WO1994017411A1 - Methodes et agents pour le diagnostic et le traitement de l'arthrite rhumatoide - Google Patents

Methodes et agents pour le diagnostic et le traitement de l'arthrite rhumatoide Download PDF

Info

Publication number
WO1994017411A1
WO1994017411A1 PCT/US1994/001077 US9401077W WO9417411A1 WO 1994017411 A1 WO1994017411 A1 WO 1994017411A1 US 9401077 W US9401077 W US 9401077W WO 9417411 A1 WO9417411 A1 WO 9417411A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
rheumatoid arthritis
antibodies
fibronectin
igg
Prior art date
Application number
PCT/US1994/001077
Other languages
English (en)
Inventor
Salvatore V. Pizzo
Mario Gonzalez-Gronow
Bruce A. Clinton
Original Assignee
Duke University
Trinity Laboratories, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Duke University, Trinity Laboratories, Inc. filed Critical Duke University
Priority to AU60989/94A priority Critical patent/AU6098994A/en
Publication of WO1994017411A1 publication Critical patent/WO1994017411A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3153—Streptokinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints

Definitions

  • the present invention relates generally to the field of immunology and, more particularly, to the disease of rheumatoid arthritis, and concerns the diagnosis and treatment of rheumatoid arthritis and the development of effective pharmaceutical agents and therapies thereagainst.
  • Rheumatoid arthritis is a destructive disease of the joints.
  • fibronectin (FN) is a normal constituent of synovial fluid
  • levels of synovM FN are greatly elevated in the inflamed joints of arthritic patients (1).
  • FN is known to be a chemoattractant for inflammatory cells (2) and plasmin (Pm) induces adherence of these types of cells to membranes (3).
  • FN is an effective Pm substrate (4), and the FN fragments thus generated may regulate the expression of collagenase and stromelysin at the transcriptional level via interaction with FN integrin receptors (5).
  • plasminogen activators are elevated in rheumatoid synovial fluid (6) while the levels of plasminogen activator inhibitors are decreased (7).
  • Pg and tissue plasminogen activator (t-PA) bind to FN with high affinity, making possible an extension of the proteolytic activity of Pm beyond cell boundaries (8). Taken together, these factors may contribute to the connective tissue destruction characteristic of rheumatoid arthritis.
  • Streptokinase a predominant protein secreted by streptococci, has the potential to disrupt the functional integrity of normal synovium by inducing Pg activation with subsequent degradation of FN.
  • SK is a potent immunogen in humans (10) and the presence of anti-SK antibodies has been associated with several autoimmune diseases.
  • Cross-reactivity of anti-SK antibodies with local antigens may be responsible for localized tissue injury in both membranous glomerulonephritis (11,12) and myocardial injury (13).
  • the present invention relates broadly to methods for diagnosing and treating rheumatoid arthritis (R-A), all predicated on the discovery of the existence of a factor herein referred to as an R-A predictive factor, that comprises a material selected from a region on fibronectin that is common to both fibronectin and steptokinase and, more particularly, a protein fragment from that region, an epitope defined within the region, and the pathogenic antibodies, and more particularly the asialylated antibodies, reactive therewith.
  • the region of the invention is defined by a 90 kD fragment of fibronectin formed by the digestive degradation of fibronectin by plasmin (Pm).
  • the region in turn, defines within it the particular epitope comprising the amino acids, LTSRPA, or Leu-Thr-Ser-Arg- Pro-Ala.
  • This sequence is presented in SEQ ID NO:l, in accordance with 37 CFR 1.821-1.825.
  • the invention is derived from the findings that fibronectin (FN) and streptokinase (SK) share an epitope of six amino acids which is recognized by a rabbit anti-SK IgG as well as by a purified anti-SK IgG from the serum of a rheumatoid arthritis patient, and that data suggesting that plasmin (Pm) proteolysis of FN may be critical in the exposure of this epitope.
  • FN fibronectin
  • SK streptokinase
  • the invention is further based on the discovery that a significant portion of the elevated levels of anti-LTSRPA (SEQ ID NO:l) antibodies are asialylated, as detected by direct ELISA and chromatofocusing. It has also been found that the asialylated (or deglycosylated) anti-LTSRPA (SEQ ID NO:l) IgGs isolated from arthritis patients have a much larger capacity to activate complement.
  • the invention includes within its scope diagnostic applications following a variety of protocols, and corresponding assays including a drug screen, and corresponding test kits.
  • the methods comprise the measurement of auto-antibodies specific for R-A predictive factor, and the consequent detection of the onset, or monitoring of the course and intensity of rheumatoid arthritis thereby.
  • the stated methods also include the detection and measurement of antibodies (IgG) such as anti-SK antibodies, that bind with the epitope and are implicated in the development of rheumatoid arthritis as well as other autoimmune diseases.
  • the invention relates to the detection of asialyl-antibodies specific for the R-A factor, and to detecting antibodies specific for the R-A factor that have increased capacity to activate complement.
  • the invention relates to detection of R-A-factor specific antibodies after adsorption or depletion of sialylated antibodies in the sample, i.e. , detection of such antibodies in a sample enriched for asialyl- antibodies.
  • the increased capacity of the antibody to activate complement is used in a diagnostic assay of the invention.
  • the invention extends to antagonists, including antibodies and non-antibody agents, that are capable of recognizing and/or binding to the R-A predictive factor- specific antibodies as defined herein, to thereby inhibit the development or intensification of the condition of rheumatoid arthritis.
  • antagonists include, but are not limited to, antibodies, particularly non-IgGl and IgG3 isotype antibodies, peptides, peptidomimetics, and drugs.
  • Antibodies include polyclonal, monoclonal and chimeric (including bispecific) antibodies, and single chain or natural F(v) region-containing portions thereof (including F(ab), F(ab') 2 , etc.), as well as anti-idiotypic antibodies, all prepared by techniques known in the art.
  • Suitable antagonists extend to drugs that may be identified by their efficacy displayed in an assay such as the ELISA prepared and illustrated herein.
  • a peptide containing the epitope sequence LTSRPA (SEQ ID NO: 1) can be used as a competitive inhibitor of antibody binding.
  • Other diagnostic procedures such as receptor assays and assays using either colorimetric or radiometric labels and measurement are also contemplated.
  • compositions including one or more of the antagonists as an active agent, in combination with a pharmaceutically acceptable carrier.
  • Such compositions may be formulated alone, or may include other known therapeutic agents for the treatment of rheumatoid arthritis.
  • the present compositions may be co-administered with other pharmaceutical compositions for the treatment of R-A or related pathology, with such co-administration following a predetermined schedule, including concurrent or sequential dosing. Specific dosages and dosing schedules will be determined by the qualified physician.
  • the invention also includes methods of inhibiting or treating rheumatoid arthritis by administering a therapeutically effective amount of a pharmaceutical composition of the invention.
  • the inhibition or prevention of R-A may proceed by the preparation of a vaccine and the inoculation of a patient therewith, where the vaccine comprises an active agent in turn, comprising or based on the R-A predictive factor of the invention.
  • Such vaccine may, for example be prepared with anti-idiotypic antibodies to the anti-SK antibodies, or alternatively, may be prepared with antisense RNA to the 90 kD region and/or the epitope as found in fibronectin.
  • the invention further extends to the preparation of antibodies capable of recognizing and binding to the 90 kD region and/or the epitope defined herein, or that are otherwise capable of neutralizing the activity thereof, such preparations including by recombinant means.
  • antisense RNA may be prepared against the sequence of the epitope exposed by the digestion of fibronectin by plasmin, and suitably engineered molecules serving anti-idiotypic antibodies may be prepared to block the reactions of the epitope with pathogenic IgG.
  • the R-A predictive factor, and particularly the polypeptide represented by SEQ ID NO: 1 may be coupled to a carrier and employed either as a diagnostic agent, or as part of a vaccine or other therapeutic modality.
  • compositions including vaccines, and therapeutic methods that operate by recognizing, reacting with and/or neutralizing the R-A predictive factor defined herein.
  • Yet another object of the invention is to provide for the first time natural asialyl- antibodies of defined antigen specificity.
  • FIGURE 1 depicts the alignment of the regions of FN (amino acids 1741-1790; SEQ ID NO:3) and SK (amino acids 6-62: SEQ ID NO:4) with sequence similarity.
  • FIGURES 2A and 2B are nitrocellulose blots presenting the results of SDS-PAGE analysis of the fragments generated by Pm digestion of human FN with different anti-SK IgGs. Protein samples were resolved in a discontinuous
  • Panel A lanes 1-3 show the nitrocellulose paper incubated with rabbit anti-SK IgG followed by reaction with an alkaline phosphatase conjugated secondary antibody. Lanes 4-6 show the nitrocellulose paper incubated with the IgG used in lanes 1-3 after filtration through LTSRPAHG (SEQ ID NO:2)-ovalbumin-SEPHAROSE. Lanes 7-9 show the Coomassie blue stained gel.
  • Panel B same as in panel A except that the nitrocellulose paper was incubated with a human anti-SK IgG. Lanes 1,4,7, native human FN (30 ⁇ g); lanes 2,5,8, FN (30 ⁇ g) incubated with 1 ⁇ g Pm; lanes 3,6,9, SK (5 ⁇ g).
  • FIGURE 3 is a blot of the results of gel electiophoresis analysis of the Pg binding region of human FN after digestion with Pm. Protein samples were resolved in a discontinuous SDS-polyacrylamide gel and electroblotted to nitrocellulose paper as described under Materials and Methods. Lanes 1 and 2 show the nitrocellulose paper incubated with 125 I-Glu-Pg. Lanes 3 and 4 show the Coomassie blue stained gel. Lanes 1 and 3 native human FN (30 ⁇ g); lanes 2 and 4 FN (30 ⁇ g) incubated with 1 ⁇ g Pm.
  • FIGURE 4 is a graph having two panels, depicting the effect of native FN and a Pm-generated 90 kD FN fragment on the rate of activation of Pg.
  • Pg (1 ⁇ g) was activated with t-PA (0.1 ⁇ /ml) or SK (1 ⁇ g) as described under Methods.
  • Panel A rate of Pm formation by t-PA in the presence of increasing concentrations of native FN (•) or the FN fragment (A).
  • Panel B rate of Pm formation by SK in the presence of increasing concentrations of native FN (•) or the FN fragment
  • FIGURE 6 is a graph depicting binding of native and asialyl rabbit anti-LTSRPA IgG to LTSRPAHG (SEQ ID NO:2)-Ovalbumin.
  • Increasing concentrations of anti-LTSRPA (anti-OP) IgG were added to 96-microwell plates coated with LTSRPAHG (SEQ ID NO:2)-Ovalbumin and incubated for 2 h at 37 * C as described under Example 2, Methods.
  • the plates were washed with PBS-Tween followed by a 1 h incubation at 37 * C with an alkaline phosphatase-conjugated anti-rabbit IgG secondary antibody. Binding was detected by monitoring the hydrolysis of the alkaline phosphatase substrate p-nitrophenylphosphate at 405 nm (•) native anti-OP IgG, (A) asialyl anti-OP IgG.
  • FIGURE 7 is a graph depicting quantification of total and asialyl anti-LTSRPA IgG.
  • Panel A The plates were washed with PBS-Tween followed by incubation with an alkaline phosphatase-conjugated anti-human IgG secondary antibody for 1 h at 37 * C.
  • Bound antibody was detected by monitoring the hydrolysis of the alkaline phosphatase substrate p-nitrophenylphosphate at 405 nm. Concentrations were calculated from standard curves made with purified rabbit anti-LTSRPA IgG as described under Example 2, Methods. Panel B; plates were incubated with biotin-conjugated Ricin for 1 h at 37 'C followed by incubation with alkaline phosphatase-conjugated avidin for 1 h at 37 * C as described under Example 2, Methods. Concentrations of asialyl-IgGs were calculated from standard curves with rabbit asialyl anti-LTSRPA IgG as described under Methods. The solid bars indicate results with RA IgG, and the stippled bars indicate the results with Normal IgG.
  • FIGURE 8 is a graph showing the distribution of IgG subclasses of the anti-LTSRPA IgG in RA patients and normal subjects.
  • Panel A IgG subclass distribution of anti-LTSRPA IgG in RA sera.
  • Panel B IgG subclass distribution of anti-LTSRPA IgG in normal subjects.
  • FIGURE 9 is a graph depicting the binding of RA and normal anti-LTSRPA IgG to FN, Pm-degraded FN and LTSRPAHG (SEQ ID NO:2)-Ovalbumin.
  • Increasing concentrations of purified RA and normal anti-LTSRPA IgGs from RA patient and control number 3 were added to 96-microwell plates coated with the antigen and incubated for 2 h at 37 * C as described under Example 2, Methods. The plates were washed with PBS-Tween followed by 1 h incubation at 37 * C with an alkaline phosphatase-conjugated anti-human IgG secondary antibody.
  • Binding was detected by monitoring the hydrolysis of the alkaline phosphatase substrate p-nitrophenylphosphate at 405 nm.
  • FIGURE 10 is a graph depicting C5 activation assay of anti-LTSRPA IgG complexes.
  • the samples and experimental conditions are similar to those of Figure 9.
  • the generation of C5a was measured by radioimmunoassay as described under Example 2, Methods. The results are expressed as the average ng of C5a generated by a given ng of bound anti-LTSRPA IgG per ml of serum.
  • A Normal IgG.
  • Panel A C5 des Arg generated by RA and normal anti-LTSRPA IgG bound to FN coated plates.
  • Panel B C5 des Arg generated by RA and normal anti-LTSRPA IgG bound to Pm-degraded FN coated plates.
  • Panel C C5 des Arg generated by RA and normal anti-LTSRPA IgG bound to LTSRPAHG (SEQ ID NO:2)-Ovalbumin coated plates.
  • FIGURE 12 is a chromatograph showing the separation of the anti-LTSRPA IgGs from a RA patient and a normal control by chromatofocusing.
  • Purified anti-LTSRPA IgG from the pair RA and normal control subjects designated as number 12 were analyzed by preparative chromatofocusing as described under Example 2, Methods.
  • Panel A Chromatofocusing profile of RA IgG (1.2 mg).
  • Panel B Chromatofocusing profile of normal IgG (0.55 mg).
  • NH2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxyl group present at the carboxyl terminus of a polypeptide.
  • amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of a ino- terminus to carboxyl-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
  • the above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.
  • R-A predictive factor protein fragment, region, and epitopes are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.
  • antibody refers to immunoglobulins, including whole antibodies as well as fragments thereof, and includes particularly such antibodies (IgGs) to the region, epitope or protein fragment as described herein; such as F(ab), F(ab') 2 or single chain antibodies containing the Fv region, that recognize or bind to specific epitopes.
  • the term thus encompasses, inter alia, polyclonal, monoclonal and chimeric antibodies, the last mentioned being described in detail in U.S. Pat. Nos. 4,816,397 and 4,816,567, which are incorporated herein by reference, as well as anti-idiotypic antibodies described in greater detail later on herein.
  • An antibody "preparation” thus contains such
  • .antibodies or fragments thereof which are reactive with an antigen when at least a portion of the individual immunoglobulin molecules in the preparation recognize (i.e., bind to) the antigen.
  • An antibody preparation is therefore termed "non- reactive" with the antigen when the binding of the individual immunoglobulin molecules to the antigen is not detectable by commonly used methods.
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
  • an antibody is said to "recognize” an epitope if it binds to the epitope.
  • “recognition” involves the .antibody binding reaction with an epitope, which may include the typical binding mechanisms and methods.
  • Binding is thus used in the conventional sense, and does not require the formation of chemical bonds.
  • epitope if generally used herein, is intended to identify one or more portions of an antigen or an immunogen which is recognized or recognizable by antibodies or other immune system components.
  • the "epitope region”, if appearing herein, refers to the epitope and the surrounding area in the vicinity of the epitope, taking into account three dimensional space. Hence, this may take into account the tertiary and quaternary structure of the antigen.
  • Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the a disease condition. According to the present invention, this may include a reduction in one or more of the symptoms of rheumatoid arthritis, e.g. , one or more of the American College of
  • Rheumatology' s revised criteria for the classification of rheumatoid arthritis a reduction in the number of painful joins; a change in Less's functional index; or a change in fibrin and erythrocyte sedimentation rates.
  • the present invention comprises the discovery of an R-A predictive factor selected from a region defined by a protein fragment of fibronectin that contains an epitope in common with siteptokinase, which epitope has the amino acid sequence LTSRPA (SEQ ID NO:l).
  • the invention further relates to the pathogenic auto-antibodies reactive with the R-A predictive factor, and more particularly to partly asialylated or deglycosylated glycoforms of such antibodies. It is the increase in the levels of such antibodies that correlates with the onset or progression of rheumatoid arthritis.
  • auto-antibody in any of its grammatical forms, refers to an antibody reactive with the R-A predictive factor.
  • Such antibodies are also refened to herein as “anti-streptokinase” antibodies, as it is believed that the antibodies develop in response to a streptococcal infection, and subsequently react with an auto-antigen (the R-A predictive factor epitope found on proteolytically modified fibronectin).
  • the R-A predictive factor- specific antibodies are at least partly asialylated.
  • such antibodies demonstrate a larger set of isoforms with isoionic points in the range of pH 6-8 than antibodies from normal individuals.
  • the antibodies from rheumatoid arthritis patients are less acidic.
  • the term "asialyl-antibody" in any of its grammatical form refers to an antibody that is less acidic than normal antibodies, e.g. , as determined by chromatofocusing.
  • asialyl-antibodies may, but need not, completely lack sialic acid, neuraminic acid, or other acidic carbohydrate functionalities.
  • deglycosylated is also used herein to refer to the asialylated or asialyl-antibodies according to the invention.
  • the IgG isolated from rheumatoid arthritis patients has a much larger capacity to activate complement.
  • the enhanced complement- activating potential of such antibodies from rheumatoid arthritis patients provides further evidence of the role of such antibodies in the onset and exacerbation of rheumatoid arthritis.
  • the identification of the R-A predictive factor of the invention grew out of the observation of a common region or epitope shared by sitesptokinase (SK) and fibronectin (FN), that is recognized by anti-SK antibodies taken from patients suffering from rheumatoid arthritis, and that accordingly is believed to play a major role in the onset and intensification of this disease.
  • the invention also extends to antagonists to the R-A predictive factor, including antibodies, peptides, peptidomimetics, and non-antibody agents, that are capable of recognizing and/or binding to the R-A predictive factor as defined herein or to the auto-antibodies reactive therewith, to thereby inhibit the development or intensification of the condition of rheumatoid arthritis.
  • Antibodies include polyclonal, monoclonal and chimeric (bispecific) antibodies, including fragments thereof containing the Fv region (such as F(ab), F(ab') 2 , and single chain Fv region antibodies), as well as anti-idiotypic antibodies, all prepared by techniques known in the art. Suitable antagonists extend to drugs that may be identified by their efficacy against the R-A predictive factor.
  • Panels of monoclonal antibodies produced against the present factor, or particular peptides can be screened for various properties; i.e., isotype, epitope, affinity, etc.
  • monoclonal antibodie- s that neutralize the activity of the R-A predictive factor or its subunits. Such monoclonals can be readily identified in activity assays for the factor. High affinity antibodies are also useful when immunoaffinity purification of native or recombinant factor is possible.
  • Prefened monoclonal antibodies should display an immunoreactivity for the antigen that is similar to that of those produced by the above-described hybridomas.
  • immunoreactivity in its various grammatical forms refers to the concentration of antigen required to achieve a 50% inhibition of the immunoreaction between a given amount of the antibody and a given amount of the R-A predictive factor antigen. That is, immunoreactivity is the concentration of antigen required to achieve a B/B 0 value of 0.5, where B 0 is the maximum amount of antibody bound in the absence of competing antigen and B is the amount of antibody bound in the presence of competing antigen, and both B 0 and B have been adjusted for background. See Robard, Clin. Chem. , 20: 1255- 1270 (1974). ,
  • the therapeutically useful antibodies of the invention lack the capacity to activate complement.
  • IgG sub-classes that do not activate complement IgG sub-classes that do not activate complement, non-IgG isotype antibodies, chimeric antibodies containing the antigen binding site of the antibody fused with a non-immunoglobulin protein, and Fv fragments of antibodies as defined hereinabove are prefened.
  • Such antibodies can competitively inhibit binding of the pathogenic auto-antibodies to the R-A predictive factor epitope on fibronectin.
  • antibodies specifically reactive with the auto-antibodies against the R-A predictive factor can be used therapeutically to neutralize such auto-antibodies.
  • such therapeutic antibodies may be anti- ideotypic antibodies, as defined herein.
  • the auto- antibodies, or immunologically equivalent analogs thereof can be used in a vaccine to generate the appropriate anti-ideotypic antibodies in an affected individual, e.g., an individual suffering rheumatoid arthritis.
  • the resulting antibodies could also be prepared in a suitable pharmaceutical composition and administered to avert or treat rheumatoid arthritis or other pathology responsive to this therapy.
  • the exact quantities, intervals of administration and administrative techniques respecting such pharmaceutical compositions may vary in accordance with those known in the medical arts, and upon the specific instruction of a qualified physician or veterinarian.
  • the existence of antibodies against the R-A predictive factor(s) makes possible another method for isolating other similar factor(s) and ligands, as well as an effective therapeutic strategy against pathogenic antibodies.
  • the method takes advantage of an antibody characteristic known as idiotype.
  • Each antibody contains a unique region that is specific for an antigen. This region is called the idiotype.
  • Antibodies themselves contain antigenic determinants; the idiotype of an antibody is an antigenic determinant unique to that molecule. By immunizing an organism with antibodies, one can raise "anti-antibodies" that recognize them, including antibodies that recognize the idiotype. Antibodies that recognize the idiotype of another antibody are called anti-idiotype antibodies.
  • anti-idiotypic antibodies mimic the shape of the original antigen that the antibody recognizes and are said to bear the "internal image" of the antigen (Kennedy, 1986).
  • antigen is a ligand
  • certain anti-idiotypes can bind to that ligand's receptor.
  • anti-idiotype antibodies already identified include anti- idiotypes that bind to receptors for insulin, angiotensin II, adenosine I, ⁇ - adrenalin, and rat brain nicotine and opiate receptors.
  • Anti-idiotypic antibodies may be used to screen for molecules binding to the original antigen. For example, one may use this technique to identify other factor ligands.
  • anti-ideotypic antibodies can be used in passive immunotherapy for the treatment of rheumatoid arthritis, as described above.
  • the present invention relates to certain therapeutic methods which would be based upon the activity of the present factor(s), antibodies or other antagonists to the factor(s), or upon agents or other drugs determined to possess an antagonistic activity.
  • such antagonists are peptides containing the amino acid sequence conesponding to the R-A predictive factor epitope, i.e. , LTSRPA, or derivatives or analogs thereof that are able to bind to the anti-LTSRPA auto-antibodies found in rheumatoid arthritis patients.
  • Such analogs include, but are not limited to, peptidomimetics and the like.
  • Peptidomimetics are molecules having some structural and functional characteristic in common with peptides, but that do not contain peptide bonds.
  • the invention relates to an analog of the peptide LTSRPA containing subunit amino acids or amino acid analogs.
  • the term analog refers to peptidomimetics.
  • the subunits may be linked by peptide bonds.
  • the subunit(s) may be linked by other bonds, e.g. , ester, ether, etc.
  • amino acid refers to natural amino acids, including glycine, unnatural or synthetic amino acids, both the D or L optical isomers of amino acids, and amino acid analogs, including mimics of di-, tri-, and larger peptides.
  • subunits of "peptides” that confer useful chemical and structural properties on the molecule in which such peptides are incorporated can be chosen.
  • peptides comprising D-amino acids will be resistant to L-amino acid-specific proteases in vivo.
  • the present invention envisions preparing peptides that have more well defined structural properties, and the use of peptidomimetics, and peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties.
  • a peptide may be synthesized that incorporates a reduced peptide bond, i.e., R r CH 2 -NH-R 2 , where R, and R 2 are amino acid residues or sequences.
  • a reduced peptide bond may be introduced as a dipeptide subunit.
  • Such a molecule would be resistant to peptide bond hydrolysis, e.g. , protease activity.
  • Such peptides would demonstrate unique function and activity, such as extended half-lives in vivo due to resistance to metabolic breakdown or endogenous protease activity.
  • constrained peptides show enhanced functional activity (Hruby, 1982, Life Sciences 31:189-199; Hruby et al., 1990, Biochem. J. 268:249-262); the present invention contemplates use of constrained peptides.
  • a therapeutic protocol is associated with the inhibition of the manifestations of conditions following from the production and activity of the R-A predictive factor, and comprises administering an antagonist capable of inhibiting the production and/or activity of the factor, in an amount effective to inhibit or neutralize R-A predictive factor activity (i.e. , the onset or intensification of rheumatoid arthritis) in the host.
  • a subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and an antagonist to the R-A predictive factor, appropriate polypeptide analogue thereof or fragment thereof, as described herein as an active ingredient.
  • the composition may contain an active ingredient derived from the amino acid sequence depicted in SEQ ID NO: 1 or an antibody thereto, in a pharmaceutically acceptable carrier.
  • the peptides of the invention may be crosslinked to themselves, or coupled to a carrier protein or a target protein to be rendered non-immunogenic, by techniques well known in the art as illustrated by U.S. Patent No. 4,822,606, the disclosure of which is incorporated herein by reference for this purpose.
  • compositions which contain polypeptides, analogs or active fragments as active ingredients are well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • a particular member of the group of R-A predictive factors or suitable antagonists of the invention such as peptides, analogues or active fragments can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
  • the present therapeutic compositions are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of inhibition of factor activity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual.
  • suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration.
  • Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration.
  • continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.
  • pg means picogram
  • ng means nanogram
  • ug means microgram
  • mg means milligram
  • ul means microliter
  • ml means milliliter
  • 1 means liter.
  • the diagnostic method of the present invention comprises examining a biological sample, such as but not limited to, blood, plasma, serum, synovial fluid, or tissue, by means of an assay including an effective amount of an means for detecting anti-R-A predictive factor auto-antibodies, preferably an affinity-purified polyclonal antibody, and more preferably a monoclonal antibody (mAb).
  • a biological sample such as but not limited to, blood, plasma, serum, synovial fluid, or tissue
  • an assay including an effective amount of an means for detecting anti-R-A predictive factor auto-antibodies, preferably an affinity-purified polyclonal antibody, and more preferably a monoclonal antibody (mAb).
  • anti-R-A predictive factor antibody molecules used herein be in the form of Fab, Fab', F(ab') 2 or F(v) portions or whole antibody molecules.
  • individuals primarily capable of benefiting from this method comprise those suffering or suspected of suffering from rheumatoid arthritis, although conditions based generally on a viral or retroviral infection or other like pathological derangement may also be capable of attention.
  • the present invention provides for distinguishing rheumatoid arthritis from other arthritic conditions by detecting the presence of increased levels of anti-RA predictive factor autoantibodies, in which the presence of increased levels compared to normal individuals indicates rheumatoid arthritis.
  • the invention provides a diagnostic assay for detecting the presence or onset of rheumatoid arthritis, comprising detecting the capacity of auto-antibodies specific for the epitope LTSRPA in a biological .sample from an individual to activate complement.
  • Suitable complement activation assays include, but are not limited to, activation of C5 to C5a, and activation of C3 to C3a.
  • Complement activation mediated by anti-LTSRPA antibodies present in the sample can be measured by standard techniques, including but not limited to enzyme- immunoassay, fluorescent immunoassay, radioimmunoassay, latex quatitation, and the like. It is also possible, although somewhat cumbersome, to measure complement activation using biological assays, such as neutrophil activation assays and the like.
  • the invention further includes a method for detecting rheumatoid arthritis, or other diseases the basis of their ability to elicit the production and activities affected by the present R-A predictive factor(s).
  • invasive stimuli could be identified and detected by their ability to stimulate the production of autoantibodies to the R-A predictive factor(s) by the relevant factor-producing cells.
  • samples of producer cells could be treated with/exposed to a number of materials known to stimulate the production of an R-A predictive factor as a control, while parallel cellular samples could be treated with or exposed to an extract of material from a location where rheumatoid arthritis is suspected. All samples could then be incubated, and thereafter, in one embodiment, samples could be assayed for the presence of the factor.
  • the method extends to the detection of antibodies or immune complexes associated with rheumatoid arthritis, wherein the immune complexes or antibodies bind to the R-A predictive factor epitope of the invention.
  • a purified quantity of the present factor may be radiolabeled, after which binding studies would be carried out using biological samples containing such antibodies or immune complexes having a possible affinity for the factor and a conesponding immunoreactivity thereto. Solutions would then be prepared that contain various quantities of labeled factor and samples would then be contacted with the labeled factor and thereafter incubated. The resulting samples are then washed, solubilized and then counted in a gamma counter for a length of time sufficient to yield a standard enor of ⁇ 5%.
  • the biological sample is a sample from an organ or tissue directly or indirectly affected by the presence of the autoantibodies or immune complexes specific for the R-A predictive factor epitope.
  • the biological sample may be tissue or synovial from an affected joint.
  • the present invention also relates to a method for detecting the presence of rheumatoid arthritis or other pathological states in mammals, by measuring the presence and activity of autoantibodies to the R-A predictive factor of the present invention. More particularly, the activity of the factor may be followed directly by assay techniques such as those discussed herein, through the use of an appropriately labeled quantity of the factor. Alternately, reagents that detect the presence of autoantibodies, such as a mouse-anti-human antibody, can be labeled and introduced into a medium to test for the presence and amount of such antibodies therein, and to thereby assess the state of the host from which the medium was drawn. In another embodiment, binding of IgG autoantibodies to the R-A predictive factor can be detected by the ability of such antibodies complexed with the factor to activate complement.
  • both the factor(s) and any antibodies or other antagonists that may be prepared thereagainst are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay, using for example, an antagonist to the factor(s) that has been labeled by either radioactive addition, reduction with sodium borohydride, or radioiodination.
  • immunoassays such as a radioimmunoassay, using for example, an antagonist to the factor(s) that has been labeled by either radioactive addition, reduction with sodium borohydride, or radioiodination.
  • a control quantity of the R-A predictive factor, an antibody or antagonist thereto, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cell-containing fluid sample of a mammal believed to be undergoing e.g. retioviral invasion. After the labeled material or its binding partner(s) has had an opportunity to react
  • a radioactive label such as the isotopes 3 H, ,4 C, 32 P, 35 S, 36 C1, 5, Cr, 57 Co, 58 Co, 59 Fe, ⁇ Y, 125 I, 131 I, and ,86 Re
  • known cunentiy available counting procedures may be utilized.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
  • the label is a fluorophore, it can be detected by fluorescence spectrometry.
  • the label is a particle, such as but not limited to a dyed latex bead or colloidal gold, it can be detected visually.
  • a particle such as but not limited to a dyed latex bead or colloidal gold.
  • the present invention includes an assay system which may be prepared in the form of a test kit adapted to the foregoing or following procedures for the quantitative analysis of the extent of the presence of the factor.
  • the system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the factor; and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s).
  • commercial test kits suitable for use by a medical specialist may be prepared to determine the presence or absence of the factor(s) or their cognates in a mammal.
  • kits will contain at least R-A predictive factor, which may or may not be labelled, and may also contain a binding partner thereto, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., "competitive", “sandwich”, “DASP” and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • test kit may be prepared for the demonstration of the presence and activity of the R-A predictive factor, comprising:
  • the diagnostic test kit may comprise:
  • test kit may be prepared and used for the purposes stated above, which operates according to a predetermined protocol (e.g. "competitive”, “sandwich”, “double antibody”, etc.), and comprises:
  • a labeled component which has been obtained by coupling the factor to a detectable label
  • one or more additional immunochemical reagents of which at least one reagent is a ligand or an immobilized ligand, which ligand is selected from the group consisting of:
  • a rapid diagnostic test for rheumatoid arthritis based on the detection of the unusual immunoglobulin reactivity to the defined R-A predictive factor epitope, wherein the activity of asialyl- immunoglobulins is detected.
  • the method comprises removing a significant portion of the sialylated antibodies from a biological sample. Removal can proceed by abso ⁇ tion of the sample on a lectin absorbent, such as a filter, chromatography column, separation cartridge, or other separation device known in the art.
  • a lectin absorbent such as a filter, chromatography column, separation cartridge, or other separation device known in the art.
  • the sample can be centrifuged through a lectin absorbent.
  • the resulting sample contains predominantly asialyl-immunoglobulins. Preferably, greater than about 95 % of the sialylated-immunoglobulins .are absorbed in this step.
  • an asialyl-enriched sample greatly increases the sensitivity and accuracy of the assay, since the asialyl- anti-R-A predictive factor antibodies are already increased in rheumatoid arthritis patients.
  • Any of the various techniques known in the art for detecting .antigen-specific antibodies can then be used to detect the asialyl-anti-LTSRPA antibodies in the sample.
  • an ELISA assay can be used, in which the LTSRPA epitope is adsorbed to the solid phase, and the asialyl-immunoglobulins detected by specific secondary antibodies.
  • the antibodies are contacted with a molecule containing the R-A predictive factor epitope for sufficient time to allow for reaction between the antibody and the epitope.
  • the antibodies are then evaluated for their ability to activate complement, which depends on the conformational change that occurs in the Fc region of the antibody upon binding to antigen, as well as the antibody isotype (IgGl and IgG3 activate complement) and the glycosylation state of the antibody (e.g. , asialylated-IgGs bind to a mannose- binding protein that activates complement through the classical pathway).
  • the antibody-epitope complexes are adsorbed to a solid phase support, such as an affinity bead, and washed to remove potential contaminants prior to the complement activation assay.
  • Non-specific complement activation can also be prevented by purifying the IgGs or immune complexes, although this is not essential.
  • Complement activation can be detected or determined by one of several methods available for the quatitation and measurement of complement components, including but not limited to, enzyme-immunoassay, fluorescent immunoassay, radioimmunoassay, latex quatitation, and the like, or biological assays for complement activity such as neutrophil chemotaxis or activation.
  • activation of C3 to produce C3a can be determined.
  • activation of C5 to produce C5a can be determined.
  • C5a is detected and measured with a 125 I-labeled human C5a des Arg radioimmunoassay kit. Accordingly, the present invention provides a test kit comprising:
  • (c) means for detecting binding of asialylated antibodies in the sample with the R-A predictive factor epitope.
  • a test kit of the invention includes:
  • Pg was purified from human plasma by affinity chromatography on 1-Lys-Sepharose (14) and separated into isoforms 1 and 2 by affinity chromatography on concaiiavalin A-Sepharose (15). Isoform 2 was used for all experiments. The purity of Pg was assessed by gel electiophoresis and contained always the Glu-Pg form as confirmed by the terminal sequence analysis NH 2 -EPLDDYVNTQGA (SEQ ID NO:5), analogous to that found in native Pg (16). SK was purified from Kabikinase ® (Kabi Co., Sweden) as described by Castellino et al. (17).
  • FN was purified from pooled plasma according to the method of Vuento and Vaheri (18). The concentration of proteins was determined spectrophotometrically at 280 nm using the A 1%/lcm of 7.5 for SK (19) and 12.9 for FN (18). Pm was generated by incubating 500 ⁇ g of Pg with 100 ⁇ l of urokinase-Sepharose in 10 mM Hepes, pH 7.4, for 1 h at 25 °C followed by centrifugation to remove the resin. FN peptide (90 kD) was purified by affinity chromatography using the heavy chain of Pm (residues 75-560) covalently linked to Sepharose-4B. The synthetic octapeptide LTSRPAHG (SEQ ID NO:2) was purchased from Multiple Peptide Systems, San Diego, CA.
  • Radioiodination of proteins was carried out by the method of Markwell (20). Radioactivity was measured in a LKB 1272 gamma counter. Incorporation of ,25 I was approximately 8 x 10 6 cpm/nmol of protein.
  • Antibodies to SK were prepared in rabbits according to standard protocols. The polyclonal anti-SK IgG was purified by affinity chromatography on a SK-Sepharose resin. Human anti-SK IgG was isolated from 40 ml of serum obtained from a rheumatoid arthritis patient and purified by affinity chromatography on SK-Sepharose as described above.
  • the octapeptide LTSRPAHG (SEQ ID NO:2) was coupled to ovalbumin with glutaraldehyde using the method of Kagen and Glick (21).
  • the octapeptide (10 mg) was dissolved in 1.5 ml of 0.4 M phosphate, pH 7.5, and added to 10 mg ovalbumin dissolved in 1 ml water.
  • 1 ml of 20 mM glutaraldehyde was added dropwise with stirring over the course of 10 min at room temperature.
  • the excess unreacted glutaraldehyde was blocked by adding 0.25 ml 1 M glycine and the mixture stined for an additional 30 min.
  • the excess peptide and reagent were removed by exhaustive dialysis versus phosphate-buffered NaCl, pH 7.2.
  • Protein sequence analysis The proteins (100 pmol) were sequenced by automated Edman degradation in a gas/liquid phase sequencer (model 477A; Applied Biosystems, Inc., Foster City, CA) with on-line PTH analysis using HPLC (model 477A; Applied Biosystems, Inc., Foster City, CA) with on-line PTH analysis using HPLC (model 477A; Applied Biosystems, Inc., Foster City, CA) with on-line PTH analysis using HPLC (model
  • FN and SK were analyzed with purified rabbit and human IgGs followed by reaction with an alkaline phosphatase-labeled secondary antibody (Sigma Chemical Co., St. Louis, MO).
  • VLK-pNA D-val-leu-lys-p-nitroanilide
  • Activation of Pg (1 ⁇ g) was initiated by addition of tPA (0.1 ⁇ /ml) or SK (1 ⁇ g) and the Pm hydrolysis of VLK-pNA was monitored continuously at 410 nm.
  • ELISA assays were performed using proteins passively adsorbed to 96-well culture plates. Plates were coated with 200 ⁇ l of 5 ⁇ g/ml of octapeptide LTSRPAHG (SEQ ID NO:2) coupled to ovalbumin in 0.1M sodium carbonate, pH 9.6, 0.2% NaN 3 and incubated overnight at 4°C. After coating, plates were washed with 200 ⁇ l 0.01 M sodium phosphate, 0.1 M sodium chloride, pH 7.4, containing 0.05% Tween-80 (PBS-Tween) to remove unbound protein. Non-specific sites were blocked by incubating with PBS-Tween
  • Proteolytic digestion by Pm also allows for a more efficient binding of Pg to FN, as evidenced by the strong interaction of Glu-Pg with the 90 kD FN peptide relative to the intact molecule.
  • Glu-Pg binds very poorly to immobilized FN
  • proteolytically modified Ly -Pg binds considerably better (8).
  • Analyses of this specific antibody indicates that liters of the cross-reacting SK-FN IgG in the sera of rheumatoid arthritis patients is significantly higher than those found in normal control sera.
  • the data supports an epitope-specific cross-reactive autoimmunity model as a potential component in the development of rheumatoid arthritis.
  • the factor that distinguishes rheumatoid arthritis from many other forms of inflammation is the persistence of the immune response and its localization within the joint. It is apparent from the above data that for the immune response to be initiated, there must be an initial proteolytic digestion of FN. This would result in the exposure of the epitope cross-reacting with anti-SK antibodies. Since proteolytic modification of FN is part of the normal cell remodelling process (30), the circulating anti-SK antibodies, originally elicited by streptococcal infections, can eventually form immune complexes on the cell surface enhancing the inflammatory process.
  • SK is a major product of these organisms.
  • sequence LTSRPA is conserved in all FN species so far sequenced including rat (31).
  • SK may also interact with cell-bound Pg, resulting in the formation of active Pm-SK complexes on the cell surface, thereby promoting local Pm generation and FN degradation.
  • Pg human umbilical vein endothelial cells
  • LTSRPA epitope
  • the epitope LTSRPA is not reactive in native fibronectin and reacts with anti-streptokinase antibodies only after plasmin digestion of the protein.
  • the present Example shows that levels of circulating anti-LTSRPA antibodies are not only abnormally elevated in the sera of rheumatoid arthritis patients, but that a significant proportion of these antibodies is asialylated. This defect in glycosylation was confirmed by direct ELISA assay and preparative chromatofocusing. No significant differences between the complement activating subclasses IgGl and IgG3 were found between anti-LTSRPA IgG of rheumatoid arthritis patients and normal controls.
  • Plasminogen Pg was purified from human plasma by affinity chromatography on 1-Lys-Sepharose (Deutsch and Mertz, 1970, Science 170: 1095- 96).
  • Fibronectin FN was purified from pooled plasmas according to the method of Vuento and Vaheri (1979, Biochem. J. 183:331-337). The concentration of FN was determined spectrophotometrically at 280 nm using the A l%n remind of 12.9 (Vuento and Vaheri, supra).
  • Plasmin was generated by incubating 500 mg of Pg with 100 ml of urokinase-Sepharose in 10 mM Hepes, pH 7.4, for 1 h at 25 °C, followed by centrifugation to remove the resin.
  • the synthetic octapeptide LTSRPAHG (SEQ ID NO:2) was purchased from Multiple Peptide Systems, San Diego, CA. Coupling of the octapeptide to ovalbumin was performed as described in Example 1, supra, and in Gonzalez-Gronow et al. (1993, Biochim. Biophys. Acta 1180:283-288).
  • RA patients and normal controls were analyzed by preparative chromatofocusing on a Mono P HR5/5 column (Pharmacia) attached to a Pharmacia FPLC System.
  • the column was equilibrated with 75 mM Tris-acetate, pH 9.3. The sample was applied to the column in the same buffer.
  • a pH gradient (9.3 -6) was generated using a 1:10 dilution of polybuffer 96 (Pharmacia) according to specifications from the manufacturer.
  • the column was eluted at 1 ml/min and fractions of 2 ml were collected.
  • ELISA assays were performed using proteins passively adsorbed to 96-well culture plates as described in Example 1 and in Gonzalez- Gronow et al., supra. Plates were coated with 200 ml of 5 mg/ml of octapeptide LTSRPAHG (SEQ ID NO:2)-coupled to ovalbumin in 0.1 M sodium carbonate, pH 9.6, 0.2% NaNj and incubated overnight at 4 * C. After coating, plates were washed with 200 ml 0.01 M sodium phosphate, 0.1 M sodium chloride, pH 7.4, containing 0.055 Tween-80 (PBS-Tween) to remove unbound proteins.
  • PBS-Tween PBS-Tween
  • Non-specific sites were blocked by incubating with PBS-Tween containing 2% bovine serum albumin (PBS-Tween-BSA) at 25 * C for 60 min. Plates were washed again with 200 ml PBS-Tween, dried, and stored at 4 * C. Calibration curves were made with a rabbit asialyl-anti-LTSRPA IgG. Purification of the mono-specific anti-LTSRPA IgG was performed by affinity chromatography on LTSRPAHG-ovalbumin-Sepharose as described in Example 1 and Gonzalez- Gronow et al., supra.
  • Asialyl IgG was prepared by incubating the purified rabbit anti-LTSRPA IgG (5 mg/ml) in 0.1 M sodium acetate, 2 mM CaCl 2 , pH 5.4, containing 100 units of Vibrio cholerae neuraminidase for 10 h at 37 * C. The solution was then dialyzed overnight against 50 mM Tris-HCl, pH 8.0, and repurified by chromatography on Protein A-Sepharose (Harlow and Lane, supra). The functional integrity of the in vitro asialylated IgG was assessed by ELISA assays on LTSRPAHG-Ovalbumin coated culture plates. No difference was observed in the binding capacity of the asialyl-IgG when compared with native IgG by this assay.
  • the concentration of asialyl-IgG was determined in triplicate by assaying increasing amounts of IgG in a 200 ml final volume of PBS-Tween-BSA incubated for 2 h at 37 * C. Plates were then washed three times with PBS-Tween and incubated for 1 h with biotin conjugated Ricinus communis lectin Ricin (1:1000 dilution). The plates were then washed three times with PBS-Tween and incubated with alkaline phosphatase conjugated to avidin (1: 1000 dilution) in PBS-Tween for 1 h.
  • IgG subclass ELISA of native human anti-LTSRPA antibodies Microtiter plates were coated with 200 ml of 5 mg/ml of octapeptide LTSRPAHG (SEQ ID NO:2) coupled to ovalbumin as described above and incubated for 2 h at 25 * C with patient sera (1:100 dilution) in a 200 ml final volume of PBS-Tween-BSA.
  • mice monoclonal anti-human IgG subclass reagents (Sigma): a) anti-human IgGl, clone HP-6091 (Fc specific); b) anti-human IgG2, clone HP-6002 (Fc specific); c) anti-human IgG3, clone HP-6050 (Fc specific); and d) anti-human IgG4, clone HP-6023 (Fc specific).
  • These antibodies are highly specific for their respective IgG subclasses and have been recommended by the IUIS/WHO study for use in several assay protocols (Hamilton, 1987, Clin. Chem. 33:1707-11).
  • FN fibronectin plates.
  • FN was passively adsorbed to 96-well culture plates as described above. Plasmin degraded FN plates were prepared by incubating the FN-coated wells with 2 mg of Pm in a 200 ml final volume of 10 mM Hepes, pH 7.4, overnight at 25 * C. To remove the Pm, the wells were washed three times with 200 ml of 100 mM 6-aminohexanoic acid in 50 mM Tris-HCl, pH 7.5, and then two times with 200 ml of 50 mM glycine-HCl, pH 3.0.
  • Binding of the anti-LTSRPA IgGs was performed in triplicate adding increasing amounts of the IgG in a 200 ml final volume of PBS-Tween-BSA, and incubated for 2 h at 37 * C. Plates were then washed three times with PBS-Tween and stored at 4' C.
  • Human complement C5a des Arg assay system Human complement C5a des Arg assay system.
  • the assay was performed in 96- well microtiter plates prepared as described above.
  • the FN-IgG, Pm-degraded FN-IgG and LTSRPA-IgG complexes were prepared as described above.
  • the antigen-antibody complexes were then incubated with 200 ml of fresh human serum as a source of C5a.
  • the serum was prepared from the blood of a single donor collected by venipuncture into siliconized vacutainer tubes.
  • the anti-LTSRPA IgGs were analyzed by an ELISA method based on the ability of Ricinus communis lectin (RC1) to bind to terminal galactose residues in oligosaccharide chains (Parkinnen, 1988, Clin. Chem. 35:1638-43).
  • the quantification of the asialylated patient IgGs was performed using a calibration curve with enzymatically asialylated rabbit anti-LTSRPA IgG purified as described under Methods.
  • the antigen-binding capacity of the asialyl IgG was compared with native IgG.
  • Figure 6 shows the binding curves obtained by an ELISA technique and demonstrates that both IgGs bind to the antigen with similar affinity.
  • a group of 44 paired samples of both RA and normal control sera was analyzed for total anti-LTSRPA IgG and by the method described to measure asialyl IgG.
  • the anti-LTSRPA IgG titers in sera from RA patients are higher than those found in a similar sample of normal sera used as controls as seen in Figure 7A (p ⁇ 0.006 by student's West).
  • levels of anti-LTSRPA asialyl-IgGs are significantly higher in RA sera when compared with normal controls as seen in Figure 7B (p ⁇ 0.001).
  • the antigen-antibody complexes were then evaluated for their capacity to activate C5 to C5a.
  • the response was poor with intact FN (Fig. 10A), but was enhanced on Pm-degraded FN (Fig. 10B).
  • a similar high response was observed with complexes bound to the LTSRPA (SEQ ID NO: l) peptide (Fig. IOC).
  • Fig. 9 Although similar amounts of Ag-Ab complexes from both normal and RA IgGs were bound (Fig. 9), the complement activating capacity was different, suggesting that the RA IgG possesses a much larger capacity to activate C5 to C5a.
  • a similar analysis was performed with purified anti-LTSRPA IgGs from 10 RA patients and normal controls. The results shown in Figure 11 indicate that the responses to the RA IgGs were significantly higher (p ⁇ 0.001) than those of the normal controls.
  • FIG. 12A shows the chromatofocusing profile of 1.2 mg of purified RA anti-LTSRPA IgG.
  • Figure 12B shows the profile of 0.55 mg of purified control anti-LTSRPA IgG.
  • Comparison of the distributions of isoforms demonstrates that the RA IgG showed a much larger set of forms with isoionic points in the range of pH 6-8 than the normal IgG. Therefore, the RA IgG contained a larger number of asialyl-IgGs (less acidic forms) than the normal IgG directed against the same epitope. These differences may account for the differential capacity to activate the complement shown for 10 patients in Figure 11.
  • Table I summarizes the data from the 10 RA patients and controls from Figure 11. As clearly shown, the RA IgGs show a much larger capacity to activate C5 to C5a than the normal IgGs. TABLE I Comparison of the anti-LTSRPA IgG titers, IgG subclass distribution and complement activiating capaity of 10 paired rheumatoid arthritis and normal subjects.
  • Example 1 documented the presence of IgG antibodies in sera from RA patients showing cross-reactivity with the epitope LTSRPA (SEQ ID NO: 1) present in both SK and a Pm-generated FN 90 kDa fragment.
  • This Example analyzed the IgG subclasses and the amount of asialyl antibodies in a paired population of RA patients and normal subjects. The majority of the anti-LTSRPA IgGs in both the RA and normal populations are of the IgGl and IgG3 subclasses. No significant differences in the distribution of these IgG subclasses between both populations was found.
  • IgG3 is the most effective complement-activating IgG subclass in humans (Natvig and Kunkel, 1973, Adv. Immunol. 16: 1-59).
  • C5a is the most potent chemotactic peptide released during the activation of the complement cascade (Fernandez et al., 1978, J. Immunol. 120: 109-115) and would therefore be a strong stimulus for the migration of inflammatory cells into the joint.
  • Recent studies show a conelation between the degree of glycosylation of IgG and RA activity indicated by both clinical and serological markers (Casburn-Budd et al, 1992, J. Rheum. 19:1070-74).
  • the presence of these non-reducing saccharides may have an additional role in acting as a ligand for the serum mannose-binding protein, which activates complement through the classical pathway (Ikada et al., 1987, J. Biol. Chem. 262:7451-54).
  • the amount of anti-LTSRPA IgG isolated from the small amount of serum obtained from each patient precluded the ability to further analyze the N-linked oligosaccharides of the Fc region at this time, although additional serum can be obtained for analysis of N-linked oligosaccharides of the Fc region of IgGs using standard techniques.
  • Radioimmunoassay Jaffe, B.B. & Behrman, H.R., eds. pp 328-329, Academic Press, New York.
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Rheumatology (AREA)
  • Rehabilitation Therapy (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une méthode de détection ou de diagnostic et de traitement de l'arthrite rhumatoïde se basant sur la présence d'auto-anticorps spécifiques à un facteur prédictif de l'arthrite rhumatoïde (A-R). Le facteur prédictif de l'A-R représente un épitope commun identifié sur la streptokinase (SK) et la fibronectine (FN) et reconnu par une immunoglobuline (IgG) anti-SK de lapin et une IgG anti-SK humaine isolée du sérum d'un patient atteint d'arthrite rhumatoïde. L'analyse des séquences de FN et de SK indique une région d'homologie contenant la séquence LTSRPA (No. ID SEQ:1). La LTSRPA (No. ID SEQ:1) couplée à une protéine vectrice réagit avec les anticorps anti-SK prélevés sur un lapain ou dans le plasma de patients atteints d'arthrite rhumatoïde. Les anticorps humains spécifiques à l'épitope LTSRPA (No. ID SEQ:1) présentent une capacité accrue à activer le complément, et un pourcentage élevé des ces anticorps est sialylé. Ces études révèlent que le fragment de FN de 90 kD généré par Pm peut réagir avec des anticorps circulants initialement produits par des infections streptococciques. Ces complexes immuns peuvent jouer un rôle dans l'étiologie de l'arthrite rhumatoïde. Des moyens diagnostiques et thérapeutiques impliquant lesdits facteurs et anticorps sont également décrits.
PCT/US1994/001077 1993-01-27 1994-01-27 Methodes et agents pour le diagnostic et le traitement de l'arthrite rhumatoide WO1994017411A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU60989/94A AU6098994A (en) 1993-01-27 1994-01-27 Methods and agents for the diagnosis and treatment of rheumatoid arthritis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US947193A 1993-01-27 1993-01-27
US08/009,471 1993-01-27

Publications (1)

Publication Number Publication Date
WO1994017411A1 true WO1994017411A1 (fr) 1994-08-04

Family

ID=21737862

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/001077 WO1994017411A1 (fr) 1993-01-27 1994-01-27 Methodes et agents pour le diagnostic et le traitement de l'arthrite rhumatoide

Country Status (2)

Country Link
AU (1) AU6098994A (fr)
WO (1) WO1994017411A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5861157A (en) * 1994-01-28 1999-01-19 Neutec Pharma Plc Diagnosis and treatment of infections due to Streptococci and Enterococci
FR2773078A1 (fr) * 1997-12-30 1999-07-02 Univ Toulouse Utilisation de peptides citrullines derives de la filaggrine pour le traitement de la polyarthrite rhumatoide
US20100248262A1 (en) * 2009-03-31 2010-09-30 Takara Bio Inc. Anti-Fibronectin Fragment Monoclonal Antibody
CN103627690A (zh) * 2008-03-31 2014-03-12 科学与工业研究委员会 链激酶突变体及其共价修饰形式
US10385121B2 (en) 2007-10-30 2019-08-20 Philogen S.P.A. Antigen associated with rheumatoid arthritis

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986004588A1 (fr) * 1985-02-01 1986-08-14 Charles Hurwitz Proteine caracteristique de l'arthrite rhumatoide
DE3513915A1 (de) * 1985-04-17 1986-10-23 Heyl Chemisch-pharmazeutische Fabrik GmbH & Co KG, 1000 Berlin Immunologischer testkit
US4980279A (en) * 1986-04-15 1990-12-25 Scripps Clinic And Research Foundation Cellular fibronectin: polypeptides, anti-polypeptide antibodies and assay methods

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986004588A1 (fr) * 1985-02-01 1986-08-14 Charles Hurwitz Proteine caracteristique de l'arthrite rhumatoide
US4645748A (en) * 1985-02-01 1987-02-24 Charles Hurwitz Protein which is characteristic of rheumatoid arthritis
DE3513915A1 (de) * 1985-04-17 1986-10-23 Heyl Chemisch-pharmazeutische Fabrik GmbH & Co KG, 1000 Berlin Immunologischer testkit
EP0199261A2 (fr) * 1985-04-17 1986-10-29 Heyl Chemisch-pharmazeutische Fabrik GmbH & Co. KG Trousse de réactifs pour test immunologique
US4980279A (en) * 1986-04-15 1990-12-25 Scripps Clinic And Research Foundation Cellular fibronectin: polypeptides, anti-polypeptide antibodies and assay methods

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ANN M. GRIFFITHS ET AL.: "Fragmented fibronectin and other synovial fluid proteins in chronic arthritis: their relation to immune complexes", CLINICA CHIMICA ACTA, vol. 184, no. 2, 29 September 1989 (1989-09-29), AMSTERDAM, NL, pages 133 - 146 *
GIUSEPPINA DE PETRO ET AL.: "Transformation-enhancing activity of gelatin binding fragments of fibronectin", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 78, no. 8, August 1981 (1981-08-01), WASHINGTON US, pages 4965 - 4969 *
HIRAKU KIKUCHI ET AL.: "Plasminogen activator in Synovial Fluid from Patients with Rheumatoid Arthritis", THE JOURNAL OF RHEUMATOLOGY, vol. 14, 1987, TORONTO, CA, pages 439 - 445 *
IVAN M. ROITT ET AL.: "The Role of Antigen in Autoimmune Responses with Special Reference to Changes in Carbohydrate Structure of IgG in Rheumatoid Arthritis", JOURNAL OF AUTOIMMUNITY, vol. 1, 1988, NEW YORK, NY, US, pages 499 - 506, XP022993471, DOI: doi:10.1016/0896-8411(88)90043-1 *
MARIO GONZALEZ-GRONOW, JAN J. ENGHILD AND SALVATORE V. PIZZO: "Streptokinase and human fibronectin share a common epitope: implications for regulation of fibrinolysis and rheumatoid arthritis", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1180, no. 3, 22 January 1993 (1993-01-22), AMSTERDAM, NL, pages 283 - 288, XP000431525 *
R. B. PAREKH, R.A. DWEK AND T.W. RADEMACHER: "Rheumatoid Arthritis as a Glycosylation Disorder", BRITISH JOURNAL OF RHEUMATOLOGY (SUPPLEMENT), vol. 27, no. II, 1988, LONDON, GB, pages 162 - 169 *
ROGER CASBURN-BUDD ET AL.: "Asialylated IgG in the Serum and Synovial Fluid of Patients with Rheumatoid Arthritis", THE JOURNAL OF RHEUMATOLOGY, vol. 19, no. 7, July 1992 (1992-07-01), TORONTO, CA, pages 1070 - 1074, XP000431526 *
ZENA WERB ET AL.: "Signal Transduction through the Fibronectin Receptor Induces Collagenase and Stromeysin Gene Expression", THE JOURNAL OF CELL BIOLOGY, vol. 109, no. 2, August 1989 (1989-08-01), NEW YORK, NY, US, pages 877 - 889, XP000431527, DOI: doi:10.1083/jcb.109.2.877 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5861157A (en) * 1994-01-28 1999-01-19 Neutec Pharma Plc Diagnosis and treatment of infections due to Streptococci and Enterococci
FR2773078A1 (fr) * 1997-12-30 1999-07-02 Univ Toulouse Utilisation de peptides citrullines derives de la filaggrine pour le traitement de la polyarthrite rhumatoide
WO1999034819A2 (fr) * 1997-12-30 1999-07-15 Universite Paul Sabatier - Toulouse Iii Utilisation de peptides citrullines derives de la filaggrine dans le cadre du traitement de maladies autoimmunes
WO1999034819A3 (fr) * 1997-12-30 1999-11-04 Univ Toulouse Utilisation de peptides citrullines derives de la filaggrine dans le cadre du traitement de maladies autoimmunes
US10385121B2 (en) 2007-10-30 2019-08-20 Philogen S.P.A. Antigen associated with rheumatoid arthritis
CN103627690A (zh) * 2008-03-31 2014-03-12 科学与工业研究委员会 链激酶突变体及其共价修饰形式
US20100248262A1 (en) * 2009-03-31 2010-09-30 Takara Bio Inc. Anti-Fibronectin Fragment Monoclonal Antibody
US8349569B2 (en) * 2009-03-31 2013-01-08 Takara Bio Inc. Anti-fibronectin fragment monoclonal antibody

Also Published As

Publication number Publication date
AU6098994A (en) 1994-08-15

Similar Documents

Publication Publication Date Title
EP0573545B1 (fr) Anticorps monoclonaux contre des sites de liaison induits par un recepteur
Mulder et al. Activation of granulocytes by anti‐neutrophil cytoplasmic antibodies (ANCA) in Wegener's granulomatosis: a predominant role for the IgG3 subclass of ANCA
US7728115B2 (en) Compositions and methods useful for the diagnosis and treatment of heparin induced thrombocytopenia/thrombosis
US6265551B1 (en) Form of dipeptidylpeptidase IV (CD26) found in human serum, antibodies thereto, and uses thereof
Fong et al. Expression of three cross-reactive idiotypes on rheumatoid factor autoantibodies from patients with autoimmune diseases and seropositive adults.
WO1994020142A1 (fr) Dosages et methodes therapeutiques bases sur des facteurs de chimiotactisme
CA2443330A1 (fr) Anticorps dirige contre l'osteopontine, et son utilisation
KR20240019394A (ko) 인간 타우를 표적화하는 화합물 및 방법
HUE029649T2 (en) Immunoglobulin cleavage fragments as disease indicators and preparations for detecting and binding such fragments
US20110207152A1 (en) Gelsolin binding agent compositions and uses of same
US20040170631A1 (en) Heparanase activity neutralizing anti-heparanase monoclonal antibody and other anti-heparanase antibodies
EP1767543A1 (fr) Nouveaux polypeptides dérivés du vénin d'abeilles et méthodes pour les utiliser
Olsen et al. Unique antigenic determinants (idiotypes) used as markers in a patient with macroglobulinemia and urticaria: similar idiotypes demonstrated in the skin and on peripheral blood lymphocytes
EP0972781B1 (fr) Polypeptides de la protéine S et leurs utilisations
WO1994017411A1 (fr) Methodes et agents pour le diagnostic et le traitement de l'arthrite rhumatoide
US20070269437A1 (en) Pro-Inflammatory and Anti-Inflammatory Antibodies Against the Heparin-Binding Protein (Hbp)
Levy et al. Characterization of murine monoclonal anti-endothelial cell antibodies (AECA) produced by idiotypic manipulation with human AECA.
US5681700A (en) Assay for pathogenicity of anti-DNA antibodies
EP0762881B1 (fr) Utilisation d'agents actifs pour la preparation de medicaments pour le traitement de l'arthrite rhumatoide
US5837686A (en) Peptides and antibodies for treatment of rheumatoid arthritis
WO2001004159A1 (fr) Compositions et methodes utiles au diagnostic et au traitement de la thrombocytopenie/thrombose induite par l'heparine
Skubitz et al. Preparation and characterization of monoclonal antibodies to human neutrophil cathepsin G, lactoferrin, eosinophil peroxidase, and eosinophil major basic protein
EP0959900B1 (fr) Diagnostic de la rectocolite hemorragique et de sous-types de ladite maladie a l'aide de l'histone h1
US20090258024A1 (en) Compositions and methods for diagnosis and treatment of chronic inflammatory diseases
JP2792651B2 (ja) ヒト膵臓癌に対するモノクローナル抗体

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BB BG BR BY CA CZ FI HU JP KP KR KZ LK MG MN MW NO NZ PL RO RU SD SK UA US VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA