WO1995006727A2 - Utilisations de la glycoproteine de myeline d'oligodendrocytes et de portions peptidiques derivees de celle-ci dans des protocoles ayant trait a des maladies auto-immunes - Google Patents

Utilisations de la glycoproteine de myeline d'oligodendrocytes et de portions peptidiques derivees de celle-ci dans des protocoles ayant trait a des maladies auto-immunes Download PDF

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WO1995006727A2
WO1995006727A2 PCT/US1994/010257 US9410257W WO9506727A2 WO 1995006727 A2 WO1995006727 A2 WO 1995006727A2 US 9410257 W US9410257 W US 9410257W WO 9506727 A2 WO9506727 A2 WO 9506727A2
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seq
mog
peptide
amino acid
leu
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PCT/US1994/010257
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English (en)
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WO1995006727A3 (fr
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Brigitte Devaux
Jonathan B. Rothbard
Dawn Smilek
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Immulogic Pharmaceutical Corporation
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Priority to JP7508328A priority Critical patent/JPH09502346A/ja
Priority to EP94928084A priority patent/EP0716696A1/fr
Priority to NZ273813A priority patent/NZ273813A/en
Priority to KR1019960701093A priority patent/KR960705038A/ko
Priority to AU77258/94A priority patent/AU7725894A/en
Publication of WO1995006727A2 publication Critical patent/WO1995006727A2/fr
Publication of WO1995006727A3 publication Critical patent/WO1995006727A3/fr
Priority to NO960858A priority patent/NO960858L/no
Priority to FI961000A priority patent/FI961000A/fi

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention is directed to autoantigens and their relevant epitopes. More specifically, the invention concerns myehn oligodendrocyte glycoprotein (MOG) and the peptide regions thereof useful in diagnosis, treatment, and prevention of autoimmune conditions. Further, methods of screening for, and developing therapeutics useful in the treatment of, autoimmune disease are also disclosed.
  • MOG myehn oligodendrocyte glycoprotein
  • Autoimmune diseases are a significant human health problem and are relatively poorly understood. As there is no microbial or viral culprit apparently directly responsible, prevention, treatment and diagnosis of such diseases must be based on the etiology of the disease. This invariably involves a complex series of reactions of endogenous metabohc intermediates, structural components, cells, and so forth. Implicit, however, in the nature of an autoimmune condition is the notion that at least one autoantigen must be involved in creating the sequence of events that results in the symptoms. Autoimmune demyelinating diseases, such as multiple sclerosis, are no exception. Multiple sclerosis is an autoimmune disease in which T lymphocytes destroy central nervous system myehn. Multiple sclerosis is the most common cause of neurological disability associated with disease in Western countries.
  • EAE allergic encephalomyelitis
  • MBP myelin basic protein
  • this unknown antigen now designated M2
  • M2 was identified as a surface antigen and detected in mouse, rabbit, rat, bovine and human CNS tissues as well as guinea pig.
  • Monoclonal antibodies putatively specific for M2 were also reported in this paper.
  • the rat cD ⁇ A obtained by Gardinier (supra) had an open reading frame that encoded a putative signal peptide of 27 amino acids followed by mature MOG peptide of 218 amino acids with a calculated molecular weight of 24,962 daltons.
  • the ⁇ -terminal amino acid sequences obtained for the murine protein were similar to those deduced from the rat cD ⁇ A.
  • the present invention provides recombinant materials for the production of the human MOG protein as well as the complete amino acid sequence thereof. Using this information, the MOG protein or useful peptides representing portions of the amino acid sequence of MOG protein can be determined and are useful in the diagnosis and treatment of demyelinating autoimmune diseases in humans. Further, methods of screening for, and developing therapeutics compositions for, autoimmune disease is disclosed.
  • the invention provides the complete amino acid sequence of human MOG protein (SEQ ID NO: 2) as well as recombinant materials for the production of the protein and fragments thereof. Knowledge of the amino acid sequence permits design of peptide portions thereof which are useful as well in diagnosis and treatment of autoimmune diseases.
  • the invention is directed to isolated and purified human MOG protein, optionally produced recombinantly, and to fragments thereof which modulate the course of development of symptomology in demyelinating autoimmune diseases.
  • the invention is directed to recombinant materials and methods useful for the production of the MOG protein or peptide portions thereof.
  • the invention is directed to pharmaceutical compositions and methods of using them in mitigating the effects of demyelinating autoimmune diseases.
  • the invention is directed to isolated and purified human MOG protein and peptides in which conservative substitutions have been made which exhibit characteristics of T-cell epitopes of the naturally occurring MOG protein and peptides.
  • the invention discloses a method for screening for demyehnating autoimmune disease and identifying therapeutic compositions comprised of MOG or fragments thereof which are capable diagnosing, preventing or treating multiple sclerosis in mammals, preferably, humans.
  • Figure 1 shows the complete nucleotide sequence (SEQ ID NO:l) and deduced amino acid sequence (SEQ ID NO: 2) of DNA encoding human MOG protein.
  • Figure 2 shows peptides, designated by amino acid sequence, which are useful in the invention (SEQ ID NOS: 4-9, 11, 15, 16, and 42-72).
  • Figure 3 is a table showing the effect of each of the natural amino acids at certain positions on peptides binding to the MHC Class II product of DR4.
  • Figure 4 is a table showing a comparison of predicted and measured IC 50 values for 12 peptides (SEQ ID NOS: 19-32) binding to the MHC Class II product of DR4.
  • Figure 5a shows peptides (20 mers) designated by amino acid sequence which correspond to SEQ ID NOS: 73-93.and are useful in the invention.
  • Figure 5b shows a peptide of human MOG l-121,designated by amino acid sequence, which contains at least one T-cell epitope.
  • Figure 6 is a bar graph of data from Example 3 where the X-axis indicates wells containing 4 different cell lines, the 7 peptides tested are indicated by the different legends and the Y-axis indicates counts per minute where the data is expressed as 3H- thmidine incorporated (CPM) by each cell line in response to each individual peptide.
  • CPM 3H- thmidine incorporated
  • the present invention provides nucleic acid sequences encoding human MOG, an autoantigen involved in demyelinating autoimmune diseases. It also provides peptides which represent subunits of the human MOG encoded by the nucleic acid molecules of the invention which are useful in moderating the autoimmune response. These subunits, which generally include on the order of at least 12-13 amino acids, are characterized by their correspondence to the T-cell epitope regions of the human MOG. While the etiology of development of the symptoms of an autoimmune disease such as multiple sclerosis, is far from clear certain events are believed to be critical to the progression of the condition.
  • Demyehnating autoimmune diseases involve an attack by the immune system on the myelin sheath, resulting in what amounts to short circuits in the nervous system.
  • the attack by the immune system is mediated by T-cells.
  • T-cell responses to a particular antigen in this case the human MOG autoantigen which is a part of myelin, require uptake and subsequent proteolytic cleavage of the antigen by antigen presenting cells and presentation of the antigen in the context of a Class II major histocompatib ility complex (MHC) encoded protein which permits their recognition by the T-cells.
  • MHC major histocompatib ility complex
  • the T-cell epitope regions of the autoantigen are those which are presented by the Class II MHC proteins to the T-cell receptors.
  • the relevant T-cells can be rendered nonresponsive in an antigen-specific fashion in protocols by providing the T-cell epitope regions of the autoantigen, as is further described below.
  • Fig. 1 SEQ ID NO: 1
  • SEQ ED NO: 2 The encoded mature protein contains a 218 amino acids; the full length protein (including signal peptide) is 87% homologous with the rat MOG protein.
  • the availability of the deduced amino acid sequence of human MOG provides the opportunity to design peptide fragments thereof which induce immune responses in mammals and peptide fragments which are T-cell epitopes - i.e., constitute those portions of the molecule which are recognized by T-cell receptors. These peptides and fragments are also included within the invention scope.
  • a host cell transformed with a vector (PVL1393, Pharmingen, CA) containing the nucleic acid sequence encoding human MOG was deposited with the American Type Culture Collection on September 8, 1994 and has accession number 75554.
  • the scope of the invention is not limited to the human MOG protein encoded by the amino acid sequence depicted in Figure 1, or to the specific nucleic acid sequence presented.
  • Naturally occurring variants and dehberate mutations designed to modify the nucleic acid sequence per se or to modify the encoded protein are also included in the scope of the invention as further described below. With respect to naturally occurring variants, DNA sequence polymorphisms, especially those resulting in
  • “silent” mutations which do not affect the amino acid sequence of the human MOG, but also sequence polymorphisms that do lead to changes in the amino acid sequence, are expected to exist in the human population. These variations in one or more nucleotides (up to about 1% of the nucleotides) of the sequence encoding MOG are a result of natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of the invention. Furthermore, there may be one or more "family members" of MOG that are related in function and amino acid sequence to the MOG encoded by the DNA disclosed herein but encoded by separate genes. Such family members are also included within the definition of human MOG and the nucleotide sequences encoding it.
  • Isolated autoantigenic proteins or fragments thereof that are novel and that are immunologically related to human MOG or fragments thereof, other than those already identified, are within the scope of the invention. These can be identified by antibody cross-reactivity or T-cell cross-reactivity. Such proteins or fragments thereof bind antibodies specific for the protein and peptides of the invention, or stimulate T-cells specific for the protein and peptides of this invention.
  • Antigenic fragments refer to an amino acid sequence having fewer amino acid residues than the entire protein and include fragments or peptides which induce an immune response in mammals, preferably humans, such as eliciting the production of IgG and IgM antibodies, or eliciting a T-cell response such as proliferation and or lymphokine secretion and or induction of T-cell anergy and/or modification of THi and TH 2 subsets.
  • antigenic fragments that comprise T-cell epitopes.
  • Peptides can be derived from the naturally occurring MOG sequence or peptides in which conservative amino acid substitutions have been made. Examples of each are shown in Figure 2 (SEQ ID NOS: 4-9, 11, 15, 16, and 42-72).
  • Nucleic acid molecules containing a sequence encoding human MOG or an antigenic fragment thereof may be obtained by reverse transcription mRNA present in human brain or other CNS tissue, as well as from genomic DNA, and can most conveniently be prepared using standard sohd phase techniques.
  • Various methods of chemically synthesizing polydeoxyn ucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura etal. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071, incorporated by reference herein).
  • the nucleic acid molecules of the invention also include RNA which can be transcribed from the DNA prepared as above.
  • the present invention also provides expression systems and host cells transformed with these systems for production of the encoded protein.
  • Host cells include bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO).
  • bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO).
  • Suitable host cells and expression vectors containing relevant promoters, enhancers and other expression control elements may be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990).
  • Other suitable host cells and expression vectors are known to those skilled in the art.
  • yeast S. cerevisae examples include pYepSecl (Baldari. et al, (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz _t __, (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA).
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al., (1983) Mo I Cell Biol 3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M.D., (1989) Virology 170:31-39).
  • COS cells Gluzman, Y., (1981) Cell 23:175-182 are used in conjunction with such vectors as pCDM 8 (Aruffo, A.
  • Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.
  • Fusion vectors usually add a number of NH 2 terminal amino acids to the expressed target gene. These NH 2 terminal amino acids often are referred to as a reporter group.
  • reporter groups usually serve two purposes: 1) to increase the solubility of the target recombinant protein; and 2) to aid in the purification of the target recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the reporter group and the target recombinant protein to enable separation of the target recombinant protein from the reporter group subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • Inducible non-fusion expression vectors include pTrc (Amann eta , (1988) Gene 69:301-315) and pET 1 Id (Studier et al, Gene Expression TechnologyJ ⁇ eihods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). While target gene expression relies on host RNA polymerase transcription from the hybrid trp-lac fusion promoter in pTrc, expression of target genes inserted into pET lid relies on transcription from the T7 gnlO- lac 0 fusion promoter mediated by coexpressed viral RNA polymerase (T7 gnl).
  • This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident g prophage harboring a T7 gnl under the transcriptional control of the lacUN 5 promoter.
  • One strategy to maximize expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).
  • Another strategy would be to alter the coding sequence of the gene so that the individual codons for each amino acid would be those preferentially utilized in highly expressed E. coli proteins (Wada et al, (1992) Nuc Acids Res 20:2111-2118). Such alteration of nucleic acid sequences of the invention could be carried out by standard DNA synthesis techniques.
  • the recombinant protein or peptide product may be secreted and harvested from the medium.
  • the protein may be retained cytoplasmically and the cells harvested, lysed and the protein isolated and purified. Suitable media for cell culture are well known in the art.
  • the protein and peptides of the invention can be purified from cell culture medium, host cells, or both using techniques known in the art for purifying proteins and peptides including ion- exchange chromatography, gel filtration chromatography, metal affinity chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with specific antibodies.
  • isolated and purified are used interchangeably herein and refer to peptides, protein, protein fragments, and nucleic acid molecules substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Accordingly, an isolated peptide is produced recombinantly or synthetically and is substantially free of cellular material and culture medium or substantially free of chemical precursors or other chemicals. Antigenic Fragments and the "Antigenic" Response
  • Fragments of the invention protein that elicit a desired antigenic response may be obtained, for example, by screening peptides corresponding to portions of the protein. These peptides may be chemically synthesized using techniques known in the art, or produced recomb inantly, or through proteolysis. For example, the protein may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments are tested to determine their antigenicity (e.g., the ability of the fragment to induce an immune response in a mammal).
  • fragments of the protein are to be used for therapeutic purposes, then the fragments which are capable of eliciting a T-cell response, such as stimulation (i.e., proliferation or lymphokine secretion) and/or are capable of inducing T-cell anergy are particularly desirable.
  • the isolated protein or preferred antigenic fragments thereof when administered to an individual subject to demyelinating autoimmune disease, are capable of modifying die B-cell response, T-cell response, or both the B-cell and the T-cell response of the individual to the autoantigen, or can be shown to result in a diminution of symptoms.
  • a diminution in symptoms includes any reduction in demyehnation characteristic of the disease condition following a treatment regimen with a peptide or protein of the invention. This diminution in symptoms may be determined subjectively or clinically.
  • Human T-cell stimulating activity can be tested by culturing T-cells obtained from a subject having an autoimmune condition with the autoantigen and/or a peptide derived from the autoantigen and determining whether prohferation of T-cells occurs in response to the autoantigen and or peptide as measured, e.g., by cellular uptake of tritiated thymidine.
  • Stimulation indices for responses by T-cells to peptides can be calculated as the maximum CPM in response to a peptide divided by the control CPM.
  • a stimulation index (S.L) equal to or greater than two times the background level is considered "positive". Positive results are used to calculate the mean stimulation index for each peptide for the group of patients tested.
  • Preferred peptides of this invention comprise at least one T-cell epitope and have a mean T-cell stimulation index of greater than or equal to 1.5.
  • a peptide having a mean T-cell stimulation index of greater than or equal to 1.5 in a significant number of patients tested (i.e. at least 10% of patients tested) is considered useful as a therapeutic agent.
  • Preferred peptides have a mean T-cell stimulation index of at least 1.5, more preferably at least 2.0 to 3.0.
  • Preferred peptides can also be identified by their ability to effect a relatively higher frequency of T-cells in a patient. This frequency is measured by generating multiple identical cultures from one patient with limiting numbers of lymphocytes and the autoantigen and/or a peptide from the autoantigen. Individual cultures are analyzed for positive reactivity with a peptide, as defined by stimulation index (described above). The frequency of peptide-reactive T-cells is the percentage of cultures from the patient that show a positive stimulation index.
  • preferred peptides have a positivity index (P.I.) of at least about 100, more preferably at least about 200 and most preferably at least about 300.
  • the positivity index for a peptide is determined by multiplying the mean T-cell stimulation index by the percent of individuals, in a population of autoimmune patients (e.g., preferably at least 15 individuals, more preferably at least 30 individuals or more), who have a T-cell stimulation index to such peptide of at least 1.5, more preferably at least 2.0.
  • the positivity index represents both the strength of a T-cell response to a peptide (S.I.) and the frequency of a T-cell response to a peptide in a population of autoimmune individuals.
  • a peptide having T-cell stimulating activity and thus comprising at least one T-cell epitope as determined by T-cell biology techniques is modified by addition or deletion of amino acid residues at either the amino or carboxy terminus of the peptide and tested to determine a change in T-cell reactivity to the modified peptide. If two or more peptides which share an area of overlap in the native protein sequence are found to have human T-cell stimulating activity, as determined by T-cell biology techniques, additional peptides can be produced comprising all or a portion of such peptides and these additional peptides can be tested by a similar procedure.
  • peptides are selected and produced recombinantly or synthetically. Peptides are selected based on various factors, including the strength of the T-cell response to the peptide (e.g., stimulation index) and the frequency of the T-cell response to the peptide in a population of autoimmune subjects. The physical and chemical properties of these selected peptides (e.g., solubility, stability) are examined to determine whether the peptides are suitable for use in therapeutic compositions or whether the peptides require modification as described herein. The ability of the selected peptides or selected modified peptides to stimulate human T-cells (e.g., induce proliferation, lymphokine secretion) is determined.
  • a T-cell epitope-containing peptide of the invention when administered to a subject in a therapeutic treatment regimen is capable of modifying the response of the individual to the autoantigen.
  • Preferred peptides of the invention comprise at least one T-cell epitope of the full length protein and accordingly the peptide comprises at least approximately 7, preferably at least about 12-40, and more preferably 13-30 amino acid residues.
  • the peptides may contain tandem repeats of a single epitope and/or more than one epitope.
  • preferred therapeutic compositions of the invention preferably comprise at least two T-cell epitopes.
  • therapeutic compositions comprising one or more preferred isolated peptides of the invention preferably comprise a sufficient percentage of the T-cell epitopes of the entire protein such that a therapeutic regimen of administration of the composition results in amelioration of disease symptoms.
  • Synthetically produced peptides of the invention comprising less than approximately forty-five amino acid residues, and most preferably less than approximately thirty amino acid residues are particularly desirable as increases in length may result in difficulty in peptide synthesis.
  • Peptides of the invention may also be produced recombinantly as described above, and it is preferable that peptides of 45 amino acids or longer be produced recombinantly.
  • Isolated antigenic peptide fragments which have T-cell stimulating activity, and thus comprise at least one T-cell epitope are particularly desirable.
  • the epitope will be the basic element, or smallest unit of recognition by a receptor, particularly immunoglobulins, histocompatibility antigens, and T-cell receptors, where the epitope comprises amino acids of the native protein. Amino acid sequences which mimic those of the epitopes can also be used.
  • a T-cell epitope is the basic element, or smallest unit of recognition b_v. a T-cell receptor, where the epitope comprises amino acids in the autoantigen essential to receptor recognition.
  • T-cell epitopes are believed to be involved in initiation and perpetuation of the autoimmune response. These T-cell epitopes are thought to trigger early events at the level of the T helper cell by being presented by an appropriate HLA molecule on the surface of an antigen presenting cell, thereby stimulating the T-cell subpopulation with the relevant T-cell receptor for the epitope. These events lead to T-cell proliferation, lymphokine secretion, local inflammatory reactions, recruitment of additional immune cells to the site of antigen T-cell interaction, and activation of the B-cell cascade leading to the production of antibodies.
  • Exposure of a subject to a peptide or protein which comprises at least one T-cell epitope of the autoantigen may tolerize, anergize or otherwise modify appropriate T-cell subpopulations such that they become non-responsive to the autoantigen and do not participate in stimulating an immune response.
  • administration of a protein or peptide which comprises at least one T-cell epitope may modify the lymphokine secretion profile as compared with exposure to the naturally-occurring autoantigen (e.g., result in a decrease of IL-4 and or an increase in IL-2 causing a modification of TH[ and TH 2 populations).
  • T-cell subpopulations which normally participate in the response to the autoantigen such that these T-cells are drawn away from the site(s) of normal exposure to the autoantigen (e.g., tissues of the CNS) to the site(s) of therapeutic administration of the protein or peptide derived therefrom.
  • This redistribution of T-cell subpopulations may ameliorate or reduce the ability of an individual's immune system to stimulate the usual immune response at the site of normal exposure to the autoantigen, resulting in a diminution in symptoms.
  • peptides are T-cell epitope-containing peptides for a specific disease
  • those skilled in the art might approach the task in many ways each of which is acceptable.
  • the procedures selected are not meant to be limiting.
  • the selection of one mode of approaching a task is not intended to exclude other modes which can accomplish the same end by alternative means by those skilled in the art, for example, the selection of likely T-cell epitope-containing peptide from a group of peptides.
  • a protein is purified and analyzed, peptides are selected for testing, the selected peptides are produced, and the selected peptides are tested for properties characteristic of
  • Peptides derived from human MOG which moderate the response of a subject to the MOG autoantigen are also included in the invention. Likely candidates for such peptides can be tested for the effect on T-cell prohferation, as previously discussed and/or testing for a candidate's affinity for binding HLA DR proteins (procedures for obtaining HLA DR proteins is discussed in detail in the section below entitled "Purification and Analysis of HLA DR proteins") .
  • Such peptides can be identified, for example, by examining the structure and selecting appropriate regions to be produced as peptides (via recombinant expression systems, synthetically or otherwise) to be examined for ability to influence B-cell and/or T-cell responses, and selecting peptides containing epitopes recognized by these cells.
  • One method of identifying such peptides includes dividing the human MOG protein antigen into non-overlapping, or overlapping peptides of desired lengths and synthesizing, purifying and testing those peptides to determine whether the peptides comprise at least one T cell epitope using any number of assays.
  • an algorithm is used for predicting those peptides.
  • Other methods known to those skilled in the art may also be employed. In the instant application, certain of the peptides were selected using the algorithm as discussed in more detail herein.
  • These peptides are designated by amino acid sequence and represent the indicated regions of the mature amino acid sequence shown as positions 1-218 of Figure 1 (SEQ ID NO: 2). These subunit peptides are characterized by their ability to bind Class II MHC proteins so as to have the ability to be presented effectively as T-cell epitopes.(Rothbard, et al., The EMBO Journal 7: 93-100, 1988).
  • a necessary (but not necessarily sufficient) condition for binding to Class II MHC proteins is the presence of a hydrophobic side chain residue, preferably a tyrosine, phenylalanine or tryptophan residue, and less desirably isoleucine, leucine, valine or methionine residue spaced at a 4-amino acid distance from a small amino acid residue such as glycine, alanine, serine, threonine or cysteine. All of the MOG peptides shown in Figure 2 (SEQ ID NOS: 4-9, 11, 15, 16, and 42-72) fulfill these minimum conditions.
  • HLA-DR human MHC protein
  • Gorga J.Biol.Chem. 262: 16087-16094, 1987
  • Buelow Eur.J.Immunol.23:69-76, 1993
  • CHO cells transfected with the genes for the DRB1 * 0101 and DRB l * 1501.alleles (Marshall et al., J. Immunol, 152:4946-4957, 1994) were grown in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum (FCS), 2mM glutamine, and anitbiotics.
  • FCS heat inactivated fetal calf serum
  • MHC class II proteins were affinity purified using the monoclonal antibody LB3J, coupled to Sepharose CL4B. Class II proteins were eluted with 1% octyl- ⁇ -D-glucopyranoside (octyl glucoside), 50mM phosphate pH 11.5 and immediately neutralized using 1M phosphate pH 6.0. Purified ⁇ heterodimers were isolated by size exclusion using a 60 cm x 2.5 cm diameter column of Bio-Gel A0.5. The fractions containing the heterodimers were concentrated using Amicon Centri-Prep® 30 devices to a nominal concentration of 500 ⁇ g/ml.
  • the purity of the material was assayed by SDS-PAGE, high performance size exclusion chromatography (HPSEC), and Edman sequencing as previously described (Buelow et al., 1993).
  • HPSEC column was a BIOSEP SEC-S3000 (300 x 7.5 mm) (Phenomenex), eluted using a buffer system of PBS containing 1.0% octyl glucoside and 1.0% acetonitrile at a flow rate of 0.800 ml min (approximately 25 minutes per run).
  • Peptide binding assays were performed as previously described (Hill et al.J994). Briefly, affinity purified class II proteins (lOnM) were incubated with serial dilutions of the test peptide and a fixed concentration of biotinylated HA 307-319 (2 nM) in PBS containing 1.0% octyl glucoside at pH 6.5 in 96 well polypropylene plates
  • DR-peptide complexes 50 ⁇ l were transferred, in duplicate, to wells of a 96-well microtiter plate precoated with the monoclonal antibody LB3J and blocked with fetal calf serum. Excess peptide was removed by washing with PBS containing 0.02% Tween 20 and 0.05%NaN 3 . Europium labeled streptavidin (Pharmacia) was added and incubated overnight.
  • the data were analyzed by fitting the data to a binding function that calculated the concentration of test peptide to bind to a specific class II protein can be ranked by the corresponding IC 50 values, with the lower values corresponding to better binding peptides.
  • T-cell assay used to identify MOG epitopes
  • T-cell assays were performed to further refine the identification of T-cell epitopes.
  • Peripheral blood lymphocytes were isolated from the blood of a human volunteer HLA-DR2 positive donor using the techniques outlined in Example 2 herein.
  • the MOG peptides selected for testing were:
  • MOG 20-32 VELPCRISPGKNA (SEQ ID NO: 5)
  • MOG 70-82, A78 ELLKDAIGAGKVT (analog to MOG 70-82) (SEQ ID NO: 8)
  • MOG 88-100,K89,S98 VKFSDEGGFTSFF (analog to MOG 88-100) (SEQ ID NO: 9)
  • results of one such study of T-cell response to human MOG peptides is illustrated in Figure 6.
  • the results have been interpreted and indicate that MOG 1-13 (SEQ ID NO: 42) is a borderline T-cell epitope-containing peptide.
  • MOG 103-115 SEQ ID NO: 55
  • these results do not preclude the possibility that less than the entire 13 mer peptide could be a T-cell epitope-containing peptide.
  • T-cell response of a human patient suffering from Multiple Sclerosis is discussed in Example 4, where a mixture of MOG peptides (20 amino acids in length and shown in Figure 5a) was found to contain at least one T-cell epitope for the auto antigen responsible for Multiple Sclerosis. Furthermore, these results do not preclude the possibility that less than an entire 20 mer peptide could be a T-cell epitope- containing peptide.
  • the structure of the protein or peptides of the invention can be modified for such purposes as increasing solubility, enhancing therapeutic or prophylactic efficacy, or stability (e.g., shelf life ex vivo and resistance to proteolytic degradation in vivo), or generally by conservative substitutions and modifications.
  • a modified protein or peptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition, to modify immunogenicity, or to which a component has been added for the same purpose.
  • Figure 2 shows 3 such amino acids, Human MOG 70-82,A78 (SEQ ID NO: 8); Human MOG 74-86,A78 (SEQ ID NO: 53); and Human MOG 88-100, K89, S98 (SEQ ID NO: 9).
  • Modification must be made in such a way that the ability to recognize the appropriate T-cell subset is not altered. It is generally understood in the art which locations of amino acid side chains are oriented toward the upper surface of the APC and which point inward. Those that point inward are suitable candidates for modification since they clearly are less likely to affect binding to the appropriate T-cell receptor.
  • One embodiment of the present invention features a peptide which comprises at least one T-cell epitope of the protein and includes the regions of the peptides as shown in Figures 2, 5a and/or 5b that are significant for T-cell receptor binding or the modified forms thereof as above described, optionally extended at the N- and/or C-terminus with irrelevant amino acid sequence.
  • T-cell epitopes comprising at least two T-cell epitopes as described above.
  • the T-cell epitopes may be identical or may be different T-cell epitopes appropriate for human MOG.
  • the T-cell epitopes are typically at least 7 amino acids, preferably 12-40 amino acids, even more preferably 13-30 amino acids in length.
  • the amino acid sequences of the T-cell epitopes can be joined by a linker to increase sensitivity to processing by antigen-presenting cells.
  • linker can be any non-epitope amino acid sequence or other appropriate Unking or joining agent.
  • the epitopes are arranged in the same or a different configuration from a naturally-occurring configuration of the epitopes in the native human MOG protein.
  • the T-cell epitope(s) can be arranged in a contiguous or noncontiguous configuration. Noncontiguous is defined as an arrangement of T-cell epitope(s) which contains additional residues between the epitopes.
  • the T-cell epitopes can be arranged in a nonsequential order (e.g., in an order different from the order of the amino acids of the native protein from which T-cell epitope(s) are derived).
  • a peptide of the invention can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 60% or more of the T-cell epitopes of human MOG.
  • Preferred peptides comprise various combinations of two or more of the above-discussed T-cell epitopes.
  • Preferred peptides comprising a combination of two or more epitopes are those wherein the peptides include sequences selected from those in Figure 2 (SEQ ID NOS: 4-9, 11, 15, 16, and 42-72), Figure 5a and Figure 5b.
  • a protein or peptide of the invention can be modified so that it maintains the ability to induce T-cell unresponsiveness and bind MHC proteins without the ability to induce a strong or any proliferative response when administered in immunogenic form.
  • critical binding residues for T-cell receptor function can be determined using known techniques (e.g., substitution of each residue and determination of the presence or absence of T-cell reactivity).
  • those residues shown to be essential to interact with the T-cell receptor can be modified by replacing the essential amino acid with another, preferably similar amino acid residue (a conservative substitution) whose presence is shown to enhance or diminish, but not ehminate T-cell reactivity.
  • those amino acid residues which are not essential for T-cell receptor interaction can be modified by being replaced by another amino acid whose incorporation may enhance or diminish T-cell reactivity, but does not ehminate binding to relevant MHC.
  • peptides of the invention can be modified by replacing an amino acid shown to be essential to interact with the MHC protein complex with another, preferably similar amino acid residue (conservative substitution) whose presence is shown to enhance or diminish, but not to ehminate T-cell activity. It is believed that peptides that bind MHC with higher affinity should render T-cells immunopassive in vivo at lower concentrations.
  • amino acid residues which are not essential for interaction with the MHC protein complex but which are present on the bound peptide can be modified by being replaced by another amino acid whose incorporation may enhance or diminish, but not ehminate T-cell reactivity.
  • Preferred amino acid substitutions for non-essential amino acids include, but are not limited to substitutions with alanine, glutamic acid, or a methyl amino acid.
  • modification of proteins or peptides is substitution of cysteine residues preferably with alanine, serine, threonine, leucine or glutamic acid residues to minimize dimerization via disulfide linkages.
  • amino acid side chains of peptides of the invention can be chemically modified.
  • Another modification is cychzation of the peptide.
  • the protein or peptides of the invention can be modified to incorporate one or more polymorphisms in the amino acid sequence of the protein autoantigen resulting from any natural allelic variation.
  • D-amino acids, non-natural amino acids, or non-amino acid analogs can be substituted or added to produce a modified protein or peptide within the scope of this invention.
  • proteins or peptides of the present invention can be modified using polyethylene glycol (PEG) according to the method of A. Sehon and co-workers (Wie et al., supra) to produce a protein or peptide conjugated with PEG.
  • PEG polyethylene glycol
  • Modification of proteins or peptides or portions thereof can also include reduction/alkylation (Tarr in: Methods of Protein Microcharacterization, J. E. Silver ed., Humana Press, Clifton NJ 155-194 (1986)); acylation (Tarr, supra); chemical coupling to an appropriate carrier (Mishell and Shiigi, eds, Selected Methods in Cellular Immunology, WH Freeman, San Francisco, CA (1980), U.S. Patent 4,939,239; or mild formalin treatment (Marsh (1971), IntArch of Allergy andAppl Immunol 41:199-215).
  • an amino acid reporter group to the peptide backbone.
  • hexahistidine can be added to a protein or peptide for purification by immobilized metal ion affinity chromatography (Hochuli, E. et al, (1988) Bio Technology 6: 1321-1325).
  • specific endoprotease cleavage sites can be introduced between the sequences of the reporter group and the protein or peptide.
  • canonical protease sensitive sites can be engineered between regions, each comprising at least one T-cell epitope via recombinant or synthetic methods.
  • charged amino acid pairs such as KK or RR
  • KK or RR can be introduced between regions within a peptide during recombinant construction of the peptide.
  • the resulting peptide can be rendered sensitive to cleavage by cathepsin and/or other trypsin-like enzymes which would generate portions of the peptide containing one or more T-cell epitopes.
  • such charged amino acid residues can result in an increase in the solubility of a peptide.
  • Site-directed mutagenesis of DNA encoding a peptide or protein of the invention can be used to modify the structure of the peptide or protein by methods known in the art. Such methods may, among others, include polymerase chain reaction (PCR) with oligonucleotide primers bearing one or more mutations (Ho et al, (1989) Gene 77:51-59) or total synthesis of mutated genes (Hostomsky, Z. et al, (1989) Biochem Biophys Res Comm 161:1056-1063).
  • PCR polymerase chain reaction
  • the aforementioned methods can be applied to change the codons present in the cDNA sequence of the invention to those preferentially utilized by the host cell in which the recombinant protein is being expressed (Wada et al, supra).
  • the isolated protein and/or antigenic fragments can be used in methods of diagnosing, treating, and preventing demyehnating autoimmune responses.
  • the present invention provides therapeutic compositions comprising isolated human MOG (SEQ ID NOS: 1 and 2), or antigenic fragment thereof, and a pharmaceutically acceptable carrier or diluent.
  • the isolated protein and/or fragments can be used in screening for the autoimmune disease and for developing candidates for therapeutic compositions.
  • compositions of the present invention can be carried out using known procedures, at dosages and for periods of time effective to ameliorate the disease.
  • compositions containing a single peptide or protein mixtures of at least two peptides (e.g., a physical mixture of at least two peptides), each comprising at least one T-cell epitope of human MOG can also be provided such compositions can be administered in the form of a therapeutic composition with a pharmaceutically acceptable carrier of diluent.
  • the therapeutic composition may contain peptides comprising at least two regions, each region comprising at least one T-cell epitope of MOG and which regions may be arranged in a configuration different from a naturally-occurring configuration of the regions in human MOG.
  • compositions and preferred combinations of peptides which can be administered simultaneously or sequentially comprise peptides comprising amino acid sequences shown in Figures 2 (SEQ ID NOS: .4-9, 11, 15, 16, and 42-72), 5a (SEQ ID NOS. 73-80, 82,83) and 5b (the first 121 amino acids of human MOG protein SEQ ID NO. 2), more preferably, MOG 1-13 (SEQ ID NO. 42) and MOG103-115 (SEQ ID NO. 55).
  • Effective amounts of the therapeutic compositions will vary according to factors such as the degree of susceptibility of the individual, die age, sex, and weight of the individual, and the ability of the protein or fragment thereof to elicit an antigenic response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the protein, peptide or pharmaceutical composition thereof may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, sublingual administration, inhalation, transdermal apphcation, or rectal administration.
  • composition may include a coating with in a material to protect from the action of enzymes, acids and other natural conditions which may cause inactivation.
  • l ⁇ g-3mg and preferably from about 20 ⁇ g-750 ⁇ g of protein or peptide per dosage unit is typical.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and hquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabenz, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterihzation.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (i.e., protein or peptide) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the protein or fragment thereof may be administered to an individual in an appropriate diluent or adjuvant, co-administered with enzyme inhibitors or in an appropriate carrier such as hposomes.
  • Pharmaceutically acceptable diluents include sahne and aqueous buffer solutions.
  • Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon.
  • Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n- hexadecyl polyethylene ether.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
  • Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al, (1984) J. Neuroimmunol 7:27).
  • the therapeutic composition is preferably administered in non-immunogenic form, e.g., one that does not contain adjuvant.
  • die protein or peptide may be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • the protein and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into die individual's diet.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations contain an effective amount.
  • the amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • compositions or preparations according to the present invention are prepared so that an oral dosage unit contains between from about 10 ⁇ g to about 200 mg of active compound.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antif ungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammahan subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieve, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • compositions comprising at least two peptides (e.g., a physical mixture of at least two peptides), each comprising at least one T-cell epitope.
  • Such compositions can be administered to an individual in the form of a therapeutic composition with a pharmaceutically acceptable carrier or diluent as hereinbefore described.
  • a therapeutically effective amount of one or more of such compositions can be administered simultaneously or sequentially.
  • the proteins or peptides of the present invention can be used in "purified" form for standardization of reagents for the diagnosis and treatment of autoimmune disease.
  • the isolated and purified protein or peptide is also useful to prepare antisera or monoclonal antibodies for use in diagnosis.
  • An animal such as a mouse or rabbit can be immunized with an immunogenic form of the isolated protein or isolated peptide, if necessary, conferring immunogeni c ity on a protein or peptide by coupling to carriers or by other techniques well known in the art.
  • the protein or peptide can be administered in the presence of adjuvant, and progress of immunization can be monitored by detection of antibody titers in plasma or serum standard ELIS A or other immunoassay can be used witii the immunogen as antigen to assess the levels of antibodies.
  • antisera can be obtained and polyclonal antibodies isolated, if desired, from the serum.
  • antibody producing cells lymphocytes
  • hybridizing cells such as myeloma cells to yield hybridoma cells.
  • Hybridoma cells can be screened immunochemically for production of antibodies reactive with the invention protein or peptide thereof.
  • the antisera or monoclonal antibodies can be used to standardize reagents in standard assays.
  • Protein, peptides, or antibodies of the present invention can also be used for detecting and diagnosing autoimmune disease. For example, this could be done by combining blood, or blood products, obtained from an individual with an isolated antigenic peptide under conditions appropriate for binding of components in the blood (e.g., antibodies, HLA molecules, T-cells and B-cells) with the peptide(s) or protein, and determining the extent to which such binding occurs.
  • components in the blood e.g., antibodies, HLA molecules, T-cells and B-cells
  • autoimmune diseases which the protein, peptides or antibodies of the present invention can be used include paper radioimmunosorbent test (PRIST), enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA), and luminescence immunoassays (LIA).
  • PRIST paper radioimmunosorbent test
  • ELISA enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • IRMA immunoradiometric assays
  • LIA luminescence immunoassays
  • ohgonucleotides useful in therapeutic and diagnostic contexts. Modulation of the expression of the gene encoding MOG affects the progression of the autoimmune disease; in addition, progression can be monitored by monitoring expression using probes for RNA. Also, ohgomers based on d e nucleotide sequence disclosed in Figure 1 herein can be used in standard assay methods for detecting die MOG-encoding DNA or RNA.
  • ohgomers “based on” the sequence disclosed in Figure 1 (SEQ ID NO: 1) is meant ohgomers that contain portions of this sequence, that are complementary to the sequence or portions thereof, that represent primers used to amplify portions of the sequence when large amounts of DNA are desirable (such as for genetic manipulation) as well as ohgomers designed on d e basis of the disclosed sequence which effect triple helix formation with die relevant portion of the duplex representing the MOG gene.
  • Relevant design parameters for PCR primers, oligomers capable of hybridizing to single strand targets, and oligomers capable of triple helix formation with DNA duplexes are well known in the art.
  • oligomers "based on” the DNA of Figure 1 may have the same sequence as a portion of this DNA, the same sequence as the complement or portion thereof, or a different sequence but one which corresponds to tiiat disclosed in Figure 1 (SEQ ID NO: 1) through art-known design parameters.
  • the ohgomers having nucleotide sequences based on the nucleotide sequence shown in Figure 1 may be conventional RNA or DNA polymers, or may be modified forms thereof as generally known in the art.
  • the phosphodiester bonds of the oligomers may be substituted by alternative linkages such as phosphorothioates, methylphosphonates and the like.
  • alternative scaffolding for nucleotide bases has also been disclosed and such modifications are included within the scope of ohgomers claimed herein.
  • a human cDNA library was subjected to the polymerase chain reaction (PCR) using 3' and 5' primers designed from the pubhshed rat MOG coding sequence of Gardinier et al. (supra).
  • PCR polymerase chain reaction
  • the human MOG sequence could not be obtained in this manner, putatively due to insufficient homology at the 5' and/or 3' ends of the human and rat sequences. Therefore, four rat internal ohgonucleotides were designed.
  • primers 166-183 (SEQ ID NO: 34) and 538-555 (SEQ ID NO: 35) were homologous to the top strand of the gene (primers 94-111 and 166-183 (SEQ ID NO: 34), base 1 starting at the ATG) and two were homologous to the bottom strand of the gene (primers 538-555 (SEQ ID NO: 35) and 685-702).
  • the combination of primers 166-183 (SEQ ID NO: 34) and 538-555 (SEQ ID NO: 35) was successful in effecting die amplification of a fragment of the approximately 400 bp expected size from a human brain cDNA library.
  • sequence of these primers was: a) 166-183: CAGAATCCGGGAAGAATGCCACGGGC (SEQ ID NO: 34); and b) 538-555: CAGCGGCCGCACGGAGTTTTCCTCTCAG (SEQ ID NO:
  • the 400 bp PCR product was cloned into expression vector pVL1393 by digesting pVL1393 (Pharmingen CA) with EcoRI and Notl, digesting die amphfied product witii the same enzymes and hgating the resulting fragments.
  • the insert was verified by digesting several clones derived from e hgated plasmids with EcoRI and Notl and sequencing the resulting 400 bp human MOG fragment.
  • the resulting insert putatively lacks 184 bp of 5' sequence and 201 bp of 3' sequence, based on die 738 bp rat open reading frame.
  • Two primers were designed from me 400 bp insert from positions 346-363 top and bottom strands as follows:
  • the human MOG 346-363 top and bottom primers (SEQ ID NOS: 36 and 37) were used in combination with d e above-mentioned 5' and 3' rat primers, respectively, to amplify the 5' and 3' missing ends of the gene from the same human brain cDNA library as previously used. A PCR product corresponding the 3' end of the gene was obtained, but the corresponding 5' end did not result.
  • the 3' fragment obtained had the expected 400 bp size and this fragment was cloned in pVL1393 and sequenced.
  • a human brain medulla ⁇ gtlO library obtained from Clontech which had been previously amphfied and had a titer of 8x10 10 pfu/ml was screened following the protocol described by die manufacturer.
  • the library was plated onto 12 large plates at 30,000 plaques/plate and the plaques were lifted onto nitrocellulose filters (2 rephca filters/plate). Twelve filters lifted from the 12 different plates were then hybridized to a 32 P labelled probe corresponding to the human MOG internal 400 bp fragment initially cloned (positions 184-534). Twenty-two strong positives were obtained. A plug was picked for each positive from the original plates and incubated overnight with ⁇ dilution buffer to elute die phage from the agar. The tube was then centrifuged and die supernatant transferred.
  • the DNA was amphfied from each individual pool using either a ⁇ gtlO forward primer with an Sstfl site: 5'-CTTTTGAGCAAGTTCAGCCTGGTTAAG-3' (SEQ ID NO: 38) or a ⁇ gtlO reverse primer with an Xhol site:
  • S'-ATTCCfiSATCCTGAGAGTCACCTTTCCCGCACC-S' SEQ ID NO: 41.
  • the last two primers include a BamHI site (underlined in the sequences) naturally present in the human MOG sequence.
  • the primers were used in four different combinations: 1) forward top/internal MOG bottom; 2) reverse bottom/internal MOG bottom; 3) internal MOG top/reverse bottom; and 4) internal MOG top/forward top.
  • the first two combinations provided the 5' end of die gene (up to the BamHI site) and the last two, the 3' end of the gene. Both 5' and 3' portions include untranslated regions. Which of the two members of each combination actually resulted in the desired fragment depends on the orientation of the cDNAs cloned into ⁇ gtlO.
  • die fragments obtained varied from one pool to another. Five of the largest 5' fragments or 3' fragments were subcloned into the Sstll and BamHI or BamHI and Xhol sites, of die SK polylinker. Three clones from each pool were then sequenced to rule out the presence of PCR errors. This provided the complete sequence of the gene coding region as well as 174 bp of the 5' untranslated sequence.
  • the human MOG gene encodes a preprotein of 248 amino acids which has 87% homology with the 246 amino acids in the rat protein.
  • the mature protein contains 218 amino acids, numbered 1-218 in Figure 1 (SEQ ID NO: 2).
  • the mature protein begins at the glycine shown at position 1 and is derived from the 248 amino acid preprotein by cleavage from the presequence extending from the MET start codon to the alanine residue immediately preceding the glycine shown in position 1.
  • human MOG cDNA was cloned into a pET H6 vector for expression in E. coli.
  • a truncated human MOG cDNA (no leader sequence and no transmembrane domains) encoding amino acids 1-121 of human MOG (the first 121 amino acids of SEQ ID NO. 2) was amphfied by PCR using the following ohgonucleotides: 5' primer:
  • the 363 bp PCR fragment was then cloned between die unique Sstll and Xhol sites of pET.H6. After cloning, the cDNA was entirely sequenced.
  • Example 1A Expression of truncated human MOG in SF-9 Insect cells and E. coli
  • the PVL1393 transfer vector containing the truncated human MOG cDNA encoding amino acids 1-121 of human MOG was cotransfected into SF-9 cells along with Baculogold linearized Baculovirus DNA (Pharmingen, San Diego, CA).
  • the culture supernatant containing recombinant viruses was harvested after 4 days.
  • the recombinant virus was plaque purified and subjected to 3 rounds of amplification to obtain a high liter viral stock.
  • AF-9 cells were then infected witii the viral stock at a MOI of 2.0.
  • the supernatant from infected cells was harvested 48 hours after infection and applied to a NiNTA agarose column.
  • the recombinant MOG protein was eluted under non-denaturing conditions using 250 mM Imidazole, dialyzed against 5% propionic acid and H2O and subsequently lypophilized. The protein concentration was estimated by BCA. The purified MOG protein was visualized on a 12.5% polyacrylamide gel stained with Coomassie blue.
  • E. coli Expression The pET.H6 vector containing the truncated human MOG cDNA was introduced into BL21(DE3) cells (Novagen, Madison, WI.) by transformation. Several colonies were grown together in 2YT medium to an OD of 1.0. The bacteria were then induced overnight with ImM IPTG. Cells were harvested and lysed with 6M Guanidine/lOOmM Tris.HCl pH 8.0 at room temperature overnight. The lysate was centrifuged at 20.000 rpm for 30 minutes and the resulting supernatant applied to a NiNTA agarose column (Quiagen, Chatsworth, CA).
  • the protein was eluted with 6M Guanidine/lOOmM sodium phosphate pH 4.5, dialyzed first against 5% propionic acid, then against H2O and subsequently lypophilized. The protein concentration was estimated by BCA. The purified MOG protein was visualized on a 12.5% polyacrylamide gel stained with Coomassie blue.
  • Example 2 Human MOG Expressed in Insect SF-9 to Induce EAE in vivo
  • Two groups of (PLJxSJL)Fl mice were injected with 10 ⁇ g and 50 ⁇ g respectively N-terminal fragment of recombinant human MOG expressed in insect SF-9 (- TM, recombinant, SF-9) which was prepared according to Examples 1 and 1A herein.
  • the truncated MOG contains amino acids 1-121 (hereinafter intended to refer to MOG 1- 121) and was selected, in part, because of its solubility relative to the solubility of amino acids 122-218 of the MOG protein (SEQ ID NO: 2).
  • Human MOG 122-218 (residues 121-218 of SEQ ID NO. 2) is extremely hydrophobic and is believe to include two transmembrane regions.
  • MOG 1-121 was emulsified in complete Freund's adjuvant and injected subcutaneously in mice. At the same time as the MOG 1-121 injection, 200ng pertussis toxin was also injected intravenously. The 200ng pertussis toxin IN. injection was repeated 2 days later.
  • Emulsions were prepared using MOG 1-121, (-TM, recombinant, SF-9), as described above combined with 400 ⁇ g H37Ra (Difco Laboratories, Detroit, MI) in CFA. Beginning at Day 8 after initial immunization, the mice were observed for signs of paralysis and scored daily as an indicator that EAE has been contracted. Mice were scored based on clinical signs according to the following scale: 1, tail paralysis; 2, partial hind limb paralysis; 3, complete hind hmb paralysis; 4, forelimb paralysis; 5, moribund or dead. The onset of symptoms began as early as 14 days for some mice.
  • mice were observed for 31 days when die mice were sacrificed, the brains and spinal cords were harvested and histological studies were undertaken to verify the clinical observations.
  • Sixty percent (60%) of mice immunized witii 50 ⁇ g of rMOG (-TM, recombinant, SF-9) and eighty percent (80%) of mice immunized with lO ⁇ g of rMOG (- TM, recombinant, SF-9) exhibited symptoms of EAE.
  • mice induced witii EAE in this manner may be useful as an animal model for screening potential therapeutics for treatment of multiple sclerosis.
  • the following is one method for identifying therapeutic compositions for the treatment of multiple sclerosis which comprises the steps of: administering human MOG to mice in immunogenic form to cause induction of
  • EAE in said mice in said mice; treating said mice induced with EAE with therapeutic compositions comprising at least one antigenic fragment of human MOG prior to the onset of symptoms of EAE or after die onset of symptoms of EAE in said mice; and determining if said therapeutic composition prevents the onset or progression of the symtoms of EAE in said mice induced with EAE.
  • T-cell assays were performed to further refine the identification of T-cell epitopes.
  • Peripheral blood lymphocytes were isolated from the blood of a human volunteer HLA-DR2 positive donor using Ficoll Hypaque. Twenty (20) milhon lymphocytes were seeded into 96 wells of a microtiter dish at 2 x 10 5 per well in RPMI culture media supplemented witii human AB serum. A mixture of selected MOG peptides was added at a final concentration of 50 ⁇ M for each peptide.
  • the MOG peptides selected were both naturally occurring peptides and analogs of naturally occurring peptides and were: MOG 1-13 GQFRVIGPRHPIR (SEQ ID NO: 42)
  • MOG 20-32 VELPCRISPGKNA (SEQ ID NO: 5)
  • Cultures were incubated in a humidified CO 2 37°C incubator for twelve days, intermittently supplemented with human IL2 (20U/ml) and IL4 (5U/ml). A sample from each culture well was removed, washed to remove previously added peptide, and reseeded into four wells of a fresh microtiter dish (two wells with the peptide mixture and two wells without die peptide mixture for each sample). Autologous irradiated cryopreserved lymphocytes were added as antigen presenting cells. After further 3 days of incubation, 3H-thymidine incorporation was measured.
  • Positive microtiter lines were scored if the mean incorporation in the peptide wells was greater than or equal to 1.5-fold higher than the wells without peptide. Positive microtiter lines were expanded witii DL2 and IL4, and then reassayed the foUowing week by die same methods with the individual peptides rather than the peptide mix.
  • the peptides identified as T-cell epitopes-conatining peptides using this method were MOG 1-13 (SEQ ID NO: 42) and MOG 103-115 (SEQ ID NO: 55).
  • Figure 6 shows the results of the assays performed.
  • Example 3 FoUowing the procedures of Example 3, the T-cell response of a human patient suffering from Multiple Sclerosis was tested with die group of MOG 20mer peptides shown in Figure 5a and N-terminal fragment of recombinant human MOG 1-121 expressed in insect SF-9 (-TM, recombinant, SF-9), which is shown to induce EAE in mice in Example 2.
  • MOG peptides tested are suspected of containing at least one T-ceU epitope for the autoantigen responsible for Multiple Sclerosis.
  • the MS patient's PBL were cultured in microtiter weUs according to the protocol in Example 3, except tiiat both recombinant MOG 1-121 (48 of 96 weUs) and a mixture of 10 MOG fragments (20-mer peptides shown in Figure 5a) (48 of 96 weUs) were used separately to initiate the cultures.
  • a sample from each culture weU was removed, washed to remove previously added peptide, and reseeded into six weUs of a fresh microtiter dish.
  • Six microtiter wells were subsequently found to react with both rMOG and the peptide mixture (2 weUs from the cultures initiated with rMOG and 4 weUs from the cultures initiated with the peptide mixture).
  • Glu Val Glu Leu Pro Cys Arg lie Ser Pro Gly Lys Asn Ala Thr Gly 50 55 60 ATG GAG GTG GGG TGG TAC CGC CCC CCC TTC TCT AGG GTG GTT CAT CTC 420
  • Gly Arg Thr Glu Leu Leu Lys Asp Ala lie Gly Glu Gly Lys Val Thr 100 105 110 CTC AGG ATC CGG AAT GTA AGG TTC TCA GAT GAA GGA GGT TTC ACC TGC 564
  • Lys lie Thr Leu Phe Val lie Val Pro Val Leu Gly Pro Leu Val Ala 210 215 220
  • Leu lie lie Cys Tyr Asn Trp Leu His Arg Arg Leu Ala Gly Gin Phe 225 230 235
  • Val Glu Leu Pro Cys Arg lie Ser Pro Gly Lys Asn Ala Thr Gly Met 50 55 60

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Abstract

La présente invention concerne une molécule d'acide nucléique ayant une séquence de nucléotides codant pour la glycoprotéine humaine de myéline d'oligodendrocytes, qui est un auto-antigène lié aux maladies démyélisantes auto-immunes. La présente invention concerne également une glycoprotéine humaine de myéline d'oligodendrocytes ou des fragments antigéniques, produits par recombinaison. Les fragments antigéniques de la glycoprotéine de myéline d'oligodendrocytes qui peuvent être préparés par synthèse, représentent des épitopes de cellules T de la glycoprotéine humaine de myéline d'oligodendrocytes. La glycoprotéine humaine de myéline d'oligodendrocytes et les fragments de celle-ci sont utiles pour diagnostiquer et traiter des maladies auto-immunes. On décrit en outre des méthodes de dépistage et la mise au point de thérapies utiles pour le traitement de maladies auto-immunes.
PCT/US1994/010257 1993-09-03 1994-09-01 Utilisations de la glycoproteine de myeline d'oligodendrocytes et de portions peptidiques derivees de celle-ci dans des protocoles ayant trait a des maladies auto-immunes WO1995006727A2 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
JP7508328A JPH09502346A (ja) 1993-09-03 1994-09-01 自己免疫疾患に関連するプロトコールにおけるミエリン希突起神経膠細胞糖蛋白質およびそのペプチド部分の使用
EP94928084A EP0716696A1 (fr) 1993-09-03 1994-09-01 Utilisations de la glycoproteine de myeline d'oligodendrocytes et de portions peptidiques derivees de celle-ci dans des protocoles ayant trait a des maladies auto-immunes
NZ273813A NZ273813A (en) 1993-09-03 1994-09-01 Myelin oligodendrocyte glycoprotein autoantigen
KR1019960701093A KR960705038A (ko) 1993-09-03 1994-09-01 자기 면역 질환에 관련된 프로토콜에서 미엘린 올리고덴드로시트 당 단백질 및 그의 펩타이드 부분들의 용도(users of myelin oligodendrocyte glycoprotein and peptide portions thereof in protocols related to autoimmune desease)
AU77258/94A AU7725894A (en) 1993-09-03 1994-09-01 Uses of myelin oligodendrocyte glycoprotein and peptide portions thereof in protocols related to autoimmune disease
NO960858A NO960858L (no) 1993-09-03 1996-03-01 Anvendelser av myelin - oligodendrocyt-glykoprotein og peptid-deler derav i metoder i forbindelse med autoimmunsykdom
FI961000A FI961000A (fi) 1993-09-03 1996-03-04 Myeliinioligodendrosyyttiglykoproteiinin ja tämän peptidiosien käyttömuotoja autoimmuunisairauksiin liittyvissä menetelmissä

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US11682493A 1993-09-03 1993-09-03
US08/116,824 1993-09-03

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996012737A2 (fr) * 1994-10-25 1996-05-02 Immulogic Pharmaceutical Corporation Compositions et traitement pour la sclerose en plaques
WO1996034622A1 (fr) * 1995-05-02 1996-11-07 Alexion Pharmaceuticals, Inc. Molecules de proteine myelinique modifiees
WO1997035879A1 (fr) * 1996-03-28 1997-10-02 Immulogic Pharmaceutical Corporation Peptides de la glycoproteine d'oligodendrocyte de myeline et leurs utilisations
WO1998033912A1 (fr) * 1997-01-30 1998-08-06 Human Genome Sciences, Inc. PROTEINE SEMBLABLE A LA GLYCOPROTEINE D'OLIGODENDROCYTE DE MYELINE (MOGp) ET PROCEDES D'UTILISATION
WO1999012966A1 (fr) * 1997-09-11 1999-03-18 Astra Aktiebolag Peptides comportant une sequence immunodominante derivant de la glycoproteine de l'oligodendrocyte myelinique humain
WO1999023867A2 (fr) * 1997-11-07 1999-05-20 Biogen, Inc. Bmog, nouvelle proteine de la famille des glycoproteines de myeline-oligodendrocyte, et son utilisation a des fins immunomodulatrices
WO2001049739A1 (fr) * 1999-12-29 2001-07-12 Fudan University Nouveau polypeptide, la proteine po 14 de la myeline, et polynucleotide le codant
EP1115425A1 (fr) * 1998-08-26 2001-07-18 The Regents of the University of California Inhibiteurs d'auto-anticorps
WO2002036619A1 (fr) * 2000-11-02 2002-05-10 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, glucoproteine humaine 42, et polynucleotide codant ce polypeptide
WO2007008933A2 (fr) * 2005-07-11 2007-01-18 Carantech Biosciences, Inc. Compositions et procedes comprenant des variants d'epissage alternes complexes de genes de myeline/oligodendrocyte (mog) et anticorps diriges contre ceux-ci
WO2013020914A1 (fr) * 2011-08-10 2013-02-14 Celares Gmbh Peptides peg-conjugués
CN101897964B (zh) * 2009-04-27 2013-04-10 中国农业大学 一种预防自身免疫疾病的药物
KR20150105365A (ko) * 2013-01-15 2015-09-16 아피토프 인터내셔날 엔브이 펩타이드
US20170043036A1 (en) * 2014-04-24 2017-02-16 University Of Florida Research Foundation, Inc Aav-based gene therapy for multiple sclerosis
US11117963B2 (en) 2016-12-26 2021-09-14 Kyowa Hakko Kirin Co., Ltd. Antibody which binds to myelin oligodendrocyte glycoprotein
US11334352B1 (en) 2020-12-29 2022-05-17 Kpn Innovations, Llc. Systems and methods for generating an immune protocol for identifying and reversing immune disease

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY AND MOLECULAR BIOLOGY INTERNATIONAL, vol.30, no.5, August 1993 pages 945 - 958 S. ABO ET AL. 'Preparation of highly purified human myelin oligodendrocyte glycoprotein in quantities sufficient for encephalitogenicity and immunogenicity studies' *
JOURNAL OF CLINICAL INVESTIGATION, vol.92, no.6, December 1993 pages 2602 - 2608 N. KERLERO DE ROSBO ET AL. 'Reactivity to myelin antigens in multiple sclerosis' *
THE JOURNAL OF CELL BIOLOGY, vol.110, no.2, February 1990 pages 471 - 479 DANIEL D. MIKOL ET AL. 'The oligodendrocyte-myelin glycoprotein belongs to a distinct family of proteins and contains the HNK-1 carbohydrate' *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996012737A3 (fr) * 1994-10-25 1996-10-10 Immulogic Pharma Corp Compositions et traitement pour la sclerose en plaques
WO1996012737A2 (fr) * 1994-10-25 1996-05-02 Immulogic Pharmaceutical Corporation Compositions et traitement pour la sclerose en plaques
WO1996034622A1 (fr) * 1995-05-02 1996-11-07 Alexion Pharmaceuticals, Inc. Molecules de proteine myelinique modifiees
WO1997035879A1 (fr) * 1996-03-28 1997-10-02 Immulogic Pharmaceutical Corporation Peptides de la glycoproteine d'oligodendrocyte de myeline et leurs utilisations
WO1998033912A1 (fr) * 1997-01-30 1998-08-06 Human Genome Sciences, Inc. PROTEINE SEMBLABLE A LA GLYCOPROTEINE D'OLIGODENDROCYTE DE MYELINE (MOGp) ET PROCEDES D'UTILISATION
WO1999012966A1 (fr) * 1997-09-11 1999-03-18 Astra Aktiebolag Peptides comportant une sequence immunodominante derivant de la glycoproteine de l'oligodendrocyte myelinique humain
WO1999023867A2 (fr) * 1997-11-07 1999-05-20 Biogen, Inc. Bmog, nouvelle proteine de la famille des glycoproteines de myeline-oligodendrocyte, et son utilisation a des fins immunomodulatrices
WO1999023867A3 (fr) * 1997-11-07 1999-07-15 Biogen Inc Bmog, nouvelle proteine de la famille des glycoproteines de myeline-oligodendrocyte, et son utilisation a des fins immunomodulatrices
EP1115425A4 (fr) * 1998-08-26 2005-01-26 Univ California Inhibiteurs d'auto-anticorps
EP1115425A1 (fr) * 1998-08-26 2001-07-18 The Regents of the University of California Inhibiteurs d'auto-anticorps
WO2001049739A1 (fr) * 1999-12-29 2001-07-12 Fudan University Nouveau polypeptide, la proteine po 14 de la myeline, et polynucleotide le codant
WO2002036619A1 (fr) * 2000-11-02 2002-05-10 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, glucoproteine humaine 42, et polynucleotide codant ce polypeptide
WO2007008933A2 (fr) * 2005-07-11 2007-01-18 Carantech Biosciences, Inc. Compositions et procedes comprenant des variants d'epissage alternes complexes de genes de myeline/oligodendrocyte (mog) et anticorps diriges contre ceux-ci
WO2007008933A3 (fr) * 2005-07-11 2007-06-28 Carantech Biosciences Inc Compositions et procedes comprenant des variants d'epissage alternes complexes de genes de myeline/oligodendrocyte (mog) et anticorps diriges contre ceux-ci
CN101897964B (zh) * 2009-04-27 2013-04-10 中国农业大学 一种预防自身免疫疾病的药物
WO2013020914A1 (fr) * 2011-08-10 2013-02-14 Celares Gmbh Peptides peg-conjugués
KR20150105365A (ko) * 2013-01-15 2015-09-16 아피토프 인터내셔날 엔브이 펩타이드
US9862751B2 (en) 2013-01-15 2018-01-09 Apitope Technology (Bristol) Limited Myelin oligodendrocyte glycoprotein peptides
US10377800B2 (en) 2013-01-15 2019-08-13 Apitope Technology (Bristol) Limited Myelin oligodendrocyte glycoprotein (MOG) peptide
KR102169902B1 (ko) * 2013-01-15 2020-10-26 아피토프 테크놀러지 (브리스톨) 리미티드 펩타이드
US20170043036A1 (en) * 2014-04-24 2017-02-16 University Of Florida Research Foundation, Inc Aav-based gene therapy for multiple sclerosis
US11117963B2 (en) 2016-12-26 2021-09-14 Kyowa Hakko Kirin Co., Ltd. Antibody which binds to myelin oligodendrocyte glycoprotein
US11334352B1 (en) 2020-12-29 2022-05-17 Kpn Innovations, Llc. Systems and methods for generating an immune protocol for identifying and reversing immune disease

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CA2170901A1 (fr) 1995-03-09
NO960858D0 (no) 1996-03-01
FI961000A0 (fi) 1996-03-04
AU7725894A (en) 1995-03-22
NO960858L (no) 1996-03-01
KR960705038A (ko) 1996-10-09
JPH09502346A (ja) 1997-03-11
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WO1995006727A3 (fr) 1995-04-13
NZ273813A (en) 1998-05-27

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