WO2006052654A2 - Compositions and methods for the treatment of immune mediated diseases - Google Patents

Abstract

Compositions and methods for effecting multi-branched therapies for the treatment of immune-mediated diseases are described. In particular, multi-branched therapies involving selected immunomodulation by bringing about, in a purposeful manner, either a regulatory response or an inflammatory response to a given autoimmune disorder or disease.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF IMMUNE

MEDIATED DISEASES

RELATED APPLICATIONS

This invention is claims the benefit of U.S. Provisional Patent Application Numbers 60/625,904, and 60/626,143, each filed November 7, 2004.

FIELD OF THE INVENTION

[0001] This invention relates generally to compositions and methods for treating immune-mediated disease states and, more specifically, to multi-branched (i.e., combination) epitope-specific immunotherapies to treat immune-mediated diseases, including rheumatoid arthritis, infectious bowel diseases (e.g., Crohn's disease and Ulcerative colitis), and demyelinating autoimmune diseases such Multiple Sclerosis.

BACKGROUND OF THE INVENTION

1. Introduction.

[0002] The following description includes information that may be useful in understanding the present invention. It is not an admission that any such information is prior art, or relevant, to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

2. Background.

[0003] In one example of an immune-mediated disease susceptible to multi-branched epitope-specific immunotherapy of the invention, Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that affects approximately 250,000 individuals in the United States. Although the clinical course is variable, the most common form of MS manifests relapsing neurological deficits, particularly paralysis, sensory deficits, and visual problems. MS is mediated by cells of the immune system (e.g., T lymphocytes, B lymphocytes, and macrophages) attacking the myelin sheaths of nerve cell axons. MS lesions in the CNS are called "plaques" due to their macroscopic appearance. [0004] Multiple sclerosis is thought to arise from pathogenic T cells that somehow evade the immune system's complex control mechanisms that lead to self-tolerance, and attack normal tissue. In MS patients, immune-mediated demyelination frequently follows acute viral and bacterial infections. Thus, an environmental insult appears to be important for the development and progression of MS.

[0005] Heat shock proteins (hsp) are families of proteins encoded by the genomes of almost all prokaryotic and eukaryotic cells. Heat shock proteins are essential for normal cell function. They are expressed both constitutively and in increased amounts when cells are exposed to an environmental stress, for example, heat, viral infection, and anoxia, as well as following exposure to certain cytokines. Heat shock proteins are the immunodominant antigens of many pathogenic and non-pathogenic bacteria and parasites, and immune responses to these proteins are ubiquitous in normal individuals. That said, individual differences in immune responses to these proteins occurs, and these responses in some patients are thought to be pathogenic. Several possible mechanisms may trigger the autoimmune event. For example, cytokine levels increase during an infection and these could activate quiescent, anti-myelin specific T cells present in the CNS. Alternatively, an immune response to pathogen-encoded hsp could occur during the infection, leading to a cross reaction with endogenous heat shock proteins expressed in the CNS.

[0006] No definitive treatment for MS has been established to date. Historically, corticosteroids have been used to reduce inflammation via toxicity to lymphocytes. While such treatment may hasten recovery from acute exacerbations of MS, these drugs do not prevent future attacks or prevent development of additional disabilities or the chronic progression of MS. Additionally, the substantial side effects of steroid treatments make such drugs undesirable for long-term use. Similarly, other toxic compounds, such as azathioprine (a purine antagonist), cyclophosphamide, and cyclosporine have been used to treat symptoms of MS. Like corticosteroid treatment, these drugs are highly toxic and beneficial only over the short term. Moreover, side effects include increased rates of malignancy, leukopenia, toxic hepatitis, gastrointestinal problems, hypertension, and nephrotoxicity.

AND-8001-PC [0007] Several more recent therapies for treating MS have also been introduced. Each of these therapies, however, has limitations, and several are accompanied by severe side effects, among other shortcomings. One such therapy involves Interferon-beta (IFN- β) administration. Undesired side effects of IFN-β therapy include flu-like syndrome, depression, hematologic abnormalities, cardiotoxicity, and elevated hepatic enzymes. Another treatment involves administration of glatiramer acetate, also known as copolymer- 1 or COPAXONE®, a mixture of polymers made of four amino acids. COPAXONE® injection appears to promote the secretion of cytokines such as interleukin-5 (IL-5) and IL-13, which are associated with Th-2-type T cell responses. COPAXONE® appears to bind various class II major histocompatibility complex (MHC) molecules, resulting in inhibition of T cell responses that might otherwise occur against several myelin antigens. However, treatment regimens designed solely around COPAXONE® are likely to be suboptimal since the specificity of MHC blocking induced by the composition appears to be restricted in its T cell receptor antagonistic activity to the antigenic portion of myelin basic protein (MBP).

[0008] Multiple sclerosis has also been treated with monoclonal antibodies, particularly the antibody Natalizumab (ANTEGREN®), a humanized monoclonal antibody that interferes with T cell movement from the blood across the blood-brain barrier into the CNS. Specifically, the monoclonal antibody attaches to an epitope present on alpha 4-integrin expressed on the surface of white blood cells chain irrespective of its associated β-chain, thereby competitively inhibiting its association with VLA-4, also known as α4βl integrin or CD49D/CD29, an adhesion molecule expressed on the surface of endothelial cells that line the blood vessels in the CNS, Monthly intravenous ANTEGREN® infusions limit the recruitment of blood-borne T cells across the blood-brain barrier, preventing these cells from migrating to join those T cells already in the area and further contributing to inflammation, edema, and lesions. It is believed that ANTEGREN® binds to the integrin epitope on T cells educated for stimulation of any of a ThI, Th2, or Th3 response. Thus, use of such antibodies is not optimal for the reason that its non-specificity keeps T cells beneficial to an inflamed tissue, i.e., T cells educated for Th2 and Th3 responses from entering tissue needing such a regulatory, or tolerating, response. There is also the likelihood of a patient becoming sensitized to the

AND-8001-PC antibody itself and thus rendering it not useable in such a patient. ANTEGREN® treatment, like IFN- β therapy, also produces side effects that include headache, weakness, urinary tract infections, gastroenteritis, rash, back pain, and fever.

[0009] Despite the treatment options currently available for MS, and other immune- mediated diseases such as Rheumatoid Arthritis, and IBD, a need still exists for treatments that more directly modulate those portions of the immune system associated with responding and/or reacting to the disease, as opposed to simply blocking or masking an immune response characteristic of the disease presented.

SUMMARY OF THE INVENTION

Definitions

[00010] The term "combination therapy" refers to a therapeutic regimen that involves the administration of at least two chemically distinct active ingredients, for example, an immunopeptidic molecules and a neuroprotective agent, to achieve an indicated therapeutic effect. It is understood that the active ingredients may be administered as part of the same composition or as different compositions. When administered as separate compositions, the compositions comprising the different active ingredients may be administered at the same or different times, by the same or different routes, using the same or different dosing regimens, all as the particular context requires and as determined by the attending physician.

[00011] The terms "contacting", "combining" reagents to "form a reaction mixture", and the like mean that the various reagents and reactants required for a particular reaction are brought together under conditions that allow the reaction to occur. For example, in the context of T cell interactions with peptide:MHC complexes, "contacting" or "combining" means that the T cells and peptide:MHC complexes brought together under conditions that allow T cells to interact with peptide:MHC complexes that can be specifically recognized by the T cell receptors expressed on the surface of the T cells.

[00012] The term "demyelinating disease" refers to any disorder that results in a reduced level of axon myelination. Such disorders that are particularly amenable to treatment by the instant therapeutic combinations and methods are those wherein

AND-8001-PC demyelination is mediated by an immune system component. Demyelinating diseases include acute disseminated encephalomyelitis, acute demyelinating polyneuropathy (Guillain Barre syndrome), chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, Marchifava-Bignami disease, central pontine myelinolysis, Devic syndrome, BaIo disease, HIV- and HTLV-associated myelopathy, transverse myelitis, demyelinating genetic diseases, spinal cord injury, and progressive multifocal leucoencephalopathy. Demyelinating diseases also include secondary demyelinating disorders where bystander myelin loss occurs as a consequence of a pathological insult e.g., virus-induced demyelination). Examples of secondary demyelinating diseases are CNS lupus erythematodes, polyarteritis nodosa, Sjogren syndrome, sarcoidosis, and isolated cerebral vasulitis.

[00013] A "heat shock protein" or "hsp", unless specifically identified, refers to any protein within one of the three families of heat shock proteins, namely hsp60/65, hsp70, and hsp90. Also included is hsp40 comprising a family of proteins commonly referred to as the dnaj family of proteins. Heat shock proteins are highly conserved across species, and hsps are found in animals and bacteria alike. Heat shock proteins also show high levels of intra-family conservation. The expression of some hsps are not altered by stress. Herein, an hsp embraces any protein or polypeptide having at least about 35%, preferably at least about 50%, and even more preferably at least about 75-85% amino acid identity over the region of alignment with an hsp whose expression in cells increases in response to a stressful stimulus. Here, sequence "identity" can be established using any art- recognized automated process for the alignment of amino acid sequences to determine identity.

[00014] An "immunopeptidic molecule" refers to a peptide or a peptidomimetic or multimers thereof as disclosed herein. For example, a peptidomimetic peptide comprises a chemically modified small molecule, including polymers of such molecule, having the activity described in the assay. Such activity is related to the induction of immune deviation. Formulations may comprise the active compound in combination with biologies, cytokines, anti-cytokines. Formulation of a compound may also include a) instant release formulations, b) gastroprotected (enteric coated formulations) to enable

AND-8001-PC delivery to a particular location in the GI immune system, b) dimers and multimers of an active compound, etc.

[00015] In the context of this invention, a "liquid composition" refers to one that, in its filled and finished form as provided from a manufacturer to an end user (e.g., a doctor or nurse), is a liquid or solution, as opposed to a solid. Here, "solid" refers to compositions that are not liquids or solutions. For example, solids include dried compositions prepared by lyophilization, freeze-drying, precipitation, and similar procedures.

[00016] A molecule, agent, or other compound that provides neuroprotecction, i.e., a "neuroprotective agent", refers to a molecule, or collection of different species of molecules (e.g., COPAXONE®) capable of protecting nerve cells from damage, including death, demyelination of at least a portion of one or more axons, apoptosis, deleterious modifications of cellular components (e.g., alkylation of DNA), etc. when administered in a therapeutically effective amount. A particularly preferred form of neuroprotection is the protection of neurons from demyelination. Here, "protection" refers to reducing neuronal demyelination by at least 5%, preferably by at least 25%, 50%, 75%, 80%, 85%, 90%, 95% or more, as compared to the level of demyelination observed in a control assay of the same type performed in the absence of the neuroprotective agent. As will be appreciated, the term "neuroprotective agent" is not necessarily meant to characterize a mechanism of action, but is meant to connote that the molecule termed a "neuroprotective agent" is a different molecular species than an immunopeptidic molecule.

[00017] In the context of peptides, the term "pan HLA-DR binding" refers to a an immunopeptidic molecule (e.g., a peptide) capable of binding to a T cell in the context of a class II MHC molecule regardless of the HLA-DR class expressed by the cell. While such immunopeptidic molecules may interact preferentially with cells expressing particular HLA-DR class, they also exhibit appreciable binding with other HLA-DR classes. Here, what constitutes "appreciable binding" will depend on context. For example, in the context of T cell isolation, appreciable binding refers to the capacity to recover a sufficient number of T cells from a biological sample for the intended purpose. In the context of immunization, appreciable binding refers to the capacity of an immunopeptidic molecule to generate a protective T cell response in a subject to whom

AND-8001-PC the peptide is administered. In the context of modulation of regulatory T cell activity, appreciable binding refers to the capacity of an irnmunopeptidic molecule to modulate regulatory T cell activity, for example, to convert a ThI response to a Th2 response or vice versa, as may be desired in the particular context.

[00018] A "patentable" composition, process, machine, or article of manufacture according to the invention means that the subject matter satisfies all statutory requirements for patentability at the time the analysis is performed. For example, with regard to novelty, non-obviousness, or the like, if later investigation reveals that one or more claims encompass one or more embodiments that would negate novelty, non- obviousness, etc., the claim(s), being limited by definition to "patentable" embodiments, specifically exclude the unpatentable embodiment(s). Also, the claims appended hereto are to be interpreted both to provide the broadest reasonable scope, as well as to preserve their validity. Furthermore, if one or more of the statutory requirements for patentability are amended or if the standards change for assessing whether a particular statutory requirement for patentability is satisfied from the time this application is filed or issues as a patent to a time the validity of one or more of the appended claims is questioned, the claims are to be interpreted in a way that (1) preserves their validity and (2) provides the broadest reasonable interpretation under the circumstances.

[00019] A "peptide" means a polymer of two or more amino acid residues linked via peptide bonds. Peptides may include naturally occurring or synthetic amino acids. Peptides may also be derivatized, for example to include one or more additional chemical moieties at one or more positions in the molecule. For example, if desired modifications may be introduced at the amino- and/or carboxy-terminus of the peptide, as well as to one or more of the side chains of one or more of the amino acid residues comprising the peptide. While peptides are usually linear molecules, the invention also envisions cyclic peptides. Cyclic peptides include those wherein the termini of the molecule are linked directly or through a linker moiety, as well as those wherein one terminus is attached (directly or through a linker moiety) to another amino acid residue of the peptide, other than the other terminus. A peptide has a "naturally occurring amino acid sequence" when the sequence of amino acids that comprise the peptide are found as contiguous amino acid residues in a naturally occurring protein or polypeptide. Thus, a "non-naturally occurring

AND-8001-PC amino acid sequence" refers to a sequence of contiguous amino acids that is not known to exist in a naturally occurring protein or polypeptide. In the context of the invention, it will be appreciated that a peptide that has a naturally occurring amino acid sequence, by definition, contains fewer than all of the amino acid residues (in terms of length, i.e., number of residues) found in the naturally occurring protein or polypeptide from which the peptide (or its sequence) is derived.

[00020] A "peptidomimetic" refers to a synthetic molecule that mimics one or more biological activities of a peptide according to the invention. In the context of the invention, a peptidomimetic is a molecule that specifically interacts with class II MHC molecules, i.e., it is a class II MHC agonist or antagonist, that are also reactive with the peptide upon which the peptidomimetic is based. Preferably, a peptidomimetic will possess at least about 1%, 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or more of the MHC binding and/or modulating activity of the peptide upon which the particular peptidomimetic is based or derived. Peptidomimetics may provide various advantages over peptides, for example, chemical stability in the gastrointestinal tract, which can facilitate oral administration of a composition comprising the peptidomimetic.

[00021] The term "pharmaceutically acceptable salt" refers to salts which retain the biological effectiveness and properties of the compounds of this invention and which are not biologically or otherwise undesirable. In many cases, the compounds of this invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids, while pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. For a review of pharmaceutically acceptable salts see Berge, et a ((1977) J. Pharm. ScI, vol. 66, 1). The expression "non-toxic pharmaceutically acceptable salts" refers to non-toxic salts formed with nontoxic, pharmaceutically acceptable inorganic or organic acids or inorganic or organic bases. For example, the salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, fumaric, methanesulfonic, and

AND-8001-PC toluenesulfonic acid and the like. Salts also include those from inorganic bases, such as ammonia, hydroxyethylamine and hydrazine. Suitable organic bases include methylamine, ethylamine, propylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, ethylenediamine, hydroxyethylamine, morpholine, piperazine, and guanidine.

[00022] A "plurality" means more than one.

[00023] The terms "separated", "purified", "isolated", and the like mean that one or more components of a sample contained in a sample-holding vessel are or have been physically removed from, or diluted in the presence of, one or more other sample components present in the vessel. Sample components that may be removed or diluted during a separating or purifying step include, chemical reaction products, unreacted chemicals, proteins, carbohydrates, lipids, and unbound molecules.

[00024] The term "species" is used herein in various contexts, e.g., a particular species of immunopeptidic molecule, neuroprotective agent, or a T cell migration-inhibitory response-associated molecule. In each context, the term refers to a population of chemically indistinct molecules of the sort referred in the particular context. For example, a "peptide species" is a population of peptides having the same amino acid sequence and chemical composition.

[00025] "Specifically associate", "specific association," and the like refer to a specific, non-random interaction between two molecules, which interaction depends on the presence of structural, hydrophobic/hydrophilic, and/or electrostatic features that allow appropriate chemical or molecular interactions between the molecules.

[00026] Herein, "stable" refers to an interaction between two molecules (e.g. , an immunopeptidic molecule such as a peptide and an MHC molecule) that is sufficiently stable such that the molecules can be maintained for the desired purpose or manipulation. For example, a "stable" interaction between a peptide and an MHC molecule refers to one wherein the peptide remains associated with the MHC molecule in the peptide :MHC complex in a manner that allows a T cell expressing a T cell receptor specifically reactive against the particular peptide:MHC complex to bind thereto.

AND-8001-PC [00027] A "subject" or "patient" refers to an animal in need of treatment that can be effected by molecules of the invention. Animals that can be treated in accordance with the invention include vertebrates, with mammals such as bovine, canine, equine, feline, ovine, porcine, and primate (including humans and non-humans primates) animals being particularly preferred examples.

[00028] A "T cell migration inhibitor" refers to an molecule that inhibits migration of T cells, or particular subsets of T cells, (e.g., a T cell educated for either a ThI, Th2 or Th3 type response), into tissue. In preferred embodiments, T cell migration inhibitors comprise an antibody or antibody-like molecule that can bind to cell adhesion molecule (o ligand therefore) present on the surface of endothelial cells, including alpha 4-integrin, to provide for either selective or not selective inhibition of T cell migration, wherein said selectivity is in the type response a T cell is educated, (i.e. ThI, Th2, or Th3).

[00029] A "therapeutically effective amount" refers to an amount of an active ingredient sufficient to effect treatment when administered to a subject in need of such treatment. In the context of immunology, a "therapeutically effective amount" is one that produces an objectively measured change in one or more immunological parameters, including an increase or decrease in cytokine expression, expansion of one or more classes of B or T cells, production of antibodies, immunity against a pathogen, e.g., adenovirus. Of course, the therapeutically effective amount will vary depending upon the particular subject and condition being treated, the weight and age of the subject, the severity of the disease condition, the particular compound chosen, the dosing regimen to be followed, timing of administration, the manner of administration and the like, all of which can readily be determined by one of ordinary skill in the art. It will be appreciated that in the context of combination therapy, what constitutes a therapeutically effective amount of a particular active ingredient may differ from what constitutes a therapeutically effective amount of the active ingredient when administered as a monotherapy (i.e., a therapeutic regimen that employs only one chemical entity as the active ingredient).

[00030] The term "treatment" or "treating" means any treatment of a disease or disorder, including preventing or protecting against the disease or disorder (that is, causing the clinical symptoms not to develop); inhibiting the disease or disorder (i.e., arresting or suppressing the development of clinical symptoms; and/or relieving the

AND-8001-PC disease or disorder (i.e., causing the regression of clinical symptoms). As will be appreciated, it is not always possible to distinguish between "preventing" and "suppressing" a disease or disorder since the ultimate inductive event or events may be unknown or latent. Accordingly, the term "prophylaxis" will be understood to constitute a type of "treatment" that encompasses both "preventing" and "suppressing". The term "protection" thus includes "prophylaxis".

[00031] One embodiment of this invention is to provide patentable multi-branched therapies for the treatment of demyelinating and other immune-mediated diseases. In particular, these therapies involve the administration of at least two different active ingredient species, wherein at least one of which is an immunopeptidic molecule that provides for epitope-specific T cell modulation and the other of which provides neuroprotection. One or more additional active therapeutic ingredients may also be included in the instant methods, if desired. Examples of such additional therapeutic ingredients include migration inhibitors and cytokines. Using such a multi-branched therapy provides for treatment of MS, RA, IBD, and other such immune-mediated diseases (e.g., cancer, viral and bacterial infections, etc.) with improved patient outcomes and with fewer compounding side effects. Additional advantages of the invention are provided as will be understood by one of ordinary skill in the art.

[00032] Thus, in a first aspect the invention concerns patentable multi-branched therapies for treating a demyelinating disease, especially those resulting from autoimmunity, particularly MS. Such methods involve the administration to patients suffering from a demyelinating disease of a therapeutically effective amount of an immunopeptidic molecule that provides for epitope-specific T cell modulation and a therapeutically effective amount of any of a neuroprotective agent and/or a T cell migration-inhibitory response-associated molecule. If desired, additional medicines may also be delivered in conjunction with these compositions.

[00033] The compositions used to effect the therapeutic methods of the invention include those wherein the immunopeptidic molecule (or, in some embodiments, a nucleic acid encoding the same) and neuroprotective agent are present in the same pharmaceutical composition. Alternatively, the active ingredients may be prepared as separate pharmaceutical compositions, which compositions may then be administered

AND-8001-PC together or sequentially to effect treatment. In general, a pharmaceutical composition comprises the desired active ingredient(s) and a pharmaceutically acceptable carrier. Such compositions are stored in suitable containers, which are then preferably packaged, most preferably with instructions for use and such other information as may be desired or required.

[00034] In preferred embodiments, the imniunopeptidic molecule is a peptide or peptidomimetic that is capable of binding class II MHC molecules, including those such as HLA DRl class II MHC molecules, in an epitope-specific manner. In some embodiments, the immunopeptidic molecule has pan HLA-DR binding activity. In other preferred embodiments, the immunopeptidic molecule is capable of inducing innate and acquired immune responses by interacting with peptide or epitope-specific inflammatory and/or regulatory T cells. In particularly preferred embodiments, the immunopeptidic molecule, when introduced to a patient in need of a reduction in inflammation and/or a stimulation of regulatory T cells educated for a Th2 or Th3 response, elicits, alone and/or in combination with other compounds {e.g., one or more cytokine and/or anti-cytokine (i.e., a molecule that inhibits a biological activity of a specified cytokine) species), T cell responses of the regulatory (i.e., Th2 and/or Th3) type and suppressor type T cells. responses

are beneficial in treatment of MS, IBD, /I^nd RA. Examples of immunopeptidic molecules include peptides derived from stress proteins, particularly heat shock proteins such as hspόO, hsp65, hsp70, and dnaJ from various organisms, including mammals and bacteria. Representative examples of such immunopeptidic molecules peptides are described in Table I₅ below.

[00035] In preferred embodiments, the neuroprotective agent is any molecule suitable for use a therapeutic agent and that, alone or in combination with a chemically distinct molecule or compound, is neuroprotective.

[00036] T cell migration inhibitors, where used, can comprise antibodies, either polyclonal or monoclonal, capable of binding adhesion molecules such as alpha A- integrin, and, by such binding, inhibit the capability of T cells to bind to the cells lining the inner surface of blood vessels, thereby inhibiting T cell migration, either in general or with respect to particular T cell subtypes, e.g., T cells that exhibit a ThI, Th2, or Th3 activity.

AND-8001-PC [00037] In preferred embodiments, the instant methods provide for continuing regulation of immunity and control of disease manifestations through the modulation (i.e., up or down, as the context requires) regulation of T cell activity, particularly of regulatory T cell activity. In further related embodiments, the methods of the invention provide amelioration of a demyelinating disease in an antigen-specific fashion, while reducing the need for conventional therapies, many of which are toxic, non-specific, uncontrolled, and expensive.

[00038] Other embodiments contemplate regulation of cytokine-mediated responses related to inflammatory and/or tolerogenic responses associated with immunomodulatory cytokines including, but not limited to, TNFα, TNF β, IFNα, IFN β or IFN γ, consensus IFN, IL-I, IL-4, IL-6, IL-IO₅ IL-15, and IL-23.

[00039] In a further embodiment, the multi-branched therapy of the invention provides for modulating a pathogenic immune response correlated with a demyelinating or other immune-mediated disease such as RA or IBD, such that the response reverts to the point in time of immune response wherein antigen-specific events are dominant and potentially relevant to disease outcome. By "dominance" of antigenic events is meant that the influence of such events is relevant enough to trigger, perpetuate, or affect in any way disease pathogenesis and outcome. By "potentially relevant" is meant that the influence is theorized but not proven, and by "relevant" is meant that there is evidence to support a role in the pathogenesis. In a related embodiment, the combination therapy of the invention enables intervention of antigen-specific events to permanently and specifically induce desired antigen-specific modulation. In a particularly preferred embodiment, such modulation can lead to clinically detectable amelioration of the demyelinating disease (e.g., MS) or other immune-mediated disease characterized by debilitating inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

[00040] Figure 1 is a graph depicting average clinical scores in EAE Lewis rats using one of three different amounts of an M. tuberculosis hsp60-derived peptide in monotherapy, as compared to conventional IFN-β therapy or a no treatment control. Peptides were administered intranasally for five consecutive days, starting on day 7 post¬ infection. IFN-β treatment was administered subcutaneously for four days starting on day

AND-8001-PC 7 post-infection. The "no treatment" controls received 10 μL of sterile distilled water per nostril for five consecutive days. Clinical scores were evaluated daily. Mean and standard deviations are represented.

[00041] Figure 2 is a bar graph depicting 15-day post-infection histological scores from EAE Lewis rats treated with HSP-derived peptide M. tuberculosis hsp60 507-521, (peptide P7; 300μg), IFN-β, or nothing.

[00042] Figure 3 A-D depict bar graphs of induction of IL-10, IFN-γ, TGF-β, and Foxp3 gene transcription as detected by TaqMan® as a result of treatment the immunomodulatory M. tuberculosis hsp60-derived 507-521 peptide. Numbers indicate the mean induction index.

[00043] Figure 4 depicts the general experimental protocol for monotherapy and combination therapy as used in experimental examples showing induction of EAE and treatment thereof in Lewis rats using immunomodulatory peptides and Copaxone.

[00044] Figure 5 is a graph showing clinical scores following treatment with immunomodulatory peptides in monotherapy and combination therapy in EAE.

[00045] Figure 6 is a graph indicating AUC or area under the curve, of clinical scores during treatment of EAE.

[00046] Figure 7 is a graph showing histological data that indicates a reduction of lesion severity by the combination therapy of hsp peptide and Copaxone.

[00047] Figures 8 A and B is a graph showing immunological data of clinical efficacy results brought about by immuno-deviations driven by T cell regulatory pathways. The graph shows an increase in FoxP3 and decrease of IFN-γ transcription.

[00048] Figure 9 is a graph showing immunological data regarding clinical efficacy from immuno-deviations driven by T cell regulatory pathways. Here, induction of FoxP3 in mandibular lymph node CD4+CD25+ T cells following intranasal treatment with HSP- derived peptide Rat-P2 is provided.

AND-8001-PC [00049] Figures 1OA and B show a graph of immunological data regarding clinical efficacy from immuno deviation driven by T cell regulatory pathways wherein there is a decrease of inflammatory and increase of regulatory cytokine levels. Figure 1OA is TNF- α and Figure 1OB is IL-10.

[00050] Figure 11 is a graph showing that immunological data regarding humoral response to an unrelated antigen is preserved after toleragenic treatment.

DETAILED DESCRIPTION OF THE INVENTION

[00051] As those in the art will appreciate, the following description describes certain preferred embodiments of the invention in detail, and is thus only representative and does not depict the actual scope of the invention. Before describing the present invention in detail, it is understood that the invention is not limited to the particular molecules (e.g., peptides and peptidomimetics), systems, and methodologies described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention defined by the appended claims.

1. Overview.

[00052] The present invention concerns patentable multi-branched therapies for the treatment of various demyelinating and other immune-mediated diseases, particularly those that are correlated with pathogenic autoimmune activity, sometimes associated with degenerative inflammatory immune responses in select tissue. These therapies are based on the administration of at least two active ingredients, namely, at least one immunopeptidic molecule and a neuroprotective agent, to a patient suffering from or predisposed to develop a demyelinating disease, for example, MS, or another such immune-mediated disease including, but not limited to RA and IBD. These non-toxic therapies will result in the modulation of pathogenic autoimmune responses in such patients, thereby enhancing clinical outcomes. The description below describes the invention in more detail.

[00053] Science has shown certain peptides derived from heat shock proteins can play a role in amplifying autoimmune inflammation. Endogenous and bacterial hsps (as well

AND-8001-PC as those from other microorganisms that parasitize humans) are recognized by the immune system as indicative of an aberrant condition (e.g., an infection), and thus they elicit a pro-inflammatory physiologic response. While such responses contribute to clearing of a potential pathogen, hsp-induced pro-inflammatory stress responses also have the effect of inducing expression of endogenous hsps in the host, thereby increasing the availability of self-hsp proteins and subsequent presentation of peptides from such self proteins to the immune system. Indeed, in MS, for example, human heat shock proteins, including human dnaJ, are expressed in the CNS, and their expression increases under inflammatory conditions. In normal individuals, such peptides are recognized by T cells with regulatory function; however, in autoimmunity, including in MS, RA*&nd IBD, ' regulatory T cell function is impaired. Restoration of regulatory function, by delivery of immunopeptidic molecules and relief from immune-mediated attack of, for example in the case of MS, the myelin sheaths of nerve cell axons, or intestinal lining cells in the case of IBD, or tissues in joints in the case of RA, can result in the treatment and prevention of de-myelinating diseases such as MS.

2. Immunopeptidic Molecules.

[00054] The multi-branched therapy regimen of the invention relies, in part, on the use of immunopeptidic molecules specific for a demyelinating disease of interest rather than conventional methods that apply non-epitope-specific approaches. This epitope specificity achieves specific immune modulation of a host's pathophysiology that is directly related to the particular disease. By such epitope-specific therapy, a change in the absolute numbers of peptide-specific T cells can be determined, as can the type and quality of the induced T cell response. Specifically, controlled immune modulation can be engineered using a therapy regimen that provides modulation of a host's pathophysiology. "Immunopeptidic molecule" refers generally to a peptide or a peptidomimetic or multimers thereof as disclosed herein. For example, a peptidomimetic peptide comprises a chemically modified small molecule, including polymers of such molecule, having the activity described in the assay. Such activity is related to the induction of immune deviation. Formulations may comprise the active compound in combination with biologies, cytokines, anti-cytokines. Formulation of a compound may also include a) instant release formulations, b) gastroprotected (enteric coated

AND-8001-PC formulations) to enable delivery to a particular location in the GI immune system, b) dimers and multimers of an active compound, etc.

[00055] The present invention uses immunopeptidic molecules, for example, peptides, that modulate T cell responsiveness due to their binding and presentation of by one or more species of MHC class II molecules. In preferred embodiments, increased presentation of these molecules (by virtue of their delivery as part of therapeutic regimen according of the invention) results in increased immunological recognition and subsequent modulation (i.e., down-regulating, up-regulating, or shifting the ratio of Thl:Th2/Th3 effector molecules produced) of the immune response to modulate inflammation, preferably by down-regulating the immune response to decrease inflammation correlated with a demyelinating and other immune-mediated diseases, for example, MS, RA, and IBD.

[00056] In the context of this invention, immunopeptidic molecules are employed modulate, block, or inhibit inflammatory responses. The immunopeptidic molecules act to modulate (i.e., either down-regulate, up-regulate, or shift the ratio of Thl:Th2 effector molecules produced) particular lymphocytes. In either case (i.e., of up- or down- regulation of inflammation), the specific response is regulated by the intracellular processing and recognition of the particular antigen by effector T-cells. Mature cytotoxic T lymphocytes (CTLs) or T helper cells (Th) remain in a resting state unless they encounter antigens that their receptors (the so-called T cell receptors, or TCRs) recognize in the context of MHC class I or II molecules. Upon encountering a particular antigen, T- cells whose TCRs specifically recognize that antigen (when presented in the context of the appropriate MHC class I or class II molecule) proliferate and perform effector functions, the ultimate of goal of which is elimination of the reactive antigens. When the antigen is processed through the cytoplasmic route (typically due to intracellular synthesis), the resultant peptides are bound to nascent MHC class I molecules which facilitate appropriate presentation to effector T-cells. MHC class I presentation favors recognition by CTLs that carry the CD8 ligand. In contrast, intracellular processing via the endocytic route (due to endocytosis, of, for example, a virus or bacteria) results in presentation on MHC class II molecules, which mode of processing favors T helper responses involved in stimulation of the humoral arm.

AND-8001-PC [00057] T cell activation entails generating a series of chemical signals (primarily cytokines) that directly stimulate other cells of the immune system to act. In the case of activation by class I MHC-peptide complexes, CTLs proliferate and act to destroy cells (e.g., cells infected with a virus or intracellular parasite, or aberrant cells) presenting that given antigen. In the context of intracellular pathogens, destruction of the host cell prevents the pathogen from proliferating and makes it accessible to neutralizing antibodies, hence permitting its elimination. In contrast, activation of Th cells by class II MHC-peptide complexes does not destroy the antigen presenting cell but rather stimulates the Th cell to proliferate and generate signals (again, primarily cytokines) that affect various cells. Among other consequences, the signaling leads to B cell stimulation, macrophage activation, CTL differentiation, and promotion of inflammation. This concerted response is relatively specific and is usually directed to foreign elements bearing the peptide presented by the class II MHC system. Restoration of activity of antigen-specific regulatory T cells (Treg cells) activity is also believed to play a critical role in immune-mediated treatments of autoimmunity.

[00058] In order to effect the therapeutic methods of the invention, an immunopeptidic molecule must be delivered to restore regulatory T cell function, preferably via mucosal tolerization. In general, these molecules mimic antigenic epitopes presented on heat shock proteins, and they bind to T cell receptors. Preferably, these molecules are recognized by the T cells of a high percentage of patients as a pro-inflammatory T cell epitope, and thus will be expected to be useful across a large percentage of a given patient population. In some embodiments, prospective patients may be pre-screened to determine whether they exhibit immune reactivity against the immnuopeptidic molecule used in the particular therapy. A treatment according to the invention may then be administered to patients exhibiting such immune reactivity.

[00059] Additionally, induction of an epitope-specific immune response provides for the subsequent generation, in vivo and/or ex vivo, of T cells capable of modulating immune responses. Examples of approaches for the epitope-specific arm of the instant combination therapy include various approaches to induce modulation, in vivo and ex vivo, of epitope-specific responses including, but not limited to tolerization to an immunomodulatory peptide via mucosal (e.g., nasal or oral) tolerization; boosting of

AND-8001-PC epitope-specific immune responses via subcutaneous, intravenous, or intramuscular immunizations with immunopeptidic molecules (including nucleic acid molecules (e.g., expression vectors) encoding peptide-based immunopeptidic molecules; regulation of epitope-specific responses by expansion in vivo and ex vivo of epitope-specific regulatory T cells; and modulation ex vivo of epitope-specific T cells through the use of artificial antigen presenting cells with or without conditioning environment. See, for example, U.S. pat. No. 6,787,154. By "conditioning environment" is meant a microenvironment in which T cell responses may be modulated by adding to the culture medium soluble mediators (e.g., cytokines), and/or by soluble or support bound molecules (e.g., co- stimulatory molecules) capable of inducing a desired T cell response.

_3. Peptides.

[00060] In preferred embodiments, the immunopeptidic molecule used in a multi- branched therapy according to the invention is a peptide molecule. In other embodiments, they are peptidomimetic molecules. With regard to peptides, they may be identified by any suitable method. A preferred method for identifying one or more peptides that modulate, and thus restore, regulatory T cell function in the context of a particular demyelinating disease employs a series of computer-based (i.e., in silicό) and in vitro studies. Initially, a pool of one or more human and one or more bacterial heat shock protein amino acid sequences are analyzed to identify peptide sequences likely to bind to class II MHC molecules, preferably a majority (and even all) types of class II MHC molecules. As will be appreciated, because of differences between class II MHC molecules in human (where MHC molecules are referred to as "HLA" (human leukocyte antigen) molecules) and those of animals likely to be used during pre-clinical testing, different peptides may be identified for use in animal testing than would be used in the context of human treatment.

[00061] Recent developments have allowed the identification of MHC allele-specific peptide binding motifs. Accordingly, motifs for peptide binding to one or more MHC class I and class II molecules can be defined by sequence analysis of naturally processed peptides and by mutational analysis of known epitopes. MHC class I bound peptides (generally produced from proteins expressed within the antigen-presenting cell) have been found to be short (generally 8-10 amino acids in length) and to possess two

AND-8001-PC dominant MHC anchor residues. In contrast, peptides that bind to MHC class II molecules (which peptides tend to be derived from extracellular material (e.g., a bacteria or another cell) endocytosed by the antigen-presenting cell) have been determined to generally be longer and more heterogeneous in size. X-ray crystallographic analysis of co-crystallized peptides and HLA molecules has revealed that there is a dominant hydrophobic anchor residue close to the N-terminus of the MHC class II-binding peptide and that secondary anchor residues are found at several other peptide positions.

[00062] Preferably, the methods of the invention utilize peptides (and other immunopeptidic molecules) that contain an epitope that can be specifically bound by at least, preferably several, and most preferably all, of the various types of MHC class II molecules. Peptides (and other immunopeptidic molecules) that may be bound by at least two, and perhaps all, MHC class II molecule species are referred to as "pan-DR binding molecules." Pan-DR binding immunopeptidic molecules fit into the peptide binding cleft of various species of MHC class II molecules, and thus may be recognized by T cells in the vast majority of subjects. Pan-DR binding peptides can be identified computationally, as described in U.S. pat. No. 6,037,135. See also published U.S. patent application publication nos. 20020146759, 20030031679, 20030143238, and 20030147910. Briefly, such peptides may be identified, for example, by scanning heat shock proteins from bacterial and human hsps, including hsp60 and dnaJ, for regions that bind three HLA DR subtypes (e.g., DRl, DR4, and DR7). Peptides so identified, as well as other immunopeptidic molecules, can then be assayed in vitro to determine whether they have the ability to induce the proliferation of autoreactive T cells, to induce the secretion of cytokines (e.g., lymphokines) from T cells, or to induce other effector functions such as cytotoxicity. T cell activation entails generating a series of chemical signals (primarily cytokines) that directly stimulate other cells of the immune system to act. In the case of activation by class I MHC-peptide complexes, CTLs proliferate and act to destroy cells (e.g., cells infected with a virus or intracellular parasite, or aberrant cells) presenting that given antigen. In the context of intracellular pathogens, destruction of the host cell prevents the pathogen from proliferating and makes it accessible to neutralizing antibodies, hence permitting its elimination. In contrast, activation of Th cells by class II MHC-peptide complexes does not destroy the antigen presenting cell but rather stimulates the Th cell to proliferate and generate signals (again, primarily cytokines) that affect

AND-8001-PC various cells. Among other consequences, the signaling leads to B cell stimulation, macrophage activation, CTL differentiation, and promotion of inflammation. This concerted response is relatively specific and is usually directed to foreign elements bearing the peptide presented by the class II MHC system. Restoration of activity of antigen-specific regulatory T cells (Treg cells) activity is also believed to play a critical role in immune-mediated treatments of autoimmunity.

[00063] In preferred embodiments, the peptides are assayed in vitro using cells (for example, peripheral blood mononuclear cells) obtained from patients known to suffer from the disease ultimately to be treated. Immune responses are measured to assess the peptide's ability to trigger statistically significant increases in the production of cytokines correlated with the desired response, alone or in conjunction with increasing proliferation of desired cell types. For example, in the context of reducing or controlling inflammation via antigen- (e.g., peptide-) specific immunotherapy achieved via mucosal tolerization, production of cytokines that effect an pro-inflammatory response in T cells in patients suffering from the particular disease, as may be assessed, for example, by measuring the production of cytokines that effect a pro-inflammatory response, can be assessed. Examples of cytokines that effect a pro-inflammatory response are IFN-γ and TNF-α, whereas IL-4 and IL-10 are known to effect anti-inflammatory, or regulatory, responses. Other markers indicative of a particular response, i.e., an anti-inflammatory response or a pro-inflammatory response, can also be measured. The resulting data is then stratified with clinical data, including disease activity, disease subpopulation, etc., and a coefficient of correlation is then calculated for the peptide to identify whether there is a meaningful correlation between the immunological data generated using the peptide and the clinical manifestations of the disease. Peptides (and other immunopeptidic molecules) that exhibit meaningful correlations are then selected for further development.

[00064] Peptide-based immunopeptidic molecules may be prepared in a variety of ways. Conveniently, they can be synthesized by conventional techniques employing automatic synthesizers, or may be synthesized manually. Automated solid phase synthetic methods are particularly preferred. Alternatively, DNA sequences can be prepared which encode the particular peptide, which may be cloned and expressed to provide the desired peptide. In this instance a methionine may be the first amino acid. In

AND-8001-PC addition, peptides may be produced by recombinant methods, including by fusion to proteins that are one of a specific binding pair, allowing purification of the fusion protein by means of affinity reagents, followed by proteolytic cleavage, usually at an engineered site to yield the desired peptide (see for example Driscoll et al. (1993) J. MoI. Bio. 232:342-350). The peptides may also be isolated from natural sources and purified by known techniques, including, for example, chromatography on ion exchange materials, separation by size, immuno-affinity chromatography and electrophoresis.

Table 1, below, lists several peptides that useful in the practice of the invention.

Table 1

AND-SOOl-PC

4. Nucleic Acids.

[00065] When a peptide is intended to be used the immunotherapeutic molecule, certain advantages may be obtained by administering it in polynucleotide form for subsequent expression. For example, the risk of potential toxicity (e.g., anaphylactic shock) associated with protein and peptide administration may be avoided if a polynucleotide encoding the peptide is administered, after which the peptide may be expressed in vivo. Herein, "polynucleotide" refers to a single- or double-stranded DNA or RNA molecule, in the form of a separate fragment or as a component of a larger construct, for example, an expression vector. Expression vectors encoding peptides useful in practicing the invention. Such polynucleotides should also be either non- replicating or engineered by means well known in the art so as not to replicate into the host genome.

[00066] Coding nucleotide sequences for peptides of interest in the invention may be readily determined (if not known) by deduction from the amino acid sequence of the peptide, taking into account the degeneracy of the bacterial and human genomes. If desired, the coding regions may be cloned from naturally occurring sources. Alternatively, and in any event with regard to peptides that do not reflect an amino acid sequence found in nature, the coding polynucleotide may be synthesized using techniques and nucleic acid synthesis equipment well-known in the art. For references in these regards, see Ausubel, et al. , Current Protocols in Molecular Biology, chapters 2 and 4 (Wiley Interscience, 1989); and Maniatis, et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., New York, 1982).

[00067] Expression vectors are preferably plasmids or cosmids that operatively encode a peptide-encoding polynucleotide, but may also be viruses or retroviruses. Preferably, the vectors are "naked", i.e., they are not associated with a delivery vehicle (e.g.,

AND-8001-PC liposomes, colloidal particles, and the like). "Operatively encodes" refers to a recombinant vector that includes all of the regulatory sequences {e.g., promoters) required for expression of the encoded peptide in suitable host cell.

[00068] For administration, peptide-encoding expression vectors may be combined with a carrier such as saline or, less desirably, may be administered with a delivery vehicle, such as a liposome or colloidal particles. Methods for preparation and use of such delivery vehicles are well-known to those of ordinary skill in the art.

5. Peptidomimetics.

[00069] Another class of immunopeptidic molecules are peptidomimetic molecules. Such molecules can be identified, for example, by first identifying a peptide that binds to a T cell receptor presented on a T cell population involved in MS, RA, or IBD autoimmunity, for instance. The gene coding for the particular T cell receptor can then be cloned and expressed using standard techniques, and the receptor may then be used as a reagent to screen diverse chemical libraries to identify molecules that bind to the receptor. Alternatively, such molecules (or libraries of related molecules), including peptidomimetics, may be designed after performing various structure-activity relationship analyses. Indeed, moderate- to high-resolution models of a peptide and TCR can be generated using well-known techniques (for example, X-ray crystallography) to determine the approximate atomic structure that a TCR-binding molecule that mimics the peptide should possess. One or more species of such molecules may then synthesized using suitable methods.

[00070] With respect to the immunopeptidic molecule, the methods of the invention contemplate administrating to a subject in an appropriate delivery system a therapeutically affective amount of an agent that is capable of acting in an epitope- specific manner, i.e., is to a degree disease specific. For example, with respect to MS, administration to a patient of peptides comprising amino acid sequence of any of Seq. ID Nos. 1 to 31 and particularly peptides having an amino acid sequence as set forth in Seq Id. Nos. 1, Seq. ID No. 8 (AT008) or Seq. ID No. 16 provides for a regulatory response wherein a Th2 response is enhanced specifically with respect to MS relevant disease. Thus, when such an immunopeptidic molecule is administered to a patient susceptible to

AND-8001-PC MS or an MS-like clinical presentation, in combination with a neuroprotective agent or additionally, if desired, a T cell migration-inhibitory response-associated molecule, such regimens provides for enhancement of a beneficial therapeutic outcome.

6. Neuroprotective Agents.

[00071] Preferred neuroprotective agents useful in the practice of the invention include complex mixtures of synthetic copolymers, a representative example of which is copolymer- 1. Copolymer- 1 (also known as glatiramer acetate) is a random mixture of synthetic polypeptide analog of myelin basic protein (MBP), which is a natural component of the myelin sheath. Interest in copolymer- 1 as a neuroprotective agent for multiple sclerosis stems from observations first made in the 1950's that myelin components such as MBP prevent or arrest experimental autoimmune encephalomyelitis (EAE). EAE is a disease resembling multiple sclerosis that can be induced in susceptible animals. Copolymer- 1 and related compounds, synthesis methods, and compositions containing such compounds are described in a number of U.S patents, including patent nos. 6,800,285; 6,645,528; 6,620,847; 6,531,464; 6,362,161; 6,342,476; 6,214,791; 6,083,534; 6,054,430; 6,048,898; 5,981,589; 5,800,808; 5,668,117; and 3,849,550. See also Shukaliak Quandt, et al. (2004), MoI. Immunol, vol. 40: 1075-1087.

[00072] Copolymer- 1 is a random complex mixture of polypeptides composed of alanine, glutamic acid, lysine, and tyrosine in a molar ratio of approximately 6:2:5:1, respectively. U.S. patent no. 6,800,285 reports copolymer-1 to have an average molar fraction of L-glutamic acid: 0.129-0.153; L-alanine: 0.392-0.462; L-tyrosine: 0.086- 0.100; and L-lysine: 0.300-0.376. Copolymer-1 is synthesized by chemically polymerizing the four amino acids forming products with average molecular weights of 23,000 daltons. Preferably, copolymer-1 compositions are substantially free (i.e., contain less than about 5%, preferably less than about 2.5%) of species of copolymer-1 having a molecular weight of over 40 kilodaltons (kDa). In such compositions, preferably over 75% of the copolymer molecules (measured in terms of molar fractions) have a molecular weight range from about 2 kDa to about 20 kDa. Preferred average copolymer-1 average molecular weights range from about 4 kDa to about 8.6 IcDa.

AND-8001-PC [00073] Copolymer- 1 and like molecules {i.e., complex mixtures of synthetic copolymer molecules, particularly those designed based on knowledge of binding motifs of immunodominant epitopes and binding pockets of DR molecules) may be prepared by any suitable method. One such method is described U.S. pat. No. 3,849,550, wherein the N-carboxyanhydrides of tyrosine, alanine, y-benzyl glutamate, and E-N-trifluoro- acetyllysine are polymerized at ambient temperature in anhydrous dioxane with diethylamine as initiator. The deblocking of the y-carboxyl group of the glutamic acid is effected by hydrogen bromide in glacial acetic acid and is followed by the removal of the trifluoroacetyl groups from the lysine residues by IM piperidine. Here, the term "ambient temperature" means a temperature ranging from about 20-26°C.

[00074] Copolymer- 1 with a desired molecular weight profile can be obtained by any suitable method, including chromatography, either before or after, if desired, partial acid or enzymatic hydrolysis to remove the high molecular weight species with subsequent purification by dialysis or ultrafiltration. A further method to obtain copolymer- 1 with the desired molecular weight profile is by preparing the desired species while the amino acids are still protected and then obtain the correct species directly upon removing the protection. Such copolymer- 1 compositions can be formulated by conventional methods. Preferably, the composition is lyophilized and stored until just prior to use. At that time, the dry composition is then reconstituted into an aqueous solution suitable for sub¬ cutaneous injection. Alternatively, copolymer- 1 -containing compositions may be formulated in any of the forms known in the art for preparing peptide drugs, preferably in dosage forms suitable for oral, nasal, buccal, or rectal administration. Typically, copolymer- 1 is administered to patients suffering from relapsing remitting multiple sclerosis, at a dosage of 20 mg per daily subcutaneous administration.

[00075] Another class of neuroprotective molecules that may be use in practicing the invention is antibodies or antibody fragments, including minibodies, diabodies, triabodies and tetrabodies and single domains, preferably monoclonal mouse or human antibodies, or "humanized" mouse monoclonal antibodies. Preferred examples of such antibodies are antibodies raised against copolymer- 1 or myelin basic protein that promote myelin repair and/or stimulate remyelination. Techniques for making monoclonal antibodies are widely known in art, and include, but are not limited to, the creation of hybridomas, synthetic or

AND-8001-PC phage-display based antibody libraries, antibody libraries, and humanized antibodies {see. E.g., U.S. patent no. 6,800,285).

7. T cell migration-inhibitors.

[00076] Preferred T cell migration inhibitors include any species of molecule capable of providing for the inhibition of T cell migration, whether as by masking one or more species of adhesion molecules involved in T cell attachment to epitopes on endothelial cell walls, such as for example, alpha 4 integrins, or by an immune modulatory route, in which case the inhibitor is capable of specifically inhibiting migration of T cells educated for any of a ThI, Th2, or Th3 response. Such specificity of inhibition is contemplated to arise from, for example, affecting stimulation of a cytokine-mediated responses related to inflammatory and/or tolerogenic responses associated with immunomodulatory cytokines including, but not limited to, TNFα, TNF β, IFNα, IFN β or IFN γ, consensus IFN, IL-I, IL-4, IL-6, IL-10, IL-15, and IL-23.

[00077] Preferred T cell inhibitors include antibodies and antibody fragments, receptor fragments, ligands, and other molecules capable of specific interaction with molecules presented on the surface of, for example, the endothelial cells present on the luminal surface of blood vessels. Preferably, such inhibitors will be selective for one or more particular T cell sub-populations. As will be appreciated, in the context of diseases characterized by excessive immune responses, it may be desired to prevent T cells that possess pro-inflammatory activity (e.g., ThI cells) from migrating from the vasculature into neighboring tissues where excess immunity occurs. On the other hand, in diseases where enhanced immunity could be advantageous, for example, cancer, pathogenic infections, etc., it may be desirable to inhibit (i.e., reduce or eliminate) the migration of T cells exhibiting anti-inflammatory activity (e.g., Th2 and/or regulatory T cells (i.e., those that are CD25+) into the affected tissue(s).

[00078] In a particularly preferred embodiment, it is preferred that treatment of demyelinating and other immune-mediated diseases include use of immunopeptidic molecules capable of providing for a tolerogenic response which is in part associated with reduction in activation of T cells and which is further related to a reduction and/or inhibition in T cell migration. In a particularly preferred embodiment, T cell educated

AND-8001-PC specifically for a ThI, Th2 or Th3 response can be specifically inhibited from migrating into inflammation susceptible tissues.

8, Compositions.

[00079] The composition(s) used in the practice of the invention may be processed in accordance with conventional methods of pharmaceutical compounding techniques to produce medicinal agents (i.e., medicaments or therapeutic compositions) for administration to subjects, including humans and other mammals, i.e., "pharmaceutical" and "veterinary" administration, respectively. See, for example, the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Typically, the immunopeptidic molecule and T cell migration inhibitor are included, together or as separate compositions, with a pharmaceutically acceptable carrier. The composition(s) may also include one or more of the following: preserving agents; solubilizing agents; stabilizing agents; wetting agents; emulsifiers; sweeteners; colorants; odorants; salts; buffers; coating agents; and antioxidants.

[00080] The immunopeptidic molecules, neuroprotective agents, and T cell migration- inhibitory response-associated molecules used in the practice of the invention may be prepared as free acids or bases, which are then preferably combined with a suitable compound to yield a pharmaceutically acceptable salt. The expression "pharmaceutically acceptable salts" refers to non-toxic salts formed with nontoxic, pharmaceutically acceptable inorganic or organic acids or inorganic or organic bases. For example, the salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, fumaric, methanesulfonic, and toluenesulfonic acid and the like. Salts also include those from inorganic bases, such as ammonia, hydroxyethylamine and hydrazine. Suitable organic bases include methylamine, ethylamine, propylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, ethylenediamine, hydroxyethylamine, morpholine, piperazine, and guanidine.

AND-8001-PC [00081] In any event, the therapeutic compositions are preferably made in the form of a dosage unit containing a given amount of an immunopeptidic molecule according to the invention and a carrier (i.e., a physiologically acceptable excipient). What constitutes a therapeutically effective amount of any such molecule for a human or other mammal (or other animal) will depend on a variety of factors, including, among others, the type of disease or disorder, the age, weight, gender, medical condition of the subject, the severity of the condition, the route of administration, and the particular compound employed. Thus, dosage regimens may vary widely, but can be determined routinely using standard methods. In any event, an "effective amount" of an immunopeptidic molecule is an amount that elicits the desired immune modulation (e.g., induction or enhancement of an immune response sought to be induced or enhanced or, alternatively, the reduction or prevention of an immune response sought to be reduced or prevented). The quantity of such a therapeutic molecule required to achieve the desired effect will depend on numerous considerations, including the particular molecule itself, the disease or disorder to be treated, the capacity of the subject's immune system to respond to the molecule, route of administration, and degree of immune modulation desired. Precise amounts of the molecule required to achieve the desired effect will depend on the judgment of the practitioner and are peculiar to each individual subject. However, suitable dosages may range from about several nanograms (ng) to about several milligrams (mg) of active ingredient per kilogram body weight per day. For oral administration, a dosage of the immunopeptidic molecule in the range of about 5-100 mg per dosage (e.g., administered daily in the form of a capsule). Preferred dosages for a peptide-based immunopeptidic molecule include about 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, and 50 mg per dose, with about 25 mg per dose being particularly preferred. Suitable regimens for initial administration and one or more booster administrations are also variable.

[00082] The preparation of therapeutic compositions is well understood in the art. Typically, such compositions are prepared as injectable, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients that are physiologically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water for

AND-8001-PC injection, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, anti-pyretics, stabilizing agents, thickening agents, suspending agents, anesthetics, preservatives, antioxidants, bacteriostatic agents, analgesics, pH buffering agents, etc. that enhance the effectiveness of the active ingredient. Such components can provide additional therapeutic benefit, or act towards preventing any potential side effects that may be posed as a result of administration of the pharmaceutical composition.

[00083] The compositions of the invention may be administered orally, parentally, by inhalation spray, rectally, intranodally, or topically in dosage unit formulations containing conventional carriers, adjuvants, and vehicles. In the context of therapeutic compositions intended for human administration, pharmaceutically acceptable carriers are used. The terms "pharmaceutically acceptable carrier" and "physiologically acceptable carrier" refer to molecular entities and compositions that are physiologically tolerable and do not typically produce an unintended allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a subject and are thus suitable for accomplishing or enhancing the delivery of an immunopeptidic molecule as a medicament.

[00084] For oral administration, the composition may be of any suitable form, including, for example, a capsule, tablet, lozenge, pastille, powder, suspension, or liquid, among others. Liquids may be administered by injection as a composition with suitable carriers including saline, dextrose, or water. The term "parenteral" includes infusion (including continuous or intermittent infusion) and injection via a subcutaneous, intravenous, intramuscular, intrasternal, or intraperitoneal route. Suppositories for rectal administration can be prepared by mixing the active ingredient(s) with a suitable non- irritating excipient such as cocoa butter and/or polyethylene glycols that are solid at ordinary temperatures but liquid at physiological temperatures.

[00085] The compositions may also be prepared in a solid form (including granules, powders or suppositories). The compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.

AND-8001-PC Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert excipient such as sucrose, lactose, or starch. Such dosage forms may also comprise additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings. Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting sweetening, flavoring, and perfuming agents.

[00086] Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Suitable vehicles and solvents that may be employed are water for injection, Ringer's solution, and isotonic sodium chloride solution, among others. In addition, sterile, fixed oils can be employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[00087] For topical administration, a suitable topical dose of a composition may be administered one to four, and preferably two or three, times daily. The dose may also be administered with intervening days during which no dose is applied. Suitable compositions for topical delivery often comprise from 0.001% to 10% w/w of active ingredient, for example, from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1% to 1% of the formulation. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes), and drops suitable for administration to the eye, ear, or nose.

[00088] Exemplary methods for administering the compositions of the invention (e.g., so as to achieve sterile or aseptic conditions) will be apparent to the skilled artisan.

AND-800I-PC Certain methods suitable for such purposes are set forth in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 7^th Ed. (1985). The administration to the patient can be intermittent; or at a gradual, continuous, constant, or controlled rate.

[00089] The invention also contemplates kits comprising compositions containing immunopeptidic molecules, neuroprotective agents, and/or T cell migration inhibitors, separately or together, stored in suitable containers (e.g., a glass vial, ampoule, device for inhalation (e.g., a blister pack, a metered dose inhaler, etc), or disposable injection device) packaged in a box in conjunction with a package insert describing, among other things, the disease or order for which the composition is used as a treatment and how to administer the composition. Containers may be designed for a single use (e.g., as a unit dose in a sealed vial to which water for injection may be added by a needle through, for example, a sealed rubber stopper), or for multiple uses. As will be appreciated, when a composition is intended for injection, the composition may be stored in dry form and is reconstituted using a suitable diluent just prior to administration. In such instances, a kit will also preferably include a container containing a suitable carrier, diluent, or excipient for reconstitution of the dry composition. Additionally, the kit can include instructions for mixing or combining ingredients and/or administration and such other information as may be desired or required by law.

9. Administration.

[00090] The present invention provides methods of treating or preventing a demyelinating disease or disorder, for example, multiple sclerosis, or an immune- mediated disease such as RA or IBD, cancer, or infection by a virus or bacteria in a subject, preferably wherein aberrant epitope-specific immune responses have been identified, or in which a genetic pre-disposition to such responses has been detected.

[00091] Administration of the composition(s) of the present invention to a host may be accomplished using any of a variety of techniques known to those of skill in the art. When administered as two or more compositions, the compositions may be administered simultaneously, separately, or sequentially, and by the same or different routes. The combinations of the invention may be additive or synergistic.

AND-8001-PC [00092] Suitable routes of administration will depend on the particular agent, its formulation, and disease to be treated. Suitable routes include, for example, the oral (including buccal or sublingual), rectal, nasal, pulmonary topical (including buccal, sublingual, or transdermal), vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) routes. Preferably, the route of administration for the immunopeptidic molecule is via a mucosal route, for example, by oral, nasal, or pulmonary delivery. Oral delivery of immunopeptidic molecule is particularly preferred in the context of the invention. Without wishing to be bound to a particular theory, mucosal, and particularly oral, delivery is preferred in order to induce tolerance to the immunopeptidic molecule. In contrast, compositions comprising a T cell migration inhibitor, particularly biologies such as proteins (e.g., antibodies), are preferably delivered by injection of a liquid formulation.

[00093] In the context of this invention, "combination therapy" refers to the administration of an immunopeptidic molecule and a neuroprotective agent as part of a specific treatment regimen intended to provide a beneficial effect from the co-action of these therapeutic agents. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the particular combination. Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days, or weeks, depending upon the combination selected). "Combination therapy" generally does not encompass the administration of each of an immunopeptidic molecule and neuroprotective agent as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present invention. Herein, "combination therapy" embraces administration of immunopeptidic molecule and neuroprotective agent in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single dose having a fixed ratio of each therapeutic agent or in multiple, single doses for each of the therapeutic agents. Sequential or substantially simultaneous administration of each of an immunopeptidic molecule and a neuroprotective agent can be effected by any appropriate route, including, but not limited to, oral and other mucosal routes, intravenous routes,

AND-8001-PC intramuscular routes, and subcutaneous routes. The therapeutic agents can be administered by the same route or by different routes. For example, a neuroprotective agent (e.g., copolymer-1) of the combination selected may be administered by subcutaneous injection while the immunopeptidic molecule may be administered orally (e.g., in the form of a pill or capsule). Alternatively, for example, all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection. The sequence in which the therapeutic agents are administered is not critical.

[00094] In addition to immunopeptidic molecules and neuroprotective agents, the instant methods may also include the administration of one or more therapeutically active agents or non-drug therapies, for example, cell-based therapies (e.g., implantation of cells intended to be therapeutic benefit, such as embryonic stem cells). Such additional therapeutically active agents may be an immunosuppressive agent (e.g., corticotrophin, a glucocorticoid, cyclophosphamide, cyclosporine, azothioprine, mitozantrone, etc.), an interferon (IFN; IFN-β-la (e.g., REBIF® and AVONEX®); IFN-β-lb (e.g., BETASERON® and BETAFERON®); IFN-α-2a (e.g., ALPHAFERONE®); IFN-α-2b (e.g., VIRAFERON®)), a phosphodiesterase type IV inhibitor, an antibody against a leukocyte adhesion molecule (e.g., ANTEGREN®), a tissue matrix metalloproteinase (MMP) inhibitor (e.g., hydroxamic acid-based inhibitors of MMPs), or a tumor necrosis factor (TNF) inhibitor (e.g., thalidomide or TNF -receptor immunoglobulin fusion protein), or TNF- β.

10. Treatment and Prevention of Multiple Sclerosis.

[00095] As described above, preferred embodiments of the instant invention concern methods for treating and preventing multiple sclerosis. Patients suitable for such treatment include those identified by criteria establishing a diagnosis of clinically definite MS. Briefly, an individual with clinically definite MS may be defined, for example, as having had two attacks and clinical evidence of either two lesions or clinical evidence of one lesion and para-clinical evidence of another, separate lesion. Definite MS may also be diagnosed by evidence of two attacks and oligoclonal bands of IgG in cerebrospinal fluid or by combination of an attack, clinical evidence of two lesions, and oligoclonal

AND-8001-PC band of IgG in cerebrospinal fluid. Slightly lower criteria may be used for a diagnosis of clinically probable MS.

[00096] Effective treatment of multiple sclerosis may be assessed in several different ways. Satisfying any of the following criteria evidences effective treatment. Three main criteria can be used: EDSS (extended disability status scale); appearance of exacerbations; or medical imaging, such as by MRI (magnetic resonance imaging). EDSS grades eight functional clinical criteria to assess the type and severity of neurologic impairment to MS (Kurtzke, Neurology, vol 33:1444, 1983). Exacerbations are defined as the appearance of a new symptom that is attributable to MS and accompanied by an appropriate new neurologic abnormality. In addition, the exacerbation must last at least 24 hours and be preceded by stability or improvement for at least 30 days. Exacerbations are either mild, moderate, or severe according to changes in a Neurological Rating Scale (Sipe et al., Neurology 34:1368,1984). An annual exacerbation rate and proportion of exacerbation-free patients are determined. Therapy is deemed to be effective if there is a statistically significant difference in the rate or proportion of exacerbation-free patients between the treated group and the placebo group for either of these measurements. Imaging, such as by MRI, can be used to measure active lesions using gadolinium-DTPA- enhanced imaging (McDonald, et ah, Ann. Neurol., vol. 36:14, 1994) or the location and extent of lesions using T2-weighted techniques, as compared to baseline. Various analyses may be performed, including those to assess evidence of new lesions, rates of appearance of active lesions, and percentage change in lesion area {see, e.g., Paty, et al, Neurology 43:665, 1993). Improvement due to therapy is established when there is a statistically significant improvement in an individual patient compared to baseline or in a treated group versus a placebo group.

[00097] Candidate patients for prevention may be identified by the presence of genetic factors. For example, a majority of MS patients have HLA-type DR2a and DR2b. The MS patients having genetic dispositions to MS who are suitable for treatment typically fall within two groups. First are patients with early disease of the relapsing remitting type. Entry criteria would include disease duration of more than one year, an EDSS score of 1.0 to 3.5, an exacerbation rate of more than 0.5 per year, and freedom from clinical

AND-8001-PC exacerbations for at least two months prior. The second group would include people with disease progression greater than 1.0 EDSS unit/year over the previous two years.

[00098] Efficacy of therapy in the context of prevention may be judged based on any suitable criteria, including frequency of MBP-reactive T cells (as may be determined by limiting dilution), proliferation response of MB- reactive T cell lines and clones, and cytokine profiles of T cell lines and clones to MBP established from patients. Efficacy is established by decrease in frequency of reactive cells, a reduction in thymidine incorporation with altered peptide compared to native, and a reduction in the expression of pro-inflammatory cytokines. Clinical measurements include relapse rate in one and two year intervals, and a change in EDSS, including time to progression from baseline of 1.0 unit on the EDSS that persists for six months. A delay in sustained progression of disability shows efficacy. Other criteria include a change in area and volume of T2 images on MRI, and the number and volume of lesions determined by gadolinium enhanced images.

[00099] In the context of this invention, it may be preferred to identify patients (including those yet to exhibit clinical manifestations of a de-myelinating disease, for example, MS) for treatment based on a diagnostic analysis prior to initiating therapy in order to identify patients most likely to respond favorably to the instant combination therapy. Such an analysis may be premised on a single test (e.g., HLA susceptibility, immune reactivity to a specific antigen, etc.), or upon a combination of two or more tests intended to identify whether a patient is more likely than not to favorably respond to the intended therapy. One preferred test is to establish whether some portion of a patient's T cells (i.e., those responsive to the particular antigen represented by the immunopeptidic molecule) can exhibit a pro-inflammatory response to the immunopeptidic molecule to be administered in the course of the instant combination therapy. Other representative examples of immunological markers that can be used to assess whether a patient is likely to respond to a particular therapy according to the invention include an increase in CD69+ positive cells and/or an increase in the intracellular production of one or more pro- inflammatory cytokines (e.g., IFN-γ and TNF-α) in response to stimulation with the immunopeptidic molecule. If desired, such determinations based on immunological factors may be combined with other diagnostic tests (e.g., HLA-typing, MRI image

AND-SOOl-PC analysis, etc.). Those patients identified as likely responders may then be administered a combined therapy according to the invention.

[000100] If desired, responsiveness to therapy can be assessed during and after treatment, as well. For example, assessment of a change (for example, a change of 10%, 20%, 25%, 50% or more from baseline) from baseline for one or more parameters of immune function (e.g., enhanced production of anti-inflammatory cytokines, e.g., IL-10 and TGF-β) can be made, as can other assessments, such as clinical assessments, including those based on MRI and/or other brain imaging data.

EXAMPLES

[000101] The following examples are provided to illustrate certain aspects of the present invention and to aid those of skill in the art in practicing the invention. These examples are in no way to be considered to limit the scope of the invention in any manner.

Example 1 : Treatment of Multiple Sclerosis by Modulation of Pathogenic Immune Responses by Combining IFN-β Therapy with Oral Epitope Specific Immunotherapy

L Introduction.

[000102] This example describes peptide-based immunopeptidic molecules for immunotherapy of multiple sclerosis (MS) using an art-recognized preclinical animal model to assess the efficacy of peptide-based immunomodulation. Specifically, the animal model of MS used here is Experimental Autoimmune Encephalomyelitis (EAE). This model was used to the study the clinical and histological outcomes of treatment based on the administration of one of several different species of molecules to the experimental animals, as described below.

[000103] As explained above, MS is an autoimmune disease characterized by chronic demyelination of nerve cells. Without being bound to a particular theory, and while the cause of MS has not yet been categorically defined, MS is believed to result from a coordinated inflammatory attack on myelin in the central nervous system, which induces damage to underlying axons, which, in turn, can lead to paralysis and even death. At the site of demyelination there is evidence that peptides encompassing the immunodominant

AND-8001-PC epitope of myelin basic protein (MBP), amino acids 83-99, are bound to the DR2 molecule expressed on inflammatory cells.

[000104] Moreover, in brain plaques of MS patients whose cells express human leukocyte antigen DR2, there are T-cell receptor (TCR) rearrangements characteristic of T-cell clones reactive against MBP (83-99) bound to human leukocyte antigen DR2. The T-cell response is characterized by a type 1 T helper-cell (ThI) phenotype. The ability of these cells to recognize peptide antigen has been extensively studied using altered peptide ligands. Together, these data indicate that MS results from the activation of autoreactive T cells directed against the myelin components of nerve cells of the central nervous system.

[000105] Experimental Autoimmune Encephalomyelitis is well recognized as correlating with MS in humans, and results in central nervous system demyelination and paralysis. EAE is inducible in genetically susceptible animals, Lewis rats, by immunization with whole myelin, constituent proteins of myelin such as MBP, myelin proteolipid protein, and peptides derived from these neuroantigens. Alternatively, demyelination can be induced by MBP-specific, MHC class II restricted CD4+ T cells adoptively transferred from MBP-sensitized donor rats to naive syngeneic recipients. Organ-infiltrating but not circulating T lymphocytes possess limited heterogeneity and share TCR Vβ gene products, a fact confirmed also in human T cells recognizing immunodominant regions of MBP. Although activated CD4+ T cells play a key role in initiating the demyelinating response in EAE, many cell types have also been implicated in this complex regulatory process. Regulatory cells usually come from the adaptive arm of the immune response; however, several subsets of lymphocytes associated with innate immunity have been implicated in the regulatory network in EAE, including NK cells and T cells.

[000106] As already mentioned, one of the critical factors in developing effective antigen-specific immunotherapy is the identification of model antigens able to trigger pro-inflammatory responses and which could be targets of immunotherapy. Despite their high degree of evolutionary conservation and roles as molecular chaperones, heat shock proteins are remarkably immunogenic, and experimental and clinical observations of various autoimmune disease models indicate that immune responses to hsps arise

AND-8001-PC spontaneously during the disease process. At times of cellular stress, including infection and chronic inflammation, the expression of hsps is upregulated. This leads to a complex cascade of events involving both the innate and adaptive arms of the immune response.

2. Modulating Inflammation - The "Molecular Dimmer" Concept.

[000107] As shown in this example, exemplary peptides of a general class of peptides derived from bacterial hsps, particularly from bacterial and human hspόO, are active in protecting against MS. These exemplary peptides have been designed to overcome HLA differences among individuals, making their use possible regardless of the genetic makeup of a particular patient to be tested and/or treated. Immunological responses to bacterial hsps have been implicated in the pathogenesis of autoimmunity in animals and in humans, as human hsps exhibit a high degree of sequence identity with their bacterial counterparts. Hsps are present at the site of inflammation and have been described as relevant targets of T-cell responses in immune mediated diseases. Cross recognition of epitopes shared by these proteins is a physiologic phenomenon aimed at generating inflammation for defensive purposes. The immune response generated is amplified by non-specific mechanisms. Under the immune regulatory convention of the present invention, regulation of such phenomena can be exploited to modulate immune responses for therapeutic purposes. In particular, it has been discovered that recognition of uniquely human peptides by patients with autoimmune diseases is associated with a down regulation of the inflammatory process and better prognosis. On the other hand, uniquely bacterial hsp-derived peptides trigger pro-inflammatory responses. Hence, as shown by the experiments of this example, a "molecular dimmer," i.e., a molecular regulator of inflammation that can be used to regulate the level of immune inflammation in MS, has been identified and adapted for therapeutic use.

3. Experimental.

[000108] The "monotherapy" (i.e., administration of only one therapeutically effective compound) experiments described in this example were designed to compare the effects of conventional IFN-β therapy with those of peptide-based therapy. Here, the peptide employed was a synthetic peptide corresponding to amino acid residues 507-521 of M. tuberculosis hsp60/65, which contains 15 amino acid residues and has the amino acid

AND-8001-PC sequence 507-IAGLFLTTEAVVADK-521 (SEQ ID NO: 1). The results of these monotherapy experiments established the efficacy in vivo of the peptide treatment alone in treating demyelination in the EAE model, which correlates with MS in humans.

[000109] EAE was induced as follows: on day 0 Lewis rats were injected intradermally in the right hind footpad with a 50 μl inoculum containing 50 μg of guinea pig MBP peptide (containing residues 68-86 of the full length protein myelin basic protein), 100 μg of M. tuberculosis, and 50 μl of incomplete Freund's adjuvant. The date of this initial injection was deemed the date EAE was induced in the experimental animals. On day 7 after EAE induction {i.e., 7 days post-induction, or "p.i."), rats were randomly divided into several experimental groups (typically, 10 experimental groups with 5 rats per group) and treated according to the established protocol with recombinant rat IFN-β (PBL Biomedical Laboratories, New Brunswick, NJ; specific activity = 4x10⁷ U/mg) or with the immunomodulatory peptide 507-521 derived from M. tuberculosis hsp60/65. IFN-β was administered subcutaneously (100,000 U/day/rat) for four consecutive days starting from day 7 p.i. in the rats of one group. M. tuberculosis peptide 507-521 (having the amino acid sequence of SEQ ID NO:1) was dissolved in phosphate-buffered saline (PBS) and given nasally (75, 150, or 300 μg/rat/day, 15 μl per nostril) for five consecutive days starting from day 7 p.i. Placebo groups received placebo (10 μl sterile water per nostril) at the same time points as the peptide-treated groups. From day 7 onward clinical scores are assessed daily.

[000110] Histology was assessed by examining tissue sections taken from some of the experimental animals. To do this, on day 15 p.i., several rats from the 300 μg/rat/day M. tuberculosis hsp60/65 507-521 peptide group, the IFN-β group, and the "no treatment" control group were perfused with 4% paraformaldehyde. Brain and spinal cord sections from these animals were histologically evaluated according to four criteria using blind evaluation. Using these criteria, actual lesion severity was scored as: 0=normal; l=trace; 2=mild; 3=moderate; or 4=severe (actual lesion severity).

[000111] The analysis of cell phenotype, function, and cytokine and transcription factor expression described in this example was performed by multiplex real-time quantitative PCR. This data was collected from animals at day 15 p.i. Briefly, sample cells were isolated from lymph nodes and stimulated in vitro for 48 hours with the M. tuberculosis

AND-8001-PC hsp60/65 507-521 peptide and processed by TaqMan® using an ABI PRISM® 7700 thermal cycler (Perkin Elmer) capable of distinguishing and quantitating multiple fluorophores in a single tube so that more than one target cDNA species could be amplified and detected in a given sample. To make cDNA, mRNA was extracted from T lymphocytes isolated from spleen, inguinal, and mandibular lymph nodes by using Rneasy Mini Kit (Qiagen, Valencia, CA). Messenger RNA was reverse-transcribed into cDNA using an oligo dT primer and reverse transcriptase. Single-strand cDNA was then amplified with gene-specific forward and reverse primer sets for GAPDH (a housekeeping gene), IFN-γ, IL-10, and TGF-β. ThI versus Th2 phenotypes were evaluated by measuring the expression induction of the Tbet and GAT A-3 gatekeepers for the ThI and Th2 phenotypes, respectively. Foxp3 gene expression was also monitored, and was used as the marker for CD4+CD25+ regulatory T cells.

4. Results.

[000112] EAE was induced as described above and the experiments conducted and evaluated as indicated. On day 7 after disease induction, rats were randomly divided into the various experimental groups and treated according to the established protocol. Several sets of experiments were performed to study the effects on EAE exerted by three different concentrations of the M. tuberculosis hsp60/65 507-521 peptide and with rat IFN-β. Figure 1 shows the results of these experiments. In the figure, for each group the mean of the standard deviation was plotted as a point for purposes of the drawing the lines shown. The difference between the M. tuberculosis hsp60/65 507-521 peptide (75- 150 and 300 μg/rat/day) given five times and the "no treatment" group at day 14 p.i. was considered not to be statistically significant (p=0.2878).

[000113] As shown in Figure 2, the disease histology improved by administering five 300 μg doses of M. tuberculosis hsp60/65 507-521 peptide as compared to the "no treatment" control animals. The clinically effective treatment dose (300 μg for M. tuberculosis hsp60/65 507-521 peptide) was defined by comparing peptide-only therapy to IFN-γ treatment at 100,000 U (defined as optimal clinically effective dose in previous experiments).

AND-8001-PC [000114] Figures 3 A-D show the induction indices for three cytokines and the Fox3 marker following treatment with the M. tuberculosis hsp60/65 507-521 peptide, as compared to the expression level of the housekeeping gene GAPDH. As shown, as compared to GAPDH expression, IFN-γ and FoxP3 were marginally induced while TGF- P was strongly induced. These data demonstrate at the immunological level that therapy with only the hsp60/65-derived M. tuberculosis 507-521 peptide resulted in control of the expression levels of the pro-inflammatory cytokine IFN-γ. The level of the anti¬ inflammatory cytokine IL-10 remained substantially unchanged following monotherapy using 300 μg doses of the HSP-derived peptide. Moreover, there was marked increased expression of the regulatory cytokine TGF-β, as well as of Foxp3. The increased expression of these two genes further shows that peptide-based immunomodulatory therapy alone can achieve active antigen-specific T cell suppression and clinical control of EAE in vivo, supporting the clinical observation that the efficacious nature of the therapy was mediated by regulatory T cells. These peptide-based monotherapy results show that treatment of EAE using hsp-derived immunomodulatory peptides (i) down- regulates CD4+ T-helper 1 cells involved in triggering autoimmunity, and (ii) up- regulates anti-inflammatory CD4+ T-helper 2 cells having a regulatory phenotype.

5. Conclusions.

[000115] The set of experiments described in this example demonstrates the in vivo efficacy of using hsp-derived peptides in monotherapy to effect immunomodulatory treatment of EAE, which corresponds to MS in humans. Put another way, these results show that peptide-based immunomodulatory treatment can result in clinical amelioration of a demyelinating autoimmune disease, as assessed using standard histological analyses. The experiments also show that clinically relevant immunological surrogate endpoints can be used to assess the clinical efficacy of this immunomodulatory therapeutic treatment. Indeed, as already shown in another immune-mediated disease, rheumatoid arthritis, the analysis of an immunological endpoint can strongly and positively correlate with clinical efficacy. In this regard, the cytokine production of hsp-derived, peptide- specific CD4+ T cells shifted from a pro-inflammatory phenotype (e.g., IFN-γ production) to an anti-inflammatory phenotype (e.g., TGF-β production). Moreover, the functional phenotype of the peptide-specific T cells changed, as demonstrated by the

AND-8001-PC increase in expression of the Foxp3 gene, a molecular marker associated with the generation/enhancement of a T cell response of the regulatory phenotype. Therefore, the immunological analysis strongly indicates that monotherapy of EAE using hsp-derived immunomodulatory peptides down-regulates CD4+ T-helper 1 cells involved in triggering autoimmunity and up-regulates anti-inflammatory CD4+ T-helper 2 cells having a regulatory phenotype. This shift THl to TH2 shift, i.e. immunodeviation, triggers a positive clinical outcome and results from immunomodulation of this MS-like disease.

[000116] As will be appreciated, the invention also includes embodiments for treating immune-mediated diseases that involve providing regulatory T cell populations which have restored or enhanced regulatory activity, the use of such regulatory T cells to treat immune-mediated disorders, and specific peptides possessing modulatory activity in such T cell populations.

Example 2: Modulation of Pathogenic Immune Responses in MS

L Introduction.

[000117] This example describes the testing of a library of HLA pan-DR binding peptides for use as potential therapeutics for the treatment of multiple sclerosis. Briefly, a group of 16 different peptides {see Table 1, above) derived from different hsp60 and dnaJ proteins and identified to contain T cell epitopes, as described above, were each individually studied in vitro using PBMC obtained from 33 well-characterized MS patients. Levels of pro-inflammatory cytokines {i.e., IFN-γ and TNF-α) and other proteins (CD69 and CFSE) produced by these cells were assayed following exposure to a particular peptide, as was cell proliferation. Statistical significance (here, p < 0.05) was determined by comparison of these results to controls peptide. The immunological data was then stratified with clinical data, including disease activity and disease sub- population, among other data. Coefficients of correlation were then calculated to identify correlations between the immunological and clinical data. Using this process, peptide Bl {see Table 1, above) was selected from among the 16 peptides tested as the first peptide for administration to humans to treat MS, alone or in conjunction with a neuroprotective agent and/or other compounds.

AND-8001-PC 2. Experimental.

[000118] Peripheral blood mononuclear cells (PBMC) were obtained from 33 patients having clinically defined multiple sclerosis (CDMS) or possible MS according to well- recognized McDonald criteria. Specifically, patients were stratified as either having clinically isolated syndrome (CIS; n = 13), relapsing remitting (RR; n =15) MS, or secondary progressive (SP; n = 5) MS. Patients with CIS were defined as those presenting with symptoms suggestive of a first demyelinating event. CIS is highly predictive of developing further inflammation and definitive MS when the episode occurs in conjunction with lesions visible on MRI and the presence of oligoclonal bands. In CIS patients included in this study, lesions were detected in an MRI of the patient's brain and, in a subgroup, CSF oligoclonal bands. In these patients, the risk of developing CDMS con-elated with the number and extent of lesions detectable in T2-weighted MRI data and the number of lesions determined from Tl -weighted MRI data. Patients enrolled in the study were not therapeutically treated as part of the study (although the five SP MS patients were under separate heavy immunosuppressive therapy), and none of them had received steroids in the three months preceding the study.

[000119] Particular focus was given to patients in the initial and active stages of disease. Thus, information on clinical and MRI activity included time from disease onset, time from the last relapse, disease type, number, and extent of T2 -weighted lesions and the number of Tl -weighted enhancing lesions at the time of immunological analysis.

[000120] The 16 peptides used in the experiments described in this example, designated Pl, P3, P5, P7, P9-12, and Bl-8, were manufactured by using known solid state peptide synthesis technology. A control peptide, which is a pan-DR binder but does not lead to T cell activation, was used for running controls.

[000121] To perform immuno-screening, 30-50 ml of heparinized peripheral blood was collected from each of the 33 patients and several healthy (i.e., no evidence of MS or another disease) controls. PBMC were obtained from each patient sample by Ficoll- Hypaque separation, and the PBMC were then resuspended at 3 x 10⁶ cells/ml in complete RPMI medium and incubated with one of the 16 test peptides or the control peptide, which were added to each cell sample at a concentration of 20 μg/ml. After 72

AND-8001-PC hr. of incubation, intracellular production of the pro-inflammatory cytokines TNF-α and IFN-γ (using FITC-anti-TNF-α and FITC-anti-IFN-γ, assayed using the cytoFix- cytoPerm method; Pharmingen, San Diego, CA) were measured by FACS analysis and ELISA, as was the production of the anti-inflammatory, or regulatory, cytokine IL-IO. In addition, T cell proliferation/activation was assessed by measuring incorporation of 5- (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). All flow cytometry was performed using a FACScan instrument (Becton Dickinson) running CellQuest software. Dead cells were excluded from the analysis by propidium iodide identification. Statistical analyses were performed as follows: a two-tailed, paired T-test was used to compare the immunological data; a Kolmogorov-Smirnov statistical analysis was used to analyze the FACS histograms; and post-analysis statistics were performed by use of the Bonferroni test.

3. Results.

[000122] Immunological analysis showed that each of the 16 peptides tested in this study resulted in the production of TNF-α and IFN-γ, as measured by FACS. As compared to the control peptide, the B 1 peptide exhibited a statistically significant difference with regard the production of each of these pro-inflammatory cytokines. The Bl peptide also clearly induced T cell proliferation, as measured by CFSE incorporation.

[000123] A series of statistical correlations between the in vitro cell-based immunological responses obtained with the Bl peptide and clinical data revealed a significant correlation between pro-inflammatory responses to the peptide and disease in those diagnosed with CIS or RR MS. Immunological data for the SP MS patients was inconclusive, due to immuno-suppression attributed to their immunosuppressive therapy.

MRI is the most useful tool for the clinical diagnosis of MS, as it can be used to detect white matter lesions indicative of clinically active disease. The statistical analysis between induction of a pro-inflammatory response by the Bl peptide and the stratification of the 33 MS patients in this study based on lesion number revealed a strong correlation between a pro-inflammatory response (as measured by the production of TNF-α and IFN- γ) compared to the control peptide in patients having more than 40 lesions. In addition, a statistically significant difference in induction of TNF-α production, as between the B 1

AND-8001-PC and control peptides, was found for those patients who had been diagnosed within the three months prior to initiation of this study.

Example 3: Treatment of Multiple Sclerosis Comprising Modulation of Pathogenic immune response using HSP-derived peptides in oral epitope specific immunotherapy and Copaxone.

[000124] As noted above in Example 1 , EAE can be induced by MBP-specific, MHC class II-restricted CD4+ T cells adoptively transferred from MBP-sensitized donor rats into I syngeneic recipients. Organ-infiltrating but not circulating T lymphocytes possess limited heterogeneity and share TCR V gene products, a fact also confirmed in human T cells recognizing immunodominant regions of MBP. Although activated CD4+ T cells play a key role in initiating the demyelinating response in EAE, many cell types have also been implicated in this complex regulatory process. Regulatory cells, in particular regulatory T cells from the adaptive arm of the immune response, have been strongly implicated in the regulatory network in EAE.

[000125] EAE is a widely used animal model for the human equivalent demyelinating disease MS (1). Similar to the human disease, EAE is a disorder in which activation of autoreactive T cells directed against myelin constituents results in central nervous system demyelination and paralysis. EAE is inducible in genetically susceptible animals (Lewis rats) by immunization with whole myelin, constituent proteins of the myelin such as myelin proteolipid protein (MBP), or peptides derived from these neuroantigens. It has been shown that activated CD4+ T cells with limited heterogeneity and sharing TCR Vgene products play a key role in initiating the demyelinating response in EAE.

[000126] This example presents a paradigm for immune-therapy of MS by mucosal administration of HSPs derived peptides. The peptide-based immunotherapy proposed for this invention targets HSP-specific autoimmune T cells with the aim of modulating their proinflammatory capacity. This is in contrast to prior attempts at treating MS by aiming at aspecifically interfering with the inflammatory process which attempts have yielded conflicting results in MS, with the notable exception of Copaxone therapy. The major limitations of an aspecific approach, however, are transient effects of such therapy, and dependency on continuous administration of the compound to control inflammation

AND-SOOl-PC and side effects. In the experimental animal model shown here, i.e., induction and protection from EAE, Copaxone has also been shown to inhibit progression of relapsing- remitting disease.

[000127] Reafients used in the animal study.

-Copaxone: Glatiramer acetate injection Manufacturer: Teva Pharmaceuticals.

-RAT-IFN-β. Recombinant rat interferon beta. Manufacturer: PBL Biomedical

Laboratories, New Brunswick, NJ. Source: cDNA expressed in CHO cells. Molecular weight: 9,680 D. Specific Activity: 4xlO⁷ U/mg. Lot#: 1790.

-MBP pool: Guinea pig MBP 68-86 and MBP 87-99. Manufacturer: Synthetic

Biomolecules, Inc. San Diego, CA.

-HSP-PEPTIDE: RatP2. Manufacturer: Synthetic Biomolecules, Inc. San Diego CA.

[000128] EAE induction and treatment protocol. As indicated by the graph in Figure 4, on Day 0, rats were injected intradermally in the right hind footpad with 50 μl inoculum containing: 50 μg Guinea pig MBP 68-86, 100 μg of M. tuberculosis and 50 μl incomplete Freund's adjuvant. On day 7, after the injection, rats were randomly divided into four experimental groups: 1. Copaxone alone; 2. Immunomodulatory HSP-derived peptide RatP2 in monotherapy; 3. Immunomodulatory HSP-derived peptide RatP2 and Copaxone in combination therapy; 4 placebo control group.

[000129] In groups 1 and 3, Copaxone was administered subcutaneously (s.c.) for 5 consecutive days starting from day 7 after induction of EAE.

[000130] HSP-derived peptide was dissolved in PBS and given nasally at the selected dose in 15 μl per nostril for 'n' consecutive days starting from day 7 after induction of EAE.

[000131] Combination Therapy Groups received Copaxone for 5 consecutive days starting from day 7 in combination with the HSP-derived peptide at the selected optimal dose and regimen of administration.

[000132] Placebo Groups received placebo (PBS) at the same time-points of the treated groups.

AND-8001-PC [000133] TaqMan Analysis of T cell function. TaqMan (Multiplex Real-Time Quantitative PCR) analysis was performed using cells isolated from lymph nodes restimulated using HSP-derived peptides. T lymphocytes isolated from spleen, inguinal and mandibular lymph nodes were evaluated by TaqMan using an ABI PRISM® 7000 thermal cycler (Perkin Elmer). The mRNA was extracted from T cells using Rneasy Mini Kit (Qiagen, Valencia, CA) and reverse-transcribed into cDNA with an oligo dT primer. Single strand cDNA was then amplified with the cytokine specific forward and reverse primer sets for GAPDH (housekeeping gene), IFN-γ , I L -10, TGF - β and for monitoring of gene expression of other phenotypical and molecular markers characteristic of regulatory T cell function.

[000134] Figure 5 shows average disease scores in EAE rats treated with hsp-derived peptide and Copaxone®. HSP-derived peptide (Rat-P2) was administered i.n. (300 μg /day/rat) for 5 consecutive days starting on Day 7 p.i. Copaxone® treatment was administered s.c. (lOOμg/day/rat) for 5 days starting on Day 7 p.i. The Non-treated group received 10 μl of sterile distilled water per nostril for 5 consecutive days starting on day 7 p.i. Disease scores were evaluated daily. Disease scores of EAE were graded according to the following criteria: 0, asymptomatic; 1, flaccid tail; 2, loss of righting reflex with or without partial hind limb paralysis; 3, complete hind limb paralysis; 4, moribund/dead, these animals were immediately removed from the study and sacrificed. DI= Disease Induction; TX= Treatments starting day. 1-5= Number of days post treatment used to calculate the area under the curve (AUC). (*) P values refer to statistically significant differences.

[000135] Figure 6 shows the area under the curve for each experimental group (AU=Arbitrary Units) was calculated using the curves shown in Figure 5 and originated by determining the EAE clinical score of the animal at each specific day. Error bars represent Mean ± SD. P values refer to statistically significant data between treatment groups and untreated control group (*). No statistically significant differences were found when treatment groups were compared to each other. These results indicate that treatment with Copaxone and hsp derived polypeptide have the greatest therapeutic efficacy. This type analysis provides insight to the entire severity of disease presentation

AND-800I-PC over the entire course of the disease, in this case a 14 day period. It provides a quantity level of how well or poorly a subject was affected over the course of the disease.

[000136] Figure 7 is a graph showing histological scores on Day 15, p.i. wherein selected rats were perfused with 4% paraformaldehyde and sections of brain and spinal cord were histologically evaluated according to four criteria using blind evaluation. The Histology score scale was as follows: 0=normal; l=trace; 2=mild; 3=moderate; 4=severe (actual lesion severity). (n=l for each experimental group).

[000137] Figure 8 A and B are graphs showing gene transcription in lymph nodes as detected by TaqMan using the immunomodulatory hsp-derived peptides. Lymph node cells were isolated on Day 15 after the induction of EAE and cultured with the hsp- derived Rat-P2 peptide for 48 hours. Messenger RNA was extracted and the amount of gene transcription was compared to the gene transcription of the house keeping gene GAPDH. After stimulation, cells were lysed to obtain RNA. Gene expression was determined by Real Time PCR (TaqMan). Read out values are expressed as LoglO of Induction Index. Data shown are from two independent experiments and are expressed as Mean±SE. P values refer to statistically significant differences (*). The graphs indicate a that the treatment resulted in a decrease in IFN-γ which is a proinflammatory cytokine and an increase in FoxP3 which is a molecular marker of regulatory T cell activation, the respective decrease and increase having a direct association with the purposeful modulation of disease.

[000138] In Figure 9, FoxP3 protein expression as detected by intracellular flow cytometry staining in lymph node cells is disclosed. Lymph node cells from mandibular (MLN) or mesenteric (MesLN) lymph nodes were isolated on day 15 after the induction of EAE. Cells were stained for CD4PECy5/CD25PE and following the cytofix-perm protocol, were also stained for FoxP3 FITC. Intracellular FoxP3 expression was evaluated by a FACSort. Lymphocytes were gated by FSC vs SSC analysis and the percentage of CD4+FoxP3+ positive cells was plotted (mean +/- SD). All CD4+FoxP3+ cells were also found to be expressing CD25 (data not shown). UnTx = untreated; Na300 = nasal treatment with 300 μg of RatP2 peptide.

AND-8001-PC [000139] Figures 1 OA and B show Intracellular TNF-alpha and IL- 10 expression, respectively, by flow cytometry following different treatments. Lymph node cells were isolated on day 15 after the induction of EAE and cultured with the HSP-derived Rat-P2 peptide for 72 hours. After stimulation, cells were stained for CD4PE-Cy5/CD3FITC and following the cytofix-perm protocol, were also stained for TNF-alpha PE or IL-10 PE. Intracellular cytokine expression was evaluated by a FACSort. Values are expressed as CD4/CD3/TNF-alpha or IL-IO positive cells. Data shown are from two independent experiments and are expressed as Mean±SE. P values refer to statistically significant differences (*).

[000140] Figure 11 is a graph showing humoral response to unrelated antigen Beta- galactosidase, demonstrating that the treatment is not broadly immunosuppressive and preserves immunity to an unrelated antigen. Peptides were administered intranasally for five consecutive days starting on day 7 post induction of disease (p.i.) on day 0. Copaxone® treatment was administered s.c. for 5 days starting on Day 7 p.i. Beta- galactosidase was administered intraperitoneally on days 0, 10, and 21. The Non treated group received 10 μl of sterile distilled water per nostril for five consecutive days starting on day 7 p.i. and 100 μl of sterile distilled water subcutaneously for five consecutive days starting on day 7 p.i. Determination of anti-beta-galactosidase antibody titer by ELISA on days 3, 8 and 21 post-injection of beta-galactosidase. Data at day 21 post immunization are shown. Rat sera isolated from whole blood via lateral saphenous vein were tested at different dilutions and shown at 1 :400 dilution. Plates were coated with betagalactosidase at lOμg/ml. Binding was revealed using rabbit anti-rat IgG+IgM-HRP conjugated (1 :7,500). Data represent mean ± SD. This figure shows the immune response to an antigen unrelated to immunomodulatory and tolerogenic peptide Rat-P2 used for therapy. In other words, regardless of the presence of EAE disease induction or status of immune modulation brought about by the immunomodulatory treatment, the immune system is not compromised to responding to presentation of antigenic agents unrelated to the disease or disorder.

[000141] In summary, this set of experiments provides results demonstrating proof-of- principle efficacy of an hsp-derived peptide for immunotherapy of EAE in an animal model of MS which is well understood by one of skill in the art as correlating with human

AND-8001-PC MS. This therapy was effective when administered as a monotherapy as well as in combination therapy with Copaxone®. Figure 5 shows therapeutic efficacy of the immunomodulatory approach by assessing the average disease scores in EAE at the peak of the disease (day 4 post induction). We also measured therapeutic efficacy using area under the curve (AUC) analysis, as shown in Figure 6, which represents a better assessment of efficacy of the treatment protocols during the entire course of disease. In addition, therapeutic efficacy throughout the entire course of the disease is demonstrated through a histological evaluation of the brain and spinal cord, as shown in Figure 7. Observed therapeutic efficacy data are further supported by immunological data in Figure 8, 9, and 10 demonstrating an increased expression of FoxP3 (Figures 8B and 9), a molecular marker specifically expressed by regulatory T cells, and by a decrease of IFN- gamma in lymph node-derived cells (Figure 8A). Data shown in Figure 9, regarding the specific induction of FoxP3 in the mandibular lymph node (MLN) CD4+CD25+ T cells following intranasal treatment with HSP-derived peptide Rat-P2 formally confirmed the involvement of CD4+/CD25+ T regulatory cells present in the draining lymphnode. Data in Figure 10 show that cytokine levels of TNF-α and IL-IO are appropriately affected. Altogether, these data support the theory that therapeutic efficacy of HSP-derived peptides in this animal model of EAE results from an immuno-deviation that is driven by the T regulatory cell pathway. Finally, as shown in Figure 11, immune responses to an unrelated antigen were not altered over time, demonstrating absence of general immunosuppression.

[000142] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the appended claims.

[000143] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of

AND-8001-PC the method described herein without departing from the spirit and scope of the invention as defined by the appended claims.

[000144] All patents, patent applications, and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents, patent applications, and publications, including those to which priority or another benefit is claimed, are herein incorporated by reference in their entirity to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[000145] The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of, and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

AND-SOOl-PC

Claims

What is claimed is:

1. A composition comprising a therapeutically effective amount of each of an immunopeptidic molecule and a neuroprotective agent.

2. A composition according to claim 1 wherein the immunopeptidic molecule comprises a peptide having a naturally occurring amino acid sequence.

3. A composition according to claim 1 wherein the immunopeptidic molecule is a peptide having the amino acid sequence GDL YVQ VQ VKQHPIF.

4. A composition according to claim 1 wherein the neuroprotective agent is coplymer-1.

5. A composition according to claim 1 wherein the immunopeptidic molecule is a peptide having the amino acid sequence GDLYVQVQVKQHPIF and the neuroprotective agent is coplymer-1.

6. A kit comprising a container containing a composition according to claim 1.

7. A kit comprising a first container containing a first composition comprising at least one therapeutic dose of an immunopeptidic molecule and a second container containing a second composition comprising at least one therapeutic dose of a neuroprotective agent.

8. A kit according to claim 7 wherein the immunopeptidic molecule comprises a peptide having a naturally occurring amino acid sequence.

9. A kit according to claim 7 wherein the immunopeptidic molecule is a peptide having the amino acid sequence GDLYVQVQVKQHPIF.

10. A kit according to claim 7 wherein the neuroprotective agent is coplymer-1.

11. A kit according to claim 7 wherein the immunopeptidic molecule is a peptide having the amino acid sequence GDLYVQVQVKQHPIF and the neuroprotective agent is coplymer-1.

AND-8001-PC

12. A method of treating a demyelinating disease, comprising administering a composition according to claim 1 to a patient having, or at risk of having, multiple sclerosis, wherein the immunopeptidic molecule provides for epitope-specific immunotherapy of the demyelinating disease.

13. A method according to claim 12 wherein the immunopeptidic molecule comprises a peptide having a naturally occurring amino acid sequence.

14. A method according to claim 12 wherein the immunopeptidic molecule is a peptide having the amino acid sequence GDL YVQ VQ VKQHPIF.

15. A method according to claim 12 wherein the neuroprotective agent is coplymer-1.

16. A method according to claim 12 wherein the immunopeptidic molecule is a peptide having the amino acid sequence GDLYV QVQVKQHP IF and the neuroprotective agent is coplymer-1.

17. A method according to claim 12 wherein the demyelinating disease is multiple sclerosis.

18. A method according to claim 17 wherein the immunopeptidic molecule comprises a peptide having a naturally occurring amino acid sequence.

19. A method according to claim 17 wherein the immunopeptidic molecule is a peptide having the amino acid sequence GDL YVQVQ VKQHPIF.

20. A method according to claim 17 wherein the neuroprotective agent is coplymer-1.

21. A method according to claim 17 wherein the immunopeptidic molecule is a peptide having the amino acid sequence GDLYV QV QVKQHPIF and the neuroprotective agent is coplymer-1.

22. A method of treating multiple sclerosis, comprising administering to a patient having, or at risk of having, multiple sclerosis a first composition comprising a therapeutically effective amount of an immunopeptidic molecule and a second composition comprising a therapeutically effective amount of a neuroprotective agent.

23. A method of treating multiple sclerosis, comprising administering to a patient having, or at risk of having, multiple sclerosis a first composition comprising a

AND-8001-PC therapeutically effective amount of a peptide having a naturally occurring amino acid sequence and a second composition comprising a therapeutically effective amount of a neuroprotective agent.

24. A method of treating multiple sclerosis, comprising administering to a patient having, or at risk of having, multiple sclerosis a first composition comprising a therapeutically effective amount of a peptide having a naturally occurring amino acid sequence of a heat shock protein and a second composition comprising a therapeutically effective amount of a neuroprotective agent.

25. A method of treating multiple sclerosis, comprising administering to a patient having, or at risk of having, multiple sclerosis a first composition comprising a therapeutically effective amount of a peptide having the amino acid sequence GDLYVQVQVKQHPIF and a second composition comprising a therapeutically effective amount of a neuroprotective agent.

26. A method of treating multiple sclerosis, comprising administering to a patient having, or at risk of having, multiple sclerosis a first composition comprising a therapeutically effective amount of an immunopeptidic molecule and a second composition comprising a therapeutically effective amount of copolymer- 1.

27. A method of treating multiple sclerosis, comprising administering to a patient having, or at risk of having, multiple sclerosis a first composition comprising a therapeutically effective amount of a peptide having a naturally occurring amino acid sequence and a second composition comprising a therapeutically effective amount of copolymer- 1.

28. A method of treating multiple sclerosis, comprising administering to a patient having, or at risk of having, multiple sclerosis a first composition comprising a therapeutically effective amount of a peptide having a naturally occurring amino acid sequence of a heat shock protein and a second composition comprising a therapeutically effective amount of copolymer- 1.

29. A method of treating multiple sclerosis, comprising administering to a patient having, or at risk of having, multiple sclerosis a first composition comprising a therapeutically effective amount of a peptide having the amino acid sequence

AND-8001-PC GDLYVQVQVKQHPIF and a second composition comprising a therapeutically effective amount of copolymer- 1.

30. A method according to any of claims 12-29, wherein the administration of the immunopeptidic molecule is via a mucosal route.

31. A method according to any of claims 30, wherein the mucosal route is selected from the group consisting of oral administration, nasal administration, and pulmonary administration.

32. A method according to claim 22 wherein said immunopeptidic molecule comprises at least one of a polypeptide having a sequence selected from the group consisting of Seq Id Nos. 1, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 23, 25, 27, 28, and 30.

33. A composition according to claim 1 wherein said immunopeptidic molecule comprises at least one of a polypeptide having a sequence selected from the group consisting of Seq Id Nos. 1, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 23, 25, 27, 28, and 30.

AND-8001-PC