measurements. Error bars are expressed as standard deviation (SD).
Figure 4. Human brain tumor explants express a HIF-2aHi phenotype which diminishes with time. Human brain tumor explants were recovered from surgery and depleted of CD1 lb+ cells. Tumor cells were plated at lxlO4 per well either alone (24 hours CD1 lb ) or with 5xl03 CD1 lb+ cells (24 hours CD1 lb+). HIF-2a expression was measured at 24 hours. In cultures from which tumor cells survived more than a week, HIF-2a was measured from lxlO4 tumor cells after 2 weeks, by which time CD1 lb+ cells had been washed away/died (2 week CD1 lb"). HIF-2a also was measured from lxlO3 separated CD1 lb+ cells 24 hours after explant. Results are representative of three separate measurements. Error bars are expressed as standard deviation (SD).
Figure 5. VSV-TAA therapy of intracranial GL261 tumors. C57BL/6 mice bearing 5 day established i.e. GL261 tumors were treated intravenously with a total of 5xl06 pfu of (VSV-HIF-2a, VSV-SOX-10, and VSV-c-MYC); (VSV-HIF-2a, VSV- SOX-10, and VSV-GFP); (VSV-N-RAS, VSV-CYT-C, and VSV-TYRP-1), or (VSV- GFP) on days 6, 8, 10, 13, 15, 17, 20, 22, 24, 27, 29, and 31. Survival with time is shown.
Figure 6. Checkpoint inhibition uncovers a repressed anti-tumor Thl IFN-γ response. A. C57BL/6 mice bearing 5 day established i.e. GL261 tumors were treated intravenously with a total of 5xl06 pfu of (VSV-GFP); (VSV-HIF-2a, VSV- SOX-10, and VSV-c-MYC), or PBS on days 6, 8, 10, 13, 15, 17, 20, 22, and 24. On days 13, 15, 17, 20, 22, and 24, these groups were treated intravenously with either PBS, control IgG antibody, or anti-PDl antibody at 10 mg/kg/mouse as shown.
Survival with time is shown. B-D. Splenocytes and lymph nodes were pooled from 3 C57BL/6 mice per group bearing 5 day established i.e. GL261 tumors treated with either (PBS/PBS); (VSV-GFP + anti-PDl antibody); (VSV-HIF-2a, VSV-SOX-10, and VSV-C-MYC+ IgG), or (VSV-HIF-2a, VSV-SOX-10, and VSV-C-MYC+ anti- PDl antibody). Cells were plated at lxlO6 cells per well and re-stimulated in vitro 3 times at 24 hour intervals with lxlO5 cells of freeze thaw lysates of GL261 tumors recovered from mice bearing i.e. GL261 tumors (B and D) or with freeze thaw lysates of in vitro cultured GL261 (C and E). 48 hours later, supernatants were assayed for IFN-γ (B and C) or IL-17 (D and E) by ELISA. F. Splenocytes and lymph nodes also were re-stimulated with the VSV-N protein derived epitope at 5 μg/mL, 3 times for 24 hours. 48 hours later, supernatants were assayed for IFN-γ. Each result is representative of 3 separate measurements. Error bars are expressed as standard deviation (SD).
Figure 7. Anti-PDl checkpoint inhibition uncovers a Thl IFN-γ anti-tumor response. A. Splenocytes and lymph nodes were pooled from 3 C57BL/6 mice per group bearing 5 day established i.e. GL261 tumors treated with either (VSV-HIF-2a, VSV-SOX-10, and VSV-C-MYC+ IgG) or (VSV-HIF-2a, VSV-SOX-10, and VSV-c- MYC+ anti-PDl antibody). Cells were plated at lxlO6 cells per well and re- stimulated in vitro 3 times at 24 hour intervals with lxlO5 cells of freeze thaw lysates of GL261 tumors recovered from mice bearing i.e. GL261 tumors (lanes 1 and 2, and 3 and 4). The same experiment also was carried out with splenocytes and lymph node cells depleted of Treg cells (lanes 5 and 6, and 7 and 8). Following 48 hours of culture, supernatants were assayed for IFN-γ (A) or IL-17 (B) by ELISA. Results are representative of 3 separate measurements. Error bars are expressed as standard deviation (SD).
Figure 8. Double checkpoint inhibition therapy enhances treatment with VSV-antigens. A. C57BL/6 mice bearing 5 day established i.e. GL261 tumors were treated intravenously with a total dose of 5xl06 pfu of (VSV-GFP); (VSV-HIF-2a, VSV-SOX-10, and VSV-c-MYC) or PBS on days 6, 8, 10, 13, 15, and 17. On days 13, 15, and 17, these groups also were treated with either anti-PDl antibody, anti- CTLA4 antibody, anti-PDl antibody plus anti-CTLA4 antibody, or PBS as shown. Survival with time is shown. B-D. Splenocytes and lymph nodes were pooled from 3 C57BL/6 mice per group bearing 5 day established i.e. GL261 tumors treated with either (VSV-GFP + anti-PDl + anti-CTLA4); (VSV-HIF-2a, VSV-SOX-10, and VSV-c-MYC + anti-PDl antibody + anti-CTLA4 antibody); (VSV-HIF-2a, VSV- SOX-10, and VSV-c-MYC + PBS); (PBS + PBS); (VSV-HIF-2a, VSV-SOX-10, and VSV-c-MYC + anti-PDl antibody); or (VSV-HIF-2a, VSV-SOX-10, and VSV-c- MYC + anti-CTLA4 antibody). Cells were plated at lxl 06 cells per well and re- stimulated in vitro 3 times at 24 hour intervals with lxlO5 cells of freeze thaw lysates of GL261 tumors recovered from mice bearing i.e. GL261 tumors (B and D) or with freeze thaw lysates of in vitro cultured GL261 (C and E). 48 hours later, supernatants were assayed for IFN-γ (B and C) or IL-17 (D and E) by ELISA.
Figure 9 is a graph plotting the percent survival of mice having s.c. B 16 tumors and treated with PBS or the indicated combinations of VSV vectors.
Figure 10 is a schematic of an in vitro assay designed to determine which specific antigens are presented by which antigen presenting cell subtypes in order to reconstitute a Thl7 response.
Figure 1 1 is a bar graph plotting IL-17 levels (pg/mL) for the indicated conditions. None/none indicates no depletion of any cells; Ly6G/Ly-V-CYT-C indicates depletion of neutrophils and the reinfection with VSV-cytochrome C;
Ly6G/Ly-V-N-RAS : indicates depletion of neutrophils and reinfection with VSV-N- RAS; Ly6G/Ly-V-TYRP- 1 indicates depletion of neutrophils and reinfection with VSV-TYRP-1 ; Ly6G/Ly-VSVGFP indicates depletion of neutrophils and the reinfection with VSV-GFP; pDC/pDC-V-CYT-C indicates depletion of plasmacytoid dendritic cells and the reinfection with VSV-cytochrome C; pDC/pDC-V-N-RAS indicates depletion of plasmacytoid dendritic cells and the reinfection with VSV-N- RAS; pDC/pDC-V-TYRP-1 indicates depletion of plasmacytoid dendritic cells and reinfection with VSV-TYRP-1 ; pDC/pDC-V-GFP indicates depletion of plasmacytoid dendritic cells and reinfection with VSV-GFP; 1 IB/1 1B-V-CYTC indicates depletion of CD1 lb+ cells and reinfection with VSV-Cytochrome C; 1 IB/11B-V-N-RAS indicates depletion of CD1 lb+ cells and reinfection with VSV-N-RAS; 1 IB/1 1B-V- TYRP-1 indicates depletion of CD1 lb+ cells and reinfection with VSV-TYRP-1; and 1 IB/11B-V-GFP indicates depletion of CD1 lb+ and reinfection with VSV-GFP.
Figure 12 is a bar graph plotting IL-17 levels (pg/mL) for the indicated conditions. None/none indicates no depletion of any cells; Ly6G/Ly-V-TGF indicates depletion of neutrophils and the reinfection with VSV-TGF ; Ly6G/Ly-V- KDR2 indicates depletion of neutrophils and reinfection with VSV-KDR2; Ly6G/Ly- V-Pglyco indicates depletion of neutrophils and reinfection with VSV-P Glyc;
Ly6G/Ly-VTYRP-l indicates depletion of neutrophils and the reinfection with VSV- TYRP-1; pDC/pDC-V-TGF indicates depletion of plasmacytoid dendritic cells and the reinfection with VSV-TGF ; pDC/pDC-V-KDR2 indicates depletion of plasmacytoid dendritic cells and the reinfection with VSV-KDR2; pDC/pDC-V P
Glyc indicates depletion of plasmacytoid dendritic cells and reinfection with VSV- P Glyc; pDC/pDC-V-TYRP-1 indicates depletion of plasmacytoid dendritic cells and reinfection with VSV-TYRP-1 ; l lB/l lB-V-TGF indicates depletion of CDl lb+ cells and reinfection with VSV-TGF ; 1 IB/11B-V-KDR2 indicates depletion of CD 1 lb+ cells and reinfection with VSV-KDR2; 1 IB/1 1B-V-Pglyc indicates depletion of CD1 lb+ cells and reinfection with VSV-P Glyc; and 1 IB/1 lB-V-TYRP-1 indicates depletion of CD1 lb+ and reinfection with VSV-TYRP-1.
Figure 13 is a proposed model of antigen presentation.
Figure 14 is contains sequence information for a truncated VSV-N-RAS virus recovered from an ASMEL.
Figure 15 is contains sequence information for a truncated VSV-CYT-C virus recovered from an ASMEL.
Figure 16 is contains sequence information for a truncated VSV-TYRP- 1 virus recovered from an ASMEL. Figure 17 is a graph plotting the percent survival of mice having s.c. B16 tumors and treated with the indicated VSV vectors.
Figure 18 is a schematic of the indicated VSV vectors.
Figure 19 is a bar graph plotting IFN-γ levels (pg/mL) for cells obtained from mice treated as indicated and stimulated with the indicated polypeptides.
Figure 20 is a schematic of an in vivo assay for assessing VSV vectors expressing IFN-β polypeptides.
Figure 21 is a graph plotting the percent survival of mice having B 16 tumors and treated with the indicated VSV vectors.
DETAILED DESCRIPTION
This document provides methods and materials for treating cancer. For example, this document provides combinations of antigens having the ability to reduce the number of cancer cells within a mammal (e.g., a human). As described herein, combinations of antigens that include a GNAQ antigen, a TYRP 1 antigen, and an N-RAS antigen, that include a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen, that include a TGF-β antigen, a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen, that include a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen, that include a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen, that include a TOPOIIa antigen and an ABCB5a antigen, that include an ABCB5a antigen, a CYT-C antigen, a N-RAS antigen, and a TYRP-1 antigen, or that include a TGF-β antigen, a KDR2 antigen, a P glycoprotein (P Glyc) antigen, and a TYRP-1 antigen can be used to treat cancer. In some cases, combinations of antigens that include a GNAQ antigen, a TYRPl antigen, and an N-RAS antigen, that include a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen, that include a TGF-β antigen, a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen, that include a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen, that include a TOPOIIa antigen, a YB- 1 antigen, a CDC7 kinase antigen, and a CD44 antigen, that include a TOPOIIa antigen and an ABCB5a antigen, that include an ABCB5a antigen, a CYT-C antigen, a N- RAS antigen, and a TYRP-1 antigen, or that include a TGF-β antigen, a KDR2 antigen, a P Glyc antigen, and a TYRP- 1 antigen can be used to reduce the number of cancer cells present within a mammal.
The methods and materials provided herein can be used to treat cancer or to reduce the number of cancer cells present within any appropriate mammal such as humans, monkeys, horses, cows, sheep, dogs, cats, mice, or rats. In addition, the methods and materials provided herein can be used to treat any appropriate cancer or to reduce the number of appropriate type of cancer cells present within a mammal. For example, the methods and materials provided herein can be used to treat melanoma (e.g., skin melanoma or uveal melanoma), non-Hodgkin lymphoma, colorectal cancer, brain tumors, papillary thyroid carcinoma, non-small-cell lung carcinoma, or adenocarcinoma of the lung or can be used to reduce the number of melanoma (e.g., skin melanoma or uveal melanoma), non-Hodgkin lymphoma, colorectal cancer, brain tumor, papillary thyroid carcinoma, non-small-cell lung carcinoma, or adenocarcinoma of the lung cancer cells present within a mammal.
In some cases, a combination of a GNAQ antigen, a TYRP1 antigen, and an N-RAS antigen can be used to treat cancer (e.g., melanoma such as uveal melanoma). In some cases, one or more viral vectors (e.g., vesicular stomatitis virus (VSV) vectors) designed to express a GNAQ antigen, a TYRPl antigen, and an N-RAS antigen can be used to treat cancer (e.g., melanoma such as uveal melanoma). For example, VSV vectors designed to express a GNAQ antigen, a TYRPl antigen, and an N-RAS antigen can be administered to a mammal (e.g., a human) with uveal melanoma to reduce the size or to prevent the additional growth of that melanoma.
In some cases, a combination of a BRAF antigen, a TOPO-IIa antigen, and a
YB-1 antigen can be used to treat cancer (e.g., melanoma such as skin melanoma). In some cases, one or more viral vectors (e.g., VSV vectors) designed to express a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen can be used to treat cancer (e.g., melanoma such as skin melanoma). For example, VSV vectors designed to express a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen can be
administered to a mammal (e.g., a human) with skin melanoma to reduce the size or to prevent the additional growth of that melanoma.
In some cases, a combination of a TGF-β antigen, a MDRl antigen, a TYRP-1 antigen, and a KDR2 antigen can be used to treat cancer (e.g., melanoma). In some cases, one or more viral vectors (e.g., VSV vectors) designed to express a TGF-β antigen, a MDRl antigen, a TYRP-1 antigen, and a KDR2 antigen can be used to treat cancer (e.g., melanoma).
In some cases, a combination of a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen can be used to treat cancer (e.g., melanoma, colorectal cancer, prostate cancer, ovarian cancer, or breast cancer). In some cases, one or more viral vectors (e.g., VSV vectors) designed to express a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen can be used to treat cancer (e.g., melanoma, colorectal cancer, prostate cancer, ovarian cancer, or breast cancer).
In some cases, a combination of a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen can be used to treat cancer (e.g., melanoma or prostate cancer). In some cases, one or more viral vectors (e.g., VSV vectors) designed to express a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen can be used to treat cancer (e.g., melanoma or prostate cancer).
In some cases, a combination of a TOPOIIa antigen and an ABCB5a antigen can be used to treat cancer (e.g., melanoma). In some cases, one or more viral vectors (e.g., VSV vectors) designed to express a TOPOIIa antigen and an ABCB5a antigen can be used to treat cancer (e.g., melanoma).
In some cases, a combination of an ABCB5a antigen, a CYT-C antigen, an N-
RAS antigen, and a TYRP-1 antigen can be used to treat cancer (e.g., melanoma). In some cases, one or more viral vectors (e.g., VSV vectors) designed to express an ABCB5a antigen, a CYT-C antigen, a NRAS antigen, and a TYRP-1 antigen can be used to treat cancer (e.g., melanoma).
In some cases, a combination of a TGF-β antigen, a KDR2 antigen, a P glycoprotein (P Glyc) antigen, and a TYRP-1 antigen can be used to treat cancer (e.g., melanoma). In some cases, one or more viral vectors (e.g., VSV vectors) designed to express a TGF-β antigen, a KDR2 antigen, a P glycoprotein (P Glyc) antigen, and a TYRP-1 antigen can be used to treat cancer (e.g., melanoma).
A GNAQ (guanine nucleotide binding protein, q polypeptide) antigen can have the amino acid sequence set forth in GenBank® Accession No. AF493896.1 (GI No. 20147684) or U40038.1 (GI No. 1181670), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
A TYRP1 (tyrosinase-related protein 1) antigen can have the amino acid sequence set forth in GenBank® Accession No. CAG2861 1 (GI No. 471 15303), NM_000550.2 (GI No. 169881242), CR407683.1 (GI No. 47115302),
XM_005251574.1 (GI No. 530390132), or X51420.1 (GI No. 37512), or a fragment of such an amino acid sequence that is between about 7 and 527 amino acid residues (e.g., between about 10 and 527 amino acid residues, between about 15 and 527 amino acid residues, between about 20 and 527 amino acid residues, between about 25 and 527 amino acid residues, between about 30 and 527 amino acid residues, or between about 30 and 200 amino acid residues) in length.
An N-RAS (neuroblastoma RAS viral oncogene homolog) antigen can have the amino acid sequence set forth in GenBank® Accession No. AAB29640 (GI No. 544859), or a fragment of such an amino acid sequence that is between about 7 and 400 amino acid residues (e.g., between about 10 and 400 amino acid residues, between about 15 and 400 amino acid residues, between about 20 and 400 amino acid residues, between about 25 and 400 amino acid residues, between about 30 and 400 amino acid residues, or between about 30 and 200 amino acid residues) in length. In some cases, an N-RAS antigen can have the amino acid sequence set forth in
GenBank® Accession No. NM_002524.4 (GI No. 334688826) or AF493919.1 (GI No. 20147730), or a fragment of such an amino acid sequence that is between about 7 and 400 amino acid residues (e.g., between about 10 and 400 amino acid residues, between about 15 and 400 amino acid residues, between about 20 and 400 amino acid residues, between about 25 and 400 amino acid residues, between about 30 and 400 amino acid residues, or between about 30 and 200 amino acid residues) in length.
A BRAF (v-raf murine sarcoma viral oncogene homolog B) antigen can have the amino acid sequence set forth in GenBank® Accession No. NM_004333.4 (GI No. 187608632), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length. In some cases, a GNAQ antigen can have the amino acid sequence set forth in
GenBank® Accession No. XM_005250045.1 (GI No. 530387105), XM 005250046.1 (GI No. 530387107), or XM_005250047.1 (GI No. 530387109), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
A TOPO-IIa (DNA topoisomerase 2-alpha) antigen can have the amino acid sequence set forth in GenBank® Accession No. NM_001067.3 (GI No. 300193028), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
A YB-1 (Y box binding protein 1) antigen can have the amino acid sequence set forth in GenBank® Accession No. NM_004559.3 (GI No. 109134359), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length. In some cases, a GNAQ antigen can have the amino acid sequence set forth in GenBank® Accession No. BC071708.1 (GI No. 47940505) or XM_005270904.1 (GI No. 530362706), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
A TGF-β antigen can have the amino acid sequence set forth in GenBank® Accession No. X02812 or J051 14 (GI No. 37092), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
A MDR1 antigen can have the amino acid sequence set forth in GenBank® Accession No. X58723 or X59732 (GI No. 34522), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
A KDR2 antigen can have the amino acid sequence set forth in GenBank® Accession No. AF063658 (GI o. 3132832), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
A CYT-C antigen can have the amino acid sequence set forth in GenBank® Accession No. NP_061820 (GI No. 1 1128019), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
An ABCB5a antigen can have the amino acid sequence set forth in GenBank® Accession Nos. NM 029961, XM_001002680, or XM_906632 (GI No. 255708374), NM 001 163941.1 (GI No. 255708476), NM_178559.5 (GI No. 255708475),
NM_001 163942.1 (GI No. 255708370), or NM_001163993.2 (GI No. 574957217), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
A CDC7 kinase antigen can have the amino acid sequence set forth in GenBank® Accession No. NM_ 009863 (GI No. 409168309), NM_001 134420.1 (GI No. 197313666), NM_003503.3 (GI No. 197313663), or NM_001 134419.1 (GI No. 197313664), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length. A CD44 antigen can have the amino acid sequence set forth in GenBank® Accession No. NM_001 177787 (GI No. 295293147), AY101 193.1 (GI No.
21429240), or AY101192.1 (GI No. 21429238), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
A P Glyc (P glycoprotein) antigen can have the amino acid sequence set forth in GenBank® Accession No. M23234.1 (GI No. 187501), AY234788.1 (GI No.
34539754), AY425006.1 (GI No. 40795902), AF399931.1 (GI No. 3330771 1), or EU854148.1 (GI No. 194740429), or a fragment of such an amino acid sequence that is between about 7 and 150 amino acid residues (e.g., between about 10 and 100 amino acid residues, between about 15 and 50 amino acid residues, between about 20 and 75 amino acid residues, between about 25 and 50 amino acid residues, between about 30 and 60 amino acid residues, or between about 30 and 50 amino acid residues) in length.
In some cases, a GNAQ, TYRPl, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF-β antigen can have the amino acid sequence set forth in one of the GenBank® Accession numbers indicated above or a fragment of such an amino acid sequence that is immunogenic and induces a robust IL-17 response. In some cases, such an antigen can include one or more mutations within the sequence provided in GenBank® provided that the mutant antigen induces a robust IL-17 response. In some cases, a GNAQ, TYRPl, N- RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF-β antigen can have the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form. For example, a GNAQ antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have one or more of the following mutations: GNAQ(209) or GNAQ(R183). A TYRPlantigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have one or more of the following mutations: 1-BP DEL of 368A (condition: albinism, oculocutaneous, type III), SER166TER (dbSNP: rsl04894130), ARG373TER, ARG356GLU, 1-BP DEL of 106T, 4-BP DEL of 1057AACA, or ARG93CYS (condition: albinism, oculocutaneous, type III). An N-RAS antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have one or more of the following mutations: Q61 (dbSNP: rsl 1554290), GLY13ASP (dbSNP: rsl21434596), GLY13ARG (dbSNP: rs l21434595),
THR50ILE, GLY60GLU (condition: Noonen syndrome 6), PR034LEU, or
GLY12ASP (condition: epidermal nevus, somatic). A BRAF antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have the following mutation: V600 (dbSNP: rsl 13488022). A TOPO-IIa antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have the following mutation: ARG486LYS (condition: resistance to inhibition of or by amsacrine). A YB-1 antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have the following mutation: YB-1(S 102). A MDR1 antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have one or more of the following mutations: G2677T, C3435T (dbSNP: rsl045642), GLY185VAL (dbSNP: rs l 128501; condition: colchicine resistance), or ALA893SER/THR (condition: inflammatory bowel disease). A KDR2 antigen having the amino acid sequence (or a fragment thereof) as found in a naturally- occurring mutated form can have one or more of the following mutations: D717V, T771R, PROl 147SER (condition: hemangioma, capillary infantile, somatic), or CYS482ARG (dbSNP: rs34231037; condition susceptibility to hemangioma or capillary infantile). An ABCB5a antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have one or more of the following mutations: G347R, M521L, P580S, or A687S. A CDC7 kinase antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have the following mutation: L2101. A CD44 antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have the following mutation: ARG46PRO (dbSNP: rs l21909545; condition: Indian blood group system polymorphism). A TGF-β antigen having the amino acid sequence (or a fragment thereof) as found in a naturally-occurring mutated form can have one or more of the following mutations: CYS225ARG (dbSNP: rs 104894719), ARG218HIS (dbSNP: rs 104894720), ARG218CYS (dbSNP: rs l04894721), TYR81HIS (dbSNP: rsl 1 1033611),
CYS223ARG (dbSNP: rsl04894722), CYS223GLY (dbSNP: rs l04894722; condition: camurati-engelmann disease), or LEU10PRO (conditions: cystic fibrosis lung disease, modifier of invasive breast cancer, or susceptibilities thereto).
In some cases, a GNAQ, TYRP1, N-RAS, BRAF, TOPO-IIa, YB-1, MDRl, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF-β antigen can have an amino acid sequence that is truncated at the C terminus. For example, a GNAQ antigen can include the N-terminal sequence of a full length GNAQ polypeptide, while lacking a portion of the C-terminal sequence of a full length GNAQ
polypeptide. In some cases, the length of the missing C-terminal sequence of a truncated antigen (e.g., a truncated GNAQ, TYRP1, N-RAS, BRAF, TOPO-IIa, YB- 1, MDRl, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF-β antigen) can be from 1 to about 300 (e.g., 1 to 275, 1 to 250, 1 to 225, 1 to 200, 1 to 175, 1 to 150, 1 to 125, 1 to 100, 1 to 75, 1 to 50, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 5 to 275, 5 to 250, 5 to 225, 5 to 200, 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 275, 10 to 250, 10 to 225, 10 to 200, 10 to 175, 10 to 150, 10 to 125, 10 to 100, 10 to 75, 10 to 50, 10 to 25, 10 to 20, or 10 to 15) amino acid residues. In some cases, the length of the missing C-terminal sequence of a truncated antigen (e.g., a truncated GNAQ, TYRP1, N-RAS, BRAF, TOPO-IIa, YB- 1, MDRl, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF-β antigen) can be between about 0.01 percent to about 85 percent (e.g., about 0.01 percent to about 85 percent, about 0.01 percent to about 75 percent, about 0.01 percent to about 65 percent, about 0.01 percent to about 55 percent, about 0.01 percent to about 45 percent, about 0.01 percent to about 35 percent, about 0.01 percent to about 25 percent, about 0.01 percent to about 15 percent, about 0.01 percent to about 10 percent, about 0.01 percent to about 5 percent, about 0.1 percent to about 85 percent, about 1 percent to about 85 percent, about 5 percent to about 85 percent, about 5 percent to about 85 percent, about 5 percent to about 75 percent, about 5 percent to about 65 percent, about 5 percent to about 55 percent, about 5 percent to about 45 percent, about 5 percent to about 35 percent, about 5 percent to about 25 percent, about 5 percent to about 15 percent, about 5 percent to about 10 percent) of the length of the full length polypeptide.
In some cases, the combination of antigens used to treat cancer or reduce the number of cancer cells within a mammal (e.g., a human) can be antigens of another species (e.g., mouse, rat, pig, monkey, sheep, cow, dog, or cat). For example, a combination of mouse, rat, or monkey antigens can be used to treat cancer or reduce the number of cancer cells within a human. Examples of GNAQ sequences from mouse are set forth in GenBank® Accession Nos. NM_008139.5 (GI No. 145966786) and BC057583.1 (GI No. 34785834). Examples of TYRP-1 sequences from mouse are set forth in GenBank® Accession Nos. NM_001282014.1 (GI No. 530537243), NM_031202.3 (GI No. 530537240), and NM_001282015.1 (GI No. 530537245). Examples of N-RAS sequences from mouse are set forth in GenBank® Accession Nos. NM_010937.2, NC_000069.6 (GI No. 372099107), and AC_000025.1 (GI No. 83280973). An example of a BRAF sequence from mouse is set forth in GenBank® Accession No. NM_139294.5 (GI No. 153791903). An example of a TOPO-IIa sequence from mouse is set forth in GenBank® Accession No. NM011623. An example of a YB-1 sequence from mouse is set forth in GenBank® Accession No. M62867 (GI No. 199820). An example of a TGF-β sequence from mouse is set forth in GenBank® Accession No. M13177.1 (GI No. 201952). An example of a MDRl sequence from mouse is set forth in GenBank® Accession No. NM_011075 (GI No. 161 169006). An example of a KDR2 sequence from mouse is set forth in GenBank® Accession No. EU884114.1 (GI No. 215400615). An example of a YB-1 sequence from mouse is set forth in GenBank® Accession No. X57621.1 (GI No. 55450), C061634.1 (GI No. 38197294), or NM_01 1732.2 (GI No. 1 13205058). An example of a P Glyc sequence from mouse is set forth in GenBank® Accession No. M33581.1 (GI No. 199104), JQ655148.1 (GI No. 406817019), M24417.1 (GI No. 2000329), or AY864315.1 (GI No. 57791235).
Any appropriate vector (e.g. a viral vector) can be used to deliver nucleic acid encoding a GNAQ, TYRP1, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT- C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF-β antigen (or combination thereof) to cells of a mammal to treat cancer as described herein. For example, viral vectors for administering nucleic acids (e.g., a nucleic acid encoding a GNAQ, TYRP1, N- RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF-β antigen (or combination thereof)) to a mammal can be prepared using standard materials (e.g., packaging cell lines, helper viruses, and vector constructs). See, for example, Gene Therapy Protocols (Methods in Molecular Medicine), edited by Jeffrey R. Morgan, Humana Press, Totowa, NJ (2002) and Viral Vectors for Gene Therapy: Methods and Protocols, edited by Curtis A. Machida, Humana Press, Totowa, NJ (2003). A viral vector for delivering nucleic acid encoding a GNAQ, TYRP1, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT- C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF-β antigen (or combination thereof) can be derived from, for example, animal viruses such as adenoviruses, adeno- associated viruses, retroviruses, lentiviruses, vaccinia viruses, vesicular stomatitis virus, herpes viruses, maraba virus, or papilloma viruses. In some cases, lentiviral vectors, vesicular stomatitis viral vectors, adenoviral vectors, adeno-associated viral vectors, or maraba viral vectors can be used to deliver nucleic acid encoding a GNAQ, TYRP1, N-RAS, BRAF, ΤΌΡΟ-Πα, YB-1, MDR1, KDR2, CYT-C,
ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF-β antigen (or combination thereof) to cells of a mammal to treat cancer as described herein. In some cases, VSV-IFN (e.g., human interferon) viral vectors such as those described elsewhere (Obuchi et al, J. Virol, 77(16):8843-56 (2003) and Jenks et al, Hum. Gene Ther., 21(4):451-62 (2010)) can be used to deliver nucleic acid encoding a GNAQ, TYRPl, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF-β antigen (or combination thereof) to cells of a mammal to treat cancer.
Any appropriate method can be used to insert nucleic acid encoding a GNAQ, TYRPl, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF-β antigen into a viral vector (e.g., a VSV vector). For example, the methods and materials described elsewhere (Kottke et al, Nature Med., 17:854-9 (2011); and Pulido et al, Nat. Biotechnol, 30:337-43 (2012)) can be used to insert nucleic acid encoding a GNAQ, TYRP l, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF-β antigen into a VSV vector such that the antigen (e.g., the GNAQ, TYRPl, N-RAS, BRAF, TOPO-IIa, YB-1, MDR1, KDR2, CYT-C, ABCB5a, P Glyc, CDC7 kinase, CD44, or TGF-β antigen) is expressed in mammalian cells. Once obtained, a combination of VSV vectors having the ability to express a GNAQ antigen, a TYRP 1 antigen, and an N-RAS antigen, a BRAF antigen, a TOPO-IIa antigen, and a YB- 1 antigen, a TGF-β antigen, a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen, a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen, a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen, a TOPOIIa antigen and an ABCB5a antigen, a TGF-β antigen, a KDR2 antigen, a P Glyc antigen, and a TYRP-1 antigen, or an ABCB5a antigen, a CYT-C antigen, a N- RAS antigen, and a TYRP-1 antigen (e.g., a combination of VSV-GNAQ, VSV- TYRP1, and VSV-N-RAS vectors, a combination of VSV-BRAF, VSV-ΤΟΡΟ-Πα, and VSV-YB-1 vectors, a combination of VSV-TGF-β, VSV-MDR1, VSV-TYRP-1, and VSV-KDR2 vectors, a combination of VSV-ΤΟΡΟΙΙα, VSV-YB-1, VSV-CDC7 kinase, and VSV-BRAF vectors, a combination of VSV-ΤΟΡΟΙΙα, VSV-YB-1, VSV- CDC7 kinase, and VSV-CD44 vectors, a combination of VSV-ΤΟΡΟΙΙα and VSV- ABCB5a vectors, a combination of VSV-TGF-β, VSV-KDR2, VSV-PGlyc, and VSV-TYRP-1 vectors, or a combination of VSV-ABCB5a, VSV-CYT-C, VSV-N- RAS, and VSV-TYRP- 1 vectors) can be administered to a mammal to treat cancer (e.g., melanoma such as uveal melanoma) or to reduce the number of cancer cells (e.g., melanoma cells such as uveal melanoma cells) present within a mammal. For example, once obtained, a combination of VSV vectors having the ability to express a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen (e.g., a combination of VSV- BRAF, VSV-ΤΟΡΟ-ΙΙα, and VSV-YB-1 vectors) can be administered to a mammal to treat cancer (e.g., melanoma such as skin melanoma) or to reduce the number of cancer cells (e.g., melanoma cells such as skin melanoma cells) present within a mammal.
Any appropriate method can be used to administer viral vectors (e.g., VSV vectors) designed to express a GNAQ antigen, a TYRPl antigen, and an N-RAS antigen, a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen, a TGF-β antigen, a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen, a TOPOIIa antigen, a YB- 1 antigen, a CDC7 kinase antigen, and a BRAF antigen, a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen, a TOPOIIa antigen and an ABCB5a antigen, a TGF-β antigen, a KDR2 antigen, a P Glyc antigen, and a TYRP-1 antigen, or an ABCB5a antigen, a CYT-C antigen, a N-RAS antigen, and a TYRP-1 antigen to a mammal having cancer. For example, intratumoral, subcutaneous, intravenous, intraperitoneal, and intradermal administrations can be used to administer viral vectors (e.g., VSV vectors) designed to express a GNAQ antigen, a TYRPl antigen, and an N-RAS antigen to a mammal having cancer (e.g., uveal melanoma) or viral vectors (e.g., VSV vectors) designed to express a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen to a mammal having cancer (e.g., skin melanoma). Once the viral vectors are administered to a mammal, the mammal can be monitored to confirm a reduction in the number of cancer cells present within the mammal. For example, imaging techniques such as MRI and CT scans can be used to confirm that the number of cancer cells present within the mammal is reduced following administration of the viral vectors. In some cases, the following examination criteria can be used. A non-nodal lesion is considered measurable if its longest diameter can be accurately measured as 2.0 cm with chest x-ray, or as = 1.0 cm with CT scan or MRI. A superficial non-nodal lesion is measurable if its longest diameter is = 1.0 cm in diameter as assessed using calipers (e.g., skin nodules) or imaging. In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, can be used. A malignant lymph node is considered measurable if its short axis is >1.5 cm when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm). In physical examinations for superficial non-nodal lesions, physical examination is acceptable, but imaging is preferable. In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, can be used.
In some cases, a GNAQ antigen, a TYRP1 antigen, and an N-RAS antigen, a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen, a TGF-β antigen, a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen, a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen, a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen, a TOPOIIa antigen and an ABCB5a antigen, a TGF-β antigen, a KDR2 antigen, a P Glyc antigen, and a TYRP-1 antigen, or an ABCB5a antigen, a CYT-C antigen, a N-RAS antigen, and a TYRP-1 antigen can be administered as a combination in the form of polypeptides. For example, a GNAQ antigen, a TYRP 1 antigen, and an N-RAS antigen (each in the form of polypeptides) can be formulated with an adjuvant such as alum, monophosphoryl lipid A, liposomes, QS21, MF-59, or immunostimulating complexes (ISCOMS) and administered to a mammal having cancer (e.g., uveal melanoma). Following this administration, the number of cancer cells present within the mammal can be reduced. In some cases, a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen can be administered as a combination in the form of polypeptides to a mammal having cancer (e.g., skin melanoma). Following this administration, the number of cancer cells present within the mammal can be reduced.
In some cases, therapy with a combination of antigens provided herein can include the use of radiation. For example, when treating cutaneous melanoma, a patient can be treated with both radiation and a combination of antigens provided herein.
In some cases, therapy with a combination of antigens provided herein can include the administration of one or more immune checkpoint inhibitors. For example, a combination of viral vectors (e.g., VSV vectors) designed to express a GNAQ antigen, a TYRP1 antigen, and an N-RAS antigen, a BRAF antigen, a TOPO- Πα antigen, and a YB-1 antigen, a TGF-β antigen, a MDR1 antigen, a TYRP-1 antigen, and a KDR2 antigen, a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen, a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen, a TOPOIIa antigen and an ABCB5a antigen, a TGF-β antigen, a KDR2 antigen, a P Glyc antigen, and a TYRP-1 antigen, or an ABCB5a antigen, a CYT-C antigen, a N-RAS antigen, and a TYRP- 1 antigen can be administered in combination with one or more immune checkpoint inhibitors to treat a mammal having cancer. Examples of immune checkpoint inhibitors include, without limitation, anti-PDl antibodies, anti-CTLA4 antibodies, anti-PDLl antibodies, anti- PDL2 antibodies, anti-CD40 ligand antibodies, and anti-KIR antibodies.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1 - Treating melanoma using VSV vectors designed to express BRAF,
ΤΟΡΟ-Πα. and YB-1 antigens
C57BL/6 mice seeded with B 16tk tumors 5 days previously were treated with GCV i.p. at 50 mg/mL for 5 consecutive days, followed by 2 days' rest, followed by 5 further consecutive injections of GCV (days 6-10 and days 13-17). Mice were injected i.v. with combinations of VSV expressing different cDNAs starting on day 20, by which time primary tumors had regressed. Subsequent i.v. injections were given on days 22, 24, 27, 29, and 31. Survival of mice (n = 7/8 per group) treated sequentially with GCV, then with combinations of VSV-cDNA with i.v. injections of VSV-BRAF+VSV-YB-l+VSV-GFP; VSV-TOPOIIa+VSV-BRAF+VSV-GFP; VSV- TOPOIIa+VSV-YB-l+VSV-GFP; or VSV-BRAF+VSV-TOPOIIa+VSV-YB-1 (3xl06 pfu/virus/injection).
The combination of VSV expressing BRAF, TOPOIIa, and YB-1 generated significant survival benefit over any of the other combinations (Figure 1). Example 2 - Treating uveal melanoma patients using VSV vectors designed to express GNAQ. TYRP1. and -RAS antigens
Human uveal melanoma patients are administered a combination of three VSV vectors: (a) a VSV vector designed to express a GNAQ antigen, (b) a VSV vector designed to express a TYRP1 antigen, and (c) a VSV vector designed to express a N- RAS antigen. VSV-IFN-β with -RAS, GNAQ, or TYRP1 is administered in one single tumor location using a 21- or 22-gauge needle, whose length may range between 15 to 20 cm under CT or ultrasound guidance. Volume of injection (Vi) = (a2)(b)(0.5) [where a = the shorter diameter and b = the longer diameter] of injectable product. A maximum volume of 15 cc is used to prepare the investigational product. The three forms of VSV-hIFN-β (i.e., VSV-IFN-N-RAS, VSV-IFN-GNAQ, and VVS-IFN-TYRPl) are mixed together in a 1 : 1 : 1 dilution. The combination of three VSV vectors is administered as a mixture via a single intratumoral injection. The injection occurs slowly. If the tumor size is over 2 cm, this may require multiple injections. These injection sites are at least 2 cm apart from one another. Depending on the size and location of the tumor, it is estimated that the procedure will take anywhere from 30 to 60 minutes in duration.
The concentration (pfus) for each of the three VSV vectors in the mixture is between 105 and 109. The injection is given on day 1, and the length of the study is 28 days.
Follow-up tumor measurements are repeated every 8 weeks, or as deemed appropriate by the investigator, through the observation period. Tissue specimens are collected and submitted on days 1, 2, and 8 on patients that allow for another biopsy. Example 3 - Treating skin melanoma patients using VSV vectors designed to express
BRAF. ΤΟΡΟ-Πα. and YB-1 antigens
Human skin melanoma patients with stage II and III melanoma are administered adjuvantly and stage IV melanoma patients with oligometastatic melanoma (1-5 metastatic deposits) are administered a combination of three VSV vectors: (a) a VSV vector designed to express a BRAF antigen, (b) a VSV vector designed to express a TOPO-IIa antigen, and (c) a VSV vector designed to express a YB-1 antigen. Patients with stage IV melanoma receive VSV (i.e., VSV-BRAF, VSV YB-1, and VSV-ΤΟΡΟ-Πα) in combination with ablative radiation. VSV-BRAF, VSV-YB-1, and VSV-ΤΟΡΟ-ΙΙα are administered intratumorly or intravenously or subcutaneously using a 21- or 22-gauge needle, whose length may range between 15 to 20 cm under CT or ultrasound guidance. Volume of injection (Vi) = (a2)(b)(0.5) [where a = the shorter diameter and b = the longer diameter] of injectable product. The three forms of VSV (i.e., VSV-BRAF, VSV-YB-1, and VSV-ΤΌΡΟ-Πα) are mixed together in a 1 : 1 : 1 dilution. The combination of three VSV vectors is administered as a mixture via a single injection.
Example 4 - Combination Viroimmunotherapy with Checkpoint
Inhibition to Treat Glioma
Cell lines
Murine B16 cells (American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS; Life technologies) and L-glutamine (Life technologies). Murine GL261 cells (American Type Culture Collection, Manassas, VA) were grown in DMEM supplemented with 10% FCS. TRAMP-C2 (TC2) cells, derived from a prostate tumor that arose in a TRAMP mouse, were characterized as described elsewhere (Kottke et ah, Cancer Res., 67: 11970-9 (2007)) and were routinely grown as tumors in C57BL/6 mice in an androgen- independent manner. The K1735 melanoma cell line (Chong et ah, Hum. Gene Ther., 7: 1771-9 (1996)) was derived from H-2k C3H/He mice.
Mice
C57BL/6 and C3H mice were purchased from The Jackson Laboratory (B; Harbor, ME) at 6-8 weeks of age.
Virus
The ASMEL VSV-cDNA library was generated as described elsewhere (Kottke et ah, Nature Med., 201 1 :854-9 (2011); Pulido et ah, Nat. Biotechnoh, 30:337-43 (2012); and Alonso-Camino et al., Mol. Ther., 22: 1936-48 (2014)).
Individual viral clones (VSV expressing N-RAS, CYT-C, TYRP-1, HIF-2a, SOX-10, or c-MYC) were isolated by limiting dilution as described elsewhere (Pulido et ah, Nat. Biotechnoh, 30:337-43 (2012); and Alonso-Camino et ah, Mol. Ther., 22: 1936- 48 (2014)). These were expanded in BHK cells and purified by sucrose gradient centrifugation. VSV-GFP was manufactured by cloning the cDNA for GFP into the plasmid pVSV-XN2 as described elsewhere (Fernandez et ah, J. Virol. , 76:895-904 (2002)). Monoclonal VSV-GFP was obtained by plaque purification on BHK-21 cells and concentrated by sucrose-gradient centrifugation.
Measurement ofHIF-2a polypeptide in i.e. explants and in vitro cultures
To establish i.e. tumors, lxlO4 cells in 2 μΐ, PBS were stereotactically injected into the brain (1 mm anterior, and 2 mm lateral to the bregma) of C57B1/6 (B16, GL261, or TC2 cells) or C3H (K1735 cells) mice. Mice were sacrificed upon sign of distress, and single-cell suspensions of brain tumor explants or in vitro cultured cells (B 16, GL261, TC2 or K1735) were plated at lxlO5 per well in DMEM + 10% FCS and 1% penicillin-streptomycin. Cell-free supernatants were harvested, and HIF-2a polypeptide expression was measured by ELISA according to the manufacturer's instructions (USCN Life Sciences, Houston TX). lxlO5 cells of each cell line (B16, GL261, TC2, K1735) from in vitro cultures also were plated and measured for HIF-2a polypeptide expression.
Measurement of HIF-2a polypeptide in co-cultures of GL261 and splenic/brain- derived CDllb+ cells
CD 1 lb+ cells were purified from brain-cell suspensions of multiple brains, or from the spleens of C57B1/6 mice (re-suspended in Iscove's modified Dulbecco's medium (IMDM; Gibco, Grand Island, NY) + 5% FCS + 1% penicillin-streptomycin + 40 μιηοΐ/ΐ 2 -ME) using CDl lb microbeads according to the manufacturer's instructions (Miltenyi Biotech, Auburn, CA). lxlO6 CDl lb+ cells were co-cultured in DMEM + 10% FCS and 1% penicillin-streptomycin with (lxlO5) GL261 cells. After 24 hours of co-culture, cell-free supernatants were harvested, and HIF-2a polypeptide levels were measured by ELISA. HIF-2a polypeptide also was evaluated following co-culture of GL261 cells with brain- or spleen-derived CDl lb+ cells, in the presence of 10 ng/mL recombinant TGF-β RII Fc Chimera 341-BR (R&D systems, MN).
Human tumor explants
Human primary glioblastoma brain tumor tissue was obtained following surgery. Within three hours of surgical resection, explants were depleted of CD l lb+ cells using CDl lb microbeads. Tumor cells were then seeded at lxlO4 cells per well in DMEM + 10% FCS + 1% penicillin-streptomycin ± isolated autologous CD 1 lb+ cells (5xl03 per well). HIF-2a polypeptide levels in cell-free supernatants were evaluated at 24 hours and again following 2 week's culture. HIF-2a polypeptide also was evaluated in cell-free supernatants from lxlO3 isolated CD1 lb+ cells, 24 hours after explant.
In vivo studies
To establish i.e. tumors, lxlO4 GL261 cells in 2 \L PBS were stereotactically injected using a syringe bearing a 26G needle into the brain (1 mm anterior, and 2 mm lateral to the bregma) of C57BL/6 mice (7-9 mice per treatment group unless otherwise stated). Virus, drug, or PBS control (100 μΚ) was administered intravenously following 5 days tumor establishment and occurred as dictated by each specific study. Mice were examined daily for overall health and, survival with time was documented.
For the therapeutic study evaluating the effect of anti-PD 1 antibody in combination with virus treatment, control ChromPure rat IgG antibody (Jackson Immunochemicals, West Grove, PA) or anti-PD 1 antibody were injected
intravenously at 225 μg/mouse/injection (Clone RMP 1-14, Bioxcell West Lebanon, NH). For therapy evaluating the use of two checkpoint inhibitors, intravenous anti- PD1 was administered at 225 μg/mouse/injection and anti-CTLA4 at 0.1
mg/mouse/injection (Bioxcell West Lebanon, NH).
In vitro splenic/lymph node T-cell reactivation and ELISA for IFN- y/IL-17
Spleens and lymph nodes were harvested from euthanized mice and dissociated into single-cell suspensions by crushing through a 100 μιη filter. Red blood cells were lysed with ACK lysis buffer (sterile distilled H2O containing 0.15 M NH4CI, 1.0 mM KHCO3 and 0.1 mM EDTA adjusted to pH 7.2 - 7.4) for 2 minutes. Cells were re-suspended at lxlO6 cells/mL in IMDM + 5% FCS + 1% penicillin- streptomycin + 40 μιηοΐ/ΐ 2-ME. Pooled cells (lxlO6 per well) were stimulated with freeze thaw lysates (equivalent to lxlO5 cells) of either GL261 tumors recovered from mice bearing i.e. GL261 tumors or in vitro cultured GL261 cells, every 24 hours for 3 days. Following 48 hours of culture, cell- free supernatants were collected and assayed by ELISA for IFN-γ (BD Biosciences, San Jose, CA) or IL-17 (R&D systems, Minneapolis, MN). Re-stimulation also was carried out with splenocytes and lymph node cells depleted of Treg cells using Miltenyi CD4+/CD25+ beads (Miltenyi Biotech, Auburn, CA). Splenocyte and lymph node single cell isolates also were stimulated as described herein with the VSV-N protein derived epitope peptide (VSV-N52-59:RGYVYQG at 5 μg/mL) (synthesized at a core facility) and supernatants were evaluated for IFN-γ and IL-17 response by ELISA.
Statistics
Survival data from animal experiments were analyzed using the log rank test with Graph Pad Prism 6 (Graph Pad software, La Jolla, CA). A two-sample, unequal variance Students t-test was used to evaluate in vitro data. Statistical significance was determined at the level of P < 0.05.
Results
Intra-cranial tumors of different histologies express a similar HIF-2aHi phenotype. It was hypothesized that the intra-cranial microenvironment imposes a HIF-2aHi phenotype upon different types of tumors, which is distinct from that expressed by the same tumor cells growing in culture. Consistent with this hypothesis, freshly resected i.e. tumors of different histological types, including K1735 melanoma (in C3H mice), as well as B16 melanoma, GL261 glioma, and TC2 prostate cancer (C57B1/6 mice), all expressed a HIF-2aHi phenotype. In contrast, the same cell lines grown in culture, from which the tumors were initially derived by i.e. implantation, expressed low or undetectable levels of HIF-2a (Figure 2).
CD1 lb+ cells in intact brain homogenate impose a HIF-2aHi phenotype on GL261 cells in vitro in part through TGF-β. The HIF-2aHi phenotype of i.e. B16-ova tumors was imposed by brain-associated, but not spleen-derived, CD1 lb+ cells. In vitro co-culture of GL261cells with CD1 lb+ cells purified from intact brain homogenate, mediated a similar HIF-2aLo to HIF-2aHi phenotypic transition (Figure 3). As for the B16 model, splenic CD1 lb+ cells were unable to impose a HIF-2aHi phenotype on in vitro cultured glioma cells (Figure 3). Whilst neutralization of neither TNF-a, VEGF, nor interferon-γ prevented induction of the HIF-2aHi phenotype in GL261 and brain-associated CD1 lb+ cell co-cultures, blocking TGF-β significantly reduced HIF-2a expression (p=0.000173) (Figure 3). These results demonstrate that CD1 lb+ cells of the brain micro-environment impose the HIF-2aHi phenotype upon tumors growing i.e., mediated, at least in part, through TGF-β. Human tumor explants express a HIF-2aHi phenotype, which is reduced over time. To investigate how the murine model might reflect the patient situation, the HIF-2a phenotype of primary human brain tumor samples was studied. Freshly resected tumors cultured with their own autologous CD1 lb+ cells exhibited a HIF- 2aHi phenotype, although levels of HIF-2a were consistently lower than in resected murine tumors (Figure 4). Brain tumor explants depleted of CD1 lb+ cells expressed lower levels of HIF-2a after 24 hours of culture, although this did not reach statistical significance (p=0.101) (Figure 4). The CD1 lb+ cells themselves did not express significant levels of HIF-2a (Figure 4). After 2 weeks, CD1 lb+ cells within these co- cultures were lost, and the level of tumor cell associated HIF-2a was significantly reduced compared to levels seen at 24 hours post explant (p=0.017) (Figure 4).
Therefore, human brain tumors also express a HIF-2aHi phenotype, which is maintained, at least in part, by immune cells within the brain microenvironment. Intracranial GL261 can be treated with VSV-tumor-associated antigen therapy and enhanced by addition of checkpoint inhibitors
Although mice bearing s.c. B16 tumors were treated successfully with a combination of VSV-expressed N-RAS, CYT-C, and TYRP-1, i.e. B16 tumors were only successfully treated with a combination of VSV expressed HIF-2a, SOX- 10, c- MYC, and TYRP-1. The hypothesis that effective immunotherapy of an i.e. tumor of a different histological type could be targeted against this common i.e. tumor phenotype imposed by the brain microenvironment was tested further. Consistent with this, systemic delivery of VSV expressed HIF-2a, SOX- 10, and c-MYC generated significant therapy over control treatment (p=0.0001) (Figure 4). Although a combination of just two of the VSV-antigen gave significant therapy compared to control treatment (p=0.0001), optimal therapy required the combination of all three (HIF-2a, SOX- 10, c-MYC) antigens ((VSV-HIF-2a/SOX-10/c-MYC) versus (VSV- HIF-2a/SOX-10+VSV-GFP) p=0.0414). Unlike in the B16 i.e. model, addition of the VSV-TYRP-1 virus gave no added therapeutic benefit to treatment with VSV- expressed HIF-2a, SOX- 10, and c-MYC (data not shown). Consistent with our previous data with B 16 i.e., as opposed to s.c. tumors, the combination of VSV expressed N-RAS, CYT-C and TYRP-1 was ineffective against i.e. GL261 tumors and offered no significant therapeutic advantage over control therapy (p=0.1432) (Figure 4). To investigate whether the viroimmunotherapy associated with VSV-antigen therapy of i.e. GL261 could be enhanced through combination with immune checkpoint inhibition, mice bearing i.e. GL261 tumors were treated with 9 (instead of the 12 of Figure 5) systemic injections of VSV expressed HIF-2a, SOX-10, and c- MYC plus the checkpoint inhibitor antibody anti-PD 1. Addition of anti-PD 1 antibody significantly extended survival compared to the virus combination alone (p=0.0006) (Figure 6A).
Taken together, these results demonstrate that the brain micro-environment- imposed antigenic signature of HIF-2a, SOX-10, and c-MYC can be immunologically targeted to treat i.c tumors of different histologies (glioma and melanoma) and that effective immunotherapy of tumors should take into account immunological profiles imposed upon tumors by their anatomical location.
Anti-PD- 1 antibody uncovers a Thl response against intra-cranial GL261. The therapeutic anti-tumor response to self antigens induced by VSV-cDNA library treatment is Thl 7 CD4+ T cell mediated and no Thl IFN-γ T cell responses could be detected. Mixed splenocytes and lymph node cultures from mice bearing i.c. GL261 tumors following treatment with VSV-HIF-2a, VSV-SOX-10, and VSV-c-MYC did not secrete IFN-γ in response to challenge with freeze/thaw lysates of explanted i.c. GL261 tumors (Figure 6B). In contrast, similar mixed cultures from mice treated with the same VSV-HIF-2a, VS V-SOX- 10, and VSV-c-MYC plus anti-PD 1 antibody, secreted significant levels of IFN-γ (p<0.05), suggesting that checkpoint inhibition through the PD1 axis uncovered a Thl response to poorly immunogenic self antigens (Figure 6B). Consistent with the distinct antigenic nature of GL261 cells growing in situ in the brain, compared to the same cells growing in culture (Figures 2 and 3), splenocyte and lymph node cultures from mice treated with VSV-HIF-
2a/SOX-10/c-MYC + anti-PD 1 did not secrete IFN-γ in response to challenge with freeze/thaw lysates derived from GL261 cells cultured in vitro (Figure 6C). These results demonstrate that a Thl response to a unique antigenic profile associated with i.c. GL261 tumors is generated following VSV-antigen viroimmunotherapy, but that it is suppressed in vivo and can be de-repressed upon checkpoint inhibition.
Anti-PD 1 antibody therapy does not enhance the Thl 7 response against intracranial GL261. Interestingly, despite enhancing therapeutic efficacy in vivo (Figure 6A), checkpoint inhibition with anti-PD 1 did not significantly enhance the Thl 7 response generated by VSV-HIF-2a/SOX-10/c-MYC treatment (p=0.674) (against either i.e. explanted, or cultured, GL261 freeze thaw lysates), however, addition of anti-PD-1 enhanced a robust Thl, IFN-γ response (Figures 6D and 6E). A robust immune response of both Thl IFN-γ, and Thl7, anti-i.e. GL261 responses were only induced when VSV expressed tumor antigens VSV-HIF-2a/SOX-10/c-MYC, as opposed to VSV-GFP, (Figures 6B and 6D, respectively), indicating that virally- mediated expression of tumor antigens was required for an effective immune response.
Anti-PDl antibody enhances the Thl response against VSV. VSV-HIF-
2a/SOX-10/c-MYC treatment reproducibly induced a Thl response against VSV antigens (Figure 6F). This anti-VSV Thl response also was significantly enhanced in mice treated with checkpoint inhibition compared with VSV-antigen treatment alone (p=0.00375) (Figure 6F).
Taken together, these results demonstrate that combination of VSV-HIF-
2a/SOX-10/c-MYC viroimmunotherapy with anti-PDl checkpoint inhibition de- represses an anti -tumor Thl IFN-γ T cell response against both self antigens and against foreign viral antigens, but has no significant effect on the anti-tumor Thl 7 response.
Anti-PDl checkpoint inhibition mimics depletion ofTreg
As before (Figure 6B), the addition of anti-PDl to VSV-HIF-2a/SOX-10/c- MYC therapy uncovered an anti-tumor Thl response (lanel and 2 compared to 3 and 4, Figure 7A). In vitro depletion of Treg from the mixed splenocyte/LN cultures prior to re-stimulation with freeze/thaw lysates also de-repressed the Thl IFN-γ T cell response against i.e. GL261, compared to Treg-intact cultures (lanes 1 and 2 compared to 5 and 6, Figure 7A). However, Treg depletion from splenocyte/LN cultures of mice treated with VSV-HIF-2a/SOX-10/c-MYC + anti-PDl did not further enhance the Thl IFN-γ T cell response already uncovered by anti-PDl therapy (lanes 3 and 4 compared to 7 and 8, Figure 7A). Neither anti-PDl, nor in vitro Treg depletion, enhanced IL-17 responses generated by VSV-antigen therapy (Figure 7B). These results demonstrate that anti-PDl immune checkpoint inhibition may operate in vivo, to de-repress an anti-tumor Thl IFN-γ T cell response and that this may be effected, at least in part, by affecting Treg function. Combination checkpoint inhibition further improves VSV-antigen therapy
Given the success with enhancing VSV-antigen (e.g., VSV-HIF-2a/SOX-10/c- MYC) therapy with single checkpoint inhibitor therapy, a combination of anti-PD 1 and anti-CTLA-4 checkpoint inhibition to target separate stages of the T cell activation/repression pathway was tested in combination with VSV-antigen (e.g., VSV-HIF-2a/SOX-10/c-MYC) therapy. As before, anti-PDl treatment resulted in a significant improvement in survival in combination with VSV-HIF-2a/SOX-10/c- MYC therapy (Figure 8A), in mice treated with a sub-optimal dose of 6 injections of VSV-VSV-HIF-2a/SOX-10/c-MYC (as opposed to the 12 of Figure 5, and 9 of Figure 6A). In contrast, anti-CTLA4 as a mono-supportive therapy for VSV-HIF- 2a/SOX-10/c-MYC gave no added therapeutic benefit to VSV-HIF-2a/SOX-10/c- MYC alone (Figure 8A). However, when used together, anti-PDl and anti-CTLA4 significantly improved VSV-HIF-2a/SOX-10/c-MYC therapy alone (p=0.0015) and also was more effective than VSV-HIF-2a/SOX-10/c-MYC + anti-PDl (p=0.0184) or anti-CTLA4 (p=0.0016) alone.
As before (Figure 6), addition of anti-PDl therapy to VSV-HiF-2a/SOX-10/c- MYC uncovered a Thl IFN-γ T cell response to i.e. GL261 explants that was not detected from mice treated with VSV-HIF-2a/SOX-10/c-MYC alone (Figure 8B). This also was true of anti-CTLA4 therapy in combination with VSV-HIF-2a/SOX- 10/c-MYC, although to a lesser extent than with anti-PDl (Figure 8B). However, splenocyte/LN cultures from mice treated with VSV-HIF-2a/SOX-10/c-MYC and both anti-PDl and anti-CTLA4 checkpoint inhibition displayed enhanced Thl IFN-γ T cell response against i.e. GL261 compared to VSV-HIF-2a/SOX-10/c-MYC therapy in combination with either checkpoint inhibitor alone, although this only reached statistical significance when compared to the anti-CTLA4 treatment group (p=0.0282) (Figure 8B).
With respect to the Th-17 recall response, VSV-HIF-2a/SOX-10/c-MYC therapy in combination with anti-CTLA4 exhibited a strong trend to enhancing the Thl 7 response to i.e. GL261 responses (Figure 8D) compared to VSV-HIF-2a/SOX- 10/c-MYC therapy alone, or in combination with anti-PDl. Interestingly, splenocyte/LN cultures from mice treated with VSV-HIF-2a/SOX-10/c-MYC therapy combined with both anti-CTLA4 and anti-PDl therapy generated the strongest Thl 7 recall responses against i.c GL261 (Figure 8D). Taken together, these results demonstrate that addition of checkpoint inhibitors, either singly or in combination, can enhance therapeutic responses to VSV- antigen (e.g., VSV-HIF-2a/SOX-10/c-MYC) treatment and that these increases in therapy are associated with the de-repression of an anti-tumor Thl IFN-γ T cell response (anti-PDl, anti-CTLA4, or both) and of the anti-tumor Thl 7 response (anti- PD1 plus anti-CTLA4).
Example 5 - Treating melanoma using VSV vectors designed to express TGF-β,
KDR2. P-Glvcoprotein. and TYRP-1 antigens
In a first experiment, C3H mice bearing 7 day established s.c. B16 tumors were treated i.v. with three cycles (9 doses total) of (1) VSV-TGF-β + VSV-KDR2 + VSV-P Glyc + VSV TYRP-1 (5xl06 pfu/100 L), (2) VSV-NRAS + VSV-TYRP-1 + VSV-CYT-C, or (3) PBS (days 7, 10, 12). Survival of tumor-bearing C3H mice (n=8 mice per group) was determined. The VSV-combination (VSV-TGF-β + VSV-KDR2 + VSV-P Glyc + VSV TYRP-1) cured 6/6 mice which did not experience toxicities associated with i.v. VSV treatment (Figure 9). Two of the 8 mice treated died of hind limb paralysis, associated with a high i.v. dose (2xl07 pfu/injection; 9 injections) of the VSV combination.
In a repeat experiment, 3/5 mice were cured of K 1735 tumors with a lower dose of VSV-combination (VSV-TGF-β + VSV-KDR2 + VSV-P Glyc + VSV TYRP- 1) (5xl06 pfu/injection; 9 injections). 5/5 mice were cured with the same dose of the total library (ASMEL). In a control group (mice treated with VSV-GFP), 1 of 5 mice never developed a tumor and 1 mouse developed a tumor which never grew above 0.3 cm in diameter.
In another experiment, an in vitro assay (Figure 10) was performed where splenocytes were depleted of different types of antigen presenting cells by Miltenyi beads. When re-stimulated with the VSV-combination (VSV-TGF-β + VSV-KDR2 + VSV-P Glyc + VSV TYRP-1), depletion of certain subsets of APC abrogated IL-17 secretion. Those APC were then infected with a single VSV-TAA (VSV-TGF-β, VSV-KDR2, VSV-P Glyc, or VSV TYRP-1) and re-introduced into the antigen presentation assay. The results were used to determine which specific antigens were presented by which APC subtype in order to reconstitute the Thl 7 response. P- glycoprotein and N-RAS antigens were presented by macrophages; the TGF-β, KDR2, and CYT-C antigens were presented by neutrophils; and the KDR2 and TYRP1 antigens were presented by plasmacytoid DC (Figures 11-13).
Example 6 - Treating melanoma using VSV vectors designed to express truncated antigens
VSV vectors having nucleic acid that encodes truncated versions of antigens were recovered from the ASMEL cDNA library. The nucleic acids were sequenced to identify the location of the 3' truncations. For the truncated version of VSV-N-RAS, the VSV vector contained an N-RAS cDNA that encodes an N-RAS polypeptide lacking the following C-terminus: YRMKKLNSSDDGTQGCMGLPCVVM (SEQ ID NO: 1). See, also, Figure 14. For the truncated version of VSV-CYT-C, the VSV vector contained a CYT-C cDNA that encodes a CYT-C polypeptide lacking the following C-terminus: YTIKRHKWSVLKSRKLAYRPPK (SEQ ID NO:2). See, also, Figure 15. For the truncated version of VSV-TYRP-1, the VSV vector contained a TYRP- 1 cDNA that encodes a TYRP- 1 polypeptide lacking the following C-terminus: YQCYAEEYEKLQNPNQSVV (SEQ ID NO:3). See, also, Figure 16.
For the truncated version of VSV-TGF-β, the VSV vector contained a TGF-β cDNA that encodes a TGF-β polypeptide lacking the following C-terminus: YYV- GRKPKVEQLSNMTVRSCKCS (SEQ ID NO:4). For the truncated version of VSV- KDR2, the VSV vector contained a KDR2 cDNA that encodes a KDR2 polypeptide lacking the following C-terminus: YSSEEAELLKLIEIGVQTGSTAQILQPD- SGTTLSSPPV (SEQ ID NO:5). For the truncated version of VSV-P-glycoprotein, the VSV vector contained a P-glycoprotein cDNA that encodes a P-glycoprotein polypeptide lacking the following C-terminus: YFSMVSVQAGTKRQ (SEQ ID NO:6).
C57BL/6 mice bearing 7 day established s.c. B16 tumors were treated i.v. with 9 doses of (1) VSV encoding library derived, truncated VSV- N-RAS + VSV-CYT-C + VSV TYRP-1 (5xl06 pfu/100 μΕ), (2) VSV encoding full length polypeptides: VSV-NRAS + VSV-TYRP-1 + VSV-CYT-C, or (3) VSV-GFP. Survival of tumor- bearing C57BL/6 (n=8 mice per group) was determined. The results were representative of two separate experiments.
The combination of truncated cDNA for Cytochrome C (CYT-C), N-RAS, and TYRP-1 was more immunogenic against B16 tumors than the full length versions, when expressed from VSV (Figure 17). The full Length VSV-cDNA combination improved survival of C57B1/6 mice with s.c. B 16 tumors, and the truncated virus combination appeared to cure the mice.
These results demonstrate that truncated antigens (e.g., antigens lacking a portion of their C terminus) can be used to treat cancer.
Example 7 - Treating cancer in dogs
Dogs (e.g., 5-10 dogs) with a solitary intracranial mass consistent with a gliomas based on MRI that is surgically accessible are recruited. The diagnosis is confirmed as a high-grade (III-IV) glioma by histopathology. The dogs are otherwise in good health and able to undergo anesthesia for surgical excision and virus delivery.
The dogs are treated by surgical removal of the tumor and administration of either single VSV vectors (e.g., VSV-HIF-2a only) or a combination of different VSV vectors (e.g., VSV-HIF-2a + VSV-SOX-10 + VSV-cMYC). For example, any particular combination of VSV vectors provided herein is administered to a dog having cancer. In some cases, a VSV-cDNA library such as an ASMEL is administered to a dog having cancer.
Toxicities are assessed using a standard veterinary scale of grade I-V events based on owner diaries, serial blood tests, and neurological examinations. Surgical resection of the tumor is performed using the appropriate approach based on MRI. Each dog is administered a standard drug regimen before craniotomy to minimize cerebral edema. After surgical debulking, each dog is administered 5xl08 pfu of Reolysin (reovirus) injected in 5-μί aliquots around the resection cavity. A postoperative MRI is performed to assess the extent of resection, and then each dog is allowed to recover from anesthesia and is monitored in an intensive care unit. After surgery, each dog is administered prednisone (1 mg/kg body weight) PO every 12 hours for 2 days, and then the dose is tapered and discontinued over 7 days.
Adjustments are made to the dose of steroids depending on the clinical signs, such as changes in mentation or neurological function (i.e., hemiparesis), of each individual dog. The dogs are examined by MRI of the brain performed immediately after surgery and then 4, 8, and 12 months after therapy. The scans are evaluated, and the surgical resection of the tumor is defined as gross total resection (GTR) if there is complete resection of the preoperative fluid-attenuated inversion recovery signal abnormality, near total resection (NTR) if a thin (< 3 mm) residual fluid-attenuated inversion recovery signal abnormality remains around the rim of the resection cavity, or subtotal resection (STR) if there is residual nodular fluid-attenuated inversion recovery signal abnormality. The sequential MRI scans are evaluated for volume of tumor in individual dogs to measure response to treatment. Clinical response is considered as complete response (CR) if there is no evidence of the target lesion, partial response (PR) if the tumor is < 25% of the original longest diameter of the tumor, progressive disease if there is > 25% increase in the original longest diameter of the tumor, or stable disease (SD) if there are small changes that do not meet the previously defined criteria. If a dog develops recurrent or worsening neurologic signs before a scheduled MRI, an unscheduled MRI is performed at that time.
As the immunological boost, each dog is treated with intravenous injections of 5xl06 pfu of VSV-TAA (e.g., a single VSV vector such as VSV-HIF-2a only or a combination of different VSV vectors such as VSV-HIF-2a + VSV-SOX-10 + VSV- cMYC) on days 10, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, and 360 after surgery, or until tumor recurrence. For example, any particular combination of VSV vectors provided herein is administered to a dog having cancer as an immunological boost. In some cases, a VSV-cDNA library such as an ASMEL is administered to a dog having cancer as an immunological boost.
Dogs are monitored for 30 minutes following each injection for any immediate adverse reactions, such as severe wheals, dyspnea, or other signs of anaphylaxis. Dogs suffering from an acute severe reaction are given dexamethasone (0.01 mg/kg SC) and diphenhydramine (0.5 mg/kg IM). Dogs are followed over a 12-month period by imaging or until euthanasia. Dogs are assessed with complete physical and neurological examinations and quality of life assessments at suture removal and each VSV-TAA injection.
Peripheral blood mononuclear cells (PBMC) are collected prior to surgery and on days 10, 60, 120, 180, 240, 300, and 360 after surgery to determine immunological response by re-stimulating the PBMC in vitro to measure T cell responses against autologous tumor cells by flow cytometry. In some cases, CTL assays and Western blots on serum are performed.
Example 8 - Treating cancer using VSV designed to express IFN-β
VSV encoding TYRP-1 (full length) and IFN-β (VSV-mIFN-mTYRP-1) was generated by inserting mTYRP-1 in the vector backbone containing IFN-β (IFN-β) located between the M and G genes of VSV (Figure 18). PCR amplification of niTYRP-1 cDNA was prepared from B 16 cells using forward (5 ' -CTCGAGATG- AAATCTTACAACGTCC-3 ' ; SEQ ID NO: 7) and reverse (5'- CTAGCTAGCTCA- GACCATGGAGTGGTTA-3 ' ; SEQ ID NO: 8) primers. The PCR product was then digested and inserted into the Xhol and Nhel site (between genes G and L of VSV) of the VSV-IFN-β vector. VSV-mTYRP-1 was generated by inserting TYRP-1 between the G and L genes. Viruses were generated from BHK cells by co-transfection of pVSV-XN2-cDNA library DNA along with plasmids encoding viral genes as described elsewhere (Fernandez et ah, J. Virol. , 76:895-904 (2002)). Virus was expanded by a single round of infection of BHK cells and purified by sucrose gradient centrifugation.
IFN gamma assay
Splenocytes/LN from C57BL/6 mice bearing s.c. B16 tumors and treated with PBS alone or with either VSV-GFP, VSV-mTYRP-1, VSV-mIFN-p-TYRP-l, or VSV-mIFN-β were harvested. Splenocytes were re-stimulated in vitro with PBS, VSV N peptide VSV-N52-59 (RGYVYQGL; SEQ ID NO:9) or with synthetic H- 2Kb-restricted melanoma peptides: murine TRP- 1222-229 (TAYRYHLL, SEQ ID NO: 10; or TWYRYHLL SEQ ID NO: l 1; TAY, TWY, respectively), TRP-2180-188 (SVYDFFVWL, SEQ ID NO: 12; TRP2), murine gplOO (EGSRNQDWL, SEQ ID NO: 13; mgplOO), or human gpl0025-33 (KVPRNQDWL, SEQ ID NO:14; hgplOO). Forty eight hours later, supernatants were assayed for IFN-γ by ELISA (Figure 19).
In vivo results
5xl05 B16-ova tumor cells in 100 \L of PBS were injected into the flanks of C57BL/6 mice (7 mice per treatment group). Seven days later, mice were treated intra-tumorally (IT) with PBS, VSV encoding antigens, or VSV-GFP at 7xl08/50 for three days every other day (Figure 20). Survival times were determined (Figure 21).
These results demonstrate that the combined use of a VSV vector encoding an antigen (e.g., TYRP-1) with IFN-β results in prolonged cancer survival and also an enhanced IFN-γ response.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A composition comprising nucleic acid encoding a GNAQ antigen, a TYRPl antigen, and an N-RAS antigen, wherein said composition comprises less than 100 separate nucleic acid molecules.
2. The composition of claim 1, wherein said composition comprises a nucleic acid molecule encoding said GNAQ antigen, a nucleic acid molecule encoding said TYRP 1 antigen, and a nucleic acid molecule encoding said N-RAS antigen.
3. The composition of claim 1, wherein said composition comprises a VSV vector comprising nucleic acid encoding said GNAQ antigen.
4. The composition of claim 1, wherein said composition comprises a VSV vector comprising nucleic acid encoding said TYRP 1 antigen.
5. The composition of claim 1, wherein said composition comprises a VSV vector comprising nucleic acid encoding said N-RAS antigen.
6. The composition of claim 1, wherein said composition comprises less than 50 separate nucleic acid molecules.
7. The composition of claim 1, wherein said composition comprises less than 10 separate nucleic acid molecules.
8. The composition of claim 1, wherein said composition comprises less than 5 separate nucleic acid molecules.
9. A method of treating cancer within a mammal, wherein said method comprises administering to said mammal a composition comprising nucleic acid encoding a GNAQ antigen, a TYRPl antigen, and an N-RAS antigen, wherein said composition comprises less than 100 separate nucleic acid molecules.
10. The method of claim 9, wherein said cancer is a melanoma.
11. The method of claim 9, wherein said mammal is a human.
12. The method of claim 9, wherein said composition comprises a nucleic acid molecule encoding said GNAQ antigen, a nucleic acid molecule encoding said TYRP 1 antigen, and a nucleic acid molecule encoding said N-RAS antigen.
13. The method of claim 9, wherein said composition comprises a VSV vector comprising nucleic acid encoding said GNAQ antigen.
14. The method of claim 9, wherein said composition comprises a VSV vector comprising nucleic acid encoding said TYRP 1 antigen.
15. The method of claim 9, wherein said composition comprises a VSV vector comprising nucleic acid encoding said N-RAS antigen.
16. The method of claim 9, wherein said composition comprises less than 50 separate nucleic acid molecules.
17. The method of claim 9, wherein said composition comprises less than 10 separate nucleic acid molecules.
18. The method of claim 9, wherein said composition comprises less than 5 separate nucleic acid molecules.
19. A composition comprising nucleic acid encoding a BRAF antigen, a TOPO- Πα antigen, and a YB-1 antigen, wherein said composition comprises less than 100 separate nucleic acid molecules.
20. The composition of claim 19, wherein said composition comprises a nucleic acid molecule encoding said BRAF antigen, a nucleic acid molecule encoding said TOPO-IIa antigen, and a nucleic acid molecule encoding said YB- 1 antigen.
21. The composition of claim 19, wherein said composition comprises a VSV vector comprising nucleic acid encoding said BRAF antigen.
22. The composition of claim 19, wherein said composition comprises a VSV vector comprising nucleic acid encoding said TOPO-IIa antigen.
23. The composition of claim 19, wherein said composition comprises a VSV vector comprising nucleic acid encoding said YB-1 antigen.
24. The composition of claim 19, wherein said composition comprises less than 50 separate nucleic acid molecules.
25. The composition of claim 19, wherein said composition comprises less than 10 separate nucleic acid molecules.
26. The composition of claim 19, wherein said composition comprises less than 5 separate nucleic acid molecules.
27. A method of treating cancer within a mammal, wherein said method comprises administering to said mammal a composition comprising nucleic acid encoding a BRAF antigen, a TOPO-IIa antigen, and a YB-1 antigen, wherein said composition comprises less than 100 separate nucleic acid molecules.
28. The method of claim 27, wherein said cancer is a melanoma.
29. The method of claim 27, wherein said mammal is a human.
30. The method of claim 27, wherein said composition comprises a nucleic acid molecule encoding said BRAF antigen, a nucleic acid molecule encoding said TOPO- IIa antigen, and a nucleic acid molecule encoding said YB- 1 antigen.
31. The method of claim 27, wherein said composition comprises a VSV vector comprising nucleic acid encoding said BRAF antigen.
32. The method of claim 27, wherein said composition comprises a VSV vector comprising nucleic acid encoding said TOPO-IIa antigen.
33. The method of claim 27, wherein said composition comprises a VSV vector comprising nucleic acid encoding said YB-1 antigen.
34. The method of claim 27, wherein said composition comprises less than 50 separate nucleic acid molecules.
35. The method of claim 27, wherein said composition comprises less than 10 separate nucleic acid molecules.
36. The method of claim 27, wherein said composition comprises less than 5 separate nucleic acid molecules.
37. A composition comprising nucleic acid encoding:
(a) a TGF-β antigen, a MDRl antigen, a TYRP-1 antigen, and a KDR2 antigen,
(b) a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a BRAF antigen,
(c) a TOPOIIa antigen, a YB-1 antigen, a CDC7 kinase antigen, and a CD44 antigen,
(d) a TOPOIIa antigen and an ABCB5a antigen, or
(e) an ABCB5a antigen, a CYT-C antigen, a -RAS antigen, and a TYRP-1 antigen,
wherein said composition comprises less than 100 separate nucleic acid molecules.
38. The composition of claim 37, wherein said composition comprises:
(a) a nucleic acid molecule encoding a TGF-β antigen, a nucleic acid molecule encoding a MDRl antigen, a nucleic acid molecule encoding a TYRP-1 antigen, and a nucleic acid molecule encoding a KDR2 antigen,
(b) a nucleic acid molecule encoding a TOPOIIa antigen, a nucleic acid molecule encoding a YB-1 antigen, a nucleic acid molecule encoding a CDC7 kinase antigen, and a nucleic acid molecule encoding a BRAF antigen,
(c) a nucleic acid molecule encoding a TOPOIIa antigen, a nucleic acid molecule encoding a YB-1 antigen, a nucleic acid molecule encoding a CDC7 kinase antigen, and a nucleic acid molecule encoding a CD44 antigen,
(d) a nucleic acid molecule encoding a TOPOIIa antigen and a nucleic acid molecule encoding an ABCB5a antigen, or
(e) a nucleic acid molecule encoding an ABCB5a antigen, a nucleic acid molecule encoding a CYT-C antigen, a nucleic acid molecule encoding a N-RAS antigen, and a nucleic acid molecule encoding a TYRP- 1 antigen.
39. The composition of claim 37, wherein said composition comprises a VSV vector comprising nucleic acid encoding said TGF-β antigen, said MDR1 antigen, said TYRP-1 antigen, said KDR2 antigen, said TOPOIIa antigen, said YB-1 antigen, said CDC7 kinase antigen, said BRAF antigen, said CD44 antigen, said ABCB5a antigen, said CYT-C antigen, or said N-RAS antigen.
40. The composition of claim 37, wherein said composition comprises less than 50 separate nucleic acid molecules.
41. The composition of claim 37, wherein said composition comprises less than 10 separate nucleic acid molecules.
42. The composition of claim 37, wherein said composition comprises less than 6 separate nucleic acid molecules.
43. A method of treating cancer within a mammal, wherein said method comprises administering to said mammal a composition of any one of claims 37-42.
44. The method of claim 43, wherein said cancer is a melanoma.
45. The method of claim 43, wherein said mammal is a human.
46. A composition comprising nucleic acid encoding a TGF-β antigen, a KDR2 antigen, a P Glyc antigen, and a TYRP- 1 antigen, wherein said composition comprises less than 100 separate nucleic acid molecules.
47. The composition of claim 46, wherein said composition comprises a nucleic acid molecule encoding said TGF-β antigen, a nucleic acid molecule encoding said KDR2 antigen, a nucleic acid molecule encoding said P Glyc antigen, and a nucleic acid molecule encoding said TYRP-1 antigen.
48. The composition of claim 46, wherein said composition comprises a VSV vector comprising nucleic acid encoding said TGF-β antigen.
49. The composition of claim 46, wherein said composition comprises a VSV vector comprising nucleic acid encoding said KDR2 antigen.
50. The composition of claim 46, wherein said composition comprises a VSV vector comprising nucleic acid encoding said P Glyc antigen.
51. The composition of claim 46, wherein said composition comprises a VSV vector comprising nucleic acid encoding said TYRP 1 antigen.
52. The composition of claim 46, wherein said composition comprises less than 50 separate nucleic acid molecules.
53. The composition of claim 46, wherein said composition comprises less than 10 separate nucleic acid molecules.
54. The composition of claim 46, wherein said composition comprises less than 5 separate nucleic acid molecules.
55. A method of treating cancer within a mammal, wherein said method comprises administering to said mammal a composition comprising nucleic acid encoding a TGF-β antigen, a KDR2 antigen, a P Glyc antigen, and a TYRP-1 antigen, wherein said composition comprises less than 100 separate nucleic acid molecules.
56. The method of claim 55, wherein said cancer is a melanoma.
57. The method of claim 55, wherein said mammal is a human.
58. The composition of any one of claims 1-8, 19-26, 37-42, and 46-54, wherein said composition comprises an immune checkpoint inhibitor.
59. The composition of claim 46, wherein said immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-CTLA4 antibody.
60. The method of any one of claims 9-18, 27-36, 43-45, and 55-57, wherein said method comprises administering an immune checkpoint inhibitor to said mammal.
61. The method of claim 60, wherein said immune checkpoint inhibitor is an anti- PD-1 antibody or an anti-CTLA4 antibody.
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