Novel proteins with anti-inflammatory properties

Abstract

The name of the invention is novel proteins with anti-inflammatory properties. The present invention provides an isolated or recombinant protein consisting of the amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4 and its use for the prevention or treatment of inflammatory conditions.

Description

[0001] This application is a divisional application. The application date of the original application is June 12, 2019, the application number is 2019800473881, and the name of the invention is “Novel protein with anti-inflammatory properties”. Technical Field

[0002] The present invention relates to a novel protein, its use in medicine (particularly its use in preventing or treating inflammatory conditions) and a method for preparing the protein. Background Art

[0003] Anti-inflammatory properties of a human chaperone protein variously referred to as immunoglobulin binding protein (BiP) or glucose-regulated protein 78 (Grp78) have been reported (Corrigall VM, Bodman-Smith MD, Brunst M, Cornell H, Panayi GS, The stress protein, BiP, stimulates human peripheral blood mononuclear cells to express an anti-inflammatory cytokine profile and to inhibit antigen presenting cell function: relevance to the treatment of inflammatory arthritis, Arthritis Rheum, 2004;50:1167–1171). The amino acid sequence of the bacterially expressed recombinant human protein differs from the known sequence of the naturally occurring protein (SEQ ID NO:5).

[0004] BiP is a ubiquitous endogenously expressed protein residing in the endoplasmic reticulum (ER), and as an intracellular protein, is necessary for the correct folding of nascent polypeptides and for protecting cells from the effects of accumulated misfolded proteins during ER stress. Therefore, BiP is also defined as a stress protein and a member of the heat shock protein 70 family. BiP, which is upregulated during cellular stress, is expressed on the cell surface and secreted into the extracellular matrix, so that free BiP can be secreted into the synovial fluid of patients with rheumatoid arthritis (Corrigall VM et al., supra).

[0005] The gene p78 encoding this native (naturally occurring) BiP has been cloned and recombinant human protein has been expressed (WO 2000 / 21995).

[0006] WO2000 / 21995 discloses novel protein analogs of BiP (SEQ. 1 and SEQ. 2) and their utility in treating inflammatory diseases.

[0007] WO2006 / 111720 discloses the use of the same two analogs in the treatment and prevention of bone loss and resorption.

[0008] WO2002 / 072133 discloses the use of BiP (particularly SEQ.1 and SEQ.2 from WO2000 / 21995) in the treatment or prevention of unwanted immune responses, including the treatment of immune-mediated diseases such as autoimmune diseases, type I diabetes, thyroiditis, multiple sclerosis, lupus, Crohn's disease, hepatitis or unwanted immune responses associated with transplant organ rejection.

[0009] Although BiP analogs (SEQ.1 and SEQ.2) have demonstrated results in multiple in vivo and in vitro animal models of inflammation, extrapolation from these models to predict what may occur in the clinic is problematic, and challenges remain in manufacturing proteins of appropriate purity and efficacy for administration to patients.

[0010] BiP analogs SEQ.1 and SEQ.2 (from now on referred to as SEQ ID NO:1 and SEQ ID NO:2) are produced using bacterial cells, but are not suitable as clinical products for human administration for a variety of reasons. It is reported that the 6x histidine tag on SEQ ID NO:1 (used for protein separation by affinity chromatography) can change the physical and functional properties of the protein. (Santiago FW et al., Antigenic and immunogenic properties of recombinant hemagglutinin proteins from H1N1 when produced in various protein expression systems, Vaccine, Vol. 30, No. 31, June 29, 2012, pp. 4606-4616) and the metal ions used for chelation may seep into the bloodstream without being completely removed. Therefore, SEQ ID NO:1 cannot be used clinically.

[0011] Although using bacterial cells as a production method is easier and cheaper than using transformed mammalian cells, the problem is that the proteins produced are not glycosylated and may not have the same folding as mammalian equivalents. In addition, the endotoxin load in bacterial products is still high and may cause adverse effects if applied to humans. It is mainly the technical difficulties in removing endotoxins from the products that have led to the increase in the popularity of transformed mammalian cell protein production (although it is costly). Purification during the manufacturing process to remove endotoxins is technically complex, expensive and time-consuming. In addition, the scalability of the production and purification process may be a problem.

[0012] Therefore, there is a need for novel BiP-based proteins with anti-inflammatory and / or immunomodulatory properties that are suitable for human administration but are easy to manufacture using scalable processes and are also effective in therapy. Summary of the invention

[0013] These needs are addressed by the novel proteins of the present invention.

[0014] Therefore, one aspect of the present invention provides an isolated or recombinant protein consisting of the amino acid sequence according to SEQ ID NO:3 or SEQ ID NO:4.

[0015] The present invention relates to an analog of an immunoglobulin binding protein (BiP), SEQ ID NO: 3, such as Figure 4 The predicted amino acid sequence of native BiP is shown in Figure 1 and can be found in the publicly available database NCBI Genbank under accession number X87949. The nucleotide sequence of native BiP is Figure 2 The native amino acid sequence has the signal sequence MKLSLVAAML LLLSAARA attached to the n-terminus. Cleavage of the signal sequence after the alanine residue releases the mature polypeptide beginning with "EEED..."

[0016] The amino acid sequence of SEQ ID NO:3 differs from that 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). The skilled person will see no reason to include RA at the n-terminus - the active polypeptide begins in its native form at a glutamic acid residue, and will appreciate that if the skilled person is seeking to provide a modified form of BiP, there are a number of options, including cleavage of the signal sequence at different positions, and providing any number of mutations, additions or deletions.

[0017] SEQ ID NO:4 corresponds to SEQ ID NO:3 with a polyhistidine affinity tag (from now on referred to as His tag) at the n-terminus of the protein (see Figure 3 ). The His tag facilitates protein purification by metal ion affinity chromatography.

[0018] SEQ ID NO:3 also has significant differences compared to SEQ ID NO:1 and SEQ ID NO:2 disclosed in WO2000 / 21995. As discussed above, SEQ ID NO:1 has a His tag at the c-terminus and the n-terminus begins with "MEED..." SEQ ID NO:2 corresponds to SEQ ID NO:1 but without the His tag.

[0019] It has been surprisingly found that the protein of SEQ ID NO: 3 has effective anti-inflammatory and immunomodulatory properties different from those reported in WO2000 / 21995 and WO2006 / 111720. These significant differences were not expected by the skilled person. These properties are shown here by relevant in vitro and in vivo studies. In addition, the protein of the present invention is safe for use in humans.

[0020] The key functional differences between SEQ ID NO: 3 according to the present invention and SEQ ID NO: 1 of WO2000 / 21995 and WO2006 / 111720 are as follows:

[0021] 1) Compared with SEQ ID NO: 1, in the presence of SEQ ID NO: 3, the production of the cytokine TNFα by peripheral blood mononuclear cells is greatly reduced (see Example 2);

[0022] 2) SEQ ID NO: 1 caused downregulation of CD86 and HLA-DR, while SEQ ID NO: 3 did not show significant loss of HLA-DR and CD86 expression (see Example 3);

[0023] 3) SEQ ID NO: 3 is suitable for use in humans, and no infusion reactions or severe adverse drug reactions were found in clinical trials (see Example 4). In contrast, SEQ ID NO: 1 is not suitable for administration to humans.

[0024] 4) SEQ ID NO: 3 significantly reduced the serum concentrations of CRP, VEGF and IL-8 in humans relative to the placebo group. This indicates that disease inflammation has been significantly reduced by administering SEQ ID NO: 3 (see Example 4)

[0025] 5) SEQ ID NO: 3 leads to an increase in CD39 expression on regulatory T cells relative to SEQ ID NO: 1; in the clinic, a significant increase in CD39 expression on regulatory T cells from patients who responded to SEQ ID NO: 3 was observed, and this was maintained for 12 weeks after infusion (see Example 5);

[0026] 6) SEQ ID NO: 3 inhibits osteoclast differentiation and resorption activity (see Example 6);

[0027] 7) There is evidence that SEQ ID NO:3 does not have an overall immunosuppressive effect, unlike SEQ ID NO:1, which reduces the recall antigen response to tuberculin PPD (see Example 7);

[0028] 8) Administration of SEQ ID NO: 3 resulted in longer survival of skin grafts in an animal model (see Example 8).

[0029] The present 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 a substitution in which an amino acid residue is replaced by another biologically similar amino acid residue (e.g., having a similar side chain). Families of amino acid residues with similar side chains have been defined in the art and include 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), non-polar 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). For example, replacing tyrosine with phenylalanine is a conservative substitution. Methods for identifying conservative amino acid substitutions that do not adversely affect protein function are well known in the art.

[0030] In a preferred embodiment, the isolated or recombinant BiP protein of the present invention comprises an amount of endotoxin impurities less than 50 endotoxin units (EU) / mg protein. In a preferred embodiment, the isolated or recombinant protein comprises an amount of endotoxin impurities less than 25 endotoxin units (EU) / mg protein. In a preferred embodiment, the isolated or recombinant protein comprises an amount of endotoxin impurities less than 2 endotoxin units / mg protein, and most preferably less than 1.5 endotoxin units / mg protein.

[0031] Endotoxins are tested using the Limulus amebocyte lysate (LAL) test to detect and quantify bacterial endotoxins extracted from the outer membrane of Gram-negative bacteria (Associates of Cape Cod, Liverpool, United Kingdom). The key component of the LAL reagent used in the endotoxin test is derived from the blood cells (amebocytes) of the horseshoe crab (Limulus polyphemus). The LAL test is defined in the United States Pharmacopeia (United States Pharmacopeia) (No. <85> It is described in the chapter "Bacterial Endotoxins Test" in the European Pharmacopoeia (Chapter 2.6.14) and in equivalent chapters in the Japanese Pharmacopoeia (Common Tests, No. 4.01).

[0032] In contrast to native proteins which are glycosylated, the proteins of the present invention are non-glycosylated or substantially non-glycosylated.

[0033] In another aspect, 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.

[0034] Preferably, the isolated or recombinant nucleic acid molecule according to claim 3 consists of the nucleic acid sequence according to SEQ ID NO:8.

[0035] Another aspect provides a recombinant vector comprising a nucleic acid molecule as defined above.

[0036] The vector may include a promoter and / or an operator sequence. The vector may include a non-mammalian sequence, such as a sequence from bacteria or yeast. The vector may include a non-mammalian promoter and / or an operator sequence, such as a bacterial or yeast promoter and / or an operator sequence.

[0037] In another aspect, 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 used in humans or non-human animals.

[0038] In another aspect, the invention provides a separation or recombinant protein as defined above for use in the treatment and / or prevention of inflammatory conditions. Preferably, the treatment and / or prevention of inflammatory conditions is achieved in the absence of significant immunosuppression. In one embodiment, the treatment and / or prevention of inflammatory conditions is achieved in the absence of significant immunosuppression (as measured by the activity of T lymphocytes relative to the activity before the protein is administered). In one embodiment, there is no significant inhibition of T cell proliferation to recall antigens (e.g., tuberculin-purified protein derivatives) or mitogens (e.g., phytohemagglutinin (PHA)) or anti-CD3 or anti-CD28 antibody-coated magnetic beads.

[0039] In a preferred embodiment, the inflammatory condition is selected from rheumatoid arthritis; psoriatic arthritis; juvenile idiopathic arthritis; ankylosing spondylitis; organ, skin, tissue, blood, serum, plasma or cell transplant rejection; or inflammatory bowel disease, such as Crohn's disease.

[0040] In a particularly preferred embodiment, the inflammatory condition is selected from rheumatoid arthritis, psoriatic arthritis or juvenile idiopathic arthritis.

[0041] In one embodiment, the isolated or recombinant protein is used to treat or prevent bone metabolic disorders, such as 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).

[0042] In another aspect, the isolated or recombinant protein as defined above is used for preventing prosthetic joint loosening.

[0043] In one embodiment of the isolated or recombinant protein as defined above for the use as defined above, said use comprises administering said protein in a dose of 1 mg to 1 g, optionally 1 mg to 500 mg, optionally 1 mg to 50 mg, optionally 1 mg to 15 mg.

[0044] The dose may be 1 mg, 5 mg or 15 mg.

[0045] The protein may be administered in a single dose or in multiple doses.

[0046] The dose may be administered as a single intravenous infusion over 0.5 to 3 hours, optionally 1 to 2 hours, preferably 1 hour.

[0047] In a preferred embodiment, a dose of 1 mg, 5 mg or 15 mg is administered to the patient for 1 hour.

[0048] In an alternative embodiment, multiple doses may be administered to the patient, wherein the interval between administration of each dose is at least 1 hour or at least 2 hours, or at least one day or at least 1 week.

[0049] Numerical ranges include numbers that define ranges, and any single value provided herein can be used as the endpoints of a range that includes other single values ​​provided herein. For example, a set of values ​​(e.g., 1, 2, 3, 8, 9, and 10) is also a disclosure of numerical ranges such as 1-10, 1-8, 3-9. Similarly, disclosed ranges are disclosures of each single value covered by the range. For example, the range 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10.

[0050] In another aspect, the present invention provides a pharmaceutical composition comprising an isolated or recombinant protein as defined in any of the preceding claims and one or more pharmaceutically acceptable excipients, adjuvants or carriers. There is no particular limitation on the one or more pharmaceutically acceptable excipients, adjuvants or carriers, and suitable excipients, adjuvants or carriers are known to those skilled in the art.

[0051] Any suitable route of administration may be used. For example, any of the oral, topical, parenteral, ocular, rectal, vaginal, inhalation, buccal, sublingual, and intranasal delivery routes may be suitable.

[0052] Pharmaceutical compositions for parenteral administration may be preferred. The proteins and pharmaceutical compositions of the invention may be administered parenterally, e.g., intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion techniques. Intravenous administration is particularly preferred.

[0053] The pharmaceutical composition may include a pharmaceutically acceptable carrier, such as physiological saline. Suitable pharmaceutical compositions may include one or more of a buffer (e.g., acetate, phosphate, citrate), a surfactant (e.g., polysorbate), a stabilizer (e.g., human albumin, polyols, amino acids), a preservative (e.g., sodium benzoate), and / or other conventional solubilizers or dispersants.

[0054] The pharmaceutical composition of the present invention can be in the form of a sterile aqueous solution, which may contain other substances, such as sufficient salt or glucose to make the solution isotonic with blood. If necessary, the aqueous solution should be appropriately buffered (preferably at a pH of 3 to 9). The preparation of suitable parenteral formulations under sterile conditions is easily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0055] Drugs and pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats and solutes to make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. The drugs and compositions may be present in unit dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in freeze-dried (lyophilized) conditions, which require only the addition of a sterile liquid carrier (e.g., water for injection) for immediate use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the aforementioned type.

[0056] In a preferred embodiment, 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.

[0057] In one embodiment, the pharmaceutical composition comprises the isolated or recombinant protein in an amount of 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.

[0058] Typically, the pharmaceutical composition is suitable for intravenous administration.

[0059] In another aspect, the present invention provides a method for treating and / or preventing a condition as defined above in a patient, the method 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.

[0060] Terms such as "treat / treatment / treating" refer to therapeutic measures that cure, slow down, alleviate symptoms of an undesirable physiological condition, a diagnosed pathological condition, a disease or a disorder, and / or prevent the progression of the condition. Therefore, those that require treatment include those that already suffer from the condition, disease or disorder. In certain embodiments, if the patient exhibits, for example, total, partial or temporary alleviation or elimination of symptoms associated with the condition, disease or disorder; a reduction in the extent of the condition, disease or disorder; stabilization (i.e., no worsening) of the condition, disease or disorder; a delay in the onset or slowing of progression of the condition, disease or disorder; an improvement in the condition, disease or disorder, including partial or total remission; and / or prolonged survival compared to the expected survival without treatment, the subject is successfully "treated" for the condition, disease or disorder.

[0061] "Prevent / prevention / preventing" refers to a prophylactic / preventative measure that avoids and / or slows the development of a targeted pathological condition, disease, or disorder. Thus, those that require prevention include those that are susceptible to or predisposed to the condition, disease, or disorder. In certain embodiments, the condition, disease, or disorder is successfully prevented if the patient develops, for example, fewer or less severe symptoms associated with the condition, disease, or disorder, or the onset of symptoms associated with the condition, disease, or disorder is delayed, compared to a patient not subjected to the methods of the invention.

[0062] In one embodiment, one or more therapeutic agents are further administered to the patient, or the protein is administered in combination with one or more therapeutic agents. In a preferred embodiment, the therapeutic agent is selected from disease modifying agents, analgesics, anti-inflammatory agents, immunotherapeutics, antibiotics, antibodies and steroids. In a specific embodiment, the therapeutic agent is a disease modifying anti-rheumatic drug (DMARD).

[0063] In another aspect, the present invention provides a method for preparing a recombinant protein consisting of an amino acid sequence according to SEQ ID NO: 3, the method comprising:

[0064] a) transforming a microorganism with the recombinant vector as defined in claim 6;

[0065] b) culturing the microorganism to produce the protein of SEQ ID NO: 4;

[0066] c) lysing the microorganism to release the protein;

[0067] d) treating the lysate with a detergent to remove endotoxin;

[0068] e) isolating and purifying said protein using immobilized metal affinity chromatography, wherein said immobilized metal is cobalt; and

[0069] f) contacting the purified protein with a diaminopeptidase, wherein the diaminopeptidase cleaves the histidine tag from the N-terminus of the protein; and

[0070] g) separating the cleaved protein from the histidine tag.

[0071] Preferably, the microorganism is a bacterium, most preferably Escherichia coli.

[0072] In a preferred embodiment, the microorganism is cultured in a culture medium that is free or substantially free of products of animal origin.

[0073] Typically, the method includes one or more additional steps of treating the protein with a detergent to remove endotoxins. The detergent may be 1,1,3,3-(tetramethylbutyl)phenyl-polyethylene glycol. As an alternative, the protein may be treated with arginine to remove endotoxins.

[0074] The above method is preferably used to produce a protein having less than 25 endotoxin units / mg protein, optionally less than 2 endotoxin units / mg protein.The addition of detergent or arginine achieves the removal of endotoxins.

[0075] Step f) is carried out at a temperature suitable for the activity of the diaminopeptidase, preferably at about 37°C.

[0076] Typically, step g) is performed using immobilized metal affinity chromatography, wherein the immobilized metal is cobalt. The cleaved His tag is bound to a column and the purified protein is eluted from the column.

[0077] Preferably, the method does not comprise more than one freeze-thaw step. Preferably, the protein is not frozen at any stage of the method. If storage of the protein between steps of the method is required, the protein is stored at a temperature of 2 to 8°C.

[0078] In one embodiment, the method further comprises one or more filtration, purification or concentration steps.

[0079] In one embodiment, the cells are lysed by shearing, particularly using a French Press.

[0080] In another embodiment, host cells other than bacteria are used to produce the proteins of the present invention. In one embodiment, cells derived from yeast, insects or fungi are used to produce the proteins of the present invention. In a preferred embodiment, mammalian cells are used to produce the proteins of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Non-limiting examples will now be described with reference to the following drawings:

[0082] Figure 1 The amino acid sequence of native BiP is shown. The primary structure of BiP consists of 664 amino acids. At the N' terminus, a leader sequence of 18 amino acids (underlined) is cleaved during post-translational changes.

[0083] Figure 2 The nucleotide sequence of native BiP is shown. The BiP gene is 2.5 kilobases.

[0084] Figure 3Shown is SEQ ID NO: 4, which is a protein of the invention comprising a His tag at the N-terminus prior to cleavage during purification.

[0085] Figure 4 SEQ ID NO: 3 is shown, which is a protein of the present invention (without a His tag)

[0086] Figure 5A The recombinant vector pQE-2 used for cloning the native BiP gene is shown. The schematic diagram of the vector used shows the site of the histidine tag and the cleavage site of the restriction enzyme. Figure 5B The BiP sequence (SEQ ID NO: 8) cloned into the NdeI / NotI sites of vector pQE-2 is shown.

[0087] Figure 6 The amino acid sequence of the protein of SEQ ID NO: 4 according to the present invention is aligned with the amino acid sequence of the natural protein.

[0088] Figure 7 SEQ ID NO: 1 is shown, which is SEQ 1 from WO 00 / 21995.

[0089] Figure 8 SEQ ID NO: 2 is shown, which is SEQ 2 from WO 00 / 21995.

[0090] Fig. 9 SEQ ID NO: 7 is shown.

[0091] Fig.10 A comparison of cytokine production induced by SEQ ID NO: 1 and SEQ ID NO: 3 is shown. Peripheral blood mononuclear cells were cultured for 24 hours in the presence of the indicated concentrations of SEQ ID NO: 1 (A, B) or SEQ ID NO: 3 (C, D, E and F). PBMCs from 4 healthy controls (filled symbols) and one rheumatoid arthritis patient (open symbols) were used. At 24 hours, supernatants were collected and the production of tumor necrosis factor (TNF) α and interleukin (IL) 10 was quantified by enzyme-linked immunosorbent assay (ELISA). E and F show the same data as C and D, but with separate y-axis scales to allow identification of five samples to be observed.

[0092] Fig.11Data from experiments are shown using peripheral blood mononuclear cells (PBMCs) cultured for 24 hours alone or with SEQ ID NO: 3 or SEQ ID NO: 1, followed by flow cytometry analysis using fluorescent dye-conjugated antibodies anti-CD80, phycoerythrin anti-CD86, fluorescein isothiocyanate (FITC) or HLA-DR.FITC. In all cases, PBMC samples were gated to enter only the CD14 population.

[0093] PBMCs were cultured as follows: A) untreated; B and E) in the presence of SEQ ID NO: 3; or C) and D) in the presence of SEQ ID NO: 1. SEQ ID NO: 1 showed downregulation of CD86 as well as HLA-DR. In contrast, SEQ ID NO: 3 according to the present invention did not show a significant loss of HLA-DR and CD86 expression.

[0094] Fig.12 The effect of SEQ ID NO:3 on serum C-reactive protein (CRP) levels obtained from patients in the first human clinical trial for SEQ ID NO:3 is shown. Three groups of patients are shown: placebo, active responders (R), and all patients receiving SEQ ID NO:3. Changes in serum levels of CRP compared to pre-infusion levels at 2 and 12 weeks post-infusion were measured for the placebo group, the responder group, and all patients treated with SEQ ID NO:3. At 12 weeks, a significant decrease in CRP levels was found in patients treated with SEQ ID NO:3. * This patient abandoned his concomitant methotrexate medication, which was a protocol violation.

[0095] Fig.13 Shown are changes in biomarker levels in patients treated with SEQ ID NO: 3. Serum concentrations of VEGF and IL-8 were measured by Luminex magnetic bead technology, and changes in serum concentrations compared to pre-infusion were calculated for each patient at 2 weeks and 12 weeks. (A) Changes in VEGF concentrations; (B) Changes in IL-8 concentrations. The data show that the placebo group (n = 6), the responder group (R) [n = 8 (2 weeks) and 6 (12 weeks)], and the total group of patients treated with SEQ ID NO: 3 who remained in the study at 12 weeks [n = 14 (2 weeks) and 12 (12 weeks)]. Concentration range (all patients): VEGF, 4-195pg / ml; IL-8, 0.7-19pg / ml.

[0096] Fig.14The expression of CD39, a marker for increased efficiency of regulatory T cell function, is shown to be upregulated. Peripheral blood mononuclear cells from RA patients were established in culture as follows: without stimulation (open bars); or with SEQ ID NO: 1 (10 μg / ml) (slashed bars) or SEQ ID NO: 3 (10 μg / ml) (black bars). After 24 hours, 48 ​​hours or 72 hours, cells were removed from the culture and stained with a panel of fluorescent dye-conjugated antibodies for CD45, CD3, CD4, CD25, CD127 and CD39. Cells were analyzed on a FACSCanto flow cytometer (BD Biosciences). The results were expressed as mean fluorescence intensity (MFI) for the following: (A) CD25hi and CD127lo cells after using multiple live gates to enter live, CD45+, CD3+, CD4+ cells; (B) CD45+CD3+CD4+CD25hiCD127lo populations in (A) for CD39. (C) PBMC (10 6 / ml) were pretreated with SEQ ID NO:1 (10 or 0.1 μg / ml) or SEQ ID NO:3 (10 or 0.1 μg / ml) according to the present invention in culture for 96 hours, washed and added to fresh autologous CFSE-stained T cells, and stimulated with anti-CD3 and anti-CD28 antibody-coated magnetic beads. The ratio of pretreated T cells to responding T cells was 1:10. After 3 days, the CFSE MFI reduction of the cells was analyzed on a FACSCalibur flow cytometer (BD Biosciences) using Cellquest software. No inhibition of the response was observed when T cells were pre-incubated with SEQ ID NO:1, but up to 30% reduction in the response was observed when cells were pre-incubated with SEQ ID NO:3. (D) In ​​the RAGULA clinical trial, the changes in CD39 expression on Treg cells (live, CD45+CD3+CD4+CD25hi CD127lo) in whole blood samples from patients with rheumatoid arthritis treated with placebo and SEQ ID NO: 3 (responders (R) or non-responders (NR)) were monitored over 12 weeks. The results are expressed as the change in cell surface expression at the indicated time points compared to the % change before infusion. After a single infusion, CD39+ expression was significantly increased for at least 12 weeks after infusion.

[0097] Fig.15Results of flow cytometric analysis of the expression of CD115 / c-Fms (A) and RANK (B) protein levels in M-CSF-dependent human osteoclast precursors cultured for 48 hours in the absence or presence of SEQ ID NO: 1 (2 μg / ml) are shown. Representative samples of mean fluorescence intensity are shown: dotted line, untreated control; heavy solid line, RANKL-activated control; light solid line, RANKL-activated SEQ ID NO: 3-treated cells (n=4). (C) qPCR analysis of c-fms and RANK expression after 48 hours of treatment of murine M-CSF-dependent osteoclast precursors with SEQ ID NO: 1 (2 μg / ml). Data show the mean ± SEM of replicate experiments using specific primers and normalized to β-actin. *p<0.05. (D) Western blot analysis shows the expression of pERK and pp38 in human osteoclast precursors in response to RANKL (10 ng / ml) for the indicated times in cells cultured in the absence or presence of SEQ ID NO: 1 (2 μg / ml, 48 hours). (E) Expression of RANKL-induced pERK in cultures containing mature human osteoclasts cultured in the absence or presence of SEQ ID NO: 1 (2 μg / ml, 48 hours). (F) Western blot analysis shows the expression of transcription factors c-Fos and NFATc1 in response to RANKL (10 ng / ml) in osteoclast precursors and mature osteoclasts treated in the absence or presence of BiP (2 μg / ml). Total ERK and p38 proteins and GAPDH were used as loading controls as indicated. *p<0.01. This suggests that SEQ ID NO: 1 downregulates CD115 and RANK cell surface expression and downstream signaling in human osteoclast precursors.

[0098] Fig.16SEQ ID NO: 3 is shown to inhibit the nuclear translocation of NF-κB p65 and p52 in osteoclast precursors and THP1 monocytes after TNFα and RANKL stimulation. In the absence (Co) or presence of SEQ ID NO: 3 (10 μg / ml) (A, B), M-CSF-dependent (A) human osteoclast precursors or (B) THP-1 cells were pretreated for 1 hour and then stimulated with TNFα (10 ng / ml) for 10 minutes. (C) Osteoclast precursors were cultured for 4 hours with or without RANKL (50 ng / ml) in the presence or absence of SEQ ID NO: 3 (10 μg / ml). After staining NF-κB p65 (A, B) or p52 (C) with DAPI counterstain, cells were fixed and subjected to flow cytometry, flow cytometry imaging or confocal microscopy. The graphs on the right side of each figure section show confocal images of nuclear translocation of p65 and p52 in representative single cells, demonstrating the absence of nuclear translocation in cells treated with SEQ ID NO: 3. *p<0.01, n=3.

[0099] Fig.17 : After treatment with SEQ ID NO: 3 or placebo, the patient's immune response was measured using stimulated PBMC cultures to detect responses to maximal stimulation, anti-CD3 and anti-CD28 antibody coated magnetic beads (3-day culture) ( Fig.17 A) or recall antigen, tuberculin PPD (5 days culture) ( Fig.17 B) T cell responses. Activation was measured by uptake of tritiated thymidine during the last 24 hours of culture.

[0100] Fig.18 (Top) Schematic diagram showing the protocol and results of the mouse skin transplantation experiment. Fig.18 (Bottom) Survival analysis in a Kaplan Meier plot is shown. This shows that SEQ ID NO:3 enabled the survival of 5 / 6 grafts to exceed that of the control group, with 50% of the grafts surviving approximately 30% longer than control mouse grafts.

[0101] Fig.19 A schematic diagram of a second transplantation exploratory experiment is shown. Administration of SEQ ID NO:3 again resulted in longer survival of skin grafts compared to those animals given modified dendritic cells (DCs). There was no benefit from mixing DC administration with SEQ ID NO:3.

[0102] Fig. 20Cytokine production of periprosthetic tissue cultured with and without SEQ ID NO: 3 is shown. Small pieces of similar size were cut from periprosthetic tissue obtained during revision surgery with prosthetic joint loosening and full informed consent from the patient. The tissue was cultured for 24-72 hours in the absence (control) or presence of SEQ ID NO: 3 (20 μg / ml). The cytokine tumor necrosis factor (TNF) α or interleukin (IL) 10 was quantified by commercial enzyme-linked immunosorbent assay (ELISA) (PharMingen, BD, Oxford, UK). Both figures show that although the amount of TNFα in the control culture and SEQ ID NO: 3 culture showed little change in all 4 cultures, IL-10 production was increased. DETAILED DESCRIPTION

[0103] Examples

[0104] Example 1: Preparation of the protein of the present invention

[0105] The BiP gene was modified to place a His tag at the N-terminus of the molecule. The 6x histidine tag was placed so that it could be removed by enzymatic digestion with diaminopeptidase after affinity purification of the protein on a cobalt column. Nickel was not used because it can cause allergic reactions if enough nickel remains to contaminate the preparation. A combination of temperature shifts and detergents was used to effectively remove endotoxins.

[0106] Yield and purification

[0107] The yield and protein purity are greatly improved by the removable His-tag system.

[0108] Table 1: Yield of SEQ ID NO: 3 from bacterial pellets:

[0109]

[0110] Table 2: Purity and yield of SEQ ID NO: 3

[0111] test result Protein concentration 5.45mg / ml endotoxin 1.51EU / mg protein Filter integrity testing of filters used for sterile filtration of pharmaceutical substances 3900mBar Bioburden No growth

[0112] Comparative Example: Preparation of SEQ ID NO: 7

[0113] During the development of the protein of the present invention, it was determined that the molecule must have the correct KDEL sequence at the C' terminus and must have no other tags attached to the protein. The present protein was prepared according to standard recombinant techniques known to those skilled in the art. However, there was too little protein, the purity was too low, and almost all biological activity was lost. Therefore, the protein could not be used.

[0114] SEQ ID NO:7 corresponds to SEQ ID NO:1 (SEQ 1 of WO00 / 21995) with the His tag removed and the KDEL amino acid sequence restored, but with no other changes compared to SEQ 1 of WO00 / 21995. It has been shown that this protein is difficult to purify, with a final protein purity of <90%, and an endotoxin load that is too high for clinical use. This suggests that it is difficult to provide an analog of natural BiP that is easy to prepare, stable, biologically active, and suitable for administration to humans.

[0115] Four trial batches were prepared to try and improve the yield, purity and endotoxin contamination reduction of pure protein. This proved to be impossible. The recovery of protein was about 1%, but endotoxin levels remained high.

[0116] Example 2

[0117] exist Fig.10 A comparison of TNFα production induced by the proteins of SEQ ID NO: 3 and SEQ ID NO: 1 is provided in . Peripheral blood mononuclear cells (PBMCs) from 4 healthy controls (filled symbols) and 1 rheumatoid arthritis patient (open symbols) were cultured for 24 hours in the presence of SEQ ID NO: 1 or SEQ ID NO: 3. Cytokines produced in the supernatant were detected and quantified by ELISA.

[0118] In the presence of SEQ ID NO: 3, TNFα production by PBMCs was greatly reduced compared to SEQ ID NO: 1. Furthermore, the proteins of the invention did not differentially increase TNFα production by rheumatoid arthritis PBMCs (compared to healthy controls), whereas the protein of SEQ ID NO: 1 appeared to induce greater TNFα production by RA PBMCs (compared to healthy PBMCs).

[0119] The clinical significance of TNFα in the pathogenesis of rheumatoid arthritis (RA) is well established (see Role of cytokines in rheumatoid arthritis: an education in pathophysiology and therapeutics, Feldmann M, Maini SR, Immunol Rev, 2008 Jun;223:7-19). Therefore, an important positive feature of the protein of the present invention is that it does not upregulate TNFα.

[0120] Example 3

[0121] The interactions between molecules that regulate T cell activation are complex, but since rheumatoid arthritis is a chronic inflammatory disease, these molecules and their relative expression are important. Human leukocyte antigen-antigen D related (HLA-DR) is a molecule constitutively expressed by monocytes, macrophages, and dendritic cells (commonly referred to as antigen presenting cells, each of which has antigenic peptides ready to present to the CD4+ T cell receptor). However, for complete T cell activation, two signals are required, one signal through the HLA-DR-T cell receptor ligation and the second signal through CD28 via CD86 or CD80 ligation. The second signal is provided by the costimulatory molecules CD86 and / or CD80, which are also expressed by antigen presenting cells and initially bind to CD28 expressed by CD4 T cells, which are then downregulated, while CTLA-4 is upregulated. The activation of T cells is regulated by the expression of these molecules. CD28 gives a positive activation signal to T cells, while CTLA-4 gives a negative signal to T cells. CTLA-4 also binds CD80 and CD86 with greater affinity than CD28, which has the effect of inhibiting T cell activation, thereby preventing chronic or persistent T cell activation.

[0122] The interaction of these four molecules helps to regulate the immune response. Therefore, it is noteworthy that SEQ ID NO: 1 shows downregulation of CD86 and HLA-DR. This plays a role in reducing T cell activation, as shown by the reduction in the in vitro response of BiP-treated PBMCs to recall antigens (e.g., tuberculin PPD), but also suggests the possibility of general immunosuppression, which has no clinical benefit in the long run (Michael Dandel, Hans Brendan Lehmkuhl, Christoph Knosalla, Roland Hetzer, "Impact of different long-term maintenance immunosuppressive therapy strategies on patients' outcome after heart transplantation", "Transplant Immunology", Vol. 23, No. 3, July 2010, pp. 93-103), see Fig.11 D. In contrast, SEQ ID NO: 3 according to the present invention does not induce HLA-DR ( Fig.11 E) and a significant loss of CD86 expression.

[0123] Fig.11 The results of flow cytometry experiments on CD14+ cells are shown. Fig.11 In case of B, compared with unstimulated cells ( Fig.11 A), the expression of CD80 increased, and the expression of CD86 increased. In the presence of SEQ ID NO:1 ( Fig.11 In the case of C), the expression of CD80 increased compared to unstimulated cells, but the expression of CD86 did not increase.

[0124] In summary, this indicates two important points of interest. First, although reducing T cell activation will reduce inflammation, as shown in the case of SEQ ID NO: 1, a general suppression of the immune system does not benefit the patient in the long term, leading to increased infections, etc. Second, SEQ ID NO: 3 according to the present invention has shown anti-inflammatory efficacy in in vivo models. This suggests that modulation of the immune system resolves chronic inflammation through BiP-specific activity, thereby avoiding general immunosuppressive effects.

[0125] Example 4: Clinical Data

[0126] In patients with active RA who have failed the treatment received, the results from a randomized placebo-controlled, dose-escalating double-blind Phase I / II clinical trial showed that the protein of the present invention is safe. In addition, biomarker analysis showed considerable anti-inflammatory activity and clinical benefits (see Kirkham B, Chaabo K, Hall C, Garrood T, Mant T, Allen E et al., "Safety and patient response as indicated by biomarker changes to binding immunoglobulin protein in the phase I / IIA RAGULA clinical trial in rheumatoid arthritis", "Rheumatology", 2016; 55: 1993–2000.)

[0127] Twenty-four active RA patients who were unresponsive to treatment with one or more disease-modifying antirheumatic drugs (DMARDs) were sequentially divided into three groups, and eight patients in each group were randomly assigned to receive placebo (two patients) or SEQ ID NO: 3 according to the present invention (six patients) at a dose of 1, 5 or 15 mg. Patients received a single iv infusion over 1 hour and were observed overnight as inpatients. Patients were monitored for the next 12 weeks by follow-up clinical and laboratory assessments for safety, efficacy (DAS28-ESR) and biomarker analysis.

[0128] Security

[0129] No infusion reactions or serious adverse drug reactions were observed. Adverse events were evenly distributed between the placebo and active groups with no drug-related toxicity. Hematological, renal, and metabolic parameters showed no drug-related toxicity.

[0130] effect

[0131] The disease activity score (DAS28) has been developed as a dynamic assessment tool and therapeutic response measure for clinical trials and practice. The DAS28-ESR uses the following disease indicators: joint tenderness count (28 joints), joint swelling count (28 joints), erythrocyte sedimentation rate (ESR) and patient-reported general health status on a 100 mm visual analog scale, see Prevoo, ML et al., Arthritis Rheum, 1995; 38: 44-8.

[0132] The primary efficacy endpoint was the DAS28-ESR response, which was divided into good, moderate, and no response according to the EULAR response criteria, with remission defined as a DAS28-ESR less than 2.6 (Kirkham B, Chaabo K, Hall C, Garrood T, Mant T, Allen E, et al., Safety and patient response as indicated by biomarker changes to binding immunoglobulin protein in the phase I / IIA RAGULA clinical trial in rheumatoid arthritis. Rheumatology, 2016;55:1993–2000). The biological efficacy endpoint was CRP ( Fig.12 ), IL-8 and VEGF ( Fig.13 ), see further discussion below. These are often used to monitor disease activity in clinical drug trials for the treatment of rheumatoid arthritis.

[0133] Clinically, good EULAR responses were more common in those patients treated with higher doses of SEQ ID NO:3, with persistent low DAS28 scores (weeks 3 to 12) observed in three patients receiving SEQ ID NO:3 but not in patients receiving placebo, but good DAS28-ESR responses were achieved in all treatment groups.

[0134] Serum VEGF and IL-8 concentrations

[0135] Fig.13 The change in serum levels of VEGF or IL-8 compared to pre-infusion baseline for each patient at 2 or 12 weeks in the presence of SEQ ID NO: 3 as measured by Luminex technology (Bio-Rad, Hemel Hempstead, UK) is shown. Only patients who were still on study at 12 weeks ( Fig.13 ).

[0136] It turns out that analysis of CRP, VEGF, and IL-8 can be used to distinguish subjects who received active drug (compared to placebo). Patients who responded to SEQ ID NO:3 showed a significant reduction in CRP at 2 weeks compared to the placebo and non-responder groups (pre-infusion levels were 12.7±1.7, while levels were 7.1±2.1 2 weeks after infusion; p=0.02). Serum VEGF and IL-8 are commonly used biomarkers in clinical trials because they are closely associated with measurements of synovitis and mononuclear cell infiltration, respectively. Significant changes in the levels of these biomarkers occurred in the group of patients receiving SEQ ID NO:3. In addition, the biomarkers did not support clinical improvement in placebo patients. Surprisingly, at 12 weeks, fewer patients receiving placebo showed a reduction in serum VEGF and IL-8 (17% and 50%, respectively) compared to the SEQ ID NO:3 responder group (71% and 83% of patients, respectively). Interestingly, even the SEQ ID NO: 3 non-responder group showed a decrease in serum concentrations (66% and 83% of patients, respectively), indicating a change in their disease pathology.

[0137] In conclusion, active responders showed serum CRP concentrations 2 weeks after infusion ( Fig.12 ) compared with pre-infusion levels and serum concentrations of VEGF and IL-8 ( Fig.13 ) compared to the placebo group. This indicates that the disease inflammation is much less than the pre-infusion level.

[0138] Example 5: SEQ ID NO: 3 upregulates CD39 on regulatory T cells

[0139] Peripheral blood mononuclear cells from RA patients were established in culture as follows: unstimulated (open bars); or stimulated with SEQ ID NO: 1 (10 μg / ml) (slashed bars) or SEQ ID NO: 3 (10 μg / ml) (black bars, Fig.14 ).

[0140] After 24, 48 or 72 hours, cells were removed from culture and stained with a panel of fluorochrome-conjugated antibodies to CD45, CD3, CD4, CD25, CD127 and CD39. Cells were analyzed on a FACSCanto flow cytometer (BD Biosciences).

[0141] Fig.14 The results were expressed as mean fluorescence intensity (MFI) for:

[0142] (A) CD25hi and CD127lo cells after using multiple gates to enter live, CD45+, CD3+, CD4+ cells;

[0143] (B) Expression of CD39 on the CD45+CD3+CD4+CD25hiCD127lo population in (A).

[0144] (C) PBMC (10 6 / ml) were pretreated with SEQ ID NO: 1 (10 or 0.1 μg / ml) or SEQ ID NO: 3 (10 or 0.1 μg / ml) according to the present invention for 96 hours in culture, washed and added to fresh autologous CFSE-stained (cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester) T cells, and stimulated with anti-CD3 and anti-CD28 antibody-coated magnetic beads. The ratio of pretreated T cells to responding T cells was 1:10. After 3 days, the CFSE MFI of the cells was analyzed on FACS Calibur using Cellquest software to reduce. As cells proliferate, the content of CFSE decreases with each division, while non-proliferating cells remain highly stained.

[0145] (D) In ​​a clinical trial, the changes in CD39 expression on Treg cells (live, CD45+CD3+CD4+CD25hiCD127lo) in whole blood samples from rheumatoid arthritis patients (responders (R) or non-responders (NR)) treated with placebo and SEQ ID NO: 3 were monitored over 12 weeks. The results are expressed as the % change in cell surface expression at the indicated time points compared to pre-infusion.

[0146] The in vitro comparison between SEQ ID NO:1 and SEQ ID NO:3 according to the present invention showed that although the actual increase in Treg numbers was low, thus confirming previous studies on SEQ ID NO:1, there was a larger difference in the % CD39 expression on Tregs in the case of SEQ ID NO:3 when compared directly to SEQ ID NO:1. Interestingly, at 72 hours, CD39 expression on Tregs from SEQ ID NO:1 cultures was lower than on control cells. In the clinic, a significant increase in CD39 expression on Treg cells from patients who responded to SEQ ID NO:3 was observed, and this was maintained for 12 weeks after infusion.

[0147] Example 6: SEQ ID NO: 3 inhibits osteoclast differentiation signal transduction pathway

[0148] To investigate the potential mechanism by which SEQ ID NO:1 and SEQ ID NO:3 inhibit osteoclastogenesis, we analyzed specific cytokine signaling and downstream signaling pathways known to be essential for osteoclast differentiation. Flow cytometric analysis of CD115 / c-Fms and RANK (receptors for M-CSF and RANKL, respectively) in human peripheral blood-derived M-CSF-dependent osteoclast precursors showed that SEQ ID NO:3 downregulated the expression of CD115 by 63±16% (inhibition range, 43-79%) ( Fig.15 A). SEQ ID NO: 1 similarly inhibited RANK protein expression by 51±29% (inhibition range, 22–90%) ( Fig.15 B). Inhibition of c-Fms and RANK expression was also observed at the mRNA level, as qPCR analysis showed a significant decrease in c-fms and RANK RNA ( Fig.15 C). To address whether reduced receptor expression leads to reduced responsiveness to osteoclastogenic cytokines, we analyzed the effect of SEQ ID NO:1 treatment on RANKL-dependent MAPK signaling. Pretreatment of human PBMC-derived osteoclast precursors with SEQ ID NO:1 significantly inhibited RANKL-induced ERK and p38 phosphorylation compared to untreated cells ( Fig.15 D). Similar results were obtained using M-CSF-dependent murine bone marrow-derived osteoclast precursors (data not shown). Further examination of RANKL responsiveness in late cultures similarly showed that RANKL-induced pERK levels were also attenuated in cultures enriched for mature osteoclasts following treatment with SEQ ID NO: 1 ( Fig.15 E).

[0149] Next, we investigated the effect of SEQ ID NO:1 on the expression of transcription factors c-Fos and NFATc1, which are essential for osteoclast differentiation and downstream of RANK and TNFα signaling in osteoclast precursors and monocytes. Pre-incubation of human osteoclast precursors with SEQ ID NO:1 significantly reduced the activation of c-Fos protein after RANKL treatment ( Fig.15 F). RANKL stimulation of the c-Fos target gene NFATc1 was similarly blocked in osteoclast precursors treated with SEQ ID NO: 1 when compared to control cell lysates ( Fig.15 F). Mature osteoclast cultures treated with SEQ ID NO: 1 also showed a significant reduction in endogenous expression of c-Fos and NFATc1 transcription factors ( Fig.15 F).

[0150] Since SEQ ID NO: 1 inhibits a signaling pathway required for monocyte differentiation into osteoclasts, we investigated whether SEQ 3 has a similar effect on NF-KB, one of the transcription factors that drives inflammation, but RANK-RANKL requires an alternative pathway to drive downstream differentiation.

[0151] Imaging flow cytometry showed that SEQ ID NO:3 treatment inhibited TNFα-induced p65NF-κB nuclear translocation in osteoclast precursors ( Fig.16 A). Similar results were obtained in response to RANKL stimulation (data not shown). Since RANKL also stimulates cells via the non-classical NF-κB pathway, we investigated the nuclear translocation of p52 NF-κB following RANKL stimulation. Confocal microscopy and image analysis showed that although RANKL stimulated efficient nuclear translocation of p52 in untreated cells, this was inhibited by pretreatment with SEQ ID NO:3 ( Fig.16 B) These results indicate that SEQ ID NO: 3 blocks canonical and non-canonical NF-κB signaling in monocytes and osteoclast precursors following TNFα and RANKL treatment.

[0152] Taken together, these data demonstrate that treatment of monocytes and osteoclast precursors with SEQ ID NO:3 reduces M-CSF- and RANKL-induced signaling and activation of essential osteoclast transcription factors NF-κB, c-Fos, and NFATc1, providing insights into the mechanism by which SEQ ID NO:3 inhibits osteoclast differentiation and resorptive activity.

[0153] The present data provide evidence that SEQ ID NO: 3 can be used to treat diseases with bone metabolism disorders.

[0154] Example 7

[0155] After treatment with SEQ ID NO: 3 or placebo, the patient's immune response was measured using stimulated PBMC cultures to detect responses to maximal stimulation, anti-CD3 and anti-CD28 antibody coated magnetic beads (3-day culture) ( Fig.17 A) or recall antigen, tuberculin PPD (5 days culture) ( Fig.17 B) T cell responses.

[0156] Activation was measured by the uptake of tritiated thymidine by proliferating cells in the last 24 hours of culture. The data show that there was no change in the response to mitogens or recall antigens within 12 weeks of the clinical trial. This indicates that SEQ ID NO: 3 has no overall immunosuppressive effect, which is different from SEQ ID NO: 1, which has been disclosed as reducing the recall antigen response to tuberculin PPD (Corrigall VM, Bodman-Smith MD, Brunst M, Cornell H, Panayi GS, "The stress protein BiP stimulates human peripheral blood mononuclear cells to express an anti-inflammatory cytokine profile and inhibit antigen presenting cell function: relevance to the treatment of inflammatory arthritis", "Arthritis and Rheumatism (Arthritis Rheum)", 2004; 50: 1167-1171).

[0157] Example 8: Transplantation; Skin Graft Mouse Model

[0158] Fig.18 Schematic diagram showing the protocol and results of the mouse skin transplantation experiment: Eight days before transplantation, anti-CD8 antibodies were administered to all recipient mice to deplete endogenous dendritic cells. There were 6 mice in each of the four groups. Seven days before transplantation, control mice received only the vehicle. SEQ ID NO:3 (20 μg / mouse) was administered intravenously. The other two groups received immature or mature plasmacytoid dendritic cells from H2Kb matched mice. One week later, small pieces of skin from the tails of H2Kd mismatched mice were transplanted to the backs of recipient mice.

[0159] Graft survival analysis by Kaplan Meier plots showed that SEQ ID NO:3 allowed 5 / 6 grafts to survive beyond the control group, with 50% of the grafts surviving approximately 30% longer than control mouse grafts.

[0160] The data presented demonstrate that SEQ ID NO:3 is effective in maintaining graft survival in a skin graft mouse model that is reported to be very difficult to maintain graft survival.

[0161] A second transplantation exploratory experiment was also performed (see Fig.19 ). Administration of SEQ ID NO:3 again resulted in longer survival of skin grafts compared to those animals given modified dendritic cells (DCs). There was no benefit from mixing administration of DCs with SEQ ID NO:3.

[0162] Example 9: Loosening of prosthetic joint

[0163] When the prosthesis is loose, the tissue developed around the prosthetic joint, i.e., the periprosthetic tissue, is very similar to the synovium that can be inflamed during RA. This tissue may cause the loosening of the prosthetic joint. During revision surgery, periprosthetic tissue (PPT) is collected to perform prosthetic joint replacement. The tissue is 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 are collected between 24 hours and 72 hours, and TNFα proinflammatory cytokines or interleukin-10 anti-inflammatory cytokines are quantified by commercial enzyme-linked immunosorbent assay (ELISA) (PharMingen, BD, Oxford, UK).

[0164] Fig. 20 It was shown that although the protein of the present invention had little effect on the production of TNFα, IL-10 was significantly increased.

[0165] The data provided by the inventors show that SEQ ID NO: 3 has a significant anti-inflammatory effect, which indicates that SEQ ID NO: 3 can be used to treat inflammatory bowel diseases, such as Crohn's disease.

[0166] Example 10

[0167] The physical and functional differences between the protein of the invention, SEQ ID NO: 1 and the native BiP are summarized in Tables 4 and 5. This clearly summarizes the significant differences between the protein of the invention, the previously disclosed recombinant BiP SEQ ID NO: 1 and the native protein.

[0168]

[0169] Table 4

[0170]

[0171] Chick collagen model improves severe arthritis n=1; **DAS28 reduction in clinical trial, CIA=collagen-induced arthritis Table 5.

Claims

1. An isolated or recombinant protein consisting of the amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO:

4.

2. The isolated or recombinant protein according to claim 1, wherein the amount of endotoxin impurities contained therein is less than 50 endotoxin units / mg protein, optionally less than 25 endotoxin units / mg protein or less than 2 endotoxin units / mg protein.

3. The isolated or recombinant protein of claim 1 or claim 2, wherein the protein is non-glycosylated.

4. An isolated or recombinant nucleic acid molecule encoding a recombinant protein consisting of the amino acid sequence according to SEQ ID NO:

4.

5. The isolated or recombinant nucleic acid molecule according to claim 4, which consists of the nucleic acid sequence according to SEQ ID NO:

8. A recombinant vector comprising the nucleic acid molecule according to claim 4 or claim 5.

7. An isolated or recombinant protein according to claim 1 or claim 2 for use in medicine or veterinary medicine.

8. An isolated or recombinant protein according to claim 1 or claim 2 for use in treating and / or preventing an inflammatory condition, optionally in treating and / or preventing an inflammatory condition in a human subject.

9. The isolated or recombinant protein according to claim 8 for use according to claim 8, wherein the treatment and / or prevention of inflammatory conditions is achieved without significant immunosuppression.

10. The isolated or recombinant protein according to claim 8 for use according to claim 9, wherein the treatment and / or prevention of inflammatory conditions is achieved without significant immunosuppression, as measured by T lymphocyte activity relative to the activity before administration of the protein.

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