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WO2007054658A1 - Contrôle de réponses immunitaires - Google Patents

Contrôle de réponses immunitaires Download PDF

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Publication number
WO2007054658A1
WO2007054658A1 PCT/GB2006/000916 GB2006000916W WO2007054658A1 WO 2007054658 A1 WO2007054658 A1 WO 2007054658A1 GB 2006000916 W GB2006000916 W GB 2006000916W WO 2007054658 A1 WO2007054658 A1 WO 2007054658A1
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WO
WIPO (PCT)
Prior art keywords
hsp70
peptide
tnf
cells
peptides
Prior art date
Application number
PCT/GB2006/000916
Other languages
English (en)
Inventor
Thomas Lehner
Charles Kelly
Yufei Wang
Original Assignee
King's College London
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0523243A external-priority patent/GB0523243D0/en
Priority claimed from GB0523242A external-priority patent/GB0523242D0/en
Application filed by King's College London filed Critical King's College London
Publication of WO2007054658A1 publication Critical patent/WO2007054658A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to peptides and fragments thereof, methods for producing such peptides and uses of such peptides, especially in the control of immune responses.
  • TNF-a The significance of TNF-a has emphasised by the therapeutic trials with anti-TNF- ⁇ : antibodies in rheumatoid arthritis and Crohn's disease carried out in many centres. TNF-o; is central among numerous other cytokines in the control of autoimmune diseases.
  • TNF-ce Antibodies to TNF-ce have been successfully used to treat acute episodes of rheumatoid arthritis, Crohn's disease, ankylosing spondylitis, or uveitis.
  • repeated administration of TNF- ⁇ antibodies induce corresponding antibodies, with loss of clinical efficacy, the development of serum sickness-like reaction, antibodies to double-stranded DNA and lymphomas (Bell and Kamm 2000). Allograft and xenograft acceptance have also been significantly prolonged by antibodies to CD40L.
  • CD40L antibodies, so with CD40L, antibodies are elicited.
  • the inventors aimed to prevent the production of TNF-O! (and IL- 12), instead of neutralising TNF- ⁇ : once it has been formed and is contributing to the immunopathogenesis of a disease.
  • Chemokines such as RANTES, play an essential role in attracting macrophages, immature DC, T, B and NK cells (Sozanni et al 1996, Kim et al 1998) and these may initiate the pathogenesis, as well as maintain autoimmunity.
  • the inventors also aimed to control as many of these components of the immune response as possible.
  • CD40 functions as a receptor not only to CD40L but also to the 7OkD mycobacterial heat shock protein (HSP70) (Wang et al 2001). Indeed, both CD40L and the C-terminal portion of HSP70 stimulate CD40 + monocytes and dendritic cells to produce IL- 12, TNF- ⁇ , and chemokines. The inventors have surprisingly found that these cytokines and chemokines can be inhibited.
  • the inventors have found it possible to block the critical costimulatory pathways in monocytes or dendritic cells between CD40 and CD40L or CD40 and HSP70. Without being bound a particular theory, it is suggested that the peptide blocking agents bind to the receptor but do not activate it.
  • an isolated peptide comprising an amino acid sequence having substantial homology selected from the group consisting of: a) KEETKKENSFEMQKGDQN; b) FTSFGLLKL; c) SLSQPGQKLVSDSTEFTETE; d) REKQYLINSQ; e) IVHVTAKDKGTGKENTIRIQ; f) EGSGLSKEDIDRMIKDAEAH; g) QPSVQIQVYQGEREIAAHN;and h) IVHVTAKDKGTGKENTIRIQEGSGLSKEDIDRMIKDAEA H;
  • a peptide is a molecule comprising a chain of amino acids.
  • the term peptide encompasses peptides of any size, but preferably refers to peptides of between 10 and 200 amino acids in length. The term does not exclude modifications of peptides such as glycosylation, acetylation and phosphorylation.
  • the definition also includes peptides containing amino acid analogues and unnatural amino acids.
  • the definition also includes peptides comprising the levorotary or dextrorotary amino acids.
  • substantial homology preferably means at least 80% homology, more preferably at least 85% homology, even more preferably at least 90% homology, most preferably at least 95% homology.
  • the peptides of the invention may comprise amino acid sequences including minor modifications from the amino acid sequences that have been specifically defined. Typically, the minor modifications are conservative, including, for example, replacement of one or more amino acids with one or more similar amino acids, such as those having similar side chains or characteristics. For example, a hydrophilic amino acid is likely to be replaced with another hydrophilic amino acid.
  • a functional fragment is a portion of a peptide that functions in the same way as the whole peptide.
  • a functional fragment of a peptide of the invention modulates the immune response in the same way as the whole peptide.
  • the peptide comprises an amino acid sequence having substantial homology to a sequence selected from: a) KEETKKENSFEMQKGDQN; b) FTSFGLLKL; c) SLSQPGQKLVSDSTEFTETE;and d) REKQYLINSQ.
  • the peptide comprises an amino acid sequence having substantial homology to a sequence selected from: e) IVHVTAKDKGTGKENTIRIQ; f) EGSGLSKEDIDRMIKDAEAH; g) QPSVQIQVYQGEREIAAHN;and h) IVHVTAKDKGTGKENTIRIQEGSGLSKEDIDRMIKDAEA H.
  • the peptide of the invention may comprise an amino acid sequence having substantial homology to one or more of the defined sequences. It may consist of that sequence or may comprise additional amino acids. Alternatively, the peptide may consist of one of the above specifically defined amino acid sequences.
  • Peptides according to the invention may be made by any known means.
  • peptides may be synthesised chemically. ' Alternatively they may be produced by using a cell to express the peptides.
  • the invention also provides a nucleic acid sequence coding for a peptide according to the invention.
  • the invention also provides a vector containing such a nucleic acid sequence and a cell transformed with such a vector.
  • the nucleic acid sequence can be synthesized using any standard synthesis method.
  • the present invention also provides an expression vector comprising the nucleic acid of the present invention.
  • Expression vectors are well known for expressing nucleic acids in a variety of different organisms, including mammalian cells, insect cells, bacteria and eukaryotic microorganisms such as yeasts. All such expression vectors are well known to those skilled in the art and the use of expression vectors in order to express the nucleic acid sequence is a standard technique well known to those skilled in the art.
  • the expression vector is a baculovirus expression vector.
  • the expression vector of the present invention comprises a promoter and the nucleic acid molecule of the present invention.
  • the vector leads to the production of the peptide of the present invention. It is further preferred that the vector comprises any other regulatory sequences required to obtain expression of the nucleic acid molecule.
  • the present invention also provides a host cell transformed with the vector of the present invention.
  • the host cell is a eukaryotic cell, more preferably a mammalian cell, such as Chinese hamster ovary (CHO) cells, HPMCs, HeLa cells, baby hamster kidney (BDH) cells, cells of hepatic origin such as HepG2 cells, and myeloma or hybridoma cell lines.
  • the host cells is a prokaryotic cell such as E. coli.
  • the present invention further provides a method for producing the peptide of the present invention comprising transfecting a host cell with the vector of the present invention, culturing the transfected host cell under suitable conditions in order to lead to the expression of the nucleic acid molecule and production of the peptide of the present invention.
  • the peptide may then be harvested from the transfected cells or from the cell growth media, depending on whether the peptide is secreted, using standard techniques.
  • a peptide or nucleic acid according to the invention for use in therapy.
  • the invention provides the use of a peptide according to the invention in the preparation of a medicament to modulate an immune response.
  • the peptides according to the invention are particularly useful because they can modulate the immune response by causing increased or decreased production of cytokines, especially TNF- a, IL- 12, RANTES, MIP- l ⁇ and MIP-I ⁇ . Whether the production of cytokines is increased or decreased depends on the peptide used.
  • Cytokines are proteins that mediate the induction and regulation of the immune system. They have a variety of actions, including initiation of inflammatory responses, and activation of inflammatory cells. They also act on lymphocytes, stimulating growth, activation and differentiation. Cytokines are secreted by a range of cells, including activated lymphocytes and macrophages. They also have a wide range of target cells. For example, Interleukin-12 is secreted by B cells and macrophages, and acts on activated T cells, natural killer (NK) cells and Lymphokine-activated killer (LAK) cells. Cytokines may be subdivided into groups such as lymphokines, monokines and chemokines.
  • Chemokines are proteins that have chemoattractant and proinflammatory properties i.e. they recruit cells required for an immune response. They are produced by a variety of cell types including natural killer (NK) cells as well as antigen presenting cells such as macrophages and dendritic cells. Chemokines exert function by binding to cell surface receptors that are members of the seven transmembrane domain G protein-coupled receptors.
  • the ⁇ - chemokines RANTES, MlP-l ⁇ and MIP-1/3 bind to the CCR5 receptor and attract antigen processing and presenting macrophages, dendritic cells (DC) and effector T and B cells.
  • the peptides of the invention have been found to modulate cytokine and/or chemokine production. This means that the peptides cause cells to produce either more or less cytokines than would normally be produced by those cells in that situation (depending on the specific peptide). For example, cells from a section of mucosa from a patient with Crohn's disease treated with the peptides of the invention will produce more or less chemokines and/or cytokines than cells from that mucosa that have not been treated with the peptides.
  • the level of cytokine and chemokine production is preferably significantly different from normal levels (in statistical analysis, p is preferably 0.1 or less, more preferably 0.075 or less, even more preferably 0.05 or less).
  • Ih particular, for example, there is preferably a change in production of at least 10%, more preferably at least 20%, more preferably at least 30%, most preferably at least 40%.
  • the production of cytokines and/or chemokines may be analysed by any method known in the art. Example methods are provided in the detailed description.
  • DC Dendritic cells
  • Maturation of DC may be identified by the presence and/or increased expression of markers on mature DC that are not present or are only present in small numbers on immature DC, such as CD83, CD80, CD86 and CD40. The presence of the markers may be analysed by any known method. Mature DC also produce cytokines and/or chemokines that are not produced by immature DC.
  • the peptides of the invention have been found to modulate DC maturation. This means that the peptides promote or inhibit DC maturation, when compared with normal DC maturation in the same circumstances.
  • the level of DC maturation is preferably significantly different (in statistical analysis, p is preferably 0.1 or less, more preferably 0.075 or less, even more preferably 0.05 or less). In particular, for example, there is preferably a change in maturation of at least 10%, more preferably at least 20%, more preferably at least 30%, most preferably at least 40%.
  • peptides according to the invention such as those comprising an amino acid sequence substantially homologous to the following sequences: a) KEETKKENSFEMQKGDQN; b) FTSFGLLKL; c) SLSQPGQKLVSDSTEFTETE; d) REKQYLINSQ; e) IVHVTAKDKGTGKENTIRIQ; f) EGSGLSKEDIDRMIKDAEAHand h) IVHVTAKDKGTGKENTIRIQEGSGLSKEDIDRMIKDAEA H have an inhibitory effect on immune responses.
  • these peptides may be used to inhibit dendritic cell maturation and/or cytokine and/or chemokine production, and may be used in the production of medicaments for these functions and for the prevention, treatment or suppression of autoimmune diseases or any other situation in which it is desirable to inhibit, prevent or suppress an immune response, such as to help prevent graft or transplant rejection.
  • autoimmune diseases are diseases caused by an appropriate reaction of the body's immune system against its own cells.
  • the term autoimmune disease is well known in the art and includes diseases such as Crohn's disease, arthritis and uveitis. In one embodiment of the invention the autoimmune disease is Crohn's disease.
  • QPSVQIQVYQGEREIAAHN are particularly useful for increasing immune responses and increasing cytokine and/or chemokine production and dendritic cell maturation.
  • the peptides of the invention may have an increased effect when administered in conjunction with a heat shock protein or a fragment thereof, or the CD40 ligand (CD40L) or a fragment thereof.
  • a heat shock protein is a protein which exhibits increased expression in a cell when the cell is subjected to stress.
  • the term heat shock protein is well known in the art.
  • Various heat shock proteins are known, such as HSP70, HSP65, HSP 40, HSP27, BiP, GP96, HSP60, HSP90 and HSP96.
  • the CD40L is the ligand for the CD40 receptor. It is well known in the art.
  • the medicament prepared by the use of the peptides of the invention may be for administration simultaneously, sequentially or separately from a heat shock protein or fragment thereof or CD40L or fragment thereof.
  • the medicament may additionally comprise a heat shock protein or CD40L or a fragment of either.
  • the peptides of the invention may be administered in isolation or in combination. When administered in combination, the peptides may be administered simultaneously, sequentially or separately. Accordingly, the medicament may comprise more than one peptide of the invention or may be arranged to be administered with another protein.
  • the peptides may also be administered in conjunction with any other treatment for the disease in question, such as TNF- ⁇ antibodies.
  • composition comprising a peptide or nucleic acid according to the invention.
  • the pharmaceutical composition may also include a carrier, adjuvant or vehicle.
  • Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical composition of this invention include, but are not limited to, ion exchangers, alumina, aluminium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilcate, polyvinyl pyrrolidone, celluose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminium stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such
  • the pharmaceutical composition of this invention may be administered orally, parenterally, by inhalation spray, or via an implanted reservoir.
  • the pharmaceutical or diagnostic composition is administered parenterally by injection.
  • the pharmaceutical or diagnostic composition of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical composition may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example, as a solution in 1,3 butanediol.
  • suitable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • oils such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcoholdiluent or dispersant such as Ph. HeIv or a similar alcohol.
  • the pharmaceutical composition of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions.
  • carriers which are commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried corn starch.
  • aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavouring and/or colouring agents may be added.
  • compositions of this invention may be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known in the art or pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fmorocarbons, and/or other solubilizing or dispersing agents known in the art.
  • the pharmaceutical composition may additionally comprise a heat shock protein, a CD40L or a fragment of either.
  • the pharmaceutical composition may also comprise more than one peptide of the invention.
  • a method of preventing, reducing or suppressing an immune response, inhibiting cytokine or chemokine production, inhibiting dendritic cell maturation or treating an autoimmune disease comprising administering one or more peptides comprising an amino acid sequence having substantial homology to a sequence selected from the group consisting of: a) KEETKKENSFEMQKGDQN; b) FTSFGLLKL; c) SLSQPGQKLVSDSTEFTETE; d) REKQYLINSQ; e) IVHVTAKDKGTGKENTIRIQ; f) EGSGLSKEDIDRMIKDAEAH;and h) IVHVTAKDKGTGKENTIRIQEGSGLSKEDIDRMIKDAEA H, or a functional fragment thereof, or a nucleic acid encoding such a peptide, or a pharmaceutical composition comprising such a peptide to a subject in need thereof.
  • Also provided is a method of increasing an immune response comprising administering a peptide comprising an amino acid sequence having substantial homology to the sequence: g) QPSVQIQVYQGEREIAAHN or a functional fragment thereof, or a nucleic acid encoding such a peptide, or a pharmaceutical composition comprising such a peptide to a subject in need thereof.
  • the peptides of the invention may be administered in isolation or in combination. When administered in combination, the peptides may be administered simultaneously, sequentially or separately.
  • the peptides may also be administered in conjunction with a heat shock protein, CD40L or a fragment of either of these.
  • Fig.l is a comparative stimulation of monocytes and DC with 3 HSP70 preparations.
  • Fig. 2 shows identification of a mHSP70 stimulating epitope and the critical residues.
  • Fig. 3 shows identification of a stimulating epitope within peptide 387-426 and inhibiting epitope within peptide 457-496 using DC costimulated with HSP70 and with two adjacent or overlapping 20mer peptides.
  • B Illustration of the stimulating epitope p407-426 at the base of L3, 4 and L4, 5 and the suppressive epitope (p457-496) in the beta-sheet.
  • Fig. 4 shows specificity of the suppressor epitope p457-496.
  • C and D Dose-dependent inhibition of DC by the 40mer peptide 457-496 of HSP70, CD40LT or LPS stimulated production of TNF-alpha and IL-12, and
  • E the effect of peptide binding motif NRLLLTG on HSP70 or HSP70 (359-494) stimulation of DC to produce IL-12.
  • Fig. 5 shows the effect of co-stimulation of dendritic cells with HSP70, CD40LT or LPS and peptides 407-426 or 457-496 on the expression of CD83 and CCR7; without peptide, with 407-426, or .... 457-496 peptide by flow cytometry showing percent and MFL.
  • Fig. 5 shows the effect of co-stimulation of dendritic cells with HSP70, CD40LT or LPS and peptides 407-426 or 457-496 on the expression of CD83 and CCR7; without peptide, with 407-426, or .... 457-496 peptide by flow cytometry showing percent and MFL.
  • FIG. 6 shows the effect of peptides 407-426 and 457-496 on p38 MAP kinase phosphorylation stimulated by HSP70, CD40L or LPS by ELISA of lysed dendritic cells, (A) by ELISA and (B) by Western blotting ; (C) CD40 transfected HEK 293 cells and (D) HEK 293 cells alone.
  • Fig. 7 shows the effect of ⁇ 38 MAP kinase inhibitor (SB203580) on (A) production of IL-12 by DC after treatment with HSP70 (1 ⁇ g/ml), CD40LT (1 ⁇ g/ml) or LPS (200 ng/ml) and (B) Maturation of DC evaluated by expression of MHC Class II two days after treatment with HSP70 (20 ⁇ g/ml), CD40LT (2 ⁇ g/ml) or LPS (500 ng/ml) in the presence and absence of p38 inhibitor (25 ⁇ m)
  • FIG. 11 A novel strategy for inhibition of CD40-CD40L or CD40-HSP70 interaction by peptides derived from HSP70 or CD40 and CD40L, stimulating production of TNF- ⁇ , IL-12 and CCL-5.
  • Table 1 shows a comparison of stimulation of DC with the 3 HSP70 preparations on maturation of DC and IL-12p40 production with LPS.
  • A The effect of stimulation of immature DC by HSP70, the C-terminal fragment (aa359-610), peptide binding fragment (aa359-494), CD40LT or LPS on the CD83 and CCR7 maturation phenotypes of DC.
  • B A dose-dependent effect of LPS on HSP70 (20/Ag), HSP70 359-494 (5 ⁇ g/ml) or CD40LT (lO ⁇ g/ml) stimulating production of IL-12p40 by DC.
  • Table 2 shows the effect of p407-426 on maturation of DC.
  • Table 3 shows the effect of ⁇ 457-496 on maturation of DC.
  • Table 5 Stimulation of IL- 12 production by HSP70, CD40L or LPS of mononuclear cells eluted from colonic biopsy specimens from patients with Crohn's disease or ulcerative colitis, and inhibition of HSP70 stimulated cells with ⁇ 457-496 or pCD40/CD40L.
  • the peptides according to the invention were developed from fragments of CD40, CD40L and HSP70, as follows:
  • KEETKKENSFEMQKGDQN is an 18 residue peptide found at amino acids 102-119 in CD40L
  • FTSFGLLKL is found at residues 253-261 of CD40L and shows homology to a hydrophobic motif of HSP70.
  • SLSQPGQKLVSDSTEFTETE is a 20 amino acid peptide found at residues 39-58 in. CD40. In the natural sequence, C replaces S at positions 41 and 49.
  • IVHVTAiCDKGTGKENTIMQEGSGLSKEDIDRMIKDAEAH is found at 457-496 in HSP70, This peptide may be divided in two, forming fragments 457-476 and 477-496.
  • QPSVQIQVYQGEREIAAIiNK is also from HSP70. p407-426.
  • PBMC peripheral blood mononuclear cells
  • HSP70 C-terminal fragment The effect of HSP70 C-terminal fragment on the production of the chemokine RANTES and cytokines IL-12 and TNF- ⁇ show a dose-dependent increase in their concentrations.
  • the monocytes are treated with GM-CSF (400 U/ml) and IL-4 (100 U/ml) for 5 days, which converts them to dendritic cells (CD83 + , CD80 + , CD86 + , CD40 + , CCR7 + , HLA-DR high cells) and these are then treated with HSP70 or CD40L, as the monocytes above.
  • Mycobacterial HSP70 and the C terminal fragment comprising residues 359-610 of HSP70 were supplied by Dr Mahavir Singh, Lionex GmbH, Braunschweig, Germany.
  • the materials are expressed in E. coli using the plasmid pJLA and purified by affinity chromatography using ATP-agarose for HSP70 and a Ni 2+ -agarose resin for HSP70 359-610.
  • Peptide amides were synthesised using the multiple peptide synthesis block, BT7400 (Biotech Ltd, UK) by standard Fmoc chemistry with PyBOP (benzotriazol-1-yl-oxy-tris- pyrrolidino-phosphonium hexafluorophosphate) as coupling agent and Rink amide MBHA (methylbenzhdrylamine) resin. Peptide amides were cleaved from the resin and protecting groups removed by incubation with TFA (trifluoroacetic acid). Peptide amides were recovered by precipitation with ether, dissolved in 1OmM NH 4 HCO 3 and lyophilised.
  • HSP70-derived peptide 457-496, a peptide derived from the CD40 ligand extracellular domain (aa 102-119) and peptides from the N terminal tandem repeat of CD40 (aa 27-36 and 39-58) were synthesised and studied for their effects on HSP70 stimulation of cytokines and chemokines.
  • PBMC (3xlO 6 /ml) or purified monocytes (2xlO 5 /ml) from either patients with Crohn's disease or normal controls are stimulated with HSP70 (20 ⁇ g/ml).
  • the cells are stimulated with both CD40LT (5 ⁇ g/ml) and HSP70 (5 ⁇ g/ml) which have a synergistic effect on stimulation of monocytes.
  • CD40LT 5 ⁇ g/ml
  • HSP70 5 ⁇ g/ml
  • the peptides were added at concentrations ranging from 0.5-100 ⁇ g/ml.
  • the supernatants are collected from the cultures on days 1, 2 and 3 and stored at -7O 0 C before assaying the cytokines and chemokines.
  • CC chemokines the supernatant obtained from monocytes and PBMC are diluted (x5), estimated by ELISA and the concentrations calculated against standard recombinant CC chemokines and expressed in pg/ml.
  • cytokines IL-12 and TNF- ⁇ supernatants are diluted (x2) and the concentrations of the cytokines expressed in pg/ml.
  • Specific ELISA antibody kits are purchased from R&D System (Oxon, UK) for CC chemokines and from Pharmingen (UK) for IL-12 and TNF- ⁇ .
  • HSP70-derived peptide 457-496 inhibited monocyte derived dendritic cells stimulated with HSP70 the production of IL-12 and TNF- ⁇ .
  • CD40L peptide derived from the sequence of CD40L showed a dose-dependent inhibition of RANTES in CD40 + THPl monocytes stimulated with HSP70 which was greater than that of a Mab to CD40. Furthermore, surface plasmon resonance binding of HSP70 or CD40LT (trimer) to CD40 transfected HEK 293 cells was inhibited with the CD40L peptide. Thus, functional production of RANTES and the receptor-ligand interaction between CD40 and HSP70 or CD40LT can be significantly inhibited by the CD40L peptide.
  • a combination of 2 or 3 of these peptides that block (a) HSP70 and CD40L, (b) HSP70 and CD40, (c) CD40L and CD40 or (d) all 3 molecules may elicit additive or synergistic inhibition of production of the chemokines and cytokines.
  • Inhibition of production of chemokines and cytokines may be elicited not only by mononuclear cells isolated from blood but also those eluted from mucosal tissues, such as the intestinal mucosa.
  • the choice of the route and mode of administration will depend on the disease to be treated.
  • the peptides can be administered as a solution directly to the gut mucosa (e.g. in Crohn's disease) by oral or rectal application, or by eye drops (in uveitis), or directly injected into a joint (in rheumatoid arthritis) or by systemic injection (EVI, SC or IV) in other autoimmune diseases or in tissue or organ transplantation.
  • the benefit of the invention is that the peptides will prevent the production of TNF- ⁇ , IL- 12, RANTES, MIP-lo: and MIP-IjS thereby preventing their pathogenic effects, instead of the current regime of administration of antibodies to TNF- ⁇ that neutralise already formed TNF- ⁇ .
  • the peptides may be administered by mouth, rectum, to the eye or nasally, in addition to their systemic administration.
  • the peptides may prevent or inhibit exacerbations of the autoimmune diseases or may have lasting suppressive effect on the disease and may prevent rejection of organ or tissue transplantation.
  • the peptides may have a beneficial effect in acting as an immunosuppressive sparing agent, thereby diminishing its side effects.
  • the invention described above may be defined in the following points, but is not limited to these points :- 1) The use of a peptide blocking agent to block CD40/CD40L binding or CD40/HSP70 binding, thereby inhibiting the formation of cytokines or chemokines which promote autoimmune disease or graft rejection.
  • a method of preventing, treating, or suppressing autoimmune disease or graft rejection which comprises the use of a peptide blocking agent according to any of points 1 to 4 or a combination of peptides as specified in point 5.
  • the invention also comprises blocking agents as specified in the above points, as compounds per se and in compositions suitable for administration to patients suffering from or susceptible to the diseases specified.
  • mHSP70 The 7OkD microbial heat shock protein (mHSP70) has a profound effect on the immune system, interacting with the CD40 receptor on DC and monocytes to produce cytokines and chemokines.
  • the mHSP70 also induces maturation of dendritic cells (DC) and thus acts as an alternative ligand to CD40L on T cells.
  • DC dendritic cells
  • cytokine stimulating epitope peptide 407-426
  • This peptide also significantly enhances maturation of DC stimulated by mHSP70 or CD40L.
  • the epitope is located at the base of the peptide binding groove of HSP70 and has 5 critical residues.
  • an inhibitory epitope (p457-496) was identified downstream from the peptide binding groove that inhibits cytokine production and maturation of DC stimulated by HSP70 or CD40L.
  • the p38 MAP kinase phosphorylation is critical in the alternative CD40-HSP70 pathway and is inhibited by ⁇ 457-496 but enhanced by p407-426.
  • Heat shock proteins are intracellular chaperones, present in most mammalian cells and microorganisms. Microbial HSP are of considerable interest in stimulating the production of cytokines and chemokines, and inducing maturation of DC. By virtue of these functions, they have adjuvant properties when administered with antigens by the systemic or mucosal routes. Some but not all of these properties are shared with the homologous mammalian HSP which are intracellular molecules and play an essential role as chaperones of proteins.
  • An important property of microbial HSP70 is their ability to present external proteins to HLA class II and cross-presentation to HLA class I pathways. Furthermore, the concept that the HSP groove may bind critical tumour peptides has led to protection in the development of tumours, by immunization with peptide bound HSP70 and HSP95 purified by ADP chromatography from tumour cells.
  • HSP70 like human (hu) HSP70 consists of three functionally distinct domains, an N- terminal 44 kDa ATPase portion (aal-358), followed by an 18 kDa peptide binding domain (aa359-494) and a C-terminal 1OkDa fragment (aa495-609).
  • Immunological functions of the three different domains in stimulating monocytes and DC have not been fully defined. However, the C-terminal portion (359-610) stimulates production of CC-chemokines, IL-12, TNF-alpha, and NO, induces TH-I polarization and maturation of DC and functions in vivo as an adjuvant.
  • the ATPase domain of mHSP70 largely lacks these functions and removal of the ATPase domain enhances HSP70 stimulation in the production of cytokines, chemokines and maturation of DC.
  • a sequence required for CTL stimulation following immunization with an ovalbumin-mHSP70 fusion protein was identified in the ATP-ase portion, as well as a suppressor epitope stimulating production of IL-IO and TGF-alphal.
  • HSP70 and APC Interaction between HSP70 and APC is receptor mediated.
  • CD 14 and TLR4 are implicated in binding HSP60/65 and were also reported as receptors for huHSP70, both mHSP70 and huHSP70 bind CD40, but utilize different sites of the molecule.
  • a major costimulatory pathway of interaction is that between CD40 on APC and CD40 ligand on T cells, the finding that mHSP70 binds and stimulates CD40 + cells to produce chemokines and cytokines may be important in the interphase between innate and adaptive immunity.
  • the significance of HSP70 binding and activating the CD40 costimulatory pathway has now been confirmed in vivo.
  • mature DC are involved in immunogenicity and immature or steady state DC in tolerance.
  • the aim of this study was firstly to identify an epitope within mHSP70 which is responsible for the stimulating functions of HSP70, in terms of cytokine and chemokine production and maturation of DC.
  • mHSP70 which is responsible for the stimulating functions of HSP70, in terms of cytokine and chemokine production and maturation of DC.
  • cytokine and chemokine stimulating functions and maturation of DC reside in the peptide binding portion of HSP70 (aa 359-494). Stimulation of monocytes or DC in vitro with peptide 407-426 generates IL-12, TNF-alpha and CCL-5, and enhances maturation of DC.
  • peptide 457-496 inhibits production of cytokines, chemokines and maturation of DC stimulated with HSP70 or CD40L. Consistent with these functions, investigation of phosphorylation by signalling p38 MAP kinase phosphorylation pathway revealed that p407-426 enhanced, whereas p457-496 inhibited phosphorylation in HSP70 or CD40L stimulated maturation and IL-12 production by DC.
  • Soluble CD40 ligand trimer (CD40LT) was kindly donated by Dr. F. Villinger (Atlanta, Georgia).
  • Human recombinant GM-CSF (Leucomax) was obtained from Sandoz Pharmaceuticals (Surrey, UK) and human recombinant IL-4 from R&D System (Abingdon, UK).
  • the fluorochrome-conjugated monoclonal antibodies to CCR7, CXCR4, CD14 and CD40 were purchased from hnmunotech (Oxford, UK).
  • the antibodies to human CD83 and control murine monoclonal antibody isotypes were obtained from Serotec (Oxford, UK) and DAKO (Ely, UK).
  • HSP70 Preparation of microbial HSP70 and its fragments.
  • HSP70, HSPTO 359-610 and HSP70 359-494 were prepared from the Escherichia coli pop strain using the pJLA603 vector (34). Cloned inserts were verified by DNA sequence analysis. HSP70 was expressed with no additional amino acid residues whereas HSP7 ⁇ 359-6 io and HSP70 359-494 were expressed with a C-terminal
  • HSP70 was purified by ion exchange chromatography using Q-Sepharose resin followed by ATP-affinity chromatography.
  • HSP7O 359-61 o and HSP70 359-494 recombinant polypeptides were prepared by affinity chromatography using Ni 2+ - chelating resin, and identity of the polypeptides was confirmed by N-terminal sequence analysis (10 cycles each).
  • the HSP preparations were further treated with polymixin B-coated beads (Sigma-Aldrich,
  • the LPS content of the HSP preparations was determined by the Limulus amebocyte lysate assay (Sigma-Aldrich), and showed ⁇ 0.006 U/ ⁇ g of HSP70 or 5 pg/1 ⁇ g of the HSP preparation.
  • HSP70 and fragments were synthesised by standard Fmoc chemistry (as described below) and included a "spacer" sequence C-terminal to the HSP70-binding motif and a C-terminal Cys for coupling.
  • the peptide was coupled (approximately 3,000 resonance units) to the surface of flow cell 2 of a CM-5 sensorchip (Biacore International SA, Meadway Technology Park, Stevenage, Herts, U.K.) using the thiol coupling procedure as described in the manufacturer's instructions.
  • Flow cell 1 served as a non-interacting reference surface.
  • Binding of fluid-phase HSP70, HSP70 359-6 i 0 and HSP70 359-494 were measured over a range of concentrations (50 -200 nM), using BIAcore X.
  • Buffer was 0.15M NaCl, 0.005% (v/v) Tween 20 in 0.01M HEPES, pH 7.4 (HBS).
  • the flow rate was 5 ⁇ l/min and the injection volume was 20 ⁇ l.
  • the surface was regenerated by injection of a pulse (5 ⁇ l) of 2M KSCN.
  • Human monocytic THPl cell line was obtained from the MRC (NIBSC, Porters Bar, UK). The nonadherent THPl cells were maintained on RPMIl 640 medium supplemented with 10% foetal calf serum (FCS), 2 niM glutamine, of 100IU/ml penicillin and 100 ⁇ g/ml streptomycin.
  • FCS foetal calf serum
  • Human primary monocytes were isolated from peripheral blood mononuclear cells (PBMC) prepared from healthy donors by centrifugation on a Ficoll-Hypaque density gradient (Amersham Biosciences, Little Chalfont, UK).
  • PBMC peripheral blood mononuclear cells
  • the CD14 + monocytes were enriched by depletion of CD14 " cells using Monocyte Isolation Kit (MACS 5 Miltenyi Biotec, Surrey, UK). The purity of isolated monocytes was consistently greater than 90% when analysed by fiow- cytometry with antibody to CD 14.
  • Human dendritic cells were generated by culturing monocytes with GM-CSF (400 U/ml) and IL-4 (100 U/ml) for 5 days (35). These monocyte- derived DC were generally considered to be immature DC, defined by surface expression of DC markers CD83, CD80, CD86, CD40 and were CD14 negative.
  • CC-chemokine CCL5, RANTES
  • cytokines TNF-alpha and IL-12 Human monocytes (lxlO 6 /ml) or monocyte derived DC (2xlO 5 /ml) were incubated with HSP70, HSP70 (359-610) or HSP70 (359-494) at concentrations of 0.005-0.5 ⁇ M in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 100IU/ml of penicillin and 100 ⁇ g/ml streptomycin.
  • DC were cultured in RPMI medium, conditioned with 400U/ml GM-CSF and 100U/ml of IL-4. After 48 hours incubation, the supernatant was used to assay the chemokine CCL-5 and the cytokines IL-12 and TNF-alpha.
  • the fragments or LPS was incubated with 100 ⁇ g of proteinase K-agarose beads (equivalent to 0.005 enzyme U) in a total volume of 100 ⁇ PBS buffer for 1 hour at 37 0 C.
  • the proteinase K beads were removed by centrifugation and the digested supernatants were collected and 3 concentrations of each were used to stimulate DC. After stimulation for 2 days the DC culture supernatants were collected and assayed for TNF-alpha or IL-12.
  • DC were stimulated with mHSP70 (20/ ⁇ g/ml) HSP70 359 - 494 (5/ig/ml) or CD40LT (10jUg/ml) in the presence of increasing concentrations of LPS (lOpg to lOOng/ml).
  • LPS LPS
  • Peptides were recovered by precipitation with ether, washed three times with ether and dried. The peptides were subsequently dissolved in 1OmM acetic acid or 1OmM NH 4 HCO 3 and lyophilised. Using the same procedure, a further panel of peptides was prepared in which each amino acid residue within the sequence of the identified stimulating peptide 407-426 was in turn substituted by alanine. Selected peptides were analysed by mass spectroscopy to confirm the sequence. The inhibitory 40 mer peptide 457-496 was synthesized by Bachem Ltd (Switzerland) to a purity of 93.7%, determined by high pressure liquid chromatography (Mr 4392.6).
  • the 20-mer peptides were used first singly and then by combining two adjacent 20-mers or using the single 40-mer in concentrations of 50 ⁇ g/ml with 10 ⁇ g/ml HSP70, 10 ⁇ g/ml CD40LT, or 500 ng/ml LPS. These were added to DC or monocytes, and after 3 days of culture TNF- ⁇ , IL-12, or CCL-5 was assayed in the supernatants.
  • the critical residues within p407-426 were identified by alanine substitution of each of the 20 N-terminal residues of 407-426, and these peptides were then used to stimulate DC to produce TNF- ⁇ and IL-12.
  • Monocyte-derived DC were prepared as above in 96-well flat-bottom tissue culture plates. After incubation for 5 days, 50 ⁇ l of medium with 4 or 20 ⁇ g/ml each of HSP70, HSP 359-610 , HSP 359-494 , or 0.2 or 2 ⁇ g/ml CD40LT or 5 or 50 ⁇ g/ml LPS were added. In another experiment, immature DC were treated with 50 ⁇ g/ml HSP peptide (407-426 or 457-476/477- 496 or p457-496). After 30 min. 10 ⁇ l of medium with HSP70, DC40LT, or LPS were added.
  • the cells were incubated for 2 days, before harvesting into 12- x 75-mm tubes, using PBS with 0.02% EDTA to help release of adherent cells.
  • the isolated cells were stained with fluorochrome-conjugatedAbs to DC markers or conjugated mouse IgG isotype as a control, for 30 min on ice. After two washings, the cells were fixed in 1% formaldehyde-PBS solution before analysis by flow cytometry. The cells were examined on a Coulter XL-MCL flow cytometer, and the data were analyzed using Win MDI software.
  • HSP70-binding motif NRLLLTG Effect of the HSP70-binding motif NRLLLTG on the production of TNF- ⁇ or IL-12.
  • NRLLLTG synthetic peptide, which has high affinity binding for HSP70.
  • Human monocytes or DC cultures were stimulated with 10 ⁇ g/ml HSP70 or 5 ⁇ g/mlHSP70 359-494 , in the presence of 5-50 ⁇ g/ml NRLLLTG. Two days after culture, the concentration of TNF- ⁇ or IL-12 was assayed in the supernatants.
  • the CC-chemokine CCL-5 in the culture supernatants was assayed using specific ELISA paired antibody (R&D System, Oxon, UK).
  • IL-12 p40 and TNF-alpha in the culture supernatants were detected using paired specific antibodies to IL12 p40 or to TNF-alpha (BD Pharmingen, UK), with sensitivity limits of 8pg/ml for TNF-alpha and 15pg/ml for IL- 12.
  • the IL- 12 p40 antibody detects only the p40 molecule and does not detect IL- 12 p70.
  • the supernatants were diluted X5 for CCL-5 and X2 for IL- 12 or TNF-alpha assay. The results were expressed in pg/ml.
  • phosphor-p38 assay For the phosphor-p38 assay, DC, monocytes, or HEK 293 cells (transfected or nontransfected) were treated with HSP70 CD40L or LPS in the presence or absence of p38 inhibitors (SB 203580; Sigma) for 30 min and then lysed in isotonic buffer with 6 M urea and 0.1% Triton X-100. The phosphor-p38 content was assayed by ELISA using IC duo-set reagents (R&D Systems), according to the manufacturer's instructions. Samples were also subjected to SDS-PAGE and Western blotting to demonstrate p38-specific phosphyorylation.
  • CD40 cDNA in the pcDNA plasmid vector was a gift from Dr. B. Seed.
  • pCDM8 encoding the E.coli /?-galactosidase (Lac-Z) was used as a control.
  • the HEK 293 cells were cultured in six-well plates up to 30-50% confluence and transfected using LipofectAmine Plus (Technologies), according to the manufacturer's protocol.
  • CD40 was detected by flow cytometry using FITC-conjugated CD40 mAb (Serotec). The percentage of CD40 + cells was consistently >65% at days 2 and 3 after transfection.
  • the p38 MAP kinase assay was conducted with CD40-transfected and untransfected HEK cells, which were treated for 20 min with 30 ⁇ g/ml HSP70, 10 ⁇ g/ml peptide-binding domain (359-494), 20 ⁇ g/ml CD40Lt, or 1 ⁇ g/ml LPS.
  • the 20-mer peptide 407 or the 40-mer peptide 457-496 was added to the cultures at a concentration of 50 ⁇ g/ml.
  • the cells were washed with cold PBS medium, detached from culture plates with 0.5% EDTA, centrifuged at 500 x g, and the cell pellets were lysed with 50 ⁇ l of cell lysis buffer before the p38 MAP kinase assay.
  • HSP70 peptide-binding domain 359-4944
  • HSP70 peptide-binding domain 359-4914
  • p359-610 p359-610
  • wild-type HSP70 peptide-binding domain 359-494.
  • Human monocytes were purified from PBMC to >90% purity, and these as well as the monocyte-derived DC were incubated with various concentrations (0.01-0.5 ⁇ M) of wild-type mHSP70, the C-terminal portion of mHSP70 (p359-610) or the peptide-binding domain (p359-494).
  • TNF- ⁇ or IL-12p40 was analyzed by ELISA.
  • Resting cultures of monocytes or those treated with BSA produced negligible levels of TNF- ⁇ (Fig. IA).
  • the mHSP70 stimulated only low levels of TNF- ⁇ production (61.6 ⁇ 10.4 pg/ml) in concentrations up to 0.5 ⁇ M.
  • the peptide-binding domain (p359-494) elicited greatly enhanced dose dependent increase in the production of TNF- ⁇ , in comparison with mHSP70, and the response was almost identical with that of the C-terminal portion of HSP70 (p359-610).
  • the cytokine-stimulating activity of the peptide-binding domain of HSP70 35 g -494 was then studied in monocyte-derived DC.
  • Immature DC were generated by culturing monocytes in GM-CSF/IL-4 medium for 5 days, which had no effect of TNF- ⁇ or IL- 12 production.
  • stimulation with mHSP70 induced a does-dependent increase in production of TNF- ⁇ from an undetectable level to 419 ⁇ 32.1 pg/ml (Fig. IB) and IL-12 ⁇ 40 from 4.6 ⁇ 3.5 pg/ml to 427.3 ⁇ 44.1 pg/ml.
  • HSP70 preparations was 0.001 U/ ⁇ g of HSP70 (1 U - 10 ng of LPS) the level of contamination was 10 pg, which was 1000 times lower than any IL-12 stimulating activity that was detected with LPS either alone or with HSP70.
  • IL-12p40 Production of IL- 12p40 by DC stimulated with the peptides showed that p407-426 was a dominant epitope in stimulating a 10-fold increase in IL-12p40 (275.4 ⁇ 41.2 pg/ml) compared with the unstimulated control (23.1 ⁇ 10.3pg/ml).
  • the adjacent peptides 387-406 and 417-446 showed slightly raised production of IL- 12.
  • a minor epitope was identified at the N-terminal end (aa 357-376) which yielded 107.6 ⁇ 24 pg/ml IL-12 ⁇ 40.
  • the native HSP70 elicited ⁇ 3- fold greater production of IL-12p40 (886.5 ⁇ 377.8 pg/ml) than ⁇ 407-426.
  • TNF- ⁇ production by DC showed that the 3 overlapping peptides 397- 416, 407-426, and 417-436 (or a total of 40 residues) elicited TNF- ⁇ concentrations of 57.2 ⁇ 17.7, 82.7 ⁇ 24, and 61.7 ⁇ 13 pg/ml, respectively, compared with no detectable TNF- ⁇ in the unstimulated control .
  • p407-426 stimulated maximum production of TNF- ⁇ , as was found with IL- 12 production, the adjacent N-terminal and C-terminal residues might also be involved in stimulating TNF- ⁇ production.
  • HSP70 HSP70 with two adjacent peptides (387-426) which elicited TNF- ⁇ production.
  • HSP70 elicited higher concentration of TNF- ⁇ than p407-426
  • costimulation of HSP70 with p407-426 and the three adjacent peptides further enhanced production of TNF- ⁇ .
  • stimulation of the monocytic cell line (THPl) with p407-426 also showed significantly higher production of CCL5, which was enhanced by costimulation of HSP70 with p407-426, compared with the other 20-mer peptides or HSP70 alone (not shown).
  • the stimulatory peptide epitope 407-426 is situated at the base of L3, 4 and IA, 5 of the peptide-binding domain.
  • the peptide-binding domain of mHSP70 has been identified and resides within the 18-kDa peptide 359-494. This fragment was prepared, and its peptide-binding property was confirmed by binding peptide NPvLLLTG, a high affinity binding peptide of HSP70. Chemokine- and cytokine-stimulating activities of the C-terminal fragment of HSP70 359-610 have been demonstrated previously. Here we show that the 18-kDa peptide-binding domain (p359-494) is as effective as the larger C-terminal fragment ( ⁇ 359-610) of HSP70 in stimulating human monocytes or DC to produce TNF- ⁇ ; IL- 12, and CCL-5. These results suggest that both the cytokineO and chemokine-stimulating activities reside in the 18-kDa peptide binding domain.
  • cytokine- and chemokine-stimulating epitopes by using overlapping 20-mer peptides, derived from the sequence of the 18-kDa peptide binding domain (359-494), to activate human monocytes and DC.
  • Alanine substitutions of each of the 20 residues within p407-426 revealed that four residues at the N-terminal end (Q407, P408, S409, V410) and E420 at the C-terminal end significantly inhibited production of IL- 12 and TNF- ⁇ by DC or monocytes.
  • the cytokine- and chemokine-stimulating epitope (p407-426) resides in the peptide-binding base between loops 3, 4 and 4, 5 of HSP70, whereas ligation with peptide NRLLLTG involves loops 1, 2 and 3, 4. Indeed, only 1 of the 10 anchor residues of HSP70 (V436) binding pNRLLLTG is shared with the 5 critical residues (V410) defined within the stimulating epitope p407-426.
  • HSP70 359-494 elicited maturation of DC comparable with that stimulated by native HSP70, HSP70 359-6]0 , CD40LT, or LPS.
  • the p407-426 epitope induced significant enhancement of maturation of DC when stimulated with either HSP70 or CD40LT. This was observed with CD83, CCR7, and CXCR4 phenotypic markers of DC. However, immature DC were only slightly affected by stimulation with p407-426.
  • HSP70 Contamination of HSP70 with LPS has been raised, especially stimulation with huHSP70 which may share with LPS the CD14, TLR2, and TLR4 receptors. Removal of LPS from human HSP70 seems to have abrogated activation of human DC, without affecting peptide delivery.
  • HSP70, CD40L and LPS stimulate human DC to phosphorylate p38 which is inhibited by p457-496 only in the HSP70 or CD40L but not LPS stimulated DC.
  • p407-426 slightly enhanced p38 phosphorylation.
  • Western blot analysis of p38 MAP kinase phosphorylation confirmed the results of ELISA, showing inhibition on costimulation with HSP70 and p457-496 but enhancement with p407-426.
  • SB203580 the p38 MAP kinase inhibitor showed consistently a dose- dependent inhibition of HSP70, CD40L or LPS dependent IL- 12 production.
  • HSP70 or CD40L stimulated ⁇ 38 MAP kinase phosphorylation was inhibited by p457-496 and slightly enhanced with p407-426.
  • LPS failed to activate ⁇ 38 and was not affected by either p457-496 or p407-426, as the cells lack CD 14 receptors.
  • the p38 MAP kinase phosphorylation pathway is also involved in maturation of DC stimulated by HSP70, CD40L or LPS, as demonstrated by inhibition of HLA class II expression by the p38 inhibitor. This is consistent with the p38 MAP kinase data on maturation of DC recently reported (38).
  • the p38 MAP kinase phosphorylation pathway has now been demonstrated to be involved in the alternative CD40-HSP70 pathway by ELISA of p38, by Western blot of p38 and also by inhibition of IL- 12 production and DC maturation using a p38 inhibitor.
  • the results of this investigation have identified two discontinuous peptide epitopes within the peptide binding portion of HSP70 (aa 357-494).
  • the stimulating epitope p407-426 is located at the base of the peptide binding groove of HSP70 and involves loops 3, 4 and 4, 5.
  • the inhibitory epitope p457-496 resides within the /3-pleated sheet.
  • the mechanism whereby two opposing functions in cytokine and chemokine production and DC maturation reside within two peptides, 30 residues apart, needs to be elucidated. There are three clear differences between the two functionally opposing epitopes.
  • Cytokine and chemokine stimulating functions of p407-426 are not dependent on HSP70 or CD40L activation, unlike the inhibitory functions of ⁇ 457-496 which are HSP70 or CD40L dependent.
  • the stimulating epitope resides within the peptide binding domain, sharing 1 of the 5 critical anchor residues, whereas the inhibitory epitope is down stream from this site.
  • p38 MAP kinase phosphorylation is inhibited by p457-496 but enhanced by p407-426.
  • Microbial HSP70 is one of the most commonly found molecules in Gram positive and negative organisms, parasites and some viruses. The functional significance of this molecule has been greatly enhanced by the finding that CD40 is a prime receptor for mHSP70. The importance of CD40-CD40L costimulatory pathway has been well established and plays a crucial part in the interphase between innate and adaptive immunity. The finding that microbial HSP70 may substitute CD40L in the CD40 costimulatory pathway greatly enhances the significance of mHSP70. Indeed, microbial HSP70 serves as an alternative ligand to CD4 + CD40L + helper cells in protection of M. tuberculosis infection in CD40L knock out mice.
  • Co-administration of the tolerogenic LCMV peptide with human HSP70 interacts with CD40 and may reverse tolerance and promote DC to elicit autoimmune diabetes.
  • Human ATP-ase portion of HSP70 also binds CD40 but at a different site, and it is noteworthy that it is the peptide binding (C-terminal) part of microbial HSP70 which binds the CD40 molecule.
  • the THl polarising adjuvant effect of mHSP70 has been recently utilized in prevention of SIV infection in macaques and may prove to be important in the treatment of HIV infected patients, with low CD4 + T cells and CD40L expression, that may be rectified by the alternative mHSP70-CD40 pathway.
  • HSP70 heat shock protein
  • DC dendritic cell
  • TNF-alpha and IL-12 production in patients with Crohn's disease.
  • Maturation of monocyte- derived DC evaluated by cell surface expression of CD40, CD83, CCR7 and CXCR4, was significantly impaired when DC from Crohn's disease but not ulcerative colitis or healthy controls were stimulated with HSP70 or the CD40 ligand (CD40L).
  • TNF-alpha concentration was increased when DC from Crohn's disease were stimulated with HSP70 or CD40L and this was associated with signaling the p38 and ERK1/2 phosphorylation MAP kinase pathways.
  • CD Crohn's Disease
  • HSP is found in most Gram-positive and Gram-negative intestinal microorganisms. Increased expression of host heat shock protein (HSP70) has been demonstrated in the intestinal mucosal and submucosal mononuclear cells, as well as epithelial cells in CD (Ludwig et al 1999). Antibodies and T cell responses to HSP have been reported in inflammatory bowel disease (IBD) (Elsaghier et al 1992, Pirzer et al 1991). A protective role of HSP70 in intestinal epithelial cells from oxidant and other stressful agents has been suggested (Musch 1996, Wischmeyer et al 1997).
  • IBD inflammatory bowel disease
  • IL- 12 may drive THl responses in CD by inducing THl cell differentiation; IFN-gamma (Trinchieri 1994, Parronchi et al 1997, Monteleone et al 1997, Breese et al 1993), TNF-alpha (Breese et al 1993, Reinecker et al 1993, Woynodt et al 1994, Kolias et al 1994) and nitric oxide production (Tian et al 1995, Rachmilewitz et al 1995, Singer et al 1996) in intestinal mononuclear cells.
  • a hypothesis of the mechanism of mucosal immune responses in CD proposes polarisation of a TH-I cell response, dominated by IL- 12 and IFN-gamma, countered by oral tolerance, associated with TGF-beta (Neurath et al 1995, 1996, Powrie et al 1996, Strober et al l 997).
  • TNF-alpha plays a critical role in the control of CD, rheumatoid arthritis and other autoimmune diseases (Feldman and Maini 2001).
  • HSP70 and its peptide binding portion p359-610
  • cytokine production and DC maturation in patients with CD, compared with those in UC and controls.
  • CD40 and CD40L can be inhibited by peptides derived from the sequences of HSP70, CD40 and CD40L.
  • maturation of DC from CD by stimulation with HSP70 or CD40L, but not with LPS was significantly impaired when compared with maturation of DC from patients with UC or healthy controls.
  • HSP70 or CD40L enhances significantly TNF-alpha production by DC from patients with CD, as compared with those from UC and healthy controls.
  • Human recombinant GM-CSF was obtained from Leucomax (Sandoz Pharmaceuticals, Surrey, UK) and human recombinant IL-4 from R&D System (Abingdon, UK).
  • the fluorochrome-conjugated monoclonal antibodies to CCR7, CXCR4, CD 14 and CD40 were purchased from Immunotech (Oxford, UK).
  • the antibodies to human CD83 and control murine monoclonal antibody isotypes were obtained from Serotec (Oxford, UK).
  • Soluble CD40 ligand trimer (CD40LT) was kindly donated by Dr. F. Villinger (Atlanta, Georgia).
  • HSP70 The recombinant Mycobacterium tuberculosis HSP70 was prepared from the Escherichia coli pop strain using the pJLA603 vector. HSP70 359-610 and HSP70 359-494 were expressed using PET 22b vector. Cloned inserts were verified by DNA sequence analysis. HSP70 was expressed with no additional amino acid residues whereas HSP7O 359-61 o and HSP70 3 5 9-494 were expressed with a C-terminal (His) 6 tag. HSP70 was purified by ion exchange chromatography using Q-Sepharose resin followed by ATP-affinity chromatography.
  • HSP7O 359-6 io and HSP70 359-494 recombinant polypeptides were prepared by affinity chromatography using Ni 2+ - chelating resin, and identity of the polypeptides was confirmed by N-terminal sequence analysis (10 cycles each).
  • the HSP preparations were further treated with polymixin B-coated beads (Sigma- Aldrich, Dorset, UK) to remove LPS.
  • the LPS content of the HSP preparations was determined by the Limulus amebocyte lysate assay (Sigma-Aldrich), and showed O.006 U/ ⁇ g of HSP70 or 5 pg/1 ⁇ g of the HSP preparation. Investigation of contamination of the HSP70 preparations with LPS
  • HSP70 or its peptide binding domain (aa 359-610) with LPS was examined as described previously (Wang et al 2005). Briefly, using the intracellular calcium chelator BAPTA/AM, TNF-alpha production was inhibited in a dose-dependent manner when monocytes were stimulated with HSP70 or the 2 peptide binding fragments but not with LPS. Proteinase K differentiated the inhibitory effect of TNF-alpha production by HSP70 and its two C-terminal fragments but not that of LPS. We then determined the minimal concentration of LPS which affects the stimulating activity of DC by HSP70.
  • Peptides 457-496 and 407-426 were synthesized by Bachem Ltd (Switzerland) to a purity of
  • CD40aa27-36 REKQ YLINS Q; aa39-58 S LS QP GQKL V S D S TEFTETE
  • Biopsy specimens were obtained in 13 patients with CD and 12 patients with UC, where possible from endoscopically inflamed and non-inflamed mucosa.
  • Five patients with iron deficiency or altered bowel habits showed normal mucosa on endoscopy and biopsies were taken which also failed to show any histopathological changes. Cells from these patients were used as controls.
  • PBMC peripheral blood mononuclear cells
  • CD14 + monocytes were enriched by depletion of CD 14 ' cells using
  • Monocyte Isolation Kit (MACS, Miltenyi Biotec, Surrey, UK). The purity of isolated monocytes was consistently greater than 90% when analysed by flow-cytometry with antibody to CD 14.
  • Human dendritic cells were generated by culturing monocytes with GM-CSF (400 U/ml) and IL-4 (100 U/ml) for 5 days. These monocyte-derived DC were generally considered to be immature DC, defined by surface expression of DC markers CD83,
  • Immature DC were stimulated with HSP70 or HSP70 359 . 610 (lO ⁇ g/ml each), CD40LT (lO ⁇ g/ml) or LPS (l ⁇ g/ml) and cultured for 2 days.
  • IL-12 and TNF-alpha in the culture supernatants were assayed by ELISA using specific antibodies (BD Pharmingen, UK), with sensitivity limits of 8pg/ml for TNF-alpha and 15pg/ml for IL-12.
  • the supernatants were diluted X2 for IL-12 or TNF-alpha assay. The results were expressed in pg/ml.
  • DC were treated with HSP70, HSP 359-6I0 , CD40L or LPS in the presence of absence of p38 inhibitors (SB 203580; Sigma) or ERK1/2 inhibitor (PD 098059) for 30 minutes, then lysed in isotonic buffer with 6M urea and 0.1% Triton X- 100.
  • the phospho-p38 and ERK1/2 were assayed by ELISA using IC duo-set reagents (R and D Systems, Oxford, UK), according to manufacturer's instructions. Samples were also subjected to SDS-PAGE and western blotting to demonstrate p38 specific phosphorylation.
  • Biopsy material was collected in sterile saline. The cells were passed through a 70 jam sterile nylon cell strainer (Becton Dickinson UK Ltd) and treated by collagenase digestion adapted from the method described before (Bell et al 2001). Briefly, the tissue was suspended in warm RPMI 1640 supplemented with 10% FCS, 2mM L-glutamine and penicillin/streptomycin (100U/ml), and digested with Collagenase at 2 mg/ml for 2 hrs at 37 0 C (Sigma-Aldrich UK Ltd).
  • the cells were then filtered through the cell strainer, washed in complete medium, counted using trypan blue exclusion and resuspended in the appropriate medium.
  • the cells were cultured with HSP70, HSP70 359-610 , CD40L or LPS as above.
  • PBMC wre stimulated with the same reagents cultured for 2 days and the culture supernatant used to assay TNF-alpha and IL-12.
  • the possibility that the enzyme treatment might have affected the function of eluted mucosal cells was examined by treatment of PBMC by the same procedure as the mucosal tissue and their capacity to generate TNF-alpha and IL-12 was compared with untreated PBMC.
  • Monocytes were prepared as above, suspended at 10 6 per ml in RPMI medium, supplemented with 10% FCS, GM-CSF (400 U/ml) and IL-4 (100 U/ml) and distributed at 5xlO 4 cells per well into 96-well flat-bottomed tissue culture plates. After incubation for five days 50 ⁇ l of medium and 0.8, 4 or 20 ⁇ g/ml of HSP70, 0.02, 0.2 or 2 ⁇ g/ml CD40LT or 5, 50 or 500 ng/ml LPS was added. The cells were incubated for two days, before harvesting into 12 x 75 mm tubes, using PBS with 0.02% EDTA to help release of adherent cells.
  • the isolated cells were stained with fluorochrome-conjugated antibodies to DC markers or conjugated mouse IgG isotype as a control, for 30 min on ice. After washing twice, the cells were fixed in 1% formaldehyde PBS solution before analysis by flow cytometry. The cells were examined on a Coulter XL-MCL flow cytometer and the data analysed using Win MDI software.
  • Peptide 457-496 derived from the sequence of HSP70 inhibits production of TNF-alpha and IL-12, when DC or monocytes derived from normal PBMC are stimulated with HSP70 or CD40L (Wang et al 2004). Inhibition of TNF-alpha is of special significance in CD, as the disease can be controlled with anti-TNF-alpha antibodies.
  • DC were stimulated with HSP70 and the dose-dependent inhibition of TNF-alpha and IL-12 production by p457-496 was evaluated. The results are presented for optimum inhibition of TNF-alpha or IL-12 production in healthy controls and the two groups of patients.
  • DC from patients with ulcerative colitis failed to show significant decrease in TNF-alpha production with p457-496. It is important to note that the concentrations of TNF-alpha and IL-12 in CD were inhibited by p457-496 to 18% and 31%, respectively below the levels of these cytokines in healthy controls.
  • Monocyte-derived DC from CD patients stimulated with CD40L showed a significant increase in TNF- ⁇ production, but a significant decrease in IL-12 concentration. A similar response, though not statistically significant, was also seen with UC.
  • This dissociated response between TNF- ⁇ and IL-12 production was further investigated by the ERK 1/2 and p38 phosphorylation MAP kinase pathways of DC from patients with CD.
  • DC stimulated by HSP70, CD40L or LPS elicited a dose-dependent inhibition of TNF-a production with the ERK1/2 inhibitor (PD098059), but not with the p38 inhibitor (SB023580).
  • TNF-a a significantly higher concentration of TNF- ⁇ (p ⁇ 0.05) was found with mucosal cells eluted from inflamed as compared with those from uninflamed mucosa when stimulated with CD40L but not with HSP70 or LPS. Intraepithelial and subepithelial (lamina propria) cells were, however not studied separately, as the specimens of mucosal tissues were inadequate to yield a sufficient number of cells.
  • IL- 12 production was then evaluated by stimulating the cells eluted from inflamed colonic mucosa of CD or UC.
  • concentration of IL-12 was greater in cells from CD than UC when stimulated with HSP70 or LPS but none of these differences reached significant levels.
  • HSP70 stimulated TNF-alpha and IL-12 production by cells eluted from mucosal tissue with peptides derived from HSP70 (p457-496) or CD40/CD40L (p27-36, p39-58 and 102-119)
  • HSP70- and CD40/CD40L-derived peptides inhibited not only PBMC but also cells eluted from intestinal tissues in Crohn's disease.
  • An attempt to correlate stimulation of TNF-alpha production with either HSP70 or CD40L failed to show a significant correlation between PBMC and cells eluted from inflamed mucosal tissues.
  • Dendritic cells have been accorded a central role in the interphase between immunity and tolerance.
  • CD40 is a receptor for microbial HSP70
  • the CD40L (CD 154) trimer was used as a positive control.
  • LPS is present in Gram-negative gut organisms and binds to CD 14 , and was therefore used as a relevant CD40 negative control.
  • Stimulation by HSP70 of monocyte-derived DC showed significant increases in TNF- ⁇ and IL- 12 production in CD but not UC.
  • stimulation with CD40L resulted in a significant increase in TNF- ⁇ but a decrease in IL-12 in DC from CD.
  • the dissociation between TNF- ⁇ and IL-12 production by stimulating DC from CD with CD40L might be accounted for by the ERK-1/2 phosphorylation MAP kinase pathways, as the inhibitor enhanced IL-12, whilst inhibiting TNF- ⁇ production.
  • the increase in TNF- ⁇ concentration produced by DC from CD stimulated with HSP70 or CD40L is consistent with that found in the literature.
  • CD40 + cells An increase in CD40 + cells was reported in mononuclear, endothelial and mesenchymal cells from patients with inflamed CD and UC (Battaglia et al 1999, Gelbmann et al 2003, Vogel et al 2004). CD40L + CD4 + and some CD8 + T cells were also increased in CD and UC (Vogel et al 2004, Lui et al 1999). The proportion of circulating CD40 + lymphocytes and monocytes were also increased in patients with CD (Sawada-Hase et al 2000).
  • CD40L + T cells were elevated when stimulated with anti-CD3 antibodies (Liu et al 1999) and increased platelet- bound CD40L and plasma levels of soluble CD40L were found in IBD (Danese et al 2003). Furthermore, manifestation of IBD were reported in a transgenic CD40L mouse model (Clegg et al 1997). Since HSP70 may function as an alternative to CD40L in the interaction with CD40 (Wang et al 2001, 2002, Becker 2002, Lazarevic et al 2003, Millar et al 2003), this may enhance the already heightened CD40-CD40L interaction in CD.
  • CD40-CD40L and the alternative CD40-HSP70 pathway is especially significant in the intestinal mucosa which is exposed to HSP70 containing gut bacteria.
  • These potent costimulatory pathways provide a mechanism of recruitment of T and B cells, monocytes and DC into the intestinal mucosa, the production of pro-inflammatory cytokines, all of which may maintain chronic inflammation, especially in genetically susceptible subjects.
  • HSP70 derived peptide 457-496 might inhibit TNF-alpha production of DC from CD (Fig. 6), as was demonstrated with DC from normal subjects (Wang et al 2005). Costimulation of p457-496 with HSP70 significantly inhibited DC from CD but not those from UC. It is noteworthy that p457-496 inhibits TNF-alpha production to below the level found in healthy controls but does not prevent completely TNF-alpha production, thereby allowing normal function of TNF-alpha. IL-12 production was also significantly inhibited with p457-496, and this was found both in CD and UC.
  • HSP70 mycobacterial and other microbial HSP70 show extensive homology with human HSP70 and mycobacteria have been directly implicated in the pathogenesis of CD. Furthermore, microbial HSP70 elicits chemokines and cytokines via the CD40 costimulatory molecule, and these may affect host immunity. Indeed, the alternative HSP70-CD40 costimulatory pathway has been invoked in priming T cells, in the control of tuberculosis, and in converting T-cell tolerance to autoimmunity.
  • TNF-alpha and IL- 12 stimulated by the CD40-CD40L or -HSP70 pathway were used peptides derived from the CD40, N-terminal tandem repeat (aa 27-36 and 39-58) and from the CD40L cytoplasmic tail (aa 102-119). Significant inhibition of production of both cytokines was found with these two peptides in DC from CD and healthy controls but not UC.
  • TNF-alpha and IL- 12 production by HSP70 stimulated DC from CD can be inhibited by a peptide derived from HSP70 which targets CD40 (Wang et al 2001), and by peptides from the CD40 and CD40L molecules.
  • TNF- ⁇ production was found to be stimulated by HSP70 or CD40L, with both mucosal cells and circulating monocyte derived DC.
  • the inhibition experiments, targeting the CD40-CD40L or CD40-HSP70 costimulatory pathway raise the possibility that the two sets of inhibitory peptides may offer an alternative strategy for inhibition of TNF-a production in CD, by direct rectal or oral administration of either peptide, or possibly both sets of peptides.
  • This peptide inhibitory strategy might prevent excessive TNF- ⁇ production, compared with anti-TNF-o; antibodies that neutralize any TNF- ⁇ already produced. Such an approach may complement existing treatment with monoclonal antibodies to TNF- ⁇ , which have to be given by injection, and usually with cytotoxic drugs.
  • A The effect of stimulation of immature DC by HSP70, the C-terminal fragment (aa 359-610), peptide binding fragment (aa359-494), CD40LT or LPS on the CD83 and CCR7 maturation phenotypes of DC.
  • B A dose-dependent effect of LPS on HSP70 (20 ⁇ g), HSP70 359494 (5 ⁇ g/ml) and CD40LT (10 ⁇ g/ml) stimulating production of IL-12p40 by DC
  • HSP70-stimulated DC on (A) TNF-a and (B) IL-12 production when treated with p457-496 derived from HSP70 and (C) TNF- ⁇ and (D) IL-12 production when treated with pCD40 (p27-36 / 39-58) and pCD40L (p102-
  • HSP70 stimulates cytokine production through a CD14- dependent pathway, demonstrating its dual role as a chaperone and cytokine. Nat. Med. 6, 435-442.
  • Hsp70 human heat shock protein 70
  • Kiener PA Moran-Davis P, Rankin BM, Wahl AF, Aruffo A and Hollengaugh D. 1995. Stimulation of CD40 with purified soluble gp39 induces proinflammatory responses in human monocytes. J.Immunol. 155:4917-4925.
  • CD40 ligand CD 154 stimulation of macrophages to produce HIV-I suppressive ⁇ -chemokines. Proc.Natl.Acad.Sci.USA. 95:5205-5210.
  • CD40 but not CD40L, is required for the optimal priming of T cells and control of aerosol M. tuberculosis infection. Immunity. 19:823-835.
  • Heat shock proteins 70 and 60 share common receptors which are expressed on human monocyte-derived but not epidermal dendritic cells. Eur. J. Immunol. 32, 322-332.
  • Dendritic cells produce IL- 12 and direct the development of ThI cells from naive CD4+ T cells. J.Immunol. 154:5071-5079.
  • Heat shock proteins 60 is a endogenous ligand of the toll-like receptor-4 complex. J. Immunol. 164, 558-561.
  • Pirzer U Schonhaar A 5 Fleischer B, Hermann E and Meyer E and Meyer sum Buschenfelde K-H. Reactivity infiltrating T lymphocytes with microbial antigens in Crohn's disease. Lancet. 1991. 338:1238-1239.
  • Rutgeerts P., Van Assche, G. and Vermeier, S. 2004. Optimizing anti-TNF treatment in inflammatory bowel disease. Gastroenterology. 126:1593-1610. Sallusto, F. and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granucyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179, 1109- 1118.
  • Chemokines a superfamily of chemotactic cytokines. Int.J.Clin.Lab Res. 26:69-82.
  • T cells enhance nitric oxide production by murine splenic macrophages through gp39 and LFA-I. Eur. J.Immunol. 25:306-309.
  • Interleukin-12 a cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes.
  • Endocytosed HSP60s use Toll-like receptor 2 (TLR2) and TLR4 to activate the Toll/interleukin-1 receptor signaling pathway in innate immune cells. J. Biol. Chem. 276, 31332-31339.
  • CD40 is a cellular receptor mediating mycobacterial heat shock protein 70 stimulation of CC-chemokines. Immunity. 15:971-983.

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Abstract

L'invention a pour objet des peptides et leurs fragments, en particulier provenant de protéines de choc thermique et CD40L, à utiliser pour contrôler des réponses immunitaires. L'invention concerne aussi des méthodes pour produire lesdits peptides.
PCT/GB2006/000916 2005-11-14 2006-03-15 Contrôle de réponses immunitaires WO2007054658A1 (fr)

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US8604178B2 (en) 2006-09-18 2013-12-10 The Board Of Trustees Of The University Of Arkansas Compositions and methods of enhancing immune responses
US8956849B2 (en) 2007-11-01 2015-02-17 The Board Of Trustees Of The University Of Arkansas Compositions and methods of enhancing immune responses to Eimeria
US8956618B2 (en) 2010-01-21 2015-02-17 The Texas A&M University System Vaccine vectors and methods of enhancing immune responses
US8961990B2 (en) 2010-06-09 2015-02-24 The Board Of Trustees Of The University Of Arkansas Vaccine and methods to reduce campylobacter infection
US9125854B2 (en) 2007-10-30 2015-09-08 The Board Of Trustees Of The University Of Arkansas Compositions and methods of enhancing immune responses to flagellated bacterium
US9603915B2 (en) 2013-02-14 2017-03-28 The Board of Trustees of the University of Akansas Compositions and methods of enhancing immune responses to Eimeria or limiting Eimeria infection
CN108409866A (zh) * 2018-01-23 2018-08-17 合肥瑞城生生物科技有限公司 一种用于治疗和预防人乳头瘤病毒感染及相关疾病的多表位组合肽
US10376571B2 (en) 2013-03-15 2019-08-13 The Board Of Trustees Of The University Of Arkansas Compositions and methods of enhancing immune responses to enteric pathogens
US10682398B2 (en) 2016-05-03 2020-06-16 The Texas A&M University System Yeast vaccine vector including immunostimulatory and antigenic polypeptides and methods of using the same
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US10004798B2 (en) 2006-09-18 2018-06-26 The Board Of Trustees Of The University Of Arkansas Compositions and methods of enhancing immune responses
US9226957B2 (en) 2006-09-18 2016-01-05 The Texas A&M University System Compositions and methods of enhancing immune responses
WO2009008719A3 (fr) * 2007-07-06 2009-11-12 Universiteit Utrecht Holding B.V. Traitement et prévention de maladies inflammatoires et de maladies auto-immunes
JP2010532785A (ja) * 2007-07-06 2010-10-14 ウニベルジテイト・ウトレヒト・ホールディング・ビー．ブイ． 炎症性疾患および自己免疫疾患の治療および予防
CN101801402B (zh) * 2007-07-06 2013-08-28 乌得勒支大学控股有限公司 炎性疾病和自身免疫疾病的治疗和预防
US8742068B2 (en) 2007-07-06 2014-06-03 Universiteit Utrecht Holding B.V. Treatment and prevention of inflammatory diseases and autoimmune diseases
US9125854B2 (en) 2007-10-30 2015-09-08 The Board Of Trustees Of The University Of Arkansas Compositions and methods of enhancing immune responses to flagellated bacterium
US8956849B2 (en) 2007-11-01 2015-02-17 The Board Of Trustees Of The University Of Arkansas Compositions and methods of enhancing immune responses to Eimeria
US10842858B2 (en) 2007-11-01 2020-11-24 The Board Of Trustees Of The University Of Arkansas Compositions and methods of enhancing immune responses to Eimeria
US9913893B2 (en) 2010-01-21 2018-03-13 The Board Of Trustees Of The University Of Arkansas Vaccine vectors and methods of enhancing immune responses
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US8961990B2 (en) 2010-06-09 2015-02-24 The Board Of Trustees Of The University Of Arkansas Vaccine and methods to reduce campylobacter infection
US10792351B2 (en) 2013-02-14 2020-10-06 The Board Of Trustees Of The University Of Arkansas Compositions and methods of enhancing immune responses to Eimeria or limiting Eimeria infection
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US9884099B2 (en) 2013-02-14 2018-02-06 The Board Of Trustees Of The University Of Arkansas Compositions and methods of enhancing immune responses to Eimeria or limiting Eimeria infection
US9603915B2 (en) 2013-02-14 2017-03-28 The Board of Trustees of the University of Akansas Compositions and methods of enhancing immune responses to Eimeria or limiting Eimeria infection
US11364290B2 (en) 2013-02-14 2022-06-21 The Board Of Trustees Of The University Of Arkansas Compositions and methods of enhancing immune responses to eimeria or limiting eimeria infection
US11904005B2 (en) 2013-02-14 2024-02-20 The Board Of Trustees Of The University Of Arkansas Compositions and methods of enhancing immune responses to Eimeria or limiting Eimeria infection
US10376571B2 (en) 2013-03-15 2019-08-13 The Board Of Trustees Of The University Of Arkansas Compositions and methods of enhancing immune responses to enteric pathogens
US10716840B2 (en) 2013-03-15 2020-07-21 The Board Of Trustees Of The University Of Arkansas Compositions and methods of enhancing immune responses to enteric pathogens
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US11718683B2 (en) * 2016-03-10 2023-08-08 Aperisys, Inc. Antigen-binding fusion proteins with modified HSP70 domains
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