The invention provides peptide compositions and methods of making and using therapeutic compositions comprising peptides for the treatment of a subject having a demyelinating condition.

Description of the Invention

SUMMARY

The invention in one embodiment features a composition comprising a peptide with an amino acid sequence having two tyrosine (Y) residues and a lysine (K) residue, such that in a complex of the peptide with an MHC class II HLA-DR2 protein involved in modulation of an immune response, the residues in the amino acid sequence corresponding to: (i) tyrosines located at P1 and P4 positions; and (ii) lysine located at a P5 position which contacts a T cell receptor protein. A related embodiment features a peptide with an amino acid sequence having at least a tyrosine (Y) residue, a valine residue (V), and a lysine (K) residue, such that in a complex of the peptide with an MHC class II HLA-DR2 protein involved in modulation of an immune response, the residues in the amino acid sequence corresponding to: (i) valine located at a P1 position; (ii) tyrosine located at a P4 position; and (iii) lysine located at a P5 position which contacts a T cell receptor.

The "P1" position in the peptide is named by analogy to the amino acid location in an immunodominant epitope for an MHC class II HLA-DR2 protein associated with MS, the MBP 85-99 peptide (SEQ ID NO: 1), in which a valine (V) at position 89 fits into the "P1" pocket in the groove or cleft of the protein in a complex formed between this peptide and protein, and this V is therefore identified as being located at a P1 position. Other positions in the peptide are named based on the location relative to the P1 position, i.e., a phenylalanine (F) at position 92 (further toward the carboxy terminus, or downstream from P1) of MBP is in the P4 position, and the amino acid adjacent to P1 but further toward the amino terminus, or upstream, is referred to as being in the P-1 (P minus one) position.

In examples of the above embodiments, the sequence further comprises a lysine (K) residue at a P-1 position; the sequence of the peptide further includes a plurality of alanine (A) residues at positions which are to the carboxy-terminal side of the lysine residue at P5. In further embodiments, the peptide is substantially pure; the peptide is synthetic. The composition comprises an additional therapeutic agent, for example, the additional therapeutic agent is selected from the group consisting of an interferon and a random heteropolymer of amino acids.

The invention in another embodiment provides a composition comprising a synthetic peptide, wherein the peptide has an amino acid sequence having a greater inhibitory activity for binding to the antigen binding groove of an MHC class II HLA-DR2 protein associated with multiple sclerosis, than a reference material selected from the group of: an immunodominant epitope from myelin basic protein (MBP), the epitope comprising MBP residues 85-99 ENPVVHFFKNIVTPR as shown in SEQ ID NO: 1; and a randomly polymerized amino acid heteropolymer having amino acids, tyrosine, alanine, glutamic acid, and lysine (Copaxone.RTM.), the composition further capable of inhibiting proliferation of an MBP-specific T cell.

For example, the greater inhibitory activity of the peptide than the reference material is at least 10%; or is at least 20%. Further, the peptide is about 5 to about 100 amino acids in length; for example, the peptide is about 5 to about 25 amino acids in length; for example, the peptide is about 5 to about 15 amino acids in length. In certain embodiments, the peptide further comprises at least one non-naturally occurring amino acid, in a location in the sequence and in an amount sufficient to inhibit proteolytic degradation of the peptide in a subject, in comparison with a peptide identical in sequence and consisting of naturally occurring amino acid residues. Alternatively, the peptide comprises at least one non-naturally occurring amino acid, in a location in the sequence and in an amount sufficient to increase the affinity for the antigen binding groove of the MHC class II HLA-DR2 protein, in comparison with a peptide identical in sequence and consisting of naturally occurring amino acid residues. The at least one non-naturally occurring amino acid is the presence of at least one D-amino acid within four residues of at least one of the carboxy-terminal and amino-terminal.

In further embodiments, the composition comprises a plurality of copies of the peptide as a monomer unit of an oligomer, each monomer unit being joined by a flexible linker. For example, the oligomer is a homo-oligomer. Alternatively, the oligomer is a hetero-oligomer. The peptide can further comprise the presence in the sequence of at least one proline residue. Further, the at least one proline residue is present proximal to at least one of carboxy- and amino-termini of the sequence, i.e., the at least one proline is at a position within at most four residues of at least one of carboxy and amino termini.

The peptide can further comprise at least one non-peptide bond. The non-peptide bond is selected from the group consisting of a peptide nucleic acid bond and a phosphorothioate bond.

The non-naturally occurring amino acid can be a substitution of at least one alanine (A) in the sequence with a peptidomimetic compound selected from the group consisting of: Tic, which is tetrahydroisoquinoline-(S)-3-carboxylic acid); Thiq, which is tetrahydroisoquinoline-(S)-1-carboxylic acid); Disc, which is (dihydroisoindole-(S)-2-carboxylic acid); C(Acm), which is acetamido-methyl-Cys; C(Pmm), which is propylamidomethyl-Cys; C(Ace), which is acetyl-Cys; MePhg, which is methylphenyl-Gly; and Nva, which is norvaline. The amino acid modification is N-methylation of a peptide backbone nitrogen.

The invention in another embodiment features a composition comprising a synthetic peptide having an amino acid sequence selected from the group consisting of -- see Original Patent.

Further, the peptide is substantially pure. The above selected peptide can further comprise substitution of a tyrosine (Y) in the P1 position by a valine (V). In a related embodiment, the above peptide comprises an oligomer having a plurality of monomer units having the amino acid sequence of the synthetic peptide, the units joined by a flexible linker. The invention also features a method for reducing demyelination of cells in a subject, the method comprising administering to the subject a composition as shown above.

Another embodiment of the invention features a method for obtaining a synthetic peptide having inhibitory activity for binding of an immunodominant epitope of multiple sclerosis (MS) to an MHC class II protein associated with MS, the method comprising:

designing a plurality of peptide sequences, wherein each peptide comprises a sequence of amino acids having a charge, size, and order within the sequence such that the peptide is capable of occupying features of an antigen binding site of an MHC class II protein associated with multiple sclerosis (MS); and

assaying each of the plurality of peptides for affinity for the MHC class II protein, to determine the amount of the peptide having inhibitory activity for binding of a reference compound to the MHC class II protein, wherein a lower amount of peptide able to inhibit the extent of binding compared to the reference compound indicates a greater inhibitory activity of the peptide for inhibiting binding of an immunodominant epitope of multiple sclerosis (MS) to an MHC class II protein associated with MS.

Yet another embodiment is a method for obtaining a synthetic peptide having inhibitory activity for proliferation of cells of a T cell line, the T cells restricted to an immunodominant epitope of multiple sclerosis (MS), the method comprising:

designing a plurality of peptide sequences, wherein each peptide comprises a sequence of amino acids having a charge, size, and order within the sequence such that the peptide is capable of occupying features of an antigen binding site of an MHC class II protein associated with multiple sclerosis (MS); and

assaying each of the plurality of peptides for an amount that has ability to inhibit proliferation of the T cells, wherein a lower amount of peptide able to inhibit the proliferation of the cells compared to the reference compound indicates a greater inhibitory activity of the peptide for inhibiting the T cells restricted to an immunodominant epitope of multiple sclerosis (MS).

In related embodiments of these methods, the reference compound is selected from a group consisting of Copaxone.RTM. and a peptide comprising a sequence of amino acids at positions 85-99 of myelin basic protein (MBP) as shown in SEQ ID NO: 1. The methods can further comprise: measuring an ability of each of the plurality of peptides to inhibit presentation of the reference compound to HLA restricted T cells. The methods can further comprise designing a plurality of peptide sequences having a charge, a size, and an order within the sequence, by choosing amino acids to occupy positions in the sequence of that peptide capable of contacting the antigen binding P1 and P4 pockets of the MHC class II protein associated with MS, corresponding to locations in the MBP 85-99 peptide amino acid sequence at residues 89 and 92, respectively. For example, the methods comprise selecting the amino acids contacting the P1 and P4 pockets from the group consisting of hydrophobic amino acids; for example, the hydrophobic amino acids are selected from the group consisting of a tyrosine (Y), a valine (V), a phenylalanine (F), a methionine (M), an isoleucine (I), and a leucine (L). The hydrophobic amino acids contacting the P4 pocket are selected from the group consisting of a tyrosine (Y) and a phenylalanine (F). In one example, the amino acid contacting the P1 pocket is valine (V). Further, the amino acid in the P5 position is a lysine (K). In comparing the affinity of each of the plurality of peptides, the method further comprises providing a reference compound having a detectable modification. For example, the modification is selected from the group of compounds which are radioactive, antigenic, biotinylated, fluorescent, photometric, and have a high affinity for an immobilized ligand.

A further embodiment of the method is determining the concentration of the peptide able to inhibit an extent of binding of the test compound to the MHC class II protein associated with multiple sclerosis, the method further comprises measuring an amount of proliferation of a DR2-restricted cell line of T cells exposed to the complex of the peptide with the MHC class II protein. Thus measuring the amount of proliferation further comprises determining an amount of IL-2 secretion by the T cells. Further, determining the amount of IL-2 secretion further comprises assaying culture fluid of the T cells for ability to support growth of IL-2 dependent cytotoxic T-cell interleukin-dependent lymphocytes (CTLL). In this assay, the lower the amount of IL-2 secretion, the greater the extent the peptide is able to inhibit proliferation of the T cells.

Another feature of the invention provides a method of treating a subject having a demyelinating condition, comprising: providing to the subject a composition capable of inhibiting binding of myelin basis protein (MBP) peptide to purified recombinant MHC class II DR2 molecules, wherein the composition is a peptide that comprises an amino acid sequence selected from the group consisting of: AAEAYKAYKAAAAAA (SEQ ID NO: 60), EAAAYKAYKAAAAAA (SEQ ID NO: 63), EAAKYEAYKAAAAAA (SEQ ID NO: 64), EKAKYEAYKAAAAAA (SEQ ID NO: 65), EAKKYEAYKAAAAAA (SEQ ID NO: 66), AKKEYAEYKAAAAAA (SEQ ID NO: 67), EAPAYKAYKAAAAPA (SEQ ID NO: 83), EAPKYEAYKAAAAPA (SEQ ID NO: 84), EAPKYEAYKAAAAPA (SEQ ID NO: 86), AKPEYAEYKAAAAPA (SEQ ID NO: 87), APEKAKYEAYKAAAAAA (SEQ ID NO: 88), APEKAKYEAYKAAAAAAPA (SEQ ID NO: 89), EKAKYEAYKAAAAAAPA (SEQ ID NO: 90), EKPKFEAYKAAAAPA (SEQ ID NO: 91), EKPKVEAYKAAAAPA (SEQ ID NO: 93), EKAKFEAFKAAAAAA (SEQ ID NO: 95), APEKAKFEAFKAAAAPA (SEQ ID NO: 96), and APEKAKFEAYKAAAAPA (SEQ ID NO: 97), wherein the subject having a demyelinating condition is treated. The demyelinating condition is selected from the group consisting of a post-viral encephalomyelitis, a post-vaccine demyelinating condition, a multiple sclerosis, and a side effect of administering an anti-TNF agent. The MBP peptide comprises MBP residues 85-99 as shown in SEQ ID NO: 1. In a related embodiment, the peptide further inhibits proliferation of autoantigen-specific HLA-DR2-restricted T cell clones. In yet another related embodiment, the amino acid sequence of the peptide selected above further comprises at least one amino acid analog substituted for an amino acid. Alternatively, the amino acid sequence of the peptide comprises at least one peptide bond analog.

The method further comprises formulating the composition in a pharmaceutically acceptable carrier. The method further comprises formulating the composition as a unit dose. In these methods, the MHC class II DR2 molecules are of a genotype associated with multiple sclerosis. For example, the MHC class II DR2 molecules are selected from the group consisting of DRB1*1501 and DRB1*1602.

Another featured embodiment of the invention herein is a kit comprising at least one container having a peptide capable of inhibiting binding of an immunodominant epitope of myelin basic protein to an MHC class II DR2 protein, and instructions for use. The peptide can be substantially pure. Further, the kit comprises a peptide in a pharmaceutically acceptable buffer, and instructions for use.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

Autoimmune Diseases

An autoimmune disease results when a host's immune response fails to distinguish foreign antigens from self molecules (autoantigens) thereby eliciting an aberrant immune response. The immune response towards self molecules results in a deviation from the normal state of self-tolerance, which arises when the production of T cells and B cells capable of reacting against autoantigens has been prevented by events that occur in the development of the immune system early in life. The cell surface proteins that play a central role in regulation of immune responses through their ability to bind and present processed peptides to T cells are the major histocompatibility complex (MHC) molecules (Rothbard, J. B. et al., Annu. Rev. Immunol. 9:527 (1991)).

A number of therapeutic agents have been developed to treat autoimmune diseases. For example, agents have been developed that can prevent formation of low molecular weight inflammatory compounds by inhibiting a cyclooxygenase. Also, agents are available that can function by inhibiting a protein mediator of inflammation by sequestering the inflammatory protein tumor necrosis factor (TNF) with an anti-TNF specific monoclonal, antibody fragment, or with a soluble form of the TNF receptor. Finally, agents are available that target and inhibit the function of a protein on the surface of a T cell (the CD4 receptor or the cell adhesion receptor ICAM-1) thereby preventing interaction with an antigen presenting cell (APC). However, compositions which are natural folded proteins as therapeutic agents can incur problems in production, formulation, storage, and delivery. Further, natural proteins can be contaminated with pathogenic agents such as viruses and prions.

An additional target for inhibition of an autoimmune response is the set of lymphocyte surface proteins represented by the MHC molecules. Specifically, these proteins are encoded by the MHC class II genes designated as HLA (human leukocyte antigen)-DR, -DQ and -DP. Each of the MHC genes is found in a large number of alternative or allelic forms within a mammalian population. The genomes of subjects affected with certain autoimmune diseases, for example, MS and rheumatoid arthritis (RA), are more likely to carry one or more characteristic MHC class II alleles, to which that disease is linked.

A potential source of agents for treatment of MS and other demyelinating conditions is to identify peptides that bind selectively in vitro to a purified MHC class II allele protein molecule, particularly to a protein which is a product of an MHC class II allele associated with demyelinating conditions. In addition, the agent should bind to that protein as it occurs on the surfaces of antigen presenting cells in vivo, and thereby block, anergize, or inactivate the class of T cells that are responsible for the demyelinating conditions, such as MS.

Major candidates for target antigens in MS include myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). T cells reactive with these antigens have been found both in normal blood (Wucherpfennig K. W. et al., J. Immunol. 150:5581 (1994); Steinman L. et al., Mol. Med. Today 1:79 (1995)) and in MS patients (Wucherpfennig K. W. et al., Immunol. Today 12:227 (1991); Marcovic-Plese S. et al., J. Immunol. 155:982 (1995); Correale J. et al., Neurology 45:1370 (1995); Kerlero de Rosbo N. et al., Eur. J. Immunol. 27:3059 (1997); Tsuchida T. et al., Proc. Natl. Acad. Sci. U.S.A. 91:10859 (1994)), suggesting that autoreactive T cells may be involved in the pathogenesis of the disease, such that these cells once activated can penetrate the blood-brain barrier. Microbial agents have been suggested to provide potential stimuli for induction of MS by immunological cross-reaction with MBP (Wucherpfennig K. W. et al., Cell 80:695 (1995); Brocke S. et al., Nature 365:642 (1993)).

Studies indicate that MBP is an important target antigen in the immunopathogenesis of MS. MBP-specific T cells have been shown to be clonally expanded in MS patients and in an in vivo activated state (Wucherpfennig K. W., et al., J. Immunol. 150:5581 (1994); Allegretta M. et al., Science 247:718 (1990); Ota K. et al., Nature 346:183 (1990); Zhang J. et al., J. Exp. Med. 179:973 (1994)). Reactivity with the immunodominant MBP 84-102 peptide is found predominantly in subjects carrying HLA-DR2, a genetic marker for susceptibility to MS. Structural characterization of MBP 84-102 identified residues critical for MHC class II binding and for TCR recognition (Wucherpfennig K. W. et al., J. Exp. Med. 179:279 (1994)), which have been recently confirmed by the crystal structure of HLA-DR2 complexed with MBP 85-99 peptide (Smith K. J. et al., J. Exp. Med. 19:1511 (1998)).

An agent that interacts with and binds promiscuously to several MHC class II molecules is Copolymer 1 (Cop 1; YEAK; Copaxone.RTM.). This synthetic amino acid heteropolymer is capable of suppressing experimental allergic encephalomyelitis (EAE; Sela, M. et al., Bull. Inst. Pasteur (Paris) (1990)), a condition which can be induced in the mouse and is a model for MS. Cop 1, the random heteropolymer of amino acids known as poly(Y,E,A,K), indicated using the one letter amino acid code (Y is tyrosine, E is glutamic acid, A is alanine, and K is lysine) is a therapeutic agent for MS, but does not suppress the disease entirely (Bornstein, M. B. et al., N. Engl. J. Med. 317:408 (1987); Johnson, K. P. et al., Neurology 45:1268 (1995)).

Cop 1 binds to purified human HLA-DR molecules within the peptide binding groove and inhibits the binding of a high affinity epitope of influenza virus HA 306-318, to both HLA-DR1 (DRB1*0101) and -DR4 (DRB1*0401) molecules, and the binding of MBP 84-102, a human immunodominant epitope of MBP, to HLA-DR2 (DRB1*1501) molecules (Fridkis-Hareli M, et al., J Immunol 160:43864397, 1998). Copolymers composed of only three amino acids (for example, EAK, YEA, YAK and YEK) also bind to purified HLA-DR1, -DR2 and -DR4 molecules (Fridkis-Hareli M, et al. Int Immunol 11:635, 1999; PCT/US99/16,617). Moreover, these three amino acid copolymers compete with CII 261-273 for binding to RA-associated HLA-DR1 (DRB1*0101) and -DR4 (DRB1*0401) molecules, and also inhibited CII-reactive T cell clones (Fridkis-Hareli M, et al. Proc Natl Acad Sci USA 95:12528, 1998); PCT/US99/16617 and PCT/US99/16747.

The bound fraction of Cop 1, treated with aminopeptidase I, has been isolated from recombinant "empty" HLA-DR molecules produced in insect cells, and has been sequenced. The Cop 1 binding motif for HLA-DR2 showed increases in levels of E at the first and second cycles, of K at the second and third cycles, and of Y and A (presumably at P1 of the bound peptide) at the third to fifth cycle. No preference was seen at the following cycles which were mainly A (Fridkis-Hareli M, et al. J Immunol 162:4697, 1999; PCT/US99/16,617). Recently, the characterization of the active component(s) of the mixture of random polypeptides was attempted by synthesis of a set of peptides based on Cop 1 binding properties to HLA-DR1 and -DR4 molecules (Fridkis-Hareli M, et al. Human Immunol 61: 640, 2000); PCT/US99/16,617. Several peptides inhibited binding of CII 261-273 epitope to DRB 1*0101 and -DR4 DRB1*0401 molecules and inhibited presentation of this epitope to CII-reactive DR1- and DR4-restricted T cell clones (Fridkis-Hareli M, et al. Human Immunol 61: 640, 2000).

Demyelinating conditions have been found to occur post-viral infection, post-vaccination, post-encephalomyelitis (Wucherpfenning K.W. et al., Immunol. Today 12:277-282 (1991)) and following administration of certain anti-TNF agents (FDA Talk Paper, Food and Drug Administration Public Health Service, Rockville, Md.).

Many derivatives of synthetic peptides having increased pharmacological life in vivo have been synthesized. The loading of MHC class II binding sites occurs in endosomal compartments abundant with proteases, particularly cathepsins. Peptides may be digested also by amino- or carboxy-peptidases in serum or other biological fluids. Therefore, proteolysis of the peptides may effectively remove the peptides from the subject (Bennett, K., et al., 1992, Eur. J. Immunol. 22:1519). To reduce or eliminate potential proteolysis, modification of the peptides, for example, N-methylation of backbone nitrogens in the peptides, which are not involved in essential hydrogen bonding interactions, could produce a peptide derivative that is resistant to proteolysis (Falconi, F., et al., 1999, Nature Biotechnology 17:562). In Falconi et al., N-methylation of a hemagglutinin (HA) peptide to produce a modified peptide derivative yielded a compound that was substantially less sensitive to digestion by cathepsin B. The resulting protease resistant peptide was also a substantially better inhibitor of presentation by MHC class II DR proteins to T-cell clones, compared to the original HA peptide.

In another embodiment, the invention provides derivatives of synthetic peptides having a chemical alteration in one or both of the peptide backbone or the amino acid side chains. These derivatives can have increased binding affinity to the MHC class II DR1 protein, result in increased inhibitory activity and/or resistance to proteolysis. This phenomenon was observed when a peptidomimetic compound was designed to replace a native hemagglutinin (HA) peptide in binding to an MHC class II DR1 protein (Falconi, F., et al., 1999, Nature Biotechnology 17:562). The designed peptide was comprised of suitable amino acid mimetic compounds for each of several particular amino acids. In one example, alanine (A) was substituted with one or more conformationally restricted aromatic compounds, Tic, which is tetrahydroisoquinoline-(S)-3-carboxylic acid), Thiq, which is tetrahydroisoquinoline-(S)-1-carboxylic acid), and Disc, which is (dihydroisoindole-(S)-2-carboxylic acid), and the blocked Cys compounds C(Acm), which is acetamido-methyl-Cys, C(Prm), which is propylamidomethyl-Cys, and C(Ace), which is acetyl-Cys. Furthermore, MePhg, which is methylphenyl-Gly, and Nva, which is norvaline, provided increased binding affinity. Substitution by some of the peptidomimetics resulted in improved inhibition of the immune response.

In various embodiments of the present invention, a series of peptides are designed having a sequence comprising amino acids tyrosine (Y), glutamic acid (E), alanine (A), and lysine (K), and further having replacements of Y with other hydrophobic residues, K with uncharged residues, and alanine (A) with prolines near the termini of the peptides. These additional peptides are tested for MHC class II HLA-DR2 binding by extent of inhibition of a labeled reference molecule having known affinity for HLA-DR2, and inhibition activity of presentation to T cells. Peptides are thereby obtained that show as least as great or increased binding affinity as the unmodified synthetic peptide, as well as an increased potency in inhibiting T-cell responses to processed protein antigens presented by the targeted MHC molecule.

Methods and Uses

The therapeutic compounds of the invention can be used to treat symptoms of multiple sclerosis, an MS demyelinating condition marked by patches or hardened tissue in the brain or the spinal cord; and other demyelinating conditions. Therapeutic compounds of the invention, while characterized by binding to MHC class II HLA-DR2 molecules, may have increased affinity for MHC class II molecules associated with additional autoimmune diseases.

A pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antimicrobials such as antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, transdermal, or subcutaneous administration. The active compound can be coated in a material to protect it from inactivation by the action of acids or other adverse natural conditions.

A composition of the present invention can be administered by a variety of methods known in the art as will be appreciated by the skilled artisan. Copaxone.RTM., for example, is supplied as an acetate form, and is reconstituted in aqueous solution and administered to an MS patient subcutaneously. The peptides herein can be similarly formulated and delivered. The peptide and any additional active compound as described herein to be administered in combination with the peptides can further be prepared with carriers that will protect it against rapid release, such as a controlled release formulation, including implants, transdermal patches, micro-encapsulated delivery systems. Many methods for the preparation of such formulations are patented and are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, Ed. Marcel Dekker, Inc., NY (1978).

Therapeutic compositions for delivery in a pharmaceutically acceptable carrier are sterile, and are preferably stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the disease situation.

In general, a preferred embodiment of the invention is to administer a suitable daily dose of a therapeutic synthetic peptide composition that will be the lowest effective dose to produce a therapeutic effect, for example, mitigation of symptoms. The therapeutic peptide compounds of the invention are preferably administered at a dose per subject per day of at least 2 mg, at least 5 mg, at least 10 mg or at least 20 mg as appropriate minimal starting dosages. In general, the compound of the effective dose of the composition of the invention can be administered in the range of 50 to 400 micrograms of the compound per kilogram of the subject per day.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective dose of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compound of the invention employed in the pharmaceutical composition at a level lower than that required in order to achieve the desired therapeutic effect, and increase the dosage with time until the desired effect is achieved.

A desired therapeutic effect can be determined by increased periods of remission of MS, such that fewer episodes of relapse per unit time are noted. Another desired therapeutic effect can be remission in symptoms such as pain, dizziness, fatigue, visual and cognitive disturbances as noted herein. Remissions of symptoms can be self-reported by the patient, or can be quantitatively detected by standard measurements of sensory and cognitive abilities, known to practitioners in the art of treating autoimmune conditions such as demyelinating conditions.

In another preferred embodiment, the pharmaceutical composition includes also an additional therapeutic agent. Thus in a method of the invention, the pharmaceutical composition can be administered as part of a combination therapy, i.e. in combination with an additional agent or agents. Examples of materials that can be used as combination therapeutics with the peptides for treatment of autoimmune disease and demyelinating conditions as additional therapeutic agents include: an antibody or an antibody fragment that can bind specifically to an inflammatory molecule or an unwanted cytokine such as interleukin-6, interleukin-8, granulocyte macrophage colony stimulating factor, and tumor necrosis factor-.alpha.; an enzyme inhibitor which can be a protein, such as .alpha..sub.1-antitrypsin, or aprotinin; an enzyme inhibitor which can be a cyclooxygenase inhibitor; an engineered binding protein, for example, an engineered protein that is a protease inhibitor such an engineered inhibitor of kallikrein; an antibacterial agent, which can be an antibiotic such as amoxicillin, rifampicin, erythromycin; an antiviral agent, which can be a low molecular weight chemical, such as acyclovir; a steroid, for example a corticosteroid, or a sex steroid such as progesterone; a non-steroidal anti-inflammatory agent such as aspirin, ibuprofen, or acetaminophen; an anti-cancer agent such as methotrexate or adriamycin; or a cytokine.

An additional therapeutic agent can be a cytokine, which as used herein includes without limitation agents which are naturally occurring proteins or variants and which function as growth factors, lymphokines, interferons such as .beta.-interferon, tumor necrosis factors, angiogenic or antiangiogenic factors, erythropoietins, thrombopoietins, interleukins, maturation factors, chemotactic proteins, or the like. Preferred combination therapeutic agents to be used with the composition of the invention and are .beta.-interferon and/or Copaxone.RTM.. A therapeutic agent to be used with the composition of the invention can be an engineered binding protein, known to one of skill in the art of remodeling a protein that is covalently attached to a virion coat protein by virtue of genetic fusion (Ladner, R. et al., U.S. Pat. No. 5,233,409; Ladner, R. et al., U.S. Pat. No. 5,403,484), and can be made according to methods known in the art. A protein that binds any of a variety of other targets can be engineered and used in the present invention as a therapeutic agent in combination with a peptide of the invention.

An improvement in the symptoms as a result of such administration is noted by a reduction in symptoms such as the symptoms of MS noted herein. A therapeutically effective dosage preferably reduces frequency of MS episodes, and severity of symptoms such as fatigue, pain, and visual disturbances by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and even still more preferably by at least about 80%, relative to untreated subjects. Cure of complete remission or improvement of symptoms can be noted by increased life span, elimination of relapsing episodes, and significantly improved overall health of the patient.

Another embodiment of the invention is a kit for assaying the binding of an analyte to an MHC protein associated with a demyelinating condition. This embodiment provides: a water-soluble MHC protein which is associated with a demyelinating condition and which has been recombinantly produced in a heterologous cell; a reaction chamber for containing the analyte and the MHC protein; and means for detecting binding of the analyte to the MHC protein. In a preferred embodiment, the MHC protein is produced in an invertebrate or a microbial cell, such as an insect cell or a yeast cell, and so is devoid of bound epitopes of human or mammalian origin, the bound peptide being in the antigen cleft, i.e., the MHC protein of the kit is "empty." Means for detecting binding of the analyte to the MHC protein can be radioactive, fluorimetric, ligand associating means such as biotinylated, chemiluminescent, or colorimetric means known to one of ordinary skill in the art. In a preferred embodiment of the kit, the MHC protein is a class II MHC HLA-DR1, -DR2, or -DR4 protein. Further, the kit can include also a reference material such as an autoantigenic peptide, such as a CII peptide, or a peptide derived from MBP, MOG, or a peptide from some other protein implicated in a demyelinating condition, such as a peptide comprising MBP residues at positions 85-99 (SEQ ID NO: 1).

 

Claim 1 of 4 Claims

1. A composition comprising a synthetic peptide having an amino acid sequence selected from the group consisting of -- see Original Patent.

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