WO2002072133A1 - Immunomodulatory properties of bip - Google Patents
Immunomodulatory properties of bip Download PDFInfo
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- WO2002072133A1 WO2002072133A1 PCT/GB2002/001151 GB0201151W WO02072133A1 WO 2002072133 A1 WO2002072133 A1 WO 2002072133A1 GB 0201151 W GB0201151 W GB 0201151W WO 02072133 A1 WO02072133 A1 WO 02072133A1
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- bip
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- homolog
- immune
- immune response
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to substances having immunomodulatory properties and to the uses of such substances for the treatment or prevention of an unwanted immune response.
- the present invention also relates to a pharmaceutical composition comprising a substance having immunomodulatory properties.
- Autoimmune diseases are one class of diseases of the first kind.
- Examples of the second kind of situation are responses which interfere either with the function of transplanted tissues and organs (allo- or xeno-graft rejection) or which inactivate substances used for gene therapy and which have not previously been encountered by the immune system of the recipient.
- a similar example of the latter kind is the response following the infusion of therapeutic proteins collectively known as 'biologies' of human or non-human origin, including monoclonal antibodies and other therapeutic proteins such as blood clotting factors, and enzymes.
- the therapeutic approach of the present invention is based on BiP, the 78kD endoplasmic reticulum chaperone.
- BiP the 78kD endoplasmic reticulum chaperone.
- BiP protein from human cells has a high degree of homology with BiP from other species and the term BiP is therefore used herein to embrace all such proteins which have the property of inducing EL- 10. Minor variations on the specific DNA and amino acid sequences disclosed in WO 00/21995 are also to be included provided the above property is retained as discussed further herein.
- BiP adjuvant arthritis
- the present invention provides the use of BiP or a functional fragment or homolog thereof, or a nucleic acid molecule encoding BiP or a functional fragment or homolog thereof, in the manufacture of a medicament for the treatment or prevention of an unwanted immune response.
- RA rheumatoid arthritis
- CIA collagen-induced arthritis
- the use of the present invention is for preventing an unwanted immune response.
- BiP refers to the 78kD endoplasmic reticulum ohaperone protein as defined in WO 00/21995. It has been found that BiP causes: CD 14+ cells to release EL- 10; stimulates CD8+ cells to proliferate and release EL- 10; inhibits the recall antigen response; and activates the expression of an array of anti-inflammatory genes in monocytes, including the migration inhibitory factor (MEF), the soluble TNF receptor II and TEVEPs.
- MEF migration inhibitory factor
- the BiP protein has the amino acid sequence given in WO 00/21995 as SEQ ED NO. 1 or SEQ ED NO. 2.
- a functional fragment refers to a fragment of BiP which is capable of eliciting at least part of an activity of the full BiP protein.
- the functional fragment has at least one of the following functions: causes CD 14+ cells to release EL- 10; stimulates CD8+ cells to proliferate and release EL- 10; inhibits the recall antigen response; or activates the expression of an array of anti-inflammatory genes in monocytes, including the migration inhibitory factor (MEF), the soluble TNF receptor II and TEMPs.
- the functional fragment is at least 20 amino acids, more preferably at least 50 amino acids and most preferably at least 100 amino acids in length.
- Particularly preferred fragments comprise a conserved region which has been found to be homologous to a number of naturally occurring BiP proteins. Such conserved regions are considered to have a specific function.
- a functional homolog refers to a homolog that retains at least part of an activity of the BiP protein described in WO 00/21995.
- the functional homolog has at least one of the following functions: causes CD14+ cells to release EL-10; stimulates CD8+ cells to proliferate and release EL-10; inhibits the recall antigen response; or activates the expression of an array of anti-inflammatory genes in monocytes, including the migration inhibitory factor (MEF), the soluble TNF receptor II and TEMPs.
- MEF migration inhibitory factor
- the functional homolog has at least 80%, more preferably at least 90% and most preferably at least 95% amino acid sequence homo logy with one of the BiP proteins described in WO 00/21995.
- sequence homology is measured by using BLAST analysis. It is particularly preferred that the functional homolog differs by only 1 to 20 amino acids from one of the BiP proteins described in WO 00/21995. It is further preferred that the amino acid changes are conservative. Conservative changes are those that replace one amino acid with one from the family of amino acids which are related in their side chains. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a major effect on the biological activity of the protein.
- the nucleic acid molecule used in the present invention encodes BiP or a functional fragment or homolog thereof as defined above.
- the nucleic acid molecule can be obtained by methods well known in the art. For example, naturally occurring sequences may be obtained by genomic cloning or cDNA cloning from suitable cell lines or from DNA or cDNA derived directly from the tissues of an organism, such as a human or mouse. Positive clones may be screened using appropriate probes for the nucleotide molecule desired. PCR cloning may also be used. The probes and primers can be easily generated given that the sequence of BiP is known (see WO 00/21995).
- the nucleic acid molecule has the sequence given in WO 00/21995 as SEQ ED NO. 3.
- nucleic acid or the probes and primers for identifying the positive clones.
- the nucleotide molecules probes or primers may be synthesised completely using standard oligonucleotide synthesis methods, such as the phosphoramidite method.
- the nucleic acid is preferably in the form of a vector comprising the necessary elements leading to the expression of the nucleic acid sequence encoding BiP or a functional fragment or homolog thereof.
- the vector comprises a promoter operably linked to the nucleic acid sequence and a transcription termination sequence. Suitable promoters, transcription termination sequences and other functional elements required to obtain expression of the nucleic acid are well known to those skilled in the art.
- the nucleic acid may be delivered to the individual using any method.
- the nucleic acid may be delivered as a free nucleic acid, in the form of a viral delivery vector such as an adenovirus, contained in a liposome or via any known method.
- the unwanted immune response may be any unwanted immune response. Specific unwanted immune responses are discussed in details below.
- TX allo- and xeno- transplants
- Present anti-rejection regimens are expensive, require life-long administration, may produce toxic side effects and are not universally effective.
- TX are grafted at a precisely known time. Just as the inventors have shown that BiP is able to prevent CIA and AA if given, respectively, at the time of or before the induction of arthritis, it is recommended to administer BiP just before or at the time of TX to prevent rejection.
- TX that may be beneficially treated in this way include all TX of tissues and organs, whether solid (for example, liver, kidney) or single cell (for example, blood cells, bone marrow cells or stem cells).
- a range of biologic therapies are used in clinical medicine. These include products from non-human sources and products from human sources.
- the biologies may be purified from a natural source, produced by recombinant gene technology, secreted after transfection of genes, or synthesised. These biologies may be proteins, glycoproteins or complex sugars.
- a disadvantage is that this has the potential to induce an immune response when administered to an immunologically naive individual.
- the unwanted immune response is associated with an immune-mediated disease.
- Immune-mediated diseases include auto-immune diseases.
- Specific immune-mediated diseases include type-1 diabetes, thyroididtis, multiple sclerosis, systemic lupus erythematosus, Crohn's disease and all forms of viral and autoimmune hepatitis.
- the use according the first embodiment of the present invention additionally comprises the use of an agent for enhancing the treatment or prevention of the immune-mediated disease in the manufacture of the medicament.
- the agent for enhancing the treatment or prevention of the immune-mediated disease may be any agent including EL-10, EL-4, EL-11, TGF-beta, EL- 13 and soluble cytokine receptors such as EL-lRa, EL-1 and TNF soluble receptors.
- the medicament is for administration to an individual suffering from or susceptible to developing an immune-mediated disease.
- Methods are know for determining whether an individual is suffering from an immune-mediated disease and methods are known for determining if an individual is likely to develop an immune-mediated disease. Methods for determining whether an individual is likely to develop an immune include analysing risk factors such as genetic markers or environmental influences such as diet, etc.
- the medicament obtained by the use according to the first embodiment of the present invention is preferably administered to an individual before the immune-mediated disease develops or as soon as the immune-mediated disease has been diagnosed.
- the unwanted immune response is associated with the rejection of a transplanted organ, tissue or cells.
- the rejection response is well know and occurs when donated tissue is recognised as foreign by the recipient's immune system.
- the rejection response occurs with transplanted organs, such as heart, lung, kidney, liver, etc., transplanted tissues, such as skin, muscle tissue, etc., and with transplanted cells, such as bone marrow cells and stem cells.
- the use according the second embodiment of the present invention additionally comprises the use of an agent for enhancing the treatment or prevention of the immune response associated with rejection of a transplanted organ, tissue or cells, in the manufacture of the medicament.
- the agent for enhancing the treatment or prevention of the immune response associated with the rejection of transplanted organs, tissue or cells may be any agent that suppresses the immune system including glucorticoids, cyclosporin A, azathioprine, rapamycin and tacrolimus.
- the medicament obtained by the use according to the second embodiment of the present invention is preferably administered to an individual before or at substantially the same time as the transplantation of the organ, tissue or cells.
- the unwanted immune response is the immune response to a biologic.
- a biologic is any therapeutic agent given to an individual.
- the biologic may be from non-human or human sources.
- the biolgic may be a protein molecule (i.e. an enzyme, an antibody molecule, receptor ligand, etc), a glycoprotein, a polypeptide, peptide, carbohydrate, or an organic or inorganic chemical compound.
- biologies can cause unwanted immune responses.
- an immune response can be raised against the biologic which may prevent the therapeutic activity of the biologic.
- the immune response may be so large that it lead to anaphylatic shock.
- anti-TNF ⁇ therapy has resulted in the shortening of the interval between dosing (infliximab) this increasing the cost, and its use has been limited by anaphylaxis.
- the use according the third embodiment of the present invention additionally comprises the use of an agent for enhancing the treatment or prevention of the immune response to the biologic.
- the agent for enhancing the treatment or prevention of the immune response to the biologic may be any agent that suppresses the immune system including glucorticoids, cyclosporin A, azathioprine, rapamycin and tacrolimus.
- the medicament obtained by the use according to the third embodiment of the present invention is preferably administered to an individual before or at substantially the same time as the biologic.
- the present invention also provides the use of BiP or a functional fragment or homolog thereof, or a nucleic acid molecule encoding BiP or a functional fragment or homolog thereof, for stimulating the release of EL-10 from cells capable of releasing EL-10.
- the cells are peripheral blood mononuclear cells (PBMCs).
- the PMBCs are CD 14+ monocytes and/or CD8+ T cells and/or CD4+ T cells.
- BiP or a functional fragment or homolog thereof, or a nucleic acid molecule encoding BiP or a functional fragment or homolog thereof is used to stimulate the release of EL-10 from PBMCs in vitro or ex vivo.
- BiP or a functional fragment or homolog thereof, or a nucleic acid molecule encoding BiP or a functional fragment or homolog thereof is used to additionally stimulates gene expression of at least one of monocyte migration inhibitory factor (MEP), soluble TNF receptor Et, EL-10 anti-inflammatory mediators and tissue inhibitor of matrix metalloproteinases (TEMP).
- MMPs matrix metalloproteinases
- MCP-1 monocyte chemoattractant protein
- the present invention also provides a pharmaceutical preparation comprising BiP or a functional fragment or homolog thereof, or a nucleic acid molecule encoding BiP or a functional fragment or homolog thereof, in combination with a pharmaceutically acceptable carrier for use in the treatment or prevention of an unwanted immune response.
- the pharmaceutical composition additionally comprises an agent for enhancing the treatment or prevention of the unwanted immune response.
- the pharmaceutical composition of the present invention comprises a therapeutically effective amount of BiP or a functional fragment or homolog thereof, or a nucleic acid molecule encoding BiP or a functional fragment or homolog thereof.
- therapeutically effective amount refers to an amount of a therapeutic agent needed to treat or prevent the unwanted immune response.
- the therapeutically effective dose can be estimated initially either in cell culture assays, or in animal models, usually mice, rabbits, dogs, or pigs.
- the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
- the precise effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician.
- an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg.
- compositions of this invention comprise BiP or a functional fragment or homolog thereof, or a nucleic acid molecule encoding BiP or a functional fragment or homolog thereof, with any pharmaceutically acceptable carrier, adjuvant or vehicle.
- Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxy
- compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Oral administration or administration by injection are preferred.
- the pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles.
- parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
- the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension.
- This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
- the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
- suitable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution.
- sterile, fixed oils are conventionally employed as a solvent or suspending medium.
- any bland fixed oil may be employed including synthetic mono- or diglycerides.
- Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
- These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. Helv or a similar alcohol.
- compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions.
- carriers which are commonly used include lactose and corn starch.
- Lubricating agents such as magnesium stearate, are also typically added.
- useful diluents include lactose and dried corn starch.
- aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
- compositions of this invention may also be administered in the form of suppositories for rectal administration.
- These compositions can be prepared by mixing a novel agent of this invention with a suitable non- irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
- suitable non- irritating excipient include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
- Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application.
- the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier.
- Carriers for topical administration of the novel agents of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
- the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active agent suspended or dissolved in a carrier.
- Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
- the pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches are also included in this invention.
- compositions of this invention may be administered by nasal aerosol or inhalation.
- Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
- the present invention also provides a method of treating or preventing an unwanted immune response comprising administrating to an individual in need of such treatment an effective dose of the pharmaceutical composition according to the present invention.
- Peptides of the CDR3 region of the T cell receptor Valpha and Vbeta chains can be synthesised and used as vaccines for delivery by intradermal or intramuscular injection (see Kotzin et al, Arthritis Rheum., ⁇ , 1906-1919, 1998). BiP or a functional fragment or homolog thereof can be used in the same way.
- BiP or a functional fragment or homolog thereof may be given parenterally or orally in appropriate cases either unmodified or modified by amino acid substitution and/or attachment of chemical groupings so as to block MHC and especially HLA-DR4 thereby leading to suppression of T cell activation and disease.
- BiP or a functional fragment or homolog thereof may be combined with soluble HLA-DR4 molecules and applied parenterally or orally.
- Plasmids consisting of nucleic acid coding for BiP or a functional fragment or homolog thereof, may be given by injection.
- DNA coding for human EL-10, EL-4, EL-11, or TGF-beta, incorporated singly or in any combination, may be used to deviate the immune response to BiP towards a TH2 mode so as to suppress disease.
- the present invention is now described by way of example only with reference to the following Figures:
- Figure 1 shows the prevention of adjuvant arthritis by BiP.
- adjuvant arthritis was induced by a single intradermal injection of 0.5 mg M.tuberculosis in 100 ⁇ l EFA in the base of the tail.
- Figure 2 shows the results of the investigation into BiP binding to peripheral blood mononuclear cell (PBMC) populations and fibroblast like synoviocytes by double immunofluorescence.
- PBMC peripheral blood mononuclear cell
- FITC human serum albumin
- Column B FITC conjugated BiP, the PBMC were double stained with CD14, CD20, CD4, CD8, CD56.
- Column C shows BiP.FITC binding to 2 rheumatoid arthritis fibroblast-like synoviocytes compared with the HSA.FITC negative control.
- Figure 3 shows EL-10 production following culture of peripheral blood mononuclear cells (PBMC) with BiP (20 ⁇ g/ml), beta-galactosidase (b-gal) (20 ⁇ g/ml) or lipopolysaccharide (LPS) (20 ng/ml) in the absence (A) or presence (B) of polymixin B for 24 hours. Culture supernatants were collected and EL-10 was measured by ELISA.
- PBMC peripheral blood mononuclear cells
- BiP beta-galactosidase
- LPS lipopolysaccharide
- FIG. 4 shows the proliferation of CD8 clone FC2B5 to BiP (closed circles) and control antigen ⁇ -galactosidase (open circles). This clone was generated from the peripheral blood of a normal individual. This profile is representative of other BiP responsive clones.
- FIG 5 shows the cytokine profiles of BiP responsive clones and lines.
- the cytokine levels were measured in supernatants of cells previously shown to be BiP responsive, stimulated by mitogen. The profiles are compared with irradiated feeder cells alone (first data set).
- Figure 6 shows the proliferative response of T cells to BiP stimulation.
- Figure 7 shows BiP-driven T cell cytokine production from animals immunised with BiP.
- Figure 8 shows BiP driven T cell cytokine production from control animals.
- Figure 9 shows the uptake of tritiated thymidine following an allogeneic reaction between peripheral blood monocytes (MO) either cultured for 5 days in tissue culture medium, or matured with granulocyte macrophage-colony stimulating factor (GM-CSF) + EL-4, or with GM-CSF + EL-4 + BiP, prior to irradiation and culture with allogeneic peripheral blood mononuclear cells.
- MO peripheral blood monocytes
- Figure 10 shows the proliferation response of peripheral blood mononuclear cells measured by the uptake of tritiated thymidine in either unstimulated cultures (TCM) or cultures stimulated with BiP (20 ⁇ g/ml) or tuberculin PPD (lO ⁇ g/ml) or with BiP and PPD.
- BiP or human serum albumin were prepared at a concentration of 2 mg/ml in carbonate buffer 0.1M pH9.6.
- a stock solution of fluorescein isothiocyanate (FITC) was prepared at 10 mg/ml in carbonate buffer 0.1M pH 9.6.
- 50 ⁇ g FITC/mg protein was added to the protein solution in a glass container covered in foil. The solution was placed on a circular mixer and incubated at room temperature for 2 hours.
- the FITC labelled protein was then placed in dialysis tubing (which had been boiled for 5 mins with each of three changes of fresh distilled water) and dialysed overnight in 5 litres of phosphate buffered saline (PBS) (0.15M NaCl, 4mM NaH 2 PO 4 , 0.01M Na 2 HPO 4 pH 7.2) followed by two further changes of 5 litres PBS.
- PBS phosphate buffered saline
- PBMC peripheral blood mononuclear cells
- lOO ⁇ l of cells were placed in a tube and lO ⁇ l of 1/5 normal human serum added.
- the cells are incubated on ice for 10 minutes and then washed twice at 300g in PBS/BSA/az at 4°C.
- the required amount of the FITC conjugated protein was then added to the cells in conjunction with any other protein directly conjugated to a different fluorochrome, such as phycoerythrin (PE), and the tube vortexed.
- PE phycoerythrin
- the actual amount of protein added must be determined for each conjugation by a dose response curve.
- the cells were incubated on ice for 20 minutes and then washed twice at 300g in PBS/BSA/az at 4°C.
- BiP.FITC was used at 1/50 dilution and counter-stained with anti-CD20 (B cell marker), CD3, CD4, CD8 (T cell markers), CD56 (NK cell marker), or CD14 (monocyte marker) all directly PE conjugated and used at 5 ⁇ l/100 ⁇ l (Becton Dickinson, Oxford, UK). After the final wash the cells were fixed in PBS/BSA/az with 1% paraformaldehyde in 250 ⁇ l aliquots. The cells were then analysed on a FACScan using Cellquest software (Becton Dickinson, Oxford, UK).
- Mononuclear cells were plated at lxl 0 6 cells ml "1 in 2ml culture wells in the presence of
- Lymphocult-T (LC-T) was added to the cultures (40 ⁇ lml "1 ) as a source of interleukin-2 (EL-2).
- EL-2 interleukin-2
- lxlO 6 irradiated autologous feeder cells were added to each well with 40 ⁇ lml " ' LC-T and 20 ⁇ gml '1 BiP. This regime was continued for 3 rounds of feeder cells and then the cells plated at 10 cells per well into 96U plates with lxlO 4 ⁇ -irradiated allogeneic feeder cells (4000 Rads: 137 Cesium source) and 2 ⁇ gml '1 Phytohaemagglutinin (PHA).
- lxlO 4 cloned cells were incubated for three days with lxlO 5 irradiated autologous feeder cells in the presence or absence of BiP (20 ⁇ gml-') or PHA (2 ⁇ gml 1 ).
- the cells were incubated for the last 18 hours with 3 H-thymidine (0.2 ⁇ Ci) and then harvested. Proliferation was expressed as a stimulation index (SI): proliferation in the presence of stimulant/proliferation in the presence of medium alone.
- SI stimulation index
- Phenotypic analysis was carried out on responding clones using CD3, 4 and 8 (BD). Briefly, cells were washed in FACS buffer (phosphate buffered saline containing 1% bovine serum albumin and 0.05% sodium azide) and incubated with 4 ⁇ l of the appropriate antibodies. Three-colour analysis was performed using a FACScan flow cytometer and cell-quest software. T cell receptor usage was determined using a panel of both FITC conjugated and non-conjugated antibodies. Briefly, for conjugated antibodies lxlO 4 cells were incubated with 4 ⁇ l of each of the FITC conjugated V beta specific antibodies. Clones were also stained with 4 ⁇ l of both anti-CD4 conjugated to FITC and CD8 conjugated to PE.
- FACS buffer phosphate buffered saline containing 1% bovine serum albumin and 0.05% sodium azide
- cells were incubated for 40 mins on ice with the primary antibody, washed twice in FACS buffer, then incubated for a further 40 mins with a FITC conjugated goat antibody raised against mouse immunoglobulins.
- EL interleukin 4 and EL-l, and ⁇ -interferon and tumour necrosis factor- ⁇ were determined by ELISA (Pharmingen, according to manufacturers instructions). Briefly, plates (Nunc Maxisorp) were coated with a cytokine specific capture antibody, blocked with 10% foetal calf serum (FCS: to stop non-specific binding) and the supernatants incubated on the plate overnight at 4°C. Bound cytokine was detected with a biotin conjugated detection antibody and visualised with streptavidin conjugated horseradish peroxidase and TMB.
- FCS foetal calf serum
- PBMC peripheral blood mononuclear cells
- BiP tuberculin purified protein derivative
- Tritiated thymidine was added to the cultures for the final 6 hours. The cells were then harvested and the uptake of tritiated thymidine assayed using a dry matrix beta counter (Canbarra-Packard, Pangbourne, UK).
- Cytokine expression array Monocytes (MO) were separated by negative selection using an immunomagnetic bead kit (Dynal, Wirral, UK) and placed in culture at 2.10 6 /ml for 24hours either alone or stimulated with BiP(20 ⁇ g/ml) or with PMA (lOng/ml) +IONO(250ng/ml). The supernatants were harvested for ELISA and the cells were processed for extraction of total RNA and production of cDNA using oligo d(T) primers and reverse transcriptase. The expression array (R&D Systems, Oxford, UK) was used according to the manufacturer's instructions.
- BiP The ability of BiP to prevent CIA and AA suggested to the inventors that it may have a generic ability to downregulate immune responses.
- BiP was conjugated to fluorescein isothiyocyanate (FITC) and used in flow cytometry. As can be seen from Figure 2, BiP binds to different populations of cells found in human peripheral blood but especially to human CD 14+ monocytes.
- FITC fluorescein isothiyocyanate
- EL-10 interleukin 10
- TNF tumour necrosis factor
- CD8+ cells from peripheral blood of humans subjects may be stimulated by BiP to proliferate (Figure 4). Indeed, the inventors have generated clones of such CD8+ T cells. These clones do not secrete the pro-inflammatory cytokine interferon (EFN) ⁇ but do secrete EL-10 ( Figure 5). Thus these cells have the characteristics of Tel regulatory CD8+ T cells and are able to downmodulate immune responses. C Inhibition of recall antigen responses by PBMC pre-treated with BiP
- D BiP induces monocytes to activate a more anti-inflammatory array of genes compared with activation by phorbol myristic acid (PMA) and calcium ionophore (IONO).
- PMA phorbol myristic acid
- IONO calcium ionophore
- BiP specifically induced gene activation for monocyte migration inhibitory factor (MEF), soluble TNF receptor TJ and EL-10 anti-inflammatory mediators, upregulated tissue inhibitor of matrix metalloproteinases (TEMP) but did not induce matrix metalloproteinase (MMP) or monocyte chemoattractant protein (MCP)-l, potent pathogenic mediators, or the inflammatory cytokine, TNF ⁇ .
- MMP matrix metalloproteinase
- MCP monocyte chemoattractant protein
- Tuberculin purified protein derivative (PPD) stimulation is a measure of the lymphocytes response to recall antigens (an antigen to which the subject has already been immunised).
- Figure 9 shows that allogeneic peripheral blood lymphocytes react to the resting monocytes with a low response.
- the monocytes are matured into dendritic cells, using the well established technique of adding granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (EL-4) for 5 days, the response by the allogeneic PBMC is greatly increased.
- GM-CSF granulocyte macrophage colony stimulating factor
- EL-4 interleukin-4
- Figure 10 shows that the PPD response by PBMC is significantly reduced to background levels when BiP is added to the PPD cultures.
- BiP As a therapeutic drug two important properties need to be ascertained: first, the nature of the cytokines released after parenteral administration of BiP and, second, the optimum dose of BiP needed to achieve the maximum release of cytokines
- mice Male DBA-1 mice (8-12 weeks old) were immunised subcutaneously (s.c.) with 200 ⁇ g of BiP in phosphate buffered saline (PBS). PBS alone or bovine serum albumin (BSA) were administered as controls. 14 days later, spleens and lymph nodes were removed and T cell cultures set up and stimulated with varying concentrations of BiP at 0.1, 1, 10, 20 ⁇ g/ml. After 4 days of culture, the pro-inflammatory cytokine interferon (EFN)- ⁇ and the anti-inflammatory cytokines interleukin (EL)-4, EL-5 and EL-10 were assayed. T cell proliferation was assessed by 3 H-thymidine incorporation.
- EFN pro-inflammatory cytokine interferon
- EL interleukin
- BiP is surprisingly immunogenic and immunising mice with BiP results in a raised anti-inflammatory cytokine profile with a switch towards a TH2 profile.
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- Epidemiology (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP02704972A EP1370278A1 (en) | 2001-03-13 | 2002-03-13 | Immunomodulatory properties of bip |
US10/471,572 US20040116344A1 (en) | 2001-03-13 | 2002-03-13 | Immunomodulatory properties of bip |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB0106161.3 | 2001-03-13 | ||
GBGB0106161.3A GB0106161D0 (en) | 2001-03-13 | 2001-03-13 | Immunomodulators |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2002072133A1 true WO2002072133A1 (en) | 2002-09-19 |
Family
ID=9910568
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB2002/001151 WO2002072133A1 (en) | 2001-03-13 | 2002-03-13 | Immunomodulatory properties of bip |
Country Status (4)
Country | Link |
---|---|
US (1) | US20040116344A1 (en) |
EP (1) | EP1370278A1 (en) |
GB (1) | GB0106161D0 (en) |
WO (1) | WO2002072133A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008536904A (en) * | 2005-04-19 | 2008-09-11 | キングス カレッジ ロンドン | use |
WO2019239126A1 (en) | 2018-06-13 | 2019-12-19 | Immune Regulation Limited | Novel protein with anti-inflammatory properties |
WO2023187422A1 (en) * | 2022-03-31 | 2023-10-05 | Revolo Biotherapeutics Limited | Compositions and their use in methods for treating intestinal inflammation |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997006685A1 (en) * | 1995-08-18 | 1997-02-27 | Sloan-Kettering Institute For Cancer Research | Method for treatment of cancer and infectious diseases and compositions useful in same |
EP0927757A1 (en) * | 1997-12-03 | 1999-07-07 | Leadd B.V. | Methods and means for inducing apoptosis by interfering with Bip-like proteins |
WO2000021995A1 (en) * | 1998-10-09 | 2000-04-20 | King's College London | Treatment of inflammatory disease |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5348945A (en) * | 1990-04-06 | 1994-09-20 | Wake Forest University | Method of treatment with hsp70 |
US5891653A (en) * | 1995-12-29 | 1999-04-06 | Attfield; Derrick Cecil | Method of suppressing graft rejection by means of stress proteins |
-
2001
- 2001-03-13 GB GBGB0106161.3A patent/GB0106161D0/en not_active Ceased
-
2002
- 2002-03-13 US US10/471,572 patent/US20040116344A1/en not_active Abandoned
- 2002-03-13 WO PCT/GB2002/001151 patent/WO2002072133A1/en not_active Application Discontinuation
- 2002-03-13 EP EP02704972A patent/EP1370278A1/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997006685A1 (en) * | 1995-08-18 | 1997-02-27 | Sloan-Kettering Institute For Cancer Research | Method for treatment of cancer and infectious diseases and compositions useful in same |
EP0927757A1 (en) * | 1997-12-03 | 1999-07-07 | Leadd B.V. | Methods and means for inducing apoptosis by interfering with Bip-like proteins |
WO2000021995A1 (en) * | 1998-10-09 | 2000-04-20 | King's College London | Treatment of inflammatory disease |
Non-Patent Citations (1)
Title |
---|
CORRIGALL VALERIE MARY ET AL: "A proposed immunoregulatory role for BiP through IL-10 production.", FASEB JOURNAL, vol. 15, no. 5, 8 March 2001 (2001-03-08), Annual Meeting of the Federation of American Societies for Experimental Biology on Experimental Biology 2001;Orlando, Florida, USA; March 31-April 04, 2001, pages A1061, XP001080616, ISSN: 0892-6638 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008536904A (en) * | 2005-04-19 | 2008-09-11 | キングス カレッジ ロンドン | use |
US8076293B2 (en) | 2005-04-19 | 2011-12-13 | Gabriel Stavros Panayi | Use of BiP or a variant, homologue, derivative or fragment thereof in the manufacture of a medicament for the prevention or treatment of bone loss or bone resorption |
WO2019239126A1 (en) | 2018-06-13 | 2019-12-19 | Immune Regulation Limited | Novel protein with anti-inflammatory properties |
WO2023187422A1 (en) * | 2022-03-31 | 2023-10-05 | Revolo Biotherapeutics Limited | Compositions and their use in methods for treating intestinal inflammation |
Also Published As
Publication number | Publication date |
---|---|
EP1370278A1 (en) | 2003-12-17 |
GB0106161D0 (en) | 2001-05-02 |
US20040116344A1 (en) | 2004-06-17 |
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