WO2011070172A1 - Anti-inflammatory agents directed against citrullinated epitopes - Google Patents

Anti-inflammatory agents directed against citrullinated epitopes Download PDF

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WO2011070172A1
WO2011070172A1 PCT/EP2010/069431 EP2010069431W WO2011070172A1 WO 2011070172 A1 WO2011070172 A1 WO 2011070172A1 EP 2010069431 W EP2010069431 W EP 2010069431W WO 2011070172 A1 WO2011070172 A1 WO 2011070172A1
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seq
antibodies
antibody
human
group
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PCT/EP2010/069431
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English (en)
French (fr)
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Jozef Maria Hendrik Raats
Renato Gerardus Silvano Chirivi
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Modiquest B.V.
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Priority to JP2012542572A priority Critical patent/JP5980683B2/ja
Priority to EP10788080.9A priority patent/EP2509999B1/en
Priority to AU2010329842A priority patent/AU2010329842B2/en
Priority to ES10788080.9T priority patent/ES2587083T3/es
Priority to US13/514,923 priority patent/US9109019B2/en
Priority to CA2783691A priority patent/CA2783691C/en
Application filed by Modiquest B.V. filed Critical Modiquest B.V.
Priority to KR1020127017692A priority patent/KR101786136B1/ko
Priority to US15/449,623 priority patent/USRE46990E1/en
Publication of WO2011070172A1 publication Critical patent/WO2011070172A1/en
Priority to US14/742,990 priority patent/US10233236B2/en
Priority to US16/227,047 priority patent/US20190119368A1/en
Priority to US16/984,515 priority patent/US20210087260A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • This invention is in the field of treating or preventing inflammation in humans and animals and relates to pharmaceutical compositions and methods for treating or preventing various inflammatory conditions.
  • the invention relates to compositions and methods for preventing or treating inflammatory conditions such as citrulline related diseases, preferably inflammatory diseases, more preferably
  • the invention provides specific binding molecules such as antibodies directed against citrulline-containing epitopes for use in the therapy and prevention of inflammatory conditions such as inflammatory arthritis, preferably rheumatoid arthritis.
  • Inflammatory conditions whether of a chronic or acute nature, represent a substantial problem in the healthcare industry.
  • chronic inflammation is considered to be inflammation of a prolonged duration (weeks or months) in which active inflammation, tissue destruction and attempts at healing are proceeding simultaneously (Robbins Pathological Basis of Disease by R. S. Cotran, V. Kumar, and S. L. Robbins, W. B. Saunders Co., p. 75, 1989).
  • chronic inflammation can follow an acute inflammatory episode, it can also begin as an insidious process that progresses with time, for example, as a result of a persistent infection (e.g., tuberculosis, syphilis, fungal infection) that causes a delayed hypersensitivity reaction, prolonged exposure to endogenous (e.g., elevated plasma lipids) or exogenous (e.g., silica, asbestos, cigarette tar, surgical sutures) toxins, or autoimmune reactions against the body's own tissues (e.g., rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, psoriasis).
  • a persistent infection e.g., tuberculosis, syphilis, fungal infection
  • endogenous e.g., elevated plasma lipids
  • exogenous e.g., silica, asbestos, cigarette tar, surgical sutures
  • autoimmune reactions against the body's own tissues
  • rheumatoid arthritis is a multisystem chronic, relapsing, inflammatory disease affecting 1 to 2% of the world's population.
  • RA is basically a severe form of chronic synovitis that sometimes leads to destruction and ankylosis of affected joints (Robbins Pathological Basis of Disease, by R. S. Cotran, V. Kumar, and S. L. Robbins, W.B. Saunders Co., 1989).
  • the disease is characterized by a marked thickening of the synovial membrane which forms villous projections that extend into the joint space, multilayering of the synoviocyte lining (synoviocyte proliferation), infiltration of the synovial membrane with white blood cells (macrophages, lymphocytes, plasma cells, and lymphoid follicles; called an "inflammatory synovitis"), and deposition of fibrin with cellular necrosis within the synovium.
  • the tissue formed as a result of this process is called pannus and eventually the pannus grows to fill the joint space.
  • the pannus develops an extensive network of new blood vessels through the process of angiogenesis, which is essential to the evolution of the synovitis.
  • pannus tissue Release of digestive enzymes (matrix metalloproteinases (e.g., collagenase, stromelysin)), and other mediators of the inflammatory process (e.g., hydrogen peroxide, superoxides, lysosomal enzymes, and products of arachadonic acid metabolism), from the cells of the pannus tissue leads to the progressive destruction of the cartilage tissue.
  • the pannus invades the articular cartilage leading to erosions and fragmentation of the cartilage tissue. Eventually there is erosion of the subchondral bone with fibrous ankylosis, and ultimately bony ankylosis, of the involved joint.
  • RA is an autoimmune disease and that many different arthrogenic stimuli activate the immune response in an immunogenetically susceptible host.
  • exogenous infectious agents Epstein-Barr virus, rubella virus, cytomegalovirus, herpes virus, human T-cell lymphotropic virus, Mycoplasma, and others
  • endogenous proteins such as collagen, proteoglycans, altered immunoglobulins and post-translationally modified proteins like citrullinated proteins have been implicated as a causative agent that triggers an inappropriate host immune response.
  • autoimmunity plays a role in the progression of the disease.
  • the relevant antigen is ingested by antigen-presenting cells (macrophages or dendritic cells in the synovial membrane), processed, and presented to T lymphocytes.
  • the T cells initiate a cellular immune response and stimulate the proliferation and differentiation of B lymphocytes into plasma cells. The end result is the production of an excessive amount of antigen.
  • anti-CCP antibodies have been demonstrated to be highly specific for RA. Recent evidence shows that each individual that is seropositive for these antibodies either already has RA or will develop this disease in the future. The presence of anti-CCP antibodies (especially when high titers are present) is predictive of erosive disease outcome (Nijenhuis et al., Clin. Chim. Acta, vol 350, 17-34, 2004). Furthermore, it has been demonstrated that anti-CCP antibodies are produced locally at the site of inflammation.
  • the invention provides a binding molecule specifically reactive with a citrullinated epitope on p15 and/or p17 for use in the treatment or prevention of inflammatory diseases.
  • P15 and p17 are identified herein as human PAD4 and/or PAD2 deiminated human histone 2A and/or histone 4, and/or on human PAD2 deiminated human histone H3.
  • the invention also provides a method for treating or preventing an inflammatory disease, comprising the step of administering to a patient in need thereof a therapeutically effective amount of an anti-inflammatory composition comprising a binding molecule specifically reactive with a citrulline epitope on p15 and/or p17.
  • compositions and methods of the present invention include pharmaceutically acceptable formulations of specific binding molecules reactive with citrulline residues.
  • the binding molecules are specifically reactive with citrullinated epitopes on two polypeptides as identified herein, termed p 15 and p17.
  • the invention also relates to polypeptides and nucleic acids as identified herein.
  • the invention provides a binding molecule specifically reactive with a citrullinated epitope on p15 and/or p17 for use in the treatment or prevention of inflammatory diseases.
  • binding molecule is used herein to indicate a molecule, preferably a small molecule, capable of specific binding. Specific binding in this respect is intended to mean that the molecule is capable of binding to a selected target molecule whereas it will not bind to another non-related target molecule under the same conditions. For instance, a binding molecule is said to specifically bind to serum albumin when it binds to serum albumin and less or not at all to another or preferably any other protein found in serum.
  • Preferred specific binding molecules are antibodies.
  • peptide should be interpreted as a structure that is capable of presenting the citrulline residue in the correct context for immunoreactivity with the specific binding molecules as described herein, preferably in the same context as it appears in the human or animal body, preferably in the context of a native polypeptide. It is also preferred that the citrullin residue is presented in the context of a native polypeptide that does not activate or trigger other components of the immune system such as cell activation or complement binding.
  • the "specific binding molecule” may be a molecule, preferably a small molecule composed of DNA, RNA, peptide, protein domain, whole proteins, or combinations thereof or parts thereof, that are capable of specifically binding to a target compound.
  • Preferred examples of specific binding molecules are peptides or antibodies.
  • Native antibodies also known as immunoglobulins
  • gamma globulin proteins that may be found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.
  • Native antibodies are typically made of basic structural units - each with two large heavy chains and two small light chains - to form, for example, monomers with one unit, dimers with two units or pentamers with five units.
  • Antibodies are produced by a white blood cell called a B cell. There are several different types of heavy chains, resulting in different kinds of antibodies. Antibodies may be grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter. Some animal species such as Camelids (e.g. llamas) and sharks may have aberrant antibody structures.
  • the unique part of the antigen recognized by an antibody is called an epitope.
  • These epitopes bind with their antibody in a highly specific interaction that allows antibodies to identify and bind only their unique antigen in the midst of the millions of different molecules that make up an organism.
  • Recognition of an antigen by an antibody tags it for attack by other parts of the immune system.
  • Antibodies can also neutralize targets directly, for example, by binding to a part of a pathogen that it needs to cause an infection.
  • the large and diverse population of antibodies is generated by random combinations of a set of gene segments that encode different antigen binding sites (or paratopes), followed by random mutations in this area of the antibody gene, which create further diversity.
  • Antibody genes also re-organize in a process called class switching that changes the base of the heavy chain to another, creating a different isotype of the antibody that retains the antigen specific variable region. This allows a single antibody to be used in several different isotypes by several different parts of the immune system.
  • antibodies refers to a structure, preferably a protein or polypeptide structure, capable of specific binding to a target molecule often referred to as "antigen”.
  • An antibody may be selected from the group consisting of single chain antibodies, single Chain Variable Fragments (scFvs), Fragment antigen binding regions (Fabs), recombinant antibodies, monoclonal antibodies, fusion proteins comprising the antigen-binding domain of a native antibody or an aptamer, single domains antibodies (sdabs), also known as VHH antibodies, nanobodies (Camelids derived single domain antibodies), shark IgNAR derived single domain antibody fragments called VNAR, Anticalins, aptamers (DNA or RNA) and active components or fragments thereof,
  • an antibody is a fusion protein comprising the antigen-binding domain of a native antibody or an aptamer, such as an aptamer in the form of DNA or RNA.
  • Human antibodies or fragments thereof are a preferred embodiment of the invention.
  • lgG1 e.g., IgGIA
  • antibodies having an lgG1 heavy chain and a lambda light chain may advantageously be used.
  • other human antibody isotypes are also encompassed by the invention, including lgG2, lgG3, lgG4, IgM, lgA1 , lgA2, IgAsec, IgD and IgE in combination with a kappa or lambda light chain.
  • all animal-derived antibodies of various isotypes can be used in the invention.
  • the antibodies can be full-size antibodies or antigen-binding fragments of antibodies, including Fab, F(ab')2, single chain Fv fragments, or single domain VHH, VH or VL single domains.
  • specific binding molecules reactive with a citrullinated epitope are to be interpreted as specific binding molecules that specifically react with a citrulline residue in the context of a larger structure such as a peptide or a peptide nucleic acid or an aptamer or a peptide mimicking structure.
  • Citrulline is an amino acid that is not incorporated into proteins during normal translation, however, it may be generated by post-translational modification of an arginine residue by peptidylarginine deiminase (PAD).
  • PAD peptidylarginine deiminase
  • Citrullination is the posttranslational conversion of arginine residues to citrulline residues, which is catalyzed by peptidylarginine deiminase (PAD).
  • PAD peptidylarginine deiminase
  • Peptidylarginine deiminase (PAD; EC 3.5.3.15) enzymes catalyse the conversion of arginine residues to citrulline residues in proteins. No tRNA exists for citrulline, the presence of citrulline residues in proteins is exclusively the result of post-translational modification. In mammals (humans, mice and rats) five PAD isotypes (PAD1 - PAD6; 'PAD4' and 'PAD5' are used for the same isotype), each encoded by a distinct gene, have been identified (Vossenaar et al, Bioessays 25, 1 106-1 1 18, 2003).
  • Free L-arginine can be converted to free L-citrulline by nitric oxide synthase (EC 1 .14.13.39) in eukaryotes or by arginine deiminase (EC 3.5.3.6) in bacteria. These enzymes are not Ca2+ dependent.
  • PAD1 (synonyms: PAD I, PAD type I) is involved in the citrullination of keratin filaments during the final stages of keratinocyte differentiation, which is important for the reorganization of the cornified envelope.
  • PAD3 sekunder-derived protein
  • THH trichohyalin
  • PAD6 The most recently identified PAD isotype, PAD6 (synonym: ePAD), was found in cytoplasmic sheets of mouse oocytes, which play an important role in early embryogenesis. The expression of its human orthologue was found to be restricted to ovary, testis and peripheral blood leukocytes (Chavanas et al., Gene vol 330; 19-27, 2004). Originally, this PAD isotype was designated ePAD, but based upon the systematic numbering of other PADs, this isotype was renamed PAD6 (Vossenaar et al., Bioessays vol 25 1 106-1 1 18, 2003).
  • PAD2 The most widely expressed isotype, PAD2 (synonyms PAD II, PAD type II, PAD-H19), is present in many different tissues, like skeletal muscle, brain, spleen, secretory glands and macrophages. Despite this broad expression pattern, only myelin basic protein (MBP) and vimentin have been identified as natural substrates. In multiple sclerosis (MS) patients develop an autoimmune response against MBP. MBP is an abundant protein of the myelin sheath, and its citrullination occurs during development of the central nervous system.
  • MBP myelin basic protein
  • MS multiple sclerosis
  • Substrates of PAD4 in the nucleus are histone core proteins (H2A, H3 and H4) and nucleophosmin/B23, a nucleolar protein that functions in ribosome assembly, nucleocytoplasmic transport and centrosome duplication.
  • Specific binding molecules according to the invention are directed against a citrullinated epitope on p15 and/or p17, two polypeptides characterized by their molecular weights of 15 kDa and 17 kDa, respectively.
  • Such specific binding molecules were found to be particularly suited for the treatment or prevention of inflammatory diseases.
  • Inflammatory Conditions or Inflammatory diseases as used herein refers to any of a number of conditions or diseases which are characterized by vascular changes: edema and infiltration of neutrophils (e.g., acute inflammatory reactions);
  • Citrulline related inflammatory diseases are herein defined as those diseases wherein citrullination plays a role in the
  • citrullination plays a role in the pathogenesis of the disease, may be easily determined by a skilled person using routine tests available in the art.
  • these diseases may be characterized by the presence of an abnormal level of citrullinated proteins in affected or disease related tissue. Such may be accomplished by an immunological test such as a western blot or an ELISA wherein the affected tissue is used as an antigen and citrullination of that antigen may be detected with the aid of an anti-citrullin antibody as described herein.
  • Proteomics applications such as mass spec, analysis to compare the level and type of citrullinaton in a diseased versus healthy tissue from affected patients.
  • the disease may also be characterized by the presence of an immune response against citrulline containing peptides or proteins.
  • This may be a humoral or a cellular immune response, such as a response mediated by T-cells or B-cells.
  • Tests for detecting anti-citrulline antibodies have been described in the art and are commercially available.
  • the invention therefore relates to a specific binding molecule for use in treating or preventing citrulline related inflammatory diseases.
  • Such diseases are for instance inflammatory arthritis, including rheumatoid arthritis and osteoarthritis, multiple sclerosis, psoriatic arthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, spondyloarthropathy, Down's syndrome, multiple system atrophy, Parkinson's disease and Lewy body dementia.
  • the invention therefore relates to a specific binding molecule for use in treating or preventing diseases selected from the group consisting of arthritis, rheumatoid arthritis, osteoarthritis, multiple sclerosis, psoriatic arthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, spondyloarthropathy, Down's syndrome, multiple system atrophy, Parkinson's disease and Lewy body dementia.
  • diseases selected from the group consisting of arthritis, rheumatoid arthritis, osteoarthritis, multiple sclerosis, psoriatic arthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, spondyloarthropathy, Down's syndrome, multiple system atrophy, Parkinson's disease and Lewy body dementia.
  • the invention in particular relates to specific binding molecules for the treatment or prevention of autoimmune diseases, more in particular rheumatoid arthritis or osteoarthritis.
  • MS Multiple sclerosis
  • MBP is a highly cationic protein, capable of forming strong interactions with negatively charged phospholipids such as
  • phosphatidylserine In approximately 18% of the MBP molecules of healthy adult humans 6 (out of 19) arginines are citrullinated (Wood et al., J Biol Chem, vol264, 5121 -5127, 1989, Wood et al., Ann Neurol, vol40, 18-24, 1996). The remaining MBP molecules do not contain citrulline. In MS patients the proportion of MBP-cit6 is increased to 45% of total MBP.
  • MBP-cit6 The decreased net positive charge of MBP-cit6 causes partial unfolding of MBP molecules and weakens their interaction with the phospholipids (Boggs et al., J Neurosci Res, vol57, 529-535, 1999, Pritzker et al., Biochemistry, vol39, 5374-5381 , 2000).
  • MBP-cit6 is capable of forming lipid complexes more rapidly than non- citrullinated MBP, the complexes that are formed are not as densely packed as those formed with non-citrullinated MBP (Boggs et al, J Neurosci Res, vol57, 529-535, 1999, Beniac et al, J Struct Biol, vol129, 80-95, 2000). MBP-cit6 is degraded 4 times more rapidly by cathepsin D than non-citrullinated MBP (Cao et al., Biochemistry, vol38, 6157- 6163, 1999).
  • MBPcit18 In a rare case of acute fulminating MS (Marburg type), 80% of the MBP molecules are heavily citrullinated (MBPcit18) (Wood et al., Ann Neurol, vol40, 18-24, 1996). The severely unfolded MBP-cit18 is degraded 45 times more rapidly by cathepsin D than normal MBP (Cao et al., Biochemistry, vol38, 6157-6163, 1999). Clinical trials with paclitaxel, the active component of the anti-cancer drug taxol, are in progress (O'Connor et al., Ann Neurol, vol46, 470, 1999).
  • paclitaxel can inhibit citrullination of MBP by PAD2 in vitro (Pritzker et al., Biochim Biophys Acta, vol1388, 154-160, 1998). Treatment with paclitaxel attenuates clinical symptoms and induces remyelination of damaged sheaths (Moscarello et al., Mult Scler, vol8, 130138, 2002), underlining the possible importance of PAD as a candidate factor in demyelinating disease (Moscarello et al., J Neurochem, vol81 , 335-343, 2002).
  • keratinocytes proliferate very rapidly and travel from the basal layer to the surface in only about four days. The skin can not shed these cells quickly enough so they accumulate in thick, dry patches, or plaques.
  • keratin K1 is citrullinated by PAD1 during terminal differentiation. This process causes the keratin filaments to become more compact, which is essential for the normal cornification process of the epidermis.
  • the keratinocytes in the psoriatic hyperproliferative plaques do not contain citrullinated keratin K1 (Ishida-Yamamoto et al., J Invest Dermatol, vol1 14, 701 -705, 2000).
  • the composition according to the invention is in a form selected from the group consisting of an aqueous solution, a gel, a hydrogel, a film, a paste, a cream, a spray, an ointment, or a wrap.
  • the above methods are used to administer the compositions described herein by a route selected from intra-articular, intraperitoneal, topical, rectal, intravenous, oral, ocular, or to the resection margin of tumors.
  • a pharmaceutically acceptable carrier comprises at least one carrier selected from the group consisting of a co-solvent solution, liposomes, micelles, liquid crystals, nanocrystals, nanoparticles, emulsions,
  • a polysaccharide comprises hyaluronic acid and derivatives thereof, dextran and derivatives thereof, cellulose and derivatives thereof (e.g., methylcellulose, hydroxy-propylcellulose, hydroxy-propylmethylcellulose, carboxymethylcellulose, cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate butyrate,
  • hydroxypropylmethyl-cellulose phthalate chitosan and derivative thereof, [beta]-glucan, arabinoxylans, carrageenans, pectin, glycogen, fucoidan, chondrotin, dermatan, heparan, heparin, pentosan, keratan, alginate, cyclodextrins, and salts and derivatives, including esters and sulfates, thereof.
  • the method according to the invention comprises delivering a composition according to the invention to a target site, most notably a synovial joint.
  • the specific binding molecule competes with monoclonal antibodies RhmAb2.102, RhmAb2.108,
  • histone 3 h2bb [Mus musculus]
  • histone cluster 2 H4 [ attus norvegicus]
  • histone cluster 2 H4 [Rattus norvegicus]
  • histone cluster 2 H4 [Rattus norvegicus]
  • Binding molecules or antibodies competing with the monoclonal antibodies as disclosed herein may be selected by standard procedures.
  • a binding assay such as an ELISA may be developed wherein the antigens as disclosed herein are immobilized on a solid support.
  • the monoclonal antibodies as disclosed herein may be labeled and interference with their binding to the immobilized antigens may be easily determined by routine analysis.
  • assays may easily be developed using any of the antigenic proteins according to SEQ ID NO: 21 , SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 37 and SEQ ID NO: 38 immobilized on a solid support.
  • Monoclonal antibodies selected from the group consisting of RhmAb2.102, RhmAb2.108, RhmAb2.109, RhmAb2.1 10,
  • RhmAb2.1 1 1 RhmAb2.1 12 and RmmAb 22.101 may be labeled and contacted with the immobilized antigen in the presence and the absence of a test antibody. If the test antibody interferes with the binding, i.e. lowers the signal obtained with any of the labeled antibodies, it may be concluded that the test antibodie competes with binding of the labeled antibody. Such a competing antibody would then be suitable for use in the methods of the invention.
  • the invention therefore relates to an antibody for use in the treatment or prevention of rheumatoid arthritiswherein the antibody is specifically reactive with a peptide selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 37 and SEQ ID NO: 38.
  • the primary mRNA sequences of the variable regions of monoclonal antibody RhmAb2.101 have been published and were deposited in the EMBL database under accession numbers as shown in table 1 .
  • RhmAb2.1 10, RhmAb2.1 1 1 and RhmAb2.1 12 are disclosed herein in SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 39, SEQ ID NO: 20, SEQ ID NO: 41 , SEQ ID NO: 40, SEQ ID NO: 19, SEQ ID NO: 43, and SEQ ID NO: 42
  • the invention therefore also relates to a polypeptide comprising a variable heavy or light chain selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 19, SEQ ID NO: 43, SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 45.
  • the invention also relates to a nucleic acid encoding a polypeptide selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 19, SEQ ID NO: 43, SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 45.
  • the invention also relates to a polypeptide comprising a variable heavy and light chain as is present in Rmm22.101 and RmmAb22.102.
  • the invention also relates to a nucleic acid encoding a polypeptide according a variable heavy and light chain as is present in RmmAb22.101 and
  • RmmAb22.102 which is shown in SEQ ID NO: 44 and SEQ ID NO: 45.
  • the specific binding molecule is an antibody selected from the group consisting of monoclonal antibodies RhmAb2.102, RhmAb2.108, RhmAb2.109, RhmAb2.1 10, RhmAb2.1 1 1 and RhmAb2.1 12,
  • the specific binding molecule comprises VH and /or VL domains derived or obtained from an antibody selected from the group consisting of monoclonal antibodies and RhmAb2.102, RhmAb2.108, RhmAb2.109, RhmAb2.1 10, RhmAb2.1 1 1 and RhmAb2.1 12, RmmAb22.101 , and RmmAb22.102.
  • derived in this context means that the primary structure of the VH and/or VL domains may be determined from the protein and nucleic acid sequences disclosed herein and cloned and rearranged in a different context, for instance a human antibody context displaying a mouse VH or VL domain. More in particular, the term “derived” or “obtained”in this respect means that the essential residues responsible for the specific binding properties of the VH and /or VL domains in a particular antibody are identified and that these essential residues or structural homologues thereof are then transferred into the context of another peptide.
  • Specific binding molecules according to the invention may be generated essentially in two ways. First, they may be derived from the antibodies and its sequences as presented herein. Reactivity of the antibodies may even be improved by side-directed mutagenesis, chain shuffling, sexual PCR, or by other means for antibody derivation and optimisation known to the person skilled in the art. Alternatively, specific binding molecules, in particular antibodies may be obtained by panning with any of the specifically reactive epitopes as described herein, in particular deiminated Histon 2A, peptide 1 (SEQ ID NO: 21 ) and other particularly reactive peptides.
  • a person skilled in the art may use the sequences described herein to clone or generate cDNA or genomic sequences for instance such as described in the below examples. Cloning of these sequences in an appropriate eukaryotic expression vector, like pcDNA3 (In Vitrogen), or derivates thereof, and subsequent transfection of mammalian cells (like CHO cells) with combinations of the appropriate light chain and heavy chain containing vectors will result in the expression and secretion of the listed antibodies RhmAb2.102, RhmAb2.108, RhmAb2.109, RhmAb2.1 10, RhmAb2.1 1 1 and RhmAb2.1 12, RmmAb22.101 , and RmmAb22.102. Also, mouse monoclonals
  • RmmAb22.101 and RmmAb22.102 may be directly expressed and secreted by their respective hybridoma cell lines as deposited. (DSMZ numbers ACC 3031 and ACC 3032)
  • the skilled person may also make analogues of the specific binding molecules as described herein by using the specific binding domains of the antibody sequences and express them in a different context such as a polypeptide such as a fusion protein. This is well known in the art.
  • Recombinant Human and Mouse monoclonal anti-citrulline antibodies were obtained as described in Examples 1 , 13, and 14.
  • Monoclonal antibody heavy chains RhmAb2.102, RhmAb2.108, RhmAb2.109, RhmAb2.1 10, RhmAb2.1 1 1 and RhmAb2.1 12 were obtained with a mouse leader sequence (SEQ ID NO: 12), and a human lgG1 Fc region (SEQ ID NO: 14).
  • Monoclonal antibody light chains RhmAb2.102, RhmAb2.108, RhmAb2.109, RhmAb2.1 1 1 and RhmAb2.1 12 were obtained with a mouse leader sequence (SEQ ID NO: 12) and a human Lambda constant region (SEQ ID NO: 16).
  • Monoclonal antibody RhmAb2.1 10 was obtained with a mouse leader sequence (SEQ ID NO: 12) and a human Kappa constant region (SEQ ID NO: 1 1 ).
  • Mouse monoclonal anti-citrullin-peptide antibodies RmmAb13.101 , RmmAb13.102 and RmmAb13.103 were obtained from a commercial source (ModiQuest Research BV Nijmegen, The Netherlands; Cat no, MQ13.101 , MQ13.102and MQ13.103).
  • Anti-citrullin antibodies were tested in an experimental model wherein inflammation is induced by injecting anti-collagen antibodies into a mouse. This model is known as collagen antibody induced arthritis (CAIA) (Nandakumar and Holmdahl, J Immunol Methods, vol304, 126-136, 2005). Anti collagen antibodies were obtained from a commercial source (ModiQuest Research BV Nijmegen, The Netherlands; Cat no, MQ18.101 ).
  • RmmAb13.102 and RmmAb13.103 were confirmed to enhance the severity of the collagen antibody induced arthritis, as has been described also by Kuhn et al. (J. Clin. Invest, vol1 16, 961 -871 , 2006); and Hill et al. (J Exp Med, vol 205, 967-979, 2008). This is shown in figures 1 a and 1 b.
  • RhmAb2.102 dramatically reduced the clinical signs of arthritis in the experimental CAIA model.
  • RhmAb2.102 Results obtained with RhmAb2.102 are shown in Figures 1 c and 1 d. Results obtained with RhmAb2.108, RhmAb2.109, RhmAb2.1 10, RhmAb2.1 1 1 and RhmAb2.1 12 were even better as compared to RhmAb2.102, as shown in Figure 9.
  • the human monoclonal antibody RhmAb2.101 had no effect at all on the clinical signs of arthritis at the dose applied.
  • the commercially available antibody RhmAb2.201 is used as an irrelevant antibody control in this experiment (ModiQuest Research B.V., cat no: MQR2.201 ). This antibody does not recognize citrulinated epitopes.
  • Figure 1 e and 1f show an independent CAIA experiment in which the clinical dose for RhmAb2.102 has been evaluated.
  • the lowest dose that gave maximum inhibition was 0,5 mg Ab/mouse which corresponds to 28 mg/kg at IP injection.
  • RmmAb22.101 , and RmmAb22.102 play an important role in the treatment or prevention of inflammatory diseases. Specific masking of these epitopes may therefore be an effective therapy of inflammatory diseases, in particular rheumatoid arthritis.
  • WO 2004/078098 discloses antibodies specific for citrullinated peptide/MHC class II complexes to inhibit T cell activation. These antibodies do not bind to the separate peptide or MHC class II molecule but only to the complex of the peptide and the MHC class II molecule.
  • the antibodies disclosed herein are different from the antibodies disclosed in WO 2004/078098 since they recognize the individual peptides and proteins as disclosed herein.
  • the antibodies recognize a polypeptide in a western blot that could not be a complex between a peptide and an MHC class II molecule, since the complex between an MHC molecule and a citrullinated peptide would never survive the reducing conditions of an SDS gel used in the immunoblot procedure.
  • the epitopes recognized by the binding molecules as disclosed herein are therefore different from the antibodies disclosed in WO 2004/078098.
  • the antibodies as disclosed herein are not specifically reactive with a complex of a peptide and an MHC class II molecule.
  • RmmAb22.101 may be performed using either Western blots containing deiminated COS-1 lysates or purified deiminated p15 and/or p17 proteins in Western blot or ELISA.
  • Proteins p15 and p17 were further characterized by Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) as detailed in example 6. Since the genome of the African Green Monkey is not completely sequenced we screened all other mammal genome databases for homology with the peptides found with MALDI-TOF MS. Proteins found with a high degree of homology turned out to be histones. This is shown in Table 3 (Example 6).
  • the invention therefore also relates to a binding molecule specifically reactive with a citrullinated epitope on histones for use in the treatment or prevention of inflammatory diseases.
  • citrullinated histones may very well be produced in vitro. These citrullinated histones may then be used as a substrate in an enzymatic binding assay to screen and select for other specific binding molecules such as peptides and antibodies reactive with epitopes on citrullinated p15 and p17, i.e. histones.
  • specific binding molecules are selected that compete with antibodies RhmAb2.102, RhmAb2.108, RhmAb2.109, RhmAb2.1 10, RhmAb2.1 1 1 and RhmAb2.1 12,
  • RmmAb22.101 and RmmAb22.102 for binding to p15 and/or p17.
  • RhmAb2.102 RhmAb2.108, RhmAb2.109, RhmAb2.H 0, RhmAb2.1 1 1 and RhmAb2.1 12, RmmAb22.101 , and RmmAb22.102
  • commercial available histones H1 , H2A, H2B, H3 and H4 were deiminated with human
  • PAD peptidylarginine deiminase
  • huPAD2 or huPAD4 enzymes
  • RhmAb 2.102 and RhmAb 2.101 are highest for Human PAD2 and/or PAD4 deiminated H2A, Human PAD 2 deiminated Histon 3, and Human PAD 4 and/or PAD 2 deiminated Histon 4.
  • a mimic is for instance a molecule with an acceptable level of equivalent activity, which, in this case, would include as being recognized with higher affinity by RhmAb2.102 as compared to RhmAb2.101 .
  • the invention therefore relates to a specific binding molecule as described above, reactive with a citrullinated epitope on human PAD4 or Human PAD 2 deiminated human histone 2A or histone 4, or on human PAD2 deiminated histone H3.
  • biotin labeled peptides as shown in table 4 were synthesized containing potential deimination sites of histone 2A. These peptides were coated on 96-well neutravidin-ELISA plates and incubated with serial dilutions of RhmAb2.101 and
  • RhmAb2.102. The results are shown in Figure 3.
  • Table 6A Reactivity of deiminated histones with RhmAb2.101 , shown in figure 2A
  • the specific binding molecules according to the invention may also be defined by their reactivity towards peptides 1 , 4 and 6; SEQ ID NO: 21 , SEQ ID NO: 24 and SEQ ID NO: 26 respectively.
  • Each of these citrulline containing peptides or derivatives thereof, individually, or a combination of such peptides, or structures containing on or more of such peptide sequences, may be used to generate specific binding molecules such as antibodies according to the invention. Such antibodies may then be selected towards any of the other antigens as disclosed herein for optimal reactivity.
  • Biotin labeled and citrullin containing fibrinogen and vimentin peptides were also tested for reactivity with the therapeutic antibodies. Peptides were coated on 96-well neutravidin-ELISA plates. Subsequently serial dilutions of RhmAb2.101 ,
  • RhmAb2.102 were applied to the coated plates. The results are shown in Table 8 and Figure 4.
  • the invention therefore also relates to a specific binding molecule as described above which is specifically reactive with an epitope on peptides msFib3 or msVim (SEQ ID NO: 37 or SEQ ID NO: 38) and their use.
  • citrullinated epitopes appear de novo in inflammated tissue.
  • human monoclonal antibody 102 RhmAb2.102
  • mice 3 mice per group
  • mice Three days later mice received another i.p. injection containing 25ug LPS. Scoring has been performed as described above.
  • a group of mice has been sacrificed, and paws were analyzed for citrulline presence by Western Blot analysis and Immunohistochemical techniques.
  • IP Immuniprecipitations
  • citrulline residues present on blot have been chemically modified according to Senshu et al. (Senshu et al, Anal Biochem, vol 203, 94-100, 1992).
  • the chemical modification can then be visualized using an antibody that recognizes the chemical modification of citrulline residues (Senshu et al, Anal Biochem, vol 203, 94-100, 1992).
  • Deiminated fibrinogen was used as a positive control in this experiment.
  • An immunoprecipitation without extracts was used as a negative control in these
  • mice subjected to CAIA have detectable citrulline levels in their inflamed joints.
  • anti-citrulline antibodies were injected on day 3 after anti-collagen antibody injection, when inflammation in the paws of mice was still absent or very low. This prevented the occurrence of clinical symptoms and is therefore useful as a treatment of inflation, in particular a prophylactic treatment.
  • RhmAb2.102 could also cure clinical symptoms once they had occurred. This was done by treating animals on day 7 after anti- collagen injection when mean arthritis scores of all 4 paws of all mice reached the arbitrary score of approximately 4. As is shown in figure 6A and 6B, RhmAb2.102 does not abolish the swelling observed, but rather stabilized the present inflammation/swelling. Animals were followed for 35 days after which inflammatory scores among placebo and RhmAb2.102 treated mice were equal ( Figure 6B and example 10). Figure 6A shows the Mean arthritis score of all paws of each group, while Figure 6B shows the mean arthritis score of the right hind paws of the animals that have been used for histological analysis at day 35.
  • FIG. 7A shows that macroscopical inflammation in the right hind paws between experimental groups on day 35 of the experiment were similar. Most surprisingly however, all known parameters for joint erosion were decreased.
  • D Inflammatory cell influx
  • B Cartilage erosion
  • E Cartilage PG depletion
  • F Chondrocyte death
  • C Bone erosion
  • RhmAb2.102 has been injected i.v. in order to deliver the antibody rapidly to sites of inflammation.
  • prophylactic treatment at day 3 and a non treated control group have been included.
  • Experimental procedures have been performed as in Example 10 with the only difference of injections with 1 mg RhmAb2.102 per mouse on day 3, 5 and 6.
  • RhmAb2.102 at day 3 inhibited the inflammatory response.
  • Treating mice with i.v. injections of RhmAb2.102 on day 5, 6 or 7 stabilized the inflammation ( Figure 8) as also seen in Figure 6. It is noteworthy that the signs of inflammation were not reduced whereas all parameters for joint erosion were decreased. This shows that joint erosion and inflammation are two separate entities that may be treated separately.
  • RhmAb2.102 Additional deiminated proteins that preferentially bind to RhmAb2.102 have been identified by mass spectrometry analysis. Furthermore, deiminated proteins that preferentially bind to RhmAb2.102 and not, or with to a lesser extent to RhmAb2.101 have also been identified by additional mass spectrometry analysis.
  • Human PAD4 deiminated Human Embryonic Kidney cell (HEK293) lysates have been immunoprecipitated with RhmAb2.101 or RhmAb2.102 (Example 1 1 ) and subjected to a high throughput nano-LC system coupled to an advanced, high-performance LTQ Fourier Transform Ion Cyclotron Resonance Mass spectrometer (nl_C LTQ FTMS ULTRA) (Example 12).
  • the invention also relates to a binding molecule specifically reactive with any of the proteins or polypeptides as shown in table 7 for use in the prevention or treatment of an inflammatory disease.
  • a binding molecule specifically reactive with an epitope on a molecule selected from the group consisting of p15, p17, more in particular a citrullinated epitope on human PAD4 and/or PAD2deiminated human histone 2A, a citrullinated epitope on human PAD4 deiminated human histone 4, human PAD2 deiminated human histone H4, human PAD2 deiminated human histone H3, or a protein selected from the group consisting of the proteins of table 9 and even more in particular a peptide according to SEQ ID NO: 21 , SEQ ID NO: 24, SEQ ID NO 26, SEQ ID NO: 37 and SEQ ID NO: 38 may be used in the treatment or prevention of inflammatory diseases as specified herein.
  • Whether a given binding molecule is specifically reactive with the above mentioned molecules may easily be determined by analysis of the ability of the binding molecule to compete with an antibody selected from the group consisting of RhmAb2.102, RhmAb2.108, RhmAb2.109, RhmAb2.1 10, RhmAb2.1 1 1 and RhmAb2.1 12, RmmAb22.101 , and RmmAb22.102 for binding to an epitope on p15 or p17 or any of the citrullinated epitopes mentioned above.
  • inflammatory diseases may also be treated or prevented by eliciting an immune response wherein specific binding molecules according to the invention are generated in the patient's own body (in vivo).
  • an immune response may be generated to prevent inflammatory disease from occurring (prophylaxis, prophylactic vaccines) or to ameliorate or decrease the
  • the invention also relates to a method for the prevention or treatment of inflammatory diseases by eliciting an immune response in vivo wherein specific binding molecules are generated reactive with an epitope selected from the group consisting of a citrullinated epitope on p15, p17, a citrullinated epitope on human PAD4 and/or PAD 2 deiminated human histone 2A, human PAD4 and/or PAD 2 deiminated human histone 4, human PAD2 deiminated human histone H3, and a peptide according to SEQ ID NO: 21 , SEQ ID NO: 24, SEQ ID NO 26, SEQ ID NO: 37 and SEQ ID NO: 38
  • Vaccines or therapeutics according to the invention may effectively comprise a citrullinated epitope specifically reactive with a binding molecule according to the invention.
  • the citrullinated epitope may be a citrullinated epitope on human PAD4 and/or PAD 2 deiminated human histone 2A, human PAD4 and/or PAD 2 deiminated human histone 4, human PAD2 deiminated human histone H3, or a peptide selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 24, SEQ ID NO 26, SEQ ID NO: 37 and SEQ ID NO: 38.
  • the invention also relates to a method as described above wherein the inflammatory disease is selected from the group consisting of autoimmune diseases, arthritis, rheumatoid arthritis, osteoarthritis, multiple sclerosis, psoriatic arthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, spondyloarthropathy, Down's syndrome, multiple system atrophy, Parkinson's disease and Lewy body dementia.
  • the inflammatory disease is selected from the group consisting of autoimmune diseases, arthritis, rheumatoid arthritis, osteoarthritis, multiple sclerosis, psoriatic arthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, spondyloarthropathy, Down's syndrome, multiple system atrophy, Parkinson's disease and Lewy body dementia.
  • autoimmune diseases such as rheumatoid arthritis.
  • a preferred specific binding molecule is an antibody.
  • peptides and proteins as mentioned herein may also be used as antigens for the detection of specific antibodies in order to diagnose inflammatory diseases, more preferably Rheumatoid Arthritis.
  • FIG. 1 A Collagen antibody induced arthritis (CAIA) model was used to test the effect of monoclonal antibodies on the severity of symptoms of arthritis. Mean arthritis score ( Figures 1 a, 1 c and 1 e) and arthritis incidence (figures 1 b, 1 d and 1f) are indicated.
  • CAIA Collagen antibody induced arthritis
  • mice used in the experiments shown in figure 1 a and 1 b received 1 ,6 mg anti- collagen antibody mix, whereas mice used in figure 1 c-f received 2,4 mg.
  • LPS 25 ⁇ g mouse
  • anti-citrulline or a control antibody RhmAb2.201
  • FIG. 2 An enzyme linked immunosorbend assay (ELISA) was used to test the affinity of a) RhmAb2.101 and b) RhmAb2.102 for human recombinant histones (H1 , H2A, H2B, H3 and H4) deiminated with huPAD2 or huPAD4. Deiminated as well as non-deiminated histones were immobilized on 96-well ELISA plates (O ⁇ g/well). CFC-1 and CFC-0 were coated at the same concentration and served as positive and negative controls respectively for specific anti-citrulline reactivity and as coating controls. Non coated wells were used to test for aspecific binding of the antibodies.
  • ELISA enzyme linked immunosorbend assay
  • FIG. 3 An enzyme linked immunosorbend assay (ELISA) was used to test the affinity of a) RhmAb2.101 and b) RhmAb2.102 for citrulline containing peptides derived from human histones H2A. Biotin and citrulline containing peptides derived from histone 2A were immobilized on neutravidin coated 96-well ELISA plates (0 ⁇ g/well). CFC-1 and CFC-0 were coated at the same concentration and served as positive and negative controls respectively for specific anti-citrulline reactivity and as coating controls. Non coated wells were used to test for aspecific binding of the antibodies.
  • ELISA enzyme linked immunosorbend assay
  • Coated wells were incubated with antibody dilution series ranging from 10ug/well down to 0,000128ug/well for 1 h at RT (z- axis). Detection of bound anti-citrulline antibodies was performed by incubating the wells with rabbit-anti-human-HRP (1 :2000) for 1 hour at RT followed by incubation with TMB substrate. The resulting OD (y-axis) is a measure for antibody binding.
  • Figure 4 An enzyme linked immunosorbend assay (ELISA) was used to test the affinity of a) RhmAb2.101 and b) RhmAb2.102 for citrulline containing peptides derived from fibrinogen and vimentin.
  • ELISA enzyme linked immunosorbend assay
  • Biotin and citrulline containing peptides derived from fibrinogen and vimentin were immobilized on neutravidin coated 96-well ELISA plates (0 ⁇ g/well).
  • CFC-1 and CFC-0 were coated at the same concentration and served as positive and negative controls respectively for specific anti-citrulline reactivity and as coating controls.
  • Non coated wells were used to test for aspecific binding of the antibodies. Coated wells were incubated with antibody dilution series ranging from 10ug/well down to
  • FIG. 5 A Collagen antibody induced arthritis (CAIA) model was used to investigate citrulline appearance in the paws. Groups of 3 mice were treated at day 0 with 2.8mg anti- collagen antibodies through i.p. injection, followed by an additional i.p. injection with LPS (25 ⁇ g mouse) on day 3. Mean arthritis score and arthritis incidence are shown in Figure 5A and 5B respectively.
  • CAIA Collagen antibody induced arthritis
  • FIG. 6 A Collagen antibody induced arthritis (CAIA) model was used to test the therapeutic effect of RhmAb2.102 when given on day 7 after anti-collagen antibody injection. Mean arthritis score of all paws ( Figure 6A) and Mean arthritis score of the right hind paws ( Figure 6B) are indicated. Groups of 5 mice were treated at day 0 through i.p. injection with 2,8mg anti-collagen antibodies. LPS (25 ⁇ g mouse) was administered on day 3 through i.p. injection, and RhmAb2.102 (1 mg/mouse) or placebo were injected via the same route at day 7. Animals have been scored daily until day 35. It was observed that RhmAb2.102 at least stabilized the present inflammation.
  • CAIA Collagen antibody induced arthritis
  • Figure 7 Histological analysis has been performed on Haematoxylin/eosin and safranin O stained tissue slides of right hind paws of all CAIA animals that have been treated on day 7 with RhmAb2.102 or placebo (Figure 7). The following parameters have been scored (arbitrary scale of 0-3) on the stained tissue slides: Cartilage erosion (B), Bone erosion (C), Inflammatory cell influx (D), Cartilage PG depletion (E), and Chondrocyte death (F).
  • Figure 7A shows the macroscopical inflammation in the right hind paws between experimental groups on the last day of the experiment (day 35). Each dot depicts a single animal. The horizontal lines indicate the mean score within an experimental group. It may be concluded that RhmAb2.102 injection protects the mice from permanent joint damage.
  • FIG. 8 A Collagen antibody induced arthritis (CAIA) model was used to test the therapeutic effect of RhmAb2.102 when given on day 3, 5, 6 and 7 days after injection of anti-collagen antibodies.
  • Groups of 5 mice were treated at day 0 through i.p. injection with 2,8mg anti-collagen antibodies.
  • LPS 25 ⁇ g/mouse
  • RhmAb2.102 (1 mg/mouse) was injected i.v. at day 3, 5, 6 or 7. Animals have been scored daily until day 19.
  • the graph depicts Mean arthritis score for each experimental group. It may again be concluded that RhmAb2.102 at least stabilized the inflammation at a level comparable to the level at the start of the therapy.
  • RhmAb2.1 1 1 and RhmAb2.1 12 when given on day 3 after injection of anti-collagen antibodies. Mean arthritis score of all paws are indicated. Groups of 3 mice were treated at day 0 with i.p. injection of 2,8mg anti-collagen antibodies. LPS (25 ⁇ g mouse) was administered at day 3 via i.p. injection, and RhmAb2.102, RhmAb2.108,
  • RhmAb2.109, RhmAb2.1 10, RhmAb2.1 1 1 and RhmAb2.1 12 (1 mg/mouse) or placebo were injected via i.v. injection on the same day. Animals have been scored daily until day 14.
  • RhmAb2.1 12 showed a higher anti-inflammatory effect than RhmAb2.102.
  • FIG. 10 The Collagen antibody induced arthritis (CAIA) model was used to test the anti inflammatory effect of RhmAb2.102, RmmAb22.101 , and RmmAb22.102 antibodies.
  • CAIA Collagen antibody induced arthritis
  • mice Groups of 3 mice were treated at day 0 with i.p. injection of 2,8mg anti-collagen antibodies.
  • LPS 25 g/mouse
  • RhmAb2.102 RhmAb2.102
  • RmmAb22.101 RhmAb2.102
  • RmmAb22.102 (6mg/mouse) and placebo were administered via i.p. injection on day 3. All animals have been scored for inflammation daily until day 10.
  • RhmAb2.102 protected the mice against inflammation in their paws. Only data of RhmAb2.102 and RmmAb22.101 are shown.
  • Figure 1 1 An enzyme linked immunosorbend assay (ELISA) was used to test the affinity of A) RhmAb2.102, B) RhmAb2.108, C) RhmAb2.109, D) RhmAb2.1 10, E) RhmAb2.1 1 1 , F) RhmAb 2.1 12, G) RmmAb22.101 , and H) RmmAb22.102 for human recombinant histones (H1 , H2A, H2B, H3 and H4) deiminated with huPAD2 or huPAD4. Deiminated as well as non-deiminated histones, and BSA were immobilized on 96-well ELISA plates (0,3 g/well).
  • ELISA enzyme linked immunosorbend assay
  • CFC-1 , CFC-0, SEQ ID NO: 21 were coated at the same concentration and served as positive and negative controls respectively for specific anti-citrulline reactivity and as coating controls.
  • Non coated wells were used to test for aspecific binding of the antibodies.
  • Coated wells were incubated with antibody dilution series ranging from 2,5 ug/well down to 0,004ug/well for 1 h at RT (z-axis). Detection of bound anti-citrulline antibodies was performed by incubating the wells with rabbit-anti-human-HRP (1 :2000) for 1 hour at RT followed by incubation with TMB substrate. The resulting OD (y-axis) is a measure for antibody binding.
  • Example 1 Recombinant human and mouse monoclonal antibodies.
  • Monoclonal antibodies against citrullinated antigens of patients with RA were initially selected by means of phage display, as described (Raats et al., J
  • Antibody coding sequences described by Raats et al., (J Reumatology, vol30, 1696-71 1 , 2003) were synthesized according to Stemmer et al (Gene, vol164, 49- 53, 1995), and subsequently cloned into mammalian expression vectors coding for human and mouse antibody isotypes.
  • Human antibodies were of the isotype lgG1 lambda and were named RhmAb2.101 , RhmAb2.102.
  • RhmAb2.101 was synthesized according to the protocol of Stemmer et al., (Gene, vol164, 49-53, 1995) based on the sequence of clone Ra3 (Raats et al., J Reumatology, vol30, 1696-71 1 , 2003) and consists of a VH derived from germline family 3-21 , combined with a VL derived from germline family ⁇ 1 b..
  • RhmAb2.102 was synthesized according to Stemmer et al (Gene, vol164, 49-53, 1995) and comprises an immunoglobulin heavy chain encoded by SEQ ID NO: 8, combined with an immunoglobulin light chain encoded by SEQ ID NO: 9.
  • the immunoglobulin heavy chain encoded by SEQ ID NO: 8 comprises a mouse leader globulin according to SEQ ID NO: 12, followed by the variable antibody heavy chain according to SEQ ID NO: 13, followed by the immunoglobulin constant domain human lgG1 according to SEQ ID NO: 14.
  • the immunoglobulin light chain encoded by SEQ ID NO: 9 comprises a mouse leader globulin according to SEQ ID NO: 12, followed by the variable antibody light chain according to SEQ ID NO: 15 followed by the immunoglobulin human lambda constant domain according to SEQ ID NO: 16.
  • variable domains VH and VL
  • VH and VL variable domains of monoclonal antibody RhmAb2.101
  • Table 1 The primary mRNA sequences of the variable domains (VH and VL) of monoclonal antibody RhmAb2.101 have been published and were deposited in the EMBL database under accession numbers as shown in table 1. Full size human antibody sequences were generated using identical leader and constant human domains as described for antibody RhmAb2.102. Table 1
  • CAIA collagen antibody induced arthritis
  • mice For that purpose, on day 0 male DBA/J1 mice (5-6 mice /group) of the age of 8 weeks have been injected i.p. with a mix of 8 anti-collagen antibodies. (Mice used in figure 1 a and 1 b received 1 ,6mg anti-collagen antibody mix, whereas mice used in figure 1 c-f received 2,4mg). On day 3, mice received another i.p. injection containing 25ug LPS mixed with 1 mg anti-citrulline antibodies (unless stated otherwise). LPS triggers the inflammation. Until day 13 of the experiment animals where scored daily for signs of inflammation in their paws. Scoring has been performed according to the table 2. The maximum arthritis score per animal is 8.
  • RhmAb2.102 Human monoclonal antibody RhmAb2.102, reduced or even abolished the clinical signs of arthritis in the experimental CAIA model, whereas RhmAb2.101 had no effect at all at the dose tested ( Figure 1 c and 1 d).
  • Example 3 Preparation of deiminated cell extract, SDS-page electrophoresis and western blotting.
  • COS-1 cells (8 10 5 ) were transiently transfected with 2 ⁇ g huPAD2 or huPAD4 expression vector using the AMAXA nucleofection device (program D-005) together with the V-kit, and cells were seeded in 20ml medium in a T75.
  • the cells were washed twice with PBS, trypsinized, spun down and resuspended in 15 ⁇ ice cold lysis buffer (20mM Tris pH7.4, 10mM ⁇ - mercaptoethanol, 100mM NaCI, 10% glycerol, protease inhibitors).
  • the cell samples were sonified 4 times for 15 seconds on ice.
  • the lysate was centrifuged at 3.000 rpm for 5 minutes and the supernatant transferred to a clean tube.
  • the cell lysate was deiminated for 30 minutes to 2 hours at 37°C by adding CaCI 2 and DTE at a final concentration of 10 and 5mM respectively. Deiminated cell lysates were stored at -20°C.
  • Blots as prepared in example 3 were cut in strips and blocked for 2 hours at RT with 5% (w/v) low fat dry milk in PBS-Tween (wash buffer) to block all nonspecific sites. Blots were then washed 5 times 5 minutes with wash buffer and strips were incubated for an additional 1 hour at RT with 4 ml wash buffer containing 20ug anti- citrulline antibody. Thereafter, the strips were washed 5 times for 10 min with wash buffer, and incubated with a peroxydase-conjugated rabbit anti-human IgG (Dako) (1 hour at RT) in wash buffer (1 :2000). Strips where then washed 3 times for 10min with wash buffer followed by a 2 times wash with PBS to wash away all unbound antibody.
  • wash buffer 5% (w/v) low fat dry milk in PBS-Tween
  • Immunoreactive bands were visualized using chemiluminescent substrate (PIERCE), and exposed to Kodak BioMax XAR autoradiography films (Eastman Kodak Company, Rochester, NY, USA).
  • RhmAb2.102 showed reactivity with a doublet of proteins with a molecular weight of approximately 15 and 17 kiloDalton.
  • PAD4 deiminated COS-1 lysates revealed prominent p15 and p17 protein bands which could not or hardly be detected in the RhmAb 2.101 precipitates. The rate of recognition of p15 and p17 proteins therefore correlates well with the therapeutic properties of these antibodies ( Figure 1 a-d).
  • Example 6 Mass-spectrometry analysis of p15 and p17.
  • the bands at p15 and p17 of the SDS-page gels of example 3 were excised from the gel and analyzed by MALDI-TOF MS. Briefly, excised gel pieces were washed 2 times with 50 ⁇ of 25 mM ammonium bicarbonate, and incubated 30 min for each washing step. A 15 min wash was repeated as above with the addition of 30% v/v acetonitrile. All liquid was removed and 25 ⁇ of 25 mM ammonium bicarbonate + 25 ⁇ of acetonitrile added and Incubated for 15 min. Again all liquid was removed and gels were incubated 30 min with 50 ⁇ of acetonitrile. All liquid was removed and the pieces were dehydrated by incubating for 2 h at 37°C.
  • the gel pieces were allowed to swell again by adding 5 ⁇ of trypsin solution ( ⁇ 15 ng trypsin/ ⁇ in 25 mM ammonium bicarbonate/5 mM n-octyl ⁇ -D-glucopyranoside) and incubated on ice for 1 hour. Excess trypsin solution was removed and gel pieces were incubate for 14 h at 37°C with 5 ⁇ 25 mM ammonium bicarbonate/5 mM n-octyl ⁇ -D-glucopyranoside.
  • Peptides were extracted by incubating with 4 ⁇ 50% acetonitrile/0.5% trifluoroacetic acid (TFA)/5 mM n-octyl ⁇ -D-glucopyranoside for 1 h at RT. Samples were sonicated for 2 min in a sonication water bath, the liquid transferred in a new tube and the extraction step was repeated. The sample was dried in a vacuum centrifuge and subjected to MALDI-TOF MS.
  • TFA trifluoroacetic acid
  • Therapeutic anti-citrulline antibody RhmAb2.102 recognizes H2A p4.
  • Human recombinant histones H1 , H2A, H2B, H3 and H4 (10C ⁇ g) were incubated 3 hours with or without 53,4 mU huPAD2 or huPAD4 at 37°C.
  • Deiminated as well as non-deiminated histones were coated on 96-well ELISA plates (O ⁇ g /well) by overnight incubation at 4°C.
  • Wells were washed 5 times with PBS-Tween20 (PBS-T) and blocked by a 1 hour incubation with PBS-T + 1 % Bovine serum albumin (BSA) at room temperature (RT).
  • PBS-T PBS-Tween20
  • BSA Bovine serum albumin
  • RhmAb2.101 or RhmAb2.102 were incubated for 1 hour at RT with serial dilutions of RhmAb2.101 or RhmAb2.102 in PBS-T + 1 % BSA starting at a concentration of 1 C ⁇ g/well.
  • Wells were washed 5 times with PBS-T and incubated with rabbit-anti-human-HRP (1 :2000) for 1 hour at RT followed by 5 washes with PBS-T and 3 wash steps with PBS.
  • Wells incubated with RhmAb2.101 were incubated 15min and wells incubated with RhmAb2.102 were incubated 10min with TMB substrate before stopping the reaction with 2M H 2 S0 4 .
  • Optical density was measured by 450nm and is a measure for the affinity of the antibodies used.
  • Example 8 Therapeutic anti-citrulline antibody RhmAb2.102 recognizes peptide 1.
  • 96-well ELISA plates were coated with neutravidin (0,1 ⁇ g /well) by overnight incubation at 4°C. Wells were washed 5 times with PBS-Tween20 (PBS-T) and blocked by a 1 hour incubation with PBS-T + 1 % Bovine serum albumin (BSA) at room temperature (RT). After 5 more washes with PBS-T, wells were incubated for 1 hour at RT with histone derived citrulline and biotin containing peptides (0 ⁇ g /well).
  • PBS-T PBS-Tween20
  • BSA Bovine serum albumin
  • RhmAb2.101 , RhmAb2.102 or RhmAb2.104 were incubated for 1 hour at RT with serial dilutions of RhmAb2.101 , RhmAb2.102 or RhmAb2.104 in PBS-T + 1 % BSA starting at a concentration of
  • Example 9 Therapeutic anti-citrulline antibodies recognize fibrinogen and vimentin derived citrulline peptides.
  • 96-well ELISA plates were coated with neutravidin (0,1 ⁇ g /well) by overnight incubation at 4 degrees C. Wells were washed 5 times with PBS-Tween20 (PBS-T) and blocked by a 1 hour incubation with PBS-T + 1 % Bovine serum albumin (BSA) at room temperature (RT). After 5 more washes with PBS-T, wells were incubated for 1 hour at RT with fibrinogen and vimentin derived citrulline and biotin containing peptides (O ⁇ g /well).
  • PBS-T PBS-Tween20
  • BSA Bovine serum albumin
  • RhmAb2.101 or RhmAb2.102 were incubated for 1 hour at RT with serial dilutions of RhmAb2.101 or RhmAb2.102 in PBS-T + 1 % BSA starting at a concentration of 10 ⁇ g/well.
  • Wells were washed 5 times with PBS-T and incubated with rabbit-anti-human- HRP (1 :2000) for 1 hour at RT followed by 5 washes with PBS-T and 3 wash steps with PBS.
  • Wells were incubated 5min with TMB substrate before stopping the reaction with 2M H 2 S0 4 .
  • Optical density was measured by 450nm and is a measure for the affinity of the antibodies used.
  • Example 10 Therapeutic potential of RhmAb2.102
  • CAIA collagen antibody induced arthritis
  • mice/group mice For that purpose, on day 0 male DBA/J1 mice (5 mice/group) of the age of 8 weeks have been injected i.p. with a mix of 8 anti-collagen antibodies (2,8mg/mouse). On day 3, mice received another i.p. injection containing 25ug LPS. LPS triggers the inflammation. On day 7 when the mean arthritis score was around 4 ( Figure 6A) one group received an i.v. injection containing 1 mg RhmAb2.102, whether the other group received an i.v. injection containing placebo.
  • Example 1 1 Preparation of huPAD4 deiminated HEK293 extract and immunoprecipitation with RhmAb2.101 or RhmAb2.102
  • HEK293 cells were harvested, washed once with PBS, spun down, and
  • the cell samples were sonified 4 times for 15 seconds on ice.
  • the lysate was centrifuged at 3.000 rpm for 5 minutes and the supernatant transferred to a clean tube.
  • the cell lysate was deiminated for 2 hours at 37°C by adding 1 U human PAD4 per
  • IP immunoprecipitations
  • RhmAb2.101 20 ⁇ g RhmAb2.102 or not coupled (negative control).
  • Protein A Sepharose Beads / antibody mixtures have been incubated 1 h at room temperature under constant rotation. Beads were subjected to 3 washes with 1 ml IPP500, one wash with 1 ml IPP150 (10mM Tris/HCI pH8,0, 150mM NaCI, 0,1 % NP40 and 0,1 % Tween-20), and subsequently incubated at room temperature with 300 ⁇ deiminated HEK293 lysate for 2 hours under constant rotation.
  • RhmAb2.102 and control beads have been eluted with 50 ⁇ elution buffer (100mM Na citrate pH3.0) , neutralized with 10 ⁇ 1 M Tris/HCI pH9,04 and stored at -20°C until nl_C LTQ FTMS ULTRA mass spectrometry (Example 12).
  • Protein identification validation was performed by an in-house developed script. Briefly, the software classifies protein identifications based on the number of uniquely identified peptide sequences, clusters proteins sharing the same set of peptides and validates the proteins with the following criteria:
  • Proteins with 1 peptide must have a peptide score: >49
  • Proteins with more than 1 peptide must have a peptide score: >29
  • peptides have been identified in all 3 samples (sample 1 : HEK293 precipitate with RhmAb2.101 ; sample 2: HEK293 precipitate with Rhm2.102; sample 3: HEK293 precipitate with empty beads).
  • emPAI Extraly Modified Protein Abundance Index
  • gi 45031431 ref 1 N P_001900.11 cathepsin D preproprotein [Homo sapiens] ⁇ gi 775397581 ref
  • Example 13 Generation/selection of a family of anti-inflammatory antibodies
  • Human-derived scFv libraries were panned against PAD2-, or PAD4- deiminated forms of human Histon-2A, Histon-4, peptide 1 (AAASGXGKQGGK, SEQ ID NO: 21 ) and against CFC-1 peptide in a similar method as decribed in Raats et al., 2003 (Raats, J.M.H., Wijnen, E.W, Pruijn, G.J.M., Van den Hoogen, F.H.M., and W.J. van Venrooij. 2003. J. Rheum. 30, 1696-171 1 ).
  • Antibodies that immunoprecipitated bands p15 and/or p17, and/or antibodies with ELISA reactivity profiles against citrullinated epitopes PAD2 and PAD4 deiminated human Histon isoforms, and/or CFC-1 and/ or peptide 1 (AAASGXGKQGGK, SEQ ID 21 , and/or citrullinated epitopes derived from proteins listed in 9comparable with
  • RhmAb2.102 were subsequently cloned into human lgG1 format. Full size human IgG antibodies were tested for their prophylactic and/or therapeutic anti-inflammatory potential in a CAIA mouse model, as described herein.
  • This screening procedure yielded antibodies with prophylactic and or therapeutic anti inflammatory potential in the CAIA mouse model with high frequency.
  • RhmAb2.108, RhmAb2.109, RhmAb2.1 10, RhmAb2.1 1 1 and RhmAb2.1 12 are disclosed herein in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 39, SEQ ID NO: 20, SEQ ID NO: 41 , SEQ ID NO: 40, SEQ ID NO: 19, SEQ ID NO: 43, SEQ ID NO: 42.
  • the RhmAb2.1 10 immunoglobulin light chain encoded by SEQ ID NO: 10 comprises a mouse leader globulin according to SEQ ID NO: 12, followed by the variable antibody light chain according to SEQ ID NO: 41 followed by the immunoglobulin human kappa constant domain according to SEQ ID NO: 1 1.
  • RhmAb2.1 12, and RhmAb2.102 (1 mg/mouse) and placebo were administered via i.p. injection on day 3. All animals have been scored for inflammation daily until day 10.
  • Antibodies against a synthetic citrulline containing peptide according to the invention have been raised in DBA J1 mice.
  • serum samples were taken and analyzed for a citrullin specific antigen response. All mice showed a specific antigen specific serum titre at the time points tested.
  • spleens In order to produce hybridoma cell-lines, spleens have been dissected after the last boost, splenocytes were harvested from the spleen and fused with a mouse myeloma cell-line (NS-1 ) according to ModiQuest B.V. procedures. Antibody specificity in hybridoma supernatants have been screened on cirtulline containing antigen as well as on the non-ctirullinated equivalent.
  • NS-1 mouse myeloma cell-line
  • CAIA Collagen antibody induced arthritis
  • RmmAb22.101 , RmmAb22.102 and RhmAb2.102 (6mg/mouse) and placebo were administered via i.p. injection on day 3. All animals have been scored for inflammation daily until day 10.
  • RhmAb2.102, RmmAb22.101 and RmmAb22.102 antibodies completely protected the mice against inflammation in their paws.
  • Example 15 Novel therapeutic anti-citrulline antibodies display similar recognition patterns to citrullinated epitopes compared to RhmAb 2.102.
  • RhmAb2.108, RhmAb2.109, RhmAb2.1 10, RhmAb2.1 1 1 , RhmAb2.1 12, RmmAb22.101 , and RmmAb22.102 were analysed in ELISA for their reactivity on various deiminated targets compared to RhmAb 2.102.
  • Human recombinant histones H1 , H2A, H2B, H3 and H4 (100 ⁇ g) were deiminated as described in Example 7.
  • Optical density was measured by 450nm and is a relative measure for the affinity of the antibodies used. This showed clearly that all therapeutic antibodies have a highly similar staining pattern compared to the therapeutic antibody RhmAb2.102. Only the mouse monoclonals show no reactivity with the Cfc1 - peptide. All therapeutic antibodies, have very high reactivity with the a peptide according to SEQ ID No: 21 , as well as with Histon 2A/p2 Histon 2A/p4, and histon 4/p2, and show slight reactivity with Histon 3/p2.

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