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WO2019239126A1 - Nouvelle protéine ayant des propriétés anti-inflammatoires - Google Patents

Nouvelle protéine ayant des propriétés anti-inflammatoires Download PDF

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Publication number
WO2019239126A1
WO2019239126A1 PCT/GB2019/051622 GB2019051622W WO2019239126A1 WO 2019239126 A1 WO2019239126 A1 WO 2019239126A1 GB 2019051622 W GB2019051622 W GB 2019051622W WO 2019239126 A1 WO2019239126 A1 WO 2019239126A1
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WO
WIPO (PCT)
Prior art keywords
protein
seq
isolated
recombinant protein
recombinant
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/GB2019/051622
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English (en)
Inventor
Valerie Mary Corrigall
Gabriel Stavros Panayi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Revolo Biotherapeutics Ltd
Original Assignee
Immune Regulation Ltd
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 GBGB1809703.0A external-priority patent/GB201809703D0/en
Priority claimed from US16/007,742 external-priority patent/US10858409B2/en
Priority to CA3103370A priority Critical patent/CA3103370A1/fr
Priority to MX2020013569A priority patent/MX2020013569A/es
Priority to JP2021519004A priority patent/JP7570324B2/ja
Priority to CN201980047388.1A priority patent/CN112888704B/zh
Priority to BR112020025398-7A priority patent/BR112020025398A2/pt
Priority to CN202410995156.1A priority patent/CN119285737A/zh
Priority to AU2019285831A priority patent/AU2019285831B2/en
Priority to KR1020207037960A priority patent/KR20210039339A/ko
Application filed by Immune Regulation Ltd filed Critical Immune Regulation Ltd
Priority to EA202092963A priority patent/EA202092963A1/ru
Priority to EP19732414.8A priority patent/EP3807303A1/fr
Priority to SG11202012363XA priority patent/SG11202012363XA/en
Publication of WO2019239126A1 publication Critical patent/WO2019239126A1/fr
Priority to IL279350A priority patent/IL279350A/en
Anticipated expiration legal-status Critical
Priority to CONC2020/0016749A priority patent/CO2020016749A2/es
Priority to ZA2021/00115A priority patent/ZA202100115B/en
Priority to AU2024200977A priority patent/AU2024200977A1/en
Ceased legal-status Critical Current

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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
    • C07K14/46—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • 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 present invention relates to a novel protein, its use in medicine, in particular its use in the prevention or treatment of inflammatory conditions, and processes for preparing the protein.
  • BiP binding immunoglobulin protein
  • Rhp78 glucose-regulated protein 78
  • the amino acid sequence of the bacteriaily expressed recombinant human protein differs from the known sequence of the naturally occurring protein (SEG ID NO: 5)
  • BiP is a ubiquitous, endogenously expressed protein resident in the endoplasmic reticulum (ER) and required as an intracellular protein for both the correct folding of nascent polypeptides and for protection of the cell from accumulated misfolded proteins at times of ER stress. Therefore, BiP is also defined as a stress protein and a member of the heat shock protein 70 family. Upregulated during cellular stress BiP is cell surface expressed and secreted into the extracellular matrix, such that cell- free BiP may be secreted into the synovial fluid of patients with rheumatoid arthritis (Corrigall VM et ai supra.,).
  • W02000/21995 discloses novel protein analogues of BiP (SEG.1 and SEQ.2) and their utility in the treatment of inflammatory disease.
  • W02006/111720 discloses the use of the same two analogues for the treatment and prevention of bone loss and resorption.
  • W02002/072133 discloses the use of BiP, in particular SEQ.1 and SEQ.2 from W02000/21995 for the treatment or prevention of an unwanted immune response, including the treatment of immune-mediated disease such as auto-immune disease, type ! diabetes, thyroiditis, multiple sclerosis, lupus, Crohn ’ s disease, hepatitis, or unwanted immune response associated with transplant organ rejection.
  • immune-mediated disease such as auto-immune disease, type ! diabetes, thyroiditis, multiple sclerosis, lupus, Crohn ’ s disease, hepatitis, or unwanted immune response associated with transplant organ rejection.
  • BiP analogues show results in a number of in vitro and in vivo animal models of inflammation, extrapolation from these models to predict what may happen in the clinic is problematic and there remains the challenge of manufacture of a protein of suitable purity and efficacy for administration to patients.
  • the BiP analogues SEQ.1 and SEQ.2 are produced using bacterial ceils but are not suitable as clinical products for human administration for several reasons. It has been reported that the 6x histidine tag on SEQ ID NO: 1 , (used for protein isolation by affinity chromatography) may alter the properties, physical and functional of the protein. (Santiago FW et al, Antigenic and immunogenic properties of recombinant hemagglutinin proteins from H1 N1 when produced in various protein expression systems, Vaccine, Volume 30, Issue 31 , 29 June 2012, Pages 4606-4616, ) while the metal ions used for chelation may leach into the bloodstream if not completely removed. SEQ !D NO: 1 therefore cannot be used clinically.
  • an aspect of the invention provides an isolated or recombinant protein consisting of the amino acid sequence according to SEQ ID NO: 3 or SEQ ID: NO: 4.
  • the present invention relates to an analogue of Binding Immunoglobulin Protein (BiP), SEQ ID NO: 3, shown in Figure 4.
  • BiP Binding Immunoglobulin Protein
  • the predicted amino acid sequence of native BiP is provided in Figure 1 and can be found under accession number X87949 in the publicly available database NCBI Genbank.
  • the nucleotide sequence of native BiP is shown in Figure 2.
  • the native amino acid sequence has a signal sequence MKLSLVAAML LLLSAARA attached to the n-terminus. Cleavage of the signal sequence after the alanine residue releases the mature polypeptide beginning “EEED...”
  • the amino acid sequence of SEQ ID NO:3 differs from the sequence of native BiP in that the N ⁇ terminus begins with the sequence“RAEEED...” rather than “EEED...”. This includes the two c-terminal amino acids of the native signal sequence (arginine and alanine). A skilled person would have no reason to include RA at the n-terminus - in the native form the active polypeptide begins at the glutamic acid residue, and it would be understood that if a skilled person was looking to provide a modified form of BiP there are multiple options, including cleaving the signal sequence at different positions and providing any number of mutations, additions or deletions.
  • SEQ ID NO: 4 corresponds to SEQ ID: NO: 3 with a polyhistidine affinity tag (from now onwards a His-tag) at the n-terminus of the protein (see Figure 3).
  • the His-tag facilitates purification of the protein by metal ion affinity chromatography.
  • SEQ ID NO: 3 also has significant differences from the SEQ ID NO: 1 and SEQ. ID NO: 2 disclosed in W02000/21995. As discussed above, SEQ ID NO: 1 has a His- tag at the c-terminus and the n-terminus begins“MEED....” SEQ ID NO: 2
  • SEQ ID NO:1 corresponds to SEQ ID NO:1 but without the His-tag. It has been surprisingly found that the protein of SEQ ID NO: 3 possesses potent anti-inflammatory and immunoregulatory properties that are distinct from those reported in WG20G0/21995 and W02006/111720. These significant differences would not have been expected by the skilled person. These properties are here demonstrated by relevant in-vitro and in-vivo studies. Furthermore, the protein of the invention is safe for use in humans.
  • SEQ ID NO: 1 causes downregulation of CD86 and HLA-DR, whilst SEQ ID NO: 3 showed no significant loss of HLA-DR and CD86 expression (see Example 3);
  • SEQ ID NO: 3 Is suitable for use in humans and in a clinical trial no infusion reactions or serious adverse drug reactions were noted (see Example 4). By contrast SEQ ID NO: 1 is not suitable for administration to humans.
  • SEQ ID NO:3 causes a significant reduction in serum concentrations of CRP, VEGF and IL-8 in humans relative to placebo groups. This indicates that disease inflammation has been significantly reduced by the administration of SEQ !D NO: 3 (see Example 4)
  • SEQ ID NO: 3 causes an increase in CD39 expression on regulatory T cells relative to SEQ ID NO: 1 ; in the clinic a significant increase in the expression of CD39 on regulatory T cells from patients responding to SEQ ID NO: 3 was observed, and this was maintained for 12 weeks post-infusion (see Example 5);
  • SEQ ID NO: 3 inhibits osteoclast differentiation and resorptive activity (see Example 6);
  • SEQ ID NO: 3 leads to longer survival of skin grafts in an animal model (see Example 8).
  • the invention includes isolated or recombinant proteins having one or more conservative substitutions in SEQ ID NO: 3 or SEQ ID NO: 4.
  • A“conservative substitution” is one in which an amino acid residue is replaced with another biologically similar amino acid residue, for instance, having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid
  • the isolated or recombinant BiP protein of the present invention comprises endotoxin impurities in an amount of less than 50 Endotoxin Units (EU) per mg of protein. In a preferred embodiment the isolated or recombinant protein comprises endotoxin impurities in an amount of less than 25 Endotoxin Units (EU) per mg of protein. In a preferred embodiment the isolated or recombinant protein comprises endotoxin impurities in an amount of less than 2 Endotoxin Units per mg of protein, most preferably less than 1.5 Endotoxin Units per mg of protein.
  • Endotoxin is detected using the Limuius Amebocyte Lysate (LAL) test to detect and quantify bacterial endotoxins extracted from the outer membrane of gram negative bacteria (Associates of Cape Cod, Liverpool, UK).
  • LAL Limuius Amebocyte Lysate
  • the critical component of the LAL reagents used in endotoxin tests is derived from blood cells (amebocytes) of the horseshoe crab, Limuius Polyphemus. LAL tests are described in the Bacterial Endotoxins Test chapter in the United States Pharmacopeia (Chapter ⁇ 85>) and in the equivalent chapters in the European Pharmacopoeia (Chapter 2.6.14) and the Japanese Pharmacopoeia (General Tests, No. 4.01 ).
  • the protein of the invention is non-giycosylated or substantially non-giycosylated, in contrast to the native protein which is glycosylated.
  • the present invention provides an isolated or recombinant nucleic acid molecule encoding a recombinant protein consisting of the amino acid sequence according to SEQ ID: NO. 4.
  • the isolated or recombinant nucleic acid molecule according to claim 3 consists of the nucleic acid sequence according to SEQ ID: NO 8.
  • An additional aspect provides a recombinant vector comprising the nucleic acid molecule as defined above.
  • the vector may comprise a promotor and/or an operator sequence.
  • the vector may comprise non-mammalian sequences, for example sequences from bacteria or yeast.
  • the vector may comprise a non-mammalian promotor and/or operator sequence, such as a bacterial or yeast promotor and/or operator sequence.
  • the invention provides an isolated or recombinant protein as defined above for use in medicine or veterinary medicine.
  • the protein of the invention may be for use in human or non-human animals.
  • the invention provides an isolated or recombinant protein as defined above for use in the treatment and/or prevention of an inflammatory condition.
  • the treatment and/or prevention of an inflammatory condition is achieved without significant immunosuppression.
  • the treatment and/or prevention of an inflammatory condition is achieved without significant immunosuppression as measured by T-lymphocyte activity relative to activity prior to administration of the protein.
  • there is no significant inhibition of T-cell proliferation to a recall antigen such as tuberculin purified protein derivative or a mitogen such as phytohaemagglutinin (PHA) or anti-GD3 or anti-CD2S antibody coated beads.
  • the inflammatory condition is selected from rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, rejection of a transplant of an organ, skin, tissue, blood, serum, plasma or cells, or inflammatory bowel disease such as Crohn’s disease.
  • the inflammatory condition is selected from rheumatoid arthritis, psoriatic arthritis, or juvenile idiopathic arthritis.
  • the isolated or recombinant protein is for use in treating or preventing diseases of dysreguiation of bone metabolism, for example osteoporosis, bone loss, bone resorption, Paget’s disease, breast cancer, bone cancer or bone loss associated with cancer.
  • Metastatic breast cancer is known to be associated with bone loss (http://www.nationalbreastcancer.org/metastatic-breast-cancer).
  • the isolated or recombinant protein as defined above is for use in the prevention of prosthetic joint loosening.
  • the use comprises administering the protein as a dose of from 1 mg to 1 g, optionally 1 mg to 500 mg, optionally 1 mg to 50 mg, optionally 1 to 15 mg.
  • the dose may be 1 mg, 5mg or 15 mg.
  • the protein may be administered as a single dose or as multiple doses.
  • the dose may be administered as a single intravenous infusion for a period of time of 0.5 to 3 hrs, optionally 1 to 2 hrs, preferably 1 hr.
  • a dose of 1 mg, or 5 mg or 15 mg is administered to a patient for a period of time of 1 hr.
  • multiple doses may be administered to the patient, wherein the interval between administration of each dose is at least 1 hr, or at least 2 hrs, or at least one day, or at least 1 week.
  • Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein.
  • a set of values such as 1 , 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1 -10, from 1-8, from 3-9, and so forth.
  • a disclosed range is a disclosure of each individual value encompassed by the range.
  • a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated or recombinant protein as defined in any preceding claim and one or more pharmaceutical!y-acceptab!e excipients, adjuvants or carriers.
  • the one or more pharmaceuficaily-acceptable excipients, adjuvants or carriers are not especially limited, and suitable excipients, adjuvants or carriers would be known to a person skilled in the art.
  • any suitable route of administration can be used.
  • any of oral, topical, parenteral, ocular, rectal, vaginal, inhalation, buccal, sublingual and intranasal delivery routes may be suitable.
  • compositions for parenteral administration may be preferred.
  • the proteins and pharmaceutical compositions of the invention can be administered parenteraliy, for example, intravenously, intra-arterially, intraperitoneally, intra- thecally, intraventriculariy, intrasterna!iy, intracraniaily, intra-muscularly or
  • Intravenous administration is particularly preferred.
  • compositions can comprise a pharmaceutically acceptable carrier, such as physiological saline.
  • Suitable pharmaceutical compositions can comprise one or more of a buffer (e.g. acetate, phosphate, citrate), a surfactant (e.g.
  • polysorbate polysorbate
  • a stabilizing agent e.g. human albumin, polyol, amino acid
  • a preservative e.g. sodium benzoate
  • compositions of the invention may be in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
  • Medicaments and pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile
  • suspensions which may include suspending agents and thickening agents.
  • the medicaments and compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze- dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • sterile liquid carrier for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • the pharmaceutical composition comprises phosphate buffered saline at pH 7.2 to 7.6, most preferably pH 7.4 In one embodiment the pharmaceutical composition comprises 0.9% w/v saline.
  • the pharmaceutical composition comprises the isolated or recombinant protein in an amount of from 2.0 to 50.0 mg/mL, optionally 2.0 to 10.0 mg/mL, preferably in an amount of about 5.0 mg/mL.
  • the pharmaceutical composition is suitable for intravenous administration.
  • the invention provides a method of treating and/or preventing a condition as defined above in a patient comprising the step of administering to a patient in need thereof an effective amount of an isolated or recombinant protein as defined above or a pharmaceutical composition as defined above.
  • Terms such as“treat” or“treatment” or“treating” refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of an undesired physiological condition, a diagnosed pathologic condition, a disease, or a disorder.
  • those in need of treatment include those already with the condition, disease, or disorder.
  • a subject is successfully“treated” for a condition, disease, or disorder if the patient shows, e.g., total, partial, or transient alleviation or elimination of symptoms associated with the condition, disease or disorder;
  • Prevent or“prevention” or“preventing” refer to prophylactic or preventative measures that avert and/or slow the development of a targeted pathologic condition, disease, or disorder.
  • those in need of prevention include those prone to having or susceptible to the condition, disease, or disorder.
  • a condition, disease, or disorder is successfully prevented if the patient develops, transiently or permanently, e.g., fewer or less severe symptoms associated with the condition, disease, or disorder, or a later onset of symptoms associated with the condition, disease, or disorder, than a patient who has not been subject to the methods of the invention.
  • the patient is further administered one or more therapeutic agents or when the protein is provided in combination with one or more therapeutic agents.
  • the therapeutic agent is selected from disease modifying agents, analgesics, anti-inflammatory agents, immunotherapeutic agents, antibiotics, antibodies and steroids.
  • the therapeutic agent is a disease-modifying anti-rheumatic drug (DMARD).
  • the invention provides a method for preparing a recombinant protein consisting of the amino acid sequence according to SEQ ID NO: 3, the method comprising: a) transforming a microorganism with the recombinant vector as defined in claim 6;
  • the microorganism is a bacterium, most preferably Escheri a coli.
  • the microorganism is cultured in a medium free from, or substantially free from animal-derived products.
  • the method comprises one or more further steps of treating the protein with a detergent to remove endotoxin.
  • the detergent may be 1 ,1 ,3,3- (tetramethylbutyl)phenyl-polyethylene glycol.
  • the protein may be treated with arginine in order to remove endotoxin.
  • the above-mentioned method is preferably for producing a protein having less than 25 Endotoxin Units per mg of protein, optionally less than 2 Endotoxin Units per mg of protein.
  • the addition of detergent or arginine enables removal of endotoxin.
  • Step f) occurs at a temperature suitable for the activity of the diaminopeptidase enzyme, preferably approximately 37°C.
  • step g) is carried out using immobilized metal affinity chromatography, wherein the immobilized metal is cobalt.
  • the cleaved His-tag binds to the column, and the purified protein elutes from the column.
  • the method does not include more than one freeze-thaw steps.
  • the protein is not frozen at any stage of the method. If it is necessary to store the protein between the steps of the method, the protein is stored at a temperature of from 2 to 8°C.
  • the method further comprises one or more filtration, purification or concentration steps.
  • ceils are lysed by shearing, in particular using a French press.
  • the protein of the invention is produced using host cells other than bacterial.
  • the protein of the invention is produced using ceils derived from yeast, insect or fungi.
  • the protein of the invention is produced using mammalian cells.
  • Figure 1 shows the amino acid sequence of native BiP.
  • the primary structure of BiP is composed of 664 amino acids.
  • the leader sequence of 18 amino acids (underlined) is cleaved during post-translational changes.
  • Figure 2 shows the nucleotide sequence of native BiP.
  • the BiP gene is 2.5 kilobases.
  • Figure 3 shows SEQ ID: NO 4, the protein of the invention including the His-tag at the N-terminus prior to cleavage during purification.
  • FIG. 4 shows SEQ ID: NO 3, the protein of the invention (not including the His-tag)
  • Figure 5A shows the recombinant vector pGE-2 used for cloning the native BiP gene.
  • a schematic of the vector used shows the site for the histidine tag and the cleavage sites for the restriction enzymes.
  • Figure 5B shows the BiP Sequence (SEG ID NO: 8) cloned into Ndel/Notl site of vector pGE-2.
  • Figure 8 shows an alignment of the amino acid sequence of the protein of SEQ ID NO: 4 in accordance with the invention with the amino acid sequence of the native protein.
  • Figure 7 shows SEQ ID NO: 1 , SEG 1 from WOQG/21995.
  • Figure 8 shows SEQ ID NO: 2, SEQ 2 from WOGG/21995.
  • Figure 9 shows SEQ ID NO: 7.
  • Figure 10 shows a comparison of cytokines production induced by SEQ ID NO:1 and SEQ ID NO: 3.
  • Peripheral blood mononuclear ceils were cultured for 24h in the presence of either SEQ ID NO: 1 (A, B) or SEQ ID NO: 3 (C, D, E and F) at the concentrations shown.
  • PBMC from 4 healthy controls (solid symbols) and one rheumatoid arthritis patient (open symbol) were used.
  • the supernatants were collected and the production of tumour necrosis factor (TNF) a and Interleukin (!L)10 were quantified by enzyme linked immunosorbent assay (ELISA).
  • E and F show the same data as C and D but with an individual y axis scale to allow the identification of the five samples to be observed.
  • Figure 11 shows data from experiments using peripheral blood mononuclear ceils (PBMC) cultured alone or with SEQ ID NQ:3 or SEQ ID NO: 1 for 24 hours before flow cytometric analysis using fluorochrome conjugated antibodies, anti- CDSO.phycoerythrin, anti-CD86.fluorescein isothiocyanate (FITC) or HLA-DR.FITC.
  • PBMC peripheral blood mononuclear ceils
  • PBMC peripheral blood mononuclear cells
  • SEQ ID NO: 1 showed down- regulation of CD86 and also HLA-DR.
  • SEQ ID NO: 3 in accordance with the invention showed no significant loss of HLA-DR and CD86 expression.
  • Figure 12 shows the effect of SEQ ID NO: 3 on serum C-reactive protein (CRP) levels taken from patients in the first human clinical trial for SEQ ID NO: 3.
  • CRP serum C-reactive protein
  • Figure 13 shows changes in biomarker levels in SEQ ID NO: 3 treated patients.
  • Serum concentrations of VEGF and IL-8 were measured by Luminex bead technology and the change from pre-infusion serum concentration calculated for each patient at 2 and 12 weeks
  • A Change in VEGF concentration
  • B change in IL-8 concentration.
  • Figure 14 shows upreguiated expression of CD39 a marker of increased regulatory T cell functional efficiency.
  • Peripheral blood mononuclear cells from a RA patient were set up in culture either unstimulated (open bars) or with SEQ ID NO: 1 (10pg/ml) (crosshatched bars) or SEQ ID NO: 3 (1 Opg/ml)(black bars). After 24h, 48h or 72b ceils were removed from culture and stained with a panel of fluorochrome
  • PBMC peripheral blood mononuclear cells
  • SEQ ID NO: 1 10 or 0.1 pg/ml
  • SEQ ID NO: 3 in accordance with the invention (10 or 0.1 pg/ml)
  • the ratio of pre-treated T cells to responder T ceils was 1 :10.
  • the cells were analysed for reduction in CFSE MF! using Cei!quest software on a FACSCalibur flow cytometer (BD Biosciences) after 3 days.
  • Figure 16 shows that SEQ ID NO: 3 inhibits nuclear translocation of NF-kB p65 and p52 in osteoclast precursors and THP1 monocytes following TNFa and RANKL stimulation.
  • M-CSF-dependent (A) human osteoclast precursors or (B) THP-1 cells were pre-treated for 1 h in the absence (Co) or presence of SEQ ID NO: 3 (l Opg/m! (A, B) and then stimulated with TNFa (10ng/mi) for 10min.
  • C pre-osteoclast were cultured in the presence or absence of SEQ ID NO: 3 (10pg/ml) with or without RANKL (50ng/ml) for 4h.
  • Figure 17 Following treatment with SEQ ID NO: 3 or placebo the immune response of patients was measured using PBMC culture stimulated to detect T cell responses to a maximal stimulus, anti-GD3 and anti-CD2S antibody coated beads (3 day culture) ( Figure 17A) or recall antigen, tuberculin PPD (5 day culture) ( Figure 17B). Activation was measured by uptake of tritiated thymidine for the last 24h of culture.
  • Figure 18A shows a schematic representation of the protocol and results of a murine skin transplantation experiment.
  • Figure 18B shows survival analysis in a Kaplan Meier graph This demonstrates that SEQ ID NO: 3 extended the survival of 5/6 of the grafts beyond that of the control group with 50% grafts surviving for
  • Figure 19 shows a schematic of a second transplantation exploratory experiment. Again SEQ ID NO: 3 administration leads to longer survival of skin grafts in comparison with those animals given modified dendritic cells (DC) Mixing DC administration with SEQ ID NO; 3 was not beneficial.
  • DC modified dendritic cells
  • Figure 20 shows cytokine production by periprosthetic tissue cultured with and without SEQ ID NO: 3. Small pieces of similar size were cut from periprosthetic tissue taken during revision surgery after prosthetic joint loosening and with the patients full informed consent. The tissue was culture for 24-72h either in the absence (control) or presence of SEQ ID NO: 3 (20 pg/mi). Cytokines tumour necrosis factor (TNF) a or interleukin (IL) 10 were quantified by commercial enzyme linked immunosorbent assay (ELISA) (PharMingen, BD, Oxford, UK). The two graphs show that whereas the amount of TNFa in the control cultures and the SEQ ID NO: 3 cultures show little change in all the 4 cultures, there Is an increase in IL-10 production.
  • TNF tumour necrosis factor
  • IL interleukin
  • the BiP gene was modified to place a His-tag at the N terminal end of the molecule.
  • the 8x Histidine-tag was situated so that it could be removed by enzymatic digestion by a diaminopeptidase following affinity purification of the protein on a cobalt column.
  • Nickel was not used because nickel could lead to an allergic reaction if sufficient remained to contaminate the preparation.
  • a combination of temperature changes and detergent was used to efficiently remove endotoxin.
  • the yield is greatly improved as is the purity of the protein by the removable His-tag system.
  • Table 1 Yield of SEQ ID NO: 3 from bacterial pellet:
  • the protein of the invention During the development of the protein of the invention, it was decided that the molecule must have the correct KDEL sequence at the C’ terminal and must have no other tag attached to the protein.
  • This protein was prepared in accordance with standard recombinant techniques known to a person skilled in the art. However, there was too little protein, the purity was too low and almost all of the biological activity had been lost. Accordingly, the protein could not be used.
  • SEQ ID NO: 7 corresponds to SEQ ID NO: 1 (SEQ1 of WOOO/21995) with the His- tag removed and the KDEL amino acid sequence restored, but no other changes from SEQ1 of WOOO/21995.
  • This protein proved very difficult to purify, the final protein purity was ⁇ 90% and the endotoxin load was too high for clinical use.
  • PBMC Peripheral blood mononuclear cells
  • TNFa production by PBMC is greatly reduced in the presence of SEQ ID NO: 3 as compared to SEQ ID NO: 1. Furthermore, the protein of the invention does not increase the production of TNFa by rheumatoid arthritis PBMC differently to healthy controls, whereas the protein of SEQ ID NO: 1 appears to induce greater production of TNFa by the RA PBMC than the healthy PBMC
  • TNFa rheumatoid arthritis
  • HLA-DR Human Leukocyte Antigen - antigen D Related
  • macrophages and dendritic ceils generally known as antigen presenting cells, each of which holds an antigenic peptide ready to be presented to the CD4+ T cell receptor.
  • two signals are required one through HLA-DR-T ceil receptor ligation and a simultaneous second signal through CD28 via CD86 or CD80 ligation.
  • the second signal is provided by costimulatory molecules CD86 and/or CD80, also expressed by the antigen presenting cells, initially binding to CD28, expressed by the CD4 T ceil which is later downregulated while CTLA-4 is upreguiated.
  • the activation of the T ceil is regulated by the expression of these molecules.
  • CD28 gives a positive activation signal while CTLA-4 gives a negative signal to the T ceil.
  • CTLA-4 also binds CD80 and CD88 with greater avidity than CD28, this has the effect of inhibiting T cell activation thus preventing chronic or continued T ceil activation. The interaction of these four molecules helps to regulate the immune response.
  • SEQ ID NO: 1 showed down-regulation of CD86 and also HLA-DR. This acted to reduce T cell activation, illustrated by a reduced in vitro response of BiP-freated PBMC to recall antigen, such as tuberculin PPD but also signals the possibility of generalised immunosuppression which would not be clinically beneficial in the long-term (Michael Dande!, Hans Brendan Lehmkuhl, Christoph Knosaila, Roland Hetzer, Impact of different long-term maintenance immunosuppressive therapy strategies on patients' outcome after heart
  • Figure 11 shows the results of flow cytometry experiments on CD14+ cells.
  • Figure 11 B there is an increase in the expression of CD80 and an increase in CD86 relative to unstimulated cells ( Figure 11 A).
  • Figure 11 C there is an increase in expression of CD80 but not CD86 relative to unstimu!ated ceils.
  • the disease activity score (DAS28) has been developed as a dynamic assessment tool and a therapeutic response measure for use in clinical trials and practice.
  • DAS28-ESR uses the following disease indicators: tender joint count (28 joints), swollen joint count (28 joints), Erythrocyte Sedimentation Rate (ESR) and patient- reported general health status on a 100 mm visual analogue scale, see Prevoo, ML et al, Arthritis Rheum 1995; 38: 44-8.
  • the main efficacy end point was DAS28-ESR response, graded according to the EULAR response criteria into good, moderate and non-response with remission defined as a DAS28-ESR of less than 2.6 (Kirkham B, Chaabo K, Hall C, Garrood T, Mant T, Alien E, et al. Safety and patient response as indicated by biomarker changes to binding immunoglobulin protein in the phase !/!IA RAGULA clinical trial in rheumatoid arthritis. Rheumatology 2016;55:1993-2000).
  • Biological efficacy endpoints were changes in CRP ( Figure 12), IL-S and VEGF ( Figure 13), see further discussion below. These are commonly used to monitor disease activity in clinical drug trials of treatments for rheumatoid arthritis.
  • Figure 13 shows the change in serum levels of VEGF or !L-8 in the presence of SEQ ID NO: 3 from the pre-infusion baseline for each patient at 2 weeks or 12 weeks measured by Luminex technology (Bio-Rad, Hemei Hempstead, UK). Only patients remaining in the study at 12 weeks were included in this analysis (Figure 13).
  • the active responding patients showed significantly lower serum concentrations of CRP, 2 weeks post-infusion compared with pre-infusion levels (Figure 12), and of VEGF and IL-8 (Figure 13) from the placebo group. This indicates that the disease inflammation is considerably less than pre-infusion levels.
  • Example 5 SEQ ID NO: 3 upregulates CD39 on regulatory T cells
  • Peripheral blood mononuclear cells from an RA patient were set up in culture either unstimulated (open bars) or with SEQ ID NO: 1 (10pg/ml)(crosshatched bars) or SEQ ID NO: 3 (1Gpg/mi) (black bars, Figure 14). After 24h, 48h or 72h cells were removed from culture and stained with a panel of fluorochrome conjugated antibodies for CD45, CDS, CD4, CD25, CD127 and CD39 Ceils were analysed on a FACSCanfo flow cytometer (BD Biosciences).
  • PBMC (10 6 /mi) were pre-freated for 96h in culture with SEQ ID NO: 1 (10 or 0.1 pg/ml) or SEQ ID NO: 3 in accordance with the invention (10 or 0.1 pg/mi), washed and added to fresh autologous CFSE stained (the cytoplasmic dye carboxyfiuorescein diacetate succinimidyi ester) T cells and stimulated with anti ⁇ CD3 and anti-CD28 antibody coated beads.
  • the ratio of pre-treated T cells to responder T ceils was 1 :10.
  • the cells were analysed for reduction in CFSE MFI using Ceilquest software on a FACS Caiibur after 3 days. As cells proliferate the CFSE content is reduced by each division and cells that do not proliferate remain highly stained.
  • Mature osteoclast cultures treated with SEQ ID NO: 1 also showed a marked decrease in the endogenous expression of both c-Fos and NFATd transcription factors (Figure 15F). Since SEQ ID NO: 1 inhibits the signalling pathways required for the differentiation of monocytes to osteoclasts, we looked to see if SEQ 3 would have a similar effect on NF-KB one of the transcription factor that drives inflammation but the alternative pathway is required by RANK-RANKL to drive downstream differentiation.
  • SEQ ID NO:3 can be used to treat diseases of dysregulated bone metabolism.
  • SEQ ID NO: 3 has no overall immunosuppressive effect, unlike SEQ ID NO: 1 which has already been published as reducing the recall antigen response to tuberculin PPD (Gorriga!i VM, Bodman-Smith MD, Brunst M, Cornell H, Panayi GS.
  • the stress protein, BiP stimulates human peripheral blood mononuclear ceils to express an anti- inflammatory cytokine profile and to inhibit antigen presenting cell function: relevance to the treatment of inflammatory arthritis. Arthritis Rheum 2004;50:1187-1171 ).
  • Example 8 Transplantation; Mouse model of skin grafts
  • Figure 18 shows a schematic representation of the protocol and results of a murine skin transplantation experiment: Ail recipient mice were administered anti-CD8 antibody to deplete endogenous dendritic ceils eight days before transplantation. There were 6 mice in each of four groups. Seven days prior to transplantation control mice received vehicle only. SEQ ID NO: 3 (2Gpg/mouse) was administered intravenously. Two other groups received immature or mature plasmacytoid dendritic cells from H2Kb matched mice. After 1 week small pieces of skin from the tail of H2Kd mismatched mice were transplanted onto the back of the recipient mice.
  • Graft survival analysis by Kaplan Meier graph shows that SEQ ID NO: 3 extended the survival of 5/6 of the grafts beyond that of the control group with 50% grafts surviving for approximately 30% longer than the control mice grafts.
  • SEQ ID NO: 3 is effective at maintaining graft survival in the mouse model of skin grafts, a model conceived to be highly difficult to maintain graft survival.
  • peri- prosthetic tissue On prosthesis loosening the tissue which develops around the prosthetic joints, peri- prosthetic tissue, is very similar to synovial membrane which becomes inflamed during RA. This tissue can cause loosening of the prosthetic joint.
  • Peri-prosthetic tissue PPT was collected during revision surgery for prosthetic joint replacements. Tissue was cut into small pieces of equal weight and cultured overnight in tissue culture medium (1 ml) in twenty-four well plates in the presence or absence of SEQ ID NO: 3. Culture supernatants were collected between 24h-72h and TNFa, pro- inflammatory, or interleukin-10, anti-inflammatory, cytokines were quantified by commercial enzyme linked immunosorbent assay (ELISA) (PharMingen, BD, Oxford,
  • Figure 20 shows that although the protein of the invention had little effect on the production of TNFa, IL-10 was markedly increased.
  • SEQ !D NO:3 has a significant anti-inflammatory effect. This indicates that SEQ ID NO: 3 can be used to treat inflammatory bowel disease, for example Crohn’s disease.
  • Tables 4 and 5 summarises the physical and functional differences between the protein of the invention, SEQ ID NO: 1 and native BiP. This clearly summarises the significant differences between the protein of the invention, the previously published recombinant BiP SEQ ID NO: 1 , and the native protein.

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Abstract

La présente invention concerne une protéine isolée ou recombinante constituée de la séquence d'acides aminés selon SEQ ID NO : 3 ou SEQ ID NO : 4 et son utilisation dans la prévention ou le traitement d'un état inflammatoire.
PCT/GB2019/051622 2018-06-13 2019-06-12 Nouvelle protéine ayant des propriétés anti-inflammatoires Ceased WO2019239126A1 (fr)

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SG11202012363XA SG11202012363XA (en) 2018-06-13 2019-06-12 Novel protein with anti-inflammatory properties
EA202092963A EA202092963A1 (ru) 2018-06-13 2019-06-12 Новый белок с противовоспалительными свойствами
JP2021519004A JP7570324B2 (ja) 2018-06-13 2019-06-12 抗炎症特性を有する新規タンパク質
CN201980047388.1A CN112888704B (zh) 2018-06-13 2019-06-12 具有消炎性质的新型蛋白质
BR112020025398-7A BR112020025398A2 (pt) 2018-06-13 2019-06-12 Proteína com propriedades anti-inflamatórias
CN202410995156.1A CN119285737A (zh) 2018-06-13 2019-06-12 具有消炎性质的新型蛋白质
AU2019285831A AU2019285831B2 (en) 2018-06-13 2019-06-12 Novel protein with anti-inflammatory properties
KR1020207037960A KR20210039339A (ko) 2018-06-13 2019-06-12 항-염증성 특성을 가진 신규 단백질
CA3103370A CA3103370A1 (fr) 2018-06-13 2019-06-12 Nouvelle proteine ayant des proprietes anti-inflammatoires
MX2020013569A MX2020013569A (es) 2018-06-13 2019-06-12 Proteína novedosa con propiedades antiinflamatorias.
IL279350A IL279350A (en) 2018-06-13 2020-12-10 A new protein with anti-inflammatory properties
CONC2020/0016749A CO2020016749A2 (es) 2018-06-13 2020-12-31 Proteína novedosa con propiedades antiinflamatorias
ZA2021/00115A ZA202100115B (en) 2018-06-13 2021-01-07 Novel protein with anti-inflammatory properties
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