WO2019018607A1 - Compositions immunomodulatrices et procédés d'utilisation associés - Google Patents

Compositions immunomodulatrices et procédés d'utilisation associés Download PDF

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Publication number
WO2019018607A1
WO2019018607A1 PCT/US2018/042831 US2018042831W WO2019018607A1 WO 2019018607 A1 WO2019018607 A1 WO 2019018607A1 US 2018042831 W US2018042831 W US 2018042831W WO 2019018607 A1 WO2019018607 A1 WO 2019018607A1
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Prior art keywords
protein
antigen
alpha
preselected
synuclein
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Elazar Rabbani
James J. Donegan
Jack Coleman
Ante TOCIL
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Enzo Biochem Inc
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Enzo Biochem Inc
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Priority claimed from US15/719,821 external-priority patent/US10245320B2/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Rigid containers without fluid transport within
    • B01L3/5082—Test tubes per se
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6866—Interferon
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)

Definitions

  • the invention relates to the field of antigen-specific immune modulation.
  • a healthy immune system recognizes and eliminates pathogens, pre-cancerous cells and other "non-self entities, while maintaining a state of non-reactiveness toward normal self cells and tissues.
  • autoimmune disease may result.
  • many chronic inflammatory and tissue-destructive diseases are autoimmune diseases, including, for example, age-related macular degeneration (AMD), uveitis, Crohn's disease, rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. Over eighty autoimmune diseases are known. What is needed and provided by the present invention are new compositions and methods for modulating immune activity, i.e., for promoting immune reactivity or immune
  • an immunomodulatory composition such as an immunomodulatory pharmaceutical composition, including a mixture of:
  • HLA protein at least partially purified HLA protein or fragments thereof, such as mammalian, for example human,
  • whole blood such as mammalian, for example human
  • a cellular fraction thereof such as a density gradient fraction thereof, such as but not limited to a white blood cell and/or red blood cell (erythrocytes) and/or platelet fraction/layer thereof, or a cell membrane fraction/preparation of any of the foregoing or an extract of any of the foregoing, such as a protein extract, a lipid extract, a carbohydrate extract, a small molecule extract or any
  • alpha-synuclein protein such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof;
  • At least one preselected antigen or immunogen such as at least one preselected peptide antigen, at least one preselected protein antigen, at least one preselected carbohydrate antigen, at least one preselected lipid antigen, and/or at least one preselected glycolipid antigen.
  • At least the first component or only the first component may be heat-treated, for example, heat- treated at or above 100°C for at least 1 or 2 or 3 or 4 or 5 minutes. Heat-treatment may include or consist of autoclaving.
  • the at least partially purified HLA protein or other proteins may be at least substantially denatured.
  • the proteins may at least partially, such as at least substantially, be fragmented into peptides.
  • the antigen or immunogen may be a molecule that is not an HLA molecule and/or is not alpha-synuclein (and/or is not a sequence fragment of either).
  • the composition may, for example, be a liquid composition or an at least substantially dry composition, such as a powder. Dry forms may be prepared by drying a liquid mixture of the components, for example, by lyophilization or any method known in the art.
  • the composition may include one or more pharmaceutically acceptable excipients.
  • a related embodiment provides a method for manufacturing an immunomodulatory composition, such as an immunomodulatory pharmaceutical composition, including the steps of: providing a first component including
  • HLA protein at least partially purified HLA protein or fragments thereof, such as mammalian, for example human,
  • whole blood such as mammalian, for example human
  • a cellular fraction thereof such as a density gradient fraction thereof, such as but not limited to a white blood cell and/or red blood cell (erythrocytes) and/or platelet fraction/layer thereof, or a cell membrane fraction/preparation of any of the foregoing or an extract of any of the foregoing, such as a protein extract, a lipid extract, a carbohydrate extract, a small molecule extract or any
  • alpha-synuclein protein such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof; and providing a second component including at least one preselected antigen or immunogen, such as at least one preselected peptide antigen, at least one preselected protein antigen, at least one preselected carbohydrate antigen, at least one preselected lipid antigen, and/or at least one preselected glycolipid antigen; and mixing the first component and the second component, for example, under aqueous conditions.
  • preselected antigen or immunogen such as at least one preselected peptide antigen, at least one preselected protein antigen, at least one preselected carbohydrate antigen, at least one preselected lipid antigen, and/or at least one preselected glycolipid antigen
  • the method may further include heat-treating at least the first component, such as only the first component prior to the mixing step.
  • the method may include mixing the two components and then heat-treating the mixture.
  • the method may include separately heat-treating the first and second components prior to the mixing step.
  • the method may further include at least substantially drying the liquid mixture to obtain an at least substantially dry form, such as a powder, by, for example, lyophilizing the liquid mixture or otherwise drying it.
  • One or more excipients may be admixed before and/or after the drying step.
  • the antigen may, for example, be a molecule that is not an HLA molecule and/or is not alpha-synuclein.
  • the antigen may be a peptide, such as a synthetic peptide, that is not a sequence fragment of an HLA molecule or alpha-synuclein.
  • a further embodiment of the invention provides a method for modulating the immune response in a mammal to at least one preselected antigen that includes administering to the mammal an immunomodulatory pharmaceutical composition as described within. Said administration may be parenteral or non-parenteral.
  • the antigen-specific modulation of the immune response may be immunostimulatory or immunosuppressive (tolerogenic).
  • a further embodiment of the invention provides a method for modulating the immune response in a mammal, such as a human, to at least one preselected antigen that includes: coadministering to the mammal:
  • HLA protein at least partially purified HLA protein or fragments thereof, such as mammalian, for example human,
  • whole blood such as mammalian, for example human
  • a cellular fraction thereof such as a density gradient fraction thereof, such as but not limited to a white blood cell and/or red blood cell (erythrocytes) and/or platelet fraction/layer thereof, or a cell membrane fraction/preparation of any of the foregoing or an extract of any of the foregoing, such as a protein extract, a lipid extract, a carbohydrate extract, a small molecule extract or any combination thereof, and
  • alpha-synuclein protein such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof; and (ii) at least one preselected antigen or immunogen, such as at least one preselected peptide antigen, at least one preselected protein antigen, at least one preselected carbohydrate antigen, at least one preselected lipid antigen, and/or at least one preselected glycolipid antigen.
  • preselected antigen or immunogen such as at least one preselected peptide antigen, at least one preselected protein antigen, at least one preselected carbohydrate antigen, at least one preselected lipid antigen, and/or at least one preselected glycolipid antigen.
  • compositions under (i) may be treated, such as heat-treated, in any of the manners described herein.
  • Still another embodiment of the invention provides an assay method for determining whether cells in a sample of cells mount an antigen-specific response to one or more preselected antigens and/or for quantifying the extent to which cells in a sample of cells mount an antigen- specific response to one or more preselected antigens, said method embodiment including the steps of: providing an isolated sample of cells, such as a sample of blood cells, such as whole blood, or a white blood cell fraction or PBMCs or T-cells; providing alpha-synuclein protein and/or a fragment thereof; providing at least one, such as one, preselected antigen; contacting the sample of cells with both the alpha-synuclein and/or fragments thereof and the at least one preselected antigen; and measuring the resulting cellular response, such as the release of a cytokine, to said contacting.
  • an isolated sample of cells such as a sample of blood cells, such as whole blood, or a white blood cell fraction or PBMCs or T-cells
  • FIGS. 1 A and IB show the effects of a null control treatment on healthy PBMCs (FIG. 1A) and the effect of heat-treated whole blood on PBMCS (FIG. 1A), respectively, both with no added antigens/epitopes, with the top panels showing the cell cycle distribution of treated cells and the bottom panels showing the expression of interferon-gamma (IFNg) in treated cells.
  • IFNg interferon-gamma
  • FIG. 2A shows the cell cycle distribution of cells in healthy PBMCs (control case).
  • FIG. 2B shows the cell cycle distribution of PBMCs treated with whole blood (not heat-treated).
  • FIG. 2C shows the cell cycle distribution of PBMCs treated with serum.
  • FIG. 2D shows the cell cycle distribution of PBMCs treated with a heat-treated red blood cell (RBC) density gradient fraction (referred to as the "immune enhancer fraction;" "RBC;” and "IE” herein).
  • RBC red blood cell
  • FIGS. 3A and 3B show cell cycle distribution of PBMCs treated with control and various heat-treated blood fractions for allogeneic blood (FIG. 3 A) and autologous blood (FIG. 3B) indicating that that the apoptosis-inducing activity of whole blood is predominantly present in the RBC fraction, and is independent of the donor source.
  • FIGS. 4A-C show the effect of control (FIG. 4A), recombinant HLA-B protein (rULA-B; FIG. 4B), and recombinant ULA-G protein (rULA-G; FIG. 4C) on the cell cycle distribution of PBMCs, indicating apoptosis-inducing activity of the rULA-G and rHLA-B proteins on the PBMCs.
  • FIGS. 5 A and 5B show the effect of various treatment on the percent of PBMCs in the sub-Gl phase (FIG. 5A) and the S+G2 phase (FIG. 5B).
  • FIGS. 6 A and 6B show the effect of various blocking factors on the ability of the immune enhancer (heat-treated RBC fraction from density gradient separation) to affect the percentage of PBMCs in the sub-Gl phase (FIG. 6A; indicative of apoptosis induction) and the S+G2 phase (FIG. 6B; indicative of proliferation).
  • FIG. 7 shows the effect of different doses of IE on the ability of phytohemagglutinin (PHA) to induce interferon-gamma (IFNy) expression in PBMCs, as measured by mRNA detection.
  • PHA phytohemagglutinin
  • IFNy interferon-gamma
  • FIGS. 8A-8C show the effects on cell cycle distribution of control (FIG. 8A), Hepatitis B virus S-antigen ("HBV-SA;" FIG. 8B) alone, and HBV-A plus IE (FIG. 8C) on PBMCs from healthy human subjects (top panels) and HBV patients (bottom panels).
  • HBV-SA Hepatitis B virus S-antigen
  • FIG. 8C HBV-A plus IE
  • FIGS. 9A-D show the effect of control (FIG. 9A), HBV-SA alone (FIG. 9B), IE alone (FIG. 9C), and HBV-SA plus IE (FIG. 9D) on the expression of the Foxp3 immune suppression marker in PBMCs from healthy human subjects (top panels) and HBV patients (bottom panels).
  • FIG. 10 shows the percent increase in Foxp3 mRNA for control and different treatments in the experiment shown in FIGS. 9A-D.
  • FIGS. 11 A and 1 IB show the effect of HBV-SA plus IE (FIG. 11 A) and HBV-SA plus IE plus anti-HLA class I antibody (aHLA) on the expression of the Foxp3 immune suppression marker in PBMCs from healthy human subjects (top panels) and HBV patients (bottom panels).
  • FIGS. 12A-D show the effect of control (FIG. 12A), HBV-SA alone (FIG. 12B), IE alone (FIG. 12C), and HBV-SA plus IE (FIG. 12D) on the expression of interferon-gamma in PBMCs from healthy human subjects (top panels) and HBV patients (bottom panels).
  • FIGS. 13A and 13B show the effect of HBV-SA plus IE (FIG. 13A) and HBV-SA plus IE plus anti-HLA class I antibody (aHLA) on the expression of interferon-gamma (IFNg) in PBMCs from healthy human subjects (top panels) and HBV patients (bottom panels).
  • HBV-SA plus IE HBV-SA plus IE plus anti-HLA class I antibody (aHLA)
  • IFNg interferon-gamma
  • FIG. 14 shows the antigen-dependent, immune stimulation-enhancing activity of human alpha-synuclein protein (as measured by IFN-gamma release) during antigen challenge of whole blood from a subject recently immunized with the subject antigen.
  • HTB heat-treated blood
  • Heat treatment may, for example, be performed at a temperature of at least 95°C, such as at least 100°C, such as at least 110°C, such as at least 120°C for at least 1 minute, such as but not limited to 1 minute, at least 15 minutes or 15 minutes, at least 20 minutes or 20 minutes, at least 25 minutes or 25 minutes, or at least 30 minutes or 30 minutes.
  • Heat treatment may, for example be conducted at a temperature in the range of 100-130°C.
  • Heat treatment may include or consist of autoclaving, for example, for lminute to three hours, such as 1-30 minutes, such as 5-25 minutes, such as 10-20 minutes, or any subrange or number of minutes within said ranges.
  • the dose, such as daily dose, of heat-treated blood or blood fraction may, for example, be in the range of 0.5mg to 5 grams or any amount or subrange of amounts therein, such as 0.5 to lOOmg or 1.0 to 50mg.
  • Heat treatment may, for example, be performed by autoclaving the material.
  • the dose of a protein or protein extract of the blood or blood fraction or purified or recombinant HLA protein (or fragment(s) thereof) or purified or recombinant alpha-synuclein protein (or fragment(s) thereof) may, for example, be in the range of 100 micrograms to lOOmg, such as 20mg to lOOmg or any subrange or value therein such as 0.5 to 5mg.
  • compositions including such extracts, proteins or fragments thereof and one or more preselected antigens such as protein or peptide antigens may, for example, be in the same weight ranges.
  • Compositions including such a combination of components may, for example, include them in molar ratios of 1 : 100 to 100: 1 of immune enhancer : preselected antigen or any subrange or molar ratio value therein such as 5: 1 to 1 :5.
  • Dosing may, for example, be performed thrice daily, twice daily, once daily, every other day, every three days, biweekly or weekly.
  • Blood products indicated were made from human whole blood or blood fractions. Blood fractions were isolated by ficol gradient centrifugation using Histopaque (Sigma) and collecting either the RBC or serum fractions as indicated in each experiment. The whole blood or fraction was then autoclaved for 20 minutes, then resuspended to twice their original volume using PBS and sonicated for 30 minutes to restore solubility. PBMCs which had been frozen in liquid nitrogen were thawed, washed in RPMI 1640, then resuspended in RPMI 1640 complete medium, treated as indicated, and incubated for between 16 and 72 hours for use, depending on the experiment. Cells were then collected and stained as indicated, and run in a FACS Calibur flow cytometer.
  • FIGS. 1A and IB show the effects of a null control treatment on healthy PBMCS (FIG. 1A) and the effect of heat-treated whole blood on PBMCS (FIG. 1A), respectively, both with no added antigens/epitopes, with the top panels showing the cell cycle distribution of treated cells and the bottom panels showing the expression of interferon-gamma (IFNg) in treated cells.
  • IFNg interferon-gamma
  • FIG. 2A shows the cell cycle distribution of cells in healthy PBMCs (control case).
  • FIG. 2B shows the cell cycle distribution of PBMCs treated with heat treated whole blood.
  • FIG. 2C shows the cell cycle distribution of PBMCs treated with heat treated serum.
  • FIG. 2D shows the cell cycle distribution of PBMCs treated with a heat-treated red blood cell fraction (referred to as the "immune enhancer fraction;" "RBC;” and "IE” herein).
  • FIGS. 3A and 3B show cell cycle distribution of PBMCs treated with control and various heat-treated blood fractions for allogeneic blood (FIG. 3 A) and autologous blood (FIG. 3B) indicating that that the apoptosis-inducing activity of whole blood is predominantly present in the RBC fraction and independent of donor source.
  • Heat-treated IE showed essentially the same extent of apoptosis induction as heat-treated whole blood.
  • the heat-treated serum fraction decreased cell proliferation but did not increase apoptosis.
  • the IE dose was 50 ⁇ .
  • FIGS. 4A-C show the effect of control (FIG. 4A), recombinant HLA-B protein (rHLA-B; FIG. 4B), and recombinant HLA-G protein (rHLA-G; FIG. 4C) on the cell cycle distribution of PBMCs, indicating apoptosis-inducing activity of the rHLA-G and rHLA-B proteins on the PBMCs.
  • FIGS. 5 A and 5B show the effect of various treatment on the percent of PBMCs in the sub-Gl phase (FIG. 5A) and the S+G2 phase (FIG. 5B).
  • the heat-treated cell-line was a HeLa cell line.
  • IE increased apoptosis and decreased proliferation of the lymphocytes (PBMCs).
  • Recombinant HLA proteins (65ng/mL) gave only minor induction of apoptosis, but decreased proliferation.
  • the heat-treated HeLa cells did not mimic apoptosis induction and, in fact, induced higher proliferation.
  • the IE dose was 5 ⁇ . The lower dose induced less apoptosis compared to the 50 ⁇ dose.
  • FIGS. 6 A and 6B show the effect of various blocking factors on the ability of the immune enhancer (heat-treated RBC density gradient fraction) to affect the percentage of PBMCs in the sub-Gl phase (FIG. 6A) and the S+G2 phase (FIG. 6B).
  • Anti-HLA-I and FasL blocking antibodies do not reverse IE-induced apoptosis.
  • Anti-HLA-I antibody itself induces an increase in apoptosis independent of IE.
  • Annexin V which blocks phosphatidyl serine shows no effect on apoptosis, but reduces proliferation in both control and IE treated groups.
  • IE phytohemagglutinin
  • IFNy interferon-gamma
  • FIGS. 8A-8C show the effects on cell cycle distribution of control (FIG. 8A), hepatitis B virus S-antigen ("HBV-SA;" FIG. 8B) alone, and HBV-A plus IE (FIG. 8C) on PBMCs from healthy human subjects (top panels) and HBV patients (bottom panels).
  • HBV-SA hepatitis B virus S-antigen
  • FIG. 8C HBV-A plus IE
  • FIGS. 9A-D show the effect of control (FIG. 9A), HBV-SA alone (FIG. 9B), IE alone (FIG. 9C), and HBV-SA plus IE (FIG. 9D) on the expression of the Foxp3 immune suppression (Treg) marker in PBMCs from healthy human subjects (top panels) and HBV patients (bottom panels).
  • FIG. 10 shows the percent increase in Foxp3 mRNA for control and different treatments in the experiment shown in FIGS. 9A-D.
  • Low-dose IE (5 ⁇ .) increased expression of antiinflammatory marker Foxp3 by cells from an antigen responsive patient in the presence of the antigen.
  • IE alone did not increase expression of Foxp3.
  • Foxp3 expression correlated with antigen-specific suppression in the presence of low-dose IE, in contrast to the general suppression of apoptosis seen with high-dose IE.
  • FIGS. 11 A and 1 IB show the effect of HBV-SA plus IE (FIG. 11 A) and HBV-SA plus IE plus anti-HLA class I antibody (aHLA) on the expression of the Foxp3 immune suppression marker in PBMCs from healthy human subjects (top panels) and HBV patients (bottom panels).
  • the experiment shows that an anti-HLA class I antibody partially blocks the induction of the Foxp3 marker by HBV-SA plus IE in both healthy PMBCs and PBMCs from HBV-infected subjects.
  • FIGS. 12A-D show the effect of control (FIG. 12A), HBV-SA alone (FIG. 12B), IE alone (FIG. 12C), and HBV-SA plus IE (FIG. 12D) on the expression of interferon-gamma in PBMCs from healthy human subjects (top panels) and HBV patients (bottom panels).
  • FIGS. 13A and 13B show the effect of HBV-SA plus IE (FIG. 13A) and HBV-SA plus IE plus anti-ULA class I antibody (aULA) on the expression of interferon-gamma (IFNg) in PBMCs from healthy human subjects (top panels) and HBV patients (bottom panels).
  • IFNg interferon-gamma
  • immunosuppressive cytokine production and high-dose IE further shifted the response toward inducing apoptosis.
  • FIG. 14 shows the immune stimulation-enhancing activity of human alpha-synuclein protein (as measured by IFN-gamma release) for antigen challenge of whole blood from a subject recently immunized with the subject antigen.
  • Whole blood for testing was obtained from a human subject recently immunized (approximately one week) with an approved shingles vaccine (i.e., immunized against Varicella zoster (chickenpox) virus antigens). Fresh whole blood aliquots were mixed with control and test protein/antigen compositions, and incubated at 37°C for 24 hours. IFN-gamma release was then evaluated by ELISA assay. As shown in FIG.
  • control whole blood only; no added antigen or proteins
  • AGShingles whole blood plus two antigens (Varicella zoster virus (VZV) ORF 26 recombinant protein at 3 ⁇ g/ml and Varicella zoster virus ORF 9 recombinant protein at 2 ⁇ g/ml, collectively at these concentrations "the AGShingles antigens" present in the shingles vaccine the subject received); sy (whole blood plus boiled alpha-synuclein protein at 20 ⁇ g/ml); synb (whole blood plus alpha-synuclein protein not boiled at 20 ⁇ g/ml); JJ_ (whole blood plus heat-treated whole blood at 100 ⁇ g/ml); SY+AGShingles (whole blood plus boiled
  • IFN-gamma release in the experiment is indicative of effector T-cell stimulation in the whole blood.
  • control, sy, synb and Tl alone i.e., all without added Varicella zoster virus antigen did not cause IFN- gamma release.
  • the recombinant human HLA-B used in the experiments was cat# RPC140684-5C ⁇ g from Biomatik USA, LLC (Wilmington, DE, USA).
  • the amino acid sequence of said HLA-B is shown in Table 1 (SEQ ID NO: 1).
  • the recombinant human HLA-G used in the experiments was cat# RPC140674-5C ⁇ g from Biomatik USA, LLC (Wilmington, DE, USA).
  • the amino acid sequence of said HLA-G is shown in Table 2 (SEQ ID NO: 2).
  • the recombinant human alpha-synuclein used in the experiments was cat # PRO-393 from ProSpec-Tany TechnoGene Ltd. ("ProspecBio;" East Brunswick, NJ, USA).
  • the amino acid sequence of said human alpha- synuclein is shown in Table 3 (SEQ ID NO: 3).
  • Varicella Zoster Virus ORF 26 recombinant protein used in the experiments was ProspecBio cat# Pro-233 and Varicella Zoster Virus ORF 9 recombinant protein used in the experiments was ProspecBio cat# Pro-232.
  • red blood cells The ability of red blood cells to augment immune responses has been previously described, especially with respect to immunosuppression arising from red blood cell
  • LFA-3 is a ligand on antigen-presenting cells (APCs) that interacts with the CD2 receptor on CD4+ cells and is thought to be a co-activator that works in conjunction with the interaction between ULAs and TCRs on T-cells. It should be pointed out that the LFA-3 in a blood preparation is in the context of being present on APCs and not as a free ligand.
  • APCs antigen-presenting cells
  • the invention also provides the following enumerated embodiments.
  • Embodiment 1 An immunomodulatory composition, such as an immunomodulatory
  • composition including a mixture of:
  • ULA protein or fragments thereof such as mammalian, for example human
  • whole blood such as mammalian, for example human
  • a cellular fraction thereof such as a density gradient fraction/layer thereof, such as but not limited to a white blood cell and/or red blood cell (erythrocyte) and/or platelet fraction/layer thereof, or a cell membrane fraction/preparation of any of the foregoing or a protein extract of any of the foregoing, and/or
  • alpha-synuclein protein such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof;
  • At least one preselected antigen or immunogen such as at least one preselected peptide antigen, at least one preselected protein antigen, at least one preselected carbohydrate antigen, at least one preselected lipid antigen, and/or at least one preselected glycolipid antigen.
  • the at least one preselected antigen or immunogen may be other than an HLA protein or fragment thereof and/or other than a synuclein protein or fragment thereof, such as other than a mammalian alpha-synuclein protein or fragment thereof.
  • Embodiment 2 The immunomodulatory composition of embodiment 1, in which the first component, such as at least partially purified HLA protein, is heat-treated, for example, heat-treated at or above 100°C for at least 1 or 2 or 3 or 4 or 5 minutes.
  • the first component such as at least partially purified HLA protein
  • Embodiment 3 The immunomodulatory composition of embodiment 1, in which the at least partially purified HLA protein is at least substantially denatured.
  • Embodiment 4 The immunomodulatory composition of any one of the preceding embodiments, in which the first component includes at least partially purified HLA protein is recombinant, such as full length or partial length recombinant protein.
  • Embodiment 5 The immunomodulatory composition of embodiments 1-3, in which the at least partially purified HLA protein is derived from a tissue source.
  • Embodiment 6 The immunomodulatory composition of embodiment 5, in which the tissue source includes blood cells.
  • Embodiment 7 The immunomodulatory composition of embodiment 5, in which the tissue source at least substantially or at least predominantly includes red blood cells.
  • Embodiment 8 The immunomodulatory composition of any one of the preceding embodiments, in which the at least one preselected peptide antigen includes a synthetic peptide.
  • the peptide may, for example, be 5-20 amino acids in length or any subrange thereof or number of amino acids therein.
  • Embodiment 9 The immunomodulatory composition of any one of the preceding embodiments, in which the at least one preselected antigen includes a self-antigen.
  • Embodiment 10 The immunomodulatory composition of embodiment 9, in which the self- antigen is associated with an autoimmune disease.
  • Embodiment 11 The immunomodulatory composition of any one of embodiments 1-8, in which the at least one preselected antigen is a cancer-associated antigen or an antigen preferentially expressed on cancer cells versus normal cells.
  • Embodiment 12 The immunomodulatory composition of any one of the preceding
  • the at least partially purified HLA protein includes at least partially purified mammalian HLA protein.
  • Embodiment 13 The immunomodulatory composition of embodiment 12, in which the at least partially purified mammalian HLA protein includes at least partially purified human HLA protein.
  • Embodiment 14 The immunomodulatory composition of any one of the preceding embodiments, in which the composition is in a form selected from the group consisting of a liquid form and an at least substantially dry form, such as a powder form or tableted form.
  • a dry form may, for example, be obtained by lyophilizing or otherwise drying a liquid mixture of the components.
  • Embodiment 15 The immunomodulatory composition of embodiment 14, in which the composition is a parenteral composition.
  • Embodiment 16 The immunomodulatory composition of embodiment 15, in which the composition is an injectable composition.
  • Embodiment 17 The immunomodulatory composition of any one of the preceding
  • the at least partially purified HLA protein includes one or more of HLA- A, HLA-B, HLA-C and HLA-G protein.
  • Embodiment 18 The immunomodulatory composition of embodiment 17, in which the at least partially purified HLA protein includes HLA-G protein.
  • Embodiment 19 The immunomodulatory composition of any one of the preceding
  • the at least partially purified HLA protein includes HLA Class II protein.
  • Embodiment 20 A method for manufacturing an immunomodulatory composition, such as an immunomodulatory pharmaceutical composition, including the steps of: providing a first component including (a) at least partially purified HLA protein or fragments thereof, such as mammalian, for example human,
  • whole blood such as mammalian, for example human
  • a cellular fraction thereof such as a density gradient fraction thereof, such as but not limited to a white blood cell and/or red blood cell (erythrocytes) and/or platelet fraction/layer thereof, or a cell membrane fraction/preparation of any of the foregoing or a protein extract of any of the foregoing, and/or
  • alpha-synuclein protein such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof; and providing a second component including at least one preselected antigen or immunogen, such as at least one preselected peptide antigen, at least one preselected protein antigen, at least one preselected carbohydrate antigen, at least one preselected lipid antigen, and/or at least one preselected glycolipid antigen; and mixing the first component and the second component, for example, under aqueous conditions.
  • preselected antigen or immunogen such as at least one preselected peptide antigen, at least one preselected protein antigen, at least one preselected carbohydrate antigen, at least one preselected lipid antigen, and/or at least one preselected glycolipid antigen
  • Embodiment 21 The method of embodiment 20, in which the at least partially purified HLA protein is provided and the method further includes the step of: heat-treating the at least partially purified HLA protein before the mixing step.
  • Embodiment 22 The method of embodiment 20, in which at least partially purified HLA protein is provided and the method further includes the step of: denaturing the at least partially purified HLA protein before the mixing step.
  • Embodiment 23 The method of embodiment 20, further including the step of: heat-treating the composition after the mixing step.
  • Embodiment 24 The method of embodiment 20, in which the providing step includes providing said fragments, and the mixing step includes mixing said fragments with said second component.
  • Embodiment 25 The method of any one of embodiments 20-24, in which the at least partially purified HLA protein includes recombinant HLA protein.
  • Embodiment 26 The method of any one of embodiments 20-24, in which the at least partially purified HLA protein is derived from a tissue source.
  • Embodiment 27 The method of embodiment 26, in which the tissue source includes blood cells.
  • Embodiment 28 The method of embodiment 27, in which the tissue source at least substantially includes red blood cells.
  • Embodiment 29 The method of any one of embodiments 20-28, in which the at least one preselected antigen includes a synthetic peptide.
  • Embodiment 30 The method of any one of embodiments 20-29, in which the at least one preselected antigen includes a self-antigen.
  • Embodiment 31 The method of embodiment 30, in which the self-antigen is associated with an autoimmune disease.
  • Embodiment 32 The method of any one of embodiments 20-29, in which the at least one preselected antigen, which may, for example, be or include one or more synthetic peptides, includes a cancer-associated antigen/epitope or an antigen/epitope preferentially expressed on cancer cells versus normal cells.
  • Embodiment 33 The method of any one of embodiments 20-29, in which the at least one preselected peptide antigen includes an antigen of a virus or cellular microorganism, such as a pathogenic virus or cellular microorganism, for example for a mammal such as human.
  • the at least one preselected antigen of a pathogenic virus may, for example, be or include an antigen of or associated with Hepatitis B virus, Hepatitis C virus, Influenza virus, HIV-1 or HIV-2.
  • the virus may be Hepatitis B and the at least one preselected antigen may be one or more of HBsAg (surface antigen, S-protein) such as SEQ ID NO: 37 (adw serotype) and/or SEQ ID NO: 38 (adr serotype), HB pre-Sl protein (SEQ ID NO: 39), HB pre- S2 protein (SEQ ID NO: 40), HBeAg (HepB envelope antigen; e.g., SEQ ID NO: 41), and HBcAg (HepB core antigen; e.g. SEQ ID NO: 42).
  • the at least one preselected antigen of a pathogenic cellular microorganism may, for example, be or include an antigen of or associated with a pathogenic bacteria, fungi, protozoan, or amoeba.
  • Embodiment 34 The method of any one of embodiments 20-33, in which the at least partially purified HLA protein includes one or more of HLA-A, HLA-B, HLA-C and HLA-G protein.
  • Embodiment 35 The method of embodiment 34, in which the at least partially purified HLA protein includes HLA-G.
  • Embodiment 36 The method of any one of embodiments 20-35, in which the at least partially purified HLA protein includes HLA Class II protein.
  • Embodiment 37 A method for modulating the immune response in a mammal to at least one preselected antigen or immunogen including administering to the mammal the
  • immunomodulatory pharmaceutical composition of any one of embodiments 1-19 Said administration may be parenteral or non-parenteral. Said administration may, for example be via ingestion. Where administration is via ingestion, an antacid may be co-administered.
  • the composition may, for example, be an enteric composition for ingestion. Administration may, for example, be via injection, such as intravenous injection, intra-thymic injection or injection into a lymph node of a subject.
  • Embodiment 38 The method of embodiment 36, in which said administration is parenteral.
  • Embodiment 39 The method of embodiment 36, in which said administration is via injection.
  • Embodiment 40 The method of any one of embodiments 36-38, in which the mammal is a human.
  • Embodiment 41 The method of any one of embodiments 36-39, in which the resultant modulation of the immune response is immunosuppressive (pro-regulatory cell response) with respect to the at least one preselected antigen or immunogen.
  • immunosuppressive pro-regulatory cell response
  • alpha-synuclein may be used as a pro-regulatory (-immunosuppressive) response adjuvant.
  • Embodiment 42 The method of any one of embodiments 36-39, in which the resultant modulation of the immune response is immunostimulatory (pro-effector cell response) with respect to the at least one preselected antigen or immunogen.
  • immunostimulatory pro-effector cell response
  • alpha-synuclein may be used as a pro-effector response adjuvant.
  • Embodiment 43 Use of a composition according to any one of embodiments 1-19 for modulating the immune response in a mammal, such as a human, to the at least one preselected antigen or immunogen.
  • Embodiment 44 The use of embodiment 43, in which the modulation of the immune response is immunosuppressive with respect to the at least one preselected antigen or immunogen.
  • Embodiment 45 The use of embodiment 43, in which the modulation of the immune response is immunostimulatory with respect to the at least one preselected antigen or immunogen.
  • Embodiment 46 Use of alpha-synuclein protein, such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof, as an immune stimulator or adjuvant in conjunction with a vaccination (use of a vaccine), for example, in a mammal such as but not limited to a human, such as, in conjunction with vaccination against a pathogen or a cancer antigen or in conjunction with use of a cancer vaccine.
  • a mammal such as but not limited to a human, such as, in conjunction with vaccination against a pathogen or a cancer antigen or in conjunction with use of a cancer vaccine.
  • Embodiment 47 Use of alpha-synuclein protein, such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof, as an immunization adjuvant or vaccine adjuvant, for example, in a mammal such as but not limited to a human.
  • alpha-synuclein protein such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof, as an immunization adjuvant or vaccine adjuvant, for example, in a mammal such as but not limited to a human.
  • Embodiment 48 A method for enhancing the immune response to an immunization with an immunogen in a subject such as a mammal, such as but not limited to a human, comprising the step of: coadministering to the subject alpha-synuclein protein, such as mammalian alpha- synuclein protein, such as human alpha-synuclein protein, or fragments thereof, with the immunogen.
  • alpha-synuclein protein such as mammalian alpha- synuclein protein, such as human alpha-synuclein protein, or fragments thereof
  • Embodiment 49 A method for enhancing the immune response of a mammalian subject, such as but not limited to a human, having a malignancy, such as a blood cancer/malignancy or a solid tumor, to said malignancy and/or a method for treating such a malignancy in a such a subject, comprising the step of: administering to the subject alpha-synuclein protein, such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof. Said administration may be with or without at least one preselected antigen.
  • Said blood such as but not limited to a human, having a malignancy, such as a blood cancer/malignancy or a solid tumor
  • a method for treating such a malignancy in a such a subject comprising the step of: administering to the subject alpha-synuclein protein, such as mammalian alpha-synuclein protein, such
  • cancer/malignancy may, for example, be Myelodysplastic syndrome (MDS), a leukemia, such as Acute lymphoblastic leukemia (ALL) or Acute myeloid leukemia (AML), or a lymphoma, such as a Hodgkin lymphoma, non-Hodgkin lymphoma or mantle cell lymphoma.
  • MDS Myelodysplastic syndrome
  • a leukemia such as Acute lymphoblastic leukemia (ALL) or Acute myeloid leukemia (AML)
  • a lymphoma such as a Hodgkin lymphoma, non-Hodgkin lymphoma or mantle cell lymphoma.
  • Said malignancy may, for example, be a liver cancer such as hepatocellular carcinoma (HCC) or
  • cholangiocarcinoma pancreatic cancer, breast cancer, prostate cancer, kidney cancer, melanoma, myeloma, glioblastoma, ovarian cancer, testicular cancer, bone cancer such as osteosarcoma, or lung cancer such as non-small cell lung cancer or small cell lung cancer.
  • Embodiment 50 Use of alpha-synuclein protein, such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof, in the treatment of a mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof, in the treatment of a mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof, in the treatment of a mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof, in the treatment of a mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof, in the treatment of a mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof,
  • malignancy/cancer such as a blood cancer or a solid tumor, in a mammal such as but not limited to a human.
  • Said administration may be with or without at least one preselected antigen.
  • Said blood cancer/malignancy may, for example, be Myelodysplastic syndrome (MDS), a leukemia, such as ALL or AML, or a lymphoma, such as a Hodgkin lymphoma, non-Hodgkin lymphoma or mantle cell lymphoma.
  • MDS Myelodysplastic syndrome
  • ALL or AML a leukemia
  • lymphoma such as a Hodgkin lymphoma, non-Hodgkin lymphoma or mantle cell lymphoma.
  • Said malignancy may, for example, be a liver cancer such as hepatocellular carcinoma (HCC) or cholangiocarcinoma, pancreatic cancer, breast cancer, prostate cancer, kidney cancer, melanoma, myeloma, glioblastoma, ovarian cancer, testicular cancer, bone cancer such as osteosarcoma, or lung cancer such as non-small cell lung cancer or small cell lung cancer.
  • HCC hepatocellular carcinoma
  • cholangiocarcinoma pancreatic cancer
  • breast cancer prostate cancer
  • kidney cancer melanoma
  • myeloma myeloma
  • glioblastoma glioblastoma
  • ovarian cancer testicular cancer
  • bone cancer such as osteosarcoma
  • lung cancer such as non-small cell lung cancer or small cell lung cancer.
  • Embodiment 51 Use of alpha-synuclein protein, such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof, in the preparation of a medicament for the treatment of a malignancy, such as a blood cancer or a solid tumor, in a mammal such as but not limited to a human.
  • a malignancy such as a blood cancer or a solid tumor
  • Said medicament may include or exclude at least one preselected antigen as described herein.
  • Said blood cancer/malignancy may, for example, be
  • MDS Myelodysplastic syndrome
  • a leukemia such as ALL or AML
  • a lymphoma such as a Hodgkin lymphoma, non-Hodgkin lymphoma or mantle cell lymphoma.
  • Said malignancy may, for example, be a liver cancer such as hepatocellular carcinoma (HCC) or cholangiocarcinoma, pancreatic cancer, breast cancer, prostate cancer, kidney cancer, melanoma, myeloma, glioblastoma, ovarian cancer, testicular cancer, bone cancer such as osteosarcoma, or lung cancer such as non-small cell lung cancer or small cell lung cancer.
  • HCC hepatocellular carcinoma
  • cholangiocarcinoma pancreatic cancer
  • breast cancer prostate cancer
  • kidney cancer melanoma
  • myeloma glioblastoma
  • ovarian cancer testicular cancer
  • bone cancer such as osteosarcoma
  • lung cancer such
  • Embodiment 52 A pharmaceutical composition for the treatment of a malignancy, such as a blood cancer or a solid tumor, in a mammal such as but not limited to a human, said composition comprising a therapeutically effective amount of alpha-synuclein protein, such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof.
  • the composition may further include one or more pharmaceutically acceptable excipients.
  • Said blood cancer/malignancy may, for example, be Myelodysplastic syndrome (MDS), a leukemia, such as ALL or AML, or a lymphoma, such as a Hodgkin lymphoma, non-Hodgkin lymphoma or mantle cell lymphoma.
  • MDS Myelodysplastic syndrome
  • a leukemia such as ALL or AML
  • a lymphoma such as a Hodgkin lymphoma, non-Hodgkin lymphoma or mantle cell lymphoma.
  • Said malignancy may, for example, be a liver cancer such as hepatocellular carcinoma (HCC) or cholangiocarcinoma, pancreatic cancer, breast cancer, prostate cancer, kidney cancer, melanoma, myeloma, glioblastoma, ovarian cancer, testicular cancer, bone cancer such as osteosarcoma, or lung cancer such as non-small cell lung cancer or small cell lung cancer
  • Embodiment 53 A method for enhancing the immune response of a mammalian subject, such as but not limited to a human, having an infectious disease, such as a microbial or viral infection, to said infectious disease, comprising the step of: administering to the subject alpha-synuclein protein, such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof. Said administration may be with or without at least one preselected antigen as described herein.
  • alpha-synuclein protein such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof.
  • Said administration may be with or without at least one preselected antigen as described herein.
  • Embodiment 54 Use of alpha-synuclein protein, such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof, in the treatment of an infectious disease, such as a microbial or viral infection, in a mammal such as but not limited to a human. Said use may be in combination with or exclude at least one preselected antigen as described herein.
  • Embodiment 55 Use of alpha-synuclein protein, such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof, in the treatment of an infectious disease, such as a microbial or viral infection, in a mammal such as but not limited to a human. Said use may be in combination with or exclude at least one preselected antigen as described herein.
  • Embodiment 55 Use of alpha-synuclein protein, such as mammalian alpha
  • alpha-synuclein protein such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof, in the preparation of a medicament, such as a medicament for the treatment of an infectious disease, such as a microbial or viral infection, in a mammal such as but not limited to a human.
  • Said medicament may include or exclude at least one preselected antigen as described herein.
  • Embodiment 56 A pharmaceutical composition for the treatment of an infectious disease, such as a microbial or viral infection, in a mammal such as but not limited to a human, said composition comprising a therapeutically effective amount of alpha-synuclein protein, such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof.
  • the composition may further include one or more pharmaceutically acceptable excipients.
  • the composition may include or exclude at least one preselected antigen as described herein.
  • the viral infection is Hepatitis B.
  • the viral infection is Hepatitis B and the composition includes at least one Hepatitis B protein or peptide antigen such as but not limited to one or more of HBsAg (surface antigen, S-protein) such as SEQ ID NO: 37 (adw serotype) and/or SEQ ID NO: 38 (adr serotype), HB pre-Sl protein (SEQ ID NO: 39), HB pre-S2 protein (SEQ ID NO: 40), HBeAg (HepB envelope antigen; e.g., SEQ ID NO: 41), and HBcAg (HepB core antigen; e.g. SEQ ID NO: 42).
  • HBsAg surface antigen, S-protein
  • SEQ ID NO: 37 adw serotype
  • SEQ ID NO: 38 adr serotype
  • HB pre-Sl protein SEQ ID NO: 39
  • HB pre-S2 protein SEQ ID NO: 40
  • HBeAg HepB envelope antigen
  • an immunomodulatory composition such as an immunomodulatory pharmaceutical composition, including, for example as or in a mixture:
  • At least one preselected antigen such as a peptide antigen, or immunogen, which antigen or immunogen is not a human or non-human mammalian alpha-synuclein protein or a fragment thereof.
  • the at least one preselected antigen or at least one preselected immunogen does not comprise a synuclein protein and/or does not comprise a fragment of a synuclein protein.
  • Embodiment 58 The immunomodulatory composition of embodiment 57, further including at least partially purified human or non-human mammalian HLA protein or fragments thereof, wherein the at least one preselected antigen or immunogen does not include human or non- human mammalian HLA protein or fragments thereof.
  • Embodiment 59 The immunomodulatory composition of embodiment 57, wherein the composition does not include HLA protein or fragments thereof.
  • Embodiment 60 Any of composition embodiments 57-59, further including at least one pharmaceutically acceptable excipient.
  • Embodiment 61 A method for modulating the immune response in a mammal to at least one preselected antigen or immunogen including administering to a human or non-human mammal a immunomodulatory pharmaceutical composition according to any one of embodiments 57-60.
  • Embodiment 62 A method for modulating the immune response in a mammal to at least one preselected antigen or immunogen including co-administering to a human or non-human mammal
  • At least one preselected antigen or immunogen which is not a human or non-human mammalian alpha-synuclein protein or a fragment thereof.
  • the at least one preselected antigen or at least one preselected immunogen does not comprise a synuclein protein and/or does not comprise a fragment of a synuclein protein.
  • Embodiment 63 The method embodiment 62, further including co-administering at least partially purified human or non-human mammalian HLA protein or fragments thereof to the human or non-human mammal, wherein the at least one preselected antigen or immunogen does not include human or non-human mammalian HLA protein or fragments thereof.
  • Embodiment 64 The method embodiment 62, wherein HLA protein or fragments thereof are not co-administered to the human or non-human mammal.
  • Embodiment 65 A method for manufacturing an immunomodulatory composition, such as an immunomodulatory pharmaceutical composition, including the steps of:
  • At least one preselected antigen or immunogen which is not a human or non- human mammalian alpha-synuclein protein or a fragments thereof;
  • Embodiment 66 The method embodiment 65, further including:
  • said mixing step further includes mixing the provided at least substantially pure alpha-synuclein protein or fragments thereof, the at least one preselected antigen or immunogen, and the at least partially purified human or non-human mammalian HLA protein or fragments thereof, and
  • the at least one preselected antigen or immunogen is not a human or non-human mammalian HLA protein or fragment thereof.
  • the at least one preselected antigen or immunogen is not an HLA protein or fragment thereof.
  • Embodiment 67 Either of embodiments 65 and 66, further including providing at least one pharmaceutically acceptable excipient, and mixing said at least one pharmaceutical excipient with the other mix components of said embodiments.
  • Embodiment 68 A pharmaceutical composition including a mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or at least one fragment thereof, such as a substantial fragment thereof.
  • the alpha-synuclein protein or fragment thereof may be recombinant or synthetic.
  • the pharmaceutical composition consists essentially of a mammalian alpha-synuclein, such as human alpha- synuclein protein, or a substantial fragment thereof.
  • the pharmaceutical composition consists essentially of a mammalian alpha-synuclein, such as human alpha- synuclein protein, or a substantial fragment thereof and at least one preselected antigen or immunogen.
  • compositions may further include one or more pharmaceutically-acceptable excipients.
  • the compositions may be parenteral or non-parenteral formulations.
  • the compositions may, for example, be oral pharmaceutical compositions (formulated for oral administration (ingestion)) or in formulated in any manners described in this disclosure.
  • the compositions may be in a solid dosage form, such as a powder, tablet or capsule.
  • the compositions may be in a liquid form for either parenteral or non-parental administration.
  • the compositions may be in liquid form for administration by injection.
  • Embodiment 69 Use of a mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or at least one fragment thereof, such as a substantial fragment thereof, as a medicament, for example in mammals such as humans.
  • the alpha-synuclein protein or fragment thereof may be recombinant or synthetic, or purified from a tissue source.
  • the at least one preselected antigen or immunogen of various embodiments may, for example, be or include a peptide such as a synthetic peptide.
  • the at least one preselected antigen or immunogen may, for example, be or include a protein such as an at least substantially purified protein, such as but not limited to an at least substantially purified recombinant protein.
  • the at least one preselected antigen or immunogen may be or include a recombinant protein or a recombinant protein fragment of a full-length protein.
  • an embodiment refers to a peptide antigen, it should be understood that corresponding embodiments directed to protein antigens are also intended to be disclosed and vice versa.
  • the at least one preselected peptide/protein antigen or immunogen may, for example, be or include a self-antigen of a human or non-human mammal, for example, a self-antigen associated with an autoimmune disease.
  • the at least one preselected peptide/protein antigen or immunogen may, for example, be or include a cancer-associated antigen or an antigen preferentially expressed on cancer cells versus normal cells of a human or non-human mammal.
  • the at least one preselected peptide/protein antigen may, for example, be or include a viral antigen, for example, of a pathogenic virus, such as a pathogenic virus that currently infects the subject or has previously infected the subject.
  • the virus may be Hepatitis B and the at least one preselected antigen may be one or more of HBsAg (surface antigen, S-protein) such as SEQ ID NO: 37 (adw serotype) and/or SEQ ID NO: 38 (adr serotype), HB pre-Sl protein (SEQ ID NO: 39), HB pre-S2 protein (SEQ ID NO: 40), HBeAg (HepB envelope antigen; e.g., SEQ ID NO: 41), and HBcAg (HepB core antigen; e.g. SEQ ID NO: 42).
  • HBsAg surface antigen, S-protein
  • SEQ ID NO: 37 adw serotype
  • SEQ ID NO: 38 adr serotype
  • HB pre-Sl protein SEQ ID NO: 39
  • HB pre-S2 protein SEQ ID NO: 40
  • HBeAg HepB envelope antigen
  • SEQ ID NO: 41 SEQ ID NO: 41
  • the at least one preselected antigen that is or includes a self-antigen may be or include S- antigen, such as human S-antigen (SEQ ID NO: 4 or SEQ ID NO: 5) and/or a protein or peptide fragment thereof such as a synthetic peptide fragment.
  • Immunomodulatory pharmaceutical composition embodiments of the invention including such antigens may be administered for the treatment of uveitis or AMD in a human or non-human mammal.
  • the peptide is or includes a fragment of S-antigen such as GEPIPVTVDVTNNTEKTVKK (SEQ ID NO: 6) or VTVDVTNNTEKTVKK (SEQ ID NO: 7).
  • SEQ ID NO: 6 GEPIPVTVDVTNNTEKTVKK
  • VTVDVTNNTEKTVKK SEQ ID NO: 7
  • B27PD ALNED LSSWT AADT (SEQ ID NO: 8)
  • Peptide 2 (P2): IFKKI SRDKS VTIYL (SEQ ID NO: 9)
  • P6 VKGKK VYVTL TCAFR (SEQ ID NO: 10)
  • P8 YGQED VD VIG LTFRR (SEQ ID NO: 11)
  • Peptide 29 (P29): LPLLA NNRER RGIAL (SEQ ID NO: 12)
  • Peptide 31 DTNLA SSTII KEGID (SEQ ID NO: 13)
  • the peptides may, for example, be the only preselected antigens or they may be used in any combination in the immunomodulatory compositions.
  • the patient has early AMD, characterized by medium drusen (63-125 ⁇ ) without pigmentary abnormalities thought to be related to AMD.
  • the patient has intermediate AMD, characterized by large drusen or with pigmentary abnormalities associated with at least medium drusen.
  • the patient has late AMD, characterized by lesions associated with neovascular AMD or geographic atrophy.
  • Drusen which are yellow or white accumulations of extracellular material that build up between Bruch's membrane and the retinal pigment epithelium of the eye, can be measured by any technique known by the skilled artisan.
  • drusen volumes are measured by spectral domain optical coherence tomography (SD-OCT).
  • the patient has wet AMD which may be associated with choroidal
  • CNV neovascularization
  • a related embodiment provides a method for treating AMD in a human or non-human mammalian subject that includes administering any of said immunomodulatory pharmaceutical compositions to the subject.
  • the result obtained by treatment of AMD or uveitis includes cessation and/or slowing of disease progression, for example, progression from early AMD to intermediate AMD, or progression from intermediate AMD to late AMD, or cessation or slowing of progression to wet AMD, or cessation and/or slowing of
  • Another embodiment of the invention provides immunomodulatory pharmaceutical compositions according to the invention for the treatment of multiple sclerosis in a mammalian subject, such as a human patient, in which the at least one preselected antigen is or includes myelin basis protein (MBP) such as human myelin basis protein (for example, Genbank
  • a related embodiment provides a method for treating multiple sclerosis in a human or non-human mammalian subject that includes administering said immunomodulatory composition to the subject.
  • Another embodiment of the invention provides an immunomodulatory pharmaceutical composition according to the invention for the treatment of rheumatoid arthritis in a human or non-human mammalian subject in which the at least one preeletced antigen of the composition is or includes type II collagen such as human type II collagen protein (for example, Genbank Accession No. AAC41772 alpha-1 type II collagen [Homo sapiens]; SEQ ID NO: 16 (see also amino acid sequence Table 5)) and/or one or more peptide fragments thereof, such as synthetic peptides.
  • a related embodiment provides a method for treating rheumatoid arthritis in a human or non-human mammalian subject that includes administering said immunomodulatory composition to the subject.
  • the at least one preselected antigen of the composition and method embodiments may, for example, be or include the therapeutic protein or one or more fragments thereof, or one or more peptides representing at least a portion of the amino acid sequence of the therapeutic protein.
  • At least partially purified HLA protein may, for example, be or include at least partially purified mammalian HLA protein. At least partially purified mammalian HLA protein may, for example, be or include at least partially purified human HLA protein. At least partially purified HLA protein may, for example, be or include one or more of HLA- A, HLA-B, HLA-C and HLA-G protein. At least partially purified HLA protein may, for example, be or include HLA Class II protein.
  • Alpha-synuclein and/or HLA and/or any proteins of embodiments of the invention may, for example, be recombinant or may be purified from tissue.
  • the immunomodulatory pharmaceutical composition may, for example, be in liquid form or in a solid/dry form such as in a powder or tablet form.
  • compositions may be parenteral or non-parenteral compositions.
  • immunomodulatory pharmaceutical compositions may, for example, be injectable compositions such as a liquid, for example aqueous, injectable solution or suspension.
  • the immunomodulatory pharmaceutical compositions may, for example, be orally administrable compositions.
  • Administration to a subject may be by any route, such as parenteral or non- parenteral or a combination of routes. Administration may, for example, be made via injection or oral administration (ingestion) or by direct delivery to any part/section of the alimentary canal.
  • Solid pharmaceutical compositions for oral administration via ingestion such as tablets or capsule may, for example, be enteric coated or otherwise formulated to prevent or minimize dissolution in the stomach but allow dissolution in the small intestine.
  • Compositions for oral administration via ingestion may, for example, comprise or be co-administered with an antacid or other acid-reducing agent, such as omeprazole.
  • the resultant modulation of the immune response may be immunosuppressive, e.g., at least partially tolerance-inducing, with respect to the at least one preselected antigen or immunogen, or the resultant modulation of the immune response is immunostimulatory with respect to the at least one preselected antigen or immunogen.
  • composition or mixture excludes (does not include) beta-2 microglobulin.
  • Still further embodiments of the invention are directed to methods and compositions for preventing and/or treating Hepatitis B infections, such as chronic Hepatitis B infections,
  • Hepatitis B-associated liver diseases and/or Hepatitis B-associated cancers such as hepatocellular carcinoma (HCC), in non-human mammals and human patients.
  • HCC hepatocellular carcinoma
  • HB SAg chronic Hepatitis B virus
  • Thl responses are reduced and Th2 responses increase.
  • the effects of the oral treatment described in Safadi et al. resulted in 17/27 patients showing an increases in IFN-gamma secretion (an increase in a Thl response) and 13/27 patients showing a reduction of IL10 secretion (a decrease in a Th2 response) thus showing a reversal in markers for progression towards development of hepatocellular carcinoma.
  • 21/27 of the patients showed an increased HB SAg specific T cell proliferation, a potentially further indication that the recipients were mounting an effective Thl response to HBV.
  • One embodiment of the invention provides a method for preventing or treating Hepatitis B infection, such as chronic Hepatitis B infection, Hepatitis B-associated liver disease and/or Hepatitis B-associated cancer such as hepatocellular carcinoma (HCC), in a non-human mammal and human patients, which method includes:
  • HepB antigens such as one or more preselected HepB antigens, for example HepB proteins or peptides that are recombinantly or synthetically manufactured;
  • the utilized components of (i) and (ii) may be mixed together or provided as mixed and administered as one composition or may be co-administered as separate compositions.
  • the mammal or human may, for example, be currently infected with Hepatitis B virus, such as chronically infected with the virus, or was previously but not currently infected with Hepatitis B virus.
  • Treatment of a chronic or active HepB infection can result in conversion to an inactive carrier state.
  • treatment can shift the immune response toward Thl and prevent/delay progression of the HCC.
  • treatment can prevent or delay the progression of HBV-associated liver diseases and progression to cancers such as HCC.
  • a related embodiment of the invention provides a pharmaceutical composition, such as an oral pharmaceutical composition for preventing or treating Hepatitis B infection, such as chronic Hepatitis B infection, Hepatitis B-associated liver disease and/or Hepatitis B-associated cancer such as hepatocellular carcinoma (HCC), in a non-human mammal and human patients, which composition includes:
  • HepB antigens such as one or more preselected HepB antigens, for example HepB proteins or peptides that are recombinantly or synthetically manufactured;
  • HTB heat-treated blood
  • RBC blood fraction such as autologous or heterologous (from the same species of mammal or a different species of mammal), a cell membrane fraction of the foregoing, a protein extract of any of the foregoing, alpha-synuclein protein, such as mammalian alpha-synuclein protein, such as human alpha-synuclein protein, or fragments thereof of any of said alpha-synucleins.
  • the one or more Hepatitis B antigens in the preceding embodiments may, for example, be or include one or more (in any combination) of HBsAg (surface antigen, S-protein) such as SEQ ID NO: 37 (adw serotype) and/or SEQ ID NO: 38 (adr serotype), HB pre-Sl protein (SEQ ID NO: 39), HB pre-S2 protein (SEQ ID NO: 40), HBeAg (HepB envelope antigen; e.g., SEQ ID NO: 41), and HBcAg (HepB core antigen; e.g. SEQ ID NO: 42); recombinant forms of each are well known and commercially available.
  • HBsAg surface antigen, S-protein
  • SEQ ID NO: 37 adw serotype
  • SEQ ID NO: 38 adr serotype
  • HB pre-Sl protein SEQ ID NO: 39
  • HB pre-S2 protein SEQ ID NO: 40
  • Alpha-synuclein in these embodiments may, for example, be recombinant (such as recombinant human alpha-synuclein, SEQ ID NO: 3) or purified from a tissue source.
  • the Hepatitis B antigen(s) and alpha-synuclein(s) may, for example, each be provided in an at least substantially pure form for use in the embodiments.
  • the measurement of immune reactivity to selected antigens is a common practice in either diagnosing the presence of a disease state or delineating the stage or progression of a disease state.
  • An example of the former is establishing whether an individual has been exposed to a particular antigen such as a viral, fungal or environmental agent.
  • An example of the latter is the determining the status of infection in a person exposed to Mycobacterium tuberculosis (TB) where different immune reactions are characteristic of different stages.
  • TB Mycobacterium tuberculosis
  • the enhancement of specific immune reactivity by the present invention may offer increased sensitivity where exposure to selected antigens can be detected at an earlier time point and enhanced detection of positive responses over background, thereby permitting determinations of positivity in otherwise ambiguous circumstances.
  • another embodiment of the invention provides an assay method for determining whether a sample of cells mounts an antigen-specific response to one or more preselected antigens and/or for quantifying the extent to which a sample of cells mounts an antigen-specific response to one or more preselected antigens, said method embodiment including the steps of: providing an isolated sample of cells, such as a sample of blood cells, such as whole blood, or a white blood cell fraction or PBMCs or T-cells; providing alpha- synuclein such as mammalian alpha-synuclein such as human alpha-synuclein and/or a fragment of any of the foregoing; providing at least one such as one preselected antigen, such as but not limited to an antigen which is a synthetic or recombinant peptide or protein; contacting the sample of cells with both the alpha-synuclein (any of the aforementioned varieties) and/or fragments thereof and the at least one preselected antigen; and measuring
  • the sample of cells may, for example, be obtained from a non-human mammal or a human.
  • the synuclein or fragments thereof and the at least one preselected antigen may, for example, be mixed with each other (or be premixed) before being contacted with, such being added to, the sample of cells.
  • Parallel steps may be run with all of the same components except for, i.e., excluding, the at least one preselected antigen (and optionally using a neutral "dummy antigen" in its place) as a control arm with the final measurements used as a negative control for the presumptive antigen stimulation arm.
  • a related embodiment of the invention provides an assay composition that includes, as mixture, an isolated sample of cells obtained from a subject, such as a sample of blood cells, such as whole blood, or a white blood cell fraction or PBMCs, said cells being, for example, non-human mammalian cells or human cells; alpha-synuclein such as mammalian alpha- synuclein such as human alpha-synuclein and/or a fragment of any of the foregoing; and at least one such as one preselected antigen, such as but not limited to an antigen which is a synthetic or recombinant peptide or protein.
  • Another embodiment of the invention provides a vessel having an inner surface defining a volume, such as but not limited to a blood collection tube or a well of a microwell plate, wherein said inner surface is coated (i) at least in part with at least one preselected antigen, such as a protein or peptide antigen, which antigen is not alpha-synuclein protein or a fragment thereof, and (ii) at least in part with alpha-synuclein protein such as recombinant or synthetic alpha-synuclein protein or a fragment thereof.
  • the surface part coated in (i) and (ii) may be the same or different, or partially overlap.
  • the vessel may, for example, be dry and not filled with any liquid as when stored for use and can be at least partially filled with liquid for use.
  • the antigen and the alpha-synuclein protein or a fragment thereof may be dried on the part(s) of the inner surface.
  • a further embodiment of the invention provides a composition of matter that includes: the aforementioned vessel embodiment;
  • T-cells such as a PBMC fraction
  • the quantity of whole blood or a fraction thereof including T-cells is in contact with both the at least one preselected protein or peptide antigen and the synthetic or recombinant alpha-synuclein protein or fragment thereof in the vessel.
  • the whole blood or fraction thereof may be fresh.
  • a related embodiment of the invention provides a composition of matter that includes: a vessel having an inner surface defining a volume, such as but not limited to a blood collection tube or a well of a microwell plate;
  • At least one preselected antigen such as a preselected protein or peptide antigen, which antigen is not alpha-synuclein protein or a fragment thereof, inside the vessel; and
  • the vessel may contain a quantity of whole blood or a fraction thereof wherein the quantity of whole blood or a fraction thereof including T-cells is in contact with both the at least one preselected antigen and the synthetic or recombinant alpha-synuclein protein or fragment thereof in the vessel.
  • the whole blood or fraction thereof may be fresh.
  • a further embodiment of the invention provides a cytokine release assay method, such as an interferon-gamma release assay (IGRA) method that includes the steps of:
  • IGRA interferon-gamma release assay
  • T-cells adding a quantity of whole blood or a fraction thereof including T-cells, which may be fresh, to the vessel whereby T-cells in the quantity of whole blood or the fraction thereof including T-cells are contacted with the at least one preselected antigen and the alpha-synuclein protein or fragment thereof;
  • a cytokine such as interferon-gamma
  • the method may also include an active mixing step just after the adding step and before the measuring step.
  • the method may further include after the adding step and before the measuring step, a step of incubating the vessel at a temperature permissive for viability of T-cells such as 37°C for a period of time such as 8-26 hours or any subrange or value therein.
  • An incubation time that is not sufficient to effect differentiation of precursor effector T-cells to immediate effector T-cells may be used.
  • a cytokine release assay method such as an interferon-gamma release assay (IGRA) method, for quantifying T-cell responsiveness to at least one preselected antigen that includes the steps of:
  • IGRA interferon-gamma release assay
  • an ex vivo quantity of whole blood or a fraction thereof including T-cells a quantity of at least one preselected antigen, such as a protein or peptide antigen, that is not alpha-synuclein protein or a fragment thereof, and
  • a cytokine such as interferon-gamma
  • the method may further include after the mixture-forming step and before the measuring step, a step of incubating the vessel at a temperature permissive for viability of T-cells such as 37°C for a period of time such as 8-26 hours or any subrange or value therein.
  • a step of incubating the vessel at a temperature permissive for viability of T-cells such as 37°C for a period of time such as 8-26 hours or any subrange or value therein.
  • An incubation time that is not sufficient to effect differentiation of precursor effector T-cells to immediate effector T-cells may be used.
  • a further embodiment of the invention provides a method of manufacturing a vessel, such as one or more wells of a microwell plate or a blood collection tube, for use in an antigen specific T-cell response assay, such as a cytokine release assay, such as an interferon-gamma release assay, that includes the steps of:
  • a vessel having an inside surface defining a volume, such as a blood collection tube or a well of a microwell plate;
  • a liquid composition such as an aqueous solution or suspension, including at least one preselected antigen, such as a protein or peptide antigen;
  • a liquid composition such as an aqueous solution or suspension, including alpha-synuclein or a fragment thereof,
  • liquid composition including the at least one preselected antigen and the liquid composition including alpha-synuclein or a fragment thereof may be the same liquid composition or different liquid compositions; contacting the inner surface of the vessel with the liquid composition(s) including the at least one antigen and the alpha-synuclein or fragment thereof, for example, by at least partially filling the volume of the vessel with composition(s); and
  • the vessel volume may be at least substantially emptied of the composition by pouring out (gravity), with or without agitation/tapping, and/or aspiration.
  • Blood collection tubes and microwell plates as recited in the various embodiments may, for example, be composed of glass or synthetic polymer such as polyethylene or polypropylene, as known in the art.
  • Any of the cytokine release assay method embodiments above may, for example, instead of or in addition to measuring cytokine release, measure mRNA expression of T-cell specific mRNA such as a cytokine, such as interferon-gamma.
  • a cytokine such as interferon-gamma.
  • an HLA protein or a fragment thereof as described herein may be used, added, and/or included.
  • the antigen(s) may also exclude HLA protein and/or fragments thereof.
  • the at least one preselected antigen may, for example, be or include an antigen of or associated with a pathogenic cellular organism such as a bacteria, fungi, protozoan, amoeba or a virus. Antigen- specific reactivity detected from the sample of cells by the measuring step is indicative or strongly predictive that the subject from which the sample was obtained is currently infected with the pathogen. In this manner, a diagnosis can be provided.
  • the at least one preselected antigen may, for example, be or include one or at least one protein/peptide antigen of
  • Mycobacterium tuberculosis bacterium for example, ESAT-6, or a peptide fragment of said antigen, for example, one or more fragments of ESAT-6, and may, for example, be or include one or more synthetic peptides or recombinant proteins.
  • full-length ESAT-6 protein may be used (such as SEQ ID NO: 17 herein), and/or any of the Mycobacterium tuberculosis ESAT-6 derived peptide antigens disclosed in U.S. Patent No.
  • 7,632,646 (such as SEQ ID NOS: 18-25 herein) may be used, and/or any of the non-ESAT-6 antigens in Mustafa et al, Clinical Infectious Diseases 2000;30(Suppl 3): S201-5 (such as SEQ ID NOS: 26-36 herein) may be used, each alone or in any combination.
  • Alpha-synuclein protein or fragments thereof may, for example, be added to the
  • Mycobacterium tuberculosis (TB) challenge peptides used in commercially available interferon- gamma release assays (IGRAs) for detecting M tuberculosis infection, such as the FDA- approved tests QuantiFERON-TB Gold In-Tube (QFT-GIT) (Cellestis/Qiagen, Venlo, Limburg) and T-SPOT.ZB (Oxford Immunotec, Abingdon, UK), to enhance their performance.
  • QFT-GIT QuantiFERON-TB Gold In-Tube
  • T-SPOT.ZB OFxford Immunotec, Abingdon, UK
  • the alpha- synuclein or fragment(s) thereof may, for example, be added to a concentration of 1-40 ⁇ g/ml, or any subrange or value therein, such as 10-30 ⁇ g/ml, in the incubation mixture (blood/cells plus antigens and alpha-synuclein or fragment(s) thereof) of the assays.
  • the antigens used in QFT-GIT and T-SPOT.ZB are selected from the RD1 portion of the TB genome, which is absent from BCG vaccine strains and most commonly occurring non- tuberculosis mycobacteria (NTM). Both QFT-GIT and T-SPOT.7B use ESAT-6 and CFP-10 peptide while QFT-GIT also includes TB 7.7 antigen. Both IGRAs include internal controls, termed the nil and mitogen, in addition to the stimulatory TB peptide antigen. The nil determines the amount of interferon gamma detected after incubation without antigens.
  • the result from the nil control is subtracted from the result after stimulation with the TB antigens to determine the interferon gamma that is attributable to TB.
  • the mitogen control is used to confirm that a test subject's cells are capable of responding to antigen stimulation and that the test was performed correctly.
  • Phytohemagglutinin (PHA) is used as a nonspecific antigen stimulant and failure to respond appropriately suggests an inadequate number of functional effector T-cells or an error in processing the blood or performing the test.
  • QFT-GIT and T-SPOT.TB measure interferon-gamma differently.
  • QFT-GIT employs ELISA that measures interferon-gamma produced in heparinized whole blood after stimulation. Blood is collected into 3 specialized tubes, approximately 1 ml in each of the nil, TB antigen, and mitogen tubes. The tubes are shaken after collection to ensure the antigens dried on the inner surface of the TB and mitogen tubes are adequately mixed with the blood. Tubes are then incubated for 16-24 hours at 37°C. After incubation, the tubes are centrifuged, plasma is extracted, and interferon-gamma levels are measured by ELISA.
  • T-SPOT.ZB uses an enzyme-linked immunospot (ELISPOT) method that determines the quantity of effector T cells responding to antigen stimulation. For most test subjects, 8 mL of heparinized whole blood is adequate to supply enough cells. From the blood, peripheral blood mononuclear cells (PBMCs) are separated, washed, and counted. The PBMCs are then added into microtiter wells at a concentration of 250,000 ⁇ 50,000 PBMCs per well. Each test employs 4 wells: a nil control, a mitogen-containing PHA, and 2 separate wells for ESAT-6 and CFP-10. The microtiter plates are then incubated at 37°C with 5% C0 2 for 16- 20 hours. Released interferon-gamma is captured by specific antibodies on the base of the wells and quantified by a colorimetric enzyme-linked immunoassay.
  • ELISPOT enzyme-linked immunospot
  • the alpha-synuclein or fragment(s) thereof may, for example, be present/added to a concentration of 1-40 ⁇ g/ml, or any subrange or value therein, such as 10-30 ⁇ g/ml, in the incubation mixture (blood/cells plus antigens and alpha-synuclein or fragment(s) thereof) of the assays.
  • the preselected antigens may also, for example, be present/added to a concentration of 1-40 ⁇ g/ml, or any subrange or value therein, such as 10-30 ⁇ g/ml, in the incubation mixture (blood/cells plus antigens and alpha- synuclein or fragment(s) thereof) of the assays.
  • Vessel embodiments of the invention such as blood collection tube and microwell embodiments may include/contain a sufficient amount of alpha-synuclein or fragment(s) thereof and preselected antigen(s) to obtain the aforementioned concentrations when a recommended assay volume of blood or cells is added to and contained by the vessel.
  • a vessel embodiment of the invention having an internal volume of at least 1 ml may contain, such as be internally coated with, 20 ⁇ g recombinant human alpha-synuclein protein and 5-10 ⁇ g total of one or more preselected synthetic peptide antigens, such as but not limited to one or moreM tuberculosis ESAT-6 and/or CFP-10 peptide antigens.
  • At least one preselected antigen and alpha- synuclein protein (or a fragment thereof) and/or HLA protein (or a fragment thereof) refers to one or more antigens that are, at least substantially, not provided by the cell sample itself, i.e., are exogenous with respect to the cells (and added to it), versus various possible antigens that may be endogenous to and already present in the obtained cell sample.
  • the recited alpha- synuclein protein (or a fragment thereof) and/or HLA protein (or a fragment thereof) refers to molecules that are exogenous to the cell sample (and added to it), versus those that may be endogenous to and already present in the obtained cell sample (for example, a fresh blood sample may include a small amount of endogenous alpha-synuclein protein and/or HLA protein).
  • the recited quantity of at least one preselected antigen and quantity of alpha-synuclein protein or fragment thereof recited in the assay method refer to things admixed with the quantity of blood or fraction thereof, not things provided by the quantity of blood or fraction thereof itself.
  • the proteins used in various embodiments of the invention may, for example, be recombinant or may be purified from biological tissue sources, such as blood.
  • the proteins may be at least substantially purified and/or at least substantially pure.
  • at least substantially purified and “at least substantially pure” it is intended is that the recited composition(s) need not be perfectly purified or perfectly pure.
  • a fragment of a protein may, for example, include at least 5, such as at least 10, consecutive amino acids of the amino sequence of the protein but less than the full length sequence of the protein.
  • a fragment of a protein may, for example, comprise consecutive amino sequence of the protein which is less than the full length of the protein, for example, 10-99% of the full length of the protein or any subrange of percentages therein, such as 10-90%, or any percent figures therein that correspond to any of the non-full length
  • Peptide synthesis may also be used to provide smaller fragments of larger proteins or small proteins.
  • Synthetic peptides used in the embodiments of the invention may, for example, be in the range of 5-150 amino acids long, such as 5-40 amino acids long, or any subrange therein or any number of amino acids within said ranges.
  • the synthetic peptides may be 10-30 amino acids long, 10-25 amino acids long, 10-20 amino acids long or 10-16 amino acids long.
  • antigen means a molecule that presents one or more immune epitopes. These epitopes may be recognized by immune cells such as T-cells. Such epitopes, and thus the antigens themselves, may be immune reactivity-promoting (immunostimulatory; pro-effector T-cell) or immune suppression-promoting (immunosuppressive; pro-regulatory T- cell).
  • the assay embodiments of the invention may be used to measure release of cytokines associated with antigen-specific effector T-cell responses, such as interferon-gamma (IFN- gamma), interleukin-6 (IL-6) and interleukin-8 (IL-8), as well as release of cytokines associated with antigen-specific regulatory/suppressive T-cell responses, such as interleukin-10 (IL-10).
  • cytokines associated with antigen-specific effector T-cell responses such as interferon-gamma (IFN- gamma), interleukin-6 (IL-6) and interleukin-8 (IL-8)
  • IFN- gamma interferon-gamma
  • IL-6 interleukin-6
  • IL-8 interleukin-8
  • the immunogen may, for example, be a vaccine immunogen.
  • the vaccine immunogen may, for example, be an infectious disease immunogen or a tumor/cancer antigen vaccine immunogen.
  • the infectious disease immunogen may, for example, be a vaccine immunogen against a cellular or viral pathogen, may for example be a live or killed/inactivated form of the pathogen or a derivative/extract thereof, and/or may for example include or consist of one or more purified antigens such as synthetic antigen molecules for the pathogen, such as synthetic peptides or recombinant proteins.
  • the tumor/cancer vaccine immunogen may, for example, include or consist of cancer cells, parts of cancer cells, or pure tumor/cancer antigens isolated from the cells or produced synthetically, such as, without limitation, synthetic peptides or recombinant proteins.

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Abstract

L'invention concerne des compositions pharmaceutiques et non pharmaceutiques immunomodulatrices qui comprennent de l'alpha-synucléine et au moins un antigène présélectionné, tel qu'au moins un antigène ou immunogène peptidique présélectionné. L'invention concerne également des procédés de modulation de l'activité immunitaire vers au moins un antigène présélectionné d'une manière au moins sensiblement spécifique d'un antigène qui comprennent l'administration d'une telle composition à un patient humain ou à un sujet mammifère non humain. L'invention concerne en outre des procédés de dosage améliorés pour quantifier des réponses cellulaires spécifiques d'un antigène, telles qu'une libération de cytokine, à des antigènes présélectionnés.
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