Patent Grant
12115214
The invention relates a compound comprising (a) a peptide and (b) a carrier, wherein said peptide having at least the motif X-X-X-X-X-X-X, wherein at least one amino acid residue X is glycosylated, said peptide being linked to the peptide binding protein and said carrier comprises at least a MHC binding motif being linked to said peptide as well as pharmaceutical compositions comprising said compound and the use of said compound or pharmaceutical composition for the treatment of a disease, such as an inflammatory joint disease.
There is an increasing population of humans suffering from different kinds of inflammatory joint diseases. Diseases, which sometimes are impossible to cure, where the treatment is lifelong and where the symptoms often become worse during the years. So far the focus of the treatment has been on trying to find compounds, which reduce the symptoms but not cure the disease or make the disease decline.
One example of such a disease is rheumatoid arthritis (RA), which is characterized by chronic inflammation of the articular synovial tissues initiated by leukocyte infiltration (mainly neutrophils, macrophages and T cells) and secretion of inflammatory cytokines (TNF-alpha, IFN-gamma, IL-I, IL-6), chemokines and destructive enzymes such as matrix metalloproteases. Activation of T cells is believed to be an important pathogenic factor in the disease although its exact role and potential as a therapeutic target has not yet been identified. The abnormal activation of T cells do, however, most likely occur years before the clinical diagnosis of the disease as T cell dependent IgG antibodies specific for immunoglobulin Fc (i.e rheumatoid factors) and citrullinated protein epitopes are highly predictive for disease (1, 2). Importantly, the risk for developing arthritis is dramatically increased in individuals who have both such antibodies and express certain MHC class II molecules, that share a specific peptide pocket, the so called MHC shared epitope (3, 4). The MHC class II region is also the strongest known genetic factor associated with RA. Taken together, these findings argue for a pathogenic role of MHC class II restricted autoreactive T cells. It has however been difficult to identify a single specificity of such T cells although T cell reactivity to several autoantigens, such as BiP, RA33 and GPI and also joint specific antigens such as type II collagen (CII), have been reported (5-8).
Since there is no way to cure inflammatory joint diseases today there is a need for developing a way to cure the disease.
The object of the present invention is to solve the above, discussed problem in connection with inflammatory joint diseases.
It has surprisingly been found that by the use of a compound comprising (a) a peptide and (b) a carrier, wherein said peptide having at least the motif X-X-X-X-X-X-X, wherein at least one amino acid residue X is glycosylated, said peptide being linked to the peptide binding protein and said carrier comprises at least a MHC binding motif being linked to said peptide it is possible for the first time to reduce and/or eliminate an inflammatory joint disease. Thereby a mammal suffering from such a disease will bee cured or at least the disease will be reduced.
Additionally the invention relates to a pharmaceutical composition comprising said compound as well as the use of said compound for the treatment of an inflammatory disease or disorder, such as arthritis, rheumatoid arthritis, ankylosing spondylitis, psoriasis arthritis, osteoarthritis, relapsing polychondritis and Menieres disease
In a specific embodiment, said compound is used to vaccinate a mammal and thereby cure or prevent an inflammatory disease or disorder.
Further advantages and objects with the present invention will be described in more detail, inter alia with reference to the accompanying drawings.
(A) HCQ.3 hybridoma, specific for GalOK264 CII259-273 epitope; (B) HCQ.4 hybridoma, specific for non-modified (K264) CII259-273 epitope.
In the context of the present application and invention the following definitions apply:
The term “MHC class II molecule” is intended to mean a protein consisting of an alpha and a beta chain, coded from two distinct genes. This protein is normally cell surface bound on so-called antigen presenting cells (APC) and serve as a receptor for peptides. The peptide is bound to a specific site, the peptide binding site, in the MHC molecule and the resulting protein structure (the peptide bound to the two chain MHC class II molecule) is recognized by the T cell receptor.
This interaction is the crucial molecular and antigen specific event in the immune response and has been well described.
The term “MHC binding motif” is intended to mean the amino acids of the variable region, (i.e. polymorphic) of an MHC class II molecule, which can contact and bind the peptide of the invention in the peptide-binding site. The variable region of an MHC class II molecule being defined as the first amino acid residues 1-90 of the alpha and the beta chain in the form seen on the cell surface.
The term “variable region on the MHC molecule” is intended to mean the first domain of both alpha and beta chains each encompassing amino acids 1-90.
The term “constant region on a MHC or Ig molecule” is intended to mean the part of the MHC class II molecule that is not part of the variable domains/regions.
The term “carrier” is intended to mean a compound, such as a protein, which can bind to the peptide of the invention and present that particular peptide to specific T cell receptors. T cell receptors (TCR), which normally are bound to T cells normally recognising type II collagen, a joint cartilage protein.
The term “peptide” is intended to mean a sequence of amino acid residues having from six to 50 amino acid residues.
The term “polypeptide” is intended to mean a sequence of amino acid residues having more 51 or more amino acid residues.
In the present context, amino acid names and atom names are used as defined by the Protein DataBank (PDB), which is based on the IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Amino Acids and Pep-tides (residue names, atom names etc.), Eur J Biochem., 138, 9-37 (1984) together with their corrections in Eur J Biochem., 152, 1 (1985). The term “amino acid” is intended to indicate an amino acid from the group consisting of alanine (Ala or A), cysteine (Cys or C) aspartic acid (Asp or D), glutamic acid (Glu or E), phenyl-alanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W) and tyrosine (Tyr or Y), or derivatives thereof.
Compound
The invention relates to a compound, comprising a peptide and a carrier.
Said peptide having at least the motif X-X-X-X-X-X-X, wherein at least one amino acid residue X is glycosylated, such as an O-linked galactose. Other examples of the glycosylated structures are N-acetyl galactosamine, glucose, N-acetyl glucosamine, glucose, mannose, fucose, as well as their mono- and dideoxygenated, mono- and difluorogenated, and C-glycoside derivatives. Said peptide being linked to the carrier and the linking between said peptide and said peptide binding protein may be covalent or they may be bound in another way as long as they can bind and remain bound to each other. The glycosylated amino acid residue X of said peptide may be hydroxylysine or a variant thereof. A variant of the lysine side chain could be any structure that is acceptable for recognition by a T cell receptor, such as hydroxynorvaline. Said peptide may comprise from 4 to 50 amino acid residues, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues. Said peptide may comprise at least one of the following amino acid sequences X—F-K-X-X-X-X-X-X-X (SEQ. ID. NO.: 12), X-F-K-X-E-X-X-X-X-X (SEQ. ID. NO.: 13), A-G-F-K-G-E-A (SEQ. ID. NO. 5) or A-A-A-K-A-A-A (SEQ. ID. NO.: 6) wherein K may be hydroxylysine. Additionally, said peptide also have a MHC class II binding motif, i.e., have the ability to bind to such a molecule. A MHC class II binding motif is capable of binding to a threedimensional structure formed by the alpha and the beta chains of the first domain of the MHC class II molecule. The amino acids of critical importance for this peptide binding structure are both located in a beta pleated sheath forming the bottom of the peptide binding structure (alpha 1-49, beta 1-49) and two alpha helices (alpha50-80, beta 50-90) forming the sides of these cleft-forming structure.
Peptides forming specific motifs unique for each MHC class II allele binds to this peptide binding structure. MHC class II alleles associated with rheumatoid arthritis forms peptide binding structure with many similarities encompassing them to bind peptides with certain binding motifs. One example of a peptide, according to the invention, is derived from collagen II, positions 260-273 (IAGFKGEQGPKGEP) (SEQ. ID. NO.: 11) which binds the DRB1*0401/DRA, the DRBI*1001/DRA and the DRB1*0101/DRA molecules. However, the peptide may be synthetic or semisynthetic or derived from other proteins as long as the peptide is identical or have similar structure as the peptide above. The MHC binding positions for binding to the DRB1*0401/DRA molecule has been hypothesised to be F263 and E266 in this peptide and the TCR binding positions K264, Q267 and K270. These TCR contacting positions could be of different importance for different TCR, i.e., they could be substituted into other amino acid residues.
Additionally, the above, mentioned peptide (IAGFKGEQGPKGEP) (SEQ. ID. NO. 11) may have one or more modifications such as hydroxylation, galactosylation or galactoglucosylation. Examples of positions, which could be modified, are positions K264 and K270 as well as deamidation of position Q267. Accordingly one or more of the amino acid residues may be substituted with another amino acid residue as long as it have the capability to be part of the MHC binding motif and function as a composition which can be used to prevent/reduce or treat a mammal suffering from a joint inflammatory disease or disorder or induce an immune response and thereby function as a vaccine. Examples of mammals includes human, dog, pig, sheep, cat, camel and horse.
The carrier of the compound to which the peptide is linked comprises at least the variable region of the MHC class II molecule, being defined as the first domains of the alpha and the beta chain, i.e., the first amino acid residues 1-90 of the alpha and the beta chain and a constant region. The alpha and the beta chains being linked to each other, such as by a leucine zipper domain.
The carrier comprises at least at least a MHC binding motif being linked to said peptide. However, one or more amino acid residues may be substituted as long as the carrier can bind the peptide and induce an immune response in a mammal. The variable part may be modified by up to 30 amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues. Examples of variable regions are those, which originate form MHC class II molecules DRB1*0401/DRA, DRBI*0402/DRA, DRBI*0403/DRA, DRBI*0404/DRA, DRBI*0405/DRA, DRB1*0406/DRA, DRBI*0407/DRA, DRBI*0408/DRA, DRBI*0409/DRA, DRBI*0410/DRA, DRBI*0101/DRA, DRB1*0102/DRA, DRBI*1001/DRA, DRBI*1002/DRA. Example sequences of one of the sequences to be used are depicted; SEQ NO 1, DRA V domain and SEQ NO 2, DRB1*0401 V domain.
According to one embodiment the compound comprises the above identified peptide and the carrier comprises a variable and a constant region, wherein said variable region comprises a peptide having 80% identity to the polypeptide sequence in its full length shown in SEQ ID NO 1 and a second polypeptide having 80% identity in its full length to the polypeptide sequence shown in SEQ ID NO 2, such as 85, 90, 95 or 100% identity in its foil length to one or both of the polypeptides shown in SEQ ID NO 1 and 2.
The carrier may also contain one or more constant regions, such as the constant regions of the MHC class II molecule or regions of an immunoglobulin such as the constant regions of IgG. For example, the class II first domain (i.e the variable or polymorphic domain) can be covalently linked to the IgG structure changing the first V domains of the VH and the VL chains. The constant and the variable regions may be covalently linked to each other. Said constant region may be selected from the group consisting of the constant region of an MHC class II molecule or an immunoglobulin. Example of a constant region being one comprising a peptide having 80% identity to the polypeptide sequence in its full length shown in SEQ ID NO 3 and a second polypeptide having 80% identity in its full length to the polypeptide sequence shown in SEQ ID NO 4, such as having 90% or 95% identity to the polypeptide sequence in its foil length shown in SEQ ID NO 3 and a second polypeptide having 90% or 95% identity in its foil length to the polypeptide sequence shown in SEQ ID NO 4 or being identical to the polypeptide sequence shown in SEQ ID NO 3 and 4.
In another embodiment, the constant region of the carrier may be a polypeptide sequence which is at least 25% of the polypeptide sequences SEQ ID NO 3 and 4, i.e., the constant region of the MHC class II, such as 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. Additionally there may be one or more substitutions of the polypeptides as long as the polypeptide sequences are linked together to form a carrier as defined above. The polypeptide sequences may be covalently linked to each other through a leucine zipper domain or part of a leucine zipper domain.
A Pharmaceutical Composition
According to another aspect the invention relates to a pharmaceutical composition comprising above defined compound and a pharmaceutically acceptable carrier, excipient or diluter.
Pharmaceutical formulations of the compound of the invention are typically administered in a composition that includes one or more pharmaceutically acceptable carriers and diluters. Such pharmaceutical compositions may be prepared in a manner known in the art that is sufficiently storage stable and suitable for administration to humans and animals. The pharmaceutical composition may be lyophilised.
“Pharmaceutically acceptable” means a carrier, diluent or excipient that at the dosage and concentrations employed does not cause any unwanted effects in the patients to whom it is administered. Such pharmaceutically acceptable carriers or excipients are well-known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000).
The pharmaceutical composition may be admixed with adjuvants such as lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, they may be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol, oils (such as corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilisation and/or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc., e.g., as disclosed elsewhere herein.
The pharmaceutical composition according to the invention may be administered locally or systemically such as topically, intravenously, orally, parenterally or as implants and even rectal use is possible. Suitable solid or liquid pharmaceutal preparation forms are, for example granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in the preparation of excipients, diluents, adjuvants or carriers are customarily used as described above.
The pharmaceutical composition will be administered to a patient in a pharmaceutically effective dose. By “pharmaceutically effective dose” is meant a dose that is sufficient to produce the desired effects in relation to the condition for which it is administered. The exact dose is dependent on the activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the patient and different doses may be needed. The administration of the dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals, e.g. vaccination.
The pharmaceutical composition of the invention may be administered alone or in combination with other therapeutic agents. These agents may be incorporated as part of the same pharmaceutical composition or may be administered separately. The “patient” for the purposes of the present invention includes both humans and other mammals. Thus the methods are applicable to both human therapy and veterinary applications.
Pharmaceutical formulations of the nucleotide sequence molecule or polypeptide of the invention are typically administered in a composition that includes one or more pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions may be prepared in a manner known in the art that is sufficiently storage stable and suitable for administration to humans and animals.
In a final aspect the above mentioned compound as well as pharmaceutical composition may be used for the treatment of a disease or a disorder such as an inflammatory joint disease threw vaccination. Examples of diseases are arthritis, reumatoid arthritis, anylosing spondylitis, psoriasis arthritis, osteoarthritis, relapsing polychondritis and Menieres disease. The vaccination may be performed by the use of any vaccination system such as DNA vaccination in which DNA is used that is translated into proteins in vivo corresponding to the above described structures. The said DNA can be administered as pure DNA or inserted into carrier structures.
Following examples are intended to illustrate, but not to limit, the invention in any manner, shape, or form, either explicitly or implicitly.
Design of the MHC Class II Aq Constructs
The cDNAs for Aalphaq and Abetaq were amplified from a first strand cDNA reaction (first strand cDNA, Pharmacia, Piscataway, NJ). The cDNAs were further modified to include cloning sites immediately upstream of the start codon, and the 3′ end from the transmembrane domain and downstream was replaced by an inframe cloning site. Next, DNA for the leucine zipper (13) domain from Jun including a 3′ end coding for 6 histidines was cloned in frame with the beta-chain cDNA. The DNA for the leucine zipper domain from Fos was added to the alpha chain construct. The resulting constructs were cloned separately into pMTAL (Invitrogen, La Jolla, CA) or pRmHa-3 (14) to allow for heavy metal-induced expression in insect cells. pMTAL contains the resistance gene for hygromycin. Where pRmHa-3 was used a Copia promoter-driven hygromycin gene was used as selection marker.
Transfection, Expression and Purification of Soluble Aq
The linearised Aq alpha-chain and Aq beta-chain constructs were co-transfected at equimolar ratios into Drosophila melanogaster SL2 cells (ATCC, CRL-1963) using calcium phosphate transfection. Stable transfectants were derived by hygromycin selection and kept under selection in Schneider's Drosophila medium (Gibco™, Paisley, Scotland, UK) containing 100 μg/ml of hygromycin B (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Large-scale cell cultures were prepared in Fernbach bottles using a magnetic stirrer. For expression of soluble Aq, transfected cells were grown in serum-free Insect express complete medium (PAA Laboratories GmbH, Linz, Austria) at 25° C., induced with 0.7 mM CuSO4 for three days, and the supernatants were clarified by centrifugation and filtration. The SL2 cells produced ˜2-3 mg of recombinant protein per liter of culture. The expressed soluble Aq molecules were purified from the clarified media using Ni-NTA (Qiagen GmbH, Hilden, Germany) affinity chromatography and the manufacturer's recommended protocol. The dialysed protein fractions were examined by ELISA, SDS-PAGE and Western blot analysis. Non-reducing SDS-PAGE analysis of NiNTA purified Aq on 4-20% gradient gel showed two bands with molecular weights of 29 and 33 kDa (approximately the predicted sizes of alpha and (3 beta chains), which demonstrates that the expressed proteins form heterodimers consisting of alpha and beta chains. Positive fractions were pooled, concentrated 5- to 10-fold by MICROSEP 30K OMEGA (PALL, GelmanSciences, Ann Arbor, MI) or Amicon® centrifugal filter devices (MILLIPORE Co, Billerica, MA) and loaded with a peptide to form MHC-peptide complexes. All protein concentrations were determined using a Dc protein assay (Bio-Rad Laboratories, Hercules, CA).
ELISA, SDS-PAGE and Western Blot Analyses of Aq
The alpha- and beta-chains of the purified Aq protein were detected by sandwich ELISA, using Y3P mAb (specific for the native alpha-chain) as capturing antibodies and biotinylated 7-16.17 (BD PharMingen, Los Angeles, CA) mAb (specific for the beta-chain) as detecting antibodies. Flat-bottom 96-well plates (Nunc, Roskilde, Denmark) were coated with 2.5 g/mL Y3P and incubated overnight at 4° C. The plates were then washed with PBS, blocked with 1% BSA (Sigma, St Louis, MO) in PBS for 1 h, washed again, and incubated for 2 h with 50 μL from the protein fractions at room temperature. Plates were washed again, followed by addition of 1 μg/mL biotinylated 7-16.17 for 1 h. After washing, the biotin-labeled antibody was detected by europium-labeled streptavidin using the DELFIA system (Wallac, Turku, Finland).
Protein purity was assessed by SDS-PAGE. Samples were electrophoresed in 4-20% polyacrylamide gradient ready mini-gels (Bio-Rad Laboratories, Hercules, CA) under denaturing and non-reducing conditions and the gels were silver stained according to the manufacturer's instructions. In parallel experiments, the gels were electrotransferred onto nitrocellulose membranes (0.45 μm). The membranes were blocked in 5% non-fat dry milk in PBS for 1 h and blotted with different MHC class II specific antibodies (M5/114, 7-16.17, 7-23.1, PCQ.6, 34-5-3, Y3P) at 4° C. overnight. After repeated washing, blots were incubated with peroxidase-conjugated goat anti-mouse IgG or goat anti-rat IgG (for M5/114) antibodies (Jackson) for 1 h. Immunoblots were developed using DAB (Vector Laboratories Inc., Burlingame, CA).
Preparation of Peptide/Aq Complexes
Empty soluble Aq molecules were loaded with 5 to 50-fold molar excess of GalOK264 CII259-273, non-modified CII259-273 or MOG79-90 peptides at 4° C. for 72 h. GalOK264 CII259-273 is a peptide from type II collagen (CII) position 259-273 which has a lysine at position 264, which is hydroxylated and galactosylated.
Non-modified is the same peptide but with a lysine without modifications of its side chain. MOG=myelin oligodendrocytic glycoprotein. MHC-peptide complexes were separated by anion-exchange HPLC (Resource™ Q column) using an ÄKTA™ explorer 100 Air system (Amersham Pharmacia Biotech AB, Uppsala, Sweden) with UNICORN V4.00 software. Separations were done with a loading solution of 10 mM Tris pH 8.5 (buffer A) and a gradient elution up to 1 M NaCl (buffer B) in 10 mM Tris. The eluted protein fractions were concentrated by ultrafiltration (MICROSEP 30K OMEGA), dialyzed against PBS and examined by ELISA, SDS-PAGE and T-cell hybridoma tests. MHC-peptide complexes were purified further on a Superdex 200 gel filtration column (Amersham Pharmacia Biotech AB, Uppsala, Sweden), concentrated again by Amicon® centrifugal filter devices (MILLIPORE Co, Billerica, MA) and stored at −20° C. until used.
Activation of T Cell Hybridomas
Peptide/AQ complexes were diluted in sterile PBS and coated onto plates by incubation at 4° C. for overnight or added directly in soluble form to the hybridomas. The coated plates were then washed twice with sterile PBS to remove unbound protein complexes, and 5×104 T-hybridoma cells were added per well in 200 μL of DMEM supplemented with 5% FCS, 100 IU/mL penicillin and 100 μg/mL streptomycin. T-cell hybridoma HCQ.3 and HCQ.4, specific for GalOK264 and for non-modified CII259-273 (K264), respectively (12), have been used in
After 24 h, IL-2 in the culture supernatants was measured by sandwich ELISA using DELFIA system (Wallac, Turku, Finland). Recombinant mouse IL-2 served as a positive control and standard. Data are represented as mean+−SE of triplicates
Induction and Clinical Evaluation of Arthritis
Animals
Male B10.Q, (B10.QxB10.RIII)F1 or B10.Qx(BALB/cxB10.Q)F2 mice, 8-10 weeks of age, were used in the experiments. The founders of the B10.Q and B10.RIII mice were originally provided by Dr. Jan Klein (Tubingen, Germany) and BALB/c mice purchased from The Jackson Laboratory (Bar Harbor, ME). The mice were bred and used at the animal department of Medical Inflammation Research (inflam.lu.se) and kept under standardized conditions.
In
In
In
In
Antigens
Rat type II collagen (CII) was prepared from the Swarm chondrosarcoma and bovine CII from joint cartilage, by limited pepsin digestion, and further purified as previously described (15). The CII peptides (non-modified CII259-273: GIAGFKGEQGPKGEP (SEQ. ID. NO.: 8), GalOK264 CII259-273: GIAGFK(Gal-Hyl)GEQGPKGEP (SEQ. ID. NO.: 9), and the various galactosylated peptides that were deoxygenated at OH groups on the galactose (position 2, 3 and 4 respectively) were synthesized, purified and characterized as previously described (9, 10, 11, 16, 17). The CII was dissolved in 0.1 M acetic acid. Mouse myelin oligodendrocytic glycoprotein MOG79-90 peptide (GKVTLRIQNVRF) (SEQ. ID. NO.: 10) was purchased from Schafer-N(Copenhagen, Denmark). All peptides were dissolved in PBS. The collagen and peptides were stored at 4° C. until used.
To induce CIA, each mouse was injected with 100 μg of CII (rat CII for B10.Q and bovine CII for (B10.QxB10.RIII)FI mice), emulsified 1:1 in complete Freund's adjuvant (CFA; Difco, Detroit, MI) at the base of the tail in a total volume of 100 μl. Thirty-five days later, the mice were given a booster injection of 50 μg of rat CII emulsified 1:1 in incomplete Freund's adjuvant (IFA; Difco, Detroit, MI) in a total volume of 50 μl. Development of clinical arthritis was followed through visual scoring of the animals based on the number of inflamed joints in each paw, starting two weeks postimmunisation and continuing until the end of the experiment. An extended scoring protocol (18) ranging from 1-15 for each paw with a maximum score of 60 per mouse was used. The mice were examined 2 to 4 times a week for at least 70 days after immunization.
The B10.Q(BALB/cxB10.Q)F2 mice were immunized with 100 μg of rat CII emulsified in IFA intradermally (i.d.) at the base of the tail on day 0 and boosted on day 35 i.d. with 50 μg of rat CII in IFA. The mice were scored for a minimum period of 202 days for arthritis development. Mice, which developed chronic arthritis (mice with severe arthritis for a minimum period of 120 days were considered as chronic) including the ones with clear relapses, were selected for the treatment protocol.
Peptide/Aq Complex Treatment Protocols
Animals were treated by either intravenous (i.v.) or intranasal (i.n.) administration of purified peptide/Aq complexes. In the intravenous treatment of CIA model, mice were injected with GalOK264/Aq, deoxygenated GalOK264/Aq,K264/Aq or as negative control MOG/Aq complex (100 μg in 200 μL PBS) on days 20 and 34 postimmunisation (for the chronic model on days 7, 11 and 28 after reimmunisation). Control mice were injected intravenously with 200 μL PBS on the same days. In the intranasal treatment experiments, mice were administrated with 10 μg (in 20 μL PBS) of peptide/AQ complex on the days mentioned above.
Measurement of Serum Anti-CII Antibody Levels
Mice were bled at the time of boost immunization (day 35) as well as at the termination of experiment (day 70) and sera were analyzed for anti-CII IgG antibody levels by quantitative ELISA (19). Briefly, 96-well ELISA plates (Nunc, Roskilde, Denmark) were coated overnight at 4° C. with 10 pg/mL native rat CII in PBS. The wells were washed three times with PBS-0.1% Tween20 and then 150 μL of blocking buffer (5% BSA in PBS) was added to each well and incubated for 1 h at room temperature. After washing, 50 μL of samples in serial dilutions from 1/100 to 1/105 were added and incubated for 2 h at room temperature. After three washes, peroxidase-conjugated goat anti-mouse IgG was added and incubated at room temperature for 1 h. After extensive washing, plates were developed using ABTS (Roche Diagnostics GmbH, Mannheim, Germany) as substrate and the absorbance was then measured at 405 nm in a SPECTRAMAX® Plus reader (Goteborgs Termometerfabrik, Göteborg, Sweden). A standard serum from arthritic and non-immunised syngeneic mice was added to each plate in serial dilutions as positive and negative controls, respectively.
B10.Q mice (10 mice per group) were immunised with 100 microgrammes rat CII in CFA on day 0 and boosted on day 35 with 50 microgrammesrat CII in IFA. On days 20 and 34, mice were treated by intravenous (i.v.) (100 microgrammesin 200 microlitres PBS) or intranasal (i.n.) (10 microgrammes in 20 microlitresPBS) administration of purified GalOK264/Aq complex. PBS (200 microlitres) was administrated (i.v.) as a control on the same days. Mice were monitored for clinical signs of arthritis for 70 days. Levels of IgG anti-CII were measured at days 35 and 70 by ELISA. All values are shown as mean±standard deviation. N/A denotes not applicable.
Histology
Hind paws were removed after ending the experiment, fixed in 4% neutral buffered formaldehyde overnight and then decalcified in 5% (w/v) EDTA at 4° C. until the bones were pliable. Tissues were then dehydrated in a gradient of alcohols, paraffin embedded, sectioned at 5 μm, mounted on glass slides, and stained with hematoxylin and eosin (H&E). Serial H&E-stained sections were analyzed microscopically for the degree of inflammation and for cartilage and bone destruction. Analyses were performed in a blinded fashion.
T-Cell Transfer
For the T-cell transfer experiment, 15 B10.Q mice (recipients) were immunized with CII/CFA (day 0) and boosted with CII/IFA on day 35 using the standard immunization protocol. At the same time (day 0), three groups (5 mice per each group) of other B10.Q mice (donors) were injected i.v. with 200 μg GalOK264/Aq in 100 μl PBS, 200 μg MOG/Aq in 100 μl PBS or 200 μl PBS alone, respectively. Five days later, erythrocyte free spleen and lymph node cells from each mouse were passed through 40 μm nylon cell strainer (BD Biosciences Discovery Labware, Bedford, MA) and then T cells were purified by negative selection using antibodies against MHC class II+(M5/114) and CDIIb+(MI/70) expressing cells (BD Biosciences PharMingen, San Diego, CA) and Dynabeads® (Dynal ASA, Oslo, Norway) followed by magnetic sorting. The purity of the resulting T cells was measured by flow cytometry and were found to be contaminated with <0.3% MHC II+ expressing cells. Purified T cells were analysed by FACS for expression of CD25+, CD62L+, CD45RB+ and NK 1.1+ surface markers but no differences between the individual mice or groups were found. Purified T cells (1×106) from each individual donor were resuspended in a final volume of 200 μl sterile PBS and transferred intravenously into recipient mice.
Statistics
Statistical difference in the incidence of disease between groups of mice described in the examples was determined using Chi Square test. To compare nonparametric data for statistical significance, we applied the Mann-Whitney U or Kruskal Wallis test on all clinical results and in vitro experiments using the StatView™ programme (SAS, Institute Inc., USA).
| Number | Date | Country | Kind |
|---|---|---|---|
| 0502530-9 | Nov 2005 | SE | national |
| Number | Name | Date | Kind |
|---|---|---|---|
| 6451314 | Clark | Sep 2002 | B1 |
| 6632604 | Mach | Oct 2003 | B2 |
| Number | Date | Country |
|---|---|---|
| 1996040944 | Dec 1996 | WO |
| 1998005684 | Feb 1998 | WO |
| 2001036448 | May 2001 | WO |
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