AU2019285831B2

AU2019285831B2 - Novel protein with anti-inflammatory properties

Info

Publication number: AU2019285831B2
Application number: AU2019285831A
Authority: AU
Inventors: Valerie Mary Corrigall; Gabriel Stavros Panayi
Original assignee: Revolo Biotherapeutics Ltd
Current assignee: Revolo Biotherapeutics Ltd
Priority date: 2018-06-13
Filing date: 2019-06-12
Publication date: 2023-11-16
Anticipated expiration: 2039-06-12
Also published as: ZA202100115B; CO2020016749A2; KR20210039339A; CA3103370A1; CN112888704A; AU2024200977A1; CL2020003220A1; BR112020025398A2; MX2020013569A; WO2019239126A1; JP2021528100A; AU2019285831A1; EP3807303A1; SG11202012363XA; IL279350A

Abstract

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 in the prevention or treatment of an inflammatory condition.

Description

NOVEL PROTEIN WITH ANTI-INFLAMMATORY PROPERTIES

Field of the Invention

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.

Background

The anti-inflammatory properties of a human molecular chaperone known variously as binding immunoglobulin 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 ceil function: relevance to the treatment of inflammatory arthritis. Arthritis Rheum

2004;50:1167-1171 ). 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.,).

The gene encoding this native (naturally-occurring) BiP, p78, has been cloned and the recombinant human protein has been expressed (W02000/21995).

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.

Although the BiP analogues (SEQ.1 and SEQ.2) 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 (from now onwards SEQ ID NO:1 and SEQ ID NO: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.

Although the use of bacterial ceils as a method of production is easier and cheaper than using transformed mammalian cells it has the problem of resulting in proteins that are not glycosylated and may not have the same folding as their mammalian equivalent. Furthermore, the endotoxin load in the bacterial product remains high and may cause adverse effects if administered to humans. It is principally the technical difficulty of removing the endotoxin from the product that has led to the increase in popularity of transformed mammalian cell protein production despite its cost. Purification to remove endotoxin during the manufacturing process is technically complex, expensive and time consuming. In addition, scalability of the production and purification processes can be a problem. Accordingly, there is a need for a novel protein with anti-inflammatory and/or immunoregulatory properties based on BiP that is suitable for human administration, yet is easy to manufacture using a scalable process and also effective in therapy.

Statement of Invention

These needs are addressed by the novel protein of the invention.

Accordingly, 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. 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

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.

Key functional differences between SEQ ID NO: 3 in accordance with the invention and SEQ ID NO: 1 of WO2GG0/21995 and WO2G06/111720 are as follows:

1 ) Production of the cytokine TNFa by peripheral blood mononuclear cells is

greatly reduced in the presence of SEQ ID NO: 3 as compared to SEQ ID NO: 1 (see Example 2);

2) 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);

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.

4) 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)

5) 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);

6) SEQ ID NO: 3 inhibits osteoclast differentiation and resorptive activity (see Example 6);

7) There is evidence to indicate that SEQ ID NO: 3 has no overall

immunosuppressive effect, unlike SEQ ID NO: 1 which reduces the recall antigen response to tuberculin PPD (see Example 7);

8) Administration of 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). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Methods of identifying conservative amino acid substitutions that do not adversely affect protein function are well-known in the art.

In a preferred embodiment, 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). 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. In a further 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.

Preferably 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.

In a further 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 for use in human or non-human animals.

In a yet further aspect, the invention provides an isolated or recombinant protein as defined above for use in the treatment and/or prevention of an inflammatory condition. Preferably the treatment and/or prevention of an inflammatory condition is achieved without significant immunosuppression. In one embodiment 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. In one embodiment 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.

In a preferred embodiment, 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.

In a particularly preferred embodiment the inflammatory condition is selected from rheumatoid arthritis, psoriatic arthritis, or juvenile idiopathic arthritis. In one embodiment 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).

In another aspect, the isolated or recombinant protein as defined above is for use in the prevention of prosthetic joint loosening.

In one embodiment of the isolated or recombinant protein as defined above for the use defined above, 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.

In a preferred embodiment a dose of 1 mg, or 5 mg or 15 mg is administered to a patient for a period of time of 1 hr.

In an alternative embodiment, 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. For example, 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. Likewise, a disclosed range is a disclosure of each individual value encompassed by the range. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10.

In a further aspect the invention provides 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. For example, any of oral, topical, parenteral, ocular, rectal, vaginal, inhalation, buccal, sublingual and intranasal delivery routes may be suitable.

Pharmaceutical 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

subcutaneously, or they may be administered by infusion techniques. Intravenous administration is particularly preferred.

Pharmaceutical 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), a stabilizing agent (e.g. human albumin, polyol, amino acid), a preservative (e.g. sodium benzoate), and/or other conventional solubilizing or dispersing agents

Pharmaceutical 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. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of 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. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

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.

In one embodiment 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.

Typically, the pharmaceutical composition is suitable for intravenous administration.

In a further aspect, 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. Thus, those in need of treatment include those already with the condition, disease, or disorder. In certain embodiments, 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;

diminishment of the extent of the condition, disease, or disorder; stabilization (i.e. , not worsening) of the condition, disease, or disorder; delay in onset or slowing of progression of the condition, disease, or disorder; amelioration of the condition, disease, or disorder, including partial or total remission; and/or prolonging survival, as compared to expected survival if not receiving treatment

“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. Thus, those in need of prevention include those prone to having or susceptible to the condition, disease, or disorder. In certain embodiments, 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.

In one embodiment the patient is further administered one or more therapeutic agents or when the protein is provided 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, immunotherapeutic agents, antibiotics, antibodies and steroids. In a particular embodiment the therapeutic agent is a disease-modifying anti-rheumatic drug (DMARD).

In a further aspect 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;

b) culturing the microorganism leading to the production of a protein

according to SEQ ID NO: 4;

c) lysing the microorganisms to release the protein;

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

e) isolating and purifying the protein using immobilized metal affinity

chromatography, wherein the immobilized metal is cobalt; and

f) contacting the purified protein with a diaminopeptidase enzyme, wherein the diaminopeptidase cleaves the histidine tag from the N~terminus of the protein; and

g) separating the cleaved protein from the histidine tag.

Preferably the microorganism is a bacterium, most preferably Escheri a coli.

In one preferred embodiment the microorganism is cultured in a medium free from, or substantially free from animal-derived products.

Typically, 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. As an alternative 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.

Typically 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.

Preferably the method does not include more than one freeze-thaw steps.

Preferably, 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.

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

In one embodiment the ceils are lysed by shearing, in particular using a French press.

In another embodiment the protein of the invention is produced using host cells other than bacterial. In one embodiment, the protein of the invention is produced using ceils derived from yeast, insect or fungi. In a preferred embodiment the protein of the invention is produced using mammalian cells.

Non-limiting examples will now be described with reference to the following figures:

Figure 1 shows the amino acid sequence of native BiP. The primary structure of BiP is composed of 664 amino acids. At the NT terminus 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.

Figure 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. At 24h 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.

In all cases the PBMC samples were live gated to access the CD14 population only.

PBMC were cultured either A) untreated; B and E) in the presence of SEG ID NQ:3, or C) and D) in the presence of SEQ ID NO: 1 SEQ ID NO: 1 showed down- regulation of CD86 and also HLA-DR. In contrast, 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. Three groups of patients are shown, placebo, active responders (R) and all patients who received SEQ ID NO: 3. The change in CRP serum levels from pre-infusion level at 2 weeks and 12 weeks post infusion were measured for the placebo group, the responding group and ail patients treated with SEQ ID NO: 3 At 12 weeks a significant drop in the level of CRP was noted in patients treated with SEQ ID NO: 3.

* this patient dropped her concomitant methotrexate medication, a protocol violation.

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. Data show placebo group (n = 6), responder group (R) [n = 8 (2 weeks) and 6 (12 weeks)] and the total patient group treated with SEQ ID NO: 3 [n = 14 (2 weeks) and 12 (12 weeks)] who remained in the study at 12 weeks. Range of concentration (all patients) VEGF, 4-195 pg/m!; and for IL-8, 0 7-19 pg/ml.

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

conjugated antibodies for CD45, CDS, CD4, CD25, CD127 and CD39. Ceils were analysed on a FACSCanto flow cytometer (BD Biosciences). The results are expressed as a the mean fluorescent intensify (MFI) for: (A) CD25hi and CD127lo cells after using multiple live gates to access live, CD45 -,CD3+-,CD4H- cells; (B) the expression of CD39 by the CD45 -CD3 -CD4+CD25hiCD127lo population in (A). (C) PBMC (10⁶/m!) were pre-treated 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/ml), washed and added to fresh autologous CFSE stained 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 MF! using Cei!quest software on a FACSCalibur flow cytometer (BD Biosciences) after 3 days. No inhibition of response was observed where T cells had been pre-incubated with SEQ ID NO: 1 but up to 30% reduction in response was observed with ceils pre- incubated with SEQ ID NO: 3. (D) In the RAGULA clinical trial, whole blood samples from placebo and SEQ ID NO: 3 treated rheumatoid arthritis patients, responder (R) or non-responder (NR) were monitored for the change in expression of CD39 on Treg cells (live, CD45÷CD3+CD4+CD25hi CD127!o) over 12 weeks. Results are expressed as a % change in cell surface expression at the indicated time-points, from pre-infusion. After a single infusion, CD3SM- expression was significantly raised for at least 12 weeks post-infusion.

Figure 15 shows the results of flow cytometry analysis of expression of CD115/c- Fms (A) and RANK (B) protein levels by M-CSF-dependent human osteoclast precursors cultured in the absence or presence of SEQ ID NO: 1 (2pg/ml) for 48b Representative samples showing mean fluorescence intensity: Dotted line, untreated control; Heavy solid line, RANKL activated control; light solid line, RANKL activated SEQ ID NO: 3 treated ceils (n =4). (C) qPCR analysis of c-fms and RANK expression following 48h treatment of murine M-CSF-dependent osteoclast precursors with SEQ ID NO: 1 (2pg/mi) Data show the mean ± SEM of duplicate experiments using specific primers and normalized to b-actin. * p < 0.05. (D) Western blot analysis showing expression of pERK and pp38 in human osteoclast precursors in response to RANKL (10ng/ml) for the indicated times in cells cultured in the absence or presence of SEQ ID NO: 1 (2pg/mi, 48h) (E) Expression of RANKL-induced pERK in cultures containing mature human osteoclasts cultured in the absence or presence of SEQ ID NO: 1 (2pg/mi, 48h) (F) Western blot analysis showing expression of transcription factors c-Fos and NFATci in response to RANKL (10ng/ml) in osteoclast precursors and mature osteoclasts treated in the absence or presence of BiP (2pg/ml). Total ERK and p38 proteins, and GAPDH were used as loading controls as indicated. * p < 0.01. This demonstrates that SEQ ID NO: 1

downregulates CD115 and RANK cell surface expression and downstream signalling in human osteoclast precursors.

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. Cells were fixed and processed for flow cytometry, imaging flow cytometry or confocal microscopy following staining for NF-kB p65 (A,B) or p52 (C) with DAP! counterstain. Panels on the right of each figure section show confocal images of nuclear translocation of p65 and p52 in a representative single ceil, showing the absence of nuclear translocation in SEQ ID NO: 3-treated cells. * p < 0.01 , n = 3.

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

approximately 30% longer than the control mice grafts.

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.

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.

Examples

Example 1 : Preparation of protein of the invention

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.

Yield and purification

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:

Time Point Pellet weight BiP (SEQ ID

taken (g) NO:3)Yield (mg/g)

Day 0 2.4g 19.8

2 months 60g 31.9

8 months 2.4g 24.2

Table 2: Purity and yield of SEQ ID NO: 3

Comparative Example; preparation of SEQ ID: NO 7

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. This demonstrates that it is difficult to provide an analogue of native BiP which is easy to prepare, stable and has biological activity, as well as being suitable for

administration to humans.

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

Example 2

A comparison of TNFa production induced by proteins of SEQ ID NO: 3 and SEQ ID NO: 1 is provided in Figure 10. Peripheral blood mononuclear cells (PBMC) from 4 healthy controls (solid symbols) and 1 rheumatoid arthritis patient (open symbol) were cultured for 24h in the presence of SEQ ID NO: 1 or SEQ ID NO: 3. Cytokines produced in the supernatant were defected and quantified by ELISA.

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

The clinical significance of TNFa 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). It is therefore an important positive feature of the protein of the invention that it does not upregulate TNFa

Example 3

The interaction between the molecules that regulate T cell activation is complex but since rheumatoid arthritis is a disease of chronic inflammation 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 ceils, generally known as antigen presenting cells, each of which holds an antigenic peptide ready to be presented to the CD4+ T cell receptor. However for full T cell activation 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.

Thus, it is notable that 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

transplantation, Transplant Immunology; Volume 23, Issue 3, July 2010, Pages 93- 103), see Figure 11 D. In contrast, SEQ ID NO: 3 in accordance with the invention caused no significant loss of HLA-DR (Figure 11 E) and CD86 expression.

Figure 11 shows the results of flow cytometry experiments on CD14+ cells. In the presence of SEQ ID NO: 3 (Figure 11 B there is an increase in the expression of CD80 and an increase in CD86 relative to unstimulated cells (Figure 11 A). In the presence of SEQ ID NO: 1 (Figure 11 C) there is an increase in expression of CD80 but not CD86 relative to unstimu!ated ceils.

In conclusion, this reveals two important points of interest. Firstly, that although a reduction in T ceil activation would reduce inflammation, as seen with SEQ ID NO: 1 , a general suppression of the immune system is not beneficial for the patient in the long term, leading to increased infection etc. Secondly, SEQ ID NO: 3 in accordance with the invention has already shown anti-inflammatory efficacy in in vivo models. This demonstrates modulation of the immune system to resolve chronic inflammation through BiP specific activity avoiding a generalised immunosuppressive effect.

Example 4: Clinical data

The results from a randomised placebo-controlled, dose ascending double blind phase I/ll clinical trial, in patients with active RA who had failed accepted therapies, showed that the protein of the invention is safe. Furthermore, biomarker analysis showed considerable anti-inflammatory activity with clinical benefit (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 l/!IA RAGULA clinical trial in rheumatoid arthritis, Rheumatology 2016;55:1993-2000.) Twenty-four patients with active RA who were not responsive to treatment with one or more disease-modifying anti-rheumatic drugs (DMARDs) were sequentially assigned to three groups each of eight patients randomly allocated to receive placebo (two patients) or the protein of SEQ ID NO: 3 in accordance with the invention (six patients), in doses of 1 , 5 or 15 mg. Patients received a single i.v. infusion over 1 h and were observed as inpatients overnight. Patients were monitored over the following 12 weeks with follow-up clinical and laboratory assessments for safety, efficacy (DAS28-ESR) and biomarker analysis.

No infusion reactions or serious adverse drug reactions were noted. Adverse events were evenly distributed between placebo and active groups with no drug-related toxicities. Haematological, renal and metabolic parameters showed no drug-related toxicities.

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.

Clinically, good EULAR responses were more common in those treated with higher doses of SEG ID NO: 3 with sustained low DAS28 scores (from 3 to 12 weeks) observed in three patients who received SEQ ID NO: 3, compared with no patients who received placebo, although good DAS28-ESR responses were achieved in all treatment groups.

Serum VEGF and IL-8 concentration

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).

Analysis of CRP, VEGF and IL-8 proved useful in differentiating subjects receiving active drug compared with placebo. Patients who responded to SEQ ID NO: 3 showed a significant decrease in CRP at 2 weeks (pre-infusion level, 12.7 ± 1.7 versus 2 week post infusion level, 7.1 ± 2.1 ; p = 0.02), compared with the placebo and non-responder groups. Serum VEGF and IL-8 are common biomarkers used in clinical trials because they correlate well with measurement of synovitis and monocyte infiltration respectively. Significant changes in levels of these bio-markers occurred in patient groups receiving SEQ ID NO: 3. Furthermore, biomarkers did not support clinical improvement in placebo patients. Strikingly, at week 12 significantly fewer patients who received placebo showed reduced serum VEGF and IL-8 (17 and 50%, respectively), compared with the SEQ ID NO: 3 responder group (71 and 83% of patients, respectively). Interestingly even the SEQ ID NO: 3 non- responder group showed reduced serum concentrations (68 and 83% of patients, respectively), suggesting a change in the pathology of their disease.

In summary, 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).

Results in Figure 14 are expressed as the mean fluorescent intensity (MF!) for:

(A) CD25bi and GD127lo cells after using multiple live gates to access

live,CD45+,CD3+,CD4+ cells;

(B) the expression of CD39 on the CD45n-CD3+CD4+CD25hiCD127lo population in (A).

(C) PBMC (10⁶/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.

(D) In the clinical trial, whole blood samples from placebo and SEQ ID NO: 3 treated rheumatoid arthritis patients, responder (R) or non-responder (NR), were monitored for the change in expression of CD39 on Treg cells (live, CD45+CD3+CD4+CD25hi CD127lo) over 12 weeks. Results are expressed as a % change in cell surface expression at the indicated time-points, from pre-infusion.

The in vitro comparison between SEQ ID NO: 1 and SEQ ID NO: 3 in accordance with the invention shows that although the actual increase in Treg number is low, thus confirming previous work with SEQ ID NO: 1 , there is a greater difference in the % expression of CD39 on the Treg with SEQ ID NO: 3 when compared directly with SEQ ID NO: 1. Of interest is that at 72b the CD39 expression on Treg from the SEQ ID NO:1 cultures is lower than that of the control ceils. In the clinic a significant increase in the expression of CD39 on Treg cells from patients responding to SEQ ID NO: 3 was observed and this was maintained for 12 weeks post-infusion. Example 6: SEQ ID NO: 3 suppresses osteoclast differentiation signaling pathways

To investigate the mechanisms underlying the inhibition of osteoclastogenesis by SEQ ID NO: 1 and SEQ ID NO: 3, 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, the receptors for M-CSF and RA KL respectively, in human peripheral blood derived M-CSF~dependent osteoclast precursors revealed that SEQ ID NO: 3 downregulated the expression of CD115 by 63 ± 16% (range of inhibition, 43-79%) (Figure 15A). SEQ ID NO: 1 similarly inhibited RANK protein expression by 51 ± 29% (range of inhibition, 22- 90%) (Figure 15B). The inhibition of c~Fms and RANK expression was also observed at the mRNA level, as qPCR analysis showed significant reductions in c- fms and RANK RNA (Figure 15C). To address whether the decrease in receptor expression resulted in reduced responsiveness to osteoclastogenic cytokines, we analyzed the effect of SEQ ID NO: 1 treatment on RAIMKL-dependent MARK signaling. Pre-treatment of human PBMC-derived osteoclast precursors with SEQ ID NO: 1 markedly suppressed RANKL-induced ERK and p38 phosphorylation compared to untreated cells (Figure 15D). Similar results were obtained using M~ CSF-dependent murine bone marrow-derived osteoclast precursors (data not shown). Further examination of RANKL responsiveness in late-stage cultures similarly revealed that RANKL-induced pERK levels were also attenuated in cultures enriched in mature osteoclasts following SEQ ID NO: 1 treatment (Figure 15E).

We next investigated the effect of SEQ ID NO: 1 on the expression of the transcription factors c-Fos and NFATd , which are essential for osteoclast differentiation and which lie downstream of RANK and TNFa signaling in osteoclast precursors and monocytes. Pre-incubation of human osteoclast precursors with SEQ ID NO: 1 greatly reduced the activation of c-Fos protein following RANKL treatment (Figure 15F). RANKL stimulation of NFATd , a c-Fos target gene, was similarly blocked in osteoclast precursors treated with SEQ ID NO: 1 when compared to control cell lysates (Figure 15F). 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.

Imaging flow cytometry demonstrated that SEQ ID NO: 3 treatment inhibited the TNFa-induced nuclear translocation of p65 NF-kB in osteoclast precursors (Figure 16A) Similar results were obtained in response to RANKL stimulation (data not shown). Since RANKL also stimulates cells via the non-canonical NF-kB pathway, we investigated the nuclear translocation of p52 NF-kB following RANKL stimulation. Confocal microscopy and image analysis showed that whereas RANKL stimulated efficient nuclear translocation of p52 in untreated ceils, this was inhibited by SEQ ID NO: 3 pretreatment (Figure 16B). These results suggest that SEQ ID NO: 3 blocks both canonical as well as non-canonical NF-kB signaling in monocytes and osteoclast precursors following TNFa and RANKL treatment.

Taken together, these data demonstrate that treatment of monocytes and osteoclast precursors with SEQ ID NO: 3 reduces M-CSF- and RANKL-induced signal transduction and activation of the essential osteoclastogenic transcription factors NF- KB, c-Fos and NFATd , thereby providing insights into the mechanisms whereby SEQ ID NO: 3 inhibits osteoclast differentiation and resorptive activity.

This data provides evidence that SEQ ID NO:3 can be used to treat diseases of dysregulated bone metabolism.

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~CD3 and anti-CD28 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 by proliferating cells for the last 24h of culture. The data show no change in response to mitogen or recall antigen over the 12 weeks of the clinical trial. This indicates that 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.

The data presented shows that SEQ ID NO: 3 is effective at maintaining graft survival in the mouse model of skin grafts, a model reputed to be highly difficult to maintain graft survival.

A second transplantation exploratory experiment was also carried out (see Figure 19). 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.

Example 9: Prosthetic joint loosening

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.

The data provided by the inventors demonstrates that 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.

Claims

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. An isolated or recombinant protein according to claim 1 , comprising endotoxin impurities in an amount of less than 50 Endotoxin Units per mg of protein, optionally less than 25 Endotoxin Units per mg of protein or less than 2 Endotoxin Units per mg of protein.

3. An isolated or recombinant protein according to claim 1 or claim 2, wherein the protein is non-glycosyiated.

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. An isolated or recombinant nucleic acid molecule according to claim 4

consisting of the nucleic acid sequence according to SEQ ID: NO 8.

6. A recombinant vector comprising the nucleic acid molecule of 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 as defined in claim 1 or claim 2 for use in the treatment and/or prevention of an inflammatory condition, optionally in a human subject.

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

10. An isolated or recombinant protein according to claim 8 for the use according to claim 9, wherein the treatment and/or prevention of an inflammatory condition is achieved without significant immunosuppression as measured by T-!ymphocyte activity relative to activity prior to administration of the protein.

1 1 . An isolated or recombinant protein according to any of claims 8 to 10 for the use according to any of claims 8 to 10, wherein 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.

12. An isolated or recombinant protein according to claim 1 1 for the use according to claim 10, wherein the inflammatory condition is selected from rheumatoid arthritis, psoriatic arthritis, or juvenile idiopathic arthritis.

13. An isolated or recombinant protein according to claim 1 or claim 2 for use in treating or preventing diseases of dysregulation of bone metabolism, optionally osteoporosis, bone loss, bone resorption, Paget’s disease, bone cancer, breast cancer or bone loss associated with cancer.

14. An isolated or recombinant protein as defined in claim 1 or claim 2 for use in the prevention of prosthetic joint loosening.

15. An isolated or recombinant protein as defined in any of claims 8 to 14 for the use according to any of claims 8 to 14, wherein the use comprises

administering the protein as a dose of from 1 mg to 1 g.

16. A pharmaceutical composition comprising the isolated or recombinant protein as defined in any preceding claim and one or more pharmaceutically- acceptable excipients, adjuvants or carriers.

17. An isolated or recombinant protein according to any of claims 1 to 15 or a pharmaceutical composition according to claim 16, for use in humans.

18. A pharmaceutical composition according to claim 16 or claim 17, comprising phosphate buffered saline at pH 7.2 to 7.6.

19. A pharmaceutical composition according to any of claims 16 to 18 comprising the isolated or recombinant protein in an amount of from 2.0 to 50.0 rng/ml.

20. A pharmaceutical composition according to claim 19 comprising the isolated or recombinant protein in an amount of about 5.0 mg/ml.

21 . A pharmaceutical composition according to any of claims 16 to 20 which is suitable for intravenous administration.

22. A method of treating and/or preventing a condition as defined in any

preceding claim 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 in any preceding claim or a pharmaceutical composition as defined in any of claims 16 to 21 .

23. An isolated or recombinant protein, an isolated or recombinant protein for use, a pharmaceutical composition or a method according to any preceding claim wherein the patient is further administered one or more therapeutic agents or when the peptides are provided in combination with one or more therapeutic agents.

24. An isolated or recombinant protein, an isolated or recombinant protein for use, a pharmaceutical composition or a method according to claim 23 wherein the therapeutic agent is selected from disease modifying agents, analgesics, anti inflammatory agents, immunotherapeutic agents, antibiotics, antibodies and steroids.

25. 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;

b) culturing the microorganism leading to the production of a protein

according to SEQ ID NO: 4;

c) lysing the microorganisms to release the protein;

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

e) isolating and purifying the protein using immobilized metal affinity

chromatography, wherein the immobilized metal is cobalt; and

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

g) separating the cleaved protein from the histidine tag.

26. A method according to claim 25, wherein the microorganism is a bacterium, optionally Escherichia coil.

27. A method according to claim 25 or claim 26, which method comprises one or more further steps of treating the protein with a detergent to remove endotoxin.

28. A method according to any of claims 25 to 27 wherein the method is for producing a protein having less than 25 Endotoxin Units per mg of protein, optionally less than 2 Endotoxin Units per mg of protein.

29. A method according to any of claims 25 to 28 wherein step g) is carried out using immobilized metal affinity chromatography, wherein the immobilized metal is cobalt.

30. A method according to claim 29, wherein the detergent is 1 ,1 ,3,3- (tetramethylbutyi)phenyl-poiyethylene glycol.

31. A method according to any of claims 25 to 30, wherein the method further comprises one or more filtration, purification or concentration steps.

32. A method according to any of claims 25 to 31 , wherein the method does not include more than one freeze-thaw steps.

33. A method according to any of claims 25 to 32 wherein the microorganism is cultured in a medium free from animal-derived products.

34. A method for preparing a recombinant protein consisting of the amino acid sequence according to SEQ !D NO: 3, the method comprising the use of non- bacteriai host cells

35. A method according to claim 34 wherein the protein is produced using cells derived from yeast, insect or fungi.

36. A method according to claim 34 wherein the protein is produced using

mammalian cells.