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WO1993007485A1 - Regions de structures d'immunoglobulines rares associees a la maladie chez l'homme - Google Patents

Regions de structures d'immunoglobulines rares associees a la maladie chez l'homme Download PDF

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Publication number
WO1993007485A1
WO1993007485A1 PCT/US1992/008264 US9208264W WO9307485A1 WO 1993007485 A1 WO1993007485 A1 WO 1993007485A1 US 9208264 W US9208264 W US 9208264W WO 9307485 A1 WO9307485 A1 WO 9307485A1
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framework region
sequence
human
rare
rheumatic disease
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PCT/US1992/008264
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English (en)
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Richard H. Weisbart
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The Regents Of The University Of California
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Publication of WO1993007485A1 publication Critical patent/WO1993007485A1/fr

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    • 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
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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 technical field of this invention is the diagnosis and treatment of human rheumatic disease.
  • rheumatic diseases include a broad spectrum of disease. Despite the high frequency and debilitating impact of rheumatic diseases, their pathogenesis remain largely unknown, though various genetic, infectious, and environmental factors have been proposed. Immune system dysfunction and autoimmunity may also be involved in some rheumatic diseases.
  • Diagnosis of rheumatic diseases is generally based upon clinical symptoms and radiological evidence. For some diseases, the presence of autoreactive T-cells or B-cells and their products have proved useful. As examples, RA, primary biliary cirrhosis, and rheumatic fever have been correlated with the presence of respectively, certain anti-immunoglobulin antibodies (rheumatoid factor), anti- mitochondrial antibodies, and autoantibodies to heart valve antigens. However, in none of these diseases does the correlation with autoantibody provide dispositive diagnosis: the autoantibodies are present in some healthy people and absent in some afflicted with rheumatic disease. More often than not, diagnosis requires a pattern of clinical correlates.
  • Treatments for rheumatic diseases typically address overlying symptoms rather than the source of the disease. More common approaches include: anti-inflammatory drugs (e.g. corticosteroids and nonsteroidal anti-inflammatory drugs such as aspirin) immunosuppressive drugs, cytotoxins such as the dihydofolate reductase inhibitor methotrexate, antibiotics, anti-malarial drugs, heavy metals such as gold salts, radiotherapy, and surgery.
  • anti-inflammatory drugs e.g. corticosteroids and nonsteroidal anti-inflammatory drugs such as aspirin
  • immunosuppressive drugs e.g. corticosteroids and nonsteroidal anti-inflammatory drugs such as aspirin
  • cytotoxins such as the dihydofolate reductase inhibitor methotrexate
  • antibiotics e.g. anti-malarial drugs
  • anti-malarial drugs e.g. anti-malarial drugs
  • heavy metals such as gold salts
  • rheumatoid factors are antiglobulin antibodies that bond the Fc portion of IgG immunoglobulins and are implicated in the pathogenesis of rheumatoid arthritis. Allen and Kunkel, Arthritis Racrum. 9:758-768 (1966) and Fong et al., I. Immunol.
  • rheumatoid factors in patients with rheumatoid arthritis may be different from these factors in individuals without rheumatoid arthritis, as indicated by studies of rheumatoid factor specificity and idiotype. See also Jirik et al., Proc. Natl. Acad. Sci. USA 83:2195-2199 (1986).
  • the human lymphoblastoid cell line hRF-1 is reported by Weisbart et al., J. Immunol. 139: 2925-2928 (1987).
  • U.S. Patent No. 4,863,850 discloses a method for diagnosing rheumatoid arthritis by the presence of a "rheumatoid arthritis specific protein” or RASP.
  • RASP is reported to be "extremely similar to human IgG”: comprising two light and two heavy chains with molecular weights corresponding to those of human IgG and having the chemical and immunological properties of human IgG.
  • the N-terminal 43 amino acids of the RASP light chain sequence reported by Yamanaka et al. reveals that RASP contains an IgG kappa light chain employing a class I variable region (V ⁇ I ). Kabat et al. (1987), Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services).
  • immunoglobulins comprising a rare framework region which differs at at least one position that is conserved in analogous framework regions, are associated with human rheumatic diseases or susceptibilities thereto.
  • Such immunoglobulins and nucleic acids encoding such immunoglobulins are detected by conventional methods.
  • Compositions useful for reducing the pathogenicity associated with such immunoglobulins find use as therapeutics.
  • compositions are provided for diagnosis and treatment of human rheumatic diseases and disease susceptibilities associated with immunoglobulin.
  • associated with immunoglobulin means that the relevant disease generally correlates with the presence or absence of certain immunoglobulin.
  • Human rheumatic diseases include a broad spectrum of syndromes: from highly organ specific to generalized systemic pathology these diseases include among others, Hashimoto's thyroiditis, primary myxoedema, rheumatic fever, myasthenia gravis, primary biliary cirrhosis, sympathetic ophthalmia, idiopathic leucopenia, active chronic hepatitis, Sjogren's syndrome, scleroderma, and systemic lupus erythematosus.
  • One class of rheumatic disease is characterized by erosive, inflammatory arthritis.
  • rheumatoid arthritis ankylosing spondylitis, Reiter's syndrome, psoriatic arthritis, and inflammatory bowel diseases such as ulcerative colitis and regional enteritis.
  • pathology is associated with immunoglobulin.
  • autoreactive immunoglobulin may be at least partly responsible for other human diseases, such as multiple sclerosis. Textbook of Rheumatology (Eds. Kelley, W.; Harris, E.D.; Ruddy, S.; Sledge, C.B., W.B. Saunders, Philadelphia, 1981).
  • Immunoglobulins are of five classes defined by the heavy chain type: IgG, IgM, IgD, IgA, and IgE.
  • the immunoglobulin light chains are of two classes: lambda ( ⁇ ) and kappa (K).
  • Heavy and light chains each comprise a constant and a variable region domain. From the N-terminal amino acid, each variable region domain is divisible into alternating framework (FR) and complementarity determining regions (CDR): FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the variable region may be one of four classes (V ⁇ I , V ⁇ II , V ⁇ III , V ⁇ IV ) depending on the sequence of, principally, the framework regions.
  • immunoglobulins containing a rare FR3 particularly, immunoglobulin light chains containing a rare FR3; more particularly, ⁇ chains containing a rare FR3; and most particularly, ⁇ chains with V ⁇ II variable regions containing a rare FR3.
  • the particular J chain associated with the variable region is a J5 segment.
  • the immunoglobulins of the present invention are generally of the IgG class.
  • immunoglobulins comprising a rare framework region which differs at at least one position that is conserved in analogous framework regions are associated with rheumatic diseases or susceptibilities thereto.
  • Analogous framework regions are those of the same framework region number and same variable region domain class, e.g. all FR3 regions within V ⁇ II variable region domains are analogous.
  • At a conserved position at least about 80%, preferably at least about 85% and more preferably at least about 90% of analogous framework regions have the same amino acid. For instance, Q is found at position 3 in greater than 97% of known V ⁇ I sequences while V is found at the same position in 97% of all known V ⁇ II .
  • N, V and T are found at positions 18, 19 and 20, respectively in nearly all V ⁇ I sequences whereas P, A and S are found at the same positions in virtually all known V ⁇ II .
  • Rare framework regions are found in fewer than about 20%, usually fewer than about 10%, and more usually fewer than about 5% of the total number of variable region domains.
  • Some framework region positions are also conserved across V ⁇ classes. For example at position 62, phenylalanine occurs in all four of the known kappa classes and is present in 78/80 of reported kappa light chains. Only one of these reported kappa chains (a V ⁇ I ) contains valine at position 62. Similarly, serine is present at residue 65 in 75/80 reported kappa chain sequences. Only one of these known kappa chains (a V ⁇ III ) contained arginine at position 65. Kabat et al. (1987). None of the 80 reported kappa light chain sequences differed from the conserved phenylalanine and serine at both positions 62 and 65.
  • the rare framework regions of the invention differ at at least one position that is conserved in analogous framework regions; preferably at at least one position is also conserved across classes and particularly within the FR3 region. These rare framework regions generally differ at one to three positions, preferably at one to two positions, and more preferably at 2 positions; particularly at position 62 or 65; and most particularly at both positions 62 and 65.
  • the difference between the rare framework regions of the invention and the analogous framework regions may be an addition, deletion, or, preferably a substitution of amino acids.
  • Substitutions may be conservative or non- conservative; that is, it may be an exchange of amino acids with similar chemical properties (polar, nonpolar, aliphatic, aromatic, etc.) or dissimilar chemical properties.
  • the following table indicates conservative substitutions: similar amino acids are found on the same line.
  • the rare framework regions may be a product of germline DNA or limited to the genome of a somatic cell lineage. In cases where the rare framework region is of germline origin, the rarity may result from a rare immunoglobulin recombination event. In those cases where a rare framework region is limited to the genome of a somatic cell lineage, the rarity may result from events such as somatic mutation. Of particular interest are rare germline framework regions that subsequently undergo somatic mutation. Such regions may differ at more than one site from analogous framework regions; for instance, one difference may result from a rare germline sequence and another from somatic mutation.
  • the presence of a rare framework region may be detected at the level of genomic, mRNA, or peptide sequences.
  • nucleic acid sequences encoding rare framework regions may be detected.
  • Methodologies for the reverse transcription of mRNA sequences and the amplification, cloning, and restriction and sequence analysis of DNA sequences are known to those skilled- in the art.
  • Of particular interest is identifying rare framework regions by an alteration of one or more restiction sites within the framework region; more particularly by the addition of a TthIIII or a HaeIII site; most particulary by the addition of both a TthIIII and a HaeIII site.
  • PCR polymerase chain reaction
  • Suitable primers may be based on framework region sequences or sequences flanking such regions.
  • nested PCR can be used.
  • one class of sequences are amplified, or nested up within a mixture of genomic or cDNA and then from this amplified sample, a sequence within the amplified class is secondarily amplified.
  • conserved v ⁇ II sequences are first amplified using primers derived from sequences conserved within this class and then a specific rare V ⁇ II sequence is secondarily amplified from the V ⁇ II - enriched sample.
  • rare framework region peptide sequences may be determined directly with, for example, specific polyclonal or monoclonal antibodies which can distinguish the rare framework region peptide sequences from the conserved framework region sequences.
  • these antibodies can be used to isolate light chains containing the rare framework regions of the invention for futher characterization such as peptide sequencing.
  • Immunoassays include ELISA, EMIT, CEDIA, SLIFA, and the like.
  • a number of diagnostic procedures have been described in variety of issued patents such as U.S. Patent Nos. 3,791,932; 3,817,837; 3,998,943, and references cited therein.
  • compositions are also provided for therapeutic intervention in disease associated with the presence of immunoglobulin.
  • Oligopeptides are provided which comprise at least eight amino acids of a rare framework region and will usually not exceed 100 amino acids. These molecules can be synthesized in pure form and can find many uses in diagnosis and therapy.
  • a rare framework region sequence of particular interest is an eight amino acid sequence coming within the sequence GVPDRVSGRGSGTDF, particularly including the sequence VSGR. These oligopeptides can be used, for example, to compete with immunoglobulin containing a rare framework region for binding to ligands where such immunoglobulin-ligand binding is otherwise associated with pathogenesis.
  • the oligopeptides will generally be fewer than about 60 amino acids, more usually fewer than about 36 amino acids, although large oligopeptides may be employed. If desired, an entire light chain containing a rare framework region may be employed, but it will be frequently convenient to use a fragment thereof. Such a light chain or fragment thereof may be used in purified form, generally greater than about 90%, usually greater than about 95% pure. Methods for purifying such peptides to such purities include various forms of chromatographic, chemical, and electrophoretic separations which are well known to those skilled in the art.
  • sequence of interest may be joined to amino acid sequences in a chain other than the naturally occurring sequence of the chain from which the sequence of interest was derived or may be joined to peptide molecules other than by peptide bounds or molecules other than molecules comprising amino acids, e.g. synthetic or naturally occurring organic compounds which compounds comprise other than amino acids joined by peptide binds.
  • a rare framework region target sequence of particular interest is an eight amino acid sequence coming within the sequence GVPDRVSGRGSGTDF, particularly including the sequence VSGR.
  • binding specific member oligopeptides may be prepared synthetically.
  • Other compositions can be used to bind immunoglobulins containing rare framework regions.
  • antibodies in a monovalent form such as Fab, Fab', or Fv or a monovalent anti-idiotypic antibody, where the variable region of the anti-idiotype binds the rare framework region.
  • Synthetic organic compounds which bind the sequence of interest may also be prepared.
  • These compounds can be further screened to identify those which prevent the immunoglobulin from binding to other ligands where such binding is associated with pathogenesis.
  • other ligands include DNA, saccharides, proteoglycans, glycosaminoglycans, and proteins, particularly immunoglobulins and more particularly, the Fc portion of human IgG.
  • the binding inhibitory compounds can be used individually or in combination and can be readily prepared in large number and screened for binding affinity to immunoglobulin containing a rare framework region.
  • oligo- or polypeptides may be modified in a variety of ways to enhance their stability, such as using an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxyl terminus, e.g., for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like.
  • an unnatural amino acid such as a D-amino acid, particularly D-alanine
  • the modified or unnatural amino acids are generally found at positions outside the VSGR tetrad indicated above.
  • Other methods of stabilization may include liposome formation, other forms of encapsulation, etc.
  • the subject inhibitory compounds may be prepared in a variety of ways known to those skilled in the art.
  • peptides under about 60 amino acids can be readily synthesized today using conventional commercially available automatic synthesizers.
  • DNA sequences may be prepared encoding the desired peptide and inserted into an appropriate expression vector for expression in a prokaryotic or eukaryotic host.
  • a wide variety of expression vectors are available today and may be used in conventional ways for transformation of a competent host for expression and isolation.
  • the open reading frame encoding the desired peptide may be joined to a signal sequence for secretion, so as to permit isolation from the culture medium.
  • compositions may be administered by any convenient way, preferably parenterally, conveniently- in a physiologically acceptable carrier, e.g., phosphate buffered saline, saline, deionized water, or the like.
  • a physiologically acceptable carrier e.g., phosphate buffered saline, saline, deionized water, or the like.
  • the compositions are added to a retained physiological fluid such as blood or synovial fluid, retained distinguishes from urine and feces.
  • the amount administered will be in the range of about 100 to 1000 ⁇ g/kg of the recipient.
  • a single bolus may be employed or repetitive administrations may be employed, generally not more frequently than once weekly.
  • the concentration of the peptide will generally be in the range of about 100 to 500 ⁇ g/ml in the dose administered.
  • Other additives may be included, such as stabilizers, bactericides, etc. These additives will be present in conventional amounts. The following examples are offered by way of illustration
  • Human synovium from rheumatoid arthritis patients was obtained at the time of surgery for joint replacement.
  • the inflammatory cell population containing 10 7 to 10 8 lymphoid cells was freed from stroma by digesting synovial tissue with DNAse and collagenase.
  • the B-lymphocytes in the cell population were transformed with Epstein-Barr virus according to the method of Cole et al., Molecular and Cellular Biochemistry (1984) 62:109.
  • the culture supernatant from one of ten transformed lymphoblastoid synovial primary cell cultures, hRF-1 produced detectable levels of IgG RF autoantibody by two weeks after EBV transformation.
  • the IgG rheumatoid factor (RF) was detected by an enzyme-linked immunosorbent assay (ELISA) using purified human IgG Fc fragments bound to 96 well microtiter plates (Weisbart et al., J. Immunology (1984) 132:2909-2912). Bound IgG RF was identified with peroxidase labeled rabbit antibodies specific for the F(ab') 2 of human IgC. After repeated cloning, these hRF-1 B-lymphoblastoid cells secreted only IgG (1-2 ⁇ g/ml), but not IgA (less than 20 pg/ml), or IgM (less than 10 pg/ml).
  • ELISA enzyme-linked immunosorbent assay
  • Isotype testing with subclass specific antiglobulins identified only human IgG 4 subclass gamma heavy chains.
  • Anti-light chain specific antiglobulins detected only kappa, but not lambda, light chains.
  • Initial specificity of this IgG 4 RF for human immunoglobulin was demonstrated by its binding to purified Fc fragments of human IgG, but not human serum albumin, bovine collagen, histone 2A, or denatured DNA, as shown in the following table.
  • the ELISA was performed as follows: The human monoclonal antibody hRF-1 was bound to 96-well microtiter plastic plates at 1 ⁇ g/100 ⁇ L and characterized by ELISA using peroxidase conjugated antisera specific for human immunoglobulin isotypes, IgG subclasses, and light chains.
  • the antigenic specificity of hRF-1 monoclonal was measured with the 5 antigens bound to 96-well microtiter plates at 1 ⁇ g/100 ⁇ L by adding hRF-1 at 2 ⁇ g/ml from culture media and detecting bound hRF-1 with affinity purified peroxidase-conjugated sheep antibodies specific for F(ab') 2 of human IgG. Results were recorded as absorbency at 414 nm corrected for background values using control ELISA wells coated only with poly-L-lysine.
  • the hRF-1 human monoclonal IgG 4 RF was tested for binding to different species' IgGs and shown to bind to human, rabbit, mouse and guinea pig IgG more strongly than to horse, goat, and sheep IgG.
  • the monoclonal IgG 4 RF did not bind at all to chicken (nonmammalian) IgG.
  • the observed binding pattern is analogous to the species-specific binding of Staphylococcal protein-A (SpA).
  • HRF-1 lymphoblast cells were grown in serum free medium, and 2.0 mg of mAb hRF-1 was purified from 40 liters of serum free supernatant by absorption to 4 gm of Protein A Sepharose CL-4B (Pharmacia, Piscataway, NJ), washed with 0.10 M phosphate buffer containing 0.5 M NaCl, and eluted with 0.10 M glycine, pH 2.8 into tubes containing carbonate buffer to produce a final pH of 4.0.
  • MAb hRF-1 (1 mg/ml), (see U.S. application serial no.
  • mAb hRF-1 and isolated light and heavy chains (20 ⁇ g/ml) were biotinylated in a molar ratio of 1:30 with NHS-LC-Biotin (Pierce Chemical Co., Rockford, IL) overnight at 40C in N,N-dimethylformamide adjusted to pH 8.5 with 5% Na 2 C0 3 .
  • Biotinylated peptides were dialyzed against PBS and assayed for binding human IgG and other mammalian IgG immunoglobulins by ELISA.
  • Binding was measured by adding streptavidin conjugated with alkaline phosphatase, and bound phosphatase was measured by the conversion of p-nitrophenyl phosphate to p-nitrophenol (A 405 ).
  • the binding of mAb Gm607 to human IgG and other mammalian IgG immunoglobulins was evaluated for comparison and was measured by adding supernatant from cultures of the Gm607 cell line and detecting mAb Gm607 (IgM) with affinity purified alkaline phosphatase labeled goat antisera specific for human IgM.
  • the supernatant from Gm607 was shown to contain IgM using the same preparation of goat antibodies to human IgM.
  • the amino acid sequence of the variable region of mAb hRF-1 light chain predicted from the nucleotide sequence
  • the amino acid sequence is compared to Gm607, a prototype V ⁇ II light chain produced by a lymphoblast cell line derived from a normal individual (Klobeck et al., Nature 309:73-76 (1984).
  • the Gm607 amino acid sequence was also predicted from the nucleotide sequence.
  • Six other reported V ⁇ II light chains are shown for comparison.
  • the amino acid sequence of hRF-1 is identical to Gm607 with the exception of two amino acid changes, corresponding to a change from phenylalanine to valine at residue 62 and a change from serine to arginine at residue 65. These amino acid changes occur in a relatively invariable region of FR3.
  • the gene for the immunoglobulin light chain of hRF-1 cells was cloned from a cDNA library expressed in Lambda Zap (Stratagene, La Jolla, CA), and the cDNA library was screened with a V ⁇ J chain oligonucleotide probe labeled with 32 P.
  • Lambda Zap-hRF-1 was converted to Bluescript, from which double stranded and single stranded DNA were isolated (Stratagene, La Jolla, CA), and dideoxy sequencing was performed using an Applied BioScience Sequenator. Two new restriction endonuclease cleavage sites are produced as a result of two nucleotide changes in FR3 of hRF-1 V ⁇ II
  • Tthllll site and the change from adenosine to cytidine produced a HaeIII site in hRF-1 V ⁇ II compared to the prototypi ⁇ V ⁇ II of Gm607.
  • a 156 bp fragment is amplified in hRF-1 genomic DNA, hRF-1 cDNA, and normal PBL DNA.
  • the 156 bp fragment amplified from DNA from normal peripheral blood lymphocytes (PBL) did not contain a TthIIII site. In contrast, a TthIIII site was present in hRF-1 cDNA.
  • a second HaeIII site is present in hRF-1 cDNA which produces an additional fragment.
  • a nucleotide sequence of 156 bp was amplified following 25 cycles of PCR with hRF-l genomic DNA, cDNA prepared from hRF-1 polyadenylated mRNA by reverse transcriptase, and DNA isolated from normal PBL. Oligonucleotide primers (100 ng) were used flanking the sequences in question. The primers used for PCR were TCTCCACAGCTCCTGATCT (sense) located at amino acids 43-48 and TTGTAGAGCTTGCATGCA (antisense) located at amino acids 88-93 in the 5' to 3' orientation. The antisense primer was labeled with [ ⁇ 3z P]ATP.
  • Reactions were performed in 25 mM tris-HCl, pH 8.0, 5 mM MgCl 2 , 50 mM NaCl, and 0.25 mM each of deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxythymidine triphosphate, and deoxyguanosine triphosphate in a total volume of 25 ⁇ l, cycling at 65o for 2 min and at 91° for 1 min. After amplification, 5 ⁇ l of the reaction were used for further analysis by 8% polyacrylamide gel electrophoresis, and the PCR products were visualized by autoradiography after 2 and 12 hours of exposure.
  • the autoradiograph was overexposed to enhance the TthIIII and HaeIII fragments in genomic DNA where the V ⁇ II of interest is diluted by multiple other V ⁇ II genes compared to amplification of the single hRF-1 V ⁇ II in the hRF-1 cDNA.
  • the autoradiograph showed the absence of bands in normal DNA from PBL uncut or cut with HaeIII comparable to bands of hRF-1 genomic DNA uncut or cut by HaeIII.
  • both the cDNA and genomic DNA cut with TthIIII showed a comparable band.
  • a nucleotide sequence of 273 bp encompassing nucleotides 15 to 287 containing the V ⁇ FR3 region of interest was amplified after 38 cycles of PCR with an automated thermal cycler (Perkin Elmer Cetus, Norwalk, CT) from hRF-1 cDNA prepared from hRF-1 total RNA by reverse transcriptase.
  • the reverse transcription reaction was performed with 0.1 ⁇ g total RNA, 0.2 ⁇ M PCR antisense (downstream) primer, 50 units Maloney murine leukemia virus reverse transcriptase (M-MLV RT) and 20 U RNA-sa inhibitor (Promega Biotec, Madison, WI) in 10mM Tris-HCl, pH-8.3, 5mM MgCl 2 , 50 mM KCl and 0.5mM each of deoxyadenosine triphosphate, deoxcytidine triphosphate, deoxythymidine triphosphate, and deoxyguanozine triphosphate in a total volume of 20 ⁇ l extending at 42 °C for 1 hour followed by inactivation of M-MLV reverse transcriptase at 95 ⁇ C for 5 min and then quickly cooling on ice for 5 min.
  • M-MLV RT Maloney murine leukemia virus reverse transcriptase
  • RNA-sa inhibitor Promega Biotec, Madison, WI
  • flanking primers used for PCR were a sense primer for amino acids 5-12 (5' - T C A G T C T C C A C T C T C C C T G C - 3') and an antisense primer for amino acids 84-91 )5' - G C T T G C A T G C A G T A A T A A C C C - 3') which were selected to specifically amplify V ⁇ II sequences.
  • the PCR reaction was performed with 1 ⁇ l of the reverse transcription cDNA reaction, 0.2 ⁇ M primers, 2.5 U of Taq polymerase (Perkin Elmer Cetus, Norwalk, CT) in 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl 2 , 50 mM KCl, and 0.20 mM each of deoxynucleotides in a total volume of 100 ⁇ l with cycle conditions of 1 min each at 94oC denaturation, 62° C annealing, and 72oC extension for 38 cycles.
  • Taq polymerase Perkin Elmer Cetus, Norwalk, CT
  • a nested PCR reaction was then performed using an internal primer to amplify the specific hRF-1 V ⁇ with respect to the guanine change at residue 62 associated with the new TthIII restriction site.
  • the nested primer used was a sense primer for amino acids 56 - 62 (5' C C G G G G T C C C T G A C A G G G - 3') with the antisense primer being the same as before.
  • the nested PCR reaction was performed with 1 ⁇ 1 of the initial flanking PCR reaction, 0.2 ⁇ M nested primers, 2.5U Taq polymerase in 10mM Tris-HCl, pH8.3, 1.5 mM MgCl 2 , 50 mM KCl, and 0.20 mM each of deoxynucleotides in a total volume of 100 ⁇ l with the same cycle conditions.
  • a 106-bp nested fragment encompassing nucleotides 182-287 was amplified and digested with HAEIII to produce a 81-bp fragment. Nested PCR products were visualized by electrophoresis in 3% Nusieve/1% Seakemp agarose gels stained with ethidium bromide.

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Abstract

On a découvert que des immunoglobulines de régions de structures rares sont associées à des maladies rhumatoïdes. Les séquences de régions de structures, le peptide et l'acide nucléique, sont utiles en techniques diagnostiques et thérapeutiques.
PCT/US1992/008264 1991-10-10 1992-09-29 Regions de structures d'immunoglobulines rares associees a la maladie chez l'homme WO1993007485A1 (fr)

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US77570291A 1991-10-10 1991-10-10
US775,702 1991-10-10

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WO1993007485A1 true WO1993007485A1 (fr) 1993-04-15

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WO2003072060A3 (fr) * 2002-02-27 2004-03-25 Immunex Corp Preparation de polypeptides
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6001569A (en) * 1994-05-17 1999-12-14 Cedars-Sinai Medical Center Methods of screening for Crohn's disease using TNF microsatellite alleles
US6534263B1 (en) 1994-05-17 2003-03-18 Cedars-Sinai Medical Center Methods of screening for Crohn's disease using TNF microsatellite alleles
WO1996012189A3 (fr) * 1994-10-07 1996-04-25 Cedars Sini Medical Ct PROCEDES DE DEPISTAGE DE LA RECTOCOLITE HEMORRAGIQUE ET DE LA MALADIE DE CROHN PAR DETECTION DE L'AUTOANTICORPS VH3-15 ET DE pANCA
US5691151A (en) * 1994-10-07 1997-11-25 Regents Of University Of California Methods of screening for ulcerative colitis and crohn's disease by detecting VH3-15 autoantibody and panca
WO2003072060A3 (fr) * 2002-02-27 2004-03-25 Immunex Corp Preparation de polypeptides
US11236393B2 (en) 2008-11-26 2022-02-01 Cedars-Sinai Medical Center Methods of determining responsiveness to anti-TNFα therapy in inflammatory bowel disease
US12084722B2 (en) 2008-11-26 2024-09-10 Cedars-Sinai Medical Center Methods of determining responsiveness to anti-TNFα therapy in inflammatory bowel disease
US9580752B2 (en) 2008-12-24 2017-02-28 Cedars-Sinai Medical Center Methods of predicting medically refractive ulcerative colitis (MR-UC) requiring colectomy
US10633449B2 (en) 2013-03-27 2020-04-28 Cedars-Sinai Medical Center Treatment and reversal of fibrosis and inflammation by inhibition of the TL1A-DR3 signaling pathway
US10316083B2 (en) 2013-07-19 2019-06-11 Cedars-Sinai Medical Center Signature of TL1A (TNFSF15) signaling pathway
US11312768B2 (en) 2013-07-19 2022-04-26 Cedars-Sinai Medical Center Signature of TL1A (TNFSF15) signaling pathway
US12269873B2 (en) 2013-07-19 2025-04-08 Cedars-Sinai Medical Center Signature of TL1A (TNFSF15) signaling pathway
US11186872B2 (en) 2016-03-17 2021-11-30 Cedars-Sinai Medical Center Methods of diagnosing inflammatory bowel disease through RNASET2

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