CN119285737A - Novel protein with anti-inflammatory properties - Google Patents

Abstract

The present invention is entitled "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 preventing or treating inflammatory conditions.

Description

Novel proteins with anti-inflammatory properties

The application is a divisional application, the application date of the original application is 2019, 6, 12, 2019800473881, and the name of the novel protein with anti-inflammatory property.

Technical Field

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 a process for the preparation of said protein.

Background

The anti-inflammatory properties of human chaperones, variously referred to as immunoglobulin binding proteins (BiP) or glucose regulatory proteins 78 (Grp 78), have been reported (Corrigall VM, bodman-Smith MD, brunst M, cornell H, panayi GS), stress proteins BiP stimulate human peripheral blood mononuclear cells to express an anti-inflammatory cytokine profile and inhibit antigen presenting cell function: related to inflammatory arthritis treatment (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 and rheumatism (ARTHRITIS RHEUM), 2004; 50:1167-1171). The amino acid sequence of the recombinant human protein expressed by bacteria is different from the known sequence of the naturally occurring protein (SEQ ID NO: 5).

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

The gene p78 encoding such a native (naturally occurring) BiP has been cloned and recombinant human proteins have been expressed (WO 2000/21995).

WO2000/21995 discloses novel protein analogues of BiP (seq.1 and seq.2) and their use in the treatment of inflammatory diseases.

WO2006/111720 discloses the use of the same two analogues in the treatment and prevention of bone loss and resorption.

WO2002/072133 discloses the use of BiP (in particular seq.1 and seq.2 from WO 2000/21995) for the treatment or prophylaxis of undesired 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 undesired immune responses associated with transplant rejection.

Although BiP analogs (seq.1 and seq.2) exhibit results in a variety of in vivo and in vitro animal models of inflammation, inferences are made from these models to predict what might be problematic in the clinic, and challenges still remain in making proteins of appropriate purity and efficacy for administration to patients.

BiP analogues SEQ.1 and SEQ.2 (referred to from now on as SEQ ID NO:1 and SEQ ID NO: 2) were produced using bacterial cells, but are unsuitable for a number of reasons as clinical products for human administration. It has been reported that the 6x histidine tag on SEQ ID NO. 1 (used for protein separation by affinity chromatography) can alter the physical and functional properties of the protein. (Santiago FW et al, antigen and immunogenic Properties of recombinant hemagglutinin proteins from H1N1 when produced in various protein expression systems (Antigenic and immunogenic properties of recombinant hemagglutinin proteins from H1N1 when produced in various protein expression systems)"," Vaccine (Vaccine), 30, 31, 2012, 6/29, 4606-4616) metal ions for chelation may penetrate the blood stream without being completely removed. Thus, SEQ ID NO. 1 cannot be used clinically.

Although the use of bacterial cells as a production method is easier and cheaper than the use of transformed mammalian cells, there is the problem that the produced protein is not glycosylated and may not have the same folding as mammalian equivalents. Furthermore, endotoxin loading in bacterial products is still high and may cause adverse effects if applied to humans. Mainly the technical difficulties in removing endotoxins from the products have led to an increase in the popularity of the production of proteins from transformed mammalian cells (although at high cost). Purification to remove endotoxins during the manufacturing process is technically complex, expensive and time consuming. In addition, scalability of the production and purification process can be a problem.

Thus, there is a need for new 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.

Disclosure of Invention

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

Accordingly, in one aspect the invention provides an isolated or recombinant protein consisting of an amino acid sequence according to SEQ ID NO. 3 or SEQ ID NO. 4.

The present invention relates to an analogue of immunoglobulin binding protein (BiP), SEQ ID NO 3, as shown in FIG. 4. The predicted amino acid sequence of native BiP is provided in fig. 1 and can be found in the publicly available database NCBI Genbank under accession number X87949. The nucleotide sequence of the native BiP is shown in figure 2. The natural amino acid sequence has a signal sequence MKLSLVAAML LLLSAARA attached to the n-terminus. Cleavage of the signal sequence following the alanine residue releases the mature polypeptide starting with "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 "ieee. This comprises the two c-terminal amino acids of the natural signal sequence (arginine and alanine). The skilled artisan will have no reason to include RA at the n-terminus-the active polypeptide starts at the glutamate residue in its native form, and will understand that if the skilled artisan is seeking to provide modified forms of BiP, there are a variety of options, including cleavage of 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 (see FIG. 3) with a polyhistidine affinity tag (from now on His tag) at the n-terminus of the protein. The His-tag facilitates purification of the protein by metal ion affinity chromatography.

SEQ ID NO. 3 also has a significant difference compared to SEQ ID NO. 1 and SEQ ID NO. 2 disclosed in WO 2000/21995. As discussed above, SEQ ID No. 1 has a His tag at the c-terminus and the n-terminus begins with "med. SEQ ID NO. 2 corresponds to SEQ ID NO. 1, but without the His tag.

It has surprisingly been found that the protein of SEQ ID NO. 3 has potent anti-inflammatory and immunomodulatory properties different from those reported in WO2000/21995 and WO 2006/111720. These significant differences are not expected by the skilled artisan. These properties are demonstrated here by related in vitro and in vivo studies. Furthermore, the proteins of the invention are safe for use in humans.

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:

1) Production of cytokine TNFα 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 down-regulation of CD86 and HLA-DR, whereas SEQ ID NO. 3 does not show significant loss of HLA-DR and CD86 expression (see example 3);

3) SEQ ID NO. 3 is suitable for use in humans and NO infusion reaction 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.

4) SEQ ID NO 3 significantly reduced serum concentrations of CRP, VEGF, and IL-8 in humans relative to placebo. This indicates that disease inflammation has been significantly reduced by administration of SEQ ID NO:3 (see example 4)

5) In clinic, a significant increase in CD39 expression on regulatory T cells from patients responding to SEQ ID NO. 3 was observed, and this was maintained for 12 weeks after infusion (see example 5);

6) SEQ ID NO. 3 inhibits osteoclast differentiation and resorption activity (see example 6);

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);

8) The administration of SEQ ID NO. 3 results in a longer survival of the skin grafts in animal models (see example 8).

The invention encompasses 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 with 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, 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 phenylalanine for tyrosine is a conservative substitution. Methods for identifying conservative amino acid substitutions that do not adversely affect the function of a protein are well known in the art.

In a preferred embodiment, the isolated or recombinant BiP proteins of the invention comprise an amount of endotoxin impurities of less than 50 Endotoxin Units (EU)/mg protein. In a preferred embodiment, the isolated or recombinant protein comprises less than 25 Endotoxin Units (EU)/mg protein as an amount of endotoxin impurities. In a preferred embodiment, the isolated or recombinant protein comprises less than 2 endotoxin units per mg of protein, most preferably less than 1.5 endotoxin units per mg of protein.

Endotoxin was detected using the Limulus Amoebocyte Lysate (LAL) test to detect and quantify bacterial endotoxin extracted from the outer membrane of gram-negative bacteria (Associates of Cape Cod, philips, uk). The key component of LAL reagent used in endotoxin test is derived from blood cells (amoebocytes) of horseshoe crab (Tachypleus americanus). The LAL test is described in chapter one of the "bacterial endotoxin test (Bacterial Endotoxins Test)" in the United states Pharmacopeia (United States Pharmacopeia) (chapter <85 >), and in chapter equivalent to those in the European Pharmacopeia (European Pharmacopoeia) (chapter 2.6.14) and Japanese Pharmacopeia (Japanese Pharmacopoeia) (general test, no. 4.01).

In contrast to the native protein which is glycosylated, the proteins of the invention are non-glycosylated or substantially non-glycosylated.

In another aspect, the 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.

In a further aspect there is provided a recombinant vector comprising a nucleic acid molecule as defined above.

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

In a further aspect, the present invention provides an isolated or recombinant protein as defined above for use in medicine or veterinary medicine. The proteins of the invention may be used in humans or non-human animals.

In a further aspect, the present invention provides an isolated or recombinant protein as defined above for use in the treatment and/or prophylaxis of inflammatory disorders. Preferably, the treatment and/or prevention of inflammatory conditions 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 recall antigens (e.g., tuberculin purified protein derivatives) or mitogens (e.g., phytohemagglutinin (PHA)) or anti-CD 3 or anti-CD 28 antibody coated magnetic beads by T cell proliferation.

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 graft rejection, 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 used to treat or prevent a disease of a disorder of bone metabolism, 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 loss of bone flow (http:// www.nationalbreastcancer.org/metastatic-breast-cancer).

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

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

The dose may be 1mg, 5mg or 15mg.

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

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

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

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.

Numerical ranges include numbers defining the ranges, and any single value provided herein can be used as an end point for a range that includes other single values provided herein. For example, a set of values (e.g., 1, 2, 3, 8, 9, and 10) are also disclosed as numerical ranges of 1-10, 1-8, 3-9, and the like. Also, the disclosed range is a disclosure of each individual value covered by the range. For example, the ranges 5-10 are also disclosed as 5, 6, 7, 8, 9 and 10.

In a further aspect, the present invention provides a pharmaceutical composition comprising an isolated or recombinant protein as defined in any preceding claim together with one or more pharmaceutically acceptable excipients, adjuvants or carriers. The one or more pharmaceutically acceptable excipients, adjuvants or carriers are not particularly limited and suitable excipients, adjuvants or carriers are known to those skilled in the art.

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.

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

The pharmaceutical composition may include a pharmaceutically acceptable carrier, such as physiological saline. Suitable pharmaceutical compositions may include one or more of buffers (e.g., acetate, phosphate, citrate), surfactants (e.g., polysorbate), stabilizers (e.g., human albumin, polyols, amino acids), preservatives (e.g., sodium benzoate), and/or other conventional solubilizing or dispersing agents.

The pharmaceutical compositions of the invention may be in the form of sterile aqueous solutions which may contain other substances, such as enough salts or glucose to render the solution isotonic with blood. If necessary, the aqueous solution should be suitably buffered (preferably at a pH of 3 to 9). 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 antioxidants, buffers, bacteriostats and solutes to render 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 medicaments and compositions may be presented in unit-dose or multi-dose containers, such as sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, such as water for injection, 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 a pH of 7.2 to 7.6, most preferably at a pH of 7.4. In one embodiment, the pharmaceutical composition comprises 0.9% w/v saline.

In one embodiment, the pharmaceutical composition comprises an isolated or recombinant protein in an amount of 2.0 to 50.0mg/mL, optionally 2.0 to 10.0mg/mL, preferably in an amount of about 5.0 mg/mL.

Typically, the pharmaceutical composition is suitable for intravenous administration.

In another aspect, the present 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 "treating" or "treating" refer to therapeutic measures that cure, slow, alleviate, and/or prevent the progression of an undesired physiological condition, a symptom of a diagnosed pathological condition, disease or 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 exhibits a total, partial, or temporary reduction or elimination of symptoms associated with the condition, disease, or disorder, a reduction in the extent of the condition, disease, or disorder, stabilization of the condition, disease, or disorder (i.e., without worsening), a delayed onset or progression of the condition, disease, or disorder, an improvement of the condition, disease, or disorder, including a partial or total relief, and/or prolonged survival compared to the intended survival without treatment.

"Prevention" refers to a prophylactic (prophylactic/PREVENTATIVE) measure that avoids and/or slows down the progression of a targeted pathological condition, disease or disorder. Thus, those in need of prevention include those susceptible to or suffering from the condition, disease or disorder. In certain embodiments, a 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 has delayed onset of symptoms associated with the condition, disease or disorder, briefly or permanently, as compared to a patient not subjected to the methods of the invention.

In one embodiment, one or more therapeutic agents are further administered to the patient or are administered in combination with one or more therapeutic agents when the protein is provided. In a preferred embodiment, the therapeutic agent is selected from the group consisting of disease modifying agents, analgesics, anti-inflammatory agents, immunotherapeutic agents, antibiotics, antibodies and steroids. In a particular embodiment, the therapeutic agent is a disease modifying antirheumatic drug (DMARD).

In another aspect, the invention provides a method for preparing a recombinant protein consisting of an 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 said microorganism, thereby producing the protein of SEQ ID NO. 4;

c) Lysing the microorganism to release the protein;

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

e) Separating and purifying the protein using immobilized metal affinity chromatography, wherein the immobilized metal is cobalt, and

F) Contacting said purified protein with a diaminopeptidase, wherein said diaminopeptidase cleaves said histidine tag from the N-terminus of said protein, and

G) Separating the cleaved protein from the histidine tag.

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

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

Typically, the method comprises one or more additional steps of treating the protein with a detergent to remove endotoxin. The detergent may be 1,3- (tetramethylbutyl) phenyl-polyethylene glycol. As an alternative, the protein may be treated with arginine in order to remove endotoxin.

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

Step f) is carried out at a temperature suitable for the activity of the diaminopeptidase, preferably at about 37 ℃.

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

Preferably, the method does not comprise more than one freeze-thawing step. Preferably, the protein is not frozen at any stage of the process. If the protein needs to be stored between the steps of the method, the protein is stored at a temperature of 2 to 8 ℃.

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

In one embodiment, the cells are lysed by shearing, particularly using a French press.

In another embodiment, host cells other than bacteria are used to produce the proteins of the invention. In one embodiment, the protein of the invention is produced using cells derived from yeast, insects, or fungi. In a preferred embodiment, mammalian cells are used to produce the proteins of the invention.

Drawings

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

FIG. 1 shows the amino acid sequence of native BiP. The primary structure of BiP is composed of 664 amino acids. At the N' end, 18 amino acid leader sequences are cleaved (underlined) during post-translational changes.

FIG. 2 shows the nucleotide sequence of the native BiP. The BiP gene is 2.5 kilobases.

FIG. 3 shows SEQ ID NO. 4, which is a protein of the present invention comprising a His tag at the N-terminus prior to cleavage during purification.

FIG. 4 shows SEQ ID NO.3, which is a protein of the present invention (not comprising a His tag)

FIG. 5A shows the recombinant vector pQE-2 used for cloning the native BiP gene. The schematic representation of the vector used shows the histidine-tagged site and the cleavage site for the restriction enzyme. FIG. 5B shows the BiP sequence cloned into the NdeI/NotI site of vector pQE-2 (SEQ ID NO: 8).

FIG. 6 shows an alignment of the amino acid sequence of the protein of SEQ ID NO. 4 according to the present invention with the amino acid sequence of a native protein.

FIG. 7 shows SEQ ID NO.1, which is SEQ 1 from WO 00/21995.

FIG. 8 shows SEQ ID NO. 2, which is SEQ 2 from WO 00/21995.

FIG. 9 shows SEQ ID NO. 7.

FIG. 10 shows a comparison of cytokine production induced by SEQ ID NO. 1 and SEQ ID NO. 3. Peripheral blood mononuclear cells were cultured for 24 hours in the presence of SEQ ID NO:1 (A, B) or SEQ ID NO:3 (C, D, E and F) at the indicated concentrations. PBMCs from 4 healthy subjects (filled symbols) and one rheumatoid arthritis patient (open symbols) were used. At 24 hours, supernatants were collected and Tumor Necrosis Factor (TNF) alpha and Interleukin (IL) 10 production was quantified by enzyme-linked immunosorbent assay (ELISA). E and F show the same data as C and D, but with a separate y-axis scale to allow five samples to be observed to be identified.

FIG. 11 shows data from experiments using Peripheral Blood Mononuclear Cells (PBMC) cultured alone or in combination with SEQ ID NO:3 or SEQ ID NO:1 for 24 hours followed by flow cytometry analysis using fluorochrome conjugated antibodies anti-CD 80, phycoerythrin anti-CD 86, fluorescein Isothiocyanate (FITC) or HLA-DR. FITC. In all cases, PBMC samples were controlled by a shutter (live gate) to enter only the CD14 population.

PBMC were cultured either 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 shows down-regulation of CD86 and HLA-DR. In contrast, SEQ ID NO:3 according to the present invention does not exhibit a significant loss of HLA-DR and CD86 expression.

FIG. 12 shows the effect of SEQ ID NO. 3 on serum C-reactive protein (CRP) levels obtained from patients in a first human clinical trial against SEQ ID NO. 3. Three groups of patients are shown, placebo, active responders (R) and all patients receiving SEQ ID NO 3. The change in serum levels of CRP at 2 and 12 weeks post-infusion compared to pre-infusion levels was measured for placebo, response and all patients treated with SEQ ID NO 3. At week 12, a significant decrease in CRP levels was found in patients treated with SEQ ID NO 3. * The patient abandoned his concomitant methotrexate medication, a behaviour that violated the regimen.

FIG. 13 shows the change 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 concentration at 2 and 12 weeks compared to pre-infusion were calculated for each patient. A change in VEGF concentration, and a change in IL-8 concentration. The data show that placebo group (n=6), responder group (R) [ n=8 (2 weeks) and 6 (12 weeks) ], and that the total patient group treated with SEQ ID NO:3 remaining under study at 12 weeks [ n=14 (2 weeks) and 12 (12 weeks) ]. The concentration ranges (all patients) were VEGF,4-195pg/ml, IL-8,0.7-19pg/ml.

Fig. 14 shows up-regulation of expression of the marker CD39, which increases the functional efficiency of regulatory T cells. Peripheral blood mononuclear cells from RA patients were established in culture either unstimulated (open bars), or with SEQ ID NO:1 (10. Mu.g/ml) (diagonal bars) or SEQ ID NO:3 (10. Mu.g/ml) (black bars). After 24 hours, 48 hours or 72 hours, cells were removed from the culture and stained with a set of fluorochrome conjugated antibodies to CD45, CD3, CD4, CD25, CD127 and CD 39. Cells were analyzed on a FACSCanto flow cytometer (BD Biosciences). The results were expressed as Mean Fluorescence Intensity (MFI) (A) for CD39 by the CD25hi and CD127lo cells after entry into living, CD45+, CD3+, CD4+ cells using multiple flaps (B) for the CD45+CD3+CD4+CD25hiCD127lo population in (A). (C) PBMC (10 ⁶/ml) were pretreated in culture with SEQ ID NO:1 (10 or 0.1. Mu.g/ml) or SEQ ID NO:3 (10 or 0.1. Mu.g/ml) according to the invention for 96 hours, washed and added to fresh autologous CFSE stained T cells, and stimulated with anti-CD 3 and anti-CD 28 antibodies coated magnetic beads. The ratio of pretreatment T cells to responder T cells was 1:10. After 3 days, cells were analyzed for CFSE MFI reduction on a FACSCalibur flow cytometer (BD Biosciences) using Cellquest software. In the case of pre-incubation of T cells with SEQ ID NO. 1, NO inhibition of the response was observed, but in the case of pre-incubation of cells with SEQ ID NO. 3, a decrease of up to 30% was observed. (D) In the RAGULA clinical trial, changes in CD39 expression on Treg cells (viable, CD45+CD3+CD4+CD25hi CD127 lo) in whole blood samples from placebo and SEQ ID NO 3 treated rheumatoid arthritis patients (responders (R) or Non Responders (NR)) were monitored over 12 weeks. Results were expressed as% change in cell surface expression at indicated time points compared to prior to infusion. Cd39+ expression increased significantly after a single infusion for at least 12 weeks after infusion.

FIG. 15 shows the results of flow cytometry analysis of the expression of CD115/c-Fms (A) and RANK (B) protein levels by M-CSF dependent human osteoclast precursors cultured for 48 hours in the absence or presence of SEQ ID NO:1 (2. Mu.g/ml). Representative samples of mean fluorescence intensity are shown as 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 precursor with SEQ ID NO 1 (2. Mu.g/ml). The data show the mean ± SEM of duplicate experiments using specific primers and normalized to β -actin. * p <0.05. (D) Western blot analysis showed that pERK and pp38 in human osteoclast precursors were responsive to expression of RANKL (10 ng/ml) in cells cultured in the absence or presence of SEQ ID NO:1 (2. Mu.g/ml, 48 hours) for the indicated time. (E) In a culture containing mature human osteoclasts cultured in the absence or presence of SEQ ID NO. 1 (2. Mu.g/ml, 48 hours), RANKL induced expression of pERK. (F) Western blot analysis showed that transcription factors c-Fos and NFATc1 respond to the expression of RANKL (10 ng/ml) in osteoclast precursors and mature osteoclasts treated in the absence or presence of BiP (2. Mu.g/ml). As indicated, total ERK and p38 proteins and GAPDH were used as loading controls. * p <0.01. This indicates that SEQ ID NO 1 down regulates CD115 and RANK cell surface expression and downstream signaling in human osteoclast precursors.

FIG. 16 shows that SEQ ID NO 3 inhibits nuclear translocation of NF- κ B p65 and p52 in osteoclast precursors and THP1 monocytes following TNFa and RANKL stimulation. M-CSF-dependent (A) human osteoclast precursor or (B) THP-1 cells were pretreated for 1 hour in the absence (Co) or in the presence of SEQ ID NO:3 (10. Mu.g/ml) (A, B) and then stimulated with TNF alpha (10 ng/ml) for 10 minutes. (C) The osteoclast precursor was incubated for 4 hours with or without RANKL (50 ng/ml) in the presence or absence of SEQ ID NO:3 (10. Mu.g/ml). After staining NF- κ B p65 (a, B) or p52 (C) with DAPI counterstain, cells were fixed and flow cytometry, flow cytometry imaging or confocal microscopy was performed. The graph on the right of each panel shows confocal images of nuclear translocation of p65 and p52 in representative single cells, indicating the absence of nuclear translocation in cells treated with SEQ ID NO 3. * p <0.01, n=3.

FIG. 17 measurement of immune response of patients using stimulated PBMC cultures after treatment with SEQ ID NO 3 or placebo to detect T cell responses to either the maximum stimulated, anti-CD 3 and anti-CD 28 antibodies coated magnetic beads (3 days of culture) (FIG. 17A) or to recall antigen, tuberculin PPD (5 days of culture) (FIG. 17B). Activation was measured by tritiated thymine uptake at the last 24 hours of incubation.

Fig. 18 (upper panel) shows a schematic of the protocol and results of the murine skin graft experiment. Fig. 18 (bottom panel) shows the survival analysis in KAPLAN MEIER panels. This shows that SEQ ID NO 3 allows 5/6 grafts to survive beyond the control, with 50% of the grafts survive approximately 30% longer than the control mouse grafts.

Fig. 19 shows a schematic of a second graft exploratory experiment. SEQ ID NO:3 administration again resulted in longer survival of the skin grafts compared to those animals given modified Dendritic Cells (DCs). There is NO benefit in mixing DC administration with SEQ ID NO 3.

FIG. 20 shows cytokine production by tissue surrounding the prosthesis cultured with and without SEQ ID NO 3. With the prosthetic joint loose and the patient fully informed consent, similarly sized pieces were excised from the prosthetic surrounding tissue obtained during the revision surgery. The tissue was cultured for 24-72 hours in the absence (control) or in the presence of SEQ ID NO:3 (20. Mu.g/ml). The cytokine Tumor Necrosis Factor (TNF) alpha or Interleukin (IL) 10 was quantified by a commercial enzyme-linked immunosorbent assay (ELISA) (PHARMINGEN, BD, oxford, uk). Both figures show that although the amount of TNFα in the control and SEQ ID NO:3 cultures showed little change in all 4 cultures, IL-10 production was increased.

Detailed Description

Examples

EXAMPLE 1 preparation of proteins of the invention

The BiP gene was modified to place the His tag at the N-terminus of the molecule. After the 6x histidine tag was placed so that the protein was affinity purified on a cobalt column, it could be removed by enzymatic digestion with diaminopeptidase. Nickel is not used because if enough nickel remains to contaminate the formulation, it may cause allergic reactions. A combination of temperature change and detergent is used to effectively remove endotoxin.

Yield and purification

By means of the removable His tag system, both the yield and the purity of the protein are greatly improved.

TABLE 1 yield of SEQ ID NO:3 from bacterial precipitation:

TABLE 2 purity and yield of SEQ ID NO:3

Testing	Results
		Protein concentration	5.45mg/ml
Endotoxin (endotoxin)	1.51EU/mg protein
		Filter integrity test for filter for sterile filtration of pharmaceutical substances	3900mBar
Biological load	No growth

Comparative example preparation of SEQ ID NO. 7

During the development of the protein of the invention, it was decided that the molecule had to have the correct KDEL sequence at the C' end and had to have no other tag attached to the protein. The present proteins are prepared according to standard recombinant techniques known to those skilled in the art. However, the protein is too little, the purity is too low, and almost all biological activity is lost. Therefore, the protein cannot be used.

SEQ ID NO. 7 corresponds to SEQ ID NO. 1 (SEQ 1 of WO 00/21995) with the His tag removed and the KDEL amino acid sequence restored, but there is NO other change compared to SEQ1 of WO 00/21995. It has been shown that this protein is difficult to purify, the final protein purity is <90%, and the endotoxin load is too high for clinical use. This suggests that it is difficult to provide analogues of natural BiP that are easy to prepare, stable and biologically active and suitable for administration to humans.

Four test batches were prepared to try and improve pure protein yield, purity and endotoxin contamination reduction. This has proven impossible. Protein recovery was about 1%, but endotoxin levels were still high.

Example 2

A comparison of TNF alpha production induced by the proteins of SEQ ID NO. 3 and SEQ ID NO. 1 is provided in FIG. 10. Peripheral Blood Mononuclear Cells (PBMC) 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.

In the presence of SEQ ID NO. 3, TNF alpha production by PBMC is greatly reduced compared to SEQ ID NO. 1. Furthermore, the protein of the invention did not differently increase tnfα production of rheumatoid arthritis PBMCs (compared to healthy controls), whereas the protein of SEQ ID NO:1 appears to induce greater tnfα production of RA PBMCs (compared to healthy PBMCs).

The clinical significance of TNFα in the pathogenesis of Rheumatoid Arthritis (RA) is well established (see "cytokine role in rheumatoid arthritis: pathophysiology and therapeutic education (Role of cytokines in rheumatoid arthritis:an education in pathophysiology and therapeutics)",Feldmann M、Maini SR," immune comment (Immunol Rev); month 6 of 2008; 223:7-19). Thus, an important positive feature of the protein of the invention is that it does not up-regulate tnfα.

Example 3

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-associated (HLA-DR) is a molecule constitutively expressed by monocytes, macrophages and dendritic cells (commonly referred to as antigen presenting cells, each of which has an antigenic peptide ready for presentation to the cd4+ T cell receptor). However, for complete T cell activation, two signals are required, one signal linked through HLA-DR-T cell receptor while the second signal is linked through CD28 via CD86 or CD 80. The second signal is provided by co-stimulatory molecules CD86 and/or CD80, which are also expressed by antigen presenting cells, initially binding to CD28 expressed by CD 4T cells, which are then down-regulated, while CTLA-4 is up-regulated. 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 sustained T cell activation.

The interaction of these four molecules helps to modulate the immune response. Thus, it is notable that SEQ ID NO. 1 shows down-regulation of CD86 as well as HLA-DR. This serves to reduce T cell activation as shown by a decrease in the in vitro response of BiP-treated PBMCs to recall antigen (e.g., tuberculin PPD), but also suggests the possibility of generalized immunosuppression, which is clinically not beneficial (MICHAEL DANDEL, hans Brendan Lehmkuhl, christoph Knosalla, roland Hetzer, influence of different long-term maintenance immunosuppression treatment strategies on patient prognosis after heart transplantation (Impact of different long-term maintenance immunosuppressive therapy strategies on patients'outcome after heart transplantation)"," transplantation immunology (TRANSPLANT IMMUNOLOGY), volume 23, stage 3, month 7, pages 93-103) in the long term, see fig. 11D. In contrast, SEQ ID NO:3 according to the present invention did not cause significant loss of HLA-DR (FIG. 11E) and CD86 expression.

Fig. 11 shows the results of flow cytometry experiments on cd14+ cells. In the presence of SEQ ID NO. 3 (FIG. 11B), CD80 expression was increased relative to unstimulated cells (FIG. 11A) and CD86 expression was increased in the presence of SEQ ID NO. 1 (FIG. 11C), CD80 expression was increased relative to unstimulated cells, but CD86 expression was not increased.

In summary, this suggests two important points of interest. First, while reducing T cell activation reduces inflammation, as shown in the case of SEQ ID NO:1, in the long term, general suppression of the immune system is not beneficial to the patient, which results in increased infection, etc. Second, SEQ ID NO 3 according to the present invention has shown anti-inflammatory efficacy in an in vivo model. This suggests that modulation of the immune system addresses chronic inflammation by BiP specific activity, thereby avoiding general immunosuppression.

Example 4 clinical data

In active RA patients who have failed the treatment received, the results from a randomized placebo-controlled, dose escalated double blind phase I/II clinical trial indicate that the proteins of the invention are safe. In addition, biomarker analysis showed considerable anti-inflammatory activity as well as clinical benefit (see Kirkham B, chaabo K, hall C, garrood T, mant T, allen E et al, safety of immunoglobulin-binding proteins as indicated by biomarker changes and patient response (Safety and patient response as indicated by biomarker changes to binding immunoglobulin protein in the phase I/IIARAGULA clinical trial in rheumatoid arthritis)"," Rheumatology (Rheumatology), 2016; 55:1993-2000) in phase I/IIA RAGULA clinical trial of rheumatoid arthritis

Twenty-four active RA patients who were not responsive to treatment with one or more disease modifying antirheumatic drugs (DMARDs) were sequentially divided into three groups, eight patients in each group were randomly assigned to receive placebo (two patients) or SEQ ID NO 3 according to the invention (six patients), at doses of 1, 5 or 15mg. Patients received a single i.v. infusion over 1 hour and were observed overnight as hospitalized patients. In the next 12 weeks, patients were monitored by follow-up clinical and laboratory evaluations for safety, efficacy (DAS 28-ESR) and biomarker analysis.

Safety of

Infusion reactions or severe adverse drug reactions were not observed. Adverse events were evenly distributed between placebo and active groups without drug-related toxicity. Blood, kidney and metabolic parameters did not show drug-related toxicity.

Efficacy of

Disease activity scoring (DAS 28) has been developed as a dynamic assessment tool for clinical trials and practice, as well as a therapeutic response measure. DAS28-ESR used the following disease indicators: joint tenderness count (28 joints), joint swelling count (28 joints), erythrocyte Sedimentation Rate (ESR) and general health status reported by patients on a 100mm visual analog scale, see Prevoo, ML et al, arthritis and rheumatism (ARTHRITIS RHEUM), 1995;38:44-8.

The primary efficacy endpoints were DAS28-ESR responses, classified as good, moderate and no response according to EULAR response criteria, where remission is defined as DAS28-ESR less than 2.6 (Kirkham B, chaabo K, hall C, garrood T, mant T, allen E et al, safety of immunoglobulin-binding proteins as indicated by biomarker changes in the I/IIA phase RAGULA clinical trial of rheumatoid arthritis and patient response thereto (Safety and patient response as indicated by biomarker changes to binding immunoglobulin protein in the phase I/IIARAGULA clinical trial in rheumatoid arthritis)"," Rheumatology (2016; 55:1993-2000). The biological efficacy endpoints were changes in CRP (fig. 12), IL-8 and VEGF (fig. 13), see further discussion below. These are commonly used to monitor disease activity in clinical drug trials for rheumatoid arthritis treatment.

Clinically, good EULAR responses were more prevalent in those treated with higher doses of SEQ ID NO:3, where a consistently low DAS28 score (3 to 12 weeks) was observed in three patients receiving SEQ ID NO:3, whereas by comparison, NO good DAS28-ESR response was observed in patients receiving placebo, but obtained in all treatment groups.

Serum VEGF and IL-8 concentration

FIG. 13 shows the change in serum levels of VEGF or IL-8 per patient at 2 or 12 weeks in the presence of SEQ ID NO:3 as measured by Luminex technology (Bio-Rad, hermerependde, UK) compared to the pre-infusion baseline. The analysis contained only patients who were still under study at 12 weeks (fig. 13).

It was demonstrated that analysis of CRP, VEGF and IL-8 could be used to distinguish subjects receiving active drug (compared to placebo). Patients responding to SEQ ID No. 3 showed a significant decrease in CRP at 2 weeks (pre-infusion level 12.7±1.7, and post-infusion 2 week level 7.1±2.1; p=0.02) compared to placebo and non-responder groups. Serum VEGF and IL-8 are common biomarkers in clinical trials because they are closely related to the measurement of synovitis and monocyte infiltration, respectively. Significant changes in the levels of these biomarkers occurred in the patient group receiving SEQ ID NO 3. Furthermore, biomarkers do not support clinical improvement in placebo patients. Surprisingly, fewer placebo-receiving patients exhibited a decrease in serum VEGF and IL-8 (17% and 50%, respectively) at week 12 compared to the SEQ ID NO 3 responder group (71% and 83%, respectively). Interestingly, even the group of SEQ ID NO:3 non-responders showed a decrease in serum concentration (66% and 83% of patients, respectively), indicating a change in pathology of their disease.

In summary, active-responsive patients exhibited a significant decrease in serum concentration of CRP (fig. 12) compared to pre-infusion levels and serum concentration of VEGF and IL-8 (fig. 13) compared to placebo after 2 weeks of infusion. This indicates that the disease inflammation is much less than the pre-infusion level.

Example 5 SEQ ID NO:3 upregulating CD39 on regulatory T cells

Peripheral blood mononuclear cells from RA patients were established in culture either unstimulated (open bars), or with SEQ ID NO:1 (10. Mu.g/ml) (diagonal bars) or SEQ ID NO:3 (10. Mu.g/ml) (black bars, FIG. 14).

After 24 hours, 48 hours or 72 hours, cells were removed from the culture and stained with a set of fluorochrome conjugated antibodies to CD45, CD3, CD4, CD25, CD127 and CD 39. Cells were analyzed on a FACSCanto flow cytometer (BD Biosciences).

The results in fig. 14 are expressed as Mean Fluorescence Intensity (MFI) for the following:

(A) CD25hi and CD127lo cells after entry into living, cd45+, cd3+, cd4+ cells using multiple shutters;

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

(C) PBMC (10 ⁶/ml) were pretreated in culture with SEQ ID NO:1 (10 or 0.1. Mu.g/ml) or SEQ ID NO:3 (10 or 0.1. Mu.g/ml) according to the invention for 96 hours, washed and added to fresh autologous CFSE stained (cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester) T cells, and coated with anti-CD 3 and anti-CD 28 antibodies for magnetic bead stimulation. The ratio of pretreatment T cells to responder T cells was 1:10. After 3 days, cells were analyzed for decreased CFSE MFI on FACS Calibur using Cellquest software. As cells proliferate, the content of CFSE will decrease with each division, while non-proliferated cells will remain highly stained.

(D) In clinical trials, changes in CD39 expression on Treg cells (viable, CD45+CD3+CD4+CD25hi CD127 lo) in whole blood samples from placebo and SEQ ID NO 3 treated rheumatoid arthritis patients (responders (R) or Non Responders (NR)) were monitored over 12 weeks. Results were expressed as% change in cell surface expression at indicated time points compared to prior to infusion.

In vitro comparison between SEQ ID NO. 1 and SEQ ID NO. 3 according to the present invention shows that although the actual increase in the number of tregs is very low, thus confirming previous studies on SEQ ID NO. 1, there is a large difference in% CD39 expression on tregs in the case of SEQ ID NO. 3 when compared directly with SEQ ID NO. 1. Interestingly, at 72 hours, CD39 expression on tregs from the SEQ ID NO:1 culture was lower than that of control cells. In clinic, a significant increase in CD39 expression on Treg cells from patients responding to SEQ ID NO 3 was observed and this was maintained for 12 weeks after infusion.

Example 6 SEQ ID NO:3 inhibits osteoclast differentiation signaling pathway

To investigate the potential mechanisms 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 cytometry analysis of CD115/c-Fms and RANK (receptors for M-CSF and RANKL, respectively) in human peripheral blood source M-CSF dependent osteoclast precursors showed that SEQ ID NO 3 down-regulates CD115 expression by 63.+ -.16% (inhibition range, 43-79%) (FIG. 15A). SEQ ID NO. 1 similarly inhibited RANK protein expression by 51.+ -. 29% (inhibition range, 22-90%) (FIG. 15B). 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 RNAs (fig. 15C). To address whether a decrease in receptor expression results in a decrease in responsiveness to osteoclast-generating cytokines, we analyzed the effect of the treatment of SEQ ID NO 1 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. 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 cultures similarly showed that RANKL-induced pERK levels were also attenuated in cultures enriched for mature osteoclasts after treatment with SEQ ID NO:1 (fig. 15E).

Next, we studied the effect of SEQ ID No. 1 on the expression of transcription factors c-Fos and NFATc1, which are critical for osteoclast differentiation and are located downstream of RANK and tnfα signaling in osteoclast precursors and monocytes. Pre-incubation of human osteoclast precursors with SEQ ID NO. 1 greatly reduced activation of the c-Fos protein after RANKL treatment (FIG. 15F). 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. 15F). Mature osteoclast cultures treated with SEQ ID NO. 1 also showed a significant decrease in endogenous expression of c-Fos and NFATc1 transcription factors (FIG. 15F).

Since SEQ ID NO.1 inhibits the signal transduction pathway required for monocyte differentiation into osteoclasts, we studied whether SEQ 3 has a similar effect on NF-KB, which is one of the transcription factors driving inflammation, but RANK-RANKL requires an alternative pathway to drive downstream differentiation.

Imaging flow cytometry showed that treatment with SEQ ID NO:3 inhibited TNFα -induced nuclear translocation of p65NF- κB in osteoclast precursors (FIG. 16A). Similar results were obtained in response to RANKL stimulation (data not shown). Since RANKL also stimulates cells via a non-classical NF- κb pathway, we studied 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. 16B). These results indicate that SEQ ID NO:3 blocks both classical and non-classical NF-. Kappa.B 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 basic osteoclast transcription factors NF-kappa B, c-Fos and NFATc1, thereby providing insight into the mechanism by which SEQ ID NO. 3 inhibits osteoclast differentiation and resorption activity.

The data provide evidence that SEQ ID NO 3 may be useful in the treatment of diseases of dysregulation of bone metabolism.

Example 7

Following treatment with SEQ ID NO 3 or placebo, the immune response of the patient was measured using stimulated PBMC cultures to detect T cell responses to either the maximum stimulated, anti-CD 3 and anti-CD 28 antibodies coated magnetic beads (3 days of culture) (FIG. 17A) or to recall antigen, tuberculin PPD (5 days of culture) (FIG. 17B).

Activation was measured by uptake of tritiated thymidine by proliferating cells during the last 24 hours of culture. The data indicate that there was no change in response to mitogens or recall antigens within 12 weeks of the clinical trial. This indicates that SEQ ID NO. 3 does not have an overall immunosuppressive effect, unlike SEQ ID NO.1, which has been disclosed to reduce the recall antigen response to tuberculin PPD (Corrigall VM, bodman-Smith MD, brunst M, cornell H, panayi GS, stress protein BiP stimulates human peripheral blood mononuclear cells to express an anti-inflammatory cytokine profile and inhibits antigen presenting cell function: correlation with treatment of inflammatory arthritis (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 and rheumatism (ARTHRITIS RHEUM), 2004; 50:1167-1171).

Example 8 transplantation skin graft mouse model

FIG. 18 shows a schematic of the protocol and results of murine skin grafting experiments in which anti-CD 8 antibodies were administered to all recipient mice eight days prior to transplantation to deplete endogenous dendritic cells. There were 6 mice in each of the four groups. Seven days prior to implantation, control mice received vehicle only. SEQ ID NO 3 (20. Mu.g/mouse) was administered intravenously. Two additional 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 to the back of recipient mice.

Analysis of graft survival by KAPLAN MEIER panels showed that SEQ ID NO 3 resulted in 5/6 grafts surviving more than the control group, with 50% of the grafts surviving approximately 30% longer than the control mice grafts.

The presented data indicate that SEQ ID NO 3 is effective in maintaining graft survival in a skin graft mouse model, which is said to be very difficult to maintain graft survival.

A second graft exploratory experiment was also performed (see fig. 19). SEQ ID NO:3 administration again resulted in longer survival of the skin grafts compared to those animals given modified Dendritic Cells (DCs). There is NO benefit in mixing DC administration with SEQ ID NO 3.

EXAMPLE 9 prosthetic joint loosening

Upon loosening of the prosthesis, the tissue developed around the joint of the prosthesis, i.e. the peri-prosthetic tissue, is very similar to synovium, which may be inflamed during RA. Such tissue may lead to loosening of the prosthetic joint. Prosthetic surrounding tissue (PPT) is collected during revision surgery for prosthetic joint replacement. Tissues were cut into equal weight pieces and cultured overnight in tissue culture medium (1 ml) in twenty-four well plates with or without the presence of SEQ ID NO: 3. Culture supernatants were collected between 24 hours and 72 hours and tnfα pro-inflammatory cytokines or interleukin-10 anti-inflammatory cytokines were quantified by a commercial enzyme-linked immunosorbent assay (ELISA) (PHARMINGEN, BD, oxford, uk).

FIG. 20 shows that although the proteins of the invention have little effect on TNF alpha production, IL-10 is significantly increased.

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

Example 10

Tables 4 and 5 summarize the physical and functional differences between the protein of the invention, SEQ ID NO:1 and native BiP. This clearly summarizes the significant differences between the proteins of the invention, the previously disclosed recombinant BiP SEQ ID NO:1 and the native protein.

TABLE 4 Table 4

The chicken collagen model improves severe arthritis n=1; reduction of DAS28 in clinical trials, CIA = collagen-induced arthritis

Table 5.

Claims (10)

1. An isolated or recombinant protein consisting of an amino acid sequence according to SEQ ID NO. 3 or SEQ ID NO. 4.

2. The isolated or recombinant protein of claim 1, comprising an amount of endotoxin impurities of 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 an amino acid sequence according to SEQ ID No. 4.

5. The isolated or recombinant nucleic acid molecule according to claim 4, which consists of a 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. The isolated or recombinant protein of claim 1 or claim 2 for use in medicine or veterinary medicine.

8. The isolated or recombinant protein of claim 1 or claim 2 for use in the treatment and/or prevention of an inflammatory condition, optionally for use in the treatment and/or prevention of an inflammatory condition in a human subject.

9. The isolated or recombinant protein of claim 8 for use according to claim 8, wherein the treatment and/or prevention of an inflammatory condition 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 an inflammatory condition is achieved without significant immunosuppression, as measured in terms of T lymphocyte activity relative to activity prior to administration of the protein.