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WO2016191641A2 - Procédés pour améliorer des réponses immunitaires spécifiques à un antigène à l'aide d'une polythérapie comprenant des antigènes de capside de papillomavirus - Google Patents

Procédés pour améliorer des réponses immunitaires spécifiques à un antigène à l'aide d'une polythérapie comprenant des antigènes de capside de papillomavirus Download PDF

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Publication number
WO2016191641A2
WO2016191641A2 PCT/US2016/034539 US2016034539W WO2016191641A2 WO 2016191641 A2 WO2016191641 A2 WO 2016191641A2 US 2016034539 W US2016034539 W US 2016034539W WO 2016191641 A2 WO2016191641 A2 WO 2016191641A2
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dna
antigen
combination
seq
cell
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PCT/US2016/034539
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WO2016191641A3 (fr
Inventor
Richard Roden
Tzyy-Choou Wu
Chien-Fu Hung
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The Johns Hopkins University
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Publication of WO2016191641A3 publication Critical patent/WO2016191641A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory

Definitions

  • HPV human papillomavirus
  • Cervarix based upon virus-like particles (VLP) derived from the major capsid protein LI effectively protect against infection by the two most common HPV types found in cervical cancer (for review see Roden R et al. Nat Rev Cancer 2006, 6:753-763).
  • the minor capsid antigen L2 also shows promise for preventive vaccination in animal models, although it is less immunogenic than LI VLP.
  • LI VLP nor L2-based vaccines generate therapeutic effects against established HPV infection (Schiller JT et al. Vaccine 2008, 26 Suppl 10:K53-61). Therefore, given the significant burden of HPV-associated lesions worldwide, there is an urgent need to develop therapeutic HPV vaccines for the control of existing HPV infection and associated malignancies.
  • therapeutic HPV vaccines focus on targeting the HPV E6 and E7, since only these oncoproteins are consistently expressed in HPV-associated cancers and are responsible for the malignant transformation.
  • DNA vaccines have emerged as attractive candidates for the treatment of cervical cancer and associated malignancies. Naked DNA is relatively safe, stable, and easy to produce and transport (for review see Gurunathan S et al. Annual review of immunology 2000, 18:927-974).
  • DNA vaccines are capable of sustained cellular gene expression, promote MHC class I antigen presentation, and have the capacity for repeated administration since they do not lead to the generation of neutralizing antibodies.
  • an important limitation of DNA vaccines is limited potency since they lack the intrinsic ability to amplify and spread in vivo. Therefore, it is important to consider strategies to improve DNA vaccine potency strategies (for review, see Hung CF et al. Methods in molecular medicine 2006, 127: 199-220; Tsen SW et al. Expert Rev Vaccines 2007, 6:227-239).
  • CD4 + T cells Activation and proliferation of CD4 + T cells is crucial to the success of both humoral and cell-mediated responses to viral infection.
  • E7 E7-specific CD8 T cell immunity
  • CD4 T cell help Peng S et al. Cell & bioscience 2014, 4: 11
  • approaches to boost CD4 + T cell help are likely to enhance DNA vaccine potency.
  • CD4 + T cells play a significant role in priming effector CD8 + T cells, thus augmenting the CD8 + T cell responses, as well as generation of memory T cell populations (for review see Castellino F et al. Annual review of immunology 2006, 24:519-540).
  • CD4 + T cells help to differentiate naive CD8 + T cells into effector cells by providing activation signals to dendritic cells (DCs), most notably IL-2, thus promoting CD8 + T cell proliferation.
  • DCs dendritic cells
  • strategies to induce CD4 + T helper cells at sites of CD8 + T cell priming can potentially enhance CTL immune responses.
  • Described herein are methods comprising co-administering to a mammalian an effective amount of a combination therapy comprising at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide, to thereby enhancing an antigen specific immune response.
  • the present invention provides for combine intracellular targeting strategies using
  • Co-administration of vectors containing BPV LI or L2 DNA in combination with DNA vaccines could elicit enhanced antigen-specific CD8 + in both CRT/E7 and ovalbumin (OVA) antigenic systems.
  • Co-administration of vectors containing BPV1 LI ⁇ L2 DNA with CRT/E7 DNA led to the generation of Ll/L2-specific CD4 + T cell immune responses as well as Ll-specific neutralizing antibodies.
  • Co-administration with BPV1 LI Co-administration with BPV1 LI
  • DNA encoding LI and L2 significantly enhances the therapeutic antitumor effects generated by CRT/E7 DNA vaccination.
  • the observed enhancement of CD8 + T cell immune responses by DNA encoding LI and L2 was also found to extend to HPV-16 L1/L2 system.
  • Co- administration of DNA encoding papillomavirus LI or L2 can be used to enhance antigen- specific CD8 + T cell immune responses generated by therapeutic HPV DNA vaccination for the control of HPV infection and HPV-associated tumors.
  • such methods can also generate neutralizing antibodies against papillomavirus for potential prevention against infection. This strategy also provides the opportunity to combine preventive and therapeutic approaches. Such methods may potentially be extended to other antigenic systems for the control of infection and/or cancer.
  • One aspect of the invention relates to a method of inducing or enhancing an antigen- specific immune response in a mammal, comprising co-administering to the mammal an effective amount of a combination therapy comprising at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide, to thereby enhancing the antigen specific immune response.
  • the papillomavirus is selected from the group consisting of human papilloma virus (HPV)l, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV10, HPV11, HPV12, HPV13, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV26, HPV27, HPV28, HPV29, HPV30, HPV31, HPV32, HPV33, HPV34, HPV35, HPV36, HPV37, HPV38, HPV39, HPV40, HPV41, HPV42, HPV43, HPV44, HPV45, HPV46, HPV47, HPV48, HPV49, HPV50, HPV51, HPV52, HPV53, 1-IPV54, HPV55, HPV56, HPV57, HPV
  • the papillomavirus is bovine papillomavirus type 1
  • the capsid antigen is a major and minor capsid antigen. In certain embodiments, the major capsid antigen is LI .
  • the minor capsid antigen is L2.
  • the capsid antigen LI is derived from BPV1, and said capsid antigen comprises an amino acid sequence at least 60% identical to the amino acid sequence set forth in SEQ ID NO: 123. In certain embodiments, the capsid antigen LI is derived from BPV1, and said capsid antigen comprises a nucleotide sequence at least 60% identical to the nucleotide sequence set forth in SEQ ID NO: 122.
  • the capsid antigen L2 is derived from BPV1, and said capsid antigen comprises an amino acid sequence at least 60% identical to the amino acid sequence set forth in SEQ ID NO: 125.
  • the capsid antigen L2 is derived from BPV1, and said capsid antigen comprises a nucleotide sequence at least 60% identical to the nucleotide sequence set forth in SEQ ID NO: 124.
  • the combination therapy elicits enhanced antigen-specific
  • the DNA vaccine comprises an antigenic peptide, and said antigenic peptide is selected from the group consisting of ovalbumin (OVA), HPV16 E6, HPV16 E7, and influenza NS 1.
  • OVA ovalbumin
  • HPV16 E6, HPV16 E7 and influenza NS 1.
  • the DNA vaccine comprises HPV-16 E7 (E7) antigen linked with calreticulum (CRT) (CRT/E7).
  • the E7 comprises an amino acid sequence set forth in RAHYNIVTF.
  • the DNA vaccine comprises OVA.
  • the OVA comprises an amino acid sequence set forth in ISQAVHAAHAEINEAGR.
  • the papillomavirus is human papillomavirus type 16 (HPV-1)
  • the major capsid antigen is LI .
  • the minor capsid antigen is L2.
  • the capsid antigen LI is derived from HPV16, and said capsid antigen comprises an amino acid sequence at least 60% identical to the amino acid sequence set forth in SEQ ID NO: 119.
  • the capsid antigen LI is derived from HPV16, and said capsid antigen comprises a nucleotide sequence at least 60% identical to the nucleotide sequence set forth in SEQ ID NO: 118.
  • the capsid antigen L2 is derived from HPV16, and said capsid antigen comprises an amino acid sequence at least 60% identical to the amino acid sequence set forth in SEQ ID NO: 121.
  • the capsid antigen L2 is derived from HPV16, and said capsid antigen comprises a nucleotide sequence at least 60% identical to the nucleotide sequence set forth in SEQ ID NO: 120.
  • the combination therapy elicits enhanced antigen-specific CD4 + T cell immune response.
  • the enhanced antigen-specific CD4 + T cell immune response is L1-, L2-, or Ll/L2-specific.
  • the mammal is a human.
  • the mammal is afflicted with cancer.
  • the combination therapy is administered intradermally, intraperitoneally, or intravenously via injection.
  • the combination therapy is administered alone or in a pharmaceutically acceptable carrier in a biologically-effective, and/or a therapeutically- effective amount,
  • the antigen-specific immune response is greater in magnitude than an antigen-specific immune response induced by administration of the DNA vaccine or DNA vector alone.
  • Another aspect of the invention relates to a combination for use in inducing or enhancing an antigen-specific immune response in a mammal, comprising (a) an effective amount of at least one DNA vector comprising a papillomavirus capsid antigen to be administered; (b) an effective amount of at least one DNA vaccine comprising an antigenic peptide to be administered, and the combination induces or enhances the antigen specific immune response.
  • the papillomavirus is selected from the group consisting of human papilloma virus (HPV)l, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV10, HPV11, HPV12, HPV13, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV26, HPV27, HPV28, HPV29, HPV30, HPV31, HPV32, HPV33, HPV34, HPV35, HPV36, HPV37, HPV38, HPV39, HPV40, HPV41, HPV42, HPV43, HPV44, HPV45, HPV46, HPV47, HPV48, HPV49, HPV50, HPV51, HPV52, HPV53, 1-IPV54, HPV55, HPV56, HPV57, HPV
  • the papillomavirus is bovine papillomavirus type 1 (BPV1).
  • the capsid antigen is a major and minor capsid antigen.
  • the major capsid antigen is LI .
  • the minor capsid antigen is L2.
  • the capsid antigen LI is derived from BPV1, and said capsid antigen comprises an amino acid sequence at least 60% identical to the amino acid sequence set forth in SEQ ID NO: 123.
  • the capsid antigen L2 is derived from BPV1, and said capsid antigen comprises an amino acid sequence at least 60% identical to the amino acid sequence set forth in SEQ ID NO: 125.
  • the combination elicits enhanced antigen-specific CD8+.
  • the DNA vaccine comprises an antigenic peptide, and said antigenic peptide is selected from the group consisting of ovalbumin (OVA), HPV16 E6, HPV16 E7, and influenza NS 1.
  • OVA ovalbumin
  • HPV16 E6, HPV16 E7 and influenza NS 1.
  • the DNA vaccine comprises HPV-16 E7 (E7) antigen linked with calreticulum (CRT) (CRT/E7).
  • the E7 comprises an amino acid sequence set forth in RAHYNIVTF.
  • the DNA vaccine comprises OVA.
  • the OVA comprises an amino acid sequence set forth in ISQAVHAAHAEINEAGR.
  • the papillomavirus is human papillomavirus type 16 (HPV-1)
  • the major capsid antigen is LI . In certain embodiments, the minor capsid antigen is L2.
  • the capsid antigen LI is derived from HPV16, and said capsid antigen comprises an amino acid sequence at least 60% identical to the amino acid sequence set forth in SEQ ID NO: 119.
  • the capsid antigen L2 is derived from HPV16, and said capsid antigen comprises an amino acid sequence at least 60% identical to the amino acid sequence set forth in SEQ ID NO: 121.
  • the combination elicits enhanced antigen-specific CD4+ T cell immune response.
  • the enhanced antigen-specific CD4+ T cell immune response is L1-, L2-, or Ll/L2-specific.
  • the mammal is a human.
  • the mammal is afflicted with cancer.
  • the combination is administered intradermally,
  • the combination is administered alone or in a
  • the antigen-specific immune response is greater in magnitude than an antigen-specific immune response induced by administration of the DNA vaccine or DNA vector alone.
  • Figure 1 contains two panels (A) and (B) describing characterization of antigen- specific CD8 + T cell immune responses generated by antigen-specific DNA vaccine mixed with vectors containing BPV1 LI or L2 DNA.
  • C57BL/6 mice (five per group) were vaccinated intradermally via gene gun with 2 ⁇ g/mouse of CRT/E7 or OVA DNA with or without BPVl LI or L2 DNA twice at 1-week intervals.
  • Splenocytes from vaccinated mice were collected 1 week after last immunization and the E7 or OVA-specific T cell immune responses were characterized using intracellular cytokine staining followed by flow cytometry analysis.
  • Panel (A) depicts representative flow cytometry data depicting the 5 number of E7 (upper panel) or OVA(lower panel)-specific CD8 + T cells.
  • Panel (B) depicts a bar graph representing the number of E7 (upper panel) or OVA (lower panel) -specific CD8 + T cells/3xl0 5 splenocytes (mean+ SD). Data shown are representative of two experiments performed. * indicates p ⁇ 0.05.
  • Figure 2 contains two panels (A) and (B) describing the characterization of antigenic) specific CD8 + T cell immune responses generated by antigen-specific DNA vaccine mixed with HPV-16 LI or L2 DNA. C57BL/6 mice (five per group) were vaccinated
  • Panel (A) depicts representative flow cytometry data depicting the number of E7 (upper panel) or OVA (lower panel)-specific CD8 + T cells.
  • Panel (B) depicts a bar graph representing the number of E7 (upper panel) or OVA (lower panel)- specific CD8 + T cells/3xl0 5 splenocytes (mean 1 SD). Data shown are representative of two 0 experiments performed. * indicates p ⁇ 0.05.
  • Figure 3 contains four panels (A), (B), (C), and (D) describing characterization of BPVl and HPV-16 LI or L2-specific CD4 + T cell immune responses generated by CRT/E7 or OVA DNA mixed with DNA encoding BPVl or HPV-16 LI or L2.
  • C57BL/6 mice (five per group) were vaccinated intradermally via gene gun with 2 ⁇ g/mouse of CRT/E7 with or 5 without BPVl or HPV-16 LI or L2 DNA twice at 1-week intervals.
  • mice were collected 1 week after last immunization and pulsed with 5 ⁇ g/mL of BPVl or HPV-16 L1/L2 VLPs.
  • the BPVl LI/ L2-specific CD4 + T cell immune responses were characterized using intracellular cytokine staining followed by flow cytometry analysis.
  • Panel (A) depicts representative flow cytometry data depicting the number of 30 BPVl Ll/L2-specific CD4 + T cells.
  • Panel (B) depicts a bar graph representing the number of BPVl Ll/L2-specific CD4 + T cells/3xl0 5 splenocytes (mean 1 SD).
  • Panel (C) depicts representative flow cytometry data depicting the number of HPV-16 Ll/L2-specific CD4 + T cells.
  • Panel (D) depicts a bar graph representing the number of HPV-16 Ll/L2-specific CD4 + T cells/3xl0 5 splenocytes (mean 1 SD). Data shown are representative of two experiments performed. * indicates p ⁇ 0.05.
  • Figure 4 contains two panels (A) and (B) describing in vivo tumor treatment experiments in mice vaccinated with CRT/E7 DNA mixed with BPVl LI DNA.
  • C57BL/6 mice (five per group) were first challenged with lxl0 5 /mouse of TC-1 tumor cells subcutaneously.
  • mice were treated intradermally via gene gun with 2 ⁇ g/mouse of CRT/E7 DNA alone, BPVl LI DNA alone or CRT/E7 DNA in combination with BPVl LI DNA.
  • Vaccinated mice were boosted twice at 1-week intervals with the same dose and regimen. Mice were sacrificed on day 30 after the last vaccination. Tumor growth were monitored twice weekly by caliper measurements and palpations.
  • Panel (A) depicts a line graph depicting the tumor volume over time of tumor-bearing mice treated with CRT/E7 DNA and/or BPVl LI DNA.
  • Panel (B) depicts Kaplan-Meier survival analysis of tumor-bearing mice treated with CRT/E7 DNA and/or BPVl LI DNA. The data presented are from one representative experiment of the two performed. * indicates p ⁇ 0.05.
  • Figure 5 contains one panel (A) describing the characterization of BPVl -specific neutralizing antibody responses generated by mice vaccinated with CRT/E7 and/or BPVl LI DNA.
  • C57BL/6 mice three per group were immunized on days 1, 15, and 30 intradermally using a gene gun with 2 ⁇ g of DNA per mouse of CRT/E7 and/or BPVl LI DNA.
  • In vitro neutralization assays were performed using BPVl LI pseudovirus on twofold dilutions of antisera collected from the mice 2 weeks after the final immunization. Endpoint titers achieving 50% neutralization are plotted and the means shown as horizontal lines.
  • PI Pre Immune
  • L1 BPV1 LI DNA vaccine
  • E7 CRT/E7 DNA vaccine.
  • Figure 6 contains three panels (A), (B), and (C) describing the comparison of OVA- specific CD8 + T cell responses induced by pcDNA3-OVA vaccination with or without coadministration of BPV LI or L2.
  • Panel (A) depicts a schematic illustration of the experiment. Briefly, 5-8 weeks old female C57BL/6 mice (3 mice/group) were vaccinated with 10 ⁇ g/mouse of pcDNA3-OVA, with either 10 ⁇ g/mouse of pcDNA3, or pcDNA3- BPVLl, or pcDNA3-BPVL2 via intramuscular injection. The mice were boosted with the same regimen once after one week.
  • PBMCs were collected from peripheral blood, stained with FITC-conjugated anti-mouse CD8a antibody, PE- conjugated OVA peptide (SIINFEKL) loaded H-2K b tetramer. The data were acquired with FACSCalibur and analyzed with CellQuest.
  • Panel (B) depicts representative flow cytometry image of PBMC staining.
  • Panel (C) depicts a summary of the flow cytometry data.
  • the inventors of the present invention have determined that that irradiated tumor microenvironments are ideal for triggering CD8-dependent antitumor effects by
  • the present invention is drawn to methods for enhancing an antigen-specific immune response in a mammal using combination of radiation therapy with local injection of therapeutic vaccines.
  • ANOVA analysis of variance
  • APC antigen presenting cell
  • CRT calreticulin
  • CTL cytotoxic T lymphocyte
  • DC dendritic cell
  • DLN draining lymph nodes
  • E7 HPV oncoprotein E7
  • ELISA enzyme-linked immunosorbent assay
  • HPV human papillomavirus
  • IFN ⁇ interferon- ⁇
  • i.m. intramuscular(ly); i. ,
  • PCR polymerase chain reaction
  • SD standard deviation
  • TAA tumor-associate antigen
  • Papillomaviruses encode two capsid proteins, LI and L2.
  • LI the major capsid protein
  • L2 the minor capsid protein
  • L2 is not required for VLP formation, but is required for formation of infectious virions (and pseudovirions). Up to 72 copies of L2 can be incorporated in a VLP, and viral infectivity correlates with L2 content (Buck, CB et al. 2008 J Virol 82:5190-7).
  • Certain aspects of the invention comprise codon-optimized capsid antigens derived from the LI or L2 protein of different strains of HPV or papillomavirus from a variety of non-human (animal) species as otherwise set forth herein, and is highly immunogenic.
  • the capsid antigens may be derived or isolated from a number of different strains of papillovirus, including strains 16, 45 and/or 58 as well as HPV strains 1, 5, 6, 11 and 18.
  • Additional strains include, but are not limited to, papilloma virus strains HPV 1, HPV2, HPV5, HPV6, HPV8, HPV 11, HPV 16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, CRPV and BPVl and including HPV strains 16, 45 and/or 58, as well as of HPV strains 1, 5, 6, 11 and 48, it is noted that the LI or L2 sequence of HPV16 and BPVl are relatively conserved across diverse papillomavirus isolates and the use of each of these sequences within the DNA vector according to the present invention may instill immunogenic protection and enhance antigenic immune responses.
  • L2 or LI peptide sequences as described above are from any of HPV strains HPV1, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV10, HPV11, HPV 12, HPV13, HPV14, HPV15, HPV 16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV26, HPV27, HPV28, HPV29, HPV30, HPV31, HPV32, HPV33, HPV34, HPV35, HPV36, HPV37, HPV38, HPV39, HPV40, HPV41, HPV42, HPV43, HPV44, HPV45, HPV46, HPV47, HPV48, HPV49, HPV50, HPV51, HPV52, HPV53, 1-IPV54, HPV55, HPV56, HPV57, HPV58, HPV59, HPV
  • L2 and L2 capsid antigens for use according to the present invention include the following for each of the above-referenced HPV strains and animal PVs (note that all of the amino acid numbers listed below are referenced based upon the sequence of HPV 16 L2 or LI and BPVl LI or L2, and that for each of the lengths of amino acids, each length within the length range is understood to be disclosed):
  • a LI capsid antigen of BPV1 may comprise the nucleotide sequence set forth in SEQ ID NO: 122.
  • a LI capsid antigen of BPV1 may comprise the amino acid sequence set forth in SEQ ID NO: 123.
  • a LI capsid antigen of HPV16 may comprise the nucleotide sequence set forth in SEQ ID NO: 118. In certain embodiments, a LI capsid antigen of HPV16 may comprise the amino acid sequence set forth in SEQ ID NO: 119. In certain embodiments, a L2 capsid antigen of BPV1 may comprise the nucleotide sequence set forth in SEQ ID NO: 124. In certain embodiments, a L2 capsid antigen of BPV1 may comprise the amino acid sequence set forth in SEQ ID NO: 125. In certain embodiments, a L2 capsid antigen of HPV16 may comprise the nucleotide sequence set forth in SEQ ID NO: 120. In certain embodiments, a L2 capsid antigen of HPV16 may comprise the amino acid sequence set forth in SEQ ID NO: 121.
  • % homology is used interchangeably herein with the term “% identity” herein and normally refers to the level of nucleic acid identity between the nucleic acid sequence of the DNA or RNA aptamers of the present inventions, when aligned using a sequence alignment program.
  • 80% homology means the same thing as 80% sequence identity determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence identity over a length of the given sequence.
  • Exemplary levels of sequence identity include, but are not limited to, 80, 85, 90, 95, 98% or more sequence identity to a given sequence as described herein.
  • Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly accessible at www' . neb i . nl m . ni h . go v/ L A S T .
  • the method comprising co-administering to the mammal an effective amount of a combination therapy comprising at least one DNA vector comprising a papillomavirus capsid antigen, said capsid antigen comprises at least 50% identical to a nucleotide sequence selected from the group consisting of : SEQ ID Nos. 118, 120, 122, and 124, or any combinations thereof.
  • said capsid antigen comprises a nucleotide sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, identical to a nucleotide sequence selected from the group consisting of : SEQ ID Nos. 118,120, 122, and 124, or any combinations thereof.
  • the method comprising co-administering to the mammal an effective amount of a combination therapy comprising at least one DNA vector comprising a papillomavirus capsid antigen, said capsid antigen comprises at least 50% identical to a amino acid sequence selected from the group consisting of : SEQ ID Nos. 118, 121, 123, and 125, or any combinations thereof.
  • said capsid antigen comprises a nucleotide sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%), or 99.9%, identical to a nucleotide sequence selected from the group consisting of : SEQ ID Nos. 119,121, 123, and 125, or any combinations thereof.
  • Nucleic acid e.g., DNA
  • DNA vaccines e.g., DNA vaccines
  • a nucleic acid vaccine will encode an antigen, e.g., an antigen against which an immune response is desired.
  • Other nucleic acids that may be used are those that increase or enhance an immune reaction, but which do not encode an antigen against which an immune reaction is desired. These vaccines are further described below.
  • antigens include proteins or fragments thereof from a pathogenic organism, e.g., a bacterium or virus or other microorganism, as well as proteins or fragments thereof from a cell, e.g., a cancer cell.
  • the antigen is from a virus, such as class human papillomavirus (HPV), e.g., E7 or E6.
  • HPV class human papillomavirus
  • E7 or E6 are also oncogenic proteins, which are important in the induction and maintenance of cellular transformation and co-expressed in most HPV-containing cervical cancers and their precursor lesions. Therefore, cancer vaccines that target E7 or E6 can be used to control of HPV-associated neoplasms (Wu, T-C, Curr Opin Immunol. (5:746-54, 1994).
  • the present invention is not limited to the exemplified antigen(s). Rather, one of skill in the art will appreciate that the same results are expected for any antigen (and epitopes thereof) for which a T cell-mediated response is desired.
  • the response so generated will be effective in providing protective or therapeutic immunity, or both, directed to an organism or disease in which the epitope or antigenic determinant is involved - for example as a cell surface antigen of a pathogenic cell or an envelope or other antigen of a pathogenic virus, or a bacterial antigen, or an antigen expressed as or as part of a pathogenic molecule.
  • the E7 nucleic acid sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO:2) from HPV-16 are shown herein (see GenBank Accession No. NC_001526).
  • the single letter code, the wild type E7 amino acid sequence (SEQ ID NO:2) is shown herein.
  • the E7 protein may be used in a "detoxified" form.
  • the E7 (detox) mutant sequence has the following two mutations:
  • nucleotide sequence that encodes the above E7 or E7(detox) polypeptide, or an antigenic fragment or epitope thereof, can be used in the present compositions and methods, including the E7 and E7(detox) sequences which are shown herein.
  • This polypeptide has 158 amino acids and is shown herein in single letter code as SEQ ID N0 5.
  • E6 proteins from cervical cancer-associated HPV types such as HPV-16 induce proteolysis of the p53 tumor suppressor protein through interaction with E6-AP.
  • MECs Human mammary epithelial cells
  • HPV-16 E6, as well as other cancer-related papillomavirus E6 proteins also binds the cellular protein E6BP (ERC-55).
  • E6BP cellular protein
  • E6(detox) a non-oncogenic mutated form of E6 may be used, referred to as "E6(detox).
  • E6 amino acid sequence provided herein.
  • Some studies of E6 mutants are based upon a shorter E6 protein of 151 nucleic acids, wherein the N-terminal residue was considered to be the Met at position 8 in the wild type E6. That shorter version of E6 is shown herein as SEQ ID NO:6.
  • VRP Venezuelan equine encephalitis virus replicon particle
  • Cys 106 neither binds nor facilitates degradation of p53 and is incapable of immortalizing human mammary epithelial cells (MEC), a phenotype dependent upon p53 degradation.
  • MEC human mammary epithelial cells
  • nucleotide sequence that encodes these E6 polypeptides, one of the mutants thereof, or an antigenic fragment or epitope thereof can be used in the present invention.
  • Other mutations can be tested and used in accordance with the methods described herein including those described in Cassetti et al, supra. These mutations can be produced from any appropriate starting sequences by mutation of the coding DNA.
  • the present invention also includes the use of a tandem E6-E7 vaccine, using one or more of the mutations described herein to render the oncoproteins inactive with respect to their oncogenic potential in vivo.
  • VRP vaccines (described in Cassetti et al, supra) comprised fused E6 and E7 genes in one open reading frame which were mutated at four or five amino acid positions.
  • the present constructs may include one or more epitopes of E6 and E7, which may be arranged in their native order or shuffled in any way that permits the expressed protein to bear the E6 and E7 antigenic epitopes in an immunogenic form.
  • DNA encoding amino acid spacers between E6 and E7 or between individual epitopes of these proteins may be introduced into the vector, provided again, that the spacers permit the expression or presentation of the epitopes in an immunogenic manner after they have been expressed by transduced host cells.
  • a nucleic acid sequence encoding HA is shown herein as SEQ ID NO: 7.
  • the amino acid sequence of HA is shown herein as SEQ ID NO: 8, with the immunodominant epitope underscored.
  • Ovalbumin Ovalbumin
  • SEQ ID NO:9 An amino acid sequence encoding a representative OVA is shown herein as SEQ ID NO:9.
  • antigens are epitopes of pathogenic microorganisms against which the host is defended by effector T cells responses, including CTL and delayed type
  • hypersensitivity typically include viruses, intracellular parasites such as malaria, and bacteria that grow intracellularly such as Mycobacterium and Listeria species.
  • the types of antigens included in the vaccine compositions used in the present invention may be any of those associated with such pathogens as well as tumor-specific antigens. It is noteworthy that some viral antigens are also tumor antigens in the case where the virus is a causative factor in the tumor.
  • Hepatitis B virus (HBV) (Beasley, R.P. et al, Lancet 2: 1129-1133 (1981) has been implicated as etiologic agent of hepatomas.
  • HBV Hepatitis B virus
  • HPV E6 and E7 antigens are the most promising targets for virus associated cancers in immunocompetent individuals because of their ubiquitous expression in cervical cancer.
  • virus-associated tumor antigens are also ideal candidates for prophylactic vaccines. Indeed, introduction of prophylactic HBV vaccines in Asia have decreased the incidence of hepatoma (Chang, MH et al. New Engl. J. Med. 336, 1855-1859 (1997), representing a great impact on cancer prevention.
  • HPV hepatitis C Virus
  • retroviruses such as human immunodeficiency virus (HIV- 1 and HIV-2)
  • herpes viruses such as Epstein Barr Virus (EBV), cytomegalovirus (CMV), HSV-1 and HSV-2, and influenza virus.
  • EBV Epstein Barr Virus
  • CMV cytomegalovirus
  • HSV-1 and HSV-2 influenza virus.
  • Useful antigens include HBV surface antigen or HBV core antigen; ppUL83 or pp89 of CMV; antigens of gpl20, gp41 or p24 proteins of HIV-1; ICP27, gD2, gB of HSV; or influenza hemagglutinin or nucleoprotein (Anthony, LS et al, Vaccine 1999; 17:373-83).
  • Other antigens associated with pathogens that can be utilized as described herein are antigens of various parasites, including malaria, e.g., malaria peptide based on repeats of NANP.
  • the invention includes methods using foreign antigens in which individuals may have existing T cell immunity (such as influenza, tetanus toxin, herpes etc).
  • existing T cell immunity such as influenza, tetanus toxin, herpes etc.
  • the skilled artisan would readily be able to determine whether a subject has existing T cell immunity to a specific antigen according to well known methods available in the art and use a foreign antigen to which the subject does not already have an existing T cell immunity.
  • the antigen is from a pathogen that is a bacterium, such as Bordetella pertussis; Ehrlichia chaffeensis; Staphylococcus aureus; Toxoplasma gondii; Legionella pneumophila; Brucella suis; Salmonella enterica; Mycobacterium avium;
  • a pathogen such as Bordetella pertussis; Ehrlichia chaffeensis; Staphylococcus aureus; Toxoplasma gondii; Legionella pneumophila; Brucella suis; Salmonella enterica; Mycobacterium avium;
  • Mycobacterium tuberculosis Listeria monocytogenes; Chlamydia trachomatis; Chlamydia pneumoniae; Rickettsia rickettsii; or, a fungus, such as, e.g., Paracoccidioides brasiliensis; or other pathogen, e.g., Plasmodium falciparum.
  • cancer includes, but is not limited to, solid tumors and blood borne tumors.
  • the term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • a term used to describe cancer that is far along in its growth, also referred to as “late stage cancer” or “advanced stage cancer,” is cancer that is metastatic, e.g., cancer that has spread from its primary origin to another part of the body.
  • advanced stage cancer includes stages 3 and 4 cancers. Cancers are ranked into stages depending on the extent of their growth and spread through the body; stages correspond with severity. Determining the stage of a given cancer helps doctors to make treatment recommendations, to form a likely outcome scenario for what will happen to the patient (prognosis), and to communicate effectively with other doctors.
  • Stage 0 cancer is cancer that is just beginning, involving just a few cells.
  • Stages I, II, III, and IV represent progressively more advanced cancers, characterized by larger tumor sizes, more tumors, the
  • TNM system Another popular staging system is known as the TNM system, a three dimensional rating of cancer extensiveness. Using the TNM system, doctors rate the cancers they find on each of three scales, where T stands for tumor size, N stands for lymph node
  • M stands for metastasis (the degree to which cancer has spread beyond its original locations).
  • T3 NO, MO
  • T2 mesenchymal cells
  • Ml suggesting a medium sized tumor that has spread to local lymph nodes and has just gotten started in a new organ location.
  • Cancers that may be treated by the methods of the present invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • alveolar rhabdomyosarcoma stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;
  • hemangioendothelioma malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma;
  • chondroblastoma malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma;
  • ameloblastoma malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma;
  • lymphoid leukemia plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
  • the present invention is also intended for use in treating animal diseases in the veterinary medicine context.
  • veterinary herpes virus infections including equine herpes viruses, bovine viruses such as bovine viral diarrhea virus (for example, the E2 antigen), bovine herpes viruses, Marek's disease virus in chickens and other fowl; animal retroviral and lentiviral diseases (e.g., feline leukemia, feline immunodeficiency, simian
  • TAA tumor-associated or tumor-specific antigen (or tumor cell derived epitope)
  • TAA tumor cell derived epitope
  • TAA tumor cell derived epitope
  • TAA tumor cell derived epitope
  • mutant p53, HER2/neu or a peptide thereof or any of a number of melanoma-associated antigens such as MAGE-1, MAGE-3, MART- 1 /Melan- A, tyrosinase, gp75, gplOO, BAGE, GAGE-1, GAGE-2, GnT-V, and pi 5 (see, for example, US Pat. 6, 187,306, incorporated herein by reference).
  • nucleic acid vaccine it is not necessary to include a full length antigen in a nucleic acid vaccine; it suffices to include a fragment that will be presented by MHC class I and/or II.
  • a nucleic acid may include 1, 2, 3, 4, 5 or more antigens, which may be the same or different ones.
  • Mutants of the antigens described here may be created, for example, using the
  • antigens that may be used herein may be proteins or peptides that differ from the naturally- occurring proteins or peptides but yet retain the necessary epitopes for functional activity.
  • an antigen may comprise, consist essentially of, or consist of an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
  • an antigen may also comprise, consist essentially of, or consist of an amino acid sequence that is encoded by a nucleotide sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%), 97%), 98%), or 99% identical to a nucleotide sequence encoding the naturally- occurring antigen or a fragment thereof.
  • an antigen may also comprise, consist essentially of, or consist of an amino acid sequence that is encoded by a nucleic acid that hybridizes under high stringency conditions to a nucleic acid encoding the naturally-occurring antigen or a fragment thereof. Hybridization conditions are further described herein.
  • an exemplary protein may comprise, consist essentially of, or consist of, an amino acid sequence that is at least about 50%, 55%, 60%>, 65%>, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of a viral protein, including for example E6 or E7, such as an E6 or E7 sequence provided herein.
  • the amino acid sequence of the protein may comprise, consist essentially of, or consist of an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of an E6 or E7 protein, wherein the amino acids that render the protein a "detox" protein are present.
  • nucleic acid e.g., DNA
  • IPP Immunogenicity- Potentiating Polypeptide
  • a nucleic acid vaccine encodes a fusion protein comprising an antigen and a second protein, e.g., an IPP.
  • An IPP may act in potentiating an immune response by promoting: processing of the linked antigenic polypeptide via the MHC class I pathway or targeting of a cellular compartment that increases the processing.
  • This basic strategy may be combined with an additional strategy pioneered by the present inventors and colleagues, that involve linking DNA encoding another protein, generically termed a "targeting polypeptide," to the antigen-encoding DNA.
  • the DNA encoding such a targeting polypeptide will be referred to herein as a "targeting DNA.” That strategy has been shown to be effective in enhancing the potency of the vectors carrying only antigen-encoding DNA. See for example, the following PCT publications by Wu et al: WO 01/29233; WO 02/009645; WO 02/061113; WO 02/074920; and WO 02/12281, all of which are incorporated by reference in their entirety.
  • the other strategies include the use of DNA encoding polypeptides that promote or enhance: (a) development, accumulation or activity of antigen presenting cells or targeting of antigen to compartments of the antigen presenting cells leading to enhanced antigen presentation;
  • the strategy includes use of:
  • a viral intercellular spreading protein selected from the group of herpes simplex virus- 1 VP22 protein, Marek's disease virus UL49 (see WO 02/09645 and US Patent No. 7,318,928), protein or a functional homologue or derivative thereof;
  • CRT-like molecules ER60 selected from the group of CRT-like molecules ER60, GRP94, gp96, or a functional homologue or derivative thereof (see WO 02/12281 and US Patent No. 7,3442,002);
  • cytoplasmic translocation polypeptide domains of a pathogen toxin selected from the group of domain II of Pseudomonas exotoxin ETA or a functional homologue or derivative thereof (see published US application 20040086845);
  • a polypeptide that stimulates dendritic cell precursors or activates dendritic cell activity selected from the group of GM-CSF, Flt3-ligand extracellular domain, or a functional homologue or derivative thereof;
  • a costimulatory signal such as a B7 family protein, including B7-DC (see U.S.
  • an anti-apoptotic polypeptide selected from the group consisting of (1) BCL-xL, (2) BCL2, (3) XIAP, (4) FLICEc-s, (5) dominant-negative caspase-8, (6) dominant negative caspase-9, (7) SPI-6, and (8) a functional homologue or derivative of any of (l)-(7). (See WO 2005/047501).
  • An antigen may be linked N-terminally or C-terminally to an IPP.
  • IPPs and fusion constructs encoding such are described below.
  • LAMP-1 Lysosomal Associated Membrane Protein 1
  • the nucleotide sequence of the immunogenic vector pcDNA3-Sig/E7/LAMP-l is shown herein as SEQ ID NO: 13, with the SigE7-LAMP-l coding sequence in lower case and underscored.
  • HSP70 The nucleotide sequence encoding HSP70 is shown herein as SEQ ID NO: 13) (i.e., nucleotides 10633-12510 of the M. tuberculosis genome in GenBank NC 000962).
  • SEQ ID NO: 14 The amino acid sequence of HSP70 is shown herein as SEQ ID NO: 14.
  • the nucleic acid sequences encoding the E7-Hsp70 chimera/fusion polypeptides are shown herein as SEQ ID NO: 15 and the corresponding amino acid sequence is shown herein as SEQ ID NO: 16.
  • the E7 coding sequence is shown in upper case and
  • ETA Pseudomonas aeruginosa exotoxin type A
  • SEQ ID NO: 17 GenBank Accession No. K01397
  • amino acid sequence of ETA is shown herein as SEQ ID NO: 18 (GenBank Accession No. K01397).
  • Residues 1-25 represent the signal peptide.
  • the first residue of the mature polypeptide, Ala is bolded/underscored.
  • the mature polypeptide is residues 26-638 of SEQ ID NO: 18.
  • SEQ ID NO: 19 The nucleotide construct in which ETA(dll) is fused to HPV-16 E7 is shown herein as SEQ ID NO:20.
  • the corresponding amino acid sequence is shown herein as SEQ ID NO:21.
  • the ETA(dll) sequence appears in plain font, extra codons from plasmid pcDNA3 are italicized. Nucleotides between ETA(dll) and E7 are also bolded (and result in the interposition of two amino acids between ETA(dll) and E7).
  • the E7 amino acid sequence is underscored (ends with Gin at position 269).
  • the nucleotide sequence of the pcDNA3 vector encoding E7 and HSP70 (pcDNA3- E7-Hsp70 is shown herein as SEQ ID NO: 22.
  • Calreticulin a well-characterized -46 kDa protein was described briefly above, as were a number of its biological and biochemical activities.
  • CRT Calreticulin
  • CRT refers to polypeptides and nucleic acids molecules having substantial identity to the exemplary human CRT sequences as described herein or homologues thereof, such as rabbit and rat CRT - well-known in the art.
  • a CRT polypeptide is a polypeptide comprising a sequence identical to or substantially identical to the amino acid sequence of CRT.
  • An exemplary nucleotide and amino acid sequence for a CRT used in the present compositions and methods are presented below.
  • calreticulin or “CRT” encompass native proteins as well as recombinantly produced modified proteins that, when fused with an antigen (at the DNA or protein level) promote the induction of immune responses and promote angiogenesis, including a CTL response.
  • calreticulin or “CRT” encompass homologues and allelic variants of human CRT, including variants of native proteins constructed by in vitro techniques, and proteins isolated from natural sources.
  • the CRT polypeptides used in the present invention, and sequences encoding them also include fusion proteins comprising non-CRT sequences, particularly MHC class I-binding peptides; and also further comprising other domains, e.g., epitope tags, enzyme cleavage recognition sequences, signal sequences, secretion signals and the like.
  • a human CRT coding sequence is shown herein as SEQ ID NO: 23.
  • the amino acid sequence of the human CRT protein encoded by SEQ ID NO:23 is set forth herein as SEQ ID NO:24. This amino acid sequence is highly homologous to GenBank Accession No. NM 004343.
  • the amino acid sequence of the rabbit and rat CRT proteins are set forth in
  • human CRT may be used as well as, DNA encoding any homologue of CRT from any species that has the requisite biological activity (as an IPP) or any active domain or fragment thereof, may be used in place of human CRT or a domain thereof.
  • nucleic acid ⁇ e.g., DNA
  • E7 elicited potent antigen-specific CD8+ T cell responses and antitumor immunity in mice vaccinated i.d., by gene gun administration.
  • N-CRT/E7, P- CRT/E7 or C-CRT/E7 DNA each exhibited significantly increased numbers of E7-specific CD8 + T cell precursors and impressive antitumor effects against E7-expressing tumors when compared with mice vaccinated with E7 DNA (antigen only).
  • N-CRT DNA administration also resulted in anti -angiogenic antitumor effects.
  • cancer therapy using DNA encoding N-CRT linked to a tumor antigen may be used for treating tumors through a combination of antigen-specific immunotherapy and inhibition of angiogenesis.
  • the amino acid sequences of the 3 human CRT domains are shown herein as annotations of the full length protein, SEQ ID NO:24.
  • the N domain comprises residues 1- 170 (normal text); the P domain comprises residues 171-269 (underscored); and the C domain comprises residues 270-417 (bold/italic).
  • the sequences of the three domains are further shown as separate polypeptides herein as human N-CRT (SEQ ID NO:25), as human P-CRT (SEQ ID NO:26), and as human C-CRT (SEQ ID NO:27).
  • the present vectors may comprises DNA encoding one or more of these domain sequences, which are shown by annotation of SEQ ID NO:28 herein, wherein the N-domain sequence is upper case, the P-domain sequence is lower case/italic/underscored, and the C domain sequence is lower case.
  • the stop codon is also shown but not counted.
  • the coding sequence for each separate domain is provided herein as human N-CRT DNA (SEQ ID NO:29), as human P-CRT DNA (SEQ ID NO:30), and as human C-CRT DNA (SEQ ID NO:31).
  • human N-CRT DNA SEQ ID NO:29
  • human P-CRT DNA SEQ ID NO:30
  • human C-CRT DNA SEQ ID NO:31
  • any nucleotide sequences that encodes these domains may be used in the present constructs.
  • the sequences may be further codon-optimized.
  • Constructs used in the present invention may employ combinations of one or more
  • v4g can be any antigen, including E7(detox) or E6 (detox).
  • the present invention may employ shorter polypeptide fragments of CRT or CRT domains provided such fragments can enhance the immune response to an antigen with which they are paired. Shorter peptides from the CRT or domain sequences shown above that have the ability to promote protein processing via the MHC-1 class I pathway are also included, and may be defined by routine experimentation.
  • the present invention may also employ shorter nucleic acid fragments that encode CRT or CRT domains provided such fragments are functional, e.g. , encode polypeptides that can enhance the immune response to an antigen with which they are paired ⁇ e.g. , linked). Nucleic acids that encode shorter peptides from the CRT or domain sequences shown above and are functional, e.g. , have the ability to promote protein processing via the MHC-1 class I pathway, are also included, and may be defined by routine experimentation.
  • a polypeptide fragment of CRT may include at least or about 50, 100, 200, 300, or 400 amino acids.
  • a polypeptide fragment of CRT may also include at least or about 25, 50, 75, 100, 25-50, 50-100, or 75-125 amino acids from a CRT domain selected from the group N-CRT, P-CRT, and C-CRT.
  • a polypeptide fragment of CRT may include residues 1-50, 50-75, 75-100, 100-125, 125-150, 150-170 of the N-domain (e.g., of SEQ ID NO:25).
  • a polypeptide fragment of CRT may include residues 1-50, 50-75, 75-100, 100-109 of the P- domain (e.g., of SEQ ID NO:26).
  • a polypeptide fragment of CRT may include residues 1- 50, 50-75, 75-100, 100-125, 125-138 of the C-domain (e.g., of SEQ ID NO:27).
  • a nucleic acid fragment of CRT may encode at least or about 50, 100, 200, 300, or 400 amino acids.
  • a nucleic acid fragment of CRT may also encode at least or about 25, 50, 75, 100, 25-50, 50-100, or 75-125 amino acids from a CRT domain selected from the group N-CRT, P-CRT, and C-CRT.
  • a nucleic acid fragment of CRT may encode residues 1-50, 50-75, 75-100, 100-125, 125-150, 150-170 of the N-domain (e.g., of SEQ ID NO:25).
  • a nucleic acid fragment of CRT may encode residues 1-50, 50-75, 75-100, 100-109 of the P- domain (e.g., of SEQ ID NO:26).
  • a nucleic acid fragment of CRT may encode residues 1- 50, 50-75, 75-100, 100-125, 125-138 of the C-domain (e.g., of SEQ ID NO:27).
  • polypeptide "fragments" of CRT do not include full-length CRT.
  • nucleic acid “fragments” of CRT do not include a full- length CRT nucleic acid sequence and do not encode a full-length CRT polypeptide.
  • a vector construct of a complete chimeric nucleic acid that can be used in the present invention is shown herein as SEQ ID NO:32.
  • the sequence is annotated to show plasmid-derived nucleotides (lower case letters), CRT-derived nucleotides (upper case bold letters), and HPV-E7-derived nucleotides (upper case, italicized/underlined letters ).
  • Five plasmid nucleotides are found between the CRT and E7 coding sequences and that the stop codon for the E7 sequence is double underscored. This plasmid is also referred to as pNGVL4a-CRT/E7(detox).
  • the Table below describes the structure of the above plasmid. Plasmid Position Genetic Construct Source of Construct
  • an alternative to CRT is another ER chaperone polypeptide exemplified by ER60, GRP94 or gp96, well-characterized ER chaperone polypeptide that representatives of the HSP90 family of stress-induced proteins (see WO 02/012281, incorporated herein by reference).
  • polypeptide as used herein means any polypeptide having substantially the same ER chaperone function as the exemplary chaperone proteins CRT, tapasin, ER60 or calnexin. Thus, the term includes all functional fragments or variants or mimics thereof.
  • a polypeptide or peptide can be routinely screened for its activity as an ER chaperone using assays known in the art. While the present invention is not limited by any particular mechanism of action, in vivo chaperones promote the correct folding and oligomerization of many glycoproteins in the ER, including the assembly of the MHC class I heterotrimeric molecule (heavy (H) chain, ⁇ 2 ⁇ , and peptide). They also retain incompletely assembled MHC class I heterotrimeric complexes in the ER (Hauri FEBS Lett. 476:32-37, 2000).
  • the potency of naked nucleic acid ⁇ e.g., DNA) vaccines may be enhanced by their ability to amplify and spread in vivo.
  • VP22 a herpes simplex virus type 1 (HSV-1) protein and its "homologues" in other herpes viruses, such as the avian Marek's Disease Virus (MDV) have the property of intercellular transport that provide an approach for enhancing vaccine potency.
  • MDV avian Marek's Disease Virus
  • the present inventors have previously created novel fusions of VP22 with a model antigen, human papillomavirus type 16 (HPV-16) E7, in a nucleic acid (e.g., DNA) vaccine which generated enhanced spreading and MHC class I presentation of antigen.
  • HPV-16 human papillomavirus type 16
  • the spreading protein may be a viral spreading protein, including a herpes virus VP22 protein.
  • a herpes virus VP22 protein Exemplified herein are fusion constructs that comprise herpes simplex virus-1 (HSV-1) VP22 (abbreviated HVP22) and its homologue from Marek's disease virus (MDV) termed MDV-VP22 or MVP-22.
  • HVP-1 herpes simplex virus-1
  • MDV Marek's disease virus
  • MVP-22 fusion constructs that comprise herpes simplex virus-1 (HSV-1) VP22
  • MVP-22 Marek's disease virus
  • homologues of VP22 from other members of the herpesviridae or polypeptides from nonviral sources that are considered to be homologous and share the functional characteristic of promoting intercellular spreading of a polypeptide or peptide that is fused or chemically conjugated thereto.
  • DNA encoding HVP22 has the sequence SEQ ID NO:33 of the longer sequence SEQ ID NO:34 (which is the full length nucleotide sequence of a vector that comprises HVP22).
  • DNA encoding MDV-VP22 is shown herein as SEQ ID NO:35.
  • amino acid sequence of HVP22 polypeptide is SEQ ID NO:36 as amino acid residues 1-301 of SEQ ID NO:37 (i.e., the full length amino acid encoded by the vector).
  • amino acid sequence of the MDV-VP22 is shown herein as SEQ ID NO:38.
  • a DNA clone pcDNA3 VP22/E7, that includes the coding sequence for HVP22 and the HPV-16 protein, E7 (plus some additional vector sequence) is SEQ ID NO:34.
  • the amino acid sequence of E7 (SEQ ID NO:39) is residues 308-403 of SEQ ID NO:37.
  • This particular clone has only 96 of the 98 residues present in E7.
  • the C-terminal residues of wild-type E7, Lys and Pro are absent from this construct.
  • Such deletion variants (e.g., terminal truncation of two or a small number of amino acids) of other antigenic polypeptides are examples of the embodiments intended within the scope of the fusion polypeptides that can be used in the present invention.
  • Homologues or variants of IPPs described herein may also be used, provided that they have the requisite biological activity. These include various substitutions, deletions, or additions of the amino acid or nucleic acid sequences. Due to code degeneracy, for example, there may be considerable variation in nucleotide sequences encoding the same amino acid sequence.
  • a functional derivative of an IPP retains measurable IPP-like activity, including that of promoting immunogenicity of one or more antigenic epitopes fused thereto by promoting presentation by class I pathways.
  • “Functional derivatives” encompass “variants” and “fragments” regardless of whether the terms are used in the conjunctive or the alternative herein.
  • compositions useful for the present invention is an isolated or recombinant nucleic acid molecule encoding a fusion protein comprising at least two domains, wherein the first domain comprises an IPP and the second domain comprises an antigenic epitope, e.g., an MHC class I-binding peptide epitope.
  • the "fusion” can be an association generated by a peptide bond, a chemical linking, a charge interaction (e.g., electrostatic attractions, such as salt bridges, H-bonding, etc.) or the like. If the polypeptides are recombinant, the "fusion protein" can be translated from a common mRNA. Alternatively, the compositions of the domains can be linked by any chemical or electrostatic means.
  • the chimeric molecules that can be used in the present invention e.g., targeting polypeptide fusion proteins
  • a peptide can be linked to a carrier simply to facilitate manipulation or identification/ location of the peptide.
  • a “functional derivative” of an IPP which refers to an amino acid substitution variant, a "fragment” of the protein.
  • a functional derivative of an IPP retains measurable activity that may be manifested as promoting immunogenicity of one or more antigenic epitopes fused thereto or co-administered therewith.
  • “Functional derivatives” encompass “variants” and “fragments” regardless of whether the terms are used in the conjunctive or the alternative herein.
  • a functional homologue must possess the above biochemical and biological activity.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the method of alignment includes alignment of Cys residues.
  • the length of a sequence being compared is at least 30%, at least 40%, at least 50%, at least 60%, and at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the length of the reference sequence (e.g., an IPP).
  • the amino acid residues (or nucleotides) at corresponding amino acid (or nucleotide) positions are then compared. When a position in the first sequence is occupied by the same amino acid residue (or nucleotide) as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. ⁇ 5:444-453 (1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases, for example, to identify other family members or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the respective programs ⁇ e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
  • a homologue of an IPP or of an IPP domain described above is characterized as having (a) functional activity of native IPP or domain thereof and (b) amino acid sequence similarity to a native IPP protein or domain thereof when determined as above, of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
  • the fusion protein's biochemical and biological activity can be tested readily using art-recognized methods such as those described herein, for example, a T cell proliferation, cytokine secretion or a cytolytic assay, or an in vivo assay of tumor protection or tumor therapy.
  • a biological assay of the stimulation of antigen-specific T cell reactivity will indicate whether the homologue has the requisite activity to qualify as a "functional" homologue.
  • a “variant” refers to a molecule substantially identical to either the full protein or to a fragment thereof in which one or more amino acid residues have been replaced
  • substitution variant or which has one or several residues deleted (deletion variant) or added (addition variant).
  • substitution variant or which has one or several residues deleted (deletion variant) or added (addition variant).
  • fragment of an IPP refers to any subset of the molecule, that is, a shorter polypeptide of the full-length protein.
  • a number of processes can be used to generate fragments, mutants and variants of the isolated DNA sequence.
  • Small subregions or fragments of the nucleic acid encoding the spreading protein for example 1-30 bases in length, can be prepared by standard, chemical synthesis.
  • Antisense oligonucleotides and primers for use in the generation of larger synthetic fragment.
  • a one group of variants are those in which at least one amino acid residue and in certain embodiments only one, has been substituted by different residue.
  • the types of substitutions that may be made in the protein molecule may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al. (supra) and Figure 3-9 of Creighton ⁇ supra). Based on such an analysis, conservative substitutions are defined herein as exchanges within one of the following five groups:
  • substitutions are (i) substitution of Gly and/or Pro by another amino acid or deletion or insertion of Gly or Pro; (ii) substitution of a hydrophilic residue, e.g., Ser or Thr, for (or by) a hydrophobic residue, e.g., Leu, He, Phe, Val or Ala; (iii) substitution of a Cys residue for (or by) any other residue; (iv) substitution of a residue having an electropositive side chain, e.g., Lys, Arg or His, for (or by) a residue having an electronegative charge, e.g.,, Glu or Asp; or (v) substitution of a residue having a bulky side chain, e.g., Phe, for (or by) a residue not having such a side chain, e.g., Gly.
  • a hydrophilic residue e.g., Ser or Thr
  • a hydrophobic residue e.g., Leu, He, Phe, Val
  • deletions, insertions and substitutions according to the present invention are those that do not produce radical changes in the characteristics of the wild- type or native protein in terms of its relevant biological activity, e.g., its ability to stimulate antigen specific T cell reactivity to an antigenic epitope or epitopes that are fused to the protein.
  • its relevant biological activity e.g., its ability to stimulate antigen specific T cell reactivity to an antigenic epitope or epitopes that are fused to the protein.
  • the effect can be evaluated by routine screening assays such as those described here, without requiring undue experimentation.
  • fusion proteins comprise an IPP protein or homolog thereof and an antigen.
  • a fusion protein may comprise, consist essentially of, or consist of an IPP or an IPP fragment, e.g., N-CRT, P-CRT and/or C-CRT, or an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the IPP or IPP fragment, wherein the IPP fragment is functionally active as further described herein, linked to an antigen.
  • a fusion protein may also comprise an IPP or an IPP fragment and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids, or about 1-5, 1-10, 1-15, 1-20, 1-25, 1- 30, 1-50 amino acids, at the N- and/or C-terminus of the IPP fragment.
  • additional amino acids may have an amino acid sequence that is unrelated to the amino acid sequence at the corresponding position in the IPP protein.
  • Homologs of an IPP or an IPP fragments may also comprise, consist essentially of, or consist of an amino acid sequence that differs from that of an IPP or IPP fragment by the addition, deletion, or substitution, e.g., conservative substitution, of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or from about 1-5, 1-10, 1-15 or 1-20 amino acids.
  • Homologs of an IPP or IPP fragments may be encoded by nucleotide sequences that are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence encoding an IPP or IPP fragment, such as those described herein.
  • homologs of an IPP or IPP fragments are encoded by nucleic acids that hybridize under stringent hybridization conditions to a nucleic acid that encodes an IPP or IPP fragment.
  • homologs may be encoded by nucleic acids that hybridize under high stringency conditions of 0.2 to 1 x SSC at 65 °C followed by a wash at 0.2 x SSC at 65 °C to a nucleic acid consisting of a sequence described herein.
  • Nucleic acids that hybridize under low stringency conditions of 6 x SSC at room temperature followed by a wash at 2 x SSC at room temperature to nucleic acid consisting of a sequence described herein or a portion thereof can be used.
  • Other hybridization conditions include 3 x SSC at 40 or 50 °C, followed by a wash in 1 or 2 x SSC at 20, 30, 40, 50, 60, or 65 °C.
  • Hybridizations can be conducted in the presence of formaldehyde, e.g., 10%, 20%, 30% 40%) or 50%), which further increases the stringency of hybridization.
  • formaldehyde e.g. 10%, 20%, 30% 40%
  • 50% e.g., 10%, 20%, 30% 40%
  • Theory and practice of nucleic acid hybridization is described, e.g., in S. Agrawal (ed.) Methods in Molecular Biology, volume 20; and Tijssen (1993) Laboratory Techniques in biochemistry and molecular biology-hybridization with nucleic acid probes, e.g., part I chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York provide a basic guide to nucleic acid hybridization.
  • a fragment of a nucleic acid sequence is defined as a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the full length CRT polypeptide, antigenic polypeptide, or the fusion thereof.
  • This invention includes the use of such nucleic acid fragments that encode polypeptides which retain the ability of the fusion polypeptide to induce increases in frequency or reactivity of T cells, including CD8+ T cells, that are specific for the antigen part of the fusion polypeptide.
  • Nucleic acid sequences that can be used in the present invention may also include linker sequences, natural or modified restriction endonuclease sites and other sequences that are useful for manipulations related to cloning, expression or purification of encoded protein or fragments.
  • a fusion protein may comprise a linker between the antigen and the IPP protein.
  • Other nucleic acid vaccines that may be used include single chain trimers (SCT), as further described in the Examples and in references cited therein, all of which are specifically incorporated by reference herein. Backbone of nucleic acid vaccine
  • a nucleic acid e.g., DNA vaccine may comprise an "expression vector" or
  • expression cassette i.e., a nucleotide sequence which is capable of affecting expression of a protein coding sequence in a host compatible with such sequences.
  • Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be included, e.g., enhancers.
  • operably linked means that the coding sequence is linked to a regulatory sequence in a manner that allows expression of the coding sequence.
  • Known regulatory sequences are selected to direct expression of the desired protein in an appropriate host cell. Accordingly, the term “regulatory sequence” includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in, for example, Goeddel, Gene Expression Technology. Methods in Enzymology, vol. 185, Academic Press, San Diego, Calif. (1990)).
  • a promoter region of a DNA or RNA molecule binds RNA polymerase and promotes the transcription of an "operably linked" nucleic acid sequence.
  • a "promoter sequence” is the nucleotide sequence of the promoter which is found on that strand of the DNA or RNA which is transcribed by the RNA polymerase.
  • Two sequences of a nucleic acid molecule, such as a promoter and a coding sequence are "operably linked” when they are linked to each other in a manner which permits both sequences to be transcribed onto the same RNA transcript or permits an RNA transcript begun in one sequence to be extended into the second sequence.
  • two sequences such as a promoter sequence and a coding sequence of DNA or RNA are operably linked if transcription commencing in the promoter sequence will produce an RNA transcript of the operably linked coding sequence.
  • two sequences In order to be "operably linked" it is not necessary that two sequences be immediately adjacent to one another in the linear sequence.
  • promoter sequences useful for the present invention must be operable in mammalian cells and may be either eukaryotic or viral promoters. Certain promoters are also described in the Examples, and other useful promoters and regulatory elements are discussed below. Suitable promoters may be inducible, repressible or constitutive. A “constitutive” promoter is one which is active under most conditions encountered in the cell's environmental and throughout development. An “inducible” promoter is one which is under environmental or developmental regulation. A “tissue specific” promoter is active in certain tissue types of an organism.
  • a constitutive promoter is the viral promoter MSV-LTR, which is efficient and active in a variety of cell types, and, in contrast to most other promoters, has the same enhancing activity in arrested and growing cells.
  • Other viral promoters include that present in the CMV-LTR (from cytomegalovirus) (Bashart, M. et al, Cell ⁇ 7:521, 1985) or in the RSV-LTR (from Rous sarcoma virus) (Gorman, CM., Proc. Natl. Acad. Sci. USA 79:6777, 1982).
  • CMV-LTR from cytomegalovirus
  • RSV-LTR from Rous sarcoma virus
  • Also useful are the promoter of the mouse metallothionein I gene (Hamer, D, et al., J. Mol. Appl. Gen.
  • transcriptional factor association with promoter regions and the separate activation and DNA binding of transcription factors include: Keegan et al, Nature 237:699, 1986; Fields et al, Nature 340:245, 1989; Jones, Cell 61:9, 1990; Lewin, Cell (57: 1161, 1990; Ptashne et al, Nature 346:329, 1990; Adams et al, Cell 72:306, 1993.
  • the promoter region may further include an octamer region which may also function as a tissue specific enhancer, by interacting with certain proteins found in the specific tissue.
  • the enhancer domain of the DNA construct useful for the present invention is one which is specific for the target cells to be transfected, or is highly activated by cellular factors of such target cells. Examples of vectors (plasmid or retrovirus) are disclosed, e.g., in Roy-Burman et al, U.S. Patent No. 5,112,767, incorporated by reference. For a general discussion of enhancers and their actions in transcription, see, Lewin, BM, Genes IV, Oxford University Press pp. 552-576, 1990 (or later edition).
  • retroviral enhancers e.g., viral LTR
  • the endogenous viral LTR may be rendered enhancer-less and substituted with other desired enhancer sequences which confer tissue specificity or other desirable properties such as transcriptional efficiency.
  • expression cassettes include plasmids, recombinant viruses, any form of a recombinant "naked DNA" vector, and the like.
  • a "vector” comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid.
  • the vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.).
  • Vectors include replicons (e.g., RNA replicons), bacteriophages) to which fragments of DNA may be attached and become replicated.
  • Vectors thus include, but are not limited to RNA, autonomous self- replicating circular or linear DNA or RNA, e.g., plasmids, viruses, and the like (U.S. Patent No. 5,217,879, incorporated by reference), and includes both the expression and
  • nonexpression plasmids where a recombinant cell or culture is described as hosting an "expression vector" this includes both extrachromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s).
  • the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.
  • virus vectors that may be used include recombinant adenoviruses (Horowitz, MS, In: Virology, Fields, BN et al., eds, Raven Press, NY, 1990, p. 1679;
  • Adeno-associated virus is also useful for human therapy (Samulski, RJ et al, EMBO J. 70:3941, 1991) according to the present invention.
  • a nucleic acid (e.g., DNA) vaccine may also use a replicon, e.g., an RNA replicon, a self-replicating RNA vector.
  • a replicon is one based on a Sindbis virus RNA replicon, e.g., SINrep5.
  • the present inventors tested E7 in the context of such a vaccine and showed (see Wu et al, U.S. Patent Application 10/343,719) that a Sindbis virus RNA vaccine encoding HSV-1 VP22 linked to E7 significantly increased activation of E7- specific CD8 T cells, resulting in potent antitumor immunity against E7-expressing tumors.
  • RNA replicon vaccines may be derived from alphavirus vectors, such as Sindbis virus (Hariharan, M J et a/., 1998. J Virol 72:950-8.), Semliki Forest virus
  • RNA may be administered as either (1) RNA or (2) DNA which is then transcribed into RNA replicons in cells transfected in vitro or in vivo (Berglund, P C et a/., 1998.
  • Semliki Forest virus is pSCAl (DiCiommo, D P et a/., J Biol Chem 1998; 273 : 18060-6).
  • the plasmid vector pcDNA3 or a functional homolog thereof may be used in a nucleic acid (e.g., DNA) vaccine.
  • pNGVL4a SEQ ID NO:41
  • pNGVL4a SEQ ID NO:41
  • pNGVL4a one plasmid backbone for use in the present invention, was originally derived from the pNGVL3 vector, which has been approved for human vaccine trials.
  • the pNGVL4a vector includes two immunostimulatory sequences (tandem repeats of CpG dinucleotides) in the noncoding region.
  • pNGFVLA4a may be used because of the fact that it has already been approved for human therapeutic use.
  • Hepatitis B Virus Molecular Hepatitis C Viruses, by Hagedorn, CH et a/., eds., Springer Verlag, 199 Hepatitis B Virus: Molecular
  • Plasmid DNA used for transfection or microinjection may be prepared using methods well-known in the art, for example using the Qiagen procedure (Qiagen), followed by DNA purification using known methods, such as the methods exemplified herein.
  • Such expression vectors may be used to transfect host cells ⁇ in vitro, ex vivo or in vivo) for expression of the DNA and production of the encoded proteins which include fusion proteins or peptides.
  • a nucleic acid ⁇ e.g., DNA) vaccine is administered to or contacted with a cell, e.g., a cell obtained from a subject ⁇ e.g., an antigen presenting cell), and administered to a subject, wherein the subject is treated before, after or at the same time as the cells are administered to the subject.
  • isolated when referring to a molecule or composition, such as a translocation polypeptide or a nucleic acid coding therefor, means that the molecule or composition is separated from at least one other compound (protein, other nucleic acid, etc.) or from other contaminants with which it is natively associated or becomes associated during processing.
  • An isolated composition can also be substantially pure.
  • An isolated composition can be in a homogeneous state and can be dry or in aqueous solution. Purity and homogeneity can be determined, for example, using analytical chemical techniques such as polyacrylamide gel electrophoresis (PAGE) or high performance liquid chromatography (HPLC). Even where a protein has been isolated so as to appear as a homogenous or dominant band in a gel pattern, there are trace contaminants which co-purify with it.
  • PAGE polyacrylamide gel electrophoresis
  • HPLC high performance liquid chromatography
  • Host cells transformed or transfected to express the fusion polypeptide or a homologue or functional derivative thereof are useful for the present invention.
  • the fusion polypeptide may be expressed in yeast, or mammalian cells such as Chinese hamster ovary cells (CHO) or human cells.
  • cells for expression according to the present invention are APCs or DCs.
  • Other suitable host cells are known to those skilled in the art.
  • nucleic acids for potentiating immune responses
  • Methods of administrating a chemotherapeutic drug and a vaccine may further comprise administration of one or more other constructs, e.g., to prolong the life of antigen presenting cells.
  • exemplary constructs are described in the following two sections. Such constructs may be administered simultaneously or at the same time as a nucleic acid ⁇ e.g., DNA) vaccine. Alternatively, they may be administered before or after administration of the DNA vaccine or chemotherapeutic drug.
  • a method comprises further administering to a subject an siRNA directed at an apoptotic pathway, such as described in WO 2006/073970, which is incorporated herein in its entirety.
  • siRNA sequences that hybridize to, and block expression of the activation of Bak and Bax proteins that are central players in the apoptosis signaling pathway.
  • Methods of treating tumors or hyperproliferative diseases involving the administration of siRNA molecules (sequences), vectors containing or encoding the siRNA, expression vectors with a promoter operably linked to the siRNA coding sequence that drives transcription of siRNA sequences that are "specific" for sequences Bak and Bax nucleic acid are also encompassed within the present invention.
  • siRNAs may include single stranded "hairpin" sequences because of their stability and binding to the target mRNA.
  • the present siRNA sequences may be used in conjunction with a broad range of DNA vaccine constructs encoding antigens to enhance and promote the immune response induced by such DNA vaccine constructs, particularly CD8+ T cell mediated immune responses typified by CTL activation and action. This is believed to occur as a result of the effect of the siRNA in prolonging the life of antigen-presenting DCs which may otherwise be killed in the course of a developing immune response by the very same CTLs that the DCs are responsible for inducing.
  • siRNAs designed in an analogous manner include caspase 8, caspase 9 and caspase 3.
  • the present invention includes compositions and methods in which siRNAs targeting any two or more of Bak, Bax, caspase 8, caspase 9 and caspase 3 are used in combination, optionally simultaneously
  • siRNAs may also be used to transfect DCs (along with antigen loading) to improve the immunogenicity of the DCs as cellular vaccines by rendering them resistant to apoptosis.
  • RNA interference RNA interference
  • RNA interference is the sequence-specific degradation of homologues in an mRNA of a targeting sequence in an siNA.
  • siNA small, or short, interfering nucleic acid
  • siNA small, or short, interfering nucleic acid
  • RNA interference sequence specific RNAi
  • siRNA short (or small) interfering RNA
  • dsRNA double-stranded RNA
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • siRNA short interfering oligonucleotide
  • short interfering nucleic acid short interfering modified oligonucleotide
  • chemically-modified siRNA post-transcriptional gene silencing RNA (ptgsRNA), translational silencing, and others.
  • ptgsRNA post-transcriptional gene silencing RNA
  • RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi (Khvorova, A et al., Cell 115:209-216 (2003); Schwarz, DS et al. 115: 199-208 (2003)))
  • RNAi molecule Considerations to be taken into account when designing an RNAi molecule include, among others, the sequence to be targeted, secondary structure of the RNA target and binding of RNA binding proteins. Methods of optimizing siRNA sequences will be evident to the skilled worker. Typical algorithms and methods are described in Vickers et al.
  • Candidate siRNA sequences against mouse and human Bax and Bak are selected using a process that involves running a BLAST search against the sequence of Bax or Bak (or any other target) and selecting sequences that "survive" to ensure that these sequences will not be cross matched with any other genes.
  • siRNA sequences selected according to such a process and algorithm may be cloned into an expression plasmid and tested for their activity in abrogating Bak/Bax function cells of the appropriate animal species.
  • Those sequences that show RNAi activity may be used by direct administration bound to particles, or recloned into a viral vector such as a replication-defective human adenovirus serotype 5 (Ad5).
  • constructs include the following:
  • the nucleotide sequence encoding the Bak protein (including the stop codon) (GenBank accession No. NM_007523 is shown herein as SEQ ID NO:44 with the targeted sequence in upper case, underscored.
  • the targeted sequence of Bak is shown herein as SEQ ID NO:44 with the targeted sequence in upper case, underscored.
  • TGCCTACGAACTCTTCACC is shown herein as SEQ ID NO:45.
  • the targeted sequence of Bax, TATGGAGCTGCAGAGGATG is shown herein as SEQ ID NO:49
  • the inhibitory molecule is a double stranded nucleic acid (i.e., an RNA), used in a method of RNA interference.
  • an RNA double stranded nucleic acid
  • the following show the "paired" 19 nucleotide structures of the siRNA sequences shown above, where the symbol J
  • Caspase 8 The nucleotide sequence of human caspase-8 is shown herein as SEQ ID NO:50 (GenBank Access. # NM_001228). One target sequence for RNAi is underscored. Others may be identified using methods such as those described herein (and in reference cited herein, primarily Far et al, supra and Reynolds et al, supra).
  • sequences of sense and antisense siRNA strands for targeting this sequence including dTdT 3' overhangs are:
  • Caspase 9 The nucleotide sequence of human caspase-9 is shown herein as SEQ ID NO:53 (see GenBank Access. # NM_001229). The sequence below is of "variant a" which is longer than a second alternatively spliced variant ⁇ , which lacks the underscored part of the sequence shown below (and which is anti-apoptotic).
  • Target sequences for RNAi, expected to fall in the underscored segment are identified using known methods such as those described herein and in Far et al., supra and Reynolds et al, supra) and siNAs, such as siRNAs, are designed accordingly.
  • Caspase 3 The nucleotide sequence of human caspase-3 is shown herein as SEQ ID NO: 54 (see GenBank Access. # NM_004346). The sequence below is of "variant a" which is the longer of two alternatively spliced variants, all of which encode the full protein.
  • Target sequences for RNAi are identified using known methods such as those described herein and in Far et al, supra and Reynolds et al, supra) and siNAs, such as siRNAs, are designed accordingly.
  • RNAi Long double stranded interfering RNAs, such a miRNAs, appear to tolerate mismatches more readily than do short double stranded RNAs.
  • RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, or an epigenetic
  • siNA molecules useful for the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level.
  • epigenetic regulation of gene expression by siNA molecules useful for the present invention can result from siNA mediated modification of chromatin structure and thereby alter gene expression (see, for example, Allshire Science 297: 1818-19, 2002; Volpe et al., Science 297: 1833-37 , 2002; Jenuwein, Science 297:2215- 18, 2002; and Hall et al, Science 297, 2232-2237, 2002.)
  • An siNA can be designed to target any region of the coding or non-coding sequence of an mRNA.
  • An siNA is a double-stranded polynucleotide molecule comprising self- complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary.
  • the siNA can be assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid based or non- nucleic acid-based linker(s).
  • the siNA can be a polynucleotide with a hairpin secondary structure, having self-complementary sense and antisense regions.
  • the siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi.
  • the siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (or can be an siNA molecule that does not require the presence within the siNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded
  • polynucleotide can further comprise a terminal phosphate group, such as a 5 '-phosphate (see for example Martinez et al. (2002) Cell 110, 563-574 and Schwarz et al. (2002) Molecular Cell 10, 537-568), or 5 ',3 '-diphosphate.
  • a terminal phosphate group such as a 5 '-phosphate (see for example Martinez et al. (2002) Cell 110, 563-574 and Schwarz et al. (2002) Molecular Cell 10, 537-568), or 5 ',3 '-diphosphate.
  • the siNA molecule useful for the present invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, Van der Waal's interactions, hydrophobic interactions, and/or stacking interactions.
  • siNA molecules need not be limited to those molecules containing only ribonucleotides but may also further encompass deoxyribonucleotides (as in the siRNAs which each include a dTdT dinucleotide) chemically-modified nucleotides, and non-nucleotides.
  • the siNA molecules useful for the present invention lack 2'-hydroxy (2'-OH) containing nucleotides.
  • siNAs do not require the presence of nucleotides having a 2' -hydroxy group for mediating RNAi and as such, siNAs useful for the present invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2'-OH group).
  • siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups.
  • siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions.
  • siNAs useful for the present invention can also be referred to as "short interfering modified oligonucleotides” or "siMON.”
  • Other chemical modifications e.g., as described in Int'l Patent Publications WO 03/070918 and WO 03/074654, both of which are incorporated by reference, can be applied to any siNA sequence useful for the present invention.
  • a molecule mediating RNAi has a 2 nucleotide 3' overhang (dTdT in the sequences disclosed herein). If the RNAi molecule is expressed in a cell from a construct, for example from a hairpin molecule or from an inverted repeat of the desired sequence, then the endogenous cellular machinery will create the overhangs.
  • Methods of making siRNAs are conventional. In vitro methods include processing the polyribonucleotide sequence in a cell-free system ⁇ e.g., digesting long dsRNAs with RNAse III or Dicer), transcribing recombinant double stranded DNA in vitro, and chemical synthesis of nucleotide sequences homologous to Bak or Bax sequences. See, e.g., Tuschl et al, Genes & Dev. 73:3191-3197, 1999.
  • In vivo methods include
  • RNA polymerase III RNA polymerase III promoters. See, for example, Kawasaki et al, supra; Miyagishi et al, supra; Lee et al, supra; Brummelkamp et al, supra; McManus et al, supra), Paddison et al, supra (both); Paul et al, supra, Sui et al, supra; and Yu et al, supra; and/or
  • RNA synthesis When synthesized in vitro, a typical micromolar scale RNA synthesis provides about 1 mg of siRNA, which is sufficient for about 1000 transfection experiments using a 24-well tissue culture plate format.
  • one or more siRNAs can be added to cells in culture media, typically at about 1 ng/ml to about 10 ⁇ g siRNA/ml.
  • Ribozymes and siNAs can take any of the forms, including modified versions, described for antisense nucleic acid molecules; and they can be introduced into cells as oligonucleotides (single or double stranded), or in the form of an expression vector.
  • an antisense nucleic acid, siNA ⁇ e.g., siRNA) or ribozyme comprises a single stranded polynucleotide comprising a sequence that is at least about 90% ⁇ e.g., at least about 93%, 95%, 97%, 98% or 99%) identical to a target segment (such as those indicted for Bak and Bax above) or a complement thereof.
  • a DNA and an RNA encoded by it are said to contain the same "sequence,” taking into account that the thymine bases in DNA are replaced by uracil bases in RNA.
  • Active variants ⁇ e.g., length variants, including fragments; and sequence variants) of the nucleic acid-based inhibitors discussed herein are also within the scope of the present invention.
  • An "active" variant is one that retains an activity of the inhibitor from which it is derived (i.e., the ability to inhibit expression). It is to test a variant to determine for its activity using conventional procedures.
  • an antisense nucleic acid or siRNA may be of any length that is effective for inhibition of a gene of interest.
  • an antisense nucleic acid is between about 6 and about 50 nucleotides ⁇ e.g., at least about 12, 15, 20, 25, 30, 35, 40, 45 or 50 nt), and may be as long as about 100 to about 200 nucleotides or more.
  • Antisense nucleic acids having about the same length as the gene or coding sequence to be inhibited may be used.
  • bases and base pairs (bp) are used interchangeably, and will be understood to correspond to single stranded (ss) and double stranded (ds) nucleic acids.
  • the length of an effective siNA is generally between about 15 bp and about 29 bp in length, between about 19 and about 29 bp ⁇ e.g., about 15, 17, 19, 21, 23, 25, 27 or 29 bp), with shorter and longer sequences being acceptable. Generally, siNAs are shorter than about 30 bases to prevent eliciting interferon effects.
  • an active variant of an siRNA having, for one of its strands, the 19 nucleotide sequence of any of SEQ ID NOs:42, 43, 46, and 47 herein can lack base pairs from either, or both, of ends of the dsRNA; or can comprise additional base pairs at either, or both, ends of the ds RNA, provided that the total of length of the siRNA is between about 19 and about 29 bp, inclusive.
  • One embodiment useful for the present invention is an siRNA that "consists essentially of sequences represented by SEQ ID NOs:42, 43, 46, and 47 or complements of these sequence.
  • An siRNA useful for the present invention may consist essentially of between about 19 and about 29 bp in length.
  • an inhibitory nucleic acid whether an antisense molecule, a ribozyme (the recognition sequences), or an siNA, comprises a strand that is complementary (100% identical in sequence) to a sequence of a gene that it is designed to inhibit.
  • 100% sequence identity is not required to practice the present invention.
  • the invention has the advantage of being able to tolerate naturally occurring sequence variations, for example, in human c-met, that might be expected due to genetic mutation, polymorphism, or evolutionary divergence.
  • the variant sequences may be artificially generated. Nucleic acid sequences with small insertions, deletions, or single point mutations relative to the target sequence can be effective inhibitors.
  • sequence identity may be optimized by sequence comparison and alignment algorithms well-known in the art (see Gribskov and Devereux, Sequence
  • sequence identity may be used (e.g., at least about 92%, 95%, 98% or 99%), or even 100%) sequence identity, between the inhibitory nucleic acid and the targeted sequence of targeted gene.
  • an active variant of an inhibitory nucleic acid useful for the present invention is one that hybridizes to the sequence it is intended to inhibit under conditions of high stringency.
  • the duplex region of an siRNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript under high stringency conditions (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C, hybridization for 12-16 hours), followed generally by washing.
  • DC-1 cells or BM-DCs presenting a given antigen X when not treated with the siRNAs useful for the present invention, respond to sufficient numbers X-specific CD8+ CTL by apoptotic cell death.
  • the same cells transfected with the siRNA or infected with a viral vector encoding the present siRNA sequences survive better despite the delivery of killing signals.
  • siRNA compositions useful for the present invention inhibit the death of DCs in vivo in the process of a developing T cell response, and thereby promote and stimulate the generation of an immune response induced by immunization with an antigen-encoding DNA vaccine vector.
  • these DCs When administered to subjects, these DCs generate stronger antigen-specific immune responses and manifest therapeutic effects (compared to DCs transfected with control siRNA).
  • siRNA constructs are useful as a part of the nucleic acid vaccination and
  • Administration to a subject of a DNA vaccine and a chemotherapeutic drug may also be accompanied by administration of a nucleic acid encoding an anti-apoptotic protein, as described in WO2005/047501 and in U.S. Patent Application Publication No.
  • the present inventors have designed and disclosed an immunotherapeutic strategy that combines antigen-encoding DNA vaccine compositions with additional DNA vectors comprising anti-apoptotic genes including bcl-2, bc-lxL, XIAP, dominant negative mutants of caspase-8 and caspase-9, the products of which are known to inhibit apoptosis (Wu, et al. U.S. Patent Application Publication No. 20070026076, incorporated herein by reference).
  • Serine protease inhibitor 6 SPI-6 which inhibits granzyme B, may also be employed in compositions and methods to delay apoptotic cell death of DCs.
  • the present inventors have shown that the harnessing of an additional biological mechanism, that of inhibiting apoptosis, significantly enhances T cell responses to DNA vaccines comprising antigen- coding sequences, as well as linked sequences encoding such IPPs.
  • Intradermal vaccination by gene gun efficiently delivers a DNA vaccine into DCs of the skin, resulting in the activation and priming of antigen-specific T cells in vivo.
  • DCs have a limited life span, hindering their long-term ability to prime antigen- specific T cells.
  • a strategy that combines combination therapy with methods to prolong the survival of DNA-transduced DCs enhances priming of antigen-specific T cells and thereby, increase DNA vaccine potency.
  • Serine protease inhibitor 6 also called Serpinb9, inhibits granzyme B, and may thereby delay apoptotic cell death in DCs.
  • combined methods are used that enhance MHC class I and II antigen processing with delivery of SPI-6 to potentiate immunity.
  • DNA-based alphaviral RNA replicon vectors also called suicidal DNA vectors.
  • an antigen e.g., HPV E7, a DNA-based Semliki Forest virus vector, pSCAl
  • the antigen DNA is fused with DNA encoding an anti-apoptotic polypeptide such BCL-xL, a member of the BCL-2 family.
  • pSCAl encoding a fusion protein of an antigen polypeptide and/BCL-xL delays cell death in transfected DCs and generates significantly higher antigen-specific CD8+ T-cell- mediated immunity.
  • the antiapoptotic function of BCL-xL is important for the
  • delaying cell death induced by an otherwise desirable suicidal DNA vaccine enhances its potency.
  • the present invention is also directed to combination therapies including administering a chemotherapeutic drug with a nucleic acid composition useful as an immunogen, comprising a combination of: (a) first nucleic acid vector comprising a first sequence encoding an antigenic polypeptide or peptide, which first vector optionally comprises a second sequence linked to the first sequence, which second sequence encodes an immunogenicity-potentiating polypeptide (TPP); b) a second nucleic acid vector encoding an anti-apoptotic polypeptide, wherein, when the second vector is administered with the first vector to a subject, a T cell-mediated immune response to the antigenic polypeptide or peptide is induced that is greater in magnitude and/or duration than an immune response induced by administration of the first vector alone.
  • the first vector above may comprise a promoter operatively linked to the first and/or the second sequence.
  • the anti-apoptotic polypeptide may be selected from the group consisting of (a) BCL-xL, (b) BCL2, (c) XIAP, (d) FLICEc-s, (e) dominant-negative caspase-8, (f) dominant negative caspase-9, (g) SPI-6, and (h) a functional homologue or a derivative of any of (a)-(g).
  • the anti-apoptotic DNA may be physically linked to the antigen-encoding DNA. Examples of this are provided in U.S. Patent Application publication No. 20070026076, incorporated by reference, primarily in the form of suicidal DNA vaccine vectors.
  • the anti-apoptotic DNA may be administered separately from, but in combination with the antigen-encoding DNA molecule.
  • the antigen-encoding DNA molecule may be administered separately from, but in combination with the antigen-encoding DNA molecule.
  • nucleotide and amino acid sequences of anti-apoptotic and other proteins are provided in the sequence listing.
  • Biologically active homologs of these proteins and constructs may also be used.
  • Biologically active homologs is to be understood as described herein in the context of other proteins, e.g., IPPs.
  • the coding sequence for BCL-xL as present in the pcDNA3 vector useful for the present invention is SEQ ID NO:55; the amino acid sequence of BCL-xL is SEQ ID NO:56; the sequence pcDNA3-BCL-xL is SEQ ID NO:57 (the BCL-xL coding sequence corresponds to nucleotides 983 to 1732); a pcDNA3 vector combining E7 and BCL-xL, designated pcDNA3-E7/BCL-xL is SEQ ID NO:58 (the E7 and BCL-xL sequences correspond to nucleotides 960 to 2009); the amino acid sequence of the E7 -BCL-xL chimeric or fusion polypeptide is SEQ ID NO:59; a mutant BCL-xL ("mtBCL-xL”) DNA sequence is SEQ ID NO:60; the amino acid sequence of mtBCL-xL is SEQ ID NO:61; the amino acid sequence of the E7-mtBCL-
  • E7/mtBCL-XL [SEQ ID NO:64], this sequence is inserted in the same position as the BCL- xL sequence is in SEQ ID NO:58; the sequence of the suicidal DNA vector pSCAl-BCL- xL is SEQ ID NO:65 (the BCL-xL sequence corresponds to nucleotides 7483 to 8232); the sequence of the "combined" vector, pSCAl-E7/BCL-xL is SEQ ID NO:66 (the sequence of E7 and BCL-xL corresponds to nucleotides 7461 to 8510); the sequence of pSCAl-mtBCL- xL [SEQ ID NO:67] is the same as that for the wild type BCL-xL except that the mtBCL- xL sequence is inserted in the same position as the wild type sequence in the pSCAl- mtBCL-xL vector; the sequence pSCAl-E7/mtBCL-
  • GENEBANK as NM 009256 is SEQ ID NO:84; the amino acid sequence of the SPI-6 protein is SEQ ID NO:85; the nucleic acid sequence of the mutant SPI-6 (mtSPI6) is SEQ ID NO:86; the amino acid sequence of the mutant SPI-6 protein (mtSPI-6) is SEQ ID NO:87; the sequence of the pcDNA3-Spi6 vector is SEQ ID NO:88 (the SPI-6 sequence corresponds to nucleotides 960 to 2081); and the sequence of the mutant vector pcDNA3- mtSpi6 vector [SEQ ID NO: 89] is the same as that above, except that the mtSPI-6 sequence is inserted in the same location in place of the wild type SPI-6.
  • Bioly active homologs of these nucleic acids and proteins may be used.
  • Biologically active homologs are to be understood as described in the context of other proteins, e.g., IPPs, herein.
  • a vector may encode an anti-apoptotic protein that is at least about 90%, 95%, 98% or 99% identical to that of a sequence set forth herein.
  • MHC class I/II activators refers to molecules or complexes thereof that increase immune responses by increasing MHC class I or II (" ⁇ / ⁇ ") antigen presentation, such as by increasing MHC class I, class II or class I and class II activity or gene expression.
  • an MHC class I/II activator is a nucleic acid encoding a protein that enhances MHC class I/II antigen presentation.
  • Exemplary MHC class I/II activators include nucleic acids encoding an MHC class II associated invariant chain (Ii), in which the CLIP region is replaced with a T cell epitope, e.g., a promiscuous T cell epitope, such as the Pan HLA-DR reactive epitope (PADRE), or a variant thereof.
  • Other MHC class I/II activators are nucleic acids encoding the MHC class II transactivator CIITA or a variant thereof.
  • an MHC class I/II activator is a nucleic acid, e.g., an isolated nucleic acid, encoding a protein comprising, consisting or consisting essentially of an invariant (Ii) chain, wherein the CLIP region is replaced with a promiscuous CD4+ T cell epitope.
  • a "promiscuous CD4+ T cell epitope” is used interchangeably with “universal CD4+ T cell epitope” and refers to peptides that bind to numerous histocompatibility alleles, e.g., human MHC class II molecules.
  • the promiscuous CD4+ T cell epitope is a Pan HLA-DR reactive epitope (PADRE), thereby forming an Ii-PADRE protein that is encoded by an Ii-PADRE nucleic acid.
  • a nucleic acid encodes an Ii chain, wherein amino acids 81-102 (KPVSQMRMATPLLMRPM (SEQ ID NO:92) are replaced with the PADRE sequence AKFVAAWTLKAAA (SEQ ID NO:93).
  • An exemplary human Ii-PADRE amino acid sequence is set forth as SEQ ID NO:91, and is encoded by nucleotide sequence SEQ ID NO:90.
  • a protein may comprise, consist essentially of, or consist of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:91.
  • a protein may comprise a PADRE that is identical to the PADRE of SEQ ID NO:91, i.e., consisting of SEQ ID NO:93.
  • a protein may comprise a PADRE sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:93; and/or an Ii sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the Ii sequence of SEQ ID NO:91.
  • An amino acid sequence may differ from that of SEQ ID NO:91 or the Ii or PADRE sequences thereof by the addition, deletion or substitution of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more amino acids.
  • a protein lacks one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids at the C- and/or N-terminus and/or internal relative to that of SEQ ID NO:91 or the Ii or PADRE region thereof.
  • an amino acid sequence differs from that of SEQ ID NO:93 or from that of the Ii sequence by the addition, deletion or substitution of at least about 1, 2, 3, 4, or 5 amino acids.
  • Variants of SEQ ID NO:91 or the PADRE or Ii regions thereof preferably have a biological activity. Such variants are referred to as “functional homologs" or “functional variants.” Functional homologs include variants of SEQ ID NO:91 that increase an immune response, e.g., an antigen specific immune response, in a subject to whom it is administered, or has any of the biological activities set forth in the Examples pertaining to Ii-PADRE. Variants of the PADRE sequence or the Ii sequence may have a biological activity that is associated with that of the wild type PADRE or Ii sequences, respectively. Biological activities can be determined as know in the art or as set forth in the Examples. In addition, comparison (or alignment) of the Ii and PADRE sequences from different species is expected to be helpful in determining which amino acids may be varied and which ones should preferably not be varied.
  • proteins provided herein comprise a PADRE amino acid sequence that replaces a larger portion of Ii, e.g., wherein Ii is lacking about amino acids 81-103, 81-104, 81-105, 81-106, 81-107, 81-108, 81-109, 81-110 or more; is lacking about amino acids 70- 102, 71-102, 72-102, 73-102, 74-102, 75-102, 76-102, 77-102, 78-102, 79-102, 80-102 or more.
  • NY-ESO-1 119- 143 is a promiscuous major histocompatibility complex class II T-helper epitope recognized by Thl- and Th2-type tumor-reactive CD4+ T cells. Cancer Res 62:213-218. 12. Falugi, F., R. Petracca, M. Mariani, E. Luzzi, S. Mancianti, V. Carinci, M. L. Melli,
  • the CLIP region in an Ii molecule may be replaced with any of the peptides in Table 2 or other promiscuous epitopes set forth in the references of Table 2, or functional variants thereof.
  • Preferred epitopes include those from tetanus toxin and influenza. Any other promiscuous CD4+ T cell epitopes may be used, e.g., those described in the following references:
  • 143 is a promiscuous major histocompatibility complex class II T-helper epitope recognized by Thl- and Th2-type tumor-reactive CD4+ T cells. Cancer Res 62:213-218.
  • the CLIP region of Ii is replaced with a T cell epitope, e.g,. a CD4+ T cell epitope, such as a promiscuous CD4+ T cell epitope, with the proviso that the resulting construct is not one that has been publicly disclosed previously, e.g., one year prior to the filing of the priority application of the instant application.
  • a T cell epitope e.g. a CD4+ T cell epitope, such as a promiscuous CD4+ T cell epitope
  • the epitope that replaces the CLIP region is not a promiscuous CD4+ T cell epitope from an HCV antigen, Listeria LLO antigen, ovalbumin antigen, Japanese cedar pollen allergen, MuLV env/gp70-derived helper epitope, and Heat Shock Protein 60 (described in references 16-21 above), or epitopes replacing CLIP regions that are described in publications that are referenced to in the Examples.
  • a nucleic acid comprises, consists essentially of, or consists of the nucleotide sequence set forth in SEQ ID NO: 90, or comprises a nucleotide sequence sequence encoding the PADRE or Ii portion thereof.
  • a nucleic acid may also comprise a nucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 90 and/or to the PADRE and/or to the Ii portion thereof.
  • Nucleic acids may differ by the addition, deletion or substitution of one or more, e.g., 1, 3, 5, 10, 15, 20, 25, 30 or more nucleotides, which may be located at the 5' end, 3' end, and/or internally to the sequence.
  • a nucleic acid encodes a protein that is a functional homolog of an Ii -PADRE protein, with the proviso that the Ii sequence and/or PADRE sequence is (or are) not the wild-type or a naturally-occurring sequence, e.g., the wild-type or naturally-occurring human sequence.
  • an MHC class I/II activator is a protein that enhances MHC class II expression, e.g., an MHC class II transactivator (CUT A).
  • CUT A MHC class II transactivator
  • the nucleotide and amino acid sequences of human CIITA are set forth as GenBank Accession Nos. P33076, NM_000246.3 and NP_000237.2 and set forth as SEQ ID NOs:94 and 95, respectively (GenelD: 4261)).
  • Variants of the protein may also be used.
  • Exemplary variants comprise, consist essentially of, or consist of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 95.
  • An amino acid sequence may differ from that of SEQ ID NO:95 by the addition, deletion or substitution of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more amino acids.
  • a protein lacks one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids at the C- and/or N-terminus and/or internally relative to that of SEQ ID NO:95.
  • mino acid changes i.e., deletions, additions or substitutions
  • the locations at which mino acid changes may be made may be determined by comparing, i.e., aligning, the amino acid sequences of CIITA homologues, e.g., those from various animal species.
  • Exemplary amino acids that may be changed include S286, S288 and S293. Indeed, as described in Greer et al., mutation of these amino acids results in a stronger
  • a nucleic acid comprises, consists essentially of, or consists of the nucleotide sequence set forth in SEQ ID NO:94.
  • a nucleic acid may also comprise a nucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:94.
  • Nucleic acids may differ by the addition, deletion or substitution of one or more, e.g., 1, 3, 5, 10, 15, 20, 25, 30 or more nucleotides, which may be located at the 5' end, 3' end, and/or internally to the sequence.
  • a nucleic acid encodes a protein that is a functional homolog of a CIITA protein, with the proviso that the sequence is not the wild-type or a naturally-occurring sequence, e.g., the wild-type or naturally-occurring human sequence.
  • nucleic acids encoding MHC class I/II activators include those that hybridize, e.g., under stringent hybridization conditions to a nucleic acid encoding an MHC class I/II activator described herein, e.g., consisting of SEQ ID NO:90 or 94 or portions thereof. Hybridization conditions are further described herein.
  • Nucleic acids encoding an MHC class I/II activator may be included in plasmids or expression vectors, such as those further described herein in the context of DNA vaccines.
  • a nucleic acid encoding an Ii -PADRE protein or functional homolog thereof is administered to a subject who is also receiving a nucleic acid encoding a CIITA protein or functional homolog thereof.
  • the nucleic acids may be administered simultaneously or consecutively.
  • the nucleic acids may also be linked, i.e., forming one nucleic acid molecule.
  • nucleotide sequences encoding an Ii- PADRE protein or a functional variant thereof; one or more nucleotide sequences encoding an antigen or a fusion protein comprising an antigen; one or more nucleotide sequences encoding a CIITA protein of a functional variant thereof may be linked to each other, i.e., present on one nucleic acid molecule.
  • Drugs may also further be administered to a mammal in accordance with the methods and compositions taught herein.
  • any drug that reduces the growth of cells without significantly affecting the immune system may be used, or at least not suppressing the immune system to the extent of eliminating the positive effects of a DNA vaccine that is administered to the subject.
  • the drugs are
  • chemotherapeutic drugs may be used, provided that the drug stimulates the effect of a vaccine, e.g., DNA vaccine.
  • a chemotherapeutic drug may be a drug that (a) induces apoptosis of cells, in particular, cancer cells, when contacted therewith; (b) reduces tumor burden; and/or (c) enhances CD8+ T cell-mediated antitumor immunity.
  • the drug must also be one that does not inhibit the immune system, or at least not at certain concentrations.
  • the chemotherapeutic drug is epigallocatechin-3-gallate (EGCG) or a chemical derivative or pharmaceutically acceptable salt thereof.
  • EGCG epigallocatechin-3-gallate
  • Epigallocatechin gallate is the major polyphenol component found in green tea.
  • EGCG has demonstrated antitumor effects in various human and animal models, including cancers of the breast, prostate, stomach, esophagus, colon, pancreas, skin, lung, and other sites.
  • EGCG has been shown to act on different pathways to regulate cancer cell growth, survival, angiogenesis and metastasis. For example, some studies suggest that EGCG protects against cancer by causing cell cycle arrest and inducing apoptosis. It is also reported that telom erase inhibition might be one of the major mechanisms underlying the anticancer effects of EGCG.
  • EGCG In comparison with commonly-used antitumor agents, including retinoids and doxorubicin, EGCG has a relatively low toxicity and is convenient to administer due to its oral bioavailability. Thus, EGCG has been used in clinical trials and appears to be a potentially ideal antitumor agent.
  • Exemplary analogs or derivatives of EGCG include (-)-EGCG, (+)-EGCG, (-)- EGCG-amide, (-)-GCG, (+)-GCG, (+)-EGCG-amide, (-)-ECG, (-)-CG, genistein, GTP-1, GTP-2, GTP-3, GTP-4, GTP-5, Bn-(+)-epigallocatechin gallate (US 2004/0186167, incorporated by reference), and dideoxy-epigallocatechin gallate (Furuta, et al, Bioorg. Med. Chem. Letters, 2007, 11 : 3095-3098), For additional examples, see US
  • chemotherapeutic drug that may be used is (a) 5,6 di-methylxanthenone-4- acetic acid (DMXAA), or a chemical derivative or analog thereof or a pharmaceutically acceptable salt thereof.
  • exemplary analogs or derivatives include xanthenone-4-acetic acid, flavone-8-acetic acid, xanthen-9-one-4-acetic acid, methyl (2,2-dimethyl-6-oxo-l,2- dihydro-6H-3, l l-dioxacyclopenta[a]anthracen-10-yl)acetate, methyl (2-methyl-6-oxo-l,2- dihydro-6H-3, l l-dioxacyclopenta[a]anthracen-10-yl)acetate, methyl (3,3-dimethyl-7-oxo- 3H,7H-4, 12-dioxabenzo[a]anthracen-10-yl)acetate, methyl-6-alkyloxyxanthen-9
  • a chemotherapeutic drug may also be cisplatin, or a chemical derivative or analog thereof or a pharmaceutically acceptable salt thereof.
  • exemplary analogs or derivatives include dichloro[4,4'-bis(4,4,4-trifluorobutyl)-2,2'-bipyridine]platinum (Kyler et al, Bioorganic & Medicinal Chemistry, 2006, 14: 8692-8700), cis-[Rh2( - 02CCH3)2(CH3CN)6]2+ (Lutterman et al, J. Am. Chem.
  • MOLI001226 CID: 450696
  • trichloroplatinum CID: 420479
  • amminetrichloro- ammonium
  • triammineplatinum CID: 119232
  • biocisplatinum CID: 84691
  • platiblastin CID: 2767
  • pharmaceutically acceptable salts thereof See US 5922689, US 4996337, US 4937358, US 4808730, US 6130245, US 7232919, and US 7038071, each incorporated by reference in their entirety.
  • chemotherapeutic drug that may be used is apigenin, or a chemical derivative or analog thereof or a pharmaceutically acceptable salt thereof.
  • exemplary analogs or derivatives include acacetin, chrysin, kampherol, luteolin, myricetin, naringenin, quercetin (Wang et al, Nutrition and Cancer, 2004, 48: 106-114), puerarin (US
  • doxorubicin Another chemotherapeutic drug that may be used is doxorubicin, or a chemical derivative or analog thereof or a pharmaceutically acceptable salt thereof.
  • exemplary analogs or derivatives include anthracyclines, 3'-deamino-3'-(3-cyano-4- morpholinyl)doxorubicin, WP744 (Faded, et al, Cancer Res., 2001, 21 : 3777-3784), annamycin (Zou, et al, Cancer Chemother. Pharmacol., 1993, 32: 190-196), 5-imino- daunorubicin, 2-pyrrolinodoxorubicin, DA-125 (Lim, et al, Cancer Chemother.
  • chemotherapeutic drugs that may be used are anti-death receptor 5 antibodies and binding proteins, and their derivatives, including antibody fragments, single-chain antibodies (scFvs), Avimers, chimeric antibodies, humanized antibodies, human antibodies and peptides binding death receptor 5.
  • scFvs single-chain antibodies
  • Avimers chimeric antibodies
  • humanized antibodies human antibodies and peptides binding death receptor 5.
  • chemotherapeutic drug that may be used is bortezomib, or a chemical derivative or analog thereof or a pharmaceutically acceptable salt thereof.
  • exemplary analogs or derivatives include MLN-273 and pharmaceutically acceptable salts thereof (Witola, et al, Eukaryotic Cell, 2007, doi: 10.1128/EC.00229-07). For additional possibilities, see Groll, et al, Structure, 14:451.
  • chemotherapeutic drug that may be used is 5-aza-2-deoxycytidine, or a chemical derivative or analog thereof or a pharmaceutically acceptable salt thereof.
  • Exemplary analogs or derivatives include other deoxycytidine derivatives and other nucleotide derivatives, such as deoxyadenine derivatives, deoxyguanine derivatives, deoxythymidine derivatives and pharmaceutically acceptable salts thereof.
  • Another chemotherapeutic drug that may be used is genistein, or a chemical derivative or analog thereof or a pharmaceutically acceptable salt thereof.
  • Exemplary analogs or derivatives include 7-O-modified genistein derivatives (Zhang, et al, Chem.
  • chemotherapeutic drug that may be used is celecoxib, or a chemical derivative or analog thereof or a pharmaceutically acceptable salt thereof.
  • exemplary analogs or derivatives include N-(2-aminoethyl)-4-[5-(4-tolyl)-3-(trifluoromethyl)-lH- pyrazol- 1 -yl]benzenesulfonamide, 4-[5-(4-aminophenyl)-3 -(trifluoromethyl)- lH-pyrazol- 1 - yl]benzenesulfonamide, OSU03012 (Johnson, et al, Blood, 2005, 105: 2504-2509), OSU03013 (Tong, et.
  • chemotherapeutics can be used with the methods disclosed in the present invention, including proteasome inhibitors (in addition to bortezomib) and inhibitors of DNA methylation.
  • Other drugs that may be used include Paclitaxel; selenium compounds; SN38, etoposide, 5-Fluorouracil; VP- 16, cox-2 inhibitors, Vioxx, cyclooxygenase-2 inhibitors, curcumin, MPC-6827 , tamoxifen or flutamide, etoposide, PG490, 2-methoxyestradiol, AEE-788, aglycon protopanaxadiol, aplidine, ARQ-501, arsenic tri oxide, BMS-387032, canertinib dihydrochloride,
  • canfosfamide hydrochloride canfosfamide hydrochloride, combretastatin A-4 prodrug, idronoxil, indisulam, INGN-201, mapatumumab, motexafin gadolinium, oblimersen sodium, OGX-011, patupilone, PXD- 101, rubitecan, tipifarnib, trabectedin PXD-101, methotrexate, Zerumbone, camptothecin, MG-98, VX-680, Ceflatonin, Oblimersen sodium, motexafin gadolinium, 1D09C3, PCK- 3145, ME-2 and apoptosis-inducing-ligand (TRAIL/ Apo-2 ligand).
  • Others are provided in a report entitled "competitive outlook on apoptosis in oncology, Dec. 2006, published by Bioseeker, and available, e.g., at
  • Apoptosis targets include the tumour-necrosis factor (TNF)-related apoptosis-inducing ligand
  • TRAIL apoptosis receptors
  • BCL2 family of anti-apoptotic proteins such as Bcl-2
  • IAP inhibitor of apoptosis
  • MDM2 p53
  • TRAIL apoptosis
  • targets include B- cell CLL/lymphoma 2, Caspase 3, CD4 molecule, Cytosolic ovarian carcinoma antigen 1, Eukaryotic translation elongation factor 2, Farnesyltransferase, CAAX box, alpha; Fc fragment of IgE; Histone deacetylase l;Histone deacetylase 2; Interleukin 13 receptor, alpha 1; Phosphodiesterase 2A, cGMP-stimulatedPhosphodiesterase 5 A, cGMP-specific; Protein kinase C, beta 1 ;Steroid 5-alpha-reductase, alpha polypeptide 1; 8.1.15
  • Topoisomerase (DNA) I Topoisomerase (DNA) II alpha; Tubulin, beta polypeptide; and p53 protein.
  • the compounds described herein are naturally-occurring and may, e.g., be isolated from nature. Accordingly, in certain embodiments, a compound is used in an isolated or purified form, i.e., it is not in a form in which it is naturally occurring.
  • an isolated compound may contain less than about 50%, 30%, 10%, 1%, 0.1% or 0.01% of a molecule that is associated with the compound in nature.
  • a purified preparation of a compound may comprise at least about 50%, 70%, 80%, 90%, 95%, 97%, 98% or 99% of the compound, by molecule number or by weight.
  • Compositions may comprise, consist essentially of consist of one or more compounds described herein. Some compounds that are naturally occurring may also be synthesized in a laboratory and may be referred to as "synthetic.” Yet other compounds described herein are non-naturally occurring.
  • the chemotherapeutic drug is in a preparation from a natural source, e.g., a preparation from green tea.
  • compositions comprising 1, 2, 3, 4, 5 or more chemotherapeutic drugs or pharmaceutically acceptable salts thereof are also provided herein.
  • compositions may comprise a pharmaceutically acceptable carrier.
  • a composition e.g., a pharmaceutical composition, may also comprise a vaccine, e.g., a DNA vaccine, and optionally 1, 2, 3, 4, 5 or more vectors, e.g., other DNA vaccines or other constructs, e.g., described herein.
  • Compounds may be provided with a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salts is art-recognized, and includes relatively non-toxic, inorganic and organic acid addition salts of compositions, including without limitation, therapeutic agents, excipients, other materials and the like.
  • Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like.
  • suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts.
  • the class of such organic bases may include mono-, di-, and trialkylamines, such as methylamine, dimethylamine, and triethylamine; mono-, di- or trihydroxyalkylamines such as mono-, di-, and
  • amino acids such as arginine and lysine; guanidine; N- methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; (trihydroxymethyl)aminoethane; and the like. See, for example, J. Pharm. Sci.. 66: 1-19 (1977).
  • compositions and kits comprising one or more DNA vaccines and one or more chemotherapeutic drugs, and optionally one or more other constructs described herein.
  • the methods of the present invention can be practiced by coadministering a combination therapy comprising at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide, described herein alone or in a pharmaceutically acceptable carrier in a biologically-effective and/or a therapeutically-effective amount.
  • the DNA vector may comprise major and minor capsid antigens from human papillomavirus type 16 (HPV16), bovine papillomavirus type 1, or any combination thereof.
  • HPV16 human papillomavirus type 16
  • bovine papillomavirus type 1 bovine papillomavirus type 1
  • the DNA protein may be used in combination with a DNA vaccine, wherein the DNA vaccine comprises an antigenic peptide.
  • said antigenic peptide is selected from HPV16 E7, HPV16E6, OVA, CRT, HPV16
  • compositions may be given alone or in combination with another protein or peptide such as an immunostimulatory molecule.
  • Treatment may include administration of an adjuvant, used in its broadest sense to include any nonspecific immune stimulating compound such as an interferon.
  • adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.
  • a therapeutically effective amount is a dosage that, when given for an effective period of time, achieves the desired immunological or clinical effect.
  • a therapeutically active amount of the combination therapy comprising at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the fusion protein to elicit a desired response in the individual. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a therapeutically effective amount of the protein, in cell associated form may be stated in terms of the protein or cell equivalents.
  • an effective amount of at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide may be between about 1 nanogram and about 1 gram per kilogram of body weight of the recipient, between about 0.1 ⁇ g/kg and about lOmg/kg, between about 1 ⁇ g/kg and about 1 mg/kg.
  • Dosage forms suitable for internal administration may contain (for the latter dose range) from about 0.1 ⁇ g to 100 ⁇ g of active ingredient per unit.
  • the active ingredient may vary from 0.5 to 95% by weight based on the total weight of the composition. Those skilled in the art of immunotherapy will be able to adjust these doses without undue experimentation.
  • the at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide may be administered via gene gun.
  • they combination thereapy may be packaged into retrovirus vectors using packaging cell lines that produce replication-defective retroviruses, as is well-known in the art (e.g., Cone, R.D.
  • a catheter delivery system can be used (Nabel, EG et al, Science 244: 1342 (1989)).
  • a retroviral vector or a liposome vector are particularly useful to deliver the at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide to a blood vessel wall, or into the blood circulation of a tumor.
  • the active protein may be present in the aqueous layer and in the lipidic layer, inside or outside, or, in any event, in the non-homogeneous system generally known as a liposomic suspension.
  • the hydrophobic layer, or lipidic layer generally, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surface active substances such as dicetylphosphate, stearylamine or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • phospholipids such as lecithin and sphingomyelin
  • steroids such as cholesterol
  • more or less ionic surface active substances such as dicetylphosphate, stearylamine or phosphatidic acid
  • Embodiments disclosed herein also relate to methods of administering an at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide described herein to a subject in order to contact in vivo cells with such compositions.
  • the routes of administration can vary with the location and nature of the cells to be contacted, and include, e.g., intravascular, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional, percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, direct injection, and oral administration and formulation.
  • the routes of administration of the at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide may include (a) intratumoral, peritumoral, and/or intradermal delivery using gene gun, (b) intramuscularly (i.m.) injection using a conventional syringe needle; and (c) use of a needle-free biojector such as the Biojector 2000 (Bioject Inc., Portland, OR) which is an injection device consisting of an injector and a disposable syringe. The orifice size controls the depth of penetration.
  • systemic administration refers to administration of at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide as described herein, in a manner that results in the introduction of the composition into the subject's circulatory system or otherwise permits its spread throughout the body.
  • Regular administration refers to administration into a specific, and somewhat more limited, anatomical space, such as intraperitoneal, intrathecal, subdural, or to a specific organ.
  • Local administration refers to administration of a composition or drug into a limited, or circumscribed, anatomic space, such as intratumoral injection into a tumor mass, subcutaneous injections, intradermal or intramuscular injections.
  • intravascular is understood to refer to delivery into the vasculature of a patient, meaning into, within, or in a vessel or vessels of the patient, whether for systemic, regional, and/or local
  • the administration can be into a vessel considered to be a vein (intravenous), while in others administration can be into a vessel considered to be an artery.
  • Veins include, but are not limited to, the internal jugular vein, a peripheral vein, a coronary vein, a hepatic vein, the portal vein, great saphenous vein, the pulmonary vein, superior vena cava, inferior vena cava, a gastric vein, a splenic vein, inferior mesenteric vein, superior mesenteric vein, cephalic vein, and/or femoral vein.
  • Arteries include, but are not limited to, coronary artery, pulmonary artery, brachial artery, internal carotid artery, aortic arch, femoral artery, peripheral artery, and/or ciliary artery. It is contemplated that delivery may be through or to an arteriole or capillary.
  • Injection into the tumor vasculature is specifically contemplated for discrete, solid, accessible tumors.
  • Local, regional or systemic administration also may be appropriate.
  • the volume to be administered can be about 4-10 ml (preferably 10 ml), while for tumors of less than about 4 cm, a volume of about 1-3 ml can be used (preferably 3 ml).
  • Multiple injections delivered as single dose comprise about 0.1 to about 0.5 ml volumes.
  • the at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide may advantageously be contacted by administering multiple injections to the tumor, spaced at approximately 1 cm intervals.
  • Continuous administration also may be applied where appropriate. Such continuous administration, such as intravenous injection, may take place for a period of 9 days with periodic injections every 3 days. Generally, the dose of the therapeutic composition via continuous administration will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the treatment occurs.
  • Other routes of administration include oral, intranasal or rectal or any other route known in the art.
  • the at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
  • a material to prevent its inactivation for example, an enzyme inhibitors of nucleases or proteases (e.g., pancreatic trypsin inhibitor, diisopropylfluorophosphate and trasylol) or in an appropriate carrier such as liposomes (including water-in-oil-in-water emulsions as well as
  • a chemotherapeutic drug may be administered in doses that are similar to the doses that the chemotherapeutic drug is used to be administered for cancer therapy.
  • the dose of chemotherapeutic agent is a dose that is effective to increase the effectiveness of the combination therapy comprising at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide, but less than a dose that results in significant immunosuppression or immunosuppression that essentially cancels out the effect of the combination effects of the at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide.
  • chemotherapeutic drugs may depend on the drug.
  • a chemotherapeutic drug may be used as it is commonly used in known methods.
  • the drugs will be administered orally or they may be injected.
  • the regimen of administration of the drugs may be the same as it is commonly used in known methods. For example, certain drugs are administered one time, other drugs are administered every third day for a set period of time, yet other drugs are administered every other day or every third, fourth, fifth, sixth day or weekly.
  • Examples provide exemplary regimens for administrating the drugs, as well as at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide.
  • the combination thereapy may be administered via intraperitoneal gene gun injection two times at a three day interval.
  • the intraperitoneal injection of the the combination therapy may be spread out over a period of 1 week, 2 weeks, 3 weeks, 4 weeks or longer.
  • the combination can be repeated
  • compositions of the present invention may be administered simultaneously or subsequently.
  • the different components may be administered as one composition.
  • compositions e.g., pharmaceutical compositions comprising one or more agents.
  • a subject first receives one or more doses of the at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide.
  • One may administer 1, 2, 3, 4, 5 or more doses of the at least one DNA vector comprising a papillomavirus capsid antigen and at least one DNA vaccine comprising an antigenic peptide.
  • a method may further comprise subjecting a subject to another cancer treatment, e.g., radiotherapy, an anti-angiogenesis agent and/or a hydrogel-based system.
  • another cancer treatment e.g., radiotherapy, an anti-angiogenesis agent and/or a hydrogel-based system.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • Isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride may be included in the pharmaceutical composition.
  • the composition should be sterile and should be fluid. It should be stable under the conditions of manufacture and storage and must include preservatives that prevent contamination with microorganisms such as bacteria and fungi.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms in the pharmaceutical composition can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for a mammalian subject; each unit contains a predetermined quantity of active material (e.g., DNA vector comprising a papillomavirus capsid antigen or DNA vaccine comprising an antigenic peptide) calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier.
  • active material e.g., DNA vector comprising a papillomavirus capsid antigen or DNA vaccine comprising an antigenic peptide
  • aerosolized solutions are used.
  • the active protein may be in combination with a solid or liquid inert carrier material. This may also be packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant.
  • the aerosol preparations can contain solvents, buffers, surfactants, and antioxidants in addition to the protein of the invention.
  • cancers that may be treated as described herein include hyper proliferative diseases, e.g., cancer, whether localized or having metastasized.
  • exemplary cancers include head and neck cancers and cervical cancer. Any cancer can be treated provided that there is a tumor associated antigen that is associated with the particular cancer.
  • Other cancers include skin cancer, lung cancer, colon cancer, kidney cancer, breast cancer, prostate cancer, pancreatic cancer, bone cancer, ovarian cancer, brain cancer, as well as blood cancers, e.g., myeloma, leukemia and lymphoma.
  • Treating a subject includes curing a subject or improving at least one symptom of the disease or preventing or reducing the likelihood of the disease to return.
  • treating a subject having cancer could be reducing the tumor mass of a subject, e.g., by about 10%, 30%, 50%, 75%, 90% or more, eliminating the tumor, preventing or reducing the likelihood of the tumor to return, or partial or complete remission.
  • DNA vaccines have emerged as attractive candidates for the control of HPV- associated cancers. However, DNA vaccines suffer from limited immunogenicity and strategies to enhance DNA vaccine potency are needed. It was previously shown that DNA vaccines encoding HPV-16 E7 antigen linked to calreticulin (CRT/E7) can elicit potent E7- specific CD8+ T cell antitumor immunity against E7-expressing tumors. Here, the inventors report a novel vaccine technology consisting of co-administration of DNA encoding
  • the vaccine technology consists of DNA encoding HPV-16 E7 antigen linked to calreticulin (CRT/E7) and DNA encoding papillomavirus LI or L2.
  • DNA vaccine encoding CRT/E7 has been shown to elicit potent E7-specific CD8+ T cell response and significantly enhanced the antitumor effect. It was shown that co-administration with DNA encoding LI or L2 in addition to CRT/E7 DNA induces even more potent E7-specific CD8+ T cell responses compared to vaccination with CRT/E7 alone. Furthermore, co-administration with DNA encoding L1/L2 led to the generation of antigen-specific CD4+ T cell help and Ll/L2-specific neutralizing antibodies.
  • the current vaccine technology can be applied through different routes of administration including intradermal as well as intramuscular administration.
  • Example 1 Co-administration with vectors encoding papillomavirus LI or L2 significantly enhances the antigen-specific CD8 + T cell immune responses generated by CRT E7 or OVA DNA vaccination
  • C57BL/6 mice (five per group) were vaccinated intradermally via gene gun with CRT/E7 or OVA DNA with or without BPV1 LI or L2 DNA twice at 1-week intervals.
  • Splenocytes from vaccinated mice were collected 1 week after last immunization and the E7 or OVA-specific T cell immune responses were characterized using intracellular cytokine staining followed by flow cytometry analysis.
  • mice vaccinated with CRT/E7 or OVA DNA vaccine in combination with BPV1-L1 or L2 DNA generated significantly higher E7-specific and OVA-specific CD8 + T cell immune responses compared to mice vaccinated with CRT/E7 or OVA DNA alone.
  • HPV16 LI or L2 DNA was co-administered with CRT/E7 or OVA DNA vaccination.
  • Example 2 Co-administration of papillomavirus LI or L2 DNA with CRT E7 or OVA DNA led to the generation of Ll L2-specific CD4 + T cell immune responses
  • CRT/E7 DNA will lead to the generation of LI or L2-specific CD4 + T cell immune responses
  • C57BL/6 mice (five per group) were vaccinated intradermally via gene gun with CRT/E7 DNA with BPVl LI or L2 DNA. Mice vaccinated with CRT/E7 DNA alone were used as negative controls. Splenocytes from vaccinated mice were collected 1 week after last immunization and incubated with BPVl L1/L2 virus-like particles (VLPs). The LI or L2-specific CD4 + T cell immune responses were characterized using intracellular cytokine staining followed by flow cytometry analysis.
  • mice vaccinated with BPVl LI in combination with CRT/E7 DNA led to the generation of Ll- specific CD4 + T cell immune responses.
  • vaccination with BPVl L2 with CRT/E7 DNA led to significant level of L2-specific CD4 + T cell immune responses compared to vaccination with CRT/E7 alone.
  • HPV16 LI or L2 DNA with CRT/E7 or OVA DNA vaccination were co-administered.
  • Example 3 Co-administration with BPVl LI DNA significantly enhances the therapeutic antitumor effects generated by CRT E7 DNA vaccination
  • mice In order to determine if the observed enhancement of antigen-specific CD8 + T cell immune responses by co-administration of BPVl LI DNA can translate into potent therapeutic antitumor effects, in vivo tumor treatment experiments were performed using an HPV-16 E7-expressing murine tumor cell line, TC-1. TC-1 also expresses HPV16 E6, but does not contain either LI or L2. C57BL/6 mice (five per group) were first challenged with TC-1 tumor cells subcutaneously. One week after tumor challenge, mice were treated intradermally via gene gun with CRT/E7 DNA alone, BPVl LI DNA alone or CRT/E7 DNA in combination with BPV1 LI DNA. Vaccinated mice were boosted twice at 1-week intervals with the same dose and regimen.
  • Tumor growth were monitored twice weekly by caliper measurements and palpations.
  • tumor-bearing mice treated with CRT/E7 DNA vaccine in combination with BPV1 LI DNA generated significantly reduced tumor volume and prolonged survival compared to mice treated with CRT/E7 DNA alone or BPV1 LI DNA alone.
  • co-administration with BPV1 LI DNA is capable of significantly enhancing the therapeutic antitumor effects generated by CRT/E7 DNA vaccination.
  • Example 4 The enhancement in E7-specific CD8+ T cell immune responses are contributed by CD4+ T helper cells
  • mice vaccinated with CRT/E7 DNA with or without the reverse sequence of BPV1 LI or L2 DNA twice at 1-week intervals Splenocytes from vaccinated mice were collected 1 week after last immunization and the E7-specific T cell immune responses were characterized using intracellular cytokine staining followed by flow cytometry analysis. It was found that mice vaccinated with CRT/E7 DNA vaccine in combination with the reverse sequence BPVl-Ll or L2 DNA did not lead to the increased frequency of E7-specific CD8 + T cell immune responses observed in mice vaccinated with CRT/E7 DNA with BPVl-Ll or L2 DNA (data not shown).
  • the insert sequences does not express into LI and L2 that activate CD4+ T cell help, thereby cannot enhance the E7-specific CD8+ T cell responses of the CRT/E7 vaccine.
  • the result suggested that CD4+ T cell help generated by co- expression of LI or L2 protein promoted E7-specific CD8+ T cell immune responses to CRT/E7 DNA vaccination.
  • mice In order to determine if co-administration of BPV1 LI DNA with CRT/E7 DNA will lead to the generation of BPV1 LI -specific neutralizing antibodies, C57BL/6 mice (three per group) were immunized on days 1, 15, and 30 intradermally via gene gun with CRT/E7 and/or BPV1 LI DNA. In vitro neutralization assays were performed using BPV1 LI pseudovirus on twofold dilutions of antisera collected from the mice 2 weeks after the final immunization. Mice vaccinated with CRT/E7 in combination with BPVl LI DNA were found to generate similar neutralizing antibody responses compared to mice vaccinated with BPVl LI DNA alone ( Figure 5).
  • Example 6 Co-administration of BPVl LI or L2 DNA with OVA DNA vaccination generated OVA-specific CD8+ T cell response through intramuscular administration
  • mice vaccinated intramuscularly with OVA DNA in combination with BPVl LI or L2 DNA generated significantly higher percentage of OVA- specific CD8+ T cells compared to mice vaccinated intramuscularly with OVA DNA alone.
  • CD4+ T cell help generated by LI or L2 DNA is the main contributor to the enhanced antigen-specific CD8+ T cell response observed.
  • CD4+ T cells can help generate memory T cells (Shedlock DJ et al. Science 2003, 300:337-339; Overstreet MG et al. PloS one 2011, 6:el5948).
  • the current study focuses on characterizing the antigen-specific CD8+ T cell therapeutic antitumor effect by the inventors' vaccination strategy, it will be of interest for future studies to further characterize the complete effect of CD4+ T cell and its ability to generate memory T cell responses for prolonged protection.
  • the present vaccination strategy was able to generate a potent therapeutic antitumor effect in tumor-bearing mice.
  • co-administration of BPVl LI DNA with CRT/E7 DNA generated LI -specific neutralizing antibodies, which confers prophylactic value.
  • vaccination with LI DNA induced LI -specific neutralizing antibodies in Balb/c mice Kwak K et al. PloS one 2013, 8:e60507. Therefore, the vaccination strategy of co-administration of LI DNA can generate potent antibody responses in more than one genetic background.
  • studies have shown that papillomavirus L2 is generally not as effective in generating L2-specific neutralizing antibodies (Kwak K et al. PloS one 2013, 8:e60507; Cason J et al. The Journal of general virology 1993, 74 ( Pt 12):2669-2677), thus co-administration with LI DNA should be prioritized in future translation.
  • the frequency of vaccination may be modified and further studied to determine the optimal vaccination regimen.
  • the data showed that the current vaccination strategy can generate enhanced antigen-specific CD8+ T cell responses by both intradermal and intramuscular vaccination. Since DNA vaccines are commonly applied intramuscularly in the clinic, future translation of the current vaccination technology may focus on the intramuscular route of administration.
  • the present study demonstrated that the employment of DNA encoding papillomavirus LI or L2 can lead to generation of antigen-specific CD4 + T cells and neutralizing antibodies, resulting in the improvement of therapeutic and preventive HPV DNA vaccine potency.
  • the strategy may potentially be extended to other antigenic systems for the control of infection and/or cancer.
  • mice C57BL/6 mice (6-8 weeks old) were purchased from the National Cancer institute (Frederick, MD). All animals were maintained under specific pathogen-free conditions at the Johns Hopkins Hospital (Baltimore, MD). Ail procedures were performed according to the Johns Hopkins Institutional Care and Use Committee approved protocols and in accordance with recommendations for the proper care of laboratory animals.
  • TC-1 cells were obtained by co-transformation of primary C57BL/6 mouse lung epithelial cells with HPV- 16 E6 and E7 and an activated ras oncogene as described previously (Lin KY et ai. Cancer Res 1996, 56:21-26).
  • DNA vaccination DNA-coated gold particles were prepared as described previously (Chen CH et al. Cancer Res 2000, 60: 1035-1042). DNA-coated gold particles were delivered to the shaved abdominal region of mice using a helium-driven gene gun (Bio-Rad Laboratories Inc., Hercules, CA, USA) with a discharge pressure of 400 p.s.i. C57BL/6 mice were immunized with 2 ⁇ g of plasmid DNA to each mouse encoding pcDNA3- CRT/E7 mixed with pcDNA3-BPVl-Ll or L2 or HPV-L1 or L2 delivered to the shaved abdomen. The mice received a homologous boost 1 week later.
  • mice were vaccinated with ⁇ g of pcDNA3-OVA DNA with ⁇ g of pcDNA3, pcDNA3-BPVLl, or pcDNA3-BPVL2 intramuscularly in the thigh muscle.
  • the mice received a homologous boost 1 week later.
  • Intracellular cytokine staining and flow cytometry analysis Splenocytes were harvested from mice 1 week after the last vaccination. Prior to intracellular cytokine staining, 5xl0 6 splenocytes from each vaccination group were incubated for 16 h with 1 ⁇ g ml "1 HPV-16 E7 H-2Db epitope (RAHYNIVTF) or OVA peptide
  • Intracellular IFN- ⁇ was stained with fluorescein isothiocyanate-conjugated rat anti-mouse IFN- ⁇ to identify the immune response and cytokine levels.
  • PBMCs were collected from peripheral blood 1 week after last intramuscular vaccination and stained with anti-mouse CD8a and OVA peptide (SIINFEKL) loaded H-2K b tetramer.
  • SIINFEKL anti-mouse CD8a and OVA peptide
  • mice were vaccinated with DNA constructs pcDNA3-BPVl LI or L2 or pcDNA3-HPV LI or L2 in conjunction with pCDNA3- CRT/E7 or pcDNA3-OVA or the control empty vector.
  • a homologous boost was administered 1 week after the first immunization. Mice were monitored for tumor growth by measuring diameters with calipers twice a week.
  • SEAP alkaline phosphatase
  • HPV - Human papillomavirus CRT - calreticulin; BPV - bovine papillomavirus; OVA - ovalbumin; DCs - dendritic cells; VLPs - virus-like particles CLIP - class Il-associated invariant peptide; PADRE - pan HLA-DR binding epitope SEAP - encapsulated secreted alkaline phosphatase.
  • KYVRSAKLRM VTGLRNTPSI QSRGLFGAIA GFIEGGWTGM IDGWYGYHHQ NEQGSGYAAD QKSTQNAING ITNKVNTVIE
  • IDGKKYTAPE ISARILMKLK RDAEAYLGED
  • ITDAVITTPA YFNDAQRQAT
  • KDAGQIAGLN VLRIVNEPTA AALAYGLDKG
  • gec gag gec tac etc ggt gag gac att acc gac gcg gtt ate acg acg ccc gec tac ttc
  • gag gtt gtc gcg gtg gga gec get ctg cag gec ggc gtc etc aag ggc gag gtg aaa gac
  • gac gtg gtg age ctg acc tgc ccg gtc gec gec ggt gaa tgc gcg ggc ccg gcg gac age
  • AAACACAAGT CAGATTTTGG CAAATTCGTT CTCAGTTCCG GCAAGTTCTA CGGTGACGAG

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Abstract

L'invention concerne des procédés consistant à co-administrer à un mammifère une quantité efficace d'une polythérapie comprenant au moins un vecteur d'ADN comprenant un antigène de capside de papillomavirus et au moins un vaccin d'ADN comprenant un peptide antigénique, afin d'améliorer ainsi une réponse immunitaire spécifique à un antigène.
PCT/US2016/034539 2015-05-28 2016-05-27 Procédés pour améliorer des réponses immunitaires spécifiques à un antigène à l'aide d'une polythérapie comprenant des antigènes de capside de papillomavirus WO2016191641A2 (fr)

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WO2022198094A1 (fr) * 2021-03-18 2022-09-22 Calyxt, Inc. Production d'albumine dans des parties de plante cannabaceae
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CN115919819A (zh) * 2022-11-24 2023-04-07 华中科技大学同济医学院附属同济医院 6-姜酚在抑制hpv16感染中的应用
WO2023154478A1 (fr) * 2022-02-11 2023-08-17 Washington University Procédés d'évaluation de cancer

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020039584A1 (en) * 1998-02-20 2002-04-04 Medigene Ag Papilloma virus capsomere vaccine formulations and methods of use
US20120225090A1 (en) * 2009-08-03 2012-09-06 The Johns Hopkins University Methods for enhancing antigen-specific immune responses

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WO2022198094A1 (fr) * 2021-03-18 2022-09-22 Calyxt, Inc. Production d'albumine dans des parties de plante cannabaceae
WO2022198093A1 (fr) * 2021-03-18 2022-09-22 Calyxt, Inc. Production d'albumine à l'aide de matrices de cellules végétales
WO2022198085A3 (fr) * 2021-03-18 2022-10-20 Calyxt, Inc. Matrices de cellules végétales et leurs procédés
WO2023154478A1 (fr) * 2022-02-11 2023-08-17 Washington University Procédés d'évaluation de cancer
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WO2023184861A1 (fr) * 2022-03-29 2023-10-05 深圳吉诺因生物科技有限公司 Épitope de hpv, son procédé d'identification et son utilisation
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