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EP0922057A1 - Peptides de la glycoproteine d'oligodendrocyte de myeline et leurs utilisations - Google Patents

Peptides de la glycoproteine d'oligodendrocyte de myeline et leurs utilisations

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Publication number
EP0922057A1
EP0922057A1 EP96913884A EP96913884A EP0922057A1 EP 0922057 A1 EP0922057 A1 EP 0922057A1 EP 96913884 A EP96913884 A EP 96913884A EP 96913884 A EP96913884 A EP 96913884A EP 0922057 A1 EP0922057 A1 EP 0922057A1
Authority
EP
European Patent Office
Prior art keywords
mog
seq
human
peptide
composition
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP96913884A
Other languages
German (de)
English (en)
Inventor
Brigitte Devaux
Jonathan Rothbard
Dawn Smilek
Barbara Wallner
Richard D. Garman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Immulogic Pharmaceutical Corp
Original Assignee
Immulogic Pharmaceutical Corp
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 Immulogic Pharmaceutical Corp filed Critical Immulogic Pharmaceutical Corp
Publication of EP0922057A1 publication Critical patent/EP0922057A1/fr
Withdrawn legal-status Critical Current

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the invention is directed to autoantigens and their relevant epitopes. More specifically, the invention concerns myelin 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 myelin 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 metabolic 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 (MS), are no exception. MS is the most common cause of neurological disability associated with disease in Western countries. It is an inflammatory disease of the central nervous system (CNS) characterized in part by destruction of myelin.
  • CNS central nervous system
  • Macrophages, plasma cells, antigen-presenting cells, and cytokine-secreting T lymphocytes can be found in the CNS of MS patients, but activated.
  • the etiology of MS is still unknown, but it is believed that activated T cells misdirected towards normal constituents of the nervous system are responsible for the pathology of MS. Models for MS and compositions and methods for treating MS are thus needed.
  • a commonly used animal model for MS is experimental allergic encephalomyelitis (EAE).
  • EAE is a CD4 + T cell mediated autoimmune demyelinating disease of the CNS which resembles MS in some of its clinical and histological features.
  • This disease can be induced, for example, in guinea pigs by administration of whole brain homogenate, and has been induced in mice with myelin basic protein (MBP) and complete Freund's adjuvant.
  • MBP myelin basic protein
  • the mouse model has been accepted as a model of human MS. Animals so immunized exhibit symptoms of EAE, including, but not limited to, paralysis and often death.
  • MBP has been associated with EAE and MS, the search for other factors and other autoantigens which may contribute to these diseases continues (Kuby (1994) Immunol. (2d Ed.) pp. 451-457).
  • the apparent first indication of the autoantigen which came to be known as myelin oligodendrocyte glycoprotein (MOG) was disclosed by Lebar et al. (J. Immunol. (1976) 116: 1439-1446). This work reported the results of a study which identified an IgG2 antibody in the serum of EAE guinea pigs as responsible for the complement-dependent demyelinating activity of the serum. The relevant antibody reacted with the putative autoantigen present in the homogenate of CNS myelin. This autoantigen was shown to be different from the encephalitogenic MBP of CNS myelin.
  • M2 this unknown antigen, now designated M2 was identified as a relatively minor component of CNS myelin located on oligodendrocyte surfaces and in the outermost lamellae of myelin in mouse, rabbit, rat, bovine and human CNS tissues as well as guinea pig (Lebar et al. (1986) J. Exp. Immunol. 66:423-443). M2 appeared as two glycoprotein bands at 27 and 54 kD, and monoclonal antibodies putatively specific for M2 were also reported in this paper.
  • Murine MOG in its monomeric form is a 25 kD amino acid sequence. This article also disclosed the first approximately 26 amino acids at the N-terminus of the murine protein. Gardinier et al. (J. Neurosci. Res.
  • 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, and treating autoimmune disease are 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 in diagnosis and treatment of autoimmune diseases.
  • the invention is directed to isolated and purified human MOG protein, and to peptide fragments thereof which modulate the course of development of symptomology in demyelinating autoimmune diseases.
  • the MOG peptides of the invention have an amino acid sequence selected from the group consisting of SEQ ID NOS:96-110 and 146- 164.
  • the invention provides human MOG peptides including at least one T cell epitope-containing region and comprising all or a portion of amino acid residues 1-95 (SEQ ID NO:205), 101-135 (SEQ ID NO:207) including 111-135 (SEQ ID NO: 102), or 171-215 (SEQ ID NO:206).
  • the invention is directed to recombinant materials and methods useful for the production of the MOG protein and peptide portions thereof.
  • the invention is directed to compositions including pharmaceutical compositions and methods of their use in mitigating the effects of demyelinating autoimmune diseases such as MS.
  • the invention is directed to isolated, purified, and modified 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.
  • a MOG peptide of the invention has a first amino acid substituted for a second amino acid. The peptide so modified retains a biological activity which it possessed before modification.
  • a first amino acid is substituted for a second, a third, and/or a fourth amino acid in the MOG peptide.
  • the peptide is coupled to a polyethylene glycol, a moiety that enhances the solubility of the peptide, a moiety that facilitates purification of the peptide, and/or a moiety including a proteolytic cleavage site.
  • MOG peptide of the invention including at least one T-cell epitope.
  • a MOG peptide includes at least two T cell epitopes.
  • the MOG peptide includes tandem copies of the same or different T cell epitopes.
  • the invention discloses a method for screening for demyelinating autoimmune disease and identifying therapeutic compositions comprised of MOG or fragments thereof which are capable diagnosing, preventing or treating MS in mammals, preferably, humans.
  • the invention also provides compositions including at least one MOG peptide.
  • the MOG peptide of the composition of the invention comprises at least one T cell epitope.
  • the composition further comprises a peptide of human MBP.
  • the composition includes tandem copies of a MOG peptide.
  • Multipeptide compositions are also provided by the invention. These compositions include more than one MOG peptide of the invention or a combination of a MOG peptide of the invention and any other MOG peptide or peptide of human MBP. In another aspect, such compositions and multipeptide compositions are part of therapeutic compositions, including a pharmaceutically acceptable carrier, for treating multiple sclerosis in a mammal. In other embodiments, a therapeutic composition is provided which includes a pharmaceutically acceptable excipient and a major histocompatibility complex (MHC) class II peptide complex capable of binding a T cell receptor and inducing anergy and/or apoptosis in a T cell bearing the receptor.
  • MHC major histocompatibility complex
  • the complex includes an MHC class II component comprises extracellular domains of an MHC class II molecule sufficient to form an antigenic binding pocket, is encoded by an allele associated with an autoimmune disease such as MS, and is soluble under physiological conditions in the absence of detergent or lipids.
  • the complex further includes a MOG peptide which is autoantigenic and bound to the antigen binding pocket.
  • the complex further includes at least a second MHC class II peptide complex.
  • the therapeutic formulations of the invention include MOG or MBP + MOG, or MBP peptides.
  • the invention provides a method of treating MS comprising the step of administering to a mammal suffering from MS a therapeutic composition of the invention in an amount sufficient to down-regulate an autoimmune response in the mammal.
  • the administering step is carried out by intravenous injection, subcutaneous injection, intramuscular injection, oral administration, inhalation, sublingual administration, transdermal administration, or rectal administration.
  • the composition is administered subcutaneously in non- immunogenic form in an amount sufficient to down-regulate the autoimmune response in the mammal.
  • FIG. 1 shows the complete nucleotide sequence (SEQ ID NO: l) of DNA encoding human MOG protein and the amino acid sequence deduced therefrom (SEQ ID NO: 2), wherein the arrow indicates the first amino acid of the mature MOG protein;
  • FIG. 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);
  • FIG. 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;
  • FIG. 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;
  • FIG. 5a shows peptides (20mers) designated by amino acid sequence (SEQ ID NOS: 73-93) which are useful in the invention
  • FIG. 5b shows a peptide of human MOG 1-121 , designated by amino acid sequence, which contains at least one T cell epitope;
  • FIG. 6 is a bar graph of data from Example 5 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 3 H-thymidine incorporated (CPM) by each cell line in response to each individual peptide;
  • CPM 3 H-thymidine incorporated
  • FIG. 7 is a schematic representation of the identity of representative MOG peptides compared with the complete MOG sequence
  • FIG. 8 A is a bar graph of data from Example 8 where the x axis indicates individual MOG peptides used to test short term MOG-selected T cell lines, and the y axis indicates the sum of ranks earned by each peptide;
  • FIG. 8B is a bar graph of data from Example 8 where the x axis indicates individual MOG peptides used to test short term MOG-selected T cell lines, and the y axis indicates the average value for percent of total peptide reactivity accounted for by each individual peptide; and
  • FIG. 9 is a bar graph of data from Example 9 where the x-axis indicates individual MOG peptides used to test short term MOG-selected T cell lines, and the y axis (positivity index) indicates the average value for percent of total peptide activity accounted for by each individual peptide.
  • the present invention provides novel peptides of human MOG, an autoantigen involved in demyelinating autoimmune diseases. It also compositions including such peptides which are useful in moderating the autoimmune response. Many of these peptides are characterized by their correspondence to the T-cell epitope regions of the human MOG.
  • Demyelinating autoimmune diseases involve a T cell mediated attack by the immune system on the myelin sheath, resulting in what amounts to short circuits in the nervous system.
  • T-cell responses to the human MOG autoantigen require uptake and subsequent proteolytic cleavage of the antigen by antigen presenting cells, followed by presentation of the antigen in the context of a Class II major histocompatibility complex (MHC)-encoded protein, thereby permitting their recognition by the T-cells.
  • MHC major histocompatibility 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 A nucleic acid sequence encoding human MOG is shown in FIG. 1 (SEQ ID NO: l), along with the deduced amino acid sequence (SEQ ID 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.
  • 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 human T-cell receptors. These peptides and fragments are also included within the invention scope of the invention.
  • the scope of the invention is not limited to the human MOG protein encoded by the amino acid sequence depicted in FIG. 1 , or to the specific nucleic acid sequence presented.
  • Naturally occurring variants and deliberate 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.
  • 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.
  • nucleotides up to about 1 % of the nucleotides
  • sequence encoding MOG 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 polymo ⁇ hisms are within the scope of the invention.
  • 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 irnmune 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 apoptosis and/or modification of TH, and TH 2 subsets.
  • antigenic fragments that comprise T-cell epitopes.
  • Peptides can be derived from the naturally occurring MOG sequence or and can be modified such that conservative amino acid substitutions have been made.
  • substitutions include replacing another amino acid (e.g., a second, third, fourth, or other amino acid), replacing more than one amino acid (e.g., a second and third, second, third and fourth, or other amino acids) with a first amino acid which does not interfere with a biological activity of the peptide; i.e., The peptide so modified retains a biological activity it possessed before modification.
  • Nonlimiting examples of namrally occurring and modified peptides are shown in FIG. 2 (SEQ ID NOS: 4-9, 11, 15, 16, 42-72. and 169), and in Tables 1 and 2 below.
  • MOG 70-82 A78 ELLKDAIGAGKVT (analog of MOG 70-82) 8
  • Nucleic acid molecules containing a sequence encoding human MOG or an antigenic fragment thereof may be obtained by reverse transcription of mRNA present in human brain or other CNS tissue, as well as from genomic DNA.
  • Various methods of chemically synthesizing polynucleotides are known, including standard solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al , 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).
  • the nucleic acid molecules of the invention also include RNA which can be transcribed from the DNA prepared as above or synthesized chemically.
  • 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, yeast, or mammalian cells such as Chinese hamster ovary cells (CHO).
  • bacterial cells such as E. coli, yeast, or mammalian cells such as Chinese hamster ovary cells (CHO).
  • CHO Chinese hamster ovary cells
  • 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 Vol. 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 et al. (1987) Gene 54: 113-123), and pYES2 (Invitrogen Co ⁇ . , San Diego
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow et al. (1989) Virol 170:31-39).
  • COS cells Gluzman (1981) Cell 23:175-182
  • pCDM 8 Aruffo et al. (1987) Proc. Natl Acad. Sci.
  • Vector DNA can be introduced into mammalian cells via any known 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 amino terminal amino acids to the expressed target gene. These amino terminal amino acids often are referred to as a reporter group.
  • reporter groups usually serve two pu ⁇ oses: 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 Co ⁇ . , 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 et al. , (1988) Gene 69:301-315) and pET lid (Studier et al. (1990) Meth. Enzymol. 185:60-89). While target gene expression relies on host RNA polymerase transcription from the hybrid T ⁇ -lac fusion promoter in pTrc, expression of target genes inserted into pET l id 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 lacUV 5 promoter.
  • T7 gnl coexpressed viral RNA polymerase
  • E. coli 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 (1990) Meth. Enzymol. 185:119-128). Another strategy is to alter the coding sequence of the gene so that the individual codons for each amino acid are those preferentially utilized in highly expressed E. coli proteins (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can 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 culmre are well known in the art.
  • the protein and peptides of the invention can be purified from cell culmre 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, ultraf iltration, 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 culmre medium or substantially free of chemical precursors or other chemicals. Antieenic Fragments and the "Antigenic" Response
  • Fragments of the MOG 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, produced recombinantly, or prepared through proteolysis of the whole or portion of the protein.
  • 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 (see, e.g. , FIG. 7). 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 pu ⁇ oses, 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 tolerance, anergy, and/or apoptosis are particularly desirable.
  • stimulation i.e., proliferation or lymphokine secretion
  • the isolated protein or preferred antigenic fragments thereof when administered to an individual subject to demyelinating autoimmune disease, are capable of modifying the 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 inflammation or in infiltration of leukocytes and/or arrest of demyelination 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 proliferation 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 counts per minute (CPM) in response to a peptide divided by the control CPM.
  • a stimulation index (S.I.) 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 culmres 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 culmres 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 admimstered 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. 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 different epitope.
  • compositions of the invention contain tandem copies of MOG peptides.
  • 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 45 amino acid residues, and most preferably less than approximately 30 amino acid residues are particularly desirable for ease of peptide synthesis.
  • Peptides of the invention may also be produced recombinantly as described above. Preferable, peptides of 45 amino acids or longer greater are produced recombinantly.
  • Isolated antigenic peptide fragments which have T-cell stimulating activity, and thus comprise at least one T-cell epitope are particularly desirable.
  • An “epitope” is 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 by a T-cell receptor, where the epitope comprises amino acids in the autoantigen essential to receptor recognition. Amino acid sequences which mimic those of the native T-cell epitopes are also within the scope of this invention.
  • T-cell epitopes are believed to be involved in initiation and pe ⁇ etuation 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, induce apoptosis, 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.
  • to "tolerize” is defined as to induce a state of non- responsiveness to subsequent challenge with antigen by any of a variety of mechanisms, including, but not limited to, clonal deletion or apoptosis of antigen- specific T cells, prevention of appropriate presentation of the antigen by relevant antigen presenting cells, or induction of immune cells with suppressive, killer or anti-inflammatory capabilities, having specificity for the antigen itself or for the relevant antigen-specific T cells.
  • the term “angergize” refers to the induction of a state of non-responsiveness to subsequent challenge with an antigen, despite the continued presence of the T cells bearing the relevant antigen-specific receptors.
  • apoptosis refers to the programmed cell death characterized by degradation of the cell nuclear strucmre, mediated by intracellular nucleases, and initiated by any of a variety of extra-cellular signals, including those related to activation of the antigen-specific T cell.
  • 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).
  • exposure to such a protein or peptide may influence 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 can approach the task in many acceptable ways.
  • 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 T cell epitopes.
  • 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 proliferation, 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 strucmre 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.
  • FIG. 2 Thirty-seven namrally occurring 13mers (together with three analogs of the namrally occurring peptides) are shown in FIG. 2 (SEQ ID NOS:4-9, 11, 15, 16, 42-72, and 169); the amino acid length is arbitrarily chosen. These peptides were chosen according to an algorithm that predicts optimal MHC class II binding. In order for the peptide to be able to bind an appropriate T cell receptor, it is necessary that it be able to bind class II MHC proteins. It is possible to remove at least one or two amino acids from the N- and/or C-terminus of each and still retain MHC binding activity. These peptides are designated by amino acid sequence and represent the indicated regions of the mamre amino acid sequence shown as positions 1-218 of FIG.
  • the 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. (1988) EMBO J. 7:93-100).
  • a necessary (but not necessarily sufficient) condition for binding to Class II MHC proteins is the presence of a hydrophobic side chain residue, most preferably a tyrosine, phenylalanine or tryptophan residue, and preferably isoleucine, leucine, valine or methionine residue spaced at a four amino acid distance from a small amino acid residue such as glycine, alanine, serine, threonine or cysteine.
  • All of the MOG peptides shown in FIG. 2 (SEQ ID NOS:4-9, 11, 15, 16, 42-72, and 169) fulfill these minimum conditions.
  • SEQ ID NOS:4-9, 11, 15, 16, 42-72, and 169 fulfill these minimum conditions.
  • FIG. 2 is intended to be a non-limiting, representative example of the algorithm for selection. However, other possible useful antigenic peptides may exist.
  • HLA-DR human MHC protein
  • MHC class II proteins were affinity purified using the monoclonal antibody LB3.1 , coupled to Sepharose CL4B. Class II proteins were eluted with 1 % octyl- ⁇ -D-glucopyranoside (octyl glucoside), 50 mM phosphate pH 11.5 and immediately neutralized using 1 M phosphate pH 6.0. Purified a ⁇ 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. (1994) J. Immunol. 152:2890-2898). Briefly, affinity purified class II proteins (10 nM) 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 (Costar, Cambridge, MA) for 16 hours at 37 °C.
  • the DR-peptide complexes (50 ⁇ l) were transferred, in duplicate, to wells of a 96-well microtiter plate precoated with the monoclonal antibody LB3.1 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, Piscataway, NJ) was added and incubated overnight.
  • 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 standard techniques outlined in Example 2 below.
  • the MOG peptides selected for testing were MOG 1-13 (SEQ ID NO:42), MOG 20-32 (SEQ ID NO:5), MOG 70-82, A78 (analog to MOG 70-82) (SEQ ID NO:8), MOG 88-100,K89,S98 (analog to MOG 88-100) (SEQ ID NO:9), MOG 103-115 (SEQ ID NO:55), MOG 118-130 (SEQ ID NO:56), and MOG 170- 182 (SEQ ID NO: 15).
  • results of one such study of T cell response to human MOG peptides are illustrated in FIG. 6. These results indicate that MOG 1-13 (SEQ ID NO:42) is a T cell epitope-containing peptide. Furthermore, the same data strongly indicates that MOG 103-115 (SEQ ID NO:55) contains at least one epitope. These results do not preclude the possibility that less than the entire 13mer peptide could be a T cell epitope-containing peptide.
  • a second method of identifying peptides that constitute T cell epitope regions of human MOG involves dividing the human MOG protein sequence into a series of 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 of T cell reactivity.
  • a series of peptides was prepared having equal length (e.g. , 20mers or 25mers) and spanning the entire length of the MOG protein sequence while overlapping the previous peptide by 10 or 15 amino acid residues.
  • the peptides were synthesized using standard solid-phase technology and FastMOCTM chemistry.
  • T cell epitopes were tested by two different methods. In the first, 2 x 10 5 T cells were plated into individual microtiter wells stimulated with the N-terminal fragment of recombinant human MOG or with various mixtures of the peptides.
  • the recombinant MOG contained residues 1-121 of the MOG sequence and was purified from the supernatant of insect cells infected with the MOG expression vector construct as detailed in Example 2.
  • the stimulated culmres were supplemented with the cytokines IL2 and IL4 to promote expansion of activated clones.
  • the cells in each individual microtiter culmre were washed and split into four to twelve wells of a new assay plate for a second stimulation or challenge with various controls and test antigens.
  • the proliferative response to the second antigen challenge was assessed by inco ⁇ oration of tritiated thymidine.
  • results from these studies indicate that MS patients have T cell responses to epitopes in the N-terminal half of the MOG protein, and that these epitopes can be encoded in peptides 20 or fewer amino acids in length.
  • the results of one such study are detailed in Example 6A.
  • the presence of an epitope in the first 25 amino acid of the MOG protein is confirmed by the application of this assay to a set of 21 MS patients. This peptide induced responses in 45.5% of the 11 MOG-responding patients in which it was tested.
  • the antigen stimulation and cytokine expansion steps were similar to those employed in the microwell assay above, except that these culmres were all initiated by stimulation with the full length MOG protein purified from human brain tissue as detailed in Example 7. Because the starting cell number is large, the cells from a single culmre can be washed after approximately two weeks of culmre and split into over 100 wells of a microtiter assay plate for a second stimulation or challenge with various controls and test antigens. The proliferative response to the second antigen challenge was assessed by inco ⁇ oration of tritiated thymidine, as above.
  • peptides 1-20 and 41-65 are confirmed as major targets of the T cell response to MOG.
  • the peptides flanking these two sequences (11-35, 31-55, 51- 75, 61-85, and 71-95) also induce significant reactivity, which may be directed at the same epitopes contained in 1-20 and 41-65, or may be due to additional epitopes that either overlap with or are completely separate from the epitopes contained in 1-20 and 41-65.
  • the strucmre of the protein or peptides of the invention can be modified for such pu ⁇ oses 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 pu ⁇ ose.
  • One embodiment of the present invention feamres a peptide which comprises at least one T-cell epitope of the protein and includes the regions of the peptides as shown in FIGS. 2, 5a, 5b, and/or 7 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, or peptides including such T cell epitopes are typically at least seven 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 linking 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.
  • Some preferred peptides of the invention 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 FIG. 2 (SEQ ID NOS:4-9, 11, 15, 16, 42-72, and 169), FIG. 5a, FIG. 5b, and FIG. 7.
  • a modified protein or peptide of the invention maintains its 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 eliminate 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 inco ⁇ oration may enhance or diminish T-cell reactivity, but does not eliminate 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 eliminate 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 inco ⁇ oration may enhance or diminish, but not eliminate 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 cyclization of the peptide.
  • the protein or peptides of the invention can be modified to inco ⁇ orate one or more polymo ⁇ hisms 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 substimted 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 can be added during chemical synthesis of a protein or peptide of the invention.
  • 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 (1986) pp. 155-194); 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) Int. Arch. Allergy and Appl. Immunol. 41: 199-215).
  • hexahistidine can be added via known recombinant or synthetic methods to a protein or peptide for purification by immobilized metal ion affinity chromatography (Hochuli et al. (1988) Bio/Technol. 6: 1321-1325).
  • immobilized metal ion affinity chromatography Hochuli et al. (1988) Bio/Technol. 6: 1321-1325.
  • known methods include, e.g. , cloning the nucleic acid sequences encoding the moiety to be added or synthesizing the moiety directly onto the peptide during the synthesis of the peptide.
  • specific endoprotease cleavage sites can be introduced via known recombinant or synthetic methods between the sequences of the reporter group and the protein or peptide.
  • functional groups e.g. , charged amino acids such as Glu, Asp, or Arg
  • Hydrophobic regions may be characterized by the presence of He, Leu, Val, Phe, and sometimes Tyr and T ⁇ residues.
  • canonical protease sensitive sites can be engineered between regions, each comprising at least one T-cell epitope via known 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 strucmre 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 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 demyelinating autoimmune responses.
  • the present invention provides therapeutic compositions comprising isolated human MOG (SEQ ID NOS: l and 2), or antigenic peptide fragments thereof (SEQ ID NOS: l and 2), or antigenic peptide fragments thereof (SEQ ID NOS: l and 2), or antigenic peptide fragments thereof (SEQ ID NOS: l and 2), or antigenic peptide fragments thereof (SEQ ID NOS: l and 2), or antigenic peptide fragments thereof (SEQ ID NOS: l and 2), or antigenic peptide fragments thereof (SEQ ID NOS: l and 2), or antigenic peptide fragments thereof (SEQ ID NOS: l and 2), or antigenic peptide fragments thereof (SEQ ID NOS: l and 2), or antigenic peptide fragments thereof (SEQ ID NOS: l and 2), or antigenic peptid
  • compositions Containing MOG Peptides Containing MOG Peptides
  • compositions including at least one novel MOG peptide of the invention having SEQ ID NOS:96-110 and 146-164).
  • such compositions include at least two MOG peptides, at least one of which is a novel peptide of the present invention.
  • At least one other MOG peptide having an amino acid sequence different than a novel MOG peptide of the invention may also be included.
  • Such other MOG peptides may be any MOG peptide known in the art, for example having SEQ ID NOS:2, 42, 55, 73-80, 82, 83, 111-145, and 165- 203 (see, e.g., U.S. Ser. no.
  • composition of the invention is a therapeutic composition containing at least one MOG peptide or one MBP peptide and a pharmaceutically acceptable carrier, diluent, or excipient.
  • a pharmaceutically acceptable carrier diluent, or excipient.
  • other compositions of the invention also contain mixtures of at least two peptides (e.g. , a physical mixmre of at least two identical or different MOG peptides, at least two identical or different MBP peptides, or at least one MOG peptide of the invention and at least one human MBP peptide or other peptides).
  • Therapeutic compositions may contain MOG peptides or MBP peptides containing at least one T-cell epitope of human MOG or human MBP, respectively. Still further, 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. A therapeutically effective amount of one or more of such compositions can be administered simultaneously or sequentially.
  • Some preferred therapeutic compositions and preferred combinations of MOG peptides which can be administered simultaneously or sequentially comprise peptides comprising amino acid sequences set forth in Table 2 and having SEQ ID NOS:96-110, 146-164, and 220-223.
  • compositions also include the MOG peptides shown in FIG. 2 (SEQ ID NOS:4-9, 11, 15, 16, 42-72, and 169), 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 MOG 103-115 (SEQ ID NO. 55), as set forth in Table 1.
  • MOG peptides shown in FIG. 2 SEQ ID NOS:4-9, 11, 15, 16, 42-72, and 169
  • 5a SEQ ID NOS. 73-80, 82,83
  • 5b the first 121 amino acids of human MOG protein SEQ ID NO. 2
  • MOG 1-13 SEQ ID NO. 42
  • MOG 103-115 SEQ ID NO. 55
  • compositions include at least one novel MOG peptide from Table 2 as well as at least one MOG peptide having SEQ ID NOS:96-110 and 146-164 with at least one of any other known MOG peptide (having, e.g., SEQ ID NOS:42, 55, 73-80, 82, 83, 111-145, and 165-203).
  • MOG peptides having, e.g., SEQ ID NOS:42, 55, 73-80, 82, 83, 111-145, and 165-203.
  • MBP peptides Preferably, the MBP-containing formulations and compositions include at least one of the MBP peptides listed below in Table 3. More preferably, these MBP-containing compositions include at least one of MBP-1 to MBP-5 listed below. Most preferably, MBP- 1.1 , MBP-2.1, and/or MBP-4.
  • MBP-3 (111-130) LSRFS WGAEGQRPGFGYGGR
  • MB-S (141-165) FKGVDAQGTLSKIFKLGGRDSRSGS Highly purified and isolated peptides produced as discussed above may be formulated into therapeutic compositions of the invention suitable for human therapy.
  • a therapeutic composition of the invention is to be administered by injection (e.g. subcutaneous injection, intravenous injection)
  • the highly purified peptide be soluble in an aqueous solution at a pharmaceutically acceptable pH (i.e. pH range of about 4-9) such that the composition is fluid and easy syringability exists.
  • the composition also preferably includes a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all excipients, solvents, dispersion media, coatings, antibacterial and antifungal agents, toxicity agents, buffering agents, abso ⁇ tion delaying or enhancing agents, surfactants, and micelle forming agents, lipids, liposomes, and liquid complex forming agents, stabilizing agents, and the like.
  • the use of such media and agents for pharmaceutically active substance is 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 inco ⁇ orated into the compositions.
  • compositions of the invention suitable for injectable use are preferably sterile aqueous solutions prepared by inco ⁇ orating active compound (i.e., one or more highly purified and isolated peptides as described above) in the required amount in an appropriate vehicle with one or a combination of ingredients enumerated above and below, as required, followed by filtered sterilization.
  • active compound i.e., one or more highly purified and isolated peptides as described above
  • Preferred pharmaceutically acceptable carriers include at least one excipient such as sterile water, sodium phosphate, mannitol, sorbitol, or sodium chloride or any combination thereof.
  • compositions which may be suitable include solvents or dispersion medium contaimng, for example, water, ethanol, polyol (for example glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • solvents or dispersion medium contaimng for example, water, ethanol, polyol (for example glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained for example by the use of coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosol and the like.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by including in the composition, an
  • Preferable therapeutic compositions of the invention should be sterile, stable under conditions of manufacmre, storage, distribution and use and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • a preferred means for manufacturing a therapeutic composition which maintains the integrity of the composition is to prepare the formulation of peptide and pharmaceutically acceptable carrier(s) such that the composition may be in the form of a lyophilized powder which is reconstituted in a pharmaceutically acceptable carrier, such as sterile water, just prior to use.
  • a pharmaceutically acceptable carrier such as sterile water
  • FIG. 10A and FIG. 10B show the PI for the additional MOG peptides synthesized to further define regions of high reactivity. These peptides were tested in the most recently assayed thirty -eight of the sixty-nine patients.
  • FIG. 10A shows that peptide 41-60 elicits more T cell reactivity than peptide 41-65 and FIG. 10B shows that peptide 181-195 elicits more reactivity than peptide 171-195. Based on the previously described results, most T cell reactivity is directed toward MOG peptides 1-20, 41-60, and 181-195.
  • a therapeutic composition of the invention may comprise more than one isolated peptide.
  • a therapeutic composition comprising a multipeptide formulation suitable for pharmaceutical administration to humans may be desirable for administration of several active peptides.
  • the multipeptide formulation includes at least two or more isolated peptides having a defined amino acid sequence and is capable of down regulating an antigen specific immune response. Any of the compositions described earlier which comprise at least two peptides may be suitable as a multipeptide formulation. Special considerations when preparing a multipeptide formulation include maintaining the solubility, and stability of all peptides in the formulation in an aqueous solution at a physiologically acceptable pH.
  • suitable excipients include sterile water, sodium phosphate, mannitol or both sodium phosphate and mannitol.
  • An additional consideration in a multipeptide formulation is the prevention of dimerization of the peptides if necessary.
  • Administration of the therapeutic compositions as described above to an individual, in a non-immunogenic form can be carried out using known procedures at dosages and for periods of time effective to cause down regulation of the MOG antigen specific immune response of the individual being treated for MS.
  • Down-regulation of an antigen specific immune response to an antigen associated with a disease condition in humans may be determined clinically whenever possible, or may be determined subjectively (i.e. , the patient feels as if some or all of the symptoms related to the disease condition being treated have been alleviated).
  • Effective amounts of the therapeutic compositions of the invention are in the range of 1 x IO 5 to 2.5 mg/kg of body weight per dosage.
  • dosages may vary according to factors such as the degree of sensitivity of the individual to the antigen, the age, sex, and weight of the individual, and the ability of peptide to cause down regulation of the antigen specific immune response in the individual.
  • a therapeutic composition of the invention may be administered in non-immunogenic form, in a convenient manner such as by injection
  • compositions for administration simultaneously or sequentially may comprise only one peptide or may comprise a multipeptide formulation as described above.
  • a peptide composition may be co-administered with enzyme inhibitors or in liposomes.
  • 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 peptide When a peptide is suitably protected, as described above, the peptide may be orally admimstered, for example, with an inert diluent or an assimilable edible carrier.
  • the peptide composition and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or inco ⁇ orated directly into the individual's diet.
  • the active compound For oral therapeutic administration, the active compound may be inco ⁇ orated 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 should contain at least 1 % by weight of active compound.
  • compositions and preparations may, of course, be varied and may conveniently be between about 5 to 80% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • Preferred 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.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder such as gum gragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin or a flavoring agent such as peppermint, oil of wintergreen, or
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservative, a dye and flavoring such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound may be inco ⁇ orated into sustained-release preparations and formulations.
  • compositions of the invention preferably about 1 ⁇ g - 3 mg and more preferably from about 20 ⁇ g to 1.5 mg, and even more preferably about 50 ⁇ g to 750 ⁇ g, and even more preferably about 75 ⁇ g to about 750 ⁇ g, of each active component (peptide) per dosage unit may be administered.
  • doses as high as 1500 ⁇ g or more may be used. It is especially advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage.
  • Unit dosage form refers to physically discrete units suited as unitary dosages for human subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the desired pharmaceutical carrier.
  • the specification for the novel unit dosage 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 achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of human subjects. Dosage regimen may be adjusted to provide the optimum prophylactic or therapeutic response. For example, several divided doses may be admimstered over the course of days, weeks, months or years, or the dose may be proportionally increased or reduced with each subsequent injection as indicated by the exigencies of the therapeutic simation.
  • subcutaneous injections of therapeutic compositions are given once a day during an acute phase and once every other day during remission for the lifetime of the individual suffering from the disease.
  • Alternatives would include, weekly, monthly or other periodic injections.
  • the dosage may remain constant for each injection or may increase or decrease with each subsequent injection.
  • a continual, lifetime treatment program may be most desirable.
  • a booster injection may be admimstered at intervals of about three months to about one year after an initial treatment period and may involve only a single injection or may involve another series of injections similar to that of the initial treatment.
  • EAE allergic encephalomyelitis
  • EAE induced by injecting MBP into susceptible strains of rats or mice has been extensively studied: a severe, progressive paralysis of tail and limbs develops, which in mouse strains such as (PL/J x SJL)F1 can undergo remission and relapse. It has previously been shown that the injection in aqueous solution of a T cell epitope-containing peptide derived from MBP can prevent or treat EAE induced by the entire MBP molecule. It has here been discovered that the injection of MOG, or peptides derived from MOG, as well as the injection of MBP + MOG or MBP + MOG derived peptides, can also induce EAE (Examples 3 and 4).
  • mice induced with EAE in this manner are useful as an animal model for screening potential therapies for the treatment of MS.
  • One method for identifying therapeutic compositions useful for the treatment of MS includes first administering human MOG to mice in immunogenic form to cause induction of EAE in the mice; then treating the 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 the onset of symptoms of EAE in said mice; and then determining if said therapeutic composition prevents the onset or progression of the symptoms of EAE in the mice.
  • MHC Class II glycoproteins or the portion of these glycoproteins capable of binding the peptide epitopes, complexed either covalently or noncovalently with specific peptide epitopes derived from a known autoantigen.
  • this approach suffers from the requirement of purification of MHC Class II molecules of every haplotype known to present the peptide epitopes in question. It is now well established that most peptide epitopes are not restricted to a very small number of Class II haplotypes (reviewed in Engelhard (1994) Ann. Rev.
  • the present invention overcomes these problems through the administration of high doses of peptide(s) of the invention having T cell epitopes and allowing for the namral presentation of these epitopes by endogenous Class II molecules. Such a regimen results in anergy of T cell responses, and hence tolerization.
  • MOG peptides of the instant invention can be used in conjugates as disclosed, for example, in U.S. Patent 5,130,297 (Sharma et al.) where therapeutic agents are prepared using the formula X— MHC—peptide or MHC ⁇ peptide ⁇ X, wherein X represents a functional moiety selected from a toxin and a labeling group; MHC is an effective portion of the MHC glycoprotein, the glycoprotein dissociated from the cell surface on which it normally resides; and "peptide" represents any of the MOG peptides listed herein.
  • MOG peptides include MOG 31-55 (SEQ ID NO:96), MOG 41-65 (SEQ ID NO:97), MOG 51-75 (SEQ ID NO:98), MOG 61-85 (SEQ ID NO:99), MOG 71-95 (SEQ ID NO: 100), MOG 101-125 (SEQ ID NO: 101), MOG 111-135 SEQ ID NO: 102), MOG 131-155 (SEQ ID NO: 103), MOG 151-175 (SEQ ID NO: 105), MOG 161- 185 (SEQ ID NO: 106), MOG 171-195 (SEQ ID NO: 107), MOG 191-215 (SEQ ID NO: 108), MOG 1-25 (SEQ ID NO: 109), and MOG 21-45 (SEQ ID NO: 110), MOG 1-22 (SEQ ID NO: 146), MOG 1-25(24C ⁇ 24S) (SEQ ID NO: 147), MOG 11-35 (SEQ ID NO: 148), MOG 31-
  • peptides include any known MOG peptides (see, e.g., U.S. Ser. No. 08/300,811; Bernard et al. , WO 95/07096; Linington et al. (1993) Eur. J. Immuno. 23: 1364-1372; Amor et al. (1994) 153:4349-4356; Mendel et al. (1995) Eur. J. Immunol. 25: 1951-1959; and Johns et al. (1995) J. Immunol. 154:5536-5541).
  • MOG peptides see, e.g., U.S. Ser. No. 08/300,811; Bernard et al. , WO 95/07096; Linington et al. (1993) Eur. J. Immuno. 23: 1364-1372; Amor et al. (1994) 153:4349-4356; Mendel et al. (1995) Eur. J. Immun
  • TCR T cell receptor
  • 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 immunogenicity 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 ELISA or other immunoassay can be used with 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
  • immortalizing 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
  • nucleotide sequence shown in FIG. 1 (SEQ ID NO: l) and of its complement (SEQ ID NO: 2) permit the design of various oligonucleotides 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, oligomers based on the nucleotide sequence disclosed in FIG. 1 herein can be used in standard assay methods for detecting the MOG- encoding DNA or RNA.
  • oligomers "based on” the sequence disclosed in FIG. 1 is meant oligomers 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 oligomers designed on the basis of the disclosed sequence which effect triple helix formation with the 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.
  • SEQ ID NO: l 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 that disclosed in FIG. 1 (SEQ ID NO: l) through art-known design parameters.
  • the oligomers having nucleotide sequences based on the nucleotide sequence shown in FIG. 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 substimted 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 oligomers claimed herein.
  • the following examples illustrate the preferred modes of making and practicing the present invention, but are not meant to limit the scope of the invention since alternative methods may be utilized to obtain similar results.
  • a human cDNA library was subjected to the polymerase chain reaction (PCR) using internal primers designed from the published rat MOG coding sequence of Gardinier et al. (supra).
  • the combination of primers 166-183 (SEQ ID NO:34) and 538-555 (SEQ ID NO: 35) was successful in effecting the amplification of a fragment of the approximately 400 bp expected size from a human brain cDNA library.
  • the sequence of these primers was:
  • CAGCGGCCGCACGGAGTTTTCCTCTCAG (SEQ ID NO: 35).
  • the 400 bp PCR product was cloned into expression vector pVL1393 by digesting pVL1393 (Pharmingen CA) with EcoRI and NotI, digesting the amplified product with the same enzymes and ligating the resulting fragments. The insert was verified by digesting several clones derived from the ligated plasmids with EcoRI and NotI 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 the 738 bp rat open reading frame.
  • Two primers were designed from the 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 the above-mentioned 3' rat primer to amplify the 3' missing end 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. This 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 amplified and had a titer of 8xl0 10 pfu/ml was screened following the protocol described by the manufacturer.
  • the library was plated onto 12 large plates at 30,000 plaques/plate and the plaques were lifted onto nitrocellulose filters (2 replica 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 the phage from the agar. The tube was then centrifuged and the supernatant transferred.
  • the DNA was amplified from each individual pool using either a ⁇ gtlO forward primer with an SstH site: 5'-CTTTTGAGCAAGTTCAGCCTGGTTAAG-3' (SEQ ID NO:38)
  • the last two primers include a BamHI site (underlined in the sequences) namrally 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 the 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 acmally resulted in the desired fragment depends on the orientation of the cDNAs cloned into ⁇ gtlO.
  • the size of the 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 the 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 complete DNA sequence recovered (SEQ ID NO: l) and deduced amino acid sequence (SEQ ID NO:2) are shown in FIG. 1.
  • 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 mamre protein contains 218 amino acids, numbered 1-218 in FIG. 1 (SEQ ID NO: 2).
  • the mamre 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 also cloned into a pET H6 vector Novagen, Madison, WI for expression in E. coli.
  • pET.H ⁇ contains a sequence encoding six histidines which allows for purification of any recombinant protein over a Ni2 + column.
  • 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 amplified by PCR using the following oligonucleotides:
  • 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 culmre 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 titer viral stock.
  • Sf-9 cells were then infected with 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, diaiyzed against 5% propionic acid and H 2 O and subsequently lyophilized. The protein concentration was estimated by BCA. The purified MOG protein was visualized on a 12.5% polyacrylamide gel stained with Coomassie blue.
  • the pET.H ⁇ vector containing the truncated human MOG cDNA was introduced into BL21(DE3) cells (Novagen, Madison, WI.) by transformation. Several colonies were grown together in 2 YT medium to an OD of 1.0. The bacteria were then induced overnight with 1 mM IPTG. Cells were harvested and lysed with 6 M Guanidine/100 mM Tris-HCl, pH 8.0 at room temperamre overnight. The lysate was centrifuged at 20.000 ⁇ m for 30 minutes and the resulting supernatant applied to a NiNTA agarose column (Quiagen, Chatsworth, CA).
  • the protein was eluted with 6 M Guanidine/100 mM sodium phosphate, pH 4.5, diaiyzed first against 5% propionic acid, then against H 2 O and subsequently lyophilized.
  • the protein concentration was estimated by BCA.
  • the purified MOG protein was visualized on a 12.5% polyacrylamide gel stained with Coomassie blue.
  • mice Two groups of (PL/J x SJL)F1 mice were injected with 10 ⁇ g and 50 ⁇ g respectively N-terminal fragment of recombinant human MOG expressed in insect (-TM, recombinant, Sf-9) which was prepared according to Example 2A 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. Emulsions were prepared using 1 volume of PBS or water containing the stated quantity of MOG 1-121, as described above combined with one volume of CFA (Life Technologies, Grand Island, NY) containing 400 ⁇ g H37Ra (Difco Laboratories, Detroit, MI) for a total infection volume of 100 ul per mouse. At the same time as the MOG 1- 121 injection, 200 ng pertussis toxin (JRH Biosciences, Lenexa, KS or List Biomedical Labs. , Campbell, CA) was also injected intravenously. The 200 ng pertussis toxin i.v. injection was repeated 2 days later.
  • mice were observed for signs of paralysis and scored daily as an indicator that EAE has been induced. Scoring was based on clinical signs according to the following scale: 1, tail paralysis; 2, partial hind limb paralysis; 3, complete hind limb paralysis; 4, forelimb paralysis; 5, moribund or dead.
  • mice The onset of symptoms began as early as 14 days for some mice. The mice were observed for 31 days, at which time the mice were sacrificed, the brains and spinal cords were harvested, and histological smdies were undertaken to verify the clinical observations. Sixty percent (60%) of mice immunized with 50 ⁇ g of MOG
  • mice immunized with 10 ⁇ g of recombinant MOG (-TM, recombinant, Sf-9) and eighty percent (80%) of mice immunized with 10 ⁇ g of recombinant MOG (-TM, recombinant, Sf-9) exhibited symptoms of EAE.
  • mice obtained from Jackson Lab, Bar Harbor, ME
  • PL/J x SJL mice obtained from Jackson Lab, Bar Harbor, ME
  • mice were immunized at the base of the tail with 100 ⁇ l of a 1: 1 emulsion of 400 ⁇ g recombinant MOG in PBS with Complete Freund's Adjuvant (CFA).
  • CFA Complete Freund's Adjuvant
  • Triplicate wells containing 4 x IO 5 cells were incubated with 0-100 ⁇ g/ml of a set of MOG-derived 20mers spanning the length of recombinant MOG (peptides 1-20 (SEQ ID NO:73), 11-30 (SEQ ID NO:74), 21-40 (SEQ ID NO:75), 31-50 (SEQ ID NO:76), 41-60 (SEQ ID NO:77), 51-70 (SEQ ID NO:78), 61-80 (SEQ ID NO:79), 71-90 (SEQ ID NO:80), 81-100 (SEQ ID NO: 151), 91-110 (SEQ ID NO:82), 101-120 (SEQ ID NO:83), and 111-130 (SEQ ID NO: 154)).
  • culmres were maintained for 24 hours, after which 50 ⁇ l of culmre supernatant was transferred to new wells containing 5 x 10 3 CTLL2.3 cells, an IL-2 dependent, IL-4 independent cell line (Dr. David Raulet, UCal Berkeley).
  • CTLL2.3 culmres were maintained for 48 hours.
  • One ⁇ Ci of 3 H- thymidine was added for the final 24 hr of the culmre period and inco ⁇ oration into DNA was assessed as described above for proliferative responses. Standard curves for known levels of recombinant IL-2 were run in parallel.
  • Peptides 1-20 (SEQ ID NO:73), 11-30 (SEQ ID NO;74), 31-50 (SEQ ID NO:76, and 41-60 (SEQ ID NO:77) stimulated proliferative responses at least twice the background (i.e. , had a Stimulation Index [SI] > 2) at two or more of the tested peptide concentrations. Responses to the positive controls recombinant MOG and PPD were very strong. A similar pattern of peptide effectiveness was observed for IL-2 secretion. Peptides 1-20, 11-30, 31-50 and 41-60 stimulated T cell responses with SI > 2 at at least two concentrations of tested peptide.
  • SI Stimulation Index
  • Peptide 11-30 (SEQ ID NO: 74) stimulated a T cell response with an SI of approximately 2.
  • SI approximately 2
  • each region may encompass at least one epitope. These regions are found at the N- terminus of the molecule at amino acids 1-30 and at the C-terminal end at amino acids 81-110.
  • mice Two groups of SJL mice were immunized at the base of the tail with 200 ⁇ l of a 1 : 1 emulsion of 200 ⁇ g recombinant MOG in PBS with CFA, supplemented with 400 ⁇ g H37Ra Mycobacterium tuberculosis (Difco, Detroit, MI). 200-400 ng Pertussis Toxin was also injected i.v. on day 0 and day 2.
  • One group of mice was injected i.v. with 250 nmoles MOG 91-110 (SEQ ID NO:82) in aqueous solution and the other group was injected with PBS on days 7, 9, 12 and 15 following immunization. Mice were scored daily to assess their degree of paralysis, using the scale described above.
  • mice injected with PBS developed severe EAE around day 10 which remained at a high level for 30 days (mean clinical score of 3-4).
  • mice injected with MOG 91-110 did not develop as severe a disease (mean clinical score approximately 1) and the disease remitted to a mean clinical score of almost zero.
  • MOG 91-110 decreased disease incidence (60% vs. 100%) and decreased the mortality which accompanied the severe disease (27% vs. 50%).
  • the peptide MOG 91-110 reduced the severity of EAE induced by recombinant MOG in SJL mice.
  • EAE was induced in (PL/J x SJL)F1 mice by injecting 100 ⁇ l of an emulsion contaimng 400 ⁇ g recombinant MOG in CFA supplemented with Mycobacteria and pertussis toxin, as described above.
  • groups of 10 mice were injected with 250 nmoles of the peptide MOG 41-60 (SEQ ID NO: 77) in aqueous solution or with PBS.
  • Mice injected with MOG 41-60 had delayed disease onset, reduced mortality, and lower average daily clinical scores after treatment compared to PBS-injected mice.
  • the peptide MOG 41-60 was effective at treating EAE induced by MOG.
  • mice were immunized with a mixmre of 100 ⁇ g recombinant MOG and 75 ⁇ g guinea pig MBP emulsified together in CFA, as described above.
  • MBP was prepared from guinea pig spinal cords by a modification of the method of Smith (J. Neurochem. (1969) 16:83). Briefly, MBP was extracted from isolated myelin membranes using chloroform and methanol, precipitated with potassium citrate, acid extracted and lyophilized. SDS-PAGE analysis of this material showed a major band at 18.5 kD.
  • mice injected with MBP and MOG were scored daily based on clinical signs according to the scale described above. Mice were injected i.v. with 125- 500 nmoles of MBP Acl-11 [4Y] (Sequence Ac-ASQYRPSQRSK (SEQ ID NO: 1)
  • mice received PBS. All mice were injected 7 times every other day starting around day 7 following immunization.
  • Peptides were synthesized using either an Applied Biosystems peptide synthesizer (Foster City, CA) or an Advanced Chemtech robotics system (Louisville, KY) utilizing FastMOCTM chemistry with commercially available
  • 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 million lymphocytes were seeded into 96 wells of a microtiter dish at 2 x 10 5 per well in RPMI culmre media supplemented with human AB serum. A mixmre of selected MOG peptides listed in Table 1 was added at a final concentration of 50 ⁇ M for each peptide. The MOG peptides selected were both namrally occurring peptides and analogs of namrally occurring peptides.
  • Culmres were incubated in a humidified CO 2 at 37 °C for twelve days, and were intermittently supplemented with human IL-2 (20 U/ml) and IL-4 (5 U/ml) (Collaborative Biomedical Products, Bedford, MA). A sample from each culmre well was removed, washed to remove previously added peptide, and reseeded into four wells of a fresh microtiter dish (two wells with the peptide mixmre and two wells without the peptide mixmre for each sample). Autologous irradiated cryopreserved lymphocytes were added as antigen presenting cells. After further 3 days of incubation, 3 H-thymidine inco ⁇ oration was measured.
  • Positive microtiter lines were scored if the mean inco ⁇ oration in the peptide wells was greater than or equal to 1.5 -fold higher than the wells without peptide. Positive microtiter lines were expanded with IL-2 and IL-4, and then reassayed the following week by the same methods with the individual peptides rather than the peptide mix.
  • FIG. 6 shows the results of the assays performed.
  • Peptides (20 amino acids in length) were synthesized using either an Applied Biosystems peptide synthesizer or Advanced Chemtech robotics system utilizing FastMOCTM chemistry with commercially available Wang resins, and Fmoc-protected amino acids as described previously by Hill et al. (J. Immunol. (1994) 152:2890-2898). These peptides are 20 amino acid peptides of human MOG overlapping by 10 amino acids and are shown in FIG. 5a.
  • the T cell response of a human patient suffering from MS was tested with the group of MOG 20mer peptides shown in FIG. 5a and a 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 3.
  • One or more of the MOG peptides tested are suspected of containing at least one T cell epitope for the autoantigen responsible for MS.
  • the PBLs of the patient were cultured in microtiter wells according to the protocol above, except that both recombinant MOG 1-121 (48 of 96 wells) and a mixmre of 10 MOG fragments (20mer peptides shown in FIG. 5a) (48 of 96 wells) were used separately to initiate the culmres. A sample from each culmre well was removed, washed to remove previously added peptide, and reseeded into six wells of a fresh microtiter dish.
  • the T cell responses of twenty-one MS patients were tested with eight MOG 25mer peptides derived from the N-terminal portion of MOG.
  • the peripheral blood lymphocytes (PBL) of MS patients were prepared and cultured according to the protocol above, except that culmres were initiated with 50 ⁇ g/ml of the N-terminal fragment of recombinant human MOG 1-121 (SEQ ID NO:2), prepared according to the protocol in Example 2. After the expansion of the microtiter culmres with IL-2 and IL-4, the entire contents of each well was harvested, washed to remove residual MOG, and reseeded into twelve wells of a fresh microtiter dish.
  • Peptide Test Group I included 1-25 (SEQ ID NO: 109), 11-35 (SEQ ID NO: 148), 21-45 (SEQ ID NO: 110), and 31-55 (SEQ ID NO:96).
  • Peptide Test Group II included 41-65 (SEQ ID NO:97), 51-75 (SEQ ID NO:98), 61-85 (SEQ ID NO:99), and 71-95 (SEQ ID NO: 100).
  • the red blood cell and yellow histone portion of the resulting pellet was carefully removed with a spatula and the remaining pellet was homogenised in the blender at lowest speed in about 2x original volume with cold 30% sucrose containing EDTA and protease inhibitors as above.
  • the homogenate was poured into polycarbonate ultracentrifuge bes and an overlay of 10.5% sucrose was added.
  • the myelin membranes were collected at the interface after centrifugation (68,000 x g for 50 min. at 4°C).
  • the myelin was washed once with deionized water (DDW) and pelleted at 68,000 x g for 60 min. at 4°C.
  • the resulting pellet was resuspended in a small volume of DDW and stored in aliquots at -80°C for further purification.
  • the pellet was resuspended into 50 ml CHCl 3 :MeOH (2: 1 v/v) by homogenization with a motor driven Potter-Elvehjem homogenizer. After centrifugation in Teflon mbes (14,000 x g for 20 min.), the solvent was decanted and the pellet was dried in the hood as above.
  • each of the pellets in the Teflon mbes was resuspended in 25 ml LDAO buffer (50 mM Hepes, pH 7.6, 0.5 mM DTT, 90 mM NaCl, 1.2 % lauryldimethyl amine oxide, 5 mM EDTA, 20 ⁇ g/ml leupeptin, 10 ⁇ g/ml soybean trypsin inhibitor and 1 mM phenyl-methylsulfonyl fluoride) and first tip sonicated for 3 x 10 sec. each at 4°C. Then the whole mixmre was brought to 0.5 liter with LDAO buffer and sonicated in a bath sonicator for 15-30 min.
  • LDAO buffer 50 mM Hepes, pH 7.6, 0.5 mM DTT, 90 mM NaCl, 1.2 % lauryldimethyl amine oxide, 5 mM EDTA, 20 ⁇ g/ml leupeptin, 10 ⁇
  • Anti-MOG specific monoclonal antibody, 818C5 (Abo et al. (1993) Biochem. Mol. Biol. Int. 30:945-958) was used as the major chromatographic step in the purification of MOG.
  • Purified mAb 8-18C5 was diaiyzed against Sulfolink Sample Preparation Buffer (0.1 M sodium phosphate, 5 mM EDTA, pH 6.0 at 4°C). The antibodies were reduced with 2-mercaptoethanolamine and desalted with SWIFT Desalting columns (Pierce, Rockford, IL) according to the manufacturer's specifications.
  • the IgG was coupled to the Sulfolink Gel (Pierce, Rockford, IL) at 5 mg IgG/ml resin.
  • the slurry was mixed for 15 min. at room temperamre and then incubated, without mixing, for at least 30 min., also at room temperamre. After washing the gel twice with 50 mM Tris, 5 mM EDTA, pH 8.5, the non-specific binding sites on the gel were blocked by cysteine-HCl, followed by low and high pH washes according to the manufacturer's procedures (Pierce). The resin was then equlibrated wim 0.2 M sodium borate, pH 9.0 and 18 M dimethylpimelimidate was added to the mix, which was rocked for 30 min. at room temperamre to crosslink the antibodies. The reaction was stopped by incubating the gel in 0.2 M ethanolamine, pH 8.0 for one hour.
  • MOG protein Most of the MOG protein was recovered in the precipitate. The protein in the supernatant was concentrated by lyophilization. Both the precipitate and the lyophilized material were combined together, washed a couple of times with DDW and stored as a suspension in DDW. Total proteins were determined by amino acid analysis and purity was determined by N-terminal sequencing.
  • peripheral blood mononuclear cells were isolated from the blood of 46 MS patients recruited at the Rocky Mountain MS Center (Denver, CO) and Fairview Medical Center (Minneapolis, MN). These patients were selected based on the following criteria. Newly diagnosed, relapsing-remitting, and chronic-progressive MS patients were included in the study. MS patients were included only if they had disease activity within the previous two years. Patients were excluded if they had received steroids or other immunosuppressive medications within the four months prior to the study.
  • MOG-reactive T cell lines were initiated from each patient by culturing 10 x 10° mononuclear cells in 2.5 ml of complete medium. On average, three culmres were set up for each subject, which were pooled before analysis. To ensure detection of namrally processed epitopes, the culmres were stimulated with intact MOG (6 ⁇ g/ml, >90% pure) isolated from human brain according to the protocol described in Example 7, and were supplemented after six days of culmre with IL-2 (Collaborative Biomedical Products, Becton Dickinson Labware,
  • T cell lines were assayed on day 14-18 for proliferative responses to titrating doses of MOG, an overlapping set of MOG peptides, or medium alone as a control. Twenty thousand T cells from each T cell line were challenged in triplicate in a volume of 200 ⁇ l with titrations of each MOG peptide or MOG protein in the presence of 5 x IO 4 gamma- irradiated (3500 Rads) peripheral blood mononuclear cells as antigen presenting cells (APC).
  • APC antigen presenting cells
  • each microwell received 1 ⁇ Curie titriated thymidine (ICN Radiochem Irvine, CA) for an additional 16-24 hours. Thymidine inco ⁇ oration was then measured by liquid scintillation counting.
  • the response to MOG or each MOG peptide is expressed as stimulation index (SI), which is the ratio of the tritium counts per minute (CPM) inco ⁇ orated in the presence of antigen to the tritium CPM inco ⁇ orated in medium alone as a negative control.
  • SI stimulation index
  • Overlapping peptides spanning the length of the MOG sequence were synthesized as described in Example 5, purified, and resuspended at 1 mM in PBS pH 7.2.
  • One representative set of peptides used for epitope mapping is shown in FIG. 7.
  • Eighteen peptides, 20 or 25 amino acids in length with overlaps of 5, 10 or 15 amino acids were tested for the presence of T cell epitopes by the challenge of MOG-reactive T cell lines described above. Peripheral blood T cell reactivity to MOG, and to peptides spanning the MOG sequence, was evaluated in 46 MS patients.
  • FIG. 8 A shows the rank received by each peptide.
  • FIG. 8 B shows the mean percentage of T cell reactivity accounted for by each of the peptides in this population of 31 MS patients.
  • peripheral blood mononuclear cells were isolated from an additional 49 MS patients recruited at the same medical centers and selected by the same criteria as mentioned in Example 8. These additional T cell lines were established and assayed according to Example 8. Additional peptides 20-25 amino acids in length shown below in Table 4 were tested. These peptides completed an overlapping set of 25 amino acid long peptides which span the entire MOG molecule, overlapping by 15 amino acids. Also, a few peptides 15-22 amino acid long were included to further define the major areas of T cell reactivity identified in Example 8.
  • MOG sequence was evaluated in a total of 95 MS patients, 46 patients from
  • Example 8 Sixty-nine of the 95 culmres (73%) responded to MOG protein in secondary proliferation assays, and were used to assess reactivity to MOG peptides as described in Example 8.
  • the criteria used to define a positive response included an SI of 2 or greater, difference between antigen wells and medium wells to be 500 CPM or greater and standard deviation of antigen wells less than or equal to the difference in CPM between antigen wells and medium wells.
  • Reactivity of the T cell lines to MOG peptides was evalulated by a method referred to as positivity index (PI).
  • PI for a peptide is the mean SI multiplied by the percentage of positive responses to that peptide.
  • FIG. 9 shows the PI for each of the peptides in the total population of sixty-nine MS patients.

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Abstract

La présente invention concerne des peptides de la glycoprotéine d'oligodendrocyte de myéline humaine (MOG), auto-antigène lié aux maladies auto-immunes démyélinissantes. La présente invention concerne également certaines compositions incluant des peptides MOG ou des mélanges de peptides MOG et de peptides du peptide MBP. Enfin, la présente invention concerne aussi certaines compositions thérapeutiques servant au diagnostic et au traitement de certaines maladies auto-immunes ainsi que des méthodes de traitement de la sclérose en plaques au moyen desdites compositions thérapeutiques.
EP96913884A 1996-03-28 1996-05-01 Peptides de la glycoproteine d'oligodendrocyte de myeline et leurs utilisations Withdrawn EP0922057A1 (fr)

Applications Claiming Priority (3)

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US62340696A 1996-03-28 1996-03-28
US623406 1996-03-28
PCT/US1996/006072 WO1997035879A1 (fr) 1996-03-28 1996-05-01 Peptides de la glycoproteine d'oligodendrocyte de myeline et leurs utilisations

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EP0922057A1 true EP0922057A1 (fr) 1999-06-16

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EP (1) EP0922057A1 (fr)
AU (1) AU5671196A (fr)
CA (1) CA2250361A1 (fr)
WO (1) WO1997035879A1 (fr)

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US6251396B1 (en) 1994-11-18 2001-06-26 Neurocrine Biosciences, Inc. Methods for treatment of multiple sclerosis using peptide analogs of human myelin basic protein
US6379670B1 (en) 1994-11-18 2002-04-30 Neurocrine Biosciences, Inc. Methods for treatment of multiple sclerosis using peptide analogs of human myelin basic protein
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
SE9703287D0 (sv) * 1997-09-11 1997-09-11 Astra Ab Peptides
US20020072493A1 (en) 1998-05-19 2002-06-13 Yeda Research And Development Co. Ltd. Activated T cells, nervous system-specific antigens and their uses
US20030108528A1 (en) * 1998-05-19 2003-06-12 Michal Eisenbach-Schwartz Activated t-cells, nervous system-specific antigens and their uses
EP1080110A2 (fr) * 1998-05-19 2001-03-07 Yeda Research And Development Co. Ltd. Lymphocytes t actives, antigenes specifiques du systeme nerveux et leur utilisation
DE69935603T2 (de) * 1998-08-26 2007-12-06 The Regents Of The University Of California, Oakland Inhibitoren von autoantikörpern
ITFI20010114A1 (it) * 2001-06-22 2002-12-22 Univ Firenze Glicopeptidi,loro preparazione e loro uso nella diagnosi o nel trattamento terapeutico della sclerosi multipla
CN100567987C (zh) * 2002-08-08 2009-12-09 贝勒医学院 T细胞疫苗及其制备方法
WO2010024927A2 (fr) * 2008-08-28 2010-03-04 The Research Foundation Of State University Of New York Traitement des amyloïdoses au moyen de protéines basiques de myéline ou de fragments desdites
WO2012045324A1 (fr) * 2010-10-07 2012-04-12 Mediagnost Gesellschaft Fur Forschung Und Herstellung Von Diagnostika Gmbh Procédé de détection d'une maladie de parkinson et système de test
WO2013020914A1 (fr) * 2011-08-10 2013-02-14 Celares Gmbh Peptides peg-conjugués
GB201300683D0 (en) * 2013-01-15 2013-02-27 Apitope Int Nv Peptide
GB201300684D0 (en) * 2013-01-15 2013-02-27 Apitope Int Nv Peptide
CN116323638A (zh) * 2020-07-22 2023-06-23 弗格制药有限公司 订书钉化肽及其方法

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JPH09502346A (ja) * 1993-09-03 1997-03-11 イミユロジク・フアーマシユーチカル・コーポレーシヨン 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
WO1995007096A1 (fr) * 1993-09-06 1995-03-16 La Trobe University Traitement de maladies auto-immunes
SI9520059A (en) * 1994-05-10 1997-08-31 Immulogic Pharma Corp Compositions and treatment for multiple sclerosis.

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Publication number Publication date
WO1997035879A1 (fr) 1997-10-02
CA2250361A1 (fr) 1997-10-02
AU5671196A (en) 1997-10-17

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