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WO2018204760A1 - Ctla4 antibodies and vaccine combinations and use of same for immunotherapy - Google Patents

Ctla4 antibodies and vaccine combinations and use of same for immunotherapy Download PDF

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Publication number
WO2018204760A1
WO2018204760A1 PCT/US2018/031049 US2018031049W WO2018204760A1 WO 2018204760 A1 WO2018204760 A1 WO 2018204760A1 US 2018031049 W US2018031049 W US 2018031049W WO 2018204760 A1 WO2018204760 A1 WO 2018204760A1
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WIPO (PCT)
Prior art keywords
antigen
consensus
protein
antibody
sequence
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PCT/US2018/031049
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French (fr)
Inventor
David Weiner
Kar MUTHUMANI
Elizabeth DUPERRET
Megan WISE
Original Assignee
David Weiner
Muthumani Kar
Duperret Elizabeth
Wise Megan
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Application filed by David Weiner, Muthumani Kar, Duperret Elizabeth, Wise Megan filed Critical David Weiner
Publication of WO2018204760A1 publication Critical patent/WO2018204760A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A61K39/001157—Telomerase or TERT [telomerase reverse transcriptase]

Definitions

  • the present invention relates to vaccines combined with CTLA4 antibodies, and use of such combination for immunotherapy.
  • MHC major histocompatibility complex
  • TCR T-cell receptor
  • CTLA-4, PD-1, LAG3, TIM3 are often necessary for initiation of an immune response; however, as antigen persists, these checkpoints ultimately serve to dampen the T cell effector function against the foreign antigen (Riley, 2009, Immunol Rev, 229: 114-125).
  • CTLA-4, PD-1, LAG3 and TIM3 are all expressed on T cells and limit T cell effector activity.
  • Antibodies blocking these molecules have been shown to augment the effector activity of tumor-specific T cells, and additionally inhibit regulatory T cell (Treg) activity and reduce tumor burden in preclinical models and/or clinical trials as mono-therapies (Walunas and Bluestone, 1998, J Immunol, 160:3855-3860; Anderson, 2014, Cancer Immunol Res, 2:393-398; Duraiswamy et al, 2013, Cancer Res, 73:3591-3603).
  • Treg regulatory T cell
  • Cytotoxic T-Lymphocyte Associated Protein 4 also known as CD 152 is a cell surface protein molecule that functions as a major negative regulator of T-cell responses. This protein is expressed in Tregs and is upregulated in T cells after activation. CTLA4 functions as an immune checkpoint and downregulates immune responses. CTLA4 is homologous to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86 on antigen-presenting cells.
  • Immune checkpoint blockade antibodies such as a-PD-1 (pembrolizumab) and a- CTLA-4 (e.g., ipilimumab) are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint blockade (ICB).
  • a-PD-1 pembrolizumab
  • a- CTLA-4 e.g., ipilimumab
  • blockade of the immune inhibitory checkpoints PD- 1 or CTLA-4 has shown promising results in the clinic for dozens of tumor types, and PD-1 blockade has become a standard of care for melanoma and non-small cell lung cancer (Hodi et al, 2010, N Engl J Med, 363:711-723; Postow et al, 2015, J Clin Oncol, 33: 1974-1982; Reck et al, 2016, N Engl J Med, 375: 1823-1833).
  • response rates to these monotherapies are relatively low (33.7% response for pembrolizumab— a-PD-1, and 11.9% response for ipilimumab— a-CTLA-4 in melanoma patients), leaving room for improvement (Robert et al, 2015, N Engl J Med, 372:2521-2532).
  • the lack of response for the majority of such patients may be due to a lack of pre-existing tumor-associated T cell responses.
  • enhanced T cell priming may be required to break tolerance to self-antigens for patients with poor response to immune checkpoint blockade.
  • Vaccines are used to stimulate an immune response in an individual to provide protection against and/or treatment for a particular disease.
  • Some vaccines include an antigen to induce the immune response.
  • Some antigens elicit a strong immune response while other antigens elicit a weak immune response.
  • a weak immune response to an antigen can be strengthened by including an adjuvant in the immunogenic composition.
  • Adjuvants come in many different forms, for example, aluminum salts, oil emulsions, sterile constituents of bacteria or other pathogens, cytokines, and so forth.
  • Vaccines are also administered in many different ways (e.g., injection, orally, etc.) into many different tissues (e.g., intramuscular, nasal, etc.). Not all delivery methods, however, are equal. Some delivery methods allow for greater compliance within a population of individuals while other delivery methods may affect immunogenicity and/or safety of the immunogenic composition.
  • Therapeutic peptide or DNA vaccination represents a more targeted approach for directing T cells towards specific, less variable, tumor-associated antigens.
  • peptide vaccines have been shown to synergize with immune checkpoint blockade
  • peptide vaccines are HLA-restricted and therefore cannot be used for all patients.
  • synthetic DNA vaccines are not HLA-restricted, are robustly presented on both MHCI and MHCII, and can be designed using consensus sequences in order to break tolerance (Flingai et al, 2013, Front Immunol, 4:354).
  • the invention relates to a composition for enhancing an immune response against an antigen in a subject in need thereof, comprising an anti-CTLA4 antibody, and a synthetic antigen capable of generating an immune response in the subject, or an immunogenic fragment or variant thereof.
  • the synthetic antigen is an isolated DNA that encodes for the antigen.
  • the synthetic antigen is at least one of TERT, prostate, WT1, tyrosinase, NYES01, PRAME, MAGE, CMV, herpes, HIV, HPV, HCV, HBV, EBV, MCV, and cancer causing viruses.
  • the synthetic antigen is TERT.
  • the HPV antigen is E6 and E7 domains of an HPV subtype.
  • the HPV subtype is at least one of HPV6, HPVl l, HPV 16, HPV 18, HPV31, HPV33, HPV52, and HPV58, or a combination thereof.
  • the HIV antigen is at least one of Env A, Env B, Env C, Env D, B Nef-Rev, and Gag, or a combination thereof.
  • the HCV antigen is at least one of El, E2, NS3, NS4a, NS4b, NS5a, and NS5b, and a combination thereof.
  • the HBV antigen is at least one of surface antigen type A, surface antigen type B, surface antigen type C, surface antigen type D, surface antigen type E, surface antigen type F, surface antigen type G, surface antigen type H, and core antigen, and a combination thereof.
  • the prostate antigen is at least one of PSA, PSMA, STEAP, PSCA, and PAP, or a combination thereof.
  • the synthetic antigen is a herpes antigen.
  • the herpes is HCMV, HSV1, HSV2, VZV, or CMV.
  • the herpes antigen is at least one of gB, gM, gN, gH, gL, gO, gE, gl, gK, gC, gD, UL128, UL130, UL131A, and UL83.
  • the anti-CTLA4 antibody is ipilimumab, ipilimumab-Probody Tx (BMS-986249), ipilimumab-NF (BMS-986218), tremelimumab, CS 1002, MDX-010 (NCT00140855), or AGEN-1884 (NCT02694822).
  • the composition for enhancing an immune response against an antigen in a subject in need thereof, comprising an anti-CTLA4 antibody, and a synthetic antigen further comprises an additional antibody targeting an immune checkpoint protein.
  • the immune checkpoint protein is selected from the group consisting of PD- 1, PD-L1, LAG3 and TIM3.
  • the antibody is selected from the group consisting of: nivolumab, pembrolizumab, pidilizumab, BMS-936559, MPDL3280A, MDX1105-01, MEDI4736, and MK-3475.
  • the composition for enhancing an immune response against an antigen in a subject in need thereof further comprises a pharmaceutically acceptable excipient.
  • the invention relates to a method for increasing an immune response in a subject in need thereof, the method comprising administering the composition for enhancing an immune response against an antigen in a subject in need thereof to the subject.
  • the method of administering the composition comprises an electroporating step.
  • the invention relates to a method of increasing an immune response in a subject in need thereof by administering a combination of a synthetic antigen and a CTL4A antibody, wherein the administering step comprises administering to the subject a prime vaccination and a boost vaccination of a synthetic antigen and, prior to the boost vaccination, administering to the subject a CTL4A antibody.
  • the method further comprises administering to the subject a subsequent boost vaccination of the synthetic antigen.
  • the method further comprises administering an antibody targeting one or more immune checkpoint protein prior to the boost vaccination.
  • one or more of the administering steps includes delivering electroporation to the site of administration.
  • one or more of the prime vaccination and the boost vaccination comprises a nucleic acid vaccine.
  • one or more of the prime vaccination and the boost vaccination comprises a DNA vaccine.
  • Figure 1 depicts exemplary experimental results demonstrating the capacity of a mTERT vaccine to generate immune response and break tolerance in C57B1/6 mice.
  • Figure 2 depicts exemplary experimental results demonstrating the anti -tumor activity of a mTERT vaccine.
  • Figure 3 depicts exemplary experimental results demonstrating antigen-specific intracellular cytokine production upon TERT DNA vaccination and ICB in non-tumor bearing mice.
  • Figure 3A depicts a schematic diagram of the experimental setup. Mice were immunized three times at two-week intervals. Mice were given antibody treatment (20C ⁇ g per mouse) every three days starting one day after first immunization. Mice were sacrificed one week following final vaccination, and splenocytes were isolated for analysis.
  • Figure 3B through Figure 3F depict exemplary experimental results demonstrating intracellular cytokine staining of CD8 T cells or CD4 T cells after stimulation with native mouse TERT peptides for 5 hours.
  • Figure 3G depicts exemplary experimental results demonstrating IFNy ELISpot responses to native mouse TERT peptide stimulation for 24 hours.
  • Figure 4 depicts exemplary experimental results demonstrating PD-1 expression on peripheral and spleen T cells upon TERT DNA vaccination and ICB in non-tumor bearing mice.
  • Figure 5 depicts exemplary experimental results demonstrating that delivery of aCTLA-4 or aPD-1 post-2 nd immunization has minimal effects on antigen-specific immune responses.
  • Figure 6 depicts exemplary experimental results demonstrating delivery of aCTLA-4 or aPD-1 post- 1 st vaccination synergizes with mTERT above checkpoint alone in generating anti-tumor immune response.
  • Figure 6A depicts a schematic diagram of the experimental setup. Mice were implanted with TC-1 tumor cells on day 0, then immunized four times at one week intervals starting 7 days after tumor implant. Mice were given antibodies (20C ⁇ g per mouse) every three days starting 1 day after the first immunization. Antibody delivery was continued until one week after the final vaccination.
  • Figure 6B depicts exemplary experimental results demonstrating tumor volume measurements over time for mice with mTERT vaccination and ICB.
  • Figure 6B depicts exemplary experimental results demonstrating mouse survival over time for mice with mTERT vaccination and ICB.
  • Figure 6D depicts exemplary experimental results demonstrating tumor volume measurements over time for mice with ICB alone.
  • Figure 6E depicts exemplary experimental results demonstrating mouse survival over time for mice with ICB alone. Mice were euthanized if they appeared sick or if the tumor diameter exceeded 1.5cm. Significance for tumor volume measurements was determined by two-way ANOVA followed by Tukey's HSD test. Significance for mouse survival was determined by Gehan-Breslow-Wilcoxon test. *p ⁇ 0.05, **p ⁇ 0.005, ***p ⁇ 0.0005, ****p ⁇ 0.00005.
  • N 12-13 mice per group.
  • Figure 7 depicts exemplary experimental results demonstrating antigen-specific intracellular cytokine production upon TERT DNA vaccination and ICB in tumor-bearing mice.
  • Figure 7A depicts a schematic diagram of the experimental setup. Mice were implanted with TC-1 tumor cells on day 0, then immunized on days 7 and 14. Antibody delivery was started one day after the first immunization and continued every three days. Mice were sacrificed on day 21 for immune cell analysis.
  • Figure 3B through Figure 3F depict exemplary experimental results demonstrating intracellular cytokine staining of CD8 or CD4 T cells after stimulation with native mouse TERT peptides for 5 hours. Significance was determined by two-way ANOVA followed by Tukey's HSD test. *p ⁇ 0.05, **p ⁇ 0.005, ***p ⁇ 0.0005, ****p ⁇ 0.00005.
  • N 7-10 mice per group, shown is a representative of three independent experiments.
  • Figure 8 depicts exemplary experimental results demonstrating the phenotype of peripheral and spleen T cells upon TERT DNA vaccination and ICB in tumor bearing mice.
  • Figure 8A depicts exemplary experimental results demonstrating the percentage of CD8+ splenocytes that are PD-1+.
  • Figure 8B depicts exemplary experimental results demonstrating the percentage of CD8+ peripheral blood mononuclear cells (PBMCs) that are PD-1+.
  • Figure 8C depicts exemplary experimental results demonstrating the percentage of CD4+ splenocytes that are PD-1+.
  • Figure 8D depicts exemplary experimental results demonstrating the percentage of CD4+ PBMCs that are PD- 1+.
  • Figure 8E depicts exemplary experimental results demonstrating the percentage of CD4+ splenocytes that are CD25+/FoxP3+.
  • Figure 9 depicts exemplary experimental results demonstrating the phenotype of tumor infiltrating lymphocytes (TILs) upon TERT DNA vaccination and ICB. Staining of CD8 or CD4 T cells isolated from the tumors of mice treated with the indicated therapies according to the schedule in Figure 4A.
  • Figure 9A depicts exemplary experimental results demonstrating the percentage of CD45+/CD3+ TILs that are CD4/CD25/FoxP3+ following TERT simulation.
  • Figure 9B depicts exemplary experimental results demonstrating the percentage of CD45+/CD3+/CD8+ TILs that are PD-1+ following TERT simulation.
  • Figure 9C depicts exemplary experimental results demonstrating the percentage of CD3+ TILs that are CD8/CD44+ following TERT simulation.
  • Figures 10, depicts exemplary experimental results demonstrating that delivery of aCTLA-4 + DNA vaccine is superior to Treg depletion + DNA vaccine using aCD25 antibody.
  • Figure 10A depicts exemplary experimental results demonstrating tumor volume measurements over time for mice with indicated treatment regimen. Mice were treated according to the schedule shown in Figure 7 A.
  • Figure 10B depicts exemplary experimental results demonstrating mouse survival over time for mice with indicated treatment regimen. Mice were euthanized if they appeared sick or if the tumor diameter exceeded 1.5cm. Significance for tumor volume measurements was determined by two-way ANOVA followed by Tukey's HSD test. Significance for mouse survival was determined by Gehan-Breslow-Wilcoxon test. *p ⁇ 0.05, **p ⁇ 0.005,
  • N 10 mice per group.
  • the present invention relates to a composition that can be used to increase or enhance an immune response, i.e., create a more effective immune response, by combining an immunogenic composition, in many cases a synthetic antigen, with CTLA4 antibody.
  • a CTLA4 antibody can be administered in combination with the antigen; whereas, in other instances, with CTLA4 antibody can be administered separately from the antigen of the immunogenic composition.
  • a CTLA4 antibody can be administered in combination with one or more additional antibody targeting one or more additional immune checkpoint proteins.
  • a CTLA4 antibody can be administered in combination with one or more antibodies against PD-1, PD-Ll, LAG3 and TIM3.
  • a combination of a CTLA4 antibody and one or more additional antibody targeting one or more additional immune checkpoint proteins is administered in combination with an antigen to create a more effective immune response.
  • the immunogenic composition of the present invention can increase the immune response to the antigen in the subject by increasing the CD8 + T cell response as compared to the immunogenic composition not including a CTLA4 antibody.
  • This increased CD8 + T cell response has cytolytic activity and secretes the anti-viral cytokine interferon-gamma (IFN- ⁇ ).
  • IFN- ⁇ anti-viral cytokine interferon-gamma
  • compositions for enhancing an immune response against an antigen in a subject in need thereof comprising a CTLA4 antibody in combination with a synthetic antigen capable of generating an immune response in the subject, or a immunogenic fragment or variant thereof.
  • the synthetic antigen can be an isolated DNA that encodes for the antigen.
  • the synthetic antigen can be selected from the group consisting of: hTERT, prostate, WT1, tyrosinase, NYES01, PRAME, MAGE, CMV, herpes, HIV, HPV, HCV, HBV, influenza, RSV, Plasmodium falciparum, and C. difficile.
  • the HPV antigen can be E6 and E7 domains of subtypes selected from the group consisting of: HPV6, HPVl l, HPV 16, HPV 18, HPV31, HPV33, HPV52, and HPV58, and a combination thereof.
  • the HIV antigen can be selected from the group consisting of:
  • Env A, Env B, Env C, Env D, B Nef-Rev, and Gag and a combination thereof.
  • influenza antigen can be selected from the group consisting of: HI HA, H2 HA, H3 HA, H5 HA, BHA antigen, and any combination thereof.
  • the Plasmodium falciparum antigen includes a circumsporozoite
  • the C. difficile antigen can be selected from the group consisting of: Toxin A, and Toxin B, and a combination thereof.
  • the HCV antigen can be selected from the group consisting of: El, E2, NS3, NS4a, NS4b, NS5a, and NS5b, and a combination thereof.
  • the HBV antigen can be selected from the group consisting of: surface antigen type A, surface antigen type B, surface antigen type C, surface antigen type D, surface antigen type E, surface antigen type F, surface antigen type G, surface antigen type H, and core antigen, and a combination thereof.
  • the RSV antigen can be selected from the group consisting of: F, G, NS1, NS2, N, M, M2-1, M2-2, P, SH, and L protein, and a combination thereof.
  • the synthetic antigen can be selected from the group consisting of: hTERT, WT1 antigen, tyrosinase, NYES01, or PRAME.
  • the prostate antigen can be selected from the group consisting of: PSA, PSMA, STEAP, PSCA, and PAP, and a combination thereof.
  • the herpes antigen can be selected from the group consisting of gB, gM, gN, gH, gL, gO, gE, gl, gK, gC, gD, UL128, UL130, UL131A, and UL83, and can be from any one of the following herpes family viruses: HCMV, HSV1, HSV2, VZV, or CMV.
  • the CTLA4 antibody is selected from the group consisting of: ipilimumab, ipilimumab-Probody Tx (BMS-986249), nonfucosylated ipilimumab
  • the CTLA4 antibody is administered in combination with a PD-1 or PD-L1 antibody.
  • PD-1 and PD-L1 antibodies appropriate for use in the methods of the invention include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, BMS- 936559 (See ClinicalTrials.gov Identifier NCT02028403), MPDL3280A (Roche, see ClinicalTrials.gov Identifier NCT02008227), MDX1105-01 (Bristol Myers Squibb, see ClinicalTrials.gov Identifier NCT00729664), MEDI4736 (Medlmmune, See
  • compositions provided herein can also include a
  • aspects of the invention also include methods for increasing an immune response in a subject in need thereof by administering any of the compositions provided herein to the subject.
  • the methods of increasing an immune response can also include an electroporating step.
  • adjuvant as used herein means any molecule added to the immunogenic composition described herein to enhance the immunogenicity of the antigens.
  • Coding sequence or "encoding nucleic acid” as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes a protein.
  • the coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.
  • “Complement” or “complementary” as used herein means a nucleic acid can mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.
  • Electrodeation means the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cellular membrane to the other.
  • fragment or "immunogenic fragment” as used herein means a nucleic acid sequence or a portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal.
  • the fragments can be DNA fragments selected from at least one of the various nucleotide sequences that encode protein fragments set forth below. Fragments can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the nucleic acid sequences set forth below.
  • fragments can comprise at least 20 nucleotides or more, at least 30 nucleotides or more, at least 40 nucleotides or more, at least 50 nucleotides or more, at least 60 nucleotides or more, at least 70 nucleotides or more, at least 80 nucleotides or more, at least 90 nucleotides or more, at least 100 nucleotides or more, at least 150 nucleotides or more, at least 200 nucleotides or more, at least 250 nucleotides or more, at least 300 nucleotides or more, at least 350 nucleotides or more, at least 400 nucleotides or more, at least 450 nucleotides or more, at least 500 nucleotides or more, at least 550 nucleotides or more, at least 600 nucleotides or more, at least 650 nucleotides or more, at least 700 nucleotides or more, at least 750 nucleotides or more,
  • Fragment or immunogenic fragment as used herein also means a polypeptide sequence or a portion thereof that is capable of eliciting an immune response in a mammal.
  • the fragments can be polypeptide fragments selected from at least one of the various amino acid sequence set forth below. Fragments can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the proteins set forth below.
  • fragments can comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 1 10 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 190 amino acids or more, at least 200 amino acids or more, at least 210 amino acids or more, at least 220 amino acids or more, at least 230 amino acids or more, or at least 240 amino acids or more of at least one of the proteins set forth below.
  • Geneetic construct refers to the DNA or RNA molecules that comprise a nucleotide sequence which encodes a protein.
  • the coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered.
  • expressible form refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed.
  • Identity means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity.
  • the residues of single sequence are included in the denominator but not the numerator of the calculation.
  • thymine (T) and uracil (U) can be considered equivalent.
  • Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.
  • Immuno response means the activation of a host's immune system, e.g., that of a mammal, in response to the introduction of antigen.
  • the immune response can be in the form of a cellular or humoral response, or both.
  • nucleic acid or "oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together.
  • the depiction of a single strand also defines the sequence of the complementary strand.
  • a nucleic acid also encompasses the complementary strand of a depicted single strand.
  • Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid.
  • a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.
  • a single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions.
  • a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
  • Nucleic acids can be single stranded or double stranded, or can contain portions of both double stranded and single stranded sequence.
  • the nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine.
  • Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.
  • operably linked means that expression of a gene is under the control of a promoter with which it is spatially connected.
  • a promoter can be positioned 5'
  • the distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.
  • a "peptide,” “protein,” or “polypeptide” as used herein can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.
  • Promoter means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell.
  • a promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same.
  • a promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • a promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
  • a promoter can regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.
  • promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.
  • Signal peptide and leader sequence are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a synthetic antigen, including some of the examples cited herein.
  • Signal peptides/leader sequences typically direct localization of a protein.
  • Signal peptides/leader sequences used herein may serve to facilitate secretion of the protein from the cell in which it is produced.
  • Signal peptides/leader sequences are often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell.
  • Signal peptides/leader sequences are linked at the N terminus of the protein.
  • Subject as used herein can mean a mammal that wants to or is in need of being immunized with the herein described vaccine.
  • the mammal can be a human, chimpanzee, dog, cat, horse, cow, pig, chicken mouse, or rat.
  • substantially identical as used herein can mean that a first and second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more amino acids.
  • Substantially identical can also mean that a first nucleic acid sequence and a second nucleic acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more nucleotides.
  • Treatment can mean protecting of an animal from a disease through means of preventing, suppressing, repressing, or completely eliminating the disease.
  • Preventing the disease involves administering an immunogenic composition of the present invention to an animal prior to onset of the disease.
  • Suppressing the disease involves administering an immunogenic composition of the present invention to an animal after induction of the disease but before its clinical appearance.
  • Repressing the disease involves administering an immunogenic composition of the present invention to an animal after clinical appearance of the disease.
  • nucleic acid means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.
  • Variant can further be defined as a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity.
  • biological activity include the ability to be bound by a specific antibody or to promote an immune response.
  • Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity.
  • hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function.
  • a consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity.
  • Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art.
  • Substitutions can be performed with amino acids having hydrophilicity values within ⁇ 2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
  • a variant may be a nucleic acid sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof.
  • the nucleic acid sequence may be
  • a variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof.
  • the amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.
  • Vector as used herein means a nucleic acid sequence containing an origin of replication.
  • a vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
  • a vector can be a DNA or RNA vector.
  • a vector can be a self- replicating extrachromosomal vector.
  • a vector is a DNA plasmid.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • an immunogenic composition comprising an antigen can be administered first followed by administration of a CTLA4 antibody.
  • a CTLA4 antibody can be administered first followed by an immunogenic composition comprising an antigen.
  • an immunogenic composition comprising an antigen can be administered followed by multiple CTLA4 antibody treatments.
  • an immunogenic composition comprising an antigen and a CTLA4 antibody may each be administered multiple times. The immunogenic composition can increase antigen presentation and the overall immune response to the antigen in a subject.
  • the combination of antigen and immune checkpoint antibody induces the immune system more efficiently than an immunogenic composition comprising the antigen alone.
  • This more efficient immune response provides increased efficacy in the treatment and/or prevention of any disease, in particular cancer, pathogen, or virus.
  • the antigen and CTLA4 antibody of the immunogenic composition can be administered together or separately to the subject in need thereof.
  • a CTLA4 antibody can be administered separately from the antigen of the immunogenic composition.
  • the CTLA4 antibody can be administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, or 96 hours before or after administration of the antigen to the subject.
  • the CTLA4 antibody can be administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or 90 days before or after administration of the antigen to the subject.
  • the CTLA4 antibody can be administered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks before or after administration of the antigen to the subject.
  • the CTLA4 antibody can be administered about 12 hours to about 15 weeks, about 12 hours to about 10 weeks, about 12 hours to about 5 weeks, about 12 hours to about 1 week, about 12 hours to about 60 hours, about 12 hours to about 48 hours, about 24 hours to about 15 weeks, about 60 hours to about 15 weeks, about 96 hours to about 15 weeks, about 1 day to about 15 weeks, about 5 days to about 15 weeks, about 10 days to about 15 weeks, about 15 days to about 15 weeks, about 20 days to about 15 weeks, about 25 days to about 15 weeks, about 30 days to about 15 weeks, about 1 week to about 15 weeks, about 5 weeks to about 15 weeks, or about 10 weeks to about 15 weeks before or after administration of the antigen to the subj ect.
  • the immunogenic composition of the present invention can have features required of effective vaccines such as being safe so the immunogenic composition itself does not cause illness or death; being protective against illness resulting from exposure to live pathogens such as viruses or bacteria; inducing neutralizing antibody to prevent infection of cells; inducing protective T cell against intracellular pathogens; and providing ease of
  • the immunogenic composition can accomplish some or all of these features by combining the antigen with the CTLA4 antibody as discussed below.
  • the immunogenic composition can further modify epitope presentation within the antigen to induce greater immune response to the antigen that an immunogenic composition comprising the antigen alone.
  • the immunogenic composition can further induce an immune response when administered to different tissues such as the muscle or the skin.
  • the immunogenic composition comprising an antigen and a CTLA4 antibody can further comprise one or more additional antibodies targeting one or more additional immune checkpoint proteins.
  • Additional immune checkpoint proteins that can be targeted include, but are not limited to, PD-1, PD-L1, LAG3 and TIM3.
  • the combination comprising an antigen, a CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins can be a single formulation or can be separate and administered in any combination or sequence.
  • an immunogenic composition comprising an antigen can be administered first followed by administration of a combination of a CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins.
  • a combination of a CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins can be administered first followed by an immunogenic composition comprising an antigen.
  • an immunogenic composition comprising an antigen can be administered followed by multiple administrations of a combination of a CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins.
  • an immunogenic composition comprising an antigen can be administered followed by a single administration of a combination of a CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins and followed thereafter by one or more administrations of an antibody selected from a CTLA4 antibody, one or more antibody targeting one or more additional immune checkpoint proteins, and a combination thereof.
  • the immunogenic composition can increase antigen presentation and the overall immune response to the antigen in a subject.
  • the combination of antigen and immune checkpoint antibody induces the immune system more efficiently than an immunogenic composition comprising the antigen alone. This more efficient immune response provides increased efficacy in the treatment and/or prevention of any disease, in particular cancer, pathogen, or virus.
  • the antigen, a CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins of the immunogenic composition can be administered together or separately to the subject in need thereof.
  • a CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins can be administered separately from the antigen of the immunogenic composition.
  • the CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins can be administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, or 96 hours before or after administration of the antigen to the subject.
  • the CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins can be administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or 90 days before or after administration of the antigen to the subject.
  • the CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins can be administered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks before or after administration of the antigen to the subject.
  • the CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins can be administered about 12 hours to about 15 weeks, about 12 hours to about 10 weeks, about 12 hours to about 5 weeks, about 12 hours to about 1 week, about 12 hours to about 60 hours, about 12 hours to about 48 hours, about 24 hours to about 15 weeks, about 60 hours to about 15 weeks, about 96 hours to about 15 weeks, about 1 day to about 15 weeks, about 5 days to about 15 weeks, about 10 days to about 15 weeks, about 15 days to about 15 weeks, about 20 days to about 15 weeks, about 25 days to about 15 weeks, about 30 days to about 15 weeks, about 1 week to about 15 weeks, about 5 weeks to about 15 weeks, or about 10 weeks to about 15 weeks before or after administration of the antigen to the subject.
  • a CTLA4 antibody can be administered separately from one or more antibody targeting one or more additional immune checkpoint proteins of the immunogenic composition.
  • the CTLA4 antibody can be administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, or 96 hours before or after administration of the one or more antibody targeting one or more additional immune checkpoint proteins to the subject.
  • the CTLA4 antibody can be administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or 90 days before or after administration of the one or more antibody targeting one or more additional immune checkpoint proteins to the subject.
  • the CTLA4 antibody can be administered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks before or after administration of the one or more antibody targeting one or more additional immune checkpoint proteins to the subject.
  • the CTLA4 antibody can be administered about 12 hours to about 15 weeks, about 12 hours to about 10 weeks, about 12 hours to about 5 weeks, about 12 hours to about 1 week, about 12 hours to about 60 hours, about 12 hours to about 48 hours, about 24 hours to about 15 weeks, about 60 hours to about 15 weeks, about 96 hours to about 15 weeks, about 1 day to about 15 weeks, about 5 days to about 15 weeks, about 10 days to about 15 weeks, about 15 days to about 15 weeks, about 20 days to about 15 weeks, about 25 days to about 15 weeks, about 30 days to about 15 weeks, about 1 week to about 15 weeks, about 5 weeks to about 15 weeks, or about 10 weeks to about 15 weeks before or after administration of the one or more antibody targeting one or more additional immune checkpoint proteins to the subject.
  • the one or more antibody for use in the method of the invention can be a synthetic antibody comprised of DNA sequence encoding at least the variable regions of an immunoglobulin.
  • Such antibody can be generated by identifying or screening for the antibody described above, which is reactive to or binds the antigen described above.
  • the method of identifying or screening for the antibody can use the antigen in methodologies known in those skilled in art to identify or screen for the antibody. Such methodologies can include, but are not limited to, selection of the antibody from a library (e.g., phage display) and immunization of an animal followed by isolation and/or purification of the antibody. See for example methods available in Rajan, S., and Sidhu, S., Methods in Enzymology. vol 502, Chapter One "Simplified Synthetic Antibody Libraries (2012), which is incorporated herein in its entirety.
  • the antibody can be a polyclonal or monoclonal antibody.
  • the antibody can be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody.
  • the humanized antibody can be an antibody from a non-human species that binds the desired antigen having one or more
  • CDRs complementarity determining regions
  • the antibody can be a known product such as, for example, ipilimumab, ipilimumab- Probody Tx (BMS-986249), ipilimumab-NF (BMS-986218), tremelimumab, CS1002, MDX- 010 (See ClinicalTrials.gov Identifier NCT00140855), AGEN-1884 (Agenus, see
  • ClinicalTrials.gov Identifier NCT02694822 nivolumab, pembrolizumab, pidilizumab, BMS- 936559 (See ClinicalTrials.gov Identifier NCT02028403), MPDL3280A (Roche, see ClinicalTrials.gov Identifier NCT02008227), MDX1105-01 (Bristol Myers Squibb, see ClinicalTrials.gov Identifier NCT00729664), MEDI4736 (Medlmmune, See
  • the antibody can be encoded by a nucleic acid sequence (cDNA) that encodes for the elements as follows:
  • the antibody can include a heavy chain polypeptide and a light chain polypeptide.
  • the heavy chain polypeptide can include a variable heavy chain (VH) region and/or at least one constant heavy chain (CH) region.
  • the at least one constant heavy chain region can include a constant heavy chain region 1 (CHI), a constant heavy chain region 2 (CH2), and a constant heavy chain region 3 (CH3), and/or a hinge region.
  • the heavy chain polypeptide can include a VH region and a CHI region.
  • the heavy chain polypeptide can include a VH region, a CHI region, a hinge region, a CH2 region, and a CH3 region.
  • the heavy chain polypeptide can include a complementarity determining region ("CDR") set.
  • the CDR set can contain three hypervariable regions of the VH region.
  • CDRl CDR2
  • CDR3 CDR3 of the heavy chain polypeptide
  • the light chain polypeptide can include a variable light chain (VL) region and/or a constant light chain (CL) region.
  • the light chain polypeptide can include a complementarity determining region ("CDR") set.
  • the CDR set can contain three hypervariable regions of the VL region. Proceeding from N-terminus of the light chain polypeptide, these CDRs are denoted "CDRl,” “CDR2,” and “CDR3,” respectively. CDRl, CDR2, and CDR3 of the light chain polypeptide can contribute to binding or recognition of the antigen.
  • the antibody may comprise a heavy chain and a light chain complementarity determining region ("CDR") set, respectively interposed between a heavy chain and a light chain framework (“FR") set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other.
  • the CDR set may contain three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as "CDRl,” “CDR2,” and “CDR3,” respectively.
  • An antigen-binding site therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
  • the antibody can be an immunoglobulin (Ig).
  • the Ig can be, for example, IgA, IgM, IgD, IgE, and IgG.
  • the immunoglobulin can include the heavy chain polypeptide and the light chain polypeptide.
  • the heavy chain polypeptide of the immunoglobulin can include a VH region, a CHI region, a hinge region, a CH2 region, and a CH3 region.
  • the light chain polypeptide of the immunoglobulin can include a VL region and CL region.
  • the proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site.
  • the enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab')2 fragment, which comprises both antigen-binding sites.
  • the antibody can be the Fab or F(ab')2.
  • the Fab can include the heavy chain polypeptide and the light chain polypeptide.
  • the heavy chain polypeptide of the Fab can include the VH region and the CHI region.
  • the light chain of the Fab can include the VL region and CL region. e. Antigen
  • the immunogenic composition can also comprise an antigen, or fragment or variant thereof.
  • the antigen can be anything that induces an immune response in a subject.
  • the antigen can be a nucleic acid sequence, an amino acid sequence, or a combination thereof.
  • the nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof.
  • the nucleic acid sequence can also include additional sequences that encode linker or tag sequences that are linked to the antigen by a peptide bond.
  • the amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.
  • the antigen can be contained in a protein, a nucleic acid, or a fragment thereof, or a variant thereof, or a combination thereof from any number of organisms, for example, a virus, a parasite, a bacterium, a fungus, or a mammal.
  • the antigen can be associated with an autoimmune disease, allergy, or asthma.
  • the antigen can be associated with cancer, herpes, influenza, hepatitis B, hepatitis C, human papilloma virus (HPV), or human immunodeficiency virus (HIV).
  • Some antigens can induce a strong immune response. Other antigens can induce a weak immune response. The antigen can elicit a greater immune response when combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins as described above.
  • the antigen can be a viral antigen, or fragment thereof, or variant thereof.
  • the viral antigen can be from a virus from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae,
  • the viral antigen can be from papilloma viruses, for example, human papillomoa virus (HPV), human immunodeficiency virus (HIV), polio virus, hepatitis viruses, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV), human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), herpes simplex 1 (HSV1; oral herpes), herpes simplex 2 (HSV2; genital herpes), herpes zoster (VZV; varicella-zoster, a.k.a., chickenpox), Epstein-Bar
  • the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a hepatitis virus antigen (i.e., hepatitis antigen), or fragment thereof, or variant thereof.
  • a hepatitis virus antigen i.e., hepatitis antigen
  • the hepatitis antigen can be an antigen or immunogen from hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and/or hepatitis E virus (HEV).
  • the hepatitis antigen can be a nucleic acid molecule(s), such as a plasmid(s), which encodes one or more of the antigens from HAV, HBV, HCV, HDV, and HEV.
  • the hepatitis antigen can be full-length or immunogenic fragments of full-length proteins.
  • the hepatitis antigen can comprise consensus sequences and/or modification for improved expression. Genetic modifications including codon optimization, RNA
  • the consensus hepatitis antigen may comprise a signal peptide such as an immunoglobulin signal peptide such as an IgE or IgG signal peptide, and in some embodiments, may comprise an HA tag.
  • the immunogens can be designed to elicit stronger and broader cellular immune responses than corresponding codong optimized immunogens.
  • the hepatitis antigen can be an antigen from HAV.
  • the hepatitis antigen can be a HAV capsid protein, a HAV non-structural protein, a fragment thereof, a variant thereof, or a combination thereof.
  • the hepatitis antigen can be an antigen from HCV.
  • the hepatitis antigen can be a HCV nucleocapsid protein (i.e., core protein), a HCV envelope protein (e.g., El and E2), a HCV non-structural protein (e.g., NS1, NS2, NS3, NS4a, NS4b, NS5a, and NS5b), a fragment thereof, a variant thereof, or a combination thereof.
  • the hepatitis antigen can be an antigen from HDV.
  • the hepatitis antigen can be a HDV delta antigen, fragment thereof, or variant thereof.
  • the hepatitis antigen can be an antigen from HEV.
  • the hepatitis antigen can be a HEV capsid protein, fragment thereof, or variant thereof.
  • the hepatitis antigen can be an antigen from HBV.
  • the hepatitis antigen can be a HBV core protein, a HBV surface protein, a HBV DNA polymerase, a HBV protein encoded by gene X, fragment thereof, variant thereof, or combination thereof.
  • the hepatitis antigen can be a HBV genotype A core protein, a HBV genotype B core protein, a HBV genotype C core protein, a HBV genotype D core protein, a HBV genotype E core protein, a HBV genotype F core protein, a HBV genotype G core protein, a HBV genotype H core protein, a HBV genotype A surface protein, a HBV genotype B surface protein, a HBV genotype C surface protein, a HBV genotype D surface protein, a HBV genotype E surface protein, a HBV genotype F surface protein, a HBV genotype G surface protein, a HBV genotype H surface protein, fragment thereof, variant thereof, or combination thereof.
  • the hepatitis antigen can be a consensus HBV core protein, or a consensus HBV surface protein.
  • the hepatitis antigen can be a HBV genotype A consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype A core protein, or a HBV genotype A consensus core protein sequence.
  • the hepatitis antigen can be a HBV genotype B consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype B core protein, or a HBV genotype B consensus core protein sequence.
  • the hepatitis antigen can be a HBV genotype C consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype C core protein, or a HBV genotype C consensus core protein sequence.
  • the hepatitis antigen can be a HBV genotype D consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype D core protein, or a HBV genotype D consensus core protein sequence.
  • the hepatitis antigen can be a HBV genotype E consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype E core protein, or a HBV genotype E consensus core protein sequence.
  • the hepatitis antigen can be a HBV genotype F consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype F core protein, or a HBV genotype F consensus core protein sequence.
  • the hepatitis antigen can be a HBV genotype G consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype G core protein, or a HBV genotype G consensus core protein sequence.
  • the hepatitis antigen can be a HBV genotype H consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype H core protein, or a HBV genotype H consensus core protein sequence.
  • the hepatitis antigen can be a HBV genotype A consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype A surface protein, or a HBV genotype A consensus surface protein sequence.
  • the hepatitis antigen can be a HBV genotype B consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype B surface protein, or a HBV genotype B consensus surface protein sequence.
  • the hepatitis antigen can be a HBV genotype C consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype C surface protein, or a HBV genotype C consensus surface protein sequence.
  • the hepatitis antigen can be a HBV genotype D consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype D surface protein, or a HBV genotype D consensus surface protein sequence.
  • the hepatitis antigen can be a HBV genotype E consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype E surface protein, or a HBV genotype E consensus surface protein sequence.
  • the hepatitis antigen can be a HBV genotype F consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype F surface protein, or a HBV genotype F consensus surface protein sequence.
  • the hepatitis antigen can be a HBV genotype G consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype G surface protein, or a HBV genotype G consensus surface protein sequence.
  • the hepatitis antigen can be a HBV genotype H consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype H surface protein, or a HBV genotype H consensus surface protein sequence.
  • HBV genotype H consensus surface DNA sequence construct an IgE leader sequence linked to a consensus sequence for HBV genotype H surface protein, or a HBV genotype H consensus surface protein sequence.
  • HPV Human Papilloma Virus
  • the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a human papilloma virus (HPV) antigen, or fragment thereof, or variant thereof.
  • HPV antigen can be from HPV types 16, 18, 31, 33, 35, 45, 52, and 58 which cause cervical cancer, rectal cancer, and/or other cancers.
  • HPV antigen can be from HPV types 6 and 11, which cause genital warts, and are known to be causes of head and neck cancer.
  • the HPV antigens can be the HPV E6 or E7 domains from each HPV type.
  • the HPV 16 antigen can include the HPV 16 E6 antigen, the HPV 16 E7 antigen, fragments, variants, or combinations thereof.
  • the HPV antigen can be HPV 6 E6 and/or E7, HPV 11 E6 and/or E7, HPV 18 E6 and/or E7, HPV 31 E6 and/or E7, HPV 33 E6 and/or E7, HPV 52 E6 and/or E7, or HPV 58 E6 and/or E7, fragments, variants, or combinations thereof.
  • the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can also be associated or combined with an RSV antigen or fragment thereof, or variant thereof.
  • the RSV antigen can be a human RSV fusion protein (also referred to herein as "RSV F", "RSV F protein” and "F protein”), or fragment or variant thereof.
  • the human RSV fusion protein can be conserved between RSV subtypes A and B.
  • the RSV antigen can be a RSV F protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23994.1).
  • the RSV antigen can be a RSV F protein from the RSV A2 strain (GenBank AAB59858.1), or a fragment or variant thereof.
  • the RSV antigen can be a monomer, a dimer or trimer of the RSV F protein, or a fragment or variant thereof.
  • the RSV antigen can be an optimized amino acid RSV F amino acid sequence, or fragment or variant thereof.
  • RSV F The postfusion form of RSV F elicits high titer neutralizing antibodies in immunized animals and protects the animals from RSV challenge.
  • the present invention utilizes this immunoresponse in the claimed vaccines.
  • the RSV F protein can be in a prefusion form or a postfusion form.
  • the RSV antigen can also be human RSV attachment glycoprotein (also referred to herein as "RSV G", "RSV G protein” and "G protein”), or fragment or variant thereof.
  • the human RSV G protein differs between RSV subtypes A and B.
  • the antigen can be RSV G protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23993).
  • the RSV antigen can be RSV G protein from: the RSV subtype B isolate H5601, the RSV subtype B isolate H1068, the RSV subtype B isolate H5598, the RSV subtype B isolate HI 123, or a fragment or variant thereof.
  • the RSV antigen can be an optimized amino acid RSV G amino acid sequence, or fragment or variant thereof.
  • the RSV antigen can be human RSV non-structural protein 1 ("NS 1 protein"), or fragment or variant thereof.
  • NS 1 protein human RSV non-structural protein 1
  • the RSV antigen can be RSV NS 1 protein, or fragment or variant thereof, from the RSV Long strain (GenBank
  • the RSV antigen human can also be RSV non-structural protein 2 ("NS2 protein"), or fragment or variant thereof.
  • NS2 protein RSV non-structural protein 2
  • the RSV antigen can be RSV NS2 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23988.1).
  • the RSV antigen can further be human RSV nucleocapsid ("N") protein, or fragment or variant thereof.
  • the RSV antigen can be RSV N protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23989.1).
  • the RSV antigen can be human RSV Phosphoprotein ("P”) protein, or fragment or variant thereof.
  • the RSV antigen can be RSV P protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23990.1).
  • the RSV antigen also can be human RSV Matrix protein ("M") protein, or fragment or variant thereof.
  • M human RSV Matrix protein
  • the RSV antigen can be RSV M protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23991.1).
  • the RSV antigen can be human RSV small hydrophobic ("SH") protein, or fragment or variant thereof.
  • the RSV antigen can be RSV SH protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23992.1).
  • the RSV antigen can also be human RSV Matrix protein2-l ("M2-1") protein, or fragment or variant thereof.
  • the RSV antigen can be RSV M2-1 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23995.1).
  • the RSV antigen can further be human RSV Matrix protein 2-2 ("M2-2") protein, or fragment or variant thereof.
  • the RSV antigen can be RSV M2-2 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23997.1).
  • the RSV antigen human can be RSV
  • the RSV antigen can be RSV L protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23996.1).
  • the RSV antigen can have an optimized amino acid sequence of NS1, NS2, N, P, M, SH, M2-1, M2-2, or L protein.
  • the RSV antigen can be a human RSV protein or recombinant antigen, such as any one of the proteins encoded by the human RSV genome.
  • the RSV antigen can be, but is not limited to, the RSV F protein from the RSV Long strain, the RSV G protein from the RSV Long strain, the optimized amino acid RSV G amino acid sequence, the human RSV genome of the RSV Long strain, the optimized amino acid RSV F amino acid sequence, the RSV NS1 protein from the RSV Long strain, the RSV NS2 protein from the RSV Long strain, the RSV N protein from the RSV Long strain, the RSV P protein from the RSV Long strain, the RSV M protein from the RSV Long strain, the RSV SH protein from the RSV Long strain, the RSV M2-1 protein from the RSV Long strain, the RSV M2-2 protein from the RSV Long strain, the RSV L protein from the RSV Long strain, the RSV G protein from the RSV subtype B isolate H5601, the RSV G protein from the RSV subtype B isolate H1068, the RSV G protein from the RSV subtype B isolate H5598, the RSV G protein from the RSV sub
  • the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with an influenza antigen or fragment thereof, or variant thereof.
  • the influenza antigens are those capable of eliciting an immune response in a mammal against one or more influenza serotypes.
  • the antigen can comprise the full length translation product HA0, subunit HA1, subunit HA2, a variant thereof, a fragment thereof or a combination thereof.
  • the influenza hemagglutinin antigen can be a consensus sequence derived from multiple strains of influenza A serotype HI, a consensus sequence derived from multiple strains of influenza A serotype H2, a hybrid sequence containing portions of two different consensus sequences derived from different sets of multiple strains of influenza A serotype HI or a consensus sequence derived from multiple strains of influenza B.
  • the influenza hemagglutinin antigen can be from influenza B.
  • the influenza antigen can also contain at least one antigenic epitope that can be effective against particular influenza immunogens against which an immune response can be induced.
  • the antigen may provide an entire repertoire of immunogenic sites and epitopes present in an intact influenza virus.
  • the antigen may be a consensus hemagglutinin antigen sequence that can be derived from hemagglutinin antigen sequences from a plurality of influenza A virus strains of one serotype such as a plurality of influenza A virus strains of serotype HI or of serotype H2.
  • the antigen may be a hybrid consensus hemagglutinin antigen sequence that can be derived from combining two different consensus hemagglutinin antigen sequences or portions thereof.
  • Each of two different consensus hemagglutinin antigen sequences may be derived from a different set of a plurality of influenza A virus strains of one serotype such as a plurality of influenza A virus strains of serotype HI .
  • the antigen may be a consensus hemagglutinin antigen sequence that can be derived from hemagglutinin antigen sequences from a plurality of influenza B virus strains.
  • the influenza antigen can be HI HA, H2 HA, H3 HA, H5 HA, or a BHA antigen.
  • the influenza antigen can be a consensus hemagglutinin antigen comprising a consensus HI amino acid sequence or a consensus H2 amino acid sequence.
  • the consensus hemagglutinin antigen may be a synthetic hybrid consensus HI sequence comprising portions of two different consensus HI sequences, which are each derived from a different set of sequences from the other.
  • An example of a consensus HA antigen that is a synthetic hybrid consensus HI protein is a protein comprising the U2 amino acid sequence.
  • the consensus hemagglutinin antigen may be a consensus hemagglutinin protein derived from hemagglutinin sequences from influenza B strains, such as a protein comprising the consensus BHA amino acid sequence.
  • the consensus hemagglutinin antigen may further comprise one or more additional amino acid sequence elements.
  • the consensus hemagglutinin antigen may further comprise on its N-terminal an IgE or IgG leader amino acid sequence.
  • the consensus hemagglutinin antigen may further comprise an immunogenic tag which is a unique immunogenic epitope that can be detected by readily available antibodies.
  • An example of such an immunogenic tag is the 9 amino acid influenza HA Tag which may be linked on the consensus hemagglutinin C terminus.
  • consensus hemagglutinin antigen may further comprise on its N-terminal an IgE or IgG leader amino acid sequence and on its C terminal an HA tag.
  • the consensus hemagglutinin antigen may be a consensus hemagglutinin protein that consists of consensus influenza amino acid sequences or fragments and variants thereof.
  • the consensus hemagglutinin antigen may be a consensus hemagglutinin protein that comprises non-influenza protein sequences and influenza protein sequences or fragments and variants thereof.
  • Examples of a consensus HI protein include those that may consist of the consensus HI amino acid sequence or those that further comprise additional elements such as an IgE leader sequence, or an HA Tag or both an IgE leader sequence and an HA Tag.
  • Examples of consensus H2 proteins include those that may consist of the consensus H2 amino acid sequence or those that further comprise an IgE leader sequence, or an HA Tag, or both an IgE leader sequence and an HA Tag.
  • hybrid consensus HI proteins include those that may consist of the consensus U2 amino acid sequence or those that further comprise an IgE leader sequence, or an HA Tag, or both an IgE leader sequence and an HA Tag.
  • hybrid consensus influenza B hemagglutinin proteins include those that may consist of the consensus BHA amino acid sequence or it may comprise an IgE leader sequence, or an HA Tag, or both an IgE leader sequence and an HA Tag.
  • the consensus hemagglutinin protein can be encoded by a consensus
  • the consensus hemagglutinin nucleic acid refers to a nucleic acid sequence that encodes a consensus protein sequence and the coding sequences used may differ from those used to encode the particular amino acid sequences in the plurality of different hemagglutinin sequences from which the consensus hemagglutinin protein sequence is derived.
  • the consensus nucleic acid sequence may be codon optimized and/or RNA optimized.
  • the consensus hemagglutinin nucleic acid sequence may comprise a Kozak's sequence in the 5 ' untranslated region.
  • the consensus hemagglutinin nucleic acid sequence may comprise nucleic acid sequences that encode a leader sequence.
  • the coding sequence of an N terminal leader sequence is 5' of the hemagglutinin coding sequence.
  • the N-terminal leader can facilitate secretion.
  • the N- terminal leader can be an IgE leader or an IgG leader.
  • the consensus hemagglutinin nucleic acid sequence can comprise nucleic acid sequences that encode an immunogenic tag.
  • the immunogenic tag can be on the C terminus of the protein and the sequence encoding it is 3 ' of the HA coding sequence.
  • the immunogenic tag provides a unique epitope for which there are readily available antibodies so that such antibodies can be used in assays to detect and confirm expression of the protein.
  • the immunogenic tag can be an H Tag at the C-terminus of the protein
  • HIV Human Immunodeficiency Virus
  • the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with an HIV antigen or fragment thereof, or variant thereof.
  • HIV antigens can include modified consensus sequences for immunogens. Genetic modifications including codon optimization, RNA optimization, and the addition of a high efficient immunoglobin leader sequence to increase the immunogenicity of constructs can be included in the modified consensus sequences.
  • the novel immunogens can be designed to elicit stronger and broader cellular immune responses than a corresponding codon optimized immunogens.
  • the HIV antigen can be a subtype A consensus envelope
  • DNA sequence construct an IgE leader sequence linked to a consensus sequence for Subtype
  • a envelope protein or a subtype A consensus Envelope protein sequence.
  • the HIV antigen can be a subtype B consensus envelope
  • DNA sequence construct an IgE leader sequence linked to a consensus sequence for Subtype
  • the HIV antigen can be a subtype C consensus envelope
  • DNA sequence construct an IgE leader sequence linked to a consensus sequence for subtype
  • the HIV antigen can be a subtype D consensus envelope
  • DNA sequence construct an IgE leader sequence linked to a consensus sequence for Subtype
  • the HIV antigen can be a subtype B Nef-Rev consensus envelope DNA sequence construct, an IgE leader sequence linked to a consensus sequence for Subtype B Nef-Rev protein, or a Subtype B Nef-Rev consensus protein sequence.
  • the HIV antigen can be a Gag consensus DNA sequence of subtype A, B, C and D DNA sequence construct, an IgE leader sequence linked to a consensus sequence for Gag consensus subtype A, B, C and D protein, or a consensus Gag subtype A, B, C and D protein sequence.
  • the HIV antigen can be a Pol DNA sequence or a Pol protein sequence.
  • the HIV antigen can be nucleic acid or amino acid sequences of Env A, Env B, Env C, Env D, B Nef-Rev , Gag, or any combination thereof.
  • the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a herpes antigen or fragment thereof, or variant thereof.
  • the herpes antigen is from HCMV, HSV1, HSV2, CeHVl, VZV or EBV.
  • the herpes antigens comprise immunogenic proteins including gB, gM, gN, gH, gL, gO, gE, gl, gK, gC, gD, UL128, UL130, UL-131A, UL-83 (pp65), whether from HCMV, HSV1, HSV2, CeHVl, VZV or EBV.
  • the antigens can be HSVl-gH, HSVl-gL, HSVl-gC, HSVl-gD, HSV2-gH, HSV2-gL, HSV2-gC, HSV2-gD, VZV-gH, VZV-gL, VZV-gM, VZV-gN, CeHVl -gH, CeHVl-gL, CeHVl-gC, CeHVl-gD, VZV-gE, or VZV-gl.
  • the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a parasite antigen or fragment or variant thereof.
  • the parasite can be a protozoa, helminth, or ectoparasite.
  • the helminth i.e., worm
  • the helminth can be a flatworm (e.g., flukes and tapeworms), a thorny -headed worm, or a round worm (e.g., pinworms).
  • the ectoparasite can be lice, fleas, ticks, and mites.
  • the parasite can be any parasite causing the following diseases: Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis,
  • Echinococcosis Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis, Taeniasis, Toxocariasis,
  • the parasite can be Acanthamoeba, Anisakis, Ascaris lumbricoides , Botfly, Balantidium coli, Bedbug, Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania, Linguatula serrata, Liver fluke, Loa loa, Paragonimus - lung fluke, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma gondii, Trypanosoma, Whipworm, or Wuchereria bancrofti.
  • the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a malaria antigen (i.e., Plasmodium falciparum (PF) antigen or PF immunogen), or fragment thereof, or variant thereof.
  • the antigen can be from a parasite causing malaria.
  • the malaria causing parasite can be Plasmodium falciparum.
  • the Plasmodium falciparum antigen can include the circumsporozoite (CS) antigen.
  • the malaria antigen can be nucleic acid molecules such as plasmids which encode one or more of the P. falciparum immunogens CS; LSA1 ; TRAP; CelTOS; and Amal.
  • the immunogens may be full length or immunogenic fragments of full length proteins.
  • the immunogens comprise consensus sequences and/or modifications for improved expression.
  • the malaria antigen can be a consensus sequence of TRAP, which is also referred to as SSP2, designed from a compilation of all full-length Plasmodium falciparum TRAP/SSP2 sequences in the GenBank database (28 sequences total).
  • Consensus TRAP immunogens i.e., ConTRAP immunogen
  • the malaria antigen can be CelTOS, which is also referred to as Ag2 and is a highly conserved Plasmodium antigen.
  • Consensus CelTOS antigens i.e., ConCelTOS immunogen
  • the malaria antigen can be Amal, which is a highly conserved Plasmodium antigen.
  • the malaria antigen can also be a consensus sequence of Amal (i.e., ConAmal immunogen) comprising in some instances, a signal peptide such as an immunoglobulin signal peptide such as an IgE or IgG signal peptide and in some embodiments, may comprise an HA Tag.
  • the malaria antigen can be a consensus CS antigen (i.e., Consensus CS immunogen) comprising in some instances, a signal peptide such as an immunoglobulin signal peptide such as an IgE or IgG signal peptide and in some embodiments, may comprise an HA Tag.
  • Consensus CS immunogen i.e., Consensus CS immunogen
  • a signal peptide such as an immunoglobulin signal peptide such as an IgE or IgG signal peptide and in some embodiments, may comprise an HA Tag.
  • the malaria antigen can be a fusion protein comprising a combination of two or more of the PF proteins set forth herein.
  • fusion proteins may comprise two or more of Consensus CS immunogen, ConLSAl immunogen, ConTRAP immunogen, ConCelTOS immunogen and ConAmal immunogen linked directly adjacent to each other or linked with a spacer or one or more amino acids in between.
  • the fusion protein comprises two PF immunogens; in some embodiments the fusion protein comprises three PF immunogens, in some embodiments the fusion protein comprises four PF immunogens, and in some embodiments the fusion protein comprises five PF immunogens.
  • Fusion proteins with two Consensus PF immunogens may comprise: CS and LSA1; CS and TRAP; CS and CelTOS; CS and Amal; LSA1 and TRAP; LSA1 and CelTOS; LSA1 and Amal; TRAP and CelTOS; TRAP and Amal; or CelTOS and Amal.
  • Fusion proteins with three Consensus PF immunogens may comprise: CS, LSA1 and TRAP; CS, LSA1 and CelTOS; CS, LSA1 and Amal; LSA1, TRAP and CelTOS; LSA1, TRAP and Amal; or TRAP, CelTOS and Amal.
  • Fusion proteins with four Consensus PF immunogens may comprise: CS, LSA1, TRAP and CelTOS; CS, LSA1, TRAP and Amal; CS, LSA1, CelTOS and Amal; CS, TRAP, CelTOS and Amal; or LSA1, TRAP, CelTOS and Amal.
  • Fusion proteins with five Consensus PF immunogens may comprise CS or CS-alt, LSA1, TRAP, CelTOS and Amal.
  • the fusion proteins comprise a signal peptide linked to the N terminus. In some embodiments, the fusion proteins comprise multiple signal peptides linked to the N terminal of each Consensus PF immunogen.
  • a spacer may be included between PF immunogens of a fusion protein. In some embodiments, the spacer between PF immunogens of a fusion protein may be a proteolyic cleavage site. In some embodiments, the spacer may be a proteolyic cleavage site recognized by a protease found in cells to which the immunogenic composition is intended to be administered and/or taken up.
  • a spacer may be included between PF immunogens of a fusion protein wherein the spacer is a proteolyic cleavage site recognized by a protease found in cells to which the immunogenic composition is intended to be administered and/or taken up and the fusion proteins comprises multiple signal peptides linked to the N terminal of each Consensus PF immunogens such that upon cleavage the signal peptide of each Consensus PF immunogens translocates the Consensus PF immunogen to outside the cell.
  • the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a bacterial antigen or fragment or variant thereof.
  • the bacterium can be from any one of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus- Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.
  • the bacterium can be a gram positive bacterium or a gram negative bacterium.
  • the bacterium can be an aerobic bacterium or an anerobic bacterium.
  • the bacterium can be an autotrophic bacterium or a heterotrophic bacterium.
  • the bacterium can be a mesophile, a neutrophile, an extremophile, an acidophile, an alkaliphile, a thermophile, a psychrophile, an halophile, or an osmophile.
  • the bacterium can be an anthrax bacterium, an antibiotic resistant bacterium, a disease causing bacterium, a food poisoning bacterium, an infectious bacterium, Salmonella bacterium, Staphylococcus bacterium, Streptococcus bacterium, or tetanus bacterium.
  • the bacterium can be a mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.
  • the bacterium can be Mycobacterium tuberculosis.
  • the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a Mycobacterium tuberculosis antigen (i.e., TB antigen or TB immunogen), or fragment thereof, or variant thereof.
  • the TB antigen can be from the Ag85 family of TB antigens, for example, Ag85A and Ag85B.
  • the TB antigen can be from the Esx family of TB antigens, for example, EsxA, EsxB, EsxC, EsxD, EsxE, EsxF, EsxH, EsxO, EsxQ, EsxR, EsxS, EsxT, EsxU, EsxV, and EsxW.
  • the TB antigen can be nucleic acid molecules such as plasmids which encode one or more of the Mycobacterium tuberculosis immunogens from the Ag85 family and the Esx family.
  • the immunogens can be full-length or immunogenic fragments of full-length proteins.
  • the immunogens can comprise consensus sequences and/or modifications for improved expression.
  • Consensus immunogens may comprise a signal peptide such as an immunoglobulin signal peptide such as an IgE or IgG signal peptide and in some embodiments, may comprise an HA tag.
  • the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a fungal antigen or fragment or variant thereof.
  • the fungus can be Aspergillus species, Blastomyces dermatitidis , Candida yeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or
  • tumor antigen or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder,” refers to antigens that are common to specific hyperproliferative disorders such as cancer.
  • antigens discussed herein are merely included by way of example. The list is not intended to be exclusive and further examples will be readily apparent to those of skill in the art.
  • Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses.
  • the selection of the antigen binding moiety of the invention will depend on the particular type of cancer to be treated.
  • Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), ⁇ -human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA,
  • CEA carcinoembryonic antigen
  • AFP alphafetoprotein
  • PCTA-1 prostate-carcinoma tumor antigen-1
  • MAGE prostate-carcinoma tumor antigen-1
  • ELF2M neutrophil elastase
  • ephrinB2 ephrinB2
  • CD22 insulin growth factor
  • the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor.
  • Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer.
  • Other target molecules belong to the group of transformation-related molecules such as the oncogene HER-2/Neu/ErbB-2.
  • Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA).
  • B-cell lymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor.
  • B-cell differentiation antigens such as CD 19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma.
  • Some of these antigens (CEA, HER-2, CD 19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.
  • the type of tumor antigen referred to in the invention may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).
  • TSA tumor-specific antigen
  • TAA associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen.
  • the expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen.
  • TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells.
  • TSA or TAA antigens include the following:
  • Differentiation antigens such as MART- 1/MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7.
  • MART-I MART- 1/MelanA
  • gplOO Pmel 17
  • the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a tumor antigen or fragment or variant thereof.
  • Cancer markers are known proteins that are present or upregulated vis-a-vis certain cancer cells. By methodology of generating antigens that represent such markers in a way to break tolerance to self, a cancer vaccine can be generated.
  • Such cancer vaccines can include the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins to enhance the immune response.
  • TERT is a telomerase reverse transcriptase that synthesizes a TTAGGG tag on the end of telomeres to prevent cell death due to chromosomal shortening.
  • Hyperproliferative cells with abnormally high expression of TERT may be targeted by immunotherapy.
  • Recent studies demonstrate that TERT expression in dendritic cells transfected with TERT genes can induce CD8+ cytotoxic T cells and elicit a CD4+ T cells in an antigen-specific fashion.
  • TERT can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules, including but not limited to those in the Examples, below.
  • a consensus TERT antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is a TERT antigen derived from multiple human TERT sequences.
  • a human TERT antigen comprises a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NO:2 and SEQ ID NO:6.
  • human TERT antigen comprises a nucleotide sequence selected from SEQ ID NO: l and SEQ ID NO:5.
  • a TERT antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is TERT antigen derived from non-human TERT sequences.
  • a TERT antigen is derived from multiple mouse TERT sequences.
  • a synthetic mouse TERT antigen encodes an amino acid sequence as set forth in SEQ ID NO: 10.
  • a synthetic mouse TERT antigen comprises a nucleotide sequence as set forth in SEQ ID NO:9.
  • a TERT antigen is operably linked to a sequence encoding a signal peptide.
  • a signal peptide has an amino acid sequence as set forth in SEQ ID NO: 13.
  • SEQ ID NO:3 and SEQ ID NO:7 provide exemplary hTERT nucleic acid sequences operably linked to a nucleotide sequence encoding an IgE leader sequence.
  • SEQ ID NO:4 and SEQ ID NO: 8 provide exemplary hTERT amino acid sequences operably linked to an IgE leader sequence.
  • SEQ ID NO: 11 provides an exemplary mTERT nucleic acid sequence operably linked to a nucleotide sequence encoding an IgE leader sequence.
  • SEQ ID NO: 12 provides an exemplary mTERT amino acid sequence operably linked to an IgE leader sequence.
  • the consensus antigen can comprise epitopes that make them particularly effective as immunogens against prostate cancer cells can be induced.
  • the consensus prostate antigen can comprise the full length translation product, a variant thereof, a fragment thereof or a combination thereof.
  • the prostate antigens can include one or more of the following: PSA antigen, PSMA antigen, STEAP antigen, PSCA antigen, Prostatic acid phosphatase (PAP) antigen, and other known prostate tumor antigens. Proteins may comprise sequences homologous to the prostate antigens, fragments of the prostate antigens and proteins with sequences homologous to fragments of the prostate antigens.
  • the prostate antigens can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
  • a prostate antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is a consensus antigen derived from multiple human prostate antigen sequences.
  • a consensus prostate antigen is derived from multiple non- human prostate antigen sequences.
  • a consensus prostate antigen is operably linked to a sequence encoding a signal peptide.
  • the antigen can be Wilm's tumor suppressor gene 1 (WT1), a fragment thereof, a variant thereof, or a combination thereof.
  • WT1 is a transcription factor containing at the N- terminus, a proline/glutamine-rich DNA-binding domain and at the C-terminus, four zinc finger motifs.
  • WT1 plays a role in the normal development of the urogenital system and interacts with numerous factors, for example, p53, a known tumor suppressor and the serine protease HtrA2, which cleaves WT1 at multiple sites after treatment with a cytotoxic drug.
  • Wild Type of WT1 can lead to tumor or cancer formation, for example, Wilm's tumor or tumors expressing WT1.
  • Wilm's tumor often forms in one or both kidneys before metastasizing to other tissues, for example, but not limited to, liver tissue, urinary tract system tissue, lymph tissue, and lung tissue. Accordingly, Wilm's tumor can be considered a metastatic tumor. Wilm's tumor usually occurs in younger children (e.g., less than 5 years old) and in both sporadic and hereditary forms.
  • the immunogenic composition can be used for treating subjects suffering from Wilm's tumor.
  • the immunogenic composition can also be used for treating subjects with cancers or tumors that express WTl for preventing development of such tumors in subjects.
  • the WTl antigen can differ from the native, "normal” WTl gene, and thus, provide therapy or prophylaxis against an WTl antigen-expressing tumor.
  • Proteins may comprise sequences homologous to the WTl antigens, fragments of the WTl antigens and proteins with sequences homologous to fragments of the WTl antigens.
  • the WTl antigens can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
  • a consensus WTl antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is derived from multiple human WTl sequences.
  • a consensus WTl antigen is derived from multiple non-human WTl sequences.
  • a consensus WTl antigen is operably linked to a sequence encoding a signal peptide.
  • the antigen tyrosinase (Tyr) antigen is an important target for immune mediated clearance by inducing (1) humoral immunity via B cell responses to generate antibodies that block monocyte chemoattractant protein-1 (MCP-1) production, thereby retarding myeloid derived suppressor cells (MDSCs) and suppressing tumor growth; (2) increase cytotoxic T lymphocyte such as CD8 + (CTL) to attack and kill tumor cells; (3) increase T helper cell responses; (4) and increase inflammatory responses via IFN- ⁇ and TFN-a or all of the aforementioned.
  • MCP-1 monocyte chemoattractant protein-1
  • CTL cytotoxic T lymphocyte
  • T helper cell responses increase inflammatory responses via IFN- ⁇ and TFN-a or all of the aforementioned.
  • Tyrosinase is a copper-containing enzyme that can be found in plant and animal tissues. Tyrosinase catalyzes the production of melanin and other pigments by the oxidation of phenols such as tyrosine. In melanoma, tyrosinase can become unregulated, resulting in increased melanin synthesis. Tyrosinase is also a target of cytotoxic T cell recognition in subjects suffering from melanoma. Accordingly, tyrosinase can be an antigen associated with melanoma.
  • the antigen can comprise protein epitopes that make them particularly effective as immunogens against which anti-Tyr immune responses can be induced.
  • the Tyr antigen can comprise the full length translation product, a variant thereof, a fragment thereof or a combination thereof.
  • the Tyr antigen can comprise a consensus protein.
  • the Tyr antigen induces antigen-specific T-cell and high titer antibody responses both systemically against all cancer and tumor related cells.
  • a protective immune response is provided against tumor formation by vaccines comprising the Tyr consensus antigen.
  • any user can design an immunogenic composition of the present invention to include a Tyr antigen to provide broad immunity against tumor formation, metastasis of tumors, and tumor growth.
  • Proteins may comprise sequences homologous to the Tyr antigens, fragments of the Tyr antigens and proteins with sequences homologous to fragments of the Tyr antigens.
  • the Tyr antigens can be administered in vectors described herein, and combined the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
  • a consensus Tyr antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is a consensus antigen derived from multiple human Tyr sequences.
  • a consensus Tyr antigen is derived from multiple non-human Tyr sequences.
  • a consensus Tyr antigen is operably linked to a sequence encoding a signal peptide.
  • NY-ESO-1 is a cancer-testis antigen expressed in various cancers where it can induce both cellular and humoral immunity. Gene expression studies have shown upregulation of the gene for NY-ESO-1, CTAG1B, in myxoid and round cell liposarcomas.
  • the NY-ESO-1 antigen comprises a consensus NY-ESO-1 protein or a nucleic acid molecule encoding a consensus NY-ESO-1 protein.
  • NY-ESO-1 antigens include sequences homologous to the NY-ESO-1 antigens, fragments of the NY- ESO-1 antigens and proteins with sequences homologous to fragments of the NY-ESO-1 antigens.
  • the NY-ESO-1 antigens can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
  • a consensus NY-ESO-1 antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is derived from multiple human NY-ESO-1 sequences.
  • a consensus NY-ESO-1 antigen is derived from multiple non-human NY-ESO- 1 sequences.
  • a consensus NY-ESO-1 antigen is operably linked to a sequence encoding a signal peptide.
  • PRAME antigen is a protein that in humans is encoded by the PRAME gene. This gene encodes an antigen that is predominantly expressed in human melanomas and that is recognized by cytolytic T lymphocytes. It is not expressed in normal tissues, except testis. The gene is also expressed in acute leukemias. Five altematively spliced transcript variants encoding the same protein have been observed for this gene. Proteins may comprise sequences homologous to the PRAME antigens, fragments of the PRAME antigens and proteins with sequences homologous to fragments of the PRAME antigens.
  • the PRAME antigens can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
  • a consensus PRAME antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is derived from multiple human PRAME sequences.
  • a consensus PRAME antigen is derived from multiple non-human PRAME sequences.
  • a consensus PRAME antigen is operably linked to a sequence encoding a signal peptide.
  • MAGE stands for Melanoma-associated Antigen, and in particular melanoma associated antigen 4 (MAGEA4).
  • MAGE-A4 is expressed in male germ cells and tumor cells of various histological types such as gastrointestinal, esophageal and pulmonary carcinomas. MAGE-A4 binds the oncoprotein, Gankyrin. This MAGE-A4 specific binding is mediated by its C-terminus. Studies have shown that exogenous MAGE-A4 can partly inhibit the adhesion-independent growth of Gankyrin-overexpressing cells in vitro and suppress the formation of migrated tumors from these cells in nude mice.
  • MAGEA4 Melanoma-associated antigen 4 protein
  • MAGEA4 can be involved in embryonic development and tumor transformation and/or progression. MAGEA4 is normally expressed in testes and placenta. MAGEA4, however, can be expressed in many different types of tumors, for example, melanoma, head and neck squamous cell carcinoma, lung carcinoma, and breast carcinoma. Accordingly, MAGEA4 can be antigen associated with a variety of tumors.
  • the MAGEA4 antigen can induce antigen-specific T cell and/or high titer antibody responses, thereby inducing or eliciting an immune response that is directed to or reactive against the cancer or tumor expressing the antigen.
  • the induced or elicited immune response can be a cellular, humoral, or both cellular and humoral immune responses.
  • the induced or elicited cellular immune response can include induction or secretion of interferon-gamma (IFN- ⁇ ) and/or tumor necrosis factor alpha (TNF-a).
  • IFN- ⁇ interferon-gamma
  • TNF-a tumor necrosis factor alpha
  • the induced or elicited immune response can reduce or inhibit one or more immune suppression factors that promote growth of the tumor or cancer expressing the antigen, for example, but not limited to, factors that down regulate MHC presentation, factors that up regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TFG- ⁇ , tumor associated macrophages, tumor associated fibroblasts.
  • immune suppression factors that promote growth of the tumor or cancer expressing the antigen, for example, but not limited to, factors that down regulate MHC presentation, factors that up regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TFG- ⁇ , tumor associated macrophages, tumor associated fibroblasts.
  • the MAGEA4 antigen can comprise protein epitopes that make them particularly effective as immunogens against which anti-MAGEA4 immune responses can be induced.
  • the MAGEA4 antigen can comprise the full length translation product, a variant thereof, a fragment thereof or a combination thereof.
  • the MAGEA4 antigen can comprise a consensus protein.
  • the nucleic acid sequence encoding the consensus MAGEA4 antigen can be optimized with regards to codon usage and corresponding RNA transcripts.
  • the nucleic acid encoding the consensus MAGEA4 antigen can be codon and RNA optimized for expression.
  • the nucleic acid sequence encoding the consensus MAGEA4 antigen can include a Kozak sequence (e.g., GCC ACC) to increase the efficiency of translation.
  • the nucleic acid encoding the consensus MAGEA4 antigen can include multiple stop codons (e.g., TGA TGA) to increase the efficiency of translation termination.
  • the MAGEA4 antigen can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
  • a consensus MAGEA4 antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is derived from multiple human MAGEA4 sequences.
  • a consensus MAGEA4 antigen is derived from multiple non-human MAGEA4 sequences.
  • a consensus MAGEA4 antigen is operably linked to a sequence encoding a signal peptide,
  • Follicle stimulating hormone receptor is an antigen that is selectively expressed in women in the ovarian granulosa cells (Simoni et al, Endocr Rev. 1997, 18:739- 773) and at low levels in the ovarian endothelium (Vannier et al, Biochemistry, 1996, 35: 1358-1366). Most importantly, this surface antigen is expressed in 50-70% of ovarian carcinomas.
  • the FSHR antigen comprises a consensus protein or a nucleic acid molecule encoding a consensus protein.
  • FSHR antigens include sequences homologous to the FSHR antigens, fragments of the FSHR antigens and proteins with sequences homologous to fragments of the FSHR antigens.
  • the FSHR antigens can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
  • a consensus FSHR antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is derived from multiple human FSHR sequences.
  • a consensus FSHR antigen is derived from multiple non-human FSHR sequences.
  • a consensus FSHR antigen is operably linked to a signal peptide.
  • FAP Fibroblast Activation Protein
  • PDGFR- ⁇ Platelet Derived Growth Factor Receptor Beta
  • GPCl Glypican-1
  • the tumor microenvironment antigen comprises a consensus protein or a nucleic acid molecule encoding a consensus protein.
  • Tumor microenvironment antigens include sequences homologous to the tumor microenvironment antigens, fragments of the tumor microenvironment antigens and proteins with sequences homologous to fragments of the tumor microenvironment antigens.
  • One or more tumor microenvironment antigens can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
  • a consensus tumor microenvironment antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is derived from multiple human tumor microenvironment antigen sequences.
  • a consensus tumor microenvironment antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is derived from multiple human tumor microenvironment antigen sequences.
  • microenvironment antigen is derived from multiple non-human tumor microenvironment antigen sequences.
  • a consensus tumor microenvironment antigen is operably linked to a signal peptide.
  • the immunogenic composition can comprise one or more vectors that include a nucleic acid encoding the antigen and the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins.
  • the one or more vectors can be capable of expressing the antigen and the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins.
  • the vector can have a nucleic acid sequence containing an origin of replication.
  • the vector can be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
  • the vector can be either a self-replication extra chromosomal vector, or a vector which integrates into a host genome.
  • the one or more vectors can be an expression construct, which is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular- transcription and translation machinery ribosomal complexes.
  • the plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.
  • the vectors of the present invention express large amounts of stable messenger RNA, and therefore proteins.
  • the vectors may have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).
  • expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).
  • the vector can be a circular plasmid or a linear nucleic acid.
  • the circular plasmid and linear nucleic acid are capable of directing expression of a particular nucleotide sequence in an appropriate subject cell.
  • the vector can have a promoter operably linked to the antigen- encoding nucleotide sequence, or the adjuvant-encoding nucleotide sequence, which may be operably linked to termination signals.
  • the vector can also contain sequences required for proper translation of the nucleotide sequence.
  • the vector comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus.
  • the promoter can also be specific to a particular tissue or organ or stage of development.
  • the vector may be circular plasmid, which may transform a target cell by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).
  • the vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing DNA encoding the antigen, or the adjuvant and enabling a cell to translate the sequence to an antigen that is recognized by the immune system, or the adjuvant.
  • a linear nucleic acid vaccine or linear expression cassette (“LEC”), that is capable of being efficiently delivered to a subject via electroporation and expressing one or more desired antigens, or one or more desired adjuvants.
  • the LEC may be any linear DNA devoid of any phosphate backbone.
  • the DNA may encode one or more antigens, or one or more adjuvants.
  • the LEC may contain a promoter, an intron, a stop codon, and/or a polyadenylation signal.
  • the expression of the antigen, or the adjuvant may be controlled by the promoter.
  • the LEC may not contain any antibiotic resistance genes and/or a phosphate backbone.
  • the LEC may not contain other nucleic acid sequences unrelated to the desired antigen gene expression, or the desired adjuvant expression.
  • the LEC may be derived from any plasmid capable of being linearized.
  • the plasmid may be capable of expressing the antigen, the CTLA4 antibody or one or more antibody targeting one or more additional immune checkpoint proteins.
  • the plasmid can be pNP (Puerto Rico/34) or pM2 (New Caledonia/99).
  • the plasmid may be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing DNA encoding the antigen, or encoding the adjuvant, and enabling a cell to translate the sequence to an antigen that is recognized by the immune system, or the adjuvant.
  • the LEC can be pcrM2.
  • the LEC can be pcrNP.
  • pcrNP and pcrMR can be derived from pNP (Puerto Rico/34) and pM2 (New Caledonia/99), respectively.
  • the vector may have a promoter.
  • a promoter may be any promoter that is capable of driving gene expression and regulating expression of the isolated nucleic acid. Such a promoter is a cis-acting sequence element required for transcription via a DNA dependent RNA polymerase, which transcribes the antigen sequence, or the adjuvant sequence described herein. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter may be positioned about the same distance from the transcription start in the vector as it is from the transcription start site in its natural setting. However, variation in this distance may be accommodated without loss of promoter function.
  • the promoter may be operably linked to the nucleic acid sequence encoding the antigen and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.
  • the promoter may be operably linked to the nucleic acid sequence encoding the adjuvant and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.
  • the promoter may be a CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or another promoter shown effective for expression in eukaryotic cells.
  • the vector may include an enhancer and an intron with functional splice donor and acceptor sites.
  • the vector may contain a transcription termination region downstream of the structural gene to provide for efficient termination.
  • the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
  • the immunogenic composition may further comprise a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient can be functional molecules such as vehicles, adjuvants other than the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins, carriers, or diluents.
  • the pharmaceutically acceptable excipient can be a transfection facilitating agent, which can include surface active agents, such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, poly cations, or nanoparticles, or other known transfection facilitating agents.
  • surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, poly cations, or nanoparticles, or other known trans
  • the transfection facilitating agent is a polyanion, poly cation, including poly-L- glutamate (LGS), or lipid.
  • the transfection facilitating agent is poly-L-glutamate, and the poly-L-glutamate may be present in the immunogenic composition at a concentration less than 6 mg/ml.
  • the transfection facilitating agent may also include surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the genetic construct.
  • ISCOMS immune-stimulating complexes
  • LPS analog including monophosphoryl lipid A
  • muramyl peptides muramyl peptides
  • quinone analogs and vesicles such as squalene and squalene
  • the DNA plasmid vaccines may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example W09324640), calcium ions, viral proteins, polyanions, poly cations, or nanoparticles, or other known transfection facilitating agents.
  • the transfection facilitating agent is a polyanion, poly cation, including poly-L-glutamate (LGS), or lipid.
  • Concentration of the transfection agent in the immunogenic composition is less than 4 mg/ml, less than 2 mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml, less than 0.250 mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml, or less than 0.010 mg/ml.
  • the pharmaceutically acceptable excipient can be an adjuvant in addition to CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins.
  • the additional adjuvant can be other genes that are expressed in an alternative plasmid or are delivered as proteins in combination with the plasmid above in the immunogenic composition.
  • the adjuvant may be selected from the group consisting of: a- interferon(IFN- a), ⁇ -interferon (IFN- ⁇ ), ⁇ -interferon, platelet derived growth factor (PDGF), TNFa, TNF , GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 having the signal sequence deleted and optionally including the signal peptide from IgE.
  • IFN- a interferon
  • IFN- ⁇ ⁇ -interferon
  • PDGF platelet derived growth factor
  • TNFa TNFa
  • TNF GM-CSF
  • EGF epidermal growth factor
  • CTL epidermal growth factor
  • CTACK epidermal growth factor
  • TECK epit
  • the adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet derived growth factor (PDGF), TNFa, TNFP, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.
  • genes that can be useful as adjuvants in addition to the PD1 antibody or PDLl antibody include those encoding: MCP-1, MIP-la, MIP-lp, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL- 1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase
  • the immunogenic composition may further comprise a genetic vaccine facilitator agent as described in U.S. Serial No. 021,579 filed April 1, 1994, which is fully incorporated by reference.
  • the immunogenic composition can be formulated according to the mode of administration to be used.
  • An injectable vaccine pharmaceutical composition can be sterile, pyrogen free and particulate free.
  • An isotonic formulation or solution can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose.
  • the immunogenic composition can comprise a vasoconstriction agent.
  • the isotonic solutions can include phosphate buffered saline.
  • Vaccine can further comprise stabilizers including gelatin and albumin. The stabilizers can allow the formulation to be stable at room or ambient temperature for extended periods of time, including LGS or poly cations or polyanions.
  • the present invention is also directed to a method of increasing an immune response in a subject.
  • Increasing the immune response can be used to treat and/or prevent disease in the subject.
  • the method can include administering the herein disclosed vaccine to the subject.
  • the subject administered the immunogenic composition can have an increased or boosted immune response as compared to a subject administered the antigen alone.
  • the immune response can be increased by about 0.5-fold to about 15-fold, about 0.5-fold to about 10-fold, or about 0.5-fold to about 8-fold.
  • the immune response in the subject administered the immunogenic composition can be increased by at least about 0.5-fold, at least about 1.0-fold, at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0- fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, or at least about 15.0-fold.
  • the immune response in the subject administered the immunogenic composition can be increased about 1% to about 1500%, about 1% to about 1000%, or about 1% to about 800%. In other embodiments, the immune response in the subject administered the immunogenic composition can be increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000%, at least about 1050%
  • the immunogenic composition dose can be between 1 ⁇ g to 10 mg active component kg body weight/time, and can be 20 ⁇ g to 10 mg component/kg body
  • the immunogenic composition can be administered every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days.
  • the number of vaccine doses for effective treatment can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10. a. Administration
  • the immunogenic composition can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration.
  • the subject can be a mammal, such as a human, a horse, a cow, a pig, a sheep, a cat, a dog, a rat, or a mouse.
  • the immunogenic composition can be administered prophylactically or therapeutically.
  • the immunogenic composition can be administered in an amount sufficient to induce an immune response.
  • the immunogenic compositions are administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect.
  • An amount adequate to accomplish this is defined as "therapeutically effective dose. " Amounts effective for this use will depend on, e.g., the particular composition of the immunogenic composition regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.
  • the immunogenic composition can be administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol.
  • the DNA of the immunogenic composition can be complexed to particles or beads that can be administered to an individual, for example, using an immunogenic composition gun.
  • a pharmaceutically acceptable carrier including a
  • physiologically acceptable compound depends, for example, on the route of administration of the expression vector.
  • the immunogenic composition can be delivered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes.
  • parenteral administration e.g., intradermal, intramuscular or subcutaneous delivery.
  • Other routes include oral administration, intranasal, and intravaginal routes.
  • the immunogenic composition can be delivered to the interstitial spaces of tissues of an individual (Feigner et al., U.S. Pat. Nos. 5,580,859 and 5,703,055, the contents of all of which are incorporated herein by reference in their entirety).
  • the immunogenic composition can also be administered to muscle, or can be administered via intradermal or subcutaneous injections, or
  • Epidermal administration of the immunogenic composition can also be employed. Epidermal administration can involve mechanically or chemically irritating the outermost layer of epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Pat. No. 5,679,647, the contents of which are incorporated herein by reference in its entirety).
  • the immunogenic composition can also be formulated for administration via the nasal passages.
  • Formulations suitable for nasal administration wherein the carrier is a solid, can include a coarse powder having a particle size, for example, in the range of about 10 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • the formulation can be a nasal spray, nasal drops, or by aerosol administration by nebulizer.
  • the formulation can include aqueous or oily solutions of the immunogenic composition.
  • the immunogenic composition can be a liquid preparation such as a suspension, syrup or elixir.
  • the immunogenic composition can also be a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as a sterile suspension or emulsion.
  • the immunogenic composition can be incorporated into liposomes, microspheres or other polymer matrices (Feigner et al, U.S. Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vols. Ito III (2nd ed. 1993), the contents of which are incorporated herein by reference in their entirety).
  • Liposomes can consist of phospholipids or other lipids, and can be nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • the immunogenic composition can be administered via electroporation, such as by a method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference.
  • the electroporation can be by a method and/or apparatus described in U.S. Patent Nos. 6,302,874; 5,676,646; 6,241,701 ; 6,233,482; 6,216,034; 6,208,893;
  • the electroporation may be carried out via a minimally invasive device.
  • the minimally invasive electroporation device may be an apparatus for injecting the immunogenic composition described above and associated fluid into body tissue.
  • the device may comprise a hollow needle, DNA cassette, and fluid delivery means, wherein the device is adapted to actuate the fluid delivery means in use so as to concurrently (for example, automatically) inject DNA into body tissue during insertion of the needle into the said body tissue.
  • This has the advantage that the ability to inject the DNA and associated fluid gradually while the needle is being inserted leads to a more even distribution of the fluid through the body tissue. The pain experienced during injection may be reduced due to the distribution of the DNA being injected over a larger area.
  • the MID may inject the immunogenic composition into tissue without the use of a needle.
  • the MID may inject the immunogenic composition as a small stream or jet with such force that the immunogenic composition pierces the surface of the tissue and enters the underlying tissue and/or muscle.
  • the force behind the small stream or jet may be provided by expansion of a compressed gas, such as carbon dioxide through a micro-orifice within a fraction of a second. Examples of minimally invasive electroporation devices, and methods of using them, are described in published U.S. Patent Application No. 20080234655; U.S.
  • the MID may comprise an injector that creates a high-speed jet of liquid that painlessly pierces the tissue.
  • Such needle-free injectors are commercially available. Examples of needle-free injectors that can be utilized herein include those described in U. S. Patent Nos. 3,805,783; 4,447,223; 5,505,697; and 4,342,310, the contents of each of which are herein incorporated by reference.
  • a desired vaccine in a form suitable for direct or indirect electrotransport may be introduced (e.g., inj ected) using a needle-free injector into the tissue to be treated, usually by contacting the tissue surface with the injector so as to actuate delivery of a jet of the agent, with sufficient force to cause penetration of the immunogenic composition into the tissue.
  • a needle-free injector into the tissue to be treated, usually by contacting the tissue surface with the injector so as to actuate delivery of a jet of the agent, with sufficient force to cause penetration of the immunogenic composition into the tissue.
  • the tissue to be treated is mucosa, skin or muscle
  • the agent is projected towards the mucosal or skin surface with sufficient force to cause the agent to penetrate through the stratum comeum and into dermal layers, or into underlying tissue and muscle, respectively.
  • Needle-free injectors are well suited to deliver vaccines to all types of tissues, particularly to skin and mucosa.
  • a needle-free inj ector may be used to propel a liquid that contains the immunogenic composition to the surface and into the subject's skin or mucosa.
  • Representative examples of the various types of tissues that can be treated using the invention methods include pancreas, larynx, nasopharynx, hypopharynx, oropharynx, lip, throat, lung, heart, kidney, muscle, breast, colon, prostate, thymus, testis, skin, mucosal tissue, ovary, blood vessels, or any combination thereof.
  • the MID may have needle electrodes that electroporate the tissue.
  • Electrodes for Mediated Delivery of Drugs and Genes is an array of needles wherein a plurality of pairs of needles may be pulsed during the therapeutic treatment.
  • needles were disposed in a circular array, but have connectors and switching apparatus enabling a pulsing between opposing pairs of needle electrodes.
  • a pair of needle electrodes for delivering recombinant expression vectors to cells may be used.
  • Such a device and system is described in U. S. Patent No. 6,763,264, the contents of which are herein incorporated by reference.
  • a single needle device may be used that allows injection of the DNA and electroporation with a single needle resembling a normal injection needle and applies pulses of lower voltage than those delivered by presently used devices, thus reducing the electrical sensation experienced by the patient.
  • the MID may comprise one or more electrode arrays.
  • the arrays may comprise two or more needles of the same diameter or different diameters.
  • the needles may be evenly or unevenly spaced apart.
  • the needles may be between 0.005 inches and 0.03 inches, between 0.01 inches and 0.025 inches; or between 0.015 inches and 0.020 inches.
  • the needle may be 0.0175 inches in diameter.
  • the needles may be 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more spaced apart.
  • the MID may consist of a pulse generator and a two or more-needle vaccine injectors that deliver the immunogenic composition and electroporation pulses in a single step.
  • the pulse generator may allow for flexible programming of pulse and injection parameters via a flash card operated personal computer, as well as comprehensive recording and storage of electroporation and patient data.
  • the pulse generator may deliver a variety of volt pulses during short periods of time. For example, the pulse generator may deliver three 15 volt pulses of 100 ms in duration.
  • An example of such a MID is the Elgen 1000 system by Inovio Biomedical Corporation, which is described in U.S. Patent No. 7,328,064, the contents of which are herein incorporated by reference.
  • the MID may be a CELLECTRA (Inovio Pharmaceuticals, Plymouth Meeting PA) device and system, which is a modular electrode system, that facilitates the introduction of a macromolecule, such as a DNA, into cells of a selected tissue in a body or plant.
  • the modular electrode system may comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a conductive link from a programmable constant-current pulse controller to the plurality of needle electrodes; and a power source.
  • An operator can grasp the plurality of needle electrodes that are mounted on a support structure and firmly insert them into the selected tissue in a body or plant.
  • the macromolecules are then delivered via the hypodermic needle into the selected tissue.
  • the programmable constant-current pulse controller is activated and constant-current electrical pulse is applied to the plurality of needle electrodes.
  • the applied constant-current electrical pulse facilitates the introduction of the macromolecule into the cell between the plurality of electrodes. Cell death due to overheating of cells is minimized by limiting the power dissipation in the tissue by virtue of constant- current pulses.
  • the Cellectra device and system is described in U. S. Patent No. 7,245,963, the contents of which are herein incorporated by reference. [00258]
  • the MID may be an El gen 1000 system (Inovio Pharmaceuticals).
  • the Elgen 1000 system may comprise device that provides a hollow needle; and fluid delivery means, wherein the apparatus is adapted to actuate the fluid delivery means in use so as to concurrently (for example automatically) inject fluid, the described vaccine herein, into body tissue during insertion of the needle into the said body tissue.
  • the advantage is the ability to inject the fluid gradually while the needle is being inserted leads to a more even distribution of the fluid through the body tissue. It is also believed that the pain experienced during injection is reduced due to the distribution of the volume of fluid being injected over a larger area.
  • the automatic injection of fluid facilitates automatic monitoring and registration of an actual dose of fluid injected.
  • This data can be stored by a control unit for documentation purposes if desired.
  • the rate of injection could be either linear or non-linear and that the injection may be carried out after the needles have been inserted through the skin of the subject to be treated and while they are inserted further into the body tissue.
  • Suitable tissues into which fluid may be injected by the apparatus of the present invention include tumor tissue, skin or liver tissue but may be muscle tissue.
  • the apparatus further comprises needle insertion means for guiding insertion of the needle into the body tissue.
  • the rate of fluid injection is controlled by the rate of needle insertion. This has the advantage that both the needle insertion and injection of fluid can be controlled such that the rate of insertion can be matched to the rate of injection as desired. It also makes the apparatus easier for a user to operate. If desired means for automatically inserting the needle into body tissue could be provided.
  • a user could choose when to commence injection of fluid. Ideally however, injection is commenced when the tip of the needle has reached muscle tissue and the apparatus may include means for sensing when the needle has been inserted to a sufficient depth for injection of the fluid to commence. This means that injection of fluid can be prompted to commence automatically when the needle has reached a desired depth (which will normally be the depth at which muscle tissue begins).
  • the depth at which muscle tissue begins could for example be taken to be a preset needle insertion depth such as a value of 4 mm which would be deemed sufficient for the needle to get through the skin layer.
  • the sensing means may comprise an ultrasound probe.
  • the sensing means may comprise a means for sensing a change in impedance or resistance.
  • the means may not as such record the depth of the needle in the body tissue but will rather be adapted to sense a change in impedance or resistance as the needle moves from a different type of body tissue into muscle. Either of these alternatives provides a relatively accurate and simple to operate means of sensing that injection may commence.
  • the depth of insertion of the needle can further be recorded if desired and could be used to control injection of fluid such that the volume of fluid to be injected is determined as the depth of needle insertion is being recorded.
  • the apparatus may further comprise: a base for supporting the needle; and a housing for receiving the base therein, wherein the base is moveable relative to the housing such that the needle is retracted within the housing when the base is in a first rearward position relative to the housing and the needle extends out of the housing when the base is in a second forward position within the housing.
  • a base for supporting the needle
  • a housing for receiving the base therein, wherein the base is moveable relative to the housing such that the needle is retracted within the housing when the base is in a first rearward position relative to the housing and the needle extends out of the housing when the base is in a second forward position within the housing.
  • the fluid delivery means may comprise piston driving means adapted to inject fluid at a controlled rate.
  • the piston driving means could for example be activated by a servo motor.
  • the piston driving means may be actuated by the base being moved in the axial direction relative to the housing.
  • alternative means for fluid delivery could be provided.
  • a closed container which can be squeezed for fluid delivery at a controlled or non-controlled rate could be provided in the place of a syringe and piston system.
  • the apparatus described above could be used for any type of injection. It is however envisaged to be particularly useful in the field of electroporation and so it may further comprise a means for applying a voltage to the needle. This allows the needle to be used not only for injection but also as an electrode during, electroporation. This is particularly advantageous as it means that the electric field is applied to the same area as the injected fluid.
  • electroporation There has traditionally been a problem with electroporation in that it is very difficult to accurately align an electrode with previously injected fluid and so users have tended to inject a larger volume of fluid than is required over a larger area and to apply an electric field over a higher area to attempt to guarantee an overlap between the injected substance and the electric field.
  • both the volume of fluid injected and the size of electric field applied may be reduced while achieving a good fit between the electric field and the fluid.
  • the present invention has multiple aspects, illustrated by the following non- limiting examples.
  • a DNA vaccine platform using synthetic consensus DNA molecules has been developed to help break tolerance to non-viral tumor-associated antigens, such as TERT and WT-1 (Yan et al, 2013, Cancer Immunol Res, 1 : 179-189; Walters et al, 2017, Mol Ther, 25:976-988).
  • This platform is based on the concept that introduction of xenogeneic antigens into mice can induce autoimmunity (Engelhorn et al, 2006, Nat Med, 12: 198-206; Guevara- Patino et al, 2006, J Clin Invest, 116: 1382-1390.
  • combination therapy with immune checkpoint blockade further improves synthetic consensus DNA vaccine immune responses.
  • a mouse TERT DNA vaccine with enhanced capacity to break tolerance was administered in combination with antibodies that block the immune checkpoint molecules CTLA-4 or PD-1.
  • Blockade of CTLA-4, or a combination of CTLA-4 and PD-1 effectively synergized with TERT DNA vaccine to slow tumor growth in mice.
  • peripheral and systemic immune responses did not necessarily correspond with anti-tumor activity of immune therapy combinations.
  • the synthetic consensus TERT sequence was generated by using 6 TERT sequences collected from animals including mouse, rat and hamster. The consensus sequence was obtained after alignment of these sequences using Clustal W. Two additional mutations were added to abolish telomerase activity (Weinrich et al, 1997, Nat Genet, 17:498-502; Lingner et al, 1997, Science, 276:561-56). All sequences were RNA and codon optimized with an IgE leader sequence and a Kozak sequence at the N terminus, and cloned into the modified pVAX vector (Genscript). The final mouse TERT sequences shares 94.6% sequence identity with native mouse TERT.
  • splenocyte isolation For splenocyte isolation, spleens from mice were collected in complete RPMI media containing 10% Fetal Bovine Serum (FBS). Cells were dissociated using a stomacher, and then filtered through a 40 ⁇ mesh filter. Red Blood Cells were then lysed using ACK Lysis buffer (LifeTechnologies). Cells were then filtered again through a 40 ⁇ mesh filter, and counted and plated for staining.
  • FBS Fetal Bovine Serum
  • ACK Lysis buffer LifeTechnologies
  • PBMC isolation blood was collected in 4% sodium citrate to prevent clotting. Blood was then layered on top of Histopaque 1083 (Sigma- Aldrich). Cells were spun for one hour, and then cells from the buffy coat were separated and stained for analysis.
  • TILs tumor infiltrating lymphocytes
  • Tumors were dissociated using digestion mix, which consists of: 170mg/L
  • Collagenase I, II and IV Collagenase I, II and IV (ThermoFisher), 12.5mg/L DNAse I (Roche), 25mg/L Elastase (Worthington) in a 50/50 mixture of Hyclone L-15 Leibowitz Media (ThermoFisher) and RPMI, supplemented with 10% FBS and 1% Penicillin/Streptomycin. Tumors were minced and then transferred to a 50mL conical filled with lOmL of digestion mix. Cells were then filtered twice through a 40 ⁇ mesh filter, and then counted and stained.
  • Splenocytes were stimulated in the presence of native mouse TERT peptides (15- mer peptides spanning the entire native mouse protein, overlapping by 9 amino acids, GenScript) for 24 hours in MABTECH Mouse IFN- ⁇ ELISpot PLUS plates. Spots were developed and counted according to the manufacturer's protocol.
  • mice Female 6-8 week old C57B1/6 mice were purchased from Jackson Laboratory. Animal care was in accordance with the guidelines of the NIH and with the Wistar Institute Animal Care and Use Committee (IACUC). Mice were immunized with 25 ⁇ g of each plasmid by intramuscular injection into the tibialis interior (TA) muscle, followed by electroporation (EP) using the CELLECTRA ® -3P adaptive constant current device (Inovio Pharmaceuticals). Two 0.1 Amp constant current square-wave pulses were delivered through a triangular three-electrode array. Each pulse was 52 milliseconds in length with a Is delay between pulses. For immunogenicity studies, mice were given a total of three immunizations at two-week intervals. For tumor challenge studies, mice were given a total of four immunizations at one-week intervals.
  • IACUC Wistar Institute Animal Care and Use Committee
  • a-CTLA-4 and a-PD-1 in combination with mTERT had robust slowing of tumor growth and significant improvement in survival compared to DNA alone or naive mice ( Figure 6B and Figure 6C). This synergy was greater for a-CTLA-4, in particular for survival, compared to a-PD-1 ( Figure 6B and Figure 6C).
  • PD-1 expression was examined on CD4 and CD8 T cells in the spleen and the periphery of tumor-bearing mice. While in non-tumor bearing mice all checkpoints enhanced PD-1 expression on CD8+ T cells to some degree (with the greatest enhancement from the combination or aPD-1 therapy alone), in tumor-bearing mice aPD-1 had no impact on PD-1 expression in both CD4+ and CD8+ T cells in the spleen and periphery ( Figure 8A through Figure 8D). However, both aCTLA-4 and a combination of aCTLA-4 and aPD-1 enhanced the frequency of PD-1+ CD4+ and CD8+ T cells in both the spleen and the periphery ( Figure 8A through Figure 8D).
  • CTLA-4 blockade synergizes with mTERT DNA vaccine more than Treg depletion
  • aCTLA-4 therapy is depletion of Tregs in the tumor microenvironment.
  • the impact of aCTLA-4 was compared to aCD25, a depletion antibody that systemically depletes regulatory T cells (Stephens et al, 2005, Proc Natl Acad Sci U S A, 102: 17418-17423).
  • Blockade of the PD-l/PD-Ll signaling axis can only partially reverse an exhausted T cell phenotype; therefore, exploring methods of further improving immune activation in tumor-bearing mice remains important (Pauken et al, 2016, Science, 354: 1160-1165).
  • aCTLA-4 and aPD-1 blockade may be the result of the different effects of aCTLA-4 and aPD-1 on regulatory T cells.
  • aCTLA-4 antibodies unlike aPD-1 antibodies, have demonstrated robust depletion of intra-tumoral Tregs in mice (Quezada et al., 2006, J Clin Invest, 116: 1935-1945; Selby et al, 2013, Cancer Immunol Res, 1 :32-42).
  • Example 2 exemplary hTERT vaccines
  • exemplary hTERT DNA sequences and encoded amino acid sequences that can be administered in combination with an ICB antibody of the invention.
  • SEQ ID NO:3 -hTERT nucleic acid sequence operably linked to a sequence encoding an IgE leader sequence.
  • SEQ ID NO: 5 Synthetic Consensus hTERT nucleic acid sequence (pgxl434).
  • SEQ ID NO: 7 Synthetic Consensus hTERT nucleic acid sequence (pgxl434), operably linked to a sequence encoding an IgE leader sequence.
  • SEQ ID NO: 8 Synthetic consensus hTERT amino acid sequence (pgx 1434) operably linked to an IgE leader sequence
  • SEQ ID NO: 9 synthetic, consensus mouse TERT (mTERT) nucleic acid sequence (pgxl418)
  • SEQ ID NO: 11 synthetic, consensus mTERT nucleic acid sequence (pgxl418) operably linked to a sequence encoding an IgE leader sequence.
  • SEQ ID NO: 12 synthetic, consensus mTERT amino acid sequence (pGX1418) operably linked to an IgE leader sequence

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Abstract

Disclosed herein is an immunogenic composition comprising at least an antigen and CTLA4 antibody. Also disclosed herein is a method for enhancing an immune response in a subject. The method may comprise administering the immunogenic composition to the subject in need thereof.

Description

CTLA4 ANTIBODIES AND VACCINE COMBINATIONS AND USE OF SAME FOR
IMMUNOTHERAPY
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No.
62/502,410 filed May 5, 2017 which is hereby incorporated by reference herein in its entirety.
TECHNICAL FIELD
[0001] The present invention relates to vaccines combined with CTLA4 antibodies, and use of such combination for immunotherapy.
BACKGROUND
[0002] The magnitude of an adaptive immune response to a foreign antigen is determined not only by the strength of interaction between the major histocompatibility complex (MHC) and T-cell receptor (TCR), but also through co-stimulation at the immunological synapse
(Pardoll, 2012, Nat Rev Cancer; 12:252-264). Without this co-stimulation, T cells will fail to initiate an effective immune response. Additional co-stimulatory molecules, or immune checkpoint molecules, exist to control the amplitude of T cell activation to prevent autoimmune responses. These immune checkpoints (CTLA-4, PD-1, LAG3, TIM3) are often necessary for initiation of an immune response; however, as antigen persists, these checkpoints ultimately serve to dampen the T cell effector function against the foreign antigen (Riley, 2009, Immunol Rev, 229: 114-125). CTLA-4, PD-1, LAG3 and TIM3 are all expressed on T cells and limit T cell effector activity. Antibodies blocking these molecules have been shown to augment the effector activity of tumor-specific T cells, and additionally inhibit regulatory T cell (Treg) activity and reduce tumor burden in preclinical models and/or clinical trials as mono-therapies (Walunas and Bluestone, 1998, J Immunol, 160:3855-3860; Anderson, 2014, Cancer Immunol Res, 2:393-398; Duraiswamy et al, 2013, Cancer Res, 73:3591-3603).
[0003] Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4) also known as CD 152 is a cell surface protein molecule that functions as a major negative regulator of T-cell responses. This protein is expressed in Tregs and is upregulated in T cells after activation. CTLA4 functions as an immune checkpoint and downregulates immune responses. CTLA4 is homologous to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86 on antigen-presenting cells.
[0004] Immune checkpoint blockade antibodies, such as a-PD-1 (pembrolizumab) and a- CTLA-4 (e.g., ipilimumab) are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint blockade (ICB). In particular, blockade of the immune inhibitory checkpoints PD- 1 or CTLA-4 has shown promising results in the clinic for dozens of tumor types, and PD-1 blockade has become a standard of care for melanoma and non-small cell lung cancer (Hodi et al, 2010, N Engl J Med, 363:711-723; Postow et al, 2015, J Clin Oncol, 33: 1974-1982; Reck et al, 2016, N Engl J Med, 375: 1823-1833). However, response rates to these monotherapies are relatively low (33.7% response for pembrolizumab— a-PD-1, and 11.9% response for ipilimumab— a-CTLA-4 in melanoma patients), leaving room for improvement (Robert et al, 2015, N Engl J Med, 372:2521-2532). The lack of response for the majority of such patients may be due to a lack of pre-existing tumor-associated T cell responses. Further, enhanced T cell priming may be required to break tolerance to self-antigens for patients with poor response to immune checkpoint blockade.
[0005] Vaccines are used to stimulate an immune response in an individual to provide protection against and/or treatment for a particular disease. Some vaccines include an antigen to induce the immune response. Some antigens elicit a strong immune response while other antigens elicit a weak immune response. A weak immune response to an antigen can be strengthened by including an adjuvant in the immunogenic composition. Adjuvants come in many different forms, for example, aluminum salts, oil emulsions, sterile constituents of bacteria or other pathogens, cytokines, and so forth.
[0006] Vaccines are also administered in many different ways (e.g., injection, orally, etc.) into many different tissues (e.g., intramuscular, nasal, etc.). Not all delivery methods, however, are equal. Some delivery methods allow for greater compliance within a population of individuals while other delivery methods may affect immunogenicity and/or safety of the immunogenic composition.
[0007] Therapeutic peptide or DNA vaccination represents a more targeted approach for directing T cells towards specific, less variable, tumor-associated antigens. In mouse models, peptide vaccines have been shown to synergize with immune checkpoint blockade
(Bartkowiak et al, 2015, Proc Natl Acad Sci, 112:E5290-E5299; Madan et al, 2012, Oncoimmunology, 1 : 1167-1168). However, peptide vaccines are HLA-restricted and therefore cannot be used for all patients. Unlike peptide vaccines, synthetic DNA vaccines are not HLA-restricted, are robustly presented on both MHCI and MHCII, and can be designed using consensus sequences in order to break tolerance (Flingai et al, 2013, Front Immunol, 4:354).
[0008] Accordingly, there remains a need for more effective immunotherapy using synthetic antigens by combining with CTLA4 antibody.
SUMMARY OF THE INVENTION
[0009] In one embodiment, the invention relates to a composition for enhancing an immune response against an antigen in a subject in need thereof, comprising an anti-CTLA4 antibody, and a synthetic antigen capable of generating an immune response in the subject, or an immunogenic fragment or variant thereof.
[0010] In one embodiment, the synthetic antigen is an isolated DNA that encodes for the antigen.
[0011] In one embodiment, the synthetic antigen is at least one of TERT, prostate, WT1, tyrosinase, NYES01, PRAME, MAGE, CMV, herpes, HIV, HPV, HCV, HBV, EBV, MCV, and cancer causing viruses. In one embodiment, the synthetic antigen is TERT. In one embodiment, the HPV antigen is E6 and E7 domains of an HPV subtype. In one embodiment, the HPV subtype is at least one of HPV6, HPVl l, HPV 16, HPV 18, HPV31, HPV33, HPV52, and HPV58, or a combination thereof. In one embodiment, the HIV antigen is at least one of Env A, Env B, Env C, Env D, B Nef-Rev, and Gag, or a combination thereof. In one embodiment, the HCV antigen is at least one of El, E2, NS3, NS4a, NS4b, NS5a, and NS5b, and a combination thereof. In one embodiment, the HBV antigen is at least one of surface antigen type A, surface antigen type B, surface antigen type C, surface antigen type D, surface antigen type E, surface antigen type F, surface antigen type G, surface antigen type H, and core antigen, and a combination thereof. In one embodiment, the prostate antigen is at least one of PSA, PSMA, STEAP, PSCA, and PAP, or a combination thereof. In one embodiment, the the synthetic antigen is a herpes antigen. In one embodiment, the herpes is HCMV, HSV1, HSV2, VZV, or CMV. In one embodiment, the herpes antigen is at least one of gB, gM, gN, gH, gL, gO, gE, gl, gK, gC, gD, UL128, UL130, UL131A, and UL83. [0012] In one embodiment, the anti-CTLA4 antibody is ipilimumab, ipilimumab-Probody Tx (BMS-986249), ipilimumab-NF (BMS-986218), tremelimumab, CS 1002, MDX-010 (NCT00140855), or AGEN-1884 (NCT02694822).
[0013] In one embodiment, the composition for enhancing an immune response against an antigen in a subject in need thereof, comprising an anti-CTLA4 antibody, and a synthetic antigen further comprises an additional antibody targeting an immune checkpoint protein. In one embodiment, the immune checkpoint protein is selected from the group consisting of PD- 1, PD-L1, LAG3 and TIM3. In one embodiment, the antibody is selected from the group consisting of: nivolumab, pembrolizumab, pidilizumab, BMS-936559, MPDL3280A, MDX1105-01, MEDI4736, and MK-3475.
[0014] In one embodiment, the composition for enhancing an immune response against an antigen in a subject in need thereof further comprises a pharmaceutically acceptable excipient.
[0015] In one embodiment, the invention relates to a method for increasing an immune response in a subject in need thereof, the method comprising administering the composition for enhancing an immune response against an antigen in a subject in need thereof to the subject. In one embodiment, the method of administering the composition comprises an electroporating step.
[0016] In one embodiment, the invention relates to a method of increasing an immune response in a subject in need thereof by administering a combination of a synthetic antigen and a CTL4A antibody, wherein the administering step comprises administering to the subject a prime vaccination and a boost vaccination of a synthetic antigen and, prior to the boost vaccination, administering to the subject a CTL4A antibody. In one embodiment, the method further comprises administering to the subject a subsequent boost vaccination of the synthetic antigen. In one embodiment, the method further comprises administering an antibody targeting one or more immune checkpoint protein prior to the boost vaccination.
[0017] In one embodiment, one or more of the administering steps includes delivering electroporation to the site of administration.
[0018] In one embodiment, one or more of the prime vaccination and the boost vaccination comprises a nucleic acid vaccine.
[0019] In one embodiment, one or more of the prime vaccination and the boost vaccination comprises a DNA vaccine. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 depicts exemplary experimental results demonstrating the capacity of a mTERT vaccine to generate immune response and break tolerance in C57B1/6 mice.
[0021] Figure 2 depicts exemplary experimental results demonstrating the anti -tumor activity of a mTERT vaccine.
[0022] Figure 3, comprising Figure 3A through Figure 3G, depicts exemplary experimental results demonstrating antigen-specific intracellular cytokine production upon TERT DNA vaccination and ICB in non-tumor bearing mice. Figure 3A depicts a schematic diagram of the experimental setup. Mice were immunized three times at two-week intervals. Mice were given antibody treatment (20C^g per mouse) every three days starting one day after first immunization. Mice were sacrificed one week following final vaccination, and splenocytes were isolated for analysis. Figure 3B through Figure 3F depict exemplary experimental results demonstrating intracellular cytokine staining of CD8 T cells or CD4 T cells after stimulation with native mouse TERT peptides for 5 hours. Figure 3G depicts exemplary experimental results demonstrating IFNy ELISpot responses to native mouse TERT peptide stimulation for 24 hours. Figure 3H depicts exemplary experimental results demonstrating surface staining of CD8 T cells from spleen of mice treated with indicated treatments according to schedule in Figure 3A. Significance was determined by two-way ANOVA followed by Tukey's HSD test. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.00005. N=4-5 mice per group, shown is a representative of three independent experiments.
[0023] Figure 4 depicts exemplary experimental results demonstrating PD-1 expression on peripheral and spleen T cells upon TERT DNA vaccination and ICB in non-tumor bearing mice.
[0024] Figure 5 depicts exemplary experimental results demonstrating that delivery of aCTLA-4 or aPD-1 post-2nd immunization has minimal effects on antigen-specific immune responses.
[0025] Figure 6, comprising Figure 6A through Figure 6E, depicts exemplary experimental results demonstrating delivery of aCTLA-4 or aPD-1 post- 1st vaccination synergizes with mTERT above checkpoint alone in generating anti-tumor immune response. Figure 6A depicts a schematic diagram of the experimental setup. Mice were implanted with TC-1 tumor cells on day 0, then immunized four times at one week intervals starting 7 days after tumor implant. Mice were given antibodies (20C^g per mouse) every three days starting 1 day after the first immunization. Antibody delivery was continued until one week after the final vaccination. Figure 6B depicts exemplary experimental results demonstrating tumor volume measurements over time for mice with mTERT vaccination and ICB. Figure 6B depicts exemplary experimental results demonstrating mouse survival over time for mice with mTERT vaccination and ICB. Figure 6D depicts exemplary experimental results demonstrating tumor volume measurements over time for mice with ICB alone. Figure 6E depicts exemplary experimental results demonstrating mouse survival over time for mice with ICB alone. Mice were euthanized if they appeared sick or if the tumor diameter exceeded 1.5cm. Significance for tumor volume measurements was determined by two-way ANOVA followed by Tukey's HSD test. Significance for mouse survival was determined by Gehan-Breslow-Wilcoxon test. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.00005. For Figure 6B and Figure 6C, N=12-13 mice per group. For Figure 6D and Figure 6E, N=9 mice per group. Each experiment was repeated at least once to verify results.
[0026] Figure 7, comprising Figure 7A through Figure 7F, depicts exemplary experimental results demonstrating antigen-specific intracellular cytokine production upon TERT DNA vaccination and ICB in tumor-bearing mice. Figure 7A depicts a schematic diagram of the experimental setup. Mice were implanted with TC-1 tumor cells on day 0, then immunized on days 7 and 14. Antibody delivery was started one day after the first immunization and continued every three days. Mice were sacrificed on day 21 for immune cell analysis. Figure 3B through Figure 3F depict exemplary experimental results demonstrating intracellular cytokine staining of CD8 or CD4 T cells after stimulation with native mouse TERT peptides for 5 hours. Significance was determined by two-way ANOVA followed by Tukey's HSD test. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.00005. N=7-10 mice per group, shown is a representative of three independent experiments.
[0027] Figure 8, comprising Figure 8A through Figure 8F, depicts exemplary experimental results demonstrating the phenotype of peripheral and spleen T cells upon TERT DNA vaccination and ICB in tumor bearing mice. Figure 8A depicts exemplary experimental results demonstrating the percentage of CD8+ splenocytes that are PD-1+. Figure 8B depicts exemplary experimental results demonstrating the percentage of CD8+ peripheral blood mononuclear cells (PBMCs) that are PD-1+. Figure 8C depicts exemplary experimental results demonstrating the percentage of CD4+ splenocytes that are PD-1+. Figure 8D depicts exemplary experimental results demonstrating the percentage of CD4+ PBMCs that are PD- 1+. Figure 8E depicts exemplary experimental results demonstrating the percentage of CD4+ splenocytes that are CD25+/FoxP3+. Figure 8F depicts exemplary experimental results demonstrating the percentage of CD4+ PBMCs that are CD25+/FoxP3+. Significance was determined by two-way ANOVA followed by Tukey's HSD test. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.00005. N=7-10 mice per group, shown is a representative of three independent experiments.
[0028] Figure 9, comprising Figure 9A through Figure 9D, depicts exemplary experimental results demonstrating the phenotype of tumor infiltrating lymphocytes (TILs) upon TERT DNA vaccination and ICB. Staining of CD8 or CD4 T cells isolated from the tumors of mice treated with the indicated therapies according to the schedule in Figure 4A. Figure 9A depicts exemplary experimental results demonstrating the percentage of CD45+/CD3+ TILs that are CD4/CD25/FoxP3+ following TERT simulation. Figure 9B depicts exemplary experimental results demonstrating the percentage of CD45+/CD3+/CD8+ TILs that are PD-1+ following TERT simulation. Figure 9C depicts exemplary experimental results demonstrating the percentage of CD3+ TILs that are CD8/CD44+ following TERT simulation. Figure 9D depicts exemplary experimental results demonstrating the percentage of CD3+ TILs that are CD8/CD44+ following PMA stimulation. Significance was determined by two-way ANOVA followed by Tukey's HSD test. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.00005. N=7-10 mice per group, shown is a representative of three independent experiments.
[0029] Figures 10, comprising Figure 10A through Figure 10B, depicts exemplary experimental results demonstrating that delivery of aCTLA-4 + DNA vaccine is superior to Treg depletion + DNA vaccine using aCD25 antibody. Figure 10A depicts exemplary experimental results demonstrating tumor volume measurements over time for mice with indicated treatment regimen. Mice were treated according to the schedule shown in Figure 7 A. Figure 10B depicts exemplary experimental results demonstrating mouse survival over time for mice with indicated treatment regimen. Mice were euthanized if they appeared sick or if the tumor diameter exceeded 1.5cm. Significance for tumor volume measurements was determined by two-way ANOVA followed by Tukey's HSD test. Significance for mouse survival was determined by Gehan-Breslow-Wilcoxon test. *p<0.05, **p<0.005,
***p<0.0005, ****p<0.00005. N= 10 mice per group.
DETAILED DESCRIPTION
[0030] The present invention relates to a composition that can be used to increase or enhance an immune response, i.e., create a more effective immune response, by combining an immunogenic composition, in many cases a synthetic antigen, with CTLA4 antibody. In some instances, a CTLA4 antibody can be administered in combination with the antigen; whereas, in other instances, with CTLA4 antibody can be administered separately from the antigen of the immunogenic composition.
[0031] In some instances, a CTLA4 antibody can be administered in combination with one or more additional antibody targeting one or more additional immune checkpoint proteins. In various embodiments, a CTLA4 antibody can be administered in combination with one or more antibodies against PD-1, PD-Ll, LAG3 and TIM3. In some instances, a combination of a CTLA4 antibody and one or more additional antibody targeting one or more additional immune checkpoint proteins is administered in combination with an antigen to create a more effective immune response.
[0032] The immunogenic composition of the present invention can increase the immune response to the antigen in the subject by increasing the CD8+ T cell response as compared to the immunogenic composition not including a CTLA4 antibody. This increased CD8+ T cell response has cytolytic activity and secretes the anti-viral cytokine interferon-gamma (IFN-γ).
[0033] Aspects of the present invention include compositions for enhancing an immune response against an antigen in a subject in need thereof, comprising a CTLA4 antibody in combination with a synthetic antigen capable of generating an immune response in the subject, or a immunogenic fragment or variant thereof.
[0034] The synthetic antigen can be an isolated DNA that encodes for the antigen.
[0035] In various exemplary embodiments, the synthetic antigen can be selected from the group consisting of: hTERT, prostate, WT1, tyrosinase, NYES01, PRAME, MAGE, CMV, herpes, HIV, HPV, HCV, HBV, influenza, RSV, Plasmodium falciparum, and C. difficile.
[0036] In one embodiment, the HPV antigen can be E6 and E7 domains of subtypes selected from the group consisting of: HPV6, HPVl l, HPV 16, HPV 18, HPV31, HPV33, HPV52, and HPV58, and a combination thereof.
[0037] In one embodiment, the HIV antigen can be selected from the group consisting of:
Env A, Env B, Env C, Env D, B Nef-Rev, and Gag, and a combination thereof.
[0038] In one embodiment, the influenza antigen can be selected from the group consisting of: HI HA, H2 HA, H3 HA, H5 HA, BHA antigen, and any combination thereof.
[0039] In one embodiment, the Plasmodium falciparum antigen includes a circumsporozoite
(CS) antigen.
[0040] In one embodiment, the C. difficile antigen can be selected from the group consisting of: Toxin A, and Toxin B, and a combination thereof. [0041] In one embodiment, the HCV antigen can be selected from the group consisting of: El, E2, NS3, NS4a, NS4b, NS5a, and NS5b, and a combination thereof.
[0042] In one embodiment, the HBV antigen can be selected from the group consisting of: surface antigen type A, surface antigen type B, surface antigen type C, surface antigen type D, surface antigen type E, surface antigen type F, surface antigen type G, surface antigen type H, and core antigen, and a combination thereof.
[0043] In one embodiment, the RSV antigen can be selected from the group consisting of: F, G, NS1, NS2, N, M, M2-1, M2-2, P, SH, and L protein, and a combination thereof.
[0044] In one embodiment, the synthetic antigen can be selected from the group consisting of: hTERT, WT1 antigen, tyrosinase, NYES01, or PRAME.
[0045] In one embodiment, the prostate antigen can be selected from the group consisting of: PSA, PSMA, STEAP, PSCA, and PAP, and a combination thereof.
[0046] In one embodiment, the herpes antigen can be selected from the group consisting of gB, gM, gN, gH, gL, gO, gE, gl, gK, gC, gD, UL128, UL130, UL131A, and UL83, and can be from any one of the following herpes family viruses: HCMV, HSV1, HSV2, VZV, or CMV.
[0047] In one embodiment, the CTLA4 antibody is selected from the group consisting of: ipilimumab, ipilimumab-Probody Tx (BMS-986249), nonfucosylated ipilimumab
(ipilimumab-NF) (BMS-986218), tremelimumab, CS 1002, MDX-010 (See ClinicalTrials.gov Identifier NCT00140855), and AGEN-1884 (Agenus, see ClinicalTrials.gov Identifier NCT02694822).
[0048] In one embodiment, the CTLA4 antibody is administered in combination with a PD-1 or PD-L1 antibody. PD-1 and PD-L1 antibodies appropriate for use in the methods of the invention include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, BMS- 936559 (See ClinicalTrials.gov Identifier NCT02028403), MPDL3280A (Roche, see ClinicalTrials.gov Identifier NCT02008227), MDX1105-01 (Bristol Myers Squibb, see ClinicalTrials.gov Identifier NCT00729664), MEDI4736 (Medlmmune, See
ClinicalTrials.gov Identifier NCT01693562), and MK-3475 (Merck, see ClinicalTrials.gov Identifier NCT02129556). The compositions provided herein can also include a
pharmaceutically acceptable excipient.
[0049] Aspects of the invention also include methods for increasing an immune response in a subject in need thereof by administering any of the compositions provided herein to the subject. The methods of increasing an immune response can also include an electroporating step.
1. Definitions
[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
[0051] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising," "consisting of and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not.
[0052] "Adjuvant" as used herein means any molecule added to the immunogenic composition described herein to enhance the immunogenicity of the antigens.
[0053] "Coding sequence" or "encoding nucleic acid" as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes a protein. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.
[0054] "Complement" or "complementary" as used herein means a nucleic acid can mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.
[0055] "Electroporation," "electro-permeabilization," or "electro-kinetic enhancement" ("EP") as used interchangeably herein means the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cellular membrane to the other.
[0056] "Fragment" or "immunogenic fragment" as used herein means a nucleic acid sequence or a portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal. The fragments can be DNA fragments selected from at least one of the various nucleotide sequences that encode protein fragments set forth below. Fragments can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the nucleic acid sequences set forth below. In some embodiments, fragments can comprise at least 20 nucleotides or more, at least 30 nucleotides or more, at least 40 nucleotides or more, at least 50 nucleotides or more, at least 60 nucleotides or more, at least 70 nucleotides or more, at least 80 nucleotides or more, at least 90 nucleotides or more, at least 100 nucleotides or more, at least 150 nucleotides or more, at least 200 nucleotides or more, at least 250 nucleotides or more, at least 300 nucleotides or more, at least 350 nucleotides or more, at least 400 nucleotides or more, at least 450 nucleotides or more, at least 500 nucleotides or more, at least 550 nucleotides or more, at least 600 nucleotides or more, at least 650 nucleotides or more, at least 700 nucleotides or more, at least 750 nucleotides or more, at least 800 nucleotides or more, at least 850 nucleotides or more, at least 900 nucleotides or more, at least 950 nucleotides or more, or at least 1000 nucleotides or more of at least one of the nucleic acid sequences set forth below.
[0057] Fragment or immunogenic fragment as used herein also means a polypeptide sequence or a portion thereof that is capable of eliciting an immune response in a mammal. The fragments can be polypeptide fragments selected from at least one of the various amino acid sequence set forth below. Fragments can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the proteins set forth below. In some embodiments, fragments can comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 1 10 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 190 amino acids or more, at least 200 amino acids or more, at least 210 amino acids or more, at least 220 amino acids or more, at least 230 amino acids or more, or at least 240 amino acids or more of at least one of the proteins set forth below.
[0058] "Genetic construct" as used herein refers to the DNA or RNA molecules that comprise a nucleotide sequence which encodes a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed.
[0059] "Identical" or "identity" as used herein in the context of two or more nucleic acids or polypeptide sequences, means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.
[0060] "Immune response" as used herein means the activation of a host's immune system, e.g., that of a mammal, in response to the introduction of antigen. The immune response can be in the form of a cellular or humoral response, or both.
[0061] "Nucleic acid" or "oligonucleotide" or "polynucleotide" as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
[0062] Nucleic acids can be single stranded or double stranded, or can contain portions of both double stranded and single stranded sequence. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.
[0063] "Operably linked" as used herein means that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5'
(upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.
[0064] A "peptide," "protein," or "polypeptide" as used herein can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.
[0065] "Promoter" as used herein means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter. [0066] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a synthetic antigen, including some of the examples cited herein. Signal peptides/leader sequences typically direct localization of a protein. Signal peptides/leader sequences used herein may serve to facilitate secretion of the protein from the cell in which it is produced. Signal peptides/leader sequences are often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. Signal peptides/leader sequences are linked at the N terminus of the protein.
[0067] "Subject" as used herein can mean a mammal that wants to or is in need of being immunized with the herein described vaccine. The mammal can be a human, chimpanzee, dog, cat, horse, cow, pig, chicken mouse, or rat.
[0068] "Substantially identical" as used herein can mean that a first and second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more amino acids. Substantially identical can also mean that a first nucleic acid sequence and a second nucleic acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more nucleotides.
[0069] "Treatment" or "treating," as used herein can mean protecting of an animal from a disease through means of preventing, suppressing, repressing, or completely eliminating the disease. Preventing the disease involves administering an immunogenic composition of the present invention to an animal prior to onset of the disease. Suppressing the disease involves administering an immunogenic composition of the present invention to an animal after induction of the disease but before its clinical appearance. Repressing the disease involves administering an immunogenic composition of the present invention to an animal after clinical appearance of the disease.
[0070] "Variant" used herein with respect to a nucleic acid means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.
[0071] Variant can further be defined as a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or to promote an immune response. Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A
conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The
hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
[0072] A variant may be a nucleic acid sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The nucleic acid sequence may be
80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof
[0073] "Vector" as used herein means a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self- replicating extrachromosomal vector. In one embodiment, a vector is a DNA plasmid.
[0074] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
2. Vaccine
[0075] Provided herein are vaccines comprising an antigen and CTLA4 antibody. The combination can be a single formulation or can be separate and administered in any combination or sequence. For example, in one embodiment, an immunogenic composition comprising an antigen can be administered first followed by administration of a CTLA4 antibody. In another embodiment, a CTLA4 antibody can be administered first followed by an immunogenic composition comprising an antigen. In yet another embodiment, an immunogenic composition comprising an antigen can be administered followed by multiple CTLA4 antibody treatments. In still another embodiment, an immunogenic composition comprising an antigen and a CTLA4 antibody may each be administered multiple times. The immunogenic composition can increase antigen presentation and the overall immune response to the antigen in a subject. The combination of antigen and immune checkpoint antibody induces the immune system more efficiently than an immunogenic composition comprising the antigen alone. This more efficient immune response provides increased efficacy in the treatment and/or prevention of any disease, in particular cancer, pathogen, or virus. a CTLA4 Vaccine
[0076] In one embodiment, the antigen and CTLA4 antibody of the immunogenic composition can be administered together or separately to the subject in need thereof. In some instances, a CTLA4 antibody can be administered separately from the antigen of the immunogenic composition.
[0077] In some embodiments, the CTLA4 antibody can be administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, or 96 hours before or after administration of the antigen to the subject. In other embodiments, the CTLA4 antibody can be administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or 90 days before or after administration of the antigen to the subject.
[0078] In still other embodiments, the CTLA4 antibody can be administered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks before or after administration of the antigen to the subject. In other embodiments, the CTLA4 antibody can be administered about 12 hours to about 15 weeks, about 12 hours to about 10 weeks, about 12 hours to about 5 weeks, about 12 hours to about 1 week, about 12 hours to about 60 hours, about 12 hours to about 48 hours, about 24 hours to about 15 weeks, about 60 hours to about 15 weeks, about 96 hours to about 15 weeks, about 1 day to about 15 weeks, about 5 days to about 15 weeks, about 10 days to about 15 weeks, about 15 days to about 15 weeks, about 20 days to about 15 weeks, about 25 days to about 15 weeks, about 30 days to about 15 weeks, about 1 week to about 15 weeks, about 5 weeks to about 15 weeks, or about 10 weeks to about 15 weeks before or after administration of the antigen to the subj ect.
[0079] The immunogenic composition of the present invention can have features required of effective vaccines such as being safe so the immunogenic composition itself does not cause illness or death; being protective against illness resulting from exposure to live pathogens such as viruses or bacteria; inducing neutralizing antibody to prevent infection of cells; inducing protective T cell against intracellular pathogens; and providing ease of
administration, few side effects, biological stability, and low cost per dose. The immunogenic composition can accomplish some or all of these features by combining the antigen with the CTLA4 antibody as discussed below.
[0080] The immunogenic composition can further modify epitope presentation within the antigen to induce greater immune response to the antigen that an immunogenic composition comprising the antigen alone. The immunogenic composition can further induce an immune response when administered to different tissues such as the muscle or the skin. b. Multi-antibody Vaccine
[0081] In one embodiment, the immunogenic composition comprising an antigen and a CTLA4 antibody can further comprise one or more additional antibodies targeting one or more additional immune checkpoint proteins. Additional immune checkpoint proteins that can be targeted include, but are not limited to, PD-1, PD-L1, LAG3 and TIM3. The combination comprising an antigen, a CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins can be a single formulation or can be separate and administered in any combination or sequence. For example, in one embodiment, an immunogenic composition comprising an antigen can be administered first followed by administration of a combination of a CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins. In another embodiment, a combination of a CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins can be administered first followed by an immunogenic composition comprising an antigen. In yet another embodiment, an immunogenic composition comprising an antigen can be administered followed by multiple administrations of a combination of a CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins. In still another embodiment, an immunogenic composition comprising an antigen can be administered followed by a single administration of a combination of a CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins and followed thereafter by one or more administrations of an antibody selected from a CTLA4 antibody, one or more antibody targeting one or more additional immune checkpoint proteins, and a combination thereof. The immunogenic composition can increase antigen presentation and the overall immune response to the antigen in a subject. The combination of antigen and immune checkpoint antibody induces the immune system more efficiently than an immunogenic composition comprising the antigen alone. This more efficient immune response provides increased efficacy in the treatment and/or prevention of any disease, in particular cancer, pathogen, or virus.
[0082] In one embodiment, the antigen, a CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins of the immunogenic composition can be administered together or separately to the subject in need thereof. In some instances, a CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins can be administered separately from the antigen of the immunogenic composition.
[0083] In some embodiments, the CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins can be administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, or 96 hours before or after administration of the antigen to the subject. In other embodiments, the CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins can be administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or 90 days before or after administration of the antigen to the subject.
[0084] In still other embodiments, the CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins can be administered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks before or after administration of the antigen to the subject. In other embodiments, the CTLA4 antibody and one or more antibody targeting one or more additional immune checkpoint proteins can be administered about 12 hours to about 15 weeks, about 12 hours to about 10 weeks, about 12 hours to about 5 weeks, about 12 hours to about 1 week, about 12 hours to about 60 hours, about 12 hours to about 48 hours, about 24 hours to about 15 weeks, about 60 hours to about 15 weeks, about 96 hours to about 15 weeks, about 1 day to about 15 weeks, about 5 days to about 15 weeks, about 10 days to about 15 weeks, about 15 days to about 15 weeks, about 20 days to about 15 weeks, about 25 days to about 15 weeks, about 30 days to about 15 weeks, about 1 week to about 15 weeks, about 5 weeks to about 15 weeks, or about 10 weeks to about 15 weeks before or after administration of the antigen to the subject. [0085] In some instances, a CTLA4 antibody can be administered separately from one or more antibody targeting one or more additional immune checkpoint proteins of the immunogenic composition. In some embodiments, the CTLA4 antibody can be administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, or 96 hours before or after administration of the one or more antibody targeting one or more additional immune checkpoint proteins to the subject. In other embodiments, the CTLA4 antibody can be administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or 90 days before or after administration of the one or more antibody targeting one or more additional immune checkpoint proteins to the subject.
[0086] In still other embodiments, the CTLA4 antibody can be administered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks before or after administration of the one or more antibody targeting one or more additional immune checkpoint proteins to the subject. In other embodiments, the CTLA4 antibody can be administered about 12 hours to about 15 weeks, about 12 hours to about 10 weeks, about 12 hours to about 5 weeks, about 12 hours to about 1 week, about 12 hours to about 60 hours, about 12 hours to about 48 hours, about 24 hours to about 15 weeks, about 60 hours to about 15 weeks, about 96 hours to about 15 weeks, about 1 day to about 15 weeks, about 5 days to about 15 weeks, about 10 days to about 15 weeks, about 15 days to about 15 weeks, about 20 days to about 15 weeks, about 25 days to about 15 weeks, about 30 days to about 15 weeks, about 1 week to about 15 weeks, about 5 weeks to about 15 weeks, or about 10 weeks to about 15 weeks before or after administration of the one or more antibody targeting one or more additional immune checkpoint proteins to the subject. c. Antibodies
[0087] The one or more antibody for use in the method of the invention can be a synthetic antibody comprised of DNA sequence encoding at least the variable regions of an immunoglobulin. Such antibody can be generated by identifying or screening for the antibody described above, which is reactive to or binds the antigen described above. The method of identifying or screening for the antibody can use the antigen in methodologies known in those skilled in art to identify or screen for the antibody. Such methodologies can include, but are not limited to, selection of the antibody from a library (e.g., phage display) and immunization of an animal followed by isolation and/or purification of the antibody. See for example methods available in Rajan, S., and Sidhu, S., Methods in Enzymology. vol 502, Chapter One "Simplified Synthetic Antibody Libraries (2012), which is incorporated herein in its entirety.
[0088] The antibody can be a polyclonal or monoclonal antibody. The antibody can be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. The humanized antibody can be an antibody from a non-human species that binds the desired antigen having one or more
complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.
[0089] The antibody can be a known product such as, for example, ipilimumab, ipilimumab- Probody Tx (BMS-986249), ipilimumab-NF (BMS-986218), tremelimumab, CS1002, MDX- 010 (See ClinicalTrials.gov Identifier NCT00140855), AGEN-1884 (Agenus, see
ClinicalTrials.gov Identifier NCT02694822), nivolumab, pembrolizumab, pidilizumab, BMS- 936559 (See ClinicalTrials.gov Identifier NCT02028403), MPDL3280A (Roche, see ClinicalTrials.gov Identifier NCT02008227), MDX1105-01 (Bristol Myers Squibb, see ClinicalTrials.gov Identifier NCT00729664), MEDI4736 (Medlmmune, See
ClinicalTrials.gov Identifier NCT01693562), and MK-3475 (Merck, see ClinicalTrials.gov Identifier NCT02129556). d. DNA based Antibodies
[0090] The antibody can be encoded by a nucleic acid sequence (cDNA) that encodes for the elements as follows:
[0091] The antibody can include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide can include a variable heavy chain (VH) region and/or at least one constant heavy chain (CH) region. The at least one constant heavy chain region can include a constant heavy chain region 1 (CHI), a constant heavy chain region 2 (CH2), and a constant heavy chain region 3 (CH3), and/or a hinge region. [0092] In some embodiments, the heavy chain polypeptide can include a VH region and a CHI region. In other embodiments, the heavy chain polypeptide can include a VH region, a CHI region, a hinge region, a CH2 region, and a CH3 region.
[0093] The heavy chain polypeptide can include a complementarity determining region ("CDR") set. The CDR set can contain three hypervariable regions of the VH region.
Proceeding from N-terminus of the heavy chain polypeptide, these CDRs are denoted "CDRl," "CDR2," and "CDR3," respectively. CDRl, CDR2, and CDR3 of the heavy chain polypeptide can contribute to binding or recognition of the antigen.
[0094] The light chain polypeptide can include a variable light chain (VL) region and/or a constant light chain (CL) region. The light chain polypeptide can include a complementarity determining region ("CDR") set. The CDR set can contain three hypervariable regions of the VL region. Proceeding from N-terminus of the light chain polypeptide, these CDRs are denoted "CDRl," "CDR2," and "CDR3," respectively. CDRl, CDR2, and CDR3 of the light chain polypeptide can contribute to binding or recognition of the antigen.
[0095] The antibody may comprise a heavy chain and a light chain complementarity determining region ("CDR") set, respectively interposed between a heavy chain and a light chain framework ("FR") set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. The CDR set may contain three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as "CDRl," "CDR2," and "CDR3," respectively. An antigen-binding site, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
[0096] The antibody can be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the immunoglobulin can include a VH region, a CHI region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin can include a VL region and CL region.
[0097] Additionally, the proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab')2 fragment, which comprises both antigen-binding sites. Accordingly, the antibody can be the Fab or F(ab')2. The Fab can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the Fab can include the VH region and the CHI region. The light chain of the Fab can include the VL region and CL region. e. Antigen
[0098] The immunogenic composition can also comprise an antigen, or fragment or variant thereof. The antigen can be anything that induces an immune response in a subject. The antigen can be a nucleic acid sequence, an amino acid sequence, or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence can also include additional sequences that encode linker or tag sequences that are linked to the antigen by a peptide bond. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.
[0099] The antigen can be contained in a protein, a nucleic acid, or a fragment thereof, or a variant thereof, or a combination thereof from any number of organisms, for example, a virus, a parasite, a bacterium, a fungus, or a mammal. The antigen can be associated with an autoimmune disease, allergy, or asthma. In other embodiments, the antigen can be associated with cancer, herpes, influenza, hepatitis B, hepatitis C, human papilloma virus (HPV), or human immunodeficiency virus (HIV).
[00100] Some antigens can induce a strong immune response. Other antigens can induce a weak immune response. The antigen can elicit a greater immune response when combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins as described above.
(1) Viral Antigens
[00101] The antigen can be a viral antigen, or fragment thereof, or variant thereof. The viral antigen can be from a virus from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae,
Poxviridae, Polyomaviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. The viral antigen can be from papilloma viruses, for example, human papillomoa virus (HPV), human immunodeficiency virus (HIV), polio virus, hepatitis viruses, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV), human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), herpes simplex 1 (HSV1; oral herpes), herpes simplex 2 (HSV2; genital herpes), herpes zoster (VZV; varicella-zoster, a.k.a., chickenpox), Epstein-Barr virus (EBV), Merkel cell polyoma virus (MCV), or cancer causing virus.
(a) Hepatitis Antigen
[00102] The CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a hepatitis virus antigen (i.e., hepatitis antigen), or fragment thereof, or variant thereof. The hepatitis antigen can be an antigen or immunogen from hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and/or hepatitis E virus (HEV). In some embodiments, the hepatitis antigen can be a nucleic acid molecule(s), such as a plasmid(s), which encodes one or more of the antigens from HAV, HBV, HCV, HDV, and HEV. The hepatitis antigen can be full-length or immunogenic fragments of full-length proteins.
[00103] The hepatitis antigen can comprise consensus sequences and/or modification for improved expression. Genetic modifications including codon optimization, RNA
optimization, and the addition of a high efficient immunoglobulin leader sequence to increase the immunogenicity of the constructs can be included in the modified consensus sequences. The consensus hepatitis antigen may comprise a signal peptide such as an immunoglobulin signal peptide such as an IgE or IgG signal peptide, and in some embodiments, may comprise an HA tag. The immunogens can be designed to elicit stronger and broader cellular immune responses than corresponding codong optimized immunogens.
[00104] The hepatitis antigen can be an antigen from HAV. The hepatitis antigen can be a HAV capsid protein, a HAV non-structural protein, a fragment thereof, a variant thereof, or a combination thereof.
[00105] The hepatitis antigen can be an antigen from HCV. The hepatitis antigen can be a HCV nucleocapsid protein (i.e., core protein), a HCV envelope protein (e.g., El and E2), a HCV non-structural protein (e.g., NS1, NS2, NS3, NS4a, NS4b, NS5a, and NS5b), a fragment thereof, a variant thereof, or a combination thereof.
[00106] The hepatitis antigen can be an antigen from HDV. The hepatitis antigen can be a HDV delta antigen, fragment thereof, or variant thereof.
[00107] The hepatitis antigen can be an antigen from HEV. The hepatitis antigen can be a HEV capsid protein, fragment thereof, or variant thereof. [00108] The hepatitis antigen can be an antigen from HBV. The hepatitis antigen can be a HBV core protein, a HBV surface protein, a HBV DNA polymerase, a HBV protein encoded by gene X, fragment thereof, variant thereof, or combination thereof. The hepatitis antigen can be a HBV genotype A core protein, a HBV genotype B core protein, a HBV genotype C core protein, a HBV genotype D core protein, a HBV genotype E core protein, a HBV genotype F core protein, a HBV genotype G core protein, a HBV genotype H core protein, a HBV genotype A surface protein, a HBV genotype B surface protein, a HBV genotype C surface protein, a HBV genotype D surface protein, a HBV genotype E surface protein, a HBV genotype F surface protein, a HBV genotype G surface protein, a HBV genotype H surface protein, fragment thereof, variant thereof, or combination thereof. The hepatitis antigen can be a consensus HBV core protein, or a consensus HBV surface protein.
[00109] In some embodiments, the hepatitis antigen can be a HBV genotype A consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype A core protein, or a HBV genotype A consensus core protein sequence.
[00110] In other embodiments, the hepatitis antigen can be a HBV genotype B consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype B core protein, or a HBV genotype B consensus core protein sequence.
[00111] In still other embodiments, the hepatitis antigen can be a HBV genotype C consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype C core protein, or a HBV genotype C consensus core protein sequence.
[00112] In some embodiments, the hepatitis antigen can be a HBV genotype D consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype D core protein, or a HBV genotype D consensus core protein sequence.
[00113] In other embodiments, the hepatitis antigen can be a HBV genotype E consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype E core protein, or a HBV genotype E consensus core protein sequence.
[00114] In some embodiments, the hepatitis antigen can be a HBV genotype F consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype F core protein, or a HBV genotype F consensus core protein sequence.
[00115] In other embodiments, the hepatitis antigen can be a HBV genotype G consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype G core protein, or a HBV genotype G consensus core protein sequence. [00116] In some embodiments, the hepatitis antigen can be a HBV genotype H consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype H core protein, or a HBV genotype H consensus core protein sequence.
[00117] In still other embodiments, the hepatitis antigen can be a HBV genotype A consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype A surface protein, or a HBV genotype A consensus surface protein sequence.
[00118] In some embodiments, the hepatitis antigen can be a HBV genotype B consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype B surface protein, or a HBV genotype B consensus surface protein sequence.
[00119] In other embodiments, the hepatitis antigen can be a HBV genotype C consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype C surface protein, or a HBV genotype C consensus surface protein sequence.
[00120] In still other embodiments, the hepatitis antigen can be a HBV genotype D consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype D surface protein, or a HBV genotype D consensus surface protein sequence.
[00121] In some embodiments, the hepatitis antigen can be a HBV genotype E consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype E surface protein, or a HBV genotype E consensus surface protein sequence.
[00122] In other embodiments, the hepatitis antigen can be a HBV genotype F consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype F surface protein, or a HBV genotype F consensus surface protein sequence.
[00123] In still other embodiments, the hepatitis antigen can be a HBV genotype G consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype G surface protein, or a HBV genotype G consensus surface protein sequence.
[00124] In other embodiments, the hepatitis antigen can be a HBV genotype H consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype H surface protein, or a HBV genotype H consensus surface protein sequence. (b) Human Papilloma Virus (HPV) Antigen
[00125] The CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a human papilloma virus (HPV) antigen, or fragment thereof, or variant thereof. The HPV antigen can be from HPV types 16, 18, 31, 33, 35, 45, 52, and 58 which cause cervical cancer, rectal cancer, and/or other cancers. The HPV antigen can be from HPV types 6 and 11, which cause genital warts, and are known to be causes of head and neck cancer.
[00126] The HPV antigens can be the HPV E6 or E7 domains from each HPV type. For example, for HPV type 16 (HPV 16), the HPV 16 antigen can include the HPV 16 E6 antigen, the HPV 16 E7 antigen, fragments, variants, or combinations thereof. Similarly, the HPV antigen can be HPV 6 E6 and/or E7, HPV 11 E6 and/or E7, HPV 18 E6 and/or E7, HPV 31 E6 and/or E7, HPV 33 E6 and/or E7, HPV 52 E6 and/or E7, or HPV 58 E6 and/or E7, fragments, variants, or combinations thereof.
(c) RSV Antigen
[00127] The CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can also be associated or combined with an RSV antigen or fragment thereof, or variant thereof. The RSV antigen can be a human RSV fusion protein (also referred to herein as "RSV F", "RSV F protein" and "F protein"), or fragment or variant thereof. The human RSV fusion protein can be conserved between RSV subtypes A and B. The RSV antigen can be a RSV F protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23994.1). The RSV antigen can be a RSV F protein from the RSV A2 strain (GenBank AAB59858.1), or a fragment or variant thereof. The RSV antigen can be a monomer, a dimer or trimer of the RSV F protein, or a fragment or variant thereof. The RSV antigen can be an optimized amino acid RSV F amino acid sequence, or fragment or variant thereof.
[00128] The postfusion form of RSV F elicits high titer neutralizing antibodies in immunized animals and protects the animals from RSV challenge. The present invention utilizes this immunoresponse in the claimed vaccines. According to the invention, the RSV F protein can be in a prefusion form or a postfusion form.
[00129] The RSV antigen can also be human RSV attachment glycoprotein (also referred to herein as "RSV G", "RSV G protein" and "G protein"), or fragment or variant thereof. The human RSV G protein differs between RSV subtypes A and B. The antigen can be RSV G protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23993). The RSV antigen can be RSV G protein from: the RSV subtype B isolate H5601, the RSV subtype B isolate H1068, the RSV subtype B isolate H5598, the RSV subtype B isolate HI 123, or a fragment or variant thereof. The RSV antigen can be an optimized amino acid RSV G amino acid sequence, or fragment or variant thereof.
[00130] In other embodiments, the RSV antigen can be human RSV non-structural protein 1 ("NS 1 protein"), or fragment or variant thereof. For example, the RSV antigen can be RSV NS 1 protein, or fragment or variant thereof, from the RSV Long strain (GenBank
AAX23987.1). The RSV antigen human can also be RSV non-structural protein 2 ("NS2 protein"), or fragment or variant thereof. For example, the RSV antigen can be RSV NS2 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23988.1). The RSV antigen can further be human RSV nucleocapsid ("N") protein, or fragment or variant thereof. For example, the RSV antigen can be RSV N protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23989.1). The RSV antigen can be human RSV Phosphoprotein ("P") protein, or fragment or variant thereof. For example, the RSV antigen can be RSV P protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23990.1). The RSV antigen also can be human RSV Matrix protein ("M") protein, or fragment or variant thereof. For example, the RSV antigen can be RSV M protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23991.1).
[00131] In still other embodiments, the RSV antigen can be human RSV small hydrophobic ("SH") protein, or fragment or variant thereof. For example, the RSV antigen can be RSV SH protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23992.1). The RSV antigen can also be human RSV Matrix protein2-l ("M2-1") protein, or fragment or variant thereof. For example, the RSV antigen can be RSV M2-1 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23995.1). The RSV antigen can further be human RSV Matrix protein 2-2 ("M2-2") protein, or fragment or variant thereof. For example, the RSV antigen can be RSV M2-2 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23997.1). The RSV antigen human can be RSV
Polymerase L ("L") protein, or fragment or variant thereof. For example, the RSV antigen can be RSV L protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23996.1).
[00132] In further embodiments, the RSV antigen can have an optimized amino acid sequence of NS1, NS2, N, P, M, SH, M2-1, M2-2, or L protein. The RSV antigen can be a human RSV protein or recombinant antigen, such as any one of the proteins encoded by the human RSV genome.
[00133] In other embodiments, the RSV antigen can be, but is not limited to, the RSV F protein from the RSV Long strain, the RSV G protein from the RSV Long strain, the optimized amino acid RSV G amino acid sequence, the human RSV genome of the RSV Long strain, the optimized amino acid RSV F amino acid sequence, the RSV NS1 protein from the RSV Long strain, the RSV NS2 protein from the RSV Long strain, the RSV N protein from the RSV Long strain, the RSV P protein from the RSV Long strain, the RSV M protein from the RSV Long strain, the RSV SH protein from the RSV Long strain, the RSV M2-1 protein from the RSV Long strain, the RSV M2-2 protein from the RSV Long strain, the RSV L protein from the RSV Long strain, the RSV G protein from the RSV subtype B isolate H5601, the RSV G protein from the RSV subtype B isolate H1068, the RSV G protein from the RSV subtype B isolate H5598, the RSV G protein from the RSV subtype B isolate HI 123, or fragment thereof, or variant thereof.
(d) Influenza Antigen
[00134] The CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with an influenza antigen or fragment thereof, or variant thereof. The influenza antigens are those capable of eliciting an immune response in a mammal against one or more influenza serotypes. The antigen can comprise the full length translation product HA0, subunit HA1, subunit HA2, a variant thereof, a fragment thereof or a combination thereof. The influenza hemagglutinin antigen can be a consensus sequence derived from multiple strains of influenza A serotype HI, a consensus sequence derived from multiple strains of influenza A serotype H2, a hybrid sequence containing portions of two different consensus sequences derived from different sets of multiple strains of influenza A serotype HI or a consensus sequence derived from multiple strains of influenza B. The influenza hemagglutinin antigen can be from influenza B.
[00135] The influenza antigen can also contain at least one antigenic epitope that can be effective against particular influenza immunogens against which an immune response can be induced. The antigen may provide an entire repertoire of immunogenic sites and epitopes present in an intact influenza virus. The antigen may be a consensus hemagglutinin antigen sequence that can be derived from hemagglutinin antigen sequences from a plurality of influenza A virus strains of one serotype such as a plurality of influenza A virus strains of serotype HI or of serotype H2. The antigen may be a hybrid consensus hemagglutinin antigen sequence that can be derived from combining two different consensus hemagglutinin antigen sequences or portions thereof. Each of two different consensus hemagglutinin antigen sequences may be derived from a different set of a plurality of influenza A virus strains of one serotype such as a plurality of influenza A virus strains of serotype HI . The antigen may be a consensus hemagglutinin antigen sequence that can be derived from hemagglutinin antigen sequences from a plurality of influenza B virus strains.
[00136] In some embodiments, the influenza antigen can be HI HA, H2 HA, H3 HA, H5 HA, or a BHA antigen. Alternatively, the influenza antigen can be a consensus hemagglutinin antigen comprising a consensus HI amino acid sequence or a consensus H2 amino acid sequence. The consensus hemagglutinin antigen may be a synthetic hybrid consensus HI sequence comprising portions of two different consensus HI sequences, which are each derived from a different set of sequences from the other. An example of a consensus HA antigen that is a synthetic hybrid consensus HI protein is a protein comprising the U2 amino acid sequence. The consensus hemagglutinin antigen may be a consensus hemagglutinin protein derived from hemagglutinin sequences from influenza B strains, such as a protein comprising the consensus BHA amino acid sequence.
[00137] The consensus hemagglutinin antigen may further comprise one or more additional amino acid sequence elements. The consensus hemagglutinin antigen may further comprise on its N-terminal an IgE or IgG leader amino acid sequence.The consensus hemagglutinin antigen may further comprise an immunogenic tag which is a unique immunogenic epitope that can be detected by readily available antibodies. An example of such an immunogenic tag is the 9 amino acid influenza HA Tag which may be linked on the consensus hemagglutinin C terminus. In some embodiments, consensus hemagglutinin antigen may further comprise on its N-terminal an IgE or IgG leader amino acid sequence and on its C terminal an HA tag.
[00138] The consensus hemagglutinin antigen may be a consensus hemagglutinin protein that consists of consensus influenza amino acid sequences or fragments and variants thereof. The consensus hemagglutinin antigen may be a consensus hemagglutinin protein that comprises non-influenza protein sequences and influenza protein sequences or fragments and variants thereof.
[00139] Examples of a consensus HI protein include those that may consist of the consensus HI amino acid sequence or those that further comprise additional elements such as an IgE leader sequence, or an HA Tag or both an IgE leader sequence and an HA Tag. [00140] Examples of consensus H2 proteins include those that may consist of the consensus H2 amino acid sequence or those that further comprise an IgE leader sequence, or an HA Tag, or both an IgE leader sequence and an HA Tag.
[00141] Examples of hybrid consensus HI proteins include those that may consist of the consensus U2 amino acid sequence or those that further comprise an IgE leader sequence, or an HA Tag, or both an IgE leader sequence and an HA Tag.
[00142] Examples of hybrid consensus influenza B hemagglutinin proteins include those that may consist of the consensus BHA amino acid sequence or it may comprise an IgE leader sequence, or an HA Tag, or both an IgE leader sequence and an HA Tag.
[00143] The consensus hemagglutinin protein can be encoded by a consensus
hemagglutinin nucleic acid, a variant thereof or a fragment thereof. Unlike the consensus hemagglutinin protein which may be a consensus sequence derived from a plurality of different hemagglutinin sequences from different strains and variants, the consensus hemagglutinin nucleic acid refers to a nucleic acid sequence that encodes a consensus protein sequence and the coding sequences used may differ from those used to encode the particular amino acid sequences in the plurality of different hemagglutinin sequences from which the consensus hemagglutinin protein sequence is derived. The consensus nucleic acid sequence may be codon optimized and/or RNA optimized. The consensus hemagglutinin nucleic acid sequence may comprise a Kozak's sequence in the 5 ' untranslated region. The consensus hemagglutinin nucleic acid sequence may comprise nucleic acid sequences that encode a leader sequence. The coding sequence of an N terminal leader sequence is 5' of the hemagglutinin coding sequence. The N-terminal leader can facilitate secretion. The N- terminal leader can be an IgE leader or an IgG leader. The consensus hemagglutinin nucleic acid sequence can comprise nucleic acid sequences that encode an immunogenic tag. The immunogenic tag can be on the C terminus of the protein and the sequence encoding it is 3 ' of the HA coding sequence. The immunogenic tag provides a unique epitope for which there are readily available antibodies so that such antibodies can be used in assays to detect and confirm expression of the protein. The immunogenic tag can be an H Tag at the C-terminus of the protein.
(e) Human Immunodeficiency Virus (HIV) Antigen
[00144] The CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with an HIV antigen or fragment thereof, or variant thereof. HIV antigens can include modified consensus sequences for immunogens. Genetic modifications including codon optimization, RNA optimization, and the addition of a high efficient immunoglobin leader sequence to increase the immunogenicity of constructs can be included in the modified consensus sequences. The novel immunogens can be designed to elicit stronger and broader cellular immune responses than a corresponding codon optimized immunogens.
[00145] In some embodiments, the HIV antigen can be a subtype A consensus envelope
DNA sequence construct, an IgE leader sequence linked to a consensus sequence for Subtype
A envelope protein, or a subtype A consensus Envelope protein sequence.
[00146] In other embodiments, the HIV antigen can be a subtype B consensus envelope
DNA sequence construct, an IgE leader sequence linked to a consensus sequence for Subtype
B envelope protein, or an subtype B consensus Envelope protein sequence.
[00147] In still other embodiments, the HIV antigen can be a subtype C consensus envelope
DNA sequence construct, an IgE leader sequence linked to a consensus sequence for subtype
C envelope protein, or a subtype C consensus envelope protein sequence.
[00148] In further embodiments, the HIV antigen can be a subtype D consensus envelope
DNA sequence construct, an IgE leader sequence linked to a consensus sequence for Subtype
D envelope protein, or a subtype D consensus envelope protein sequence.
[00149] In some embodiments, the HIV antigen can be a subtype B Nef-Rev consensus envelope DNA sequence construct, an IgE leader sequence linked to a consensus sequence for Subtype B Nef-Rev protein, or a Subtype B Nef-Rev consensus protein sequence.
[00150] In other embodiments, the HIV antigen can be a Gag consensus DNA sequence of subtype A, B, C and D DNA sequence construct, an IgE leader sequence linked to a consensus sequence for Gag consensus subtype A, B, C and D protein, or a consensus Gag subtype A, B, C and D protein sequence.
[00151] In still other embodiments the HIV antigen can be a Pol DNA sequence or a Pol protein sequence. The HIV antigen can be nucleic acid or amino acid sequences of Env A, Env B, Env C, Env D, B Nef-Rev , Gag, or any combination thereof.
(f) Herpes Antigen
[00152] The CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a herpes antigen or fragment thereof, or variant thereof. In one embodiment, the herpes antigen is from HCMV, HSV1, HSV2, CeHVl, VZV or EBV. The herpes antigens comprise immunogenic proteins including gB, gM, gN, gH, gL, gO, gE, gl, gK, gC, gD, UL128, UL130, UL-131A, UL-83 (pp65), whether from HCMV, HSV1, HSV2, CeHVl, VZV or EBV. In some embodiments, the antigens can be HSVl-gH, HSVl-gL, HSVl-gC, HSVl-gD, HSV2-gH, HSV2-gL, HSV2-gC, HSV2-gD, VZV-gH, VZV-gL, VZV-gM, VZV-gN, CeHVl -gH, CeHVl-gL, CeHVl-gC, CeHVl-gD, VZV-gE, or VZV-gl.
(g) Parasite Antigen
[00153] The CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a parasite antigen or fragment or variant thereof. The parasite can be a protozoa, helminth, or ectoparasite. The helminth (i.e., worm) can be a flatworm (e.g., flukes and tapeworms), a thorny -headed worm, or a round worm (e.g., pinworms). The ectoparasite can be lice, fleas, ticks, and mites.
[00154] The parasite can be any parasite causing the following diseases: Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis,
Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis, Taeniasis, Toxocariasis,
Toxoplasmosis, Trichinosis, and Trichuriasis.
[00155] The parasite can be Acanthamoeba, Anisakis, Ascaris lumbricoides , Botfly, Balantidium coli, Bedbug, Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania, Linguatula serrata, Liver fluke, Loa loa, Paragonimus - lung fluke, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma gondii, Trypanosoma, Whipworm, or Wuchereria bancrofti.
(h) Malaria Antigen
[00156] The CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a malaria antigen (i.e., Plasmodium falciparum (PF) antigen or PF immunogen), or fragment thereof, or variant thereof. The antigen can be from a parasite causing malaria. The malaria causing parasite can be Plasmodium falciparum. The Plasmodium falciparum antigen can include the circumsporozoite (CS) antigen.
[00157] In some embodiments, the malaria antigen can be nucleic acid molecules such as plasmids which encode one or more of the P. falciparum immunogens CS; LSA1 ; TRAP; CelTOS; and Amal. The immunogens may be full length or immunogenic fragments of full length proteins. The immunogens comprise consensus sequences and/or modifications for improved expression.
[00158] In other embodiments, the malaria antigen can be a consensus sequence of TRAP, which is also referred to as SSP2, designed from a compilation of all full-length Plasmodium falciparum TRAP/SSP2 sequences in the GenBank database (28 sequences total). Consensus TRAP immunogens (i.e., ConTRAP immunogen) may comprise a signal peptide such as an immunoglobulin signal peptide such as an IgE or IgG signal peptide and in some embodiments, may comprise an HA Tag.
[00159] In still other embodiments, the malaria antigen can be CelTOS, which is also referred to as Ag2 and is a highly conserved Plasmodium antigen. Consensus CelTOS antigens (i.e., ConCelTOS immunogen) may comprise a signal peptide such as an immunoglobulin signal peptide such as an IgE or IgG signal peptide and in some embodiments, may comprise an HA Tag.
[00160] In further embodiments, the malaria antigen can be Amal, which is a highly conserved Plasmodium antigen. The malaria antigen can also be a consensus sequence of Amal (i.e., ConAmal immunogen) comprising in some instances, a signal peptide such as an immunoglobulin signal peptide such as an IgE or IgG signal peptide and in some embodiments, may comprise an HA Tag.
[00161] In some embodiments, the malaria antigen can be a consensus CS antigen (i.e., Consensus CS immunogen) comprising in some instances, a signal peptide such as an immunoglobulin signal peptide such as an IgE or IgG signal peptide and in some embodiments, may comprise an HA Tag.
[00162] In other embodiments, the malaria antigen can be a fusion protein comprising a combination of two or more of the PF proteins set forth herein. For example, fusion proteins may comprise two or more of Consensus CS immunogen, ConLSAl immunogen, ConTRAP immunogen, ConCelTOS immunogen and ConAmal immunogen linked directly adjacent to each other or linked with a spacer or one or more amino acids in between. In some embodiments, the fusion protein comprises two PF immunogens; in some embodiments the fusion protein comprises three PF immunogens, in some embodiments the fusion protein comprises four PF immunogens, and in some embodiments the fusion protein comprises five PF immunogens. Fusion proteins with two Consensus PF immunogens may comprise: CS and LSA1; CS and TRAP; CS and CelTOS; CS and Amal; LSA1 and TRAP; LSA1 and CelTOS; LSA1 and Amal; TRAP and CelTOS; TRAP and Amal; or CelTOS and Amal. Fusion proteins with three Consensus PF immunogens may comprise: CS, LSA1 and TRAP; CS, LSA1 and CelTOS; CS, LSA1 and Amal; LSA1, TRAP and CelTOS; LSA1, TRAP and Amal; or TRAP, CelTOS and Amal. Fusion proteins with four Consensus PF immunogens may comprise: CS, LSA1, TRAP and CelTOS; CS, LSA1, TRAP and Amal; CS, LSA1, CelTOS and Amal; CS, TRAP, CelTOS and Amal; or LSA1, TRAP, CelTOS and Amal. Fusion proteins with five Consensus PF immunogens may comprise CS or CS-alt, LSA1, TRAP, CelTOS and Amal.
[00163] In some embodiments, the fusion proteins comprise a signal peptide linked to the N terminus. In some embodiments, the fusion proteins comprise multiple signal peptides linked to the N terminal of each Consensus PF immunogen. In some embodiments, a spacer may be included between PF immunogens of a fusion protein. In some embodiments, the spacer between PF immunogens of a fusion protein may be a proteolyic cleavage site. In some embodiments, the spacer may be a proteolyic cleavage site recognized by a protease found in cells to which the immunogenic composition is intended to be administered and/or taken up. In some embodiments, a spacer may be included between PF immunogens of a fusion protein wherein the spacer is a proteolyic cleavage site recognized by a protease found in cells to which the immunogenic composition is intended to be administered and/or taken up and the fusion proteins comprises multiple signal peptides linked to the N terminal of each Consensus PF immunogens such that upon cleavage the signal peptide of each Consensus PF immunogens translocates the Consensus PF immunogen to outside the cell.
(i) Bacterial Antigens
[00164] The CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a bacterial antigen or fragment or variant thereof. The bacterium can be from any one of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus- Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.
[00165] The bacterium can be a gram positive bacterium or a gram negative bacterium. The bacterium can be an aerobic bacterium or an anerobic bacterium. The bacterium can be an autotrophic bacterium or a heterotrophic bacterium. The bacterium can be a mesophile, a neutrophile, an extremophile, an acidophile, an alkaliphile, a thermophile, a psychrophile, an halophile, or an osmophile.
[00166] The bacterium can be an anthrax bacterium, an antibiotic resistant bacterium, a disease causing bacterium, a food poisoning bacterium, an infectious bacterium, Salmonella bacterium, Staphylococcus bacterium, Streptococcus bacterium, or tetanus bacterium. The bacterium can be a mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile. The bacterium can be Mycobacterium tuberculosis.
(j) Mycobacterium tuberculosis Antigens
[00167] The CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a Mycobacterium tuberculosis antigen (i.e., TB antigen or TB immunogen), or fragment thereof, or variant thereof. The TB antigen can be from the Ag85 family of TB antigens, for example, Ag85A and Ag85B. The TB antigen can be from the Esx family of TB antigens, for example, EsxA, EsxB, EsxC, EsxD, EsxE, EsxF, EsxH, EsxO, EsxQ, EsxR, EsxS, EsxT, EsxU, EsxV, and EsxW.
[00168] In some embodiments, the TB antigen can be nucleic acid molecules such as plasmids which encode one or more of the Mycobacterium tuberculosis immunogens from the Ag85 family and the Esx family. The immunogens can be full-length or immunogenic fragments of full-length proteins. The immunogens can comprise consensus sequences and/or modifications for improved expression. Consensus immunogens may comprise a signal peptide such as an immunoglobulin signal peptide such as an IgE or IgG signal peptide and in some embodiments, may comprise an HA tag.
(k) Fungal Antigens
[00169] The CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a fungal antigen or fragment or variant thereof. The fungus can be Aspergillus species, Blastomyces dermatitidis , Candida yeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or
Cladosporium.
(1) Tumor Antigen
[00170] In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder," refers to antigens that are common to specific hyperproliferative disorders such as cancer. The antigens discussed herein are merely included by way of example. The list is not intended to be exclusive and further examples will be readily apparent to those of skill in the art.
[00171] Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. The selection of the antigen binding moiety of the invention will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA,
Her2/neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.
[00172] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER-2/Neu/ErbB-2. Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. B-cell differentiation antigens such as CD 19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD 19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.
[00173] The type of tumor antigen referred to in the invention may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells.
[00174] Non-limiting examples of TSA or TAA antigens include the following:
Differentiation antigens such as MART- 1/MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl 85erbB2, pl 80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C- associated protein, TAAL6, TAG72, TLP, and TPS.
[00175] The CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins can be associated or combined with a tumor antigen or fragment or variant thereof. Cancer markers are known proteins that are present or upregulated vis-a-vis certain cancer cells. By methodology of generating antigens that represent such markers in a way to break tolerance to self, a cancer vaccine can be generated. Such cancer vaccines can include the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins to enhance the immune response. The following are some exemplary tumor antigens:
a. TERT
[00176] TERT is a telomerase reverse transcriptase that synthesizes a TTAGGG tag on the end of telomeres to prevent cell death due to chromosomal shortening. Hyperproliferative cells with abnormally high expression of TERT may be targeted by immunotherapy. Recent studies demonstrate that TERT expression in dendritic cells transfected with TERT genes can induce CD8+ cytotoxic T cells and elicit a CD4+ T cells in an antigen-specific fashion.
[00177] TERT can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules, including but not limited to those in the Examples, below.
[00178] In one embodiment, a consensus TERT antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is a TERT antigen derived from multiple human TERT sequences. In one embodiment, a human TERT antigen comprises a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NO:2 and SEQ ID NO:6. In one embodiment, human TERT antigen comprises a nucleotide sequence selected from SEQ ID NO: l and SEQ ID NO:5.
[00179] In one embodiment, a TERT antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is TERT antigen derived from non-human TERT sequences. In one embodiment, a TERT antigen is derived from multiple mouse TERT sequences. In one embodiment, a synthetic mouse TERT antigen encodes an amino acid sequence as set forth in SEQ ID NO: 10. In one embodiment, a synthetic mouse TERT antigen comprises a nucleotide sequence as set forth in SEQ ID NO:9.
[00180] In one embodiment, a TERT antigen is operably linked to a sequence encoding a signal peptide. In one embodiment, a signal peptide has an amino acid sequence as set forth in SEQ ID NO: 13. SEQ ID NO:3 and SEQ ID NO:7 provide exemplary hTERT nucleic acid sequences operably linked to a nucleotide sequence encoding an IgE leader sequence. SEQ ID NO:4 and SEQ ID NO: 8 provide exemplary hTERT amino acid sequences operably linked to an IgE leader sequence. SEQ ID NO: 11 provides an exemplary mTERT nucleic acid sequence operably linked to a nucleotide sequence encoding an IgE leader sequence. SEQ ID NO: 12 provides an exemplary mTERT amino acid sequence operably linked to an IgE leader sequence.
b. prostate antigens
[00181] The following are antigens capable of eliciting an immune response in a mammal against a prostate antigen. The consensus antigen can comprise epitopes that make them particularly effective as immunogens against prostate cancer cells can be induced. The consensus prostate antigen can comprise the full length translation product, a variant thereof, a fragment thereof or a combination thereof.
[00182] The prostate antigens can include one or more of the following: PSA antigen, PSMA antigen, STEAP antigen, PSCA antigen, Prostatic acid phosphatase (PAP) antigen, and other known prostate tumor antigens. Proteins may comprise sequences homologous to the prostate antigens, fragments of the prostate antigens and proteins with sequences homologous to fragments of the prostate antigens.
[00183] The prostate antigens can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
[00184] In one embodiment, a prostate antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is a consensus antigen derived from multiple human prostate antigen sequences. In one embodiment, a consensus prostate antigen is derived from multiple non- human prostate antigen sequences. In one embodiment, a consensus prostate antigen is operably linked to a sequence encoding a signal peptide.
[00185]
c. WT1
[00186] The antigen can be Wilm's tumor suppressor gene 1 (WT1), a fragment thereof, a variant thereof, or a combination thereof. WT1 is a transcription factor containing at the N- terminus, a proline/glutamine-rich DNA-binding domain and at the C-terminus, four zinc finger motifs. WT1 plays a role in the normal development of the urogenital system and interacts with numerous factors, for example, p53, a known tumor suppressor and the serine protease HtrA2, which cleaves WT1 at multiple sites after treatment with a cytotoxic drug.
[00187] Mutation of WT1 can lead to tumor or cancer formation, for example, Wilm's tumor or tumors expressing WT1. Wilm's tumor often forms in one or both kidneys before metastasizing to other tissues, for example, but not limited to, liver tissue, urinary tract system tissue, lymph tissue, and lung tissue. Accordingly, Wilm's tumor can be considered a metastatic tumor. Wilm's tumor usually occurs in younger children (e.g., less than 5 years old) and in both sporadic and hereditary forms. Accordingly, the immunogenic composition can be used for treating subjects suffering from Wilm's tumor. The immunogenic composition can also be used for treating subjects with cancers or tumors that express WTl for preventing development of such tumors in subjects. The WTl antigen can differ from the native, "normal" WTl gene, and thus, provide therapy or prophylaxis against an WTl antigen-expressing tumor. Proteins may comprise sequences homologous to the WTl antigens, fragments of the WTl antigens and proteins with sequences homologous to fragments of the WTl antigens.
[00188] The WTl antigens can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
[00189] In one embodiment, a consensus WTl antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is derived from multiple human WTl sequences. In one embodiment, a consensus WTl antigen is derived from multiple non-human WTl sequences. In one embodiment, a consensus WTl antigen is operably linked to a sequence encoding a signal peptide.
d. Tyrosinase antigen
[00190] The antigen tyrosinase (Tyr) antigen is an important target for immune mediated clearance by inducing (1) humoral immunity via B cell responses to generate antibodies that block monocyte chemoattractant protein-1 (MCP-1) production, thereby retarding myeloid derived suppressor cells (MDSCs) and suppressing tumor growth; (2) increase cytotoxic T lymphocyte such as CD8+ (CTL) to attack and kill tumor cells; (3) increase T helper cell responses; (4) and increase inflammatory responses via IFN-γ and TFN-a or all of the aforementioned.
[00191] Tyrosinase is a copper-containing enzyme that can be found in plant and animal tissues. Tyrosinase catalyzes the production of melanin and other pigments by the oxidation of phenols such as tyrosine. In melanoma, tyrosinase can become unregulated, resulting in increased melanin synthesis. Tyrosinase is also a target of cytotoxic T cell recognition in subjects suffering from melanoma. Accordingly, tyrosinase can be an antigen associated with melanoma.
[00192] The antigen can comprise protein epitopes that make them particularly effective as immunogens against which anti-Tyr immune responses can be induced. The Tyr antigen can comprise the full length translation product, a variant thereof, a fragment thereof or a combination thereof.
[00193] The Tyr antigen can comprise a consensus protein. The Tyr antigen induces antigen-specific T-cell and high titer antibody responses both systemically against all cancer and tumor related cells. As such, a protective immune response is provided against tumor formation by vaccines comprising the Tyr consensus antigen. Accordingly, any user can design an immunogenic composition of the present invention to include a Tyr antigen to provide broad immunity against tumor formation, metastasis of tumors, and tumor growth. Proteins may comprise sequences homologous to the Tyr antigens, fragments of the Tyr antigens and proteins with sequences homologous to fragments of the Tyr antigens.
[00194] The Tyr antigens can be administered in vectors described herein, and combined the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
[00195] In one embodiment, a consensus Tyr antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is a consensus antigen derived from multiple human Tyr sequences. In one embodiment, a consensus Tyr antigen is derived from multiple non-human Tyr sequences. In one embodiment, a consensus Tyr antigen is operably linked to a sequence encoding a signal peptide.
e. NY-ESO-1
[00196] NY-ESO-1 is a cancer-testis antigen expressed in various cancers where it can induce both cellular and humoral immunity. Gene expression studies have shown upregulation of the gene for NY-ESO-1, CTAG1B, in myxoid and round cell liposarcomas.
[00197] In various embodiments, the NY-ESO-1 antigen comprises a consensus NY-ESO-1 protein or a nucleic acid molecule encoding a consensus NY-ESO-1 protein. NY-ESO-1 antigens include sequences homologous to the NY-ESO-1 antigens, fragments of the NY- ESO-1 antigens and proteins with sequences homologous to fragments of the NY-ESO-1 antigens. [00198] The NY-ESO-1 antigens can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
[00199] In one embodiment, a consensus NY-ESO-1 antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is derived from multiple human NY-ESO-1 sequences. In one embodiment, a consensus NY-ESO-1 antigen is derived from multiple non-human NY-ESO- 1 sequences. In one embodiment, a consensus NY-ESO-1 antigen is operably linked to a sequence encoding a signal peptide.
[00200]
f. PRAME
[00201] Melanoma antigen preferentially expressed in tumors (PRAME antigen) is a protein that in humans is encoded by the PRAME gene. This gene encodes an antigen that is predominantly expressed in human melanomas and that is recognized by cytolytic T lymphocytes. It is not expressed in normal tissues, except testis. The gene is also expressed in acute leukemias. Five altematively spliced transcript variants encoding the same protein have been observed for this gene. Proteins may comprise sequences homologous to the PRAME antigens, fragments of the PRAME antigens and proteins with sequences homologous to fragments of the PRAME antigens.
[00202] The PRAME antigens can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
[00203] In one embodiment, a consensus PRAME antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is derived from multiple human PRAME sequences. In one embodiment, a consensus PRAME antigen is derived from multiple non-human PRAME sequences. In one embodiment, a consensus PRAME antigen is operably linked to a sequence encoding a signal peptide.
g. MAGE
[00204] MAGE stands for Melanoma-associated Antigen, and in particular melanoma associated antigen 4 (MAGEA4). MAGE-A4 is expressed in male germ cells and tumor cells of various histological types such as gastrointestinal, esophageal and pulmonary carcinomas. MAGE-A4 binds the oncoprotein, Gankyrin. This MAGE-A4 specific binding is mediated by its C-terminus. Studies have shown that exogenous MAGE-A4 can partly inhibit the adhesion-independent growth of Gankyrin-overexpressing cells in vitro and suppress the formation of migrated tumors from these cells in nude mice. This inhibition is dependent upon binding between MAGE-A4 and Gankyrin, suggesting that interactions between Gankyrin and MAGE-A4 inhibit Gankyrin-mediated carcinogenesis. It is likely that MAGE expression in tumor tissue is not a cause, but a result of tumorgenesis, and MAGE genes take part in the immune process by targeting early tumor cells for destruction.
[00205] Melanoma-associated antigen 4 protein (MAGEA4) can be involved in embryonic development and tumor transformation and/or progression. MAGEA4 is normally expressed in testes and placenta. MAGEA4, however, can be expressed in many different types of tumors, for example, melanoma, head and neck squamous cell carcinoma, lung carcinoma, and breast carcinoma. Accordingly, MAGEA4 can be antigen associated with a variety of tumors.
[00206] The MAGEA4 antigen can induce antigen-specific T cell and/or high titer antibody responses, thereby inducing or eliciting an immune response that is directed to or reactive against the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or elicited cellular immune response can include induction or secretion of interferon-gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-a). In other embodiments, the induced or elicited immune response can reduce or inhibit one or more immune suppression factors that promote growth of the tumor or cancer expressing the antigen, for example, but not limited to, factors that down regulate MHC presentation, factors that up regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor associated macrophages, tumor associated fibroblasts.
[00207] The MAGEA4 antigen can comprise protein epitopes that make them particularly effective as immunogens against which anti-MAGEA4 immune responses can be induced. The MAGEA4 antigen can comprise the full length translation product, a variant thereof, a fragment thereof or a combination thereof. The MAGEA4 antigen can comprise a consensus protein.
[00208] The nucleic acid sequence encoding the consensus MAGEA4 antigen can be optimized with regards to codon usage and corresponding RNA transcripts. The nucleic acid encoding the consensus MAGEA4 antigen can be codon and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the consensus MAGEA4 antigen can include a Kozak sequence (e.g., GCC ACC) to increase the efficiency of translation. The nucleic acid encoding the consensus MAGEA4 antigen can include multiple stop codons (e.g., TGA TGA) to increase the efficiency of translation termination.
[00209] The MAGEA4 antigen can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
[00210] In one embodiment, a consensus MAGEA4 antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is derived from multiple human MAGEA4 sequences. In one embodiment, a consensus MAGEA4 antigen is derived from multiple non-human MAGEA4 sequences. In one embodiment, a consensus MAGEA4 antigen is operably linked to a sequence encoding a signal peptide,
h. FSHR
[00211] Follicle stimulating hormone receptor (FSHR) is an antigen that is selectively expressed in women in the ovarian granulosa cells (Simoni et al, Endocr Rev. 1997, 18:739- 773) and at low levels in the ovarian endothelium (Vannier et al, Biochemistry, 1996, 35: 1358-1366). Most importantly, this surface antigen is expressed in 50-70% of ovarian carcinomas.
[00212] In various embodiments, the FSHR antigen comprises a consensus protein or a nucleic acid molecule encoding a consensus protein. FSHR antigens include sequences homologous to the FSHR antigens, fragments of the FSHR antigens and proteins with sequences homologous to fragments of the FSHR antigens.
[00213] The FSHR antigens can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
[00214] In one embodiment, a consensus FSHR antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is derived from multiple human FSHR sequences. In one embodiment, a consensus FSHR antigen is derived from multiple non-human FSHR sequences. In one embodiment, a consensus FSHR antigen is operably linked to a signal peptide. i. Tumor Microenvironment Antigens
[00215] Several proteins are overexpressed in the tumor microenvironment including, but not limited to, Fibroblast Activation Protein (FAP), Platelet Derived Growth Factor Receptor Beta (PDGFR-β), and Glypican-1 (GPCl). FAP is a membrane-bound enzyme with gelatinase and peptidase activity that is up-regulated in cancer-associated fibroblasts in over 90% of human carcinomas. PDGFR-β is a cell surface tyrosine kinase receptor that has roles in the regulation of many biological processes including embryonic development, angiogenesis, cell proliferation and differentiation. GPCl is a cell surface proteoglycan that is enriched in cancer cells.
[00216] In various embodiments, the tumor microenvironment antigen comprises a consensus protein or a nucleic acid molecule encoding a consensus protein. Tumor microenvironment antigens include sequences homologous to the tumor microenvironment antigens, fragments of the tumor microenvironment antigens and proteins with sequences homologous to fragments of the tumor microenvironment antigens.
[00217] One or more tumor microenvironment antigens can be administered in vectors described herein, and combined with the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins in various vaccination schedules.
[00218] In one embodiment, a consensus tumor microenvironment antigen that can be administered with a CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins is derived from multiple human tumor microenvironment antigen sequences. In one embodiment, a consensus tumor
microenvironment antigen is derived from multiple non-human tumor microenvironment antigen sequences. In one embodiment, a consensus tumor microenvironment antigen is operably linked to a signal peptide. f. Vector
[00219] The immunogenic composition can comprise one or more vectors that include a nucleic acid encoding the antigen and the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins. The one or more vectors can be capable of expressing the antigen and the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins. The vector can have a nucleic acid sequence containing an origin of replication. The vector can be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. The vector can be either a self-replication extra chromosomal vector, or a vector which integrates into a host genome.
[00220] The one or more vectors can be an expression construct, which is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular- transcription and translation machinery ribosomal complexes. The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The vectors of the present invention express large amounts of stable messenger RNA, and therefore proteins.
[00221] The vectors may have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).
(1) Expression Vectors
[00222] The vector can be a circular plasmid or a linear nucleic acid. The circular plasmid and linear nucleic acid are capable of directing expression of a particular nucleotide sequence in an appropriate subject cell. The vector can have a promoter operably linked to the antigen- encoding nucleotide sequence, or the adjuvant-encoding nucleotide sequence, which may be operably linked to termination signals. The vector can also contain sequences required for proper translation of the nucleotide sequence. The vector comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.
(2) Circular and Linear Vectors
[00223] The vector may be circular plasmid, which may transform a target cell by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication). [00224] The vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing DNA encoding the antigen, or the adjuvant and enabling a cell to translate the sequence to an antigen that is recognized by the immune system, or the adjuvant.
[00225] Also provided herein is a linear nucleic acid vaccine, or linear expression cassette ("LEC"), that is capable of being efficiently delivered to a subject via electroporation and expressing one or more desired antigens, or one or more desired adjuvants. The LEC may be any linear DNA devoid of any phosphate backbone. The DNA may encode one or more antigens, or one or more adjuvants. The LEC may contain a promoter, an intron, a stop codon, and/or a polyadenylation signal. The expression of the antigen, or the adjuvant may be controlled by the promoter. The LEC may not contain any antibiotic resistance genes and/or a phosphate backbone. The LEC may not contain other nucleic acid sequences unrelated to the desired antigen gene expression, or the desired adjuvant expression.
[00226] The LEC may be derived from any plasmid capable of being linearized. The plasmid may be capable of expressing the antigen, the CTLA4 antibody or one or more antibody targeting one or more additional immune checkpoint proteins. The plasmid can be pNP (Puerto Rico/34) or pM2 (New Caledonia/99). The plasmid may be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing DNA encoding the antigen, or encoding the adjuvant, and enabling a cell to translate the sequence to an antigen that is recognized by the immune system, or the adjuvant.
[00227] The LEC can be pcrM2. The LEC can be pcrNP. pcrNP and pcrMR can be derived from pNP (Puerto Rico/34) and pM2 (New Caledonia/99), respectively.
(3) Promoter, Intron, Stop Codon, and Polyadenylation Signal
[00228] The vector may have a promoter. A promoter may be any promoter that is capable of driving gene expression and regulating expression of the isolated nucleic acid. Such a promoter is a cis-acting sequence element required for transcription via a DNA dependent RNA polymerase, which transcribes the antigen sequence, or the adjuvant sequence described herein. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter may be positioned about the same distance from the transcription start in the vector as it is from the transcription start site in its natural setting. However, variation in this distance may be accommodated without loss of promoter function. [00229] The promoter may be operably linked to the nucleic acid sequence encoding the antigen and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. The promoter may be operably linked to the nucleic acid sequence encoding the adjuvant and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.
[00230] The promoter may be a CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or another promoter shown effective for expression in eukaryotic cells.
[00231] The vector may include an enhancer and an intron with functional splice donor and acceptor sites. The vector may contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes. g. Excipients and other Components of the immunogenic composition
[00232] The immunogenic composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be functional molecules such as vehicles, adjuvants other than the CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins, carriers, or diluents. The pharmaceutically acceptable excipient can be a transfection facilitating agent, which can include surface active agents, such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, poly cations, or nanoparticles, or other known transfection facilitating agents.
[00233] The transfection facilitating agent is a polyanion, poly cation, including poly-L- glutamate (LGS), or lipid. The transfection facilitating agent is poly-L-glutamate, and the poly-L-glutamate may be present in the immunogenic composition at a concentration less than 6 mg/ml. The transfection facilitating agent may also include surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the genetic construct. The DNA plasmid vaccines may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example W09324640), calcium ions, viral proteins, polyanions, poly cations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, poly cation, including poly-L-glutamate (LGS), or lipid. Concentration of the transfection agent in the immunogenic composition is less than 4 mg/ml, less than 2 mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml, less than 0.250 mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml, or less than 0.010 mg/ml.
[00234] The pharmaceutically acceptable excipient can be an adjuvant in addition to CTLA4 antibody and optionally one or more antibody targeting one or more additional immune checkpoint proteins. The additional adjuvant can be other genes that are expressed in an alternative plasmid or are delivered as proteins in combination with the plasmid above in the immunogenic composition. The adjuvant may be selected from the group consisting of: a- interferon(IFN- a), β-interferon (IFN-β), γ-interferon, platelet derived growth factor (PDGF), TNFa, TNF , GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 having the signal sequence deleted and optionally including the signal peptide from IgE. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet derived growth factor (PDGF), TNFa, TNFP, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.
[00235] Other genes that can be useful as adjuvants in addition to the PD1 antibody or PDLl antibody include those encoding: MCP-1, MIP-la, MIP-lp, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL- 1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL,
TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof. [00236] The immunogenic composition may further comprise a genetic vaccine facilitator agent as described in U.S. Serial No. 021,579 filed April 1, 1994, which is fully incorporated by reference.
[00237] The immunogenic composition can be formulated according to the mode of administration to be used. An injectable vaccine pharmaceutical composition can be sterile, pyrogen free and particulate free. An isotonic formulation or solution can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The immunogenic composition can comprise a vasoconstriction agent. The isotonic solutions can include phosphate buffered saline. Vaccine can further comprise stabilizers including gelatin and albumin. The stabilizers can allow the formulation to be stable at room or ambient temperature for extended periods of time, including LGS or poly cations or polyanions.
3. Method of Vaccination
[00238] The present invention is also directed to a method of increasing an immune response in a subject. Increasing the immune response can be used to treat and/or prevent disease in the subject. The method can include administering the herein disclosed vaccine to the subject. The subject administered the immunogenic composition can have an increased or boosted immune response as compared to a subject administered the antigen alone. In some embodiments, the immune response can be increased by about 0.5-fold to about 15-fold, about 0.5-fold to about 10-fold, or about 0.5-fold to about 8-fold. Alternatively, the immune response in the subject administered the immunogenic composition can be increased by at least about 0.5-fold, at least about 1.0-fold, at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0- fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, or at least about 15.0-fold.
[00239] In still other alternative embodiments, the immune response in the subject administered the immunogenic composition can be increased about 1% to about 1500%, about 1% to about 1000%, or about 1% to about 800%. In other embodiments, the immune response in the subject administered the immunogenic composition can be increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000%, at least about 1050%, at least about 1100%, at least about 1150%, at least about 1200%, at least about 1250%, at least about 1300%, at least about 1350%, at least about 1450%, or at least about 1500%.
[00240] The immunogenic composition dose can be between 1 μg to 10 mg active component kg body weight/time, and can be 20 μg to 10 mg component/kg body
weight/time. The immunogenic composition can be administered every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of vaccine doses for effective treatment can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10. a. Administration
[00241] The immunogenic composition can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration. The subject can be a mammal, such as a human, a horse, a cow, a pig, a sheep, a cat, a dog, a rat, or a mouse.
[00242] The immunogenic composition can be administered prophylactically or therapeutically. In prophylactic administration, the immunogenic composition can be administered in an amount sufficient to induce an immune response. In therapeutic applications, the immunogenic compositions are administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount adequate to accomplish this is defined as "therapeutically effective dose. " Amounts effective for this use will depend on, e.g., the particular composition of the immunogenic composition regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician. [00243] The immunogenic composition can be administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Feigner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Feigner (U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S. Pat. No. 5,679,647, issued Oct. 21, 1997), the contents of all of which are incorporated herein by reference in their entirety. The DNA of the immunogenic composition can be complexed to particles or beads that can be administered to an individual, for example, using an immunogenic composition gun. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a
physiologically acceptable compound, depends, for example, on the route of administration of the expression vector.
[00244] The immunogenic composition can be delivered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes. For the DNA of the immunogenic composition in particular, the immunogenic composition can be delivered to the interstitial spaces of tissues of an individual (Feigner et al., U.S. Pat. Nos. 5,580,859 and 5,703,055, the contents of all of which are incorporated herein by reference in their entirety). The immunogenic composition can also be administered to muscle, or can be administered via intradermal or subcutaneous injections, or
transdermally, such as by iontophoresis. Epidermal administration of the immunogenic composition can also be employed. Epidermal administration can involve mechanically or chemically irritating the outermost layer of epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Pat. No. 5,679,647, the contents of which are incorporated herein by reference in its entirety).
[00245] The immunogenic composition can also be formulated for administration via the nasal passages. Formulations suitable for nasal administration, wherein the carrier is a solid, can include a coarse powder having a particle size, for example, in the range of about 10 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. The formulation can be a nasal spray, nasal drops, or by aerosol administration by nebulizer. The formulation can include aqueous or oily solutions of the immunogenic composition.
[00246] The immunogenic composition can be a liquid preparation such as a suspension, syrup or elixir. The immunogenic composition can also be a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as a sterile suspension or emulsion.
[00247] The immunogenic composition can be incorporated into liposomes, microspheres or other polymer matrices (Feigner et al, U.S. Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vols. Ito III (2nd ed. 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes can consist of phospholipids or other lipids, and can be nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
[00248] The immunogenic composition can be administered via electroporation, such as by a method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference. The electroporation can be by a method and/or apparatus described in U.S. Patent Nos. 6,302,874; 5,676,646; 6,241,701 ; 6,233,482; 6,216,034; 6,208,893;
6,192,270; 6,181,964; 6,150,148; 6,120,493; 6,096,020; 6,068,650; and 5,702,359, the contents of which are incorporated herein by reference in their entirety. The electroporation may be carried out via a minimally invasive device.
[00249] The minimally invasive electroporation device ("MID") may be an apparatus for injecting the immunogenic composition described above and associated fluid into body tissue. The device may comprise a hollow needle, DNA cassette, and fluid delivery means, wherein the device is adapted to actuate the fluid delivery means in use so as to concurrently (for example, automatically) inject DNA into body tissue during insertion of the needle into the said body tissue. This has the advantage that the ability to inject the DNA and associated fluid gradually while the needle is being inserted leads to a more even distribution of the fluid through the body tissue. The pain experienced during injection may be reduced due to the distribution of the DNA being injected over a larger area.
[00250] The MID may inject the immunogenic composition into tissue without the use of a needle. The MID may inject the immunogenic composition as a small stream or jet with such force that the immunogenic composition pierces the surface of the tissue and enters the underlying tissue and/or muscle. The force behind the small stream or jet may be provided by expansion of a compressed gas, such as carbon dioxide through a micro-orifice within a fraction of a second. Examples of minimally invasive electroporation devices, and methods of using them, are described in published U.S. Patent Application No. 20080234655; U.S.
Patent No. 6,520,950; U.S. Patent No. 7,171,264; U.S. Patent No. 6,208,893; U.S. Patent NO. 6,009,347; U.S. Patent No. 6,120,493; U.S. Patent No. 7,245,963; U.S. Patent No. 7,328,064; and U. S. Patent No. 6,763,264, the contents of each of which are herein incorporated by reference.
[00251] The MID may comprise an injector that creates a high-speed jet of liquid that painlessly pierces the tissue. Such needle-free injectors are commercially available. Examples of needle-free injectors that can be utilized herein include those described in U. S. Patent Nos. 3,805,783; 4,447,223; 5,505,697; and 4,342,310, the contents of each of which are herein incorporated by reference.
[00252] A desired vaccine in a form suitable for direct or indirect electrotransport may be introduced (e.g., inj ected) using a needle-free injector into the tissue to be treated, usually by contacting the tissue surface with the injector so as to actuate delivery of a jet of the agent, with sufficient force to cause penetration of the immunogenic composition into the tissue. For example, if the tissue to be treated is mucosa, skin or muscle, the agent is projected towards the mucosal or skin surface with sufficient force to cause the agent to penetrate through the stratum comeum and into dermal layers, or into underlying tissue and muscle, respectively.
[00253] Needle-free injectors are well suited to deliver vaccines to all types of tissues, particularly to skin and mucosa. In some embodiments, a needle-free inj ector may be used to propel a liquid that contains the immunogenic composition to the surface and into the subject's skin or mucosa. Representative examples of the various types of tissues that can be treated using the invention methods include pancreas, larynx, nasopharynx, hypopharynx, oropharynx, lip, throat, lung, heart, kidney, muscle, breast, colon, prostate, thymus, testis, skin, mucosal tissue, ovary, blood vessels, or any combination thereof.
[00254] The MID may have needle electrodes that electroporate the tissue. By pulsing between multiple pairs of electrodes in a multiple electrode array, for example set up in rectangular or square patterns, provides improved results over that of pulsing between a pair of electrodes. Disclosed, for example, in U.S. Patent No. 5,702,359 entitled "Needle
Electrodes for Mediated Delivery of Drugs and Genes" is an array of needles wherein a plurality of pairs of needles may be pulsed during the therapeutic treatment. In that application, which is incorporated herein by reference as though fully set forth, needles were disposed in a circular array, but have connectors and switching apparatus enabling a pulsing between opposing pairs of needle electrodes. A pair of needle electrodes for delivering recombinant expression vectors to cells may be used. Such a device and system is described in U. S. Patent No. 6,763,264, the contents of which are herein incorporated by reference. Alternatively, a single needle device may be used that allows injection of the DNA and electroporation with a single needle resembling a normal injection needle and applies pulses of lower voltage than those delivered by presently used devices, thus reducing the electrical sensation experienced by the patient.
[00255] The MID may comprise one or more electrode arrays. The arrays may comprise two or more needles of the same diameter or different diameters. The needles may be evenly or unevenly spaced apart. The needles may be between 0.005 inches and 0.03 inches, between 0.01 inches and 0.025 inches; or between 0.015 inches and 0.020 inches. The needle may be 0.0175 inches in diameter. The needles may be 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more spaced apart.
[00256] The MID may consist of a pulse generator and a two or more-needle vaccine injectors that deliver the immunogenic composition and electroporation pulses in a single step. The pulse generator may allow for flexible programming of pulse and injection parameters via a flash card operated personal computer, as well as comprehensive recording and storage of electroporation and patient data. The pulse generator may deliver a variety of volt pulses during short periods of time. For example, the pulse generator may deliver three 15 volt pulses of 100 ms in duration. An example of such a MID is the Elgen 1000 system by Inovio Biomedical Corporation, which is described in U.S. Patent No. 7,328,064, the contents of which are herein incorporated by reference.
[00257] The MID may be a CELLECTRA (Inovio Pharmaceuticals, Plymouth Meeting PA) device and system, which is a modular electrode system, that facilitates the introduction of a macromolecule, such as a DNA, into cells of a selected tissue in a body or plant. The modular electrode system may comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a conductive link from a programmable constant-current pulse controller to the plurality of needle electrodes; and a power source. An operator can grasp the plurality of needle electrodes that are mounted on a support structure and firmly insert them into the selected tissue in a body or plant. The macromolecules are then delivered via the hypodermic needle into the selected tissue. The programmable constant-current pulse controller is activated and constant-current electrical pulse is applied to the plurality of needle electrodes. The applied constant-current electrical pulse facilitates the introduction of the macromolecule into the cell between the plurality of electrodes. Cell death due to overheating of cells is minimized by limiting the power dissipation in the tissue by virtue of constant- current pulses. The Cellectra device and system is described in U. S. Patent No. 7,245,963, the contents of which are herein incorporated by reference. [00258] The MID may be an El gen 1000 system (Inovio Pharmaceuticals). The Elgen 1000 system may comprise device that provides a hollow needle; and fluid delivery means, wherein the apparatus is adapted to actuate the fluid delivery means in use so as to concurrently (for example automatically) inject fluid, the described vaccine herein, into body tissue during insertion of the needle into the said body tissue. The advantage is the ability to inject the fluid gradually while the needle is being inserted leads to a more even distribution of the fluid through the body tissue. It is also believed that the pain experienced during injection is reduced due to the distribution of the volume of fluid being injected over a larger area.
[00259] In addition, the automatic injection of fluid facilitates automatic monitoring and registration of an actual dose of fluid injected. This data can be stored by a control unit for documentation purposes if desired.
[00260] It will be appreciated that the rate of injection could be either linear or non-linear and that the injection may be carried out after the needles have been inserted through the skin of the subject to be treated and while they are inserted further into the body tissue.
[00261] Suitable tissues into which fluid may be injected by the apparatus of the present invention include tumor tissue, skin or liver tissue but may be muscle tissue.
[00262] The apparatus further comprises needle insertion means for guiding insertion of the needle into the body tissue. The rate of fluid injection is controlled by the rate of needle insertion. This has the advantage that both the needle insertion and injection of fluid can be controlled such that the rate of insertion can be matched to the rate of injection as desired. It also makes the apparatus easier for a user to operate. If desired means for automatically inserting the needle into body tissue could be provided.
[00263] A user could choose when to commence injection of fluid. Ideally however, injection is commenced when the tip of the needle has reached muscle tissue and the apparatus may include means for sensing when the needle has been inserted to a sufficient depth for injection of the fluid to commence. This means that injection of fluid can be prompted to commence automatically when the needle has reached a desired depth (which will normally be the depth at which muscle tissue begins). The depth at which muscle tissue begins could for example be taken to be a preset needle insertion depth such as a value of 4 mm which would be deemed sufficient for the needle to get through the skin layer.
[00264] The sensing means may comprise an ultrasound probe. The sensing means may comprise a means for sensing a change in impedance or resistance. In this case, the means may not as such record the depth of the needle in the body tissue but will rather be adapted to sense a change in impedance or resistance as the needle moves from a different type of body tissue into muscle. Either of these alternatives provides a relatively accurate and simple to operate means of sensing that injection may commence. The depth of insertion of the needle can further be recorded if desired and could be used to control injection of fluid such that the volume of fluid to be injected is determined as the depth of needle insertion is being recorded.
[00265] The apparatus may further comprise: a base for supporting the needle; and a housing for receiving the base therein, wherein the base is moveable relative to the housing such that the needle is retracted within the housing when the base is in a first rearward position relative to the housing and the needle extends out of the housing when the base is in a second forward position within the housing. This is advantageous for a user as the housing can be lined up on the skin of a patient, and the needles can then be inserted into the patient's skin by moving the housing relative to the base.
[00266] As stated above, it is desirable to achieve a controlled rate of fluid injection such that the fluid is evenly distributed over the length of the needle as it is inserted into the skin. The fluid delivery means may comprise piston driving means adapted to inject fluid at a controlled rate. The piston driving means could for example be activated by a servo motor. However, the piston driving means may be actuated by the base being moved in the axial direction relative to the housing. It will be appreciated that alternative means for fluid delivery could be provided. Thus, for example, a closed container which can be squeezed for fluid delivery at a controlled or non-controlled rate could be provided in the place of a syringe and piston system.
[00267] The apparatus described above could be used for any type of injection. It is however envisaged to be particularly useful in the field of electroporation and so it may further comprise a means for applying a voltage to the needle. This allows the needle to be used not only for injection but also as an electrode during, electroporation. This is particularly advantageous as it means that the electric field is applied to the same area as the injected fluid. There has traditionally been a problem with electroporation in that it is very difficult to accurately align an electrode with previously injected fluid and so users have tended to inject a larger volume of fluid than is required over a larger area and to apply an electric field over a higher area to attempt to guarantee an overlap between the injected substance and the electric field. Using the present invention, both the volume of fluid injected and the size of electric field applied may be reduced while achieving a good fit between the electric field and the fluid.
[00268] The present invention has multiple aspects, illustrated by the following non- limiting examples.
4. Example 1
[00269] A DNA vaccine platform using synthetic consensus DNA molecules has been developed to help break tolerance to non-viral tumor-associated antigens, such as TERT and WT-1 (Yan et al, 2013, Cancer Immunol Res, 1 : 179-189; Walters et al, 2017, Mol Ther, 25:976-988). This platform is based on the concept that introduction of xenogeneic antigens into mice can induce autoimmunity (Engelhorn et al, 2006, Nat Med, 12: 198-206; Guevara- Patino et al, 2006, J Clin Invest, 116: 1382-1390. Here, it is demonstrated that combination therapy with immune checkpoint blockade further improves synthetic consensus DNA vaccine immune responses. A mouse TERT DNA vaccine with enhanced capacity to break tolerance was administered in combination with antibodies that block the immune checkpoint molecules CTLA-4 or PD-1. Blockade of CTLA-4, or a combination of CTLA-4 and PD-1, effectively synergized with TERT DNA vaccine to slow tumor growth in mice. However, peripheral and systemic immune responses did not necessarily correspond with anti-tumor activity of immune therapy combinations. These studies highlight the potential application of these DNA vaccine/immune checkpoint blockade combinations in patients that respond poorly to immune checkpoint blockade alone. Furthermore, these results suggest that this synergistic effect is due to the effect of immune checkpoint blockade on altering the tumor microenvironment rather than boosting vaccine responses in the periphery.
[00270] Materials and Methods
[00271] DNA plasmids
[00272] The synthetic consensus TERT sequence was generated by using 6 TERT sequences collected from animals including mouse, rat and hamster. The consensus sequence was obtained after alignment of these sequences using Clustal W. Two additional mutations were added to abolish telomerase activity (Weinrich et al, 1997, Nat Genet, 17:498-502; Lingner et al, 1997, Science, 276:561-56). All sequences were RNA and codon optimized with an IgE leader sequence and a Kozak sequence at the N terminus, and cloned into the modified pVAX vector (Genscript). The final mouse TERT sequences shares 94.6% sequence identity with native mouse TERT.
[00273] PBMC and splenocvte isolation
[00274] For splenocyte isolation, spleens from mice were collected in complete RPMI media containing 10% Fetal Bovine Serum (FBS). Cells were dissociated using a stomacher, and then filtered through a 40μιη mesh filter. Red Blood Cells were then lysed using ACK Lysis buffer (LifeTechnologies). Cells were then filtered again through a 40μιη mesh filter, and counted and plated for staining. For PBMC isolation, blood was collected in 4% sodium citrate to prevent clotting. Blood was then layered on top of Histopaque 1083 (Sigma- Aldrich). Cells were spun for one hour, and then cells from the buffy coat were separated and stained for analysis.
[00275] Isolation of tumor infiltrating lymphocytes (TILs)
[00276] Tumors were dissociated using digestion mix, which consists of: 170mg/L
Collagenase I, II and IV (ThermoFisher), 12.5mg/L DNAse I (Roche), 25mg/L Elastase (Worthington) in a 50/50 mixture of Hyclone L-15 Leibowitz Media (ThermoFisher) and RPMI, supplemented with 10% FBS and 1% Penicillin/Streptomycin. Tumors were minced and then transferred to a 50mL conical filled with lOmL of digestion mix. Cells were then filtered twice through a 40μιη mesh filter, and then counted and stained.
[00277] ELISpot assay
[00278] Splenocytes were stimulated in the presence of native mouse TERT peptides (15- mer peptides spanning the entire native mouse protein, overlapping by 9 amino acids, GenScript) for 24 hours in MABTECH Mouse IFN-γ ELISpotPLUS plates. Spots were developed and counted according to the manufacturer's protocol.
[00279] Intracellular Cytokine Staining and Flow cytometry
[00280] 2 million splenocytes per mouse were stimulated with native mouse TERT peptides for 5 hours in the presence of GolgiStop GolgiPlus (BD Bioscience). Phorbol myristate acetate (PMA, BD Bioscience), and complete media were used as positive and negative controls, respectively. Cells were incubated with FITC a-mouse CD107a (clone 1D4B, Biolegend) during the stimulation to stain for degranulation. After stimulation, cells were washed and stained for viability using LIVE/DEAD violet. Surface stain was then added followed by fixation and permeabilization using FoxP3/transcription factor
fixation/permeabilization kit (eBioscience). The following antibodies were used in these studies: PECy5 aCD3 (clone 145-2C11, BD Pharmingen), BV510 aCD4 (clone RM4-5, Biolegend), BV605 aTNFa (clone MP6-XT22), PE aT-bet (clone 4B10, Biolegend), BV711 aPD-1 (clone 29F.1A12, Biolegend), APC aFoxP3 (clone FJK-16s, eBioscience), APCCy7 aCD8 (clone 53-6.7, Biolegend), AF700 aCD44 (clone IM7, Biolegend), APC alFNy (clone XMG1.2, Biolegend). All data was collected on a modified LSRII flow cytometer (BD Bioscience) and analyzed using FlowJo software (TreeStar).
[00281] Animal Immunization
[00282] Female 6-8 week old C57B1/6 mice were purchased from Jackson Laboratory. Animal care was in accordance with the guidelines of the NIH and with the Wistar Institute Animal Care and Use Committee (IACUC). Mice were immunized with 25 μg of each plasmid by intramuscular injection into the tibialis interior (TA) muscle, followed by electroporation (EP) using the CELLECTRA®-3P adaptive constant current device (Inovio Pharmaceuticals). Two 0.1 Amp constant current square-wave pulses were delivered through a triangular three-electrode array. Each pulse was 52 milliseconds in length with a Is delay between pulses. For immunogenicity studies, mice were given a total of three immunizations at two-week intervals. For tumor challenge studies, mice were given a total of four immunizations at one-week intervals.
[00283] Immune checkpoint blockade antibodies
[00284] The following antibodies were used for animal studies (BioXCell): a-mouse PD-1 (clone RMP1-14), a-mouse CTLA-4 (clone 9D9), or aCD25 (clone PC-61.5.3). Mice were given 20C^g of each antibody, delivered intraperitoneally, according to the schedule in each Figure.
[00285] Tumor challenge studies
[00286] The TC-1 cell line was a gift from Dr. Yvonne Paterson from the University of Pennsylvania. This cell line was tested negative for Mycoplasma contamination prior to freezing the cells down. Cells were thawed and cultured for fewer than 5 passages prior to implantation into mice. Cells were cultured in Dulbecco's modified Eagle Media (Mediatech) supplemented with 10% fetal bovine serum (FBS). Cells were subcutaneously implanted onto the right flank of C57B1/6 mice (50,000 cells per mouse). One week following tumor implantation, mouse tumors were measured and mice were randomized to treatment groups so that the average tumor volume and standard deviation was equivalent between groups. Tumors were monitored twice a week with electronic calipers. Tumor volume was calculated using the formula Volume= (n/6)*(height)*(width2). Mice were euthanized upon any sign of distress or sickness, or when tumor diameters exceeded 1.5cm, according to IACUC guidelines.
[00287] Statistical Analysis
[00288] Statistical analyses were performed using GraphPad Prism Software. For each experiment, error bars represent the mean ± the standard error of the mean. For all experiments, statistical significance was determined by one- or two-way ANOVA, followed by Tukey's post-hoc HSD test. For mouse survival studies, significance was determined by Gehan-Breslow-Wilcoxon test.
[00289] Results
[00290] Impact of immune checkpoint blockade on antigen-specific immune responses in non-tumor bearing mice
[00291] The impact of immune checkpoint blockade on antigen-specific immune responses to the synthetic consensus mouse TERT DNA vaccine was examined. This vaccine exhibits a robust capacity to generate an immune response against matched peptides, while maintaining the capacity to break tolerance to native mouse TERT peptides in C57B1/6 mice (Figure 1 A and Figure IB). For the remaining experiments, the focus was on antigen-specific immune responses targeting native mouse TERT peptides. The ability of a mouse TERT DNA vaccine to induce anti -tumor immunity in a therapeutic setting was explored using a TC-1 mouse tumor model. This TERT vaccine alone was capable of slowing tumor growth and prolonging survival in a proportion of the mice (Figure 2A and Figure 2B).
[00292] Combination therapy using the immune checkpoint blockade antibodies aCTLA-4 and aPD-1 was then evaluated. Mice were immunized three times at two-week intervals, and checkpoint antibody treatment was initiated 1 day following the first immunization (Figure 3A). Mice were treated with checkpoint antibodies at a dose of 200μg/mouse every three days, and analyzed immune responses one week following the final vaccination. None of these antibody treatments significantly increased antigen-specific type 1 cellular immune responses compared to DNA vaccination alone (Figure 3B through Figure 3F). In fact, a- CTLA-4 and the combination therapy with a-CTLA-4 and a-PD-1 slightly decreased antigen- specific IFNy+ and TNFa+ CD8+ T cell responses (Figure 3B and Figure 3C). This trend was also true for antigen-specific CD8+ T cells co-expressing IFNy, the degranulation marker CD 107a and the T cell activation transcription factor T-bet (Figure 3D). In the CD4+ compartment, a-CTLA-4 treatment in combination with mTERT immunization appeared to slightly boost antigen-specific IFNy and TNFa immune responses; however, this change was not statistically significant (Figure 3E and Figure 3F). IFNy ELISpot responses showed no significant changes in antigen-specific immunity when mTERT DNA vaccine was combined with a-CTLA-4, a-PD-1, or a combination of the two (Figure 3G).
[00293] Next, T cell exhaustion markers were examined following delivery of the mTERT vaccine with immune checkpoint blockade. Immunization alone slightly increased the frequency of PD-1+ CD8+ T cells in both the spleen and periphery, though this increase was not statistically significant compared to naive mice (Figure 3H). Addition of a-CTLA-4, a- PD-1 or a combination of the two increased the frequency of PD-1+ CD8+ T cells in both the spleen and the periphery in both non-immunized and mTERT immunized mice (Figure 3H, Figure 4 A and Figure 4B). This boost in PD-1+ T cells was greatest for the combination therapy with a-CTLA-4 and a-PD-1, followed by a-PD-1 therapy alone (Figure 3H, Figure 4A and Figure 4B). Treatment of mice with immune checkpoint antibodies alone did not induce any TERT antigen-specific immune responses (data not shown), but had the same impact on PD-1 expression on peripheral and spleen CD8+ T cells compared to checkpoint delivery with TERT immunization (Figure 4A and Figure 4B).
[00294] Because immune checkpoint molecules are required for initiation of an immune response, whether delivery of aCTLA-4 or aPD-1 after the 2nd immunization (boost) instead of the 1st immunization (prime) could augment antigen-specific immune responses was tested, compared to delivery of DNA vaccine alone. There was no significant impact of delivering aCTLA-4, aPD-1 or a combination of the two on antigen-specific immune responses compared to DNA vaccine alone in non-tumor bearing mice when these checkpoints were delivered after the 2nd immunization (Figure 5). [00295] CTLA-4 and PD-1 blockade synergize with mTERT DNA vaccine in generating anti-tumor immunity
[00296] Next, whether aCTLA-4 or aPD-1 therapy would improve anti -tumor activity in a therapeutic tumor challenge setting was examined. Mice were implanted with TC-1 tumors and immunizations were initiated after palpable tumors formed one week following implantation (Figure 6A). Mice were immunized at one week intervals for a total of four immunizations. Checkpoint treatment began 1 day following the first immunization, and continued until one week following the final immunization (Figure 6A).
[00297] Despite the lack of boosting in antigen-specific immune responses in non- tumor bearing mice (Figure 3), both a-CTLA-4 and a-PD-1 therapy synergized with mTERT DNA immunization above vaccine therapy alone in a therapeutic TC-1 tumor challenge model when immune checkpoints were delivered 1 day post- 1st immunization (Figure 6B through Figure 6E). Both a-CTLA-4 and a-PD-1 therapy alone had no initial impact in slowing tumor growth and no significant impact on mouse survival (Figure 6D and Figure 6E). mTERT immunization alone slightly slowed tumor growth and improved survival compared to naive mice. However, a-CTLA-4 and a-PD-1 in combination with mTERT had robust slowing of tumor growth and significant improvement in survival compared to DNA alone or naive mice (Figure 6B and Figure 6C). This synergy was greater for a-CTLA-4, in particular for survival, compared to a-PD-1 (Figure 6B and Figure 6C).
[00298] Combination therapy of aCTLA-4 and a-PD-1 in both animal models and in the clinic has shown improved anti-tumor responses compared to treatment with each checkpoint alone (Shi et al, 2016, Nat Commun, 7: 12335; Wolchok et al, 2013, N Engl J Med.
2013;369: 122-133; Lussier et al, 2015, J Immunother Cancer, 3:21). In particular, combination therapy with aCTLA-4 and a-PD-1 was reported to improve the anti -tumor immunity of cell based vaccines (Duraiswamy et al, 2013, Cancer Res, 73:3591-3603; Curran et al, 2010, Proc Natl Acad Sci, 107:4275-4280). A triple-combination therapy with aCTLA-4, a-PD-1 and mTERT was examined to determine if the combination could further improve therapeutic anti -tumor activity. Combination therapy of mTERT with both aCTLA-4 and a-PD-1 slightly improved anti -tumor activity and survival of mice. This effect was greater than the combined treatment of aCTLA-4 and mTERT (Figure 6B and Figure 6C). The combination therapy alone without the DNA vaccine exhibited little impact on tumor growth or mouse survival (Figure 6D and Figure 6E). [00299] Impact of immune checkpoint blockade on immune responses in tumor-bearing mice
[00300] Since the impact of aCTLA-4 and aPD-1 therapy on tumor burden was more profound than the effect of these checkpoints on antigen-specific immune responses in non- tumor bearing mice, the immune responses in tumor-bearing mice were examined. Mice were implanted with TC-1 tumors, immunized on days 7 and 14, and sacrificed on day 21 (Figure 7A). Checkpoint inhibitor delivery was initiated one day following the first immunization, and continued every three days until day 21 (Figure 7A). Immune cell exhaustion markers in the periphery and spleen, as well as antigen-specific immune responses in the spleen were examined.
[00301] There was no significant impact of any of the checkpoint antibodies or combinations on systemic antigen-specific CD8+ or CD4+ immune responses, including expression of IFNy and TNFa in CD4+ and CD8+ T cells, or IFNy/CD107a/T-bet in CD8+ T cells compared to mTERT DNA alone (Figure 7B through Figure 7F).
[00302] PD-1 expression was examined on CD4 and CD8 T cells in the spleen and the periphery of tumor-bearing mice. While in non-tumor bearing mice all checkpoints enhanced PD-1 expression on CD8+ T cells to some degree (with the greatest enhancement from the combination or aPD-1 therapy alone), in tumor-bearing mice aPD-1 had no impact on PD-1 expression in both CD4+ and CD8+ T cells in the spleen and periphery (Figure 8A through Figure 8D). However, both aCTLA-4 and a combination of aCTLA-4 and aPD-1 enhanced the frequency of PD-1+ CD4+ and CD8+ T cells in both the spleen and the periphery (Figure 8A through Figure 8D).
[00303] The frequency of CD4+/CD25+/FoxP3+ regulatory T cells (Tregs) in the spleen and periphery were examined upon immune checkpoint blockade therapy in combination with mTERT DNA vaccine (Figure 8E and Figure 8F). aCTLA-4 therapy is known to impact Tregs in a tissue-dependent manner, with a more profound depletion of Tregs within the tumor compared to other tissue sites (Quezada et al, 2006, J Clin Invest, 116: 1935-1945; Selby et al, 2013, Cancer Immunol Res, 1 :32-42). All checkpoint therapies and
combinations decreased Treg frequency in the spleen, but slightly increased Treg frequency in the periphery (Figure 8E and Figure 8F).
[00304] The immune responses within the tumors were examined in these same mice. aCTLA-4 treatment resulted in significant depletion of intra-tumoral Tregs (Figure 9A), while aPD-1 therapy did not alter the frequency of intra-tumoral Tregs, and combination therapy with both aCTLA-4 and aPD-1 resulted in only a modest decrease of intra-tumoral Tregs (Figure 9A). Similar to the results observed in the spleen and peripheral T cells, the percentage of PD-1+ tumor infiltrating CD8+ lymphocytes also increased upon aCTLA-4 treatment, and, to a lesser extent, combination therapy with aCTLA-4 and aPD-1 treatment (Figure 9B). In addition, in tumor-bearing mice, aCTLA-4 treatment alone enhanced the frequency of CD44+ CD8+ TILs (Figure 9C). This effect was more pronounced after treatment of these TILs with PMA (Figure 9D). Interestingly, aPD-1 and the combination of aCTLA-4 and aPD-1 therapy did not significantly impact CD44 expression in the TILs (Figure 9C and Figure 9D). The TERT DNA vaccine alone did not significantly impact Tregs, PD-1 or CD44 expression in TILs (Figure 9A through Figure 9D).
[00305] CTLA-4 blockade synergizes with mTERT DNA vaccine more than Treg depletion
[00306] Without being bound by a particular theory, it has been proposed that the primary mechanism for aCTLA-4 therapy is depletion of Tregs in the tumor microenvironment. In order to test the contribution of Tregs to the synergy between TERT DNA vaccine and aCTLA-4 treatment, the impact of aCTLA-4 was compared to aCD25, a depletion antibody that systemically depletes regulatory T cells (Stephens et al, 2005, Proc Natl Acad Sci U S A, 102: 17418-17423). In these experiments, while aCD25 in combination with mTERT significantly improved anti-tumor responses, this effect was not as strong as combination therapy of aCTLA-4 and mTERT in terms of tumor growth or overall survival (Figure 10A and Figure 10B). This data suggests that the mechanism of action of aCTLA-4 extends beyond depletion of Tregs in the tumor.
[00307] In these studies, robust synergy was oberserved between mTERT DNA vaccination and aCTLA-4 immune checkpoint blockade. This combination exhibited long-term effects for prolonging mouse survival in a therapeutic tumor challenge model. Importantly, these mice did not exhibit anti-tumor immunity after delivery of immune checkpoint antibodies alone, suggesting that addition of a DNA vaccine to an immune checkpoint blockade regimen may allow non-responders to become responders.
[00308] As is observed in the clinic, in some tumor models there are objective responses to treatment with immune checkpoint blockade alone. For instance, the immunogenic SalN fibrosarcoma model is somewhat susceptible to aCTLA-4, aPD-1 or aLAG-3 therapy alone (Leach et al, 1996, Science, 271 : 1734-1736; Woo et al., 2012, Cancer Res, 72:917-927). Furthermore, some tumor cell lines that respond partially or poorly to mono-therapy with an immune checkpoint blocking antibody may respond much better to combination therapies with multiple antibodies.
[00309] Only a handful of preclinical studies have examined the efficacy of combining DNA vaccines with immune checkpoint blockade for tumor therapy, and no previous studies have examined combination therapy with DNA vaccines and aCTLA-4 blocking antibody in a therapeutic tumor challenge model.
[00310] This study showed a clear bias towards DNA vaccine synergy with a-CTLA-4 blockade over a-PD-1 blockade. Furthermore, the triple-therapy was only slightly better than double-therapy with vaccine plus aCTLA-4. This is useful information, since the double- therapy with aCTLA-4 and aPD-1 has significantly more toxicity compared to mono-therapy with either aCTLA-4 or aPD-1 alone in humans (Boutros et al, 2016, Nature Research;
13:473-486).
[00311] Interesting differences were observed in PD-1 expression on T cells after immune checkpoint blockade between tumor-bearing and non-tumor bearing mice. In both tumor- bearing and non-tumor bearing mice, aCTLA-4 treatment increased the proportion of CD8+ T cells expressing PD-1 in the spleen, periphery and tumor (Figure 6, Figure 9 and Figure 10). However, aPD-1 therapy only induced PD-1 expression on T cells in non-tumor bearing mice, and not tumor-bearing mice. The interesting differences observed between tumor- bearing and non-tumor bearing mice may reflect enhancement of T cell exhaustion and preexposure of T cells to antigens in tumor-bearing mice. Blockade of the PD-l/PD-Ll signaling axis can only partially reverse an exhausted T cell phenotype; therefore, exploring methods of further improving immune activation in tumor-bearing mice remains important (Pauken et al, 2016, Science, 354: 1160-1165).
[00312] Some differences between aCTLA-4 and aPD-1 blockade in the context of DNA vaccines may be the result of the different effects of aCTLA-4 and aPD-1 on regulatory T cells. aCTLA-4 antibodies, unlike aPD-1 antibodies, have demonstrated robust depletion of intra-tumoral Tregs in mice (Quezada et al., 2006, J Clin Invest, 116: 1935-1945; Selby et al, 2013, Cancer Immunol Res, 1 :32-42). There is also some evidence for ipilimumab-dependent ADCC ex vivo in patient samples; however, the extent to which clinical aCTLA-4 antibodies can deplete Tregs in patient tumors remains unclear (Romano et al,. 2015, Proc Natl Acad Sci U S A, 112:6140-6145). In the present study, aCTLA-4 antibody treatment was superior to depletion of Tregs (with an aCD25 depletion antibody) in combination with mTERT DNA vaccine (Figure 1). A separate study found a similar result when comparing combination therapy of GVAX or FVAX with aCD25 depletion, implying that aCTLA-4 has additional effects beyond simple depletion of Tregs (Curran et al, 2009, Cancer Res. 69:7747-7755). These additional effects may consist of up-regulation of the T cell effector marker CD44 on intra-tumoral CD8+ T cells, which was observed upon treatment with aCTLA-4 or aCTLA- 4/aPD-l combination therapy.
[00313] Importantly, an increase in antigen-specific immune responses in the spleen upon combination therapy with TERT DNA vaccine and immune checkpoint blockade was not observed, despite the synergy of these therapies in inducing anti-tumor immunity. Without being bound by a particular theory, this data suggests that immune checkpoint blockade functions to alter the immune microenvironment at the tumor site rather than acting on antigen-specific cells in the periphery. In some cases these therapeutic combinations trended towards a decrease in antigen-specific responses in the spleen, suggesting that the addition of immune checkpoint blockade antibodies may alter T cell trafficking and may drive antigen- specific immune cells to peripheral tissues in the mouse. Taken together, these studies support further combination immune therapies with synthetic TERT DNA vaccination and immune checkpoint blockade.
[00314] Example 2: exemplary hTERT vaccines
[00315] Provided are exemplary hTERT DNA sequences and encoded amino acid sequences that can be administered in combination with an ICB antibody of the invention.
[00316] SEQ ID NO: 1 - human TERT (hTERT) nucleic acid sequence
[00317] SEQ ID NO:2 - hTERT amino acid sequence
[00318] SEQ ID NO:3 -hTERT nucleic acid sequence operably linked to a sequence encoding an IgE leader sequence.
[00319] SEQ ID NO:4 - hTERT amino acid sequence operably linked to an IgE leader sequence
[00320] SEQ ID NO: 5 - Synthetic Consensus hTERT nucleic acid sequence (pgxl434).
[00321] SEQ ID NO:6 - Synthetic consensus hTERT amino acid sequence (pgx 1434)
[00322] SEQ ID NO: 7 - Synthetic Consensus hTERT nucleic acid sequence (pgxl434), operably linked to a sequence encoding an IgE leader sequence.
[00323] SEQ ID NO: 8 - Synthetic consensus hTERT amino acid sequence (pgx 1434) operably linked to an IgE leader sequence [00324] SEQ ID NO: 9 - synthetic, consensus mouse TERT (mTERT) nucleic acid sequence (pgxl418)
[00325] SEQ ID NO: 10 - synthetic, consensus mTERT amino acid sequence (pGX1418)
[00326] SEQ ID NO: 11 - synthetic, consensus mTERT nucleic acid sequence (pgxl418) operably linked to a sequence encoding an IgE leader sequence.
[00327] SEQ ID NO: 12 - synthetic, consensus mTERT amino acid sequence (pGX1418) operably linked to an IgE leader sequence
[00328] SEQ ID NO: 13 - amino acid sequence of an IgE leader sequence

Claims

CLAIMS What is claimed is:
1. A composition for enhancing an immune response against an antigen in a subject in need thereof, comprising:
a) an anti-CTLA4 antibody, and
b) a synthetic antigen capable of generating an immune response in the subject, or an immunogenic fragment or variant thereof.
2. The composition of claim 1 wherein the synthetic antigen is an isolated nucleic acid molecule comprising a nucleotide sequence that encodes for the antigen.
3. The composition of claim 2 wherein the synthetic antigen is selected from the group consisting of: TERT, prostate, WT1, tyrosinase, NYES01, PRAME, MAGE, CMV, herpes, HIV, HPV, HCV, HBV, EBV, MCV, and cancer causing viruses.
4. The composition of claim 3, wherein the synthetic antigen is TERT.
5. The composition of claim 3, wherein the HPV antigen is E6 and E7 domains of subtypes selected from the group consisting of: HPV6, HPVl l, HPV 16, HPV 18, HPV31, HPV33, HPV52, and HPV58, and a combination thereof.
6. The composition of claim 3, wherein the HIV antigen is selected from the group consisting of: Env A, Env B, Env C, Env D, B Nef-Rev, and Gag, and a combination thereof.
7. The composition of claim 3, wherein the HCV antigen is selected from the group consisting of: El, E2, NS3, NS4a, NS4b, NS5a, and NS5b, and a combination thereof.
8. The composition of claim 3, wherein the HBV antigen is selected from the group consisting of: surface antigen type A, surface antigen type B, surface antigen type C, surface antigen type D, surface antigen type E, surface antigen type F, surface antigen type G, surface antigen type H, and core antigen, and a combination thereof.
9. The composition of claim 3, wherein the prostate antigen is selected from the group consisting of: PSA, PSMA, STEAP, PSCA, and PAP, and a combination thereof.
10. The composition of claim 3, wherein the synthetic antigen is a herpes antigen, wherein the herpes is HCMV, HSV1, HSV2, VZV, or CMV, and the herpes antigen is selected from the group consisting of gB, gM, gN, gH, gL, gO, gE, gl, gK, gC, gD, UL128, UL130, UL131A, and UL83.
11. The composition of claim 1, wherein the anti-CTLA4 antibody is selected from the group consisting of ipilimumab, ipilimumab-Probody Tx (BMS-986249), ipilimumab-NF (BMS-986218), tremelimumab, CS1002, MDX-010 (NCT00140855), and AGEN-1884 (NCT02694822).
12. The composition of claim 1, further comprising an additional antibody targeting an immune checkpoint protein.
13. The composition of claim 12, wherein the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, LAG3 and TIM3.
14. The composition of claim 12, wherein the antibody is selected from the group consisting of: nivolumab, pembrolizumab, pidilizumab, BMS-936559, MPDL3280A, MDX1105-01, MEDI4736, and MK-3475.
15. The composition of claim 1, further comprising a pharmaceutically acceptable excipient.
16. A method for increasing an immune response in a subject in need thereof, the method comprising administering the composition of any one of claims 1-15 to the subject.
17. The method of claim 16, wherein administering the composition comprises an electroporating step.
18. A method of increasing an immune response in a subject in need thereof by administering a combination of a synthetic antigen and a CTL4A antibody, wherein the administering step comprises: administering to the subject a prime vaccination and a boost vaccination of a synthetic antigen, and
prior to the boost vaccination, administering to the subject a CTL4A antibody.
19. The method of claim 18, further comprising a step of further administering to the subject a subsequent boost vaccination of the synthetic antigen.
20. The method of claim 18, further comprising administering an antibody targeting one or more immune checkpoint protein prior to the boost vaccination.
21. The method of claim 18, wherein any of the administering steps include delivering electroporation to the site of administration.
22. The method of claim 18, wherein one or more of the prime vaccination and the boost vaccination comprises a nucleic acid vaccine.
23. The method of claim 22, wherein one or more of the prime vaccination and the boost vaccination comprises a DNA vaccine.
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