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WO1994021669A1 - Antigene relatif a des maladies inflammatoires - Google Patents

Antigene relatif a des maladies inflammatoires Download PDF

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Publication number
WO1994021669A1
WO1994021669A1 PCT/US1994/002911 US9402911W WO9421669A1 WO 1994021669 A1 WO1994021669 A1 WO 1994021669A1 US 9402911 W US9402911 W US 9402911W WO 9421669 A1 WO9421669 A1 WO 9421669A1
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WO
WIPO (PCT)
Prior art keywords
antigen
gene
seq
set forth
sequence set
Prior art date
Application number
PCT/US1994/002911
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English (en)
Inventor
Cynthia K. French
Karen K. Yamamoto
Phoebe M. Chow
Nemegias T. Alido
Original Assignee
Medclone, 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 Medclone, Inc. filed Critical Medclone, Inc.
Publication of WO1994021669A1 publication Critical patent/WO1994021669A1/fr

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Classifications

    • 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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the present invention relates generally to connective tissue diseases such as " rheumatoid arthritis and systemic lupus erythematosus (SLE) . More particularly, the present invention relates to antigens which are related to such diseases.
  • SLE Systemic lupus erythematosus
  • SLE is a chronic inflammatory disease which results in injury to the skin, joints, kidneys, nervous system and mucous membranes. SLE is not limited to these areas and can affect any organ of the body. SLE is an extremely debilitating diseases which is present in approximately one person in 800. The high frequency of SLE and its debilitating nature have resulted in intense study of this disease by the medical community.
  • SLE is an autoimmune connective tissue disease which is characterized by the presence of a high level of autoantibodies.
  • Patients with SLE typically have a wide variety of autoantibodies against nuclear and cytoplasmic cellular components.
  • the antinuclear antibodies are known to be directed against a variety of materials including deoxyribonucleoprotein, DNA and histone.
  • An exemplary antibody which has been associated with SLE is the 3E10 anti-DNA antibody (see Weisbart, et al.
  • an antigen has been identified and isolated which is related to SLE.
  • the antigen has been identified as HP- 8.
  • the HP-8 antigen is expressed by a gene which was identified by immunoscreening of the human placental cDNA gtll expression library with the 3E10 antibody mentioned above.
  • the isolated cDNA gene sequence (insert size 154 bp) was found to hybridize to a 3.3 kb and 1.2 kb mRNA transcript. It was found that the 3.3 kb transcript was expressed in brain, heart, placenta, lung, skeletal muscle and pancreatic tissues.
  • the 1.2 kb transcript was found to be present in brain, heart, lung, skeletal muscle and kidney.
  • proteins which include the HP-8 antigen epitope are produced by recombinant means involving culturing of transformed micro-organisms which include the gene which codes on expression for the HP-8 antigen.
  • HP-8 antigen is useful in mapping and determining the genetic origin for expression of gene products in patients with SLE.
  • the HP-8 antigen may be used in procedures for developing therapeutic rational drug designs to be used in treating SLE or other related connective tissue diseases such as rheumatoid arthritis.
  • proteins and polypeptides which include the HP-8 antigen are used to raise antibodies in animals.
  • the antibodies which are raised in response to the HP-8 antigen are useful in the study and treatment of SLE.
  • HP-8 antigen in accordance with the present invention is defined as a protein or polypeptide which includes an epitope which is substantially homologous with the amino acid sequence set forth in SEQ ID NO: 2.
  • the entire protein or polypeptide will have a molecular weight of less than 10 Kd based on mRNA size.
  • Preferred proteins will have molecular weights on the order of 60 to 100 Kd, depending on glycosylation.
  • the amino acid sequence of the epitope of the protein or polypeptide must be 90 % homologous with the amino acid sequence set forth in SEQ ID NO: 2.
  • HP-8 antigens in accordance with the present invention may be produced in accordance with any of the known processes for preparing polypeptides and proteins. It is preferred that the antigen be expressed in prokaryotic, - eukaryotic or insect viral cells by recombinant means.
  • An exemplary procedure for producing HP-8 antigen is as follows:
  • cDNA library was plated and screened according to manufacturers instructions.
  • cDNA library was a human placental cDNA GTll expression library, Catalog No: HL-1075B (Clontech Laboratories, Palo Alto, CA) .
  • Large 150 mm LB soft agar plates were used to plate and screen the library with MAb 3E10.
  • 0.6 ml of plating bacteria (Y1090) was incubated with a proper dilution of lambda gtll phage and absorbed to the cells at 37° C for 15 minutes.
  • 7.5 ml of LB soft agar was added to the culture and quickly poured onto the plates and incubated at 42° C for 3.5 hours.
  • Detection of bound antibody used an alkaline phosphatase conjugate. Filters were incubated with goat-anti-mouse conjugate (2 ul) in 5 ml of buffer A for 30 minutes. Following incubation the filters were washed 3 times with 50 ml of Buffer A (10 minutes each wash) . An additional wash was done in Buffer C for 10 minutes. Detection was performed by addition of 25 ul Nitro blue tetrazolium (NBT) (100 mg/ml) and 12 ul 5- Bromo-4-chloro-3-indolyl phosphate (BCIP) (100 mg/ml) . Filters were incubated until signals became visible under reduced illumination.
  • NBT Nitro blue tetrazolium
  • BCIP 5- Bromo-4-chloro-3-indolyl phosphate
  • the reaction was terminated by washing in 1 mM EDTA and positives selected. Six positives were identified in the screen and one was determined to be a true positive following secondary and tertiary rescreening using dilution cloning. The positive, designated HP-8, was screened against normal human sera as a negative control indicating the validity of the 3E10 reactivity. Details of the preparation of the buffers are described in the Clontech Handbook (1992) . The lambda phage was grown up on plates according to protocols supplied from Clontech (pgs. 20-22, Clontech Protocol Handbook 1992) . Isolated DNA was obtained and Eco Rl digested using standard methods described in Maniatis, T.
  • the double-stranded pBLUESCRIPT pSKII+ plasmids containing the HP-8 specific clone fragment were grown and DNA harvested using the Qiagen column purification system (Qiagen Corp., Chatsworth, CA, Catalog number: 12162) . T3 and T7 primers were used to sequence the cDNA. Procedures were followed using standard cycle sequencing conditions recommended by the manufacturers (ABI, Foster City, CA, Catalog number: 401384) .
  • the nucleotide sequence and corresponding amino " acid sequence are set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • a multiple tissue northern blot was obtained from Clontech Laboratories, Palo Alto, California (Catalog number: 7760-1) and hybridized to 32P labelled cDNA insert from HP-8.
  • the probe was prepared according to manufacturers instructions (BRL, Gaithersburg, MD, Catalog number: 8187-SA) at high specific activity. Hybridization conditions were performed as described in the Clontech handbook for Product number 7760-1. Washed filters were air dried and exposed to Kodak XR-5 x-ray film for 18 hours at -70°C.
  • the epitope of the HP-8 antigen i.e. SEQ ID NO:2
  • SEQ ID NO:2 The epitope of the HP-8 antigen (i.e. SEQ ID NO:2) is approximately 60-80 percent homologous with various proteins and polypeptides which belong to the osteonectin family (see P.T. Russell et al., THE OSTEONECTIN FAMILY OF PROTEINS, J. Biochem. , Vol. 20, No. 7, pp. 653-660, 1988).
  • Specific examples of related osteonectin proteins are Osteonectin/BM401 SPARC and SCI. These specific osteonectins are described in the following three references:
  • DNA sequences which code on expression for the HP-8 antigen epitope may be inserted into appropriate 0 expression vectors for expression in prokaryotic eukaryotic or insect viral cells.
  • a wide variety of expression vectors are available and may be used in conventional procedures to transform competent host cells for expression and isolation of the HP-8 antigen. 5 Methods for preparing gene sequences, inserting the sequences into expression vectors, transforming competent hosts and growing the host in culture for production of products are disclosed in U.S. Patent Nos. 4,710,473; 4,711,843; and 4,713,339. 0
  • the HP-8 antigen can be used to generate antibodies.
  • the HP-8 antigen can be used in any of the conventional procedures involving administering an antigen to a host animal in order to raise antibodies.
  • the administration protocols including dosage levels, 5 administration schedules and isolation and recovery of antibodies from the host animal are all well known in the art.
  • the HP-8 antigen is used in the same manner as any other antigen to elicit the production of antibodies in a host animal.
  • the HP-8 antigen includes epitopes which bind 3E10 antibodies and therefore will be useful in investigating the etiology of SLE.
  • HP-8 will be useful in developing therapeutic rational drug designs which will be effective in treating SLE and other related 5 connective tissue diseases such as rheumatoid arthritis.
  • the similarity of the HP-8 antigen epitope to osteonectin makes it amenable for use in the same manner as osteonectin.
  • ORGANISM Homo sapiens
  • TISSUE TYPE Placenta

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Rheumatology (AREA)
  • Hematology (AREA)
  • Toxicology (AREA)
  • Rehabilitation Therapy (AREA)
  • Diabetes (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Autoantigène identifié en tant que HP-8 et relatif au lupus érythémateux systémique. L'antigène HP-8 est exprimé par un gène identifié par immunocriblage de la banque d'expression de GT11 de l'ADNc du placenta humain avec l'anticorps monoclonal 3E10. L'anticorps 3E10 est un autoanticorps d'ADN anti-bicaténaire à faible affinité dérivé des modèles murins de MRL du lupus érythémateux systémique chez l'homme.
PCT/US1994/002911 1993-03-18 1994-03-17 Antigene relatif a des maladies inflammatoires WO1994021669A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US3312093A 1993-03-18 1993-03-18
US033,120 1993-03-18

Publications (1)

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WO1994021669A1 true WO1994021669A1 (fr) 1994-09-29

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996032493A1 (fr) * 1995-04-12 1996-10-17 Medclone, Inc. Auto-antigene hp-8
WO1996037225A1 (fr) * 1995-05-25 1996-11-28 Oklahoma Medical Research Foundation Procede de traitement du lupus erythemateux dissemine
US7192715B2 (en) 1993-11-30 2007-03-20 Oklahoma Medical Research Foundation Diagnostics and therapy of epstein-barr virus in autoimmune disorders
US7273613B1 (en) 1997-01-13 2007-09-25 The Board of Regents, The University of Oklahoma Diagnostics and therapy of Epstein-Barr virus in autoimmune disorders

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4711843A (en) * 1980-12-31 1987-12-08 Cetus Corporation Method and vector organism for controlled accumulation of cloned heterologous gene products in Bacillus subtilis
US4713339A (en) * 1983-01-19 1987-12-15 Genentech, Inc. Polycistronic expression vector construction
US4812397A (en) * 1987-02-10 1989-03-14 The Regents Of The University Of California MAB-anti-DNA related to nephritis

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4711843A (en) * 1980-12-31 1987-12-08 Cetus Corporation Method and vector organism for controlled accumulation of cloned heterologous gene products in Bacillus subtilis
US4713339A (en) * 1983-01-19 1987-12-15 Genentech, Inc. Polycistronic expression vector construction
US4812397A (en) * 1987-02-10 1989-03-14 The Regents Of The University Of California MAB-anti-DNA related to nephritis

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, Volume 26, issued 1987, ENGEL et al., "Calcium Binding Domains and Calcium-Induced Conformational Transition of SPARC/BM-40/Osteonectin, an Extracellular Glycoprotein Expressed in Mineralized and Nonmineralized Tissues", pages 6958-6965. *
H. MANIATIS et al., "Molecular Cloning, a Laboratory Manual", published 1982, by MACGRAW-HILL (N.Y.), pages 403-435. *
J. IMMUNOLOGY, Volume 110, Number 5, MATTIOLI et al., "Physical Association of Two Nuclear Antigens and Mutual Occurence of their Antibodies: The Relationship of the Sm and RNA Protein (MO) Systems in SLE Sera", pages 1318-1324. *
J. IMMUNOLOGY, Volume 144, Number 7, issued 01 April 1990, WEISBART et al., A Conserved Anti-DNA Antibody Idiotype Associated with Nephritis in Murine and Human Systemic Lupus Erythematosus", pages 2653-2658. *
NEURON, Volume 2, issued 1990, JOHNSTON et al., "Molecular Cloning of SC1: A Putative Brain Extracellular Matrix Glycoprotein Showing Partial Similarity to Osteonectin/BM40/SPARC", pages 165-176. *
PROC. NATL. ACAD. SCI. USA, Volume 85, issued May 1988, BOLANDER et al., "Osteonectin cDNA Sequence Reveals Potential Binding Regions for Calcium and Hydroxyapatite and Shows Homologies with Both a Basement Membrane Protein (SPARC) and a Serine Roteinase Inhibitor (Ovomucoid)", pages 2919-2923. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 263, Number 23, issued 1988, MCVEY et al., "Characterization of the Mouse SPARC/Osteonectin Gene", pages 11111-1116. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 264, Number 9, issued 1989, REEVES et al., "Molecular Cloning of cDNA Encoding the p70 (Ku) Lupus Autoantigen", pages 5047-5052. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5807993A (en) * 1993-03-18 1998-09-15 Vivorx Autoimmune, Inc. HP-8 autoantigen
US7192715B2 (en) 1993-11-30 2007-03-20 Oklahoma Medical Research Foundation Diagnostics and therapy of epstein-barr virus in autoimmune disorders
WO1996032493A1 (fr) * 1995-04-12 1996-10-17 Medclone, Inc. Auto-antigene hp-8
EP0871762A1 (fr) * 1995-04-12 1998-10-21 Medclone, Inc. Auto-antigene hp-8
EP0871762A4 (fr) * 1995-04-12 2001-04-11 Medclone Inc Auto-antigene hp-8
WO1996037225A1 (fr) * 1995-05-25 1996-11-28 Oklahoma Medical Research Foundation Procede de traitement du lupus erythemateux dissemine
US7273613B1 (en) 1997-01-13 2007-09-25 The Board of Regents, The University of Oklahoma Diagnostics and therapy of Epstein-Barr virus in autoimmune disorders

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