New compositions and procedures for the treatment of autoimmune and allergic diseases

专利摘要:
The present invention provides improved methods and compositions for treating and preventing autoimmune and allergic diseases. More specifically, the invention relates to new immunomodulating complexes that are fusion proteins comprising a mutant subunit of the A1-subunit of the cholera toxin (CTA1), a peptide capable of binding to a specific cellular receptor, and one or more epitopes associated with an autoimmune or allergic disease. In the mutant CTA1 subunit, the amino acids corresponding to the amino acid 7, arginine, and amino acid 187, cysteine, in the native CTA1 have been replaced.
公开号:SE1051122A1
申请号:SE1051122
申请日:2010-10-28
公开日:2012-04-29
发明作者:Nils Lycke
申请人:Mivac Dev Aktiebolag；
IPC主号:

专利说明:

endogenous antigen. In addition to determining whether a response should be generated or not (antigen specificity), the immune system must also select appropriate effector functions to deal with each pathogen (effector specificity). A cell of crucial importance for degrading and regulating these effector functions is the CD4-positive T cell (CD4 + T cells). Furthermore, it is the release of specific cytokines from CD4 + T cells that seems to be the main mechanism by which T cells mediate their functions. Thus, mapping the different types of cytokines produced by CD4 + T cells, as well as how their secretion is regulated, is extremely important for understanding how the immune response is regulated.
The mapping of cytokine production from long-lived CD4 + T cell clones from mice was first published more than 20 years ago (Mosmann et al. J Immunol 136: 2348-2357, 1986). In these studies, it was shown that CD4 + T cells showed two distinct patterns for cytokine production, which were designated T helper cell type 1 (Th1) and T helper cell type 2 (Th2). Th1 cells were found to produce interleukin-2 (IL-2), interferon-γ (IFN-γ) and lymphotoxin (LT), while Th2 clones mainly produced IL-4, IL-5, IL-6 and IL-13 ( Cherwinski et al. J Exp Med 169: 1229-1244, 1987). Somewhat later, additional cytokines, IL-9 and IL-10, were isolated from Th2 clones (Van Snick et al. J Exp Med 1692363-368, 1989) (Fiorentino et al. J Exp Med 170: 2081-2095, 1989). Finally, it was observed that additional cytokines such as H, -3, granulocyte / macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor-α (TNF-α) were secreted by both Th1 and Th2 cells. Recently, it has been reported that CD4 + T cells isolated from the affected joints in patients with Lyme disease contain a subset of IL-17-producing CD4 + T cells that differ from Th1 and Th2 cells (Infante-Duarte et al. J Immunol 165: 6107-61 15, 2000). These IL-17-producing CD4 + T cells are termed Thl 7. IL-17, a pro-inflammatory cytokine produced primarily by activated T cells, enhances priming of T cells and stimulates fibroblasts, endothelial cells, macrophages and epithelial cells to produce a number of pro-inflammatory cells. mediators, including IL-1, IL-6, TNF-u, NOS-2, metalloproteases and chemokines, leading to induction of inflammation. IL-17 expression is elevated in patients with a number of allergic and autoimmune diseases, such as RA (rheumatoid arthritis), MS (multiple sclerosis), inflammatory bowel disease (IBD) and asthma, indicating that IL-17 contributes to the induction and / or or the development of such diseases.
There is ample evidence to show that suppressor T cells, now referred to as regulatory T cells (Treg cells), inhibit autoreactive T cells as an active mechanism of peripheral immune tolerance. It is heretofore clearly established that Treg cells can be divided into two different subtypes, namely natural (or constitutive) and inducible (or adaptive) populations according to their origin (Mills, Nat Rev Immunol 4: 841-855, 2004). In addition, a variety of T-reg cell subgroups have been identified by their surface markers or cytokine production, such as CD4 + Treg cells (including native CD4 + CD25 + Treg cells, IL-10-producing Tr1 cells, and TGF-β-producing Th3 cells). cells), CD8 + Treg cells, 2 Veto CD8 + cells, yö T cells, NKT (NK1.1 + CD4-CD8-) cells, NK1.1- CD4-CD8 cells, etc.
Accumulating evidence has shown that naturally occurring CD4 + CD25 + Treg cells play an active role in down-regulating pathogenic autoimmune responses and in maintaining immune homeostasis (Akbari et al.
Curr Opin Immunol l5: 627-633, 2003).
Autoimmune diseases include a wide range of diseases that can affect many different organs and tissues in the body (See, e.g., Paul, W.E. (1999) Fundamental Immunology, Fourth Edition, Lippincott-Raven, New York.).
Current treatments for human autoimmune diseases include glucocorticoids, cytotoxic agents, and newly developed biological drugs. Treatment of human systemic autoimmune diseases is generally empirical and unsatisfactory. Broad immunosuppressive drugs such as corticosteroids are largely used for a large number of severe autoimmune and inflammatory diseases. In addition to corticosteroids, other immunosuppressive agents are used in the treatment of systemic autoimmune diseases. Cyclophosphamide is an alkylating agent that causes a profound depletion of both T and B lymphocytes and a decrease in cell-mediated immunity. Cyclosporine, tacrolimus and mycophenolate mofetil are natural products with specific properties such as T lymphocyte inhibition and have been used to treat systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and, to a limited extent, vasculitis and myositis.
These drugs are associated with a significant renal toxicity. Methotrexate is also used as a "second-line drug" in RA, with the aim of reducing disease progression. It is also used in polymyositis and other connective tissue diseases. Other approaches that have been tried include monoclonal antibodies with the intention of blocking the action of cytokines or depleting lymphocytes (Fox, Am J Med 99: 82-88, 1995). Treatments for multiple sclerosis (MS) include interferon-ß and copolymer-1, which reduces the relapse rate by 20-30% and has only a modest effect on disease progression. MS is also treated with immunosuppressive agents, including methylprednisolone, other steroids, methotrexate, cladribine and cyclophosphamide. These immunosuppressive agents have minimal effect in the treatment of MS. The introduction of the antibody Tysabri (natalizumab), an alpha 4 integrin antagonist, in the treatment of MS has been overshadowed by incidents of progressive multifocal leukoencephalopathy (PML) in patients receiving treatment. Current treatment for RA uses substances that non-specifically inhibit or modulate immune functions such as methotrexate, sulfasalazine, hydroxychloroquine, leu onamide, prednisone, as well as newly developed TNF-αt antagonists etanercept and iniximab (Moreland et al. J Rheumatol 28: 1431- 52, 2001). Etanercept and in fl iximab block TNF-oc throughout the body, making patients more susceptible to death through sepsis, exacerbation of chronic mycobacterial infections and the development of demyelinating episodes.
In the case of organ-specific autoimmunity, a number of different therapeutic approaches have been tried.
Soluble protein antigens have been administered systemically to inhibit the subsequent immune response to this antigen. Such treatments include administration of basal myelin proteins, its dominant peptide or a mixture of myelin proteins to animals with experimental autoimmune encephalomyelitis and people with multiple sclerosis (Brocke et al. Nature 379: 343-6, 1996; Critch fi eld et al. Science 263: 1139- 43, 1994; Weiner et al. Annu Rev Immunol 12: 809-37, 1994), administration of type II collagen or a mixture of collagen proteins to animals with collagen-induced arthritis and humans with rheumatoid arthritis (Gumanovskaya et al. Immunology 91: 466-73, 1999; McKown et al. Arthritis Rheum 42: 1204-8, 1999; Trentham et al. Science 261: 1727-30, 1993), administration of insulin to animals and humans with autoimmune diabetes (Pozzilli and Gisella Cavallo, Diabetes Metab Res Rev 16: 306-7, 2000), and administration of S-antigen to animals and humans with autoimmune uveitis (Nussenblatt et al. Am J Ophthalmol 123: 583-92, 1997).
Another approach is the attempt to design rational therapeutic strategies for the systemic administration of a peptide antigen based on the specific interaction between T cell receptors and peptides bound to MHC molecules. A study using a peptide approach in an animal model of diabetes resulted in the development of antibody production against the peptide (Hurtenbach et al. J Exp Med 177: 1499, 1993). Another approach is the administration of T cell receptor peptide immunization. (See, e.g., Vandenbark et al. Nature 3412541, 1989). Yet another approach is to induce oral tolerance by ingestion of peptide or protein antigens. (See, e.g., Weiner, Immunol Today 18: 335, 1997).
Mucosal tolerance refers to the phenomenon of systemic tolerance to provocation with an antigen that has previously been administered via a mucosa, usually orally, nasally, or via the nose and respiratory tract, but also vaginally and rectally. (Weiner et al. Annu Rev Immunol l2z809-83 7, 1994). Mucosal tolerance was discovered in the 20th century in models of delayed hypersensitivity and contact allergic reactions in guinea pigs, but the mechanisms of tolerance remained poorly defined until the age of modern immunology. The use of cell separation techniques, cytokine synthesis tests, and transgenic models in which antigen-specific T cells can be detected in vivo have gradually elucidated mechanisms of mucosal tolerance (Garside and Mowat. Crit Rev Immunol 17: 1 19-137, 1997). It has become clear that antigen administration via mucous membranes can result in different types of tolerance depending on the route of administration and antigen dosage. For example, a high dose of oral antigen induces T cell activation followed by deletion of or anergy of T cells that respond to the antigen (Chen et al. Nature 376: 177-180, 1995) analogous to parenteral administration of high doses of soluble antigen . This results in the extinction of T cells that are specific for this antigen and do not respond to a subsequent antigen challenge, ie. passive tolerance. In contrast, a low dose of oral antigen does not induce any deletion or anergy but, when administered repetitively, induces a distinct type of immune response characterized by the presence of regulatory, protective T cells, Treg cells, which secrete anti-inflammatory cytokines. i.e. active tolerance (von Herrath, Res Immunol. 148: 541-554, 1997). These Treg cells usually belong to the class of CD4-positive T helper cells.
Delivery of intact protein antigen to the nasopharyngeal mucosa also induces protective Treg cells. In this case, both CD4- and CD8-positive T cells can be induced. Regulatory Treg cells induced after oral or intranasal antigen administration produce anti-inflammatory cytokines such as IL-4, IL-10 and TGF-β. To induce mucosal tolerance, antigen can also be given in the form of an aerosol. Administration by these three routes, oral, intranasal or aerosol inhalation, results in antigen uptake and presentation in different lymphoid compartments in each case. Accordingly, oral antigens are presented mainly to T cells in mesenteric lymph nodes and to some extent in Peyer's plaques, intranasal antigens in deep-lying cervical lymph nodes, and inhaled antigens in mediastinal lymph nodes. Repeated exposure to antigen has the potential to induce regulatory T cells in each case, but the properties of these cells differ depending on the route of administration and the type of antigen. While regulatory cells induced by oral antigen are CD4-positive T cells and express T cell receptors (TCRs) consisting of αβ heterodimers, in the case of nasal and respiratory antigen administration, the regulatory cells may also be CD8-positive T cells. which expresses a γ-heterodimer TCR (ie γ-T cells). Some of these cells may also have a CD8 receptor which is an α-homodimer instead of the conventional α1-heterodimer TCR. A majority of cells carrying CD8otoc and γ-TCR populate skin or mucosal tissues.
In recent decades, there has been a significant increase in both the incidence and prevalence of allergic diseases in the Western world. Allergic rhinitis, the most common of these conditions, affects 15-20 percent of the population. The allergic reaction is activated by allergen-induced cross-linking of specific IgE on the surface of mast cells and basols, leading to the release of histamine and other mediators that cause an acute allergic reaction followed by a late-phase reaction, which is characterized by the presence of eosinophils, neutrophils and Th2 cells that synthesize IL-4, IL-5 and IL-13.
Specific immunotherapy (SIT) is recognized as an effective treatment for allergic rhinitis. SIT has traditionally been performed by repeated subcutaneous administration of small amounts of specific allergen.
Although this form of treatment may be an effective treatment option, there are concerns about the safety of this form of immune treatment, as well as regarding the difficulty of standardizing the allergen extract used as a vaccine. Consequently, there is a strong interest in developing alternative and new treatments for allergic diseases. One approach is the use of mucosal vaccines (Widermann, Curr Drug Targets In Allmy 4, 577-583, 2005). Other alternatives are based on the use of allergen derivatives with reduced or no allergenic ability as vaccines (Vrtala et al. Methods 32, 313-320, 2004). These include allergens obtained using protein techniques and synthetic peptides representing immunodominant T cell epitopes of allergens. Ole el, for example, has been identified as the most relevant allergen in olive pollen (Wheeler et al. Mol Immunol 27.63 l-636, 1990). Immune responses are now modified by administering polypeptides, alone or in combination with adjuvants (immunomodulatory agents). The hepatitis B virus vaccine contains, for example, recombinant hepatitis B virus surface antigen, a non-body antigen which has been formulated in aluminum hydroxide, which acts as an adjuvant. This vaccine induces an immune response against hepatitis B virus surface antigen to protect against infection. An alternative approach involves administering an attenuated, replication-inhibited and / or non-pathogenic form of virus or bacterium, each and every non-body-specific antigen, to initiate a protective immune response against the pathogen in the host. For example, the oral polio vaccine is composed of a live, attenuated virus, a non-native antigen, which infects cells and replicates in the vaccinated individual to induce effective immunity to poliovirus, a foreign or non-native antigen, without causing clinical disease. The inactivated polio vaccine may alternatively contain inactivated or "killed" virus which cannot infect or replicate and which is administered subcutaneously to induce protective immunity against poliovirus.
DNA therapies have been described for the treatment of autoimmune diseases. Such DNA therapies include DNA encoding the antigen-binding regions of the T cell receptor to alter levels of autoreactive T cells that drive the autoimmune response (Waisman et al. Nat Med 2: 899-905, 1996; U.S. Patent 5,939,400). DNA encoding autoantigens was applied to particles and administered with a gas gun ("gene gun") to the skin to prevent MS and collagen-induced arthritis. (WO 97/46253; Ramshaw et al. Immunol Cell Biol 752409-413, 1997). DNA-encoding adhesion molecules, cytokines (eg TNFot), chemokines (eg CC chemokines) and other immune molecules (eg Fas ligand) have been used to treat autoimmune diseases in animal models (Youssef et al. J Clin Invest l06: 36l-37l, 2000; Wildbaum et al. J Clin Invest 1062671- 679, 2000; Wildbaum et al. J Immunol 16525860-5866, 2000).
Methods for treating autoimmune diseases by administering nucleic acid encoding one or more autoantigenes are described in WO 00/53019, WO 2003/045316 and WO 2004/047734. Although these procedures have been successful, further improvements are needed.
Bacterial enterotoxins are used as immunostimulatory adjuvants in vaccines for the prevention of infectious diseases. Cholera toxin (CT) and the closely related heat-unstable LT toxin from E.coli are perhaps the most potent and best-studied mucosal adjuvants used experimentally today (Rappuoli et al. Immunol Today 202493-500) but once tested clinically, their potential 6 toxicity and association with cases of Bell's palsy (idiopathic facial paresis) led to their withdrawal from the market (Gluck et al. J Infect Dis 181: 1129-1132, 2000; Gluck et al. Vaccine 20 (Suppl.1): S42-44, 2001; Mutsch et al. N Engl J Med. 350: 896-903, 2004). The bacterial enterotoxins CT and LT have been shown to be effective immune enhancers in experimental animals as well as in humans. (Freytag et al. Curr Top Microbiol Immunol 236: 215-236, 1999). These enterotoxins are structurally ABS complex and consist of an ADP-ribosyltransferase active A1 subunit and an A2 subunit that binds the A1 subunit to a pentamer of B subunits. The holotoxins bind to most mammalian cells via the B subunit (CTB) which interacts specifically with the GM1 ganglioside receptor in the cell membrane. While holotoxins have been shown to enhance mucosal immune responses, the conjugate between CTB and antigen has been used to specifically tolerate the immune system. (Holmgren et al. Am J Trop Med Hyg 50: 42-54, 1994). Studies in mice have shown that CT and LT can accumulate in the olfactory nerve and olfactory lobe when given intranasally, a mechanism that depends on the ability of the B subunits of CT or LT to bind to GM1 ganglioside receptors present on all nucleated mammalian cells. (Fujihashi et al. Vaccine 20: 2431-2438, 2002). Although less toxic mutants of CT and LT have been developed, with significant adjuvant function, such molecules still carry a significant risk of causing side effects (Giuliani et al. J Exp Med 187: 1123-1132, 1998; Yamamoto et al. J Exp Med 185: 1203-1210, 1997) especially when one considers that the adjuvant ability of CT and LT appears to be a combination of the ADP-ribosyltransferase activity of the A subunit and the ability to bind ganglioside receptors on the target cells (Soriani et al. Microbiology 148: 667-676 , 2002). These observations and others prevent the use of CT or LT holotoxins in human vaccines.
Current observations, on the other hand, have demonstrated that it is possible to maintain adjuvant functions of these molecules, with no or greatly reduced toxicity, by introducing point-specific mutations into the gene encoding the Al subunit. Examples of mutated molecules that have been shown to be effective adjuvants are LTK63 and LTR72, (Giuliani et al. J Exp Med 187: 1123-1132, 1998) where the former has no enzymatic activity and the latter has significantly reduced ADP-ribosylating ability. . Despite this, the GM] -ganglioside receptor-dependent binding remains a problem in these mutants and consequently they can still cause nerve cell accumulation and neurotoxicity.
A better solution to this dilemma of efficacy against toxicity is the CTA1-DD molecule which has been shown to be a very effective mucosal and systemic adjuvant (Ågren et al. J Immunol 158: 3936-3946, 1997; US 5,9l7,026 ). This unique adjuvant is based on the enzymatically active Al subunit of CT, combined with a dimer of an immunoglobulin binding element from protein A from Staphylococcus aureus. The molecule thereby avoids binding to all core-bearing cells, which could result in unwanted reactions, and makes full use of the CTA1 enzyme in the holotoxin. Consequently, all studies to date have found that CTA1-DD is non-toxic and retains excellent immune-boosting functions. When administered systemically, CTA1-DD provides comparable adjuvant effects to those of intact CT and greatly enhances both cellular and humoral immunity to specific immunogens co-administered with the adjuvant. It also acts as a mucosal adjuvant and should be safe as it lacks a B subunit which is a prerequisite for CT holotoxin toxicity. CTA1-DD cannot bind to ganglioside receptors; instead, it activates B cells, limiting the CTA1 -DD adjuvant to a limited repertoire of cells with which it can interact. However, the adjuvant effect is not entirely dependent on B cells, which has been shown to strongly induce specific CD4-positive T cell immunity after intranasal immunization using the CTA1-DD adjuvant in B cell deficient mice (Eliasson et al Vaccine 25: 1243 -52, 2008, Akhiani et al., Scand J. Immunol 63: 97-105, 2006).
The adjuvant effect of CTA1-DD was not found in the mutants CTA1-E112K-DD and CTA1-R7K-DD, which lack the ADP-ribosylating enzymatic activity (Lycke, Immunol Lett 97: 193-198, 2005) WO 2009/078796 further describes immunomodulatory complexes , comprising the mutant CTA1-R7K-DD and more specifically the immunomodulatory complexes comprising CTA1-R7K-DD linked to the shared immunodominant collagen II peptide, comprising amino acids 260-273 (CII260-273).
A conjugate of CTB and a peptide derived from bovine collagen II has been shown to be able to protect mice from developing collagen-induced autoimmune ear disease as well as collagen-induced arthritis (Kim et al. Ann Otol Rhinol Laryngol 110: 646-654, 2001 Tarkowski et al.
Arthritis Rheum 42: 1628-34, 1999). However, CTB would not be suitable for use in humans due to its GM1 ganglioside binding properties and potentially neurotoxic effects as discussed above.
SUMMARY OF THE INVENTION None of the present invention relates to improved methods and compositions for the prophylaxis, prevention and / or treatment of autoimmune or allergic diseases, comprising administering immunomodulatory complexes, wherein the autoimmune complex is a fusion protein comprising a subunit of a mutated A1 subunit. from the cholera toxin (CTA1), a peptide capable of binding to a specific cellular receptor and one or more epitopes associated with the disease. Administration of a therapeutically or prophylactically effective amount of the immunomodulatory complex to an individual triggers inhibition of an immune response directed against an antigen associated with the disease, thereby treating or preventing the disease.
The epitope can be an autoimmune epitope when the disease to be treated is an autoimmune disease, the epitope can be an allergenic epitope when the disease to be treated is an allergic disease.
In one embodiment, the invention provides an immunomodulatory complex, which comprises a fusion protein comprising: (a) a mutated subunit of the ADP-ribosylating A1 subunit of the cholera toxin (CTA1) (b) a peptide capable of binding to a specific cellular receptor and (c) one or more epitopes associated with an autoimmune or allergic disease in which the amino acids of the mutated CTA] subunit, which correspond to amino acid 7, arginine and amino acid 187, cysteine, in the naturally occurring CTA1, have been replaced.
In a preferred embodiment, the amino acid lysine has been further inserted into the N-terminus of the mutated CTA1 subunit.
In a preferred embodiment, the fusion protein CTA1-R7K / C 187A mutant comprises a (SEQ ID NO.
NO: 1), where amino acid 7, arginine, in the original CTA1 sequence has been replaced by a lysine and where amino acid 187, cysteine, in the original CTA1 sequence has been replaced by an alanine.
In an even more preferred embodiment, the fusion protein comprises the K-CTA1-R7K / C1887A mutant (SEQ. 1D, NO: 2), wherein amino acid 7, arginine, in the original CTA1 sequence has been replaced by a lysine, wherein amino acid 187, cysteine, in the original CTA1 sequence has been replaced by an alanine, and where the amino acid lysine has been inserted into the N-terminus.
Replacement of amino acid 7, arginine, with a lysine knocks out the ADP-ribosylating activity, replacement of amino acid 187 cysteine with an alanine, prevents the formation of dimers and the insertion of a lysine in the N-terminal part dramatically increases the expression and production of a fusion protein .
In one embodiment, the fusion protein comprises a peptide which specifically binds to a receptor expressed on a cell capable of presenting antigen, especially cells expressing MHC class I or MHC class II antigen. The antigen presenting cell can be selected from the group consisting of lymphocytes, such as B lymphocytes, T cells, monocytes, macrophages, dendritic cells, Langerhans cells, epithelial cells and endothelial cells.
The peptide is a peptide that binds to receptors on the above cells, preferably an Ig or Fc receptor expressed by said antigen presenting cell and most preferably to receptors on B lymphocytes and dendritic cells.
Examples of specific peptides are peptides capable of binding to receptors such as: (i) granulocyte-macrophage colony stimulating factor (GM-C SF) having the ability to bind to GM-CSF receptor-on / ß heterodimers present on monocytes, neutrophils, eosinophils, fibroblasts and endothelial cells. (ii) CD4 and CD8 expressed on T cells, which together with the T cell receptor (TcR) act as co-receptors for MHC class II and MHC class I molecules, respectively. MHC class I is expressed on the most core-bearing cells, while MHC class II molecules are expressed on dendritic cells, B cells, monocytes, macrophages, myeloid and erythroid precursor cells and certain epithelial cells, (iii) CD28 and CTLA-4, two mainly homodimer proteins expressed on T cells, which bind to CD80 and CD86B7 expressed on B cells. (iv) CD4O is present mainly on the surface of mature B cells, which interact with CD40L (gp39 or CD154) expressed on T cells. cells, (v) different isotypes of the constant Ig tongue region regions, which interact with a number of high-affinity or low-affinity Fc receptors present on mast cells, basols, eosinophils, platelets, dendritic cells, macrophages, NK cells and B cells , (vi) complement receptors (CR), CR1, CR2 and CRS, which are expressed on B cells and follicular dendritic cells, have been shown to be important in the generation of normal humoral immune responses and they probably also participate in the development of autoimmune (viii) lectin receptors, C-type (CLR) such as Dectin-1 expressed on dendritic cells, (viii) DEC205, an endocytic receptor for antigen uptake and modification, which is expressed at high levels in a subset of dendritic cells, ( ix) CD1 lc, a cell surface receptor for a number of soluble factors and proteins (LPS, fibrinogen, iC3b) which is found primarily on myeloid cells, (x) the mannose receptor present on dendritic cells, macrophages and other antigen presenting cells, (xi) the specific HSP60 receptors present on macrophages. (xii) CD103, an integrin alpha chain, expressed by a subset of dendritic cells. (xii) the 33D1 antigen, which is present in dendritic cells.
According to a particularly preferred embodiment of the invention, said peptide consists of protein A or a fragment thereof, in one or two copies, such as one or two D subunits thereof. According to another particularly preferred embodiment of the invention, said peptide is constituted by an antibody fragment, such as a single chain antibody fragment, which specifically binds to a receptor expressed on a cell capable of antigen presentation.
The peptide is preferably such that the resulting fusion protein is water soluble and has the ability to direct the fusion protein towards a specific cell receptor which is different from receptors which bind to the original toxin, thereby mediating intracellular uptake of at least said subunit.
The autoantigenic epitopes can be associated with an autoimmune disease, such as insulin-dependent diabetes mellitus (IDDM), multiple sclerosis (MS), systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA), Sjögren's syndrome (SS).
In some embodiments, the autoantigenic epitopes associated with HDDM are an epitope derived from the group consisting of: preproinsulin; proinsulin, insulin, and insulin B chain; glutamic acid decarboxylase-65 and -67 (GAD), tyrosine phosphatase-IA-2; islet cell-specific glucose-6-phosphatase-related protein (IGRP) and islet cell antigen (69 kD).
In some embodiments, the autoantigen epitope associated with MS is an epitope derived from the group consisting of myelin basal protein (MBP), proteolipid protein (PLP), myelin-associated oligodendrocyte basal protein (MOBP), myelin-oligodendrocyte glycoprotein (MOGP), and myogassocyte protein (MOGP). MAG).
In some embodiments, the autoantigenic epitope associated with RA is an epitope derived from the group consisting of type I, II, III, IV, V, IX, and XI collagen, GP-39, gr laggrin and fi brin. In a preferred embodiment, the epitope is derived from type II collagen, the epitope being preferably the shared immunodominant collagen II peptide, comprising amino acids 260-273 (CII260-273).
The allergic epitopes may be associated with allergic asthma, allergic rhinitis, allergic alveolitis, atopic dermatitis or food hypersensitivity. In some embodiments, the allergic epitopes are an epitope ll derived from a plant pollen, such as Ole el allergen from olive pollen, Cry jI and Cry jII allergens from Japanese cedar pollen, timothy grass pollen nPhl p4 or the dominant birch pollen allergen Bet vl , the dominant wormwood pollen antigen Art vl, an animal allergen such as the feline allergen Fel dl or the dog allergen Can fl, the dust mite allergen Der fl, Der pl, Der ml, Blo t4, a fungal antigen such as the Alternaría antigen Alt al, the Asperígullus antigen Asp fl, Cladosporíum- the antigens ClA h1 and Cla h2, the Penicillum antigen Pen chl3; or a food allergen such as the chicken egg white allergens Gal dl, Gal d2 and Gal d3, the peanut allergen Ara h2, the soybean allergen Gly ml, Gly m5 and Gly m6, the fish allergen Gad cl or the shrimp allergen Pen al.
In a preferred embodiment, the immunomodulatory complex is the fusion protein K-CTA1-R7K / Cl87A-COL-DD (SEQ ID NO: 4), where COL is the shared immunodominant collagen II peptide, which comprises amino acids 260-273 (CII260 -273) (SEQ ID NO: 5).
The present invention provides methods and compositions for the treatment, prophylaxis and / or prevention of an autoimmune disease, such as multiple sclerosis, rheumatoid arthritis, insulin dependent diabetes mellitus, autoimmune uveitis, Behçet's disease, primary biliary cirrhosis, myasthenia gravis, Sjögren's syndrome, pem fi gus vulgaris, sclerosis vulgaris, , pemic anemia, systemic lupus erythematosus (SLE) and Graves' disease, comprising administering an immunomodulatory complex according to the invention to an individual, which comprises one or more autoantigenic epitopes associated with the disease.
In certain embodiments, the present invention provides improved methods for the treatment, prophylaxis and / or prevention of the autoimmune disease insulin dependent diabetes mellitus (IDDM), which comprises administering an immunomodulatory complex comprising one or more autoantigenic epitopes associated with the IDDM of the invention. individual. In some embodiments, the autoantigenic epitopes associated with IDMM are an epitope derived from the group consisting of: preproinsulin; proinsulin, insulin, and insulin B chain; glutamic acid decarboxylase-65 and -67 (GAD); tyrosine phosphatase IA-2; islet cell-specific glucose-6-phosphatase-related protein (IGRP) and islet cell antigen (69 kD).
In other embodiments of the present invention, there are provided improved methods of treating, prophylaxis and / or preventing multiple sclerosis (MS) comprising administering an immunomodulatory complex of the invention comprising one or more autoantigenic epitopes associated with MS to an individual. In some embodiments, the autoantigenic epitope is an epitope derived from the group consisting of myelin basal protein (MBP), proteolipid protein 12 (PLP), myelin-associated oligodendrocyte basal protein (MOBP), myelin oligodendrocyte glycoprotein (MOG) and myelin associn (MOG).
In other embodiments, improved methods of treating, prophylaxis and / or preventing rheumatoid arthritis (RA) are provided, which comprise administering an immunomodulatory complex of the present invention comprising one or more autoantigenic epitopes associated with RA to a subject. In some embodiments, the autoantigenic epitope is an epitope derived from the group consisting of type I, II, III, IV, V, IX, and XI collagen, GP-39, fi laggrin and fibrin. In a preferred embodiment, the epitope is derived from collagen type II, the epitope being preferably the divided immunodominant collagen II peptide comprising amino acids 260-273 (CII260-273).
According to a particularly preferred embodiment of the invention, said peptide consists of protein A or a fragment thereof, in one or two copies, such as one or two D subunits thereof. According to another particularly preferred embodiment of the invention, said peptide is constituted by an antibody fragment, such as a single chain antibody fragment, which binds specifically to a receptor expressed on a cell capable of presenting antigen.
Multiple immunomodulatory complexes comprising different autoantigenic epitopes may be administered as a cocktail and each individual immunomodulatory complex may also comprise multiple autoantigenic epitopes. Similarly, multiple immunomodulatory complexes comprising different allergic epitopes may be administered as a cocktail and each individual immunomodulatory complex may comprise multiple allergenic epitopes.
In certain variations, the methods and compositions for treating, prophylaxis and / or preventing an autoimmune or allergic disease may further comprise administering the immunomodulatory complex of the present invention in combination with other substances, for example polynucleotides comprising an immunomodulatory sequence, pharmacological substances, adjuvants, cytokines or vectors encoding cytokines.
Yet another embodiment of the present invention provides a pharmaceutical composition comprising an immunomodulatory complex according to the invention. The pharmaceutical composition according to the invention can be used for prophylaxis, prevention and / or treatment of an allergic or autoimmune disease. The autoimmune disease can be selected from the group consisting of insulin-dependent diabetes mellitus, multiple sclerosis, rheumatoid arthritis, autoimmune uveitis, primary biliary cirrhosis, myasthenia gravis, Sjögren's syndrome, pemphigus vulgaris, scleroderma, pemic anemia, systemic lupus erythematosus and Graves' disease. The allergic disease can be selected from the 13 group which consists of allergic asthma, allergic rhinitis, allergic alveolitis, atopic derrnatitis or food hypersensitivity.
Yet another embodiment of the present invention provides the use of an immunomodulatory complex according to the invention for the production of a medical product for the prophylaxis, prevention and / or treatment of an autoimmune or allergic disease. The autoimmune disease can be selected from the group consisting of insulin-dependent diabetes mellitus, multiple sclerosis, rheumatoid arthritis, autoimmune uveitis, primary biliary cirrhosis, myasthenia gravis, Sjögren's syndrome, pemphigus vulgaris, scleroderma, pemic anemia, systemic lupus erythematosus and Graves' disease.
The allergic disease can be selected from the group consisting of allergic asthma, allergic rhinitis, allergic alveolitis, atopic derinatitis or food hypersensitivity.
In yet another embodiment, the present invention provides isolated nucleic acid molecules encoding an immunomodulatory complex of the invention. Accordingly, the present invention provides isolated nucleic acid molecules encoding an immune complex which is a fusion protein comprising a mutated subunit of the ADP-ribosylating Al subunit of the cholera toxin (CTA1), a peptide capable of binding to a specific and specific cellular receptor. which are associated with an autoimmune or allergic disease.
In one embodiment, the nucleic acid of the invention encodes an immunomodulatory complex comprising a fusion protein comprising: (a) a mutated subunit of the ADP-ribosylating Al subunit of the cholera toxin (CTA1) (b) a peptide capable of binding to a specific cellular receptor, and (c) one or fl epitopes associated with an autoimmune or allergic disease in which the amino acids corresponding to amino acid 7, arginine, and amino acid 187, cysteine, in the original CTA1 have been replaced in the mutated CTA1 subunit.
In a preferred embodiment, the amino acid lysine has been further inserted into the N-terminal of the mutated CTA1 subunit.
In a preferred embodiment, the nucleic acid of the invention encodes a fusion protein comprising the CTA1-R7K / Cl87A mutant (SEQ ID NO: 1), wherein amino acid 7, arginine, in the original CTA1 sequence has been replaced by a lysine and wherein amino acid 187, cysteine, in the original sequence has been replaced by an alanine. In an even more preferred embodiment, the nucleic acid of the present invention encodes a fusion protein comprising the K-CTA1-R7K / Cl 87A mutant (SEQ ID NO: 2), wherein amino acid 7, arginine, in the original CTA1 sequence has been replaced with a lysine and where amino acid 187, cysteine, in the original CTA1 sequence has replaced with an alanine, and where the amino acid lysine has been inserted into the N-terminal.
In one embodiment, the nucleic acid of the invention encodes a fusion protein which comprises a peptide which specifically binds to a receptor expressed on a cell capable of presenting antigen, in particular cells expressing MHC class I or MHC class II molecules. The antigen presenting cells can be selected from the group consisting of lymphocytes, such as B lymphocytes, T cells, monocytes, macrophages, dendritic cells, Langerhans cells, epithelial cells and endothelial cells.
In one embodiment, the nucleic acid of the present invention encodes a fusion protein comprising an autoantigenic epitope associated with an autoimmune disease, such as insulin-dependent diabetes mellitus (IDDM), multiple sclerosis (MS), systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA). or Sjögren's syndrome (SS).
In another embodiment, nucleic acid of the present invention encodes a fusion protein comprising an allergic epitope associated with an allergic disease, such as allergic asthma, allergic rhinitis, allergic alveolitis, atopic dermatitis or food hypersensitivity.
In some embodiments, the autoantigenic epitopes associated with IDDM are an epitope derived from the group consisting of: preproinsulin; proinsulin, insulin, and insulin B chain; glutamic acid decarboxylase-65 and -67 (GAD); tyrosine phosphatase IA-2; islet cell-specific glucose-6-phosphatase-related protein (IGRP) and islet cell antigen 69 kD. In some embodiments, the autoantigenic epitopes associated with MS are an epitope derived from the group consisting of: myelin basal protein (MBP), proteolipid protein (PLP), myelin-associated oligodendrocyte basal protein (MOBP), myelin oligodendrocyte glycoprotein (MOBP) and myelin (MOPP). ). In some embodiments, the autoantigenic epitopes associated with RA are an epitope derived from the group consisting of type I, II, III, IV, V, IX, and XI collagen, GP-39. fi laggrin, and fi brin. In some embodiments, the autoantigenic epitopes associated with SS are an epitope derived from the group consisting of: HSP60 stress protein, fodrin, Ro-ribonucleoprotein (or SSA-ribonucleoprotein) and La-ribonucleoprotein (or SSB-ribonucleoprotein).
The nucleic acids of the present invention may be DNA or RNA. The nucleic acid of the present invention may be a nucleic acid sequence encoding the fusion protein K-CTA1-R7K / Cl87A-COL-DD, such as the nucleic acid sequence SEK. ID. NO .: 3.
In another embodiment, the invention provides a pharmaceutical composition comprising a nucleic acid of the invention. The pharmaceutical composition can be used for prophylaxis, prevention and / or treatment of an allergic or autoimmune disease. The invention further provides methods for the prophylaxis, prevention and / or treatment of an autoimmune or allergic disease in an individual, the method comprising administering to an individual an effective amount of a nucleic acid of the invention.
In a further embodiment of the present invention there are provided plasmids, vectors and expression systems comprising a nucleic acid of the invention. The recombinant expression system is preferably adapted for bacterial expression. The invention further provides transformed cells containing a plasmid, vector or expression system according to the invention. The transformed cells are preferably transformed bacterial cells.
DEFINITIONS Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. As used herein, the following themes and phrases have the meanings ascribed to them unless otherwise specified elsewhere.
The amino acid sequence of the ADP-ribosylating Al subunit of the cholera toxin (CTA1) can be found e.g. in GenBank with accession number AAM22586.1, ADG44926. 1, AAM74170.1, CAE11218.1, or AAA27514.1. The term "a subunit of the ADP-ribosylating Al subunit of the cholera toxin (CTA1)" refers to a polypeptide comprising at least one sequence corresponding to the sequence from amino acid 7, lysine, to amino acid 187, cysteine, from the sequence of the mature ADP the ribosylating Al subunit of the cholera toxin (CTA1), such as a polypeptide comprising at least one sequence corresponding to the sequence from amino acid 1, asparagine, to amino acid 187, cysteine, from the sequence of the mature ADP ribosylating Al subunit of the cholera toxin (CTA1) , or at least one sequence corresponding to the sequence from amino acid 1, asparagine, to amino acid 194, serine, from the sequence of the mature ADP-ribosylating Al subunit of the cholera toxin (CTA1).
The terms "polynucleotide" and "nucleic acid" refer to a polymer composed of a plurality of nucleotide units (ribonucleotide or deoxyribonucleotide or related structural variants) joined together via phosphodiester linkages. A polynucleotide or nucleic acid, usually any length, may be of essential essence. from between about six (6) nucleotides to 109 nucleotides or longer Polynucleotides and nucleic acids include RNA, DNA, synthetic forms and mixed polymers, both sense and antisense strands, double or single stranded, and may also be chemically or biochemically modified or may be contain non-naturally occurring or derivatized nucleotide bases, as will be apparent to one skilled in the art.
"Antigen" as used herein refers to any molecule that can be recognized by the immune system, that is, by B cells or T cells or both.
"Autoantigen" as used herein refers to an endogenous molecule, usually a polysaccharide or a protein or fragment thereof which elicits a pathological immune response. Autoantigen includes glycosylated proteins and peptides as well as proteins or peptides that carry other forms of post-translational modifications, including citrullinated peptides. When referring to the autoantigen or epitope thereof as "associated with an autoimmune disease", it is understood that the autoantigen or epitope is involved in the pathophysiology of the disease either by inducing the pathophysiology (ie associated with the etiology of the disease), contributing to or facilitating a pathophysiological process; and / or by being the target of a pathophysiological process. In the case of an autoimmune disease, for example, the immune system incorrectly targets the autoantigen, which causes damage and malfunction of cells and tissues in which the autoantigen is expressed and / or is present.
Under normal physiological conditions, autoantigen is ignored by one's own immune system through the elimination, inactivation or lack of activation of immune cells that have the capacity to recognize the autoantigen through a process called "immune tolerance".
The "allergen" as used herein refers to an exogenous molecule, usually a polysaccharide or a protein or fragment thereof, which triggers a pathological immune response. Allergens include glycosylated proteins and peptides, as well as proteins and peptides that carry other forms of post-translational modifications. The allergen can be derived from e.g. pollen, fungi, insecticides, dandruff, mold and foods. A number of food allergens have been purified and are well characterized, such as Ara hl, Ara h2, Ara h3 and Ara h6 from peanut; Gal dl, Gal d2, and Gal d3 from chicken egg white; Gly ml from soybean; Gad cl from fish; and Pen al from shrimp. The dominant cat allergen (Fel dl) and the dog allergen (Can fl), as well as the dust mite allergen Der fl and Der pl, are well characterized.
The original hourglass grass pollen nPhl p4, as well as a number of related recombinant allergens, rPhl lp, rPhl 2p, rPhl 5p, rPhl 6p, rPhl 7p, rPhl llp, rPhl l2p, the predominant birch pollen allergen Bet vl, the predominant plane tree I the olive pollen allergen Ole el, the predominant wormwood ambrosia allergen Amb al, the predominant wormwood allergen Art vl and Art V3, are well characterized. As used herein, the term "epitope" is intended to mean a portion of a polysaccharide or polypeptide that has a particular form or structure that is recognized by either B cells or T cells in an animal's immune system. An epitope may include portions of both a polysaccharide and a polypeptide, e.g. a glycosylated peptide.
"Autoantigen epitope" refers to an epitope of an autoantigen which triggers a pathological immune response.
"Allergenic epitope" refers to an epitope of an allergen that triggers a pathological immune response.
The terms "polypeptide", "peptide", and "protein" are used interchangeably and herein refer to a polymer of amino acid residues. The term refers to amino acid polymers in which one or more of the amino acid residues is an artificial chemical replica of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
"Body-specific protein", "body-specific polypeptide" or "body-specific peptide" is used interchangeably herein and refers to any protein, polypeptide or peptide or fragment or derivative thereof which is: encoded in the animal's genome; produced or generated in the animal; can be modified post-translationally at any time in the animal's life; and is present in the animal non-physiologically. The term "non-physiological" or "non-physiological", when used to describe body proteins, body polypeptides or body peptides of the present invention, means a difference or deviation from the normal role or process of the animal for this body protein, body gene. polypeptide or polypeptide peptide. When referring to the body protein, body polypeptide or body peptide as "associated with a disease" or "involved in a disease", it is to be understood that the body protein, body polypeptide or body peptide may be modified in its form or structure and consequently unable to perform its physiological role or process or may be involved in the pathophysiology of the condition or disease either by inducing the pathophysiology; cause or facilitate the pathophysiological process; and / or be the target of a pathophysiological process. In autoimmune diseases, for example, the immune system abnormally attacks the body's own proteins, causing damage and dysfunction of cells and tissues in which the body's own protein is expressed and / or is present. Alternatively, the body protein, body polypeptide or body peptide may itself be expressed in non-physiological levels and / or have a non-physiological function.
In neurodegenerative diseases, for example, the body's own proteins are expressed in a different way and aggregate in lesions in the brain, thereby causing neural dysfunction. In other cases, the body's own protein exacerbates an unwanted condition or process. In osteoarthritis, for example, the body's own proteins, including collagenases and matrix metalloproteinases, break down cartilage that covers the joint surfaces in joints in a different way. Examples of post-translational modifications of proprietary proteins or proprietary polypeptides or proprietary peptides are glycosylation, addition of lipid groups, reversible phosphorylation, addition of dimethylarginine residues, citrullination and proteolysis and more specifically citrullination of fillagrin and fi brine with peptidylpharinemine, citrullination of MBP and SLE autoantigen proteolysis of caspases and spruce enzymes.
Immunologically, the body's own proteins, polypeptides and peptides are all considered to be host-specific self-antigens and under normal physiological conditions these are ignored by the immune system through the elimination, inactivation or lack of activation of immune cells capable of recognizing self-antigen through a process called "immune tolerance". A body protein, polypeptide or peptide does not include immune proteins, polypeptides or peptides which are molecules that are physiologically expressed only by cells of the immune system for the purpose of regulating immune function.
The immune system is the defense mechanism that provides the means to create a rapid, highly specific and protective response to the myriad of potentially pathogenic microorganisms present in the animal world. Examples of immunoproteins, polypeptides or peptides are proteins including the T cell receptor, immunoglobulins, cytokines, including type I interleukins and type II cytokines, including the interferons and IL-10, TNF, lymphotoxin and chemokines such as macrophage protein amatorial protein-1-alpha and beta. , monocyte chemotactic protein and RANTES and other molecules directly involved in immune function, such as Fas ligand. There are certain immune proteins, polypeptides or peptides that are included in the body protein, polypeptide or peptide of the present invention and they are: MHC class I membrane glycoproteins, MHC class II glycoproteins and osteopontin. Physical proteins, polypeptides or peptides do not include proteins, polypeptides or peptides that are missing in the individual, either in whole or in part, due to a genetic or acquired deficiency, which gives a metabolic or functional disease and is replaced either by administration of said protein, polypeptide or peptide or by administering a polynucleotide encoding said protein, polypeptide or peptide (gene therapy). Examples of such diseases include Duchenne muscular dystrophy, Becker muscular dystrophy, cystic fibrosis, phenylketonuria, galactosemia, maple syrupy disease and homocystinuria.
"Modulation of", "modulation" or "altering an immune response" as used herein means any alteration of an existing or potential immune response to an autoimmune or allergenic epitope, including e.g. nucleic acids, lipids, phospholipids, carbohydrates, proprietary polypeptides, protein complexes or ribonucleoprotein complexes, which occur as a result of administration of an immunomodulatory complex or polynucleotide encoding an immunomodulatory complex.
Such modulation includes any change in the presence, capacity or function of any immune cell that is involved in or has the ability to participate in an immune response. Immune cells include 19 B cells, T cells, NK cells, NK T cells, professional antigen presenting cells, non-professional either presenting cells, inflammatory cells or any other cell involved in or affecting an immune response. "Modulation" includes any change that is transmitted to an existing immune response, a maturing immune response, a potential immune response, or the ability to induce, regulate, influence, or respond to an immune response. Modulation includes all changes in the expression and / or function of genes, proteins and / or other molecules in immune cells that are part of an immune response.
"Modulation of an immune response" includes, for example, the following; elimination, deletion or sequestration of immune cells; induction or generation of immune cells that can modulate the fi functional function of other cells, such as autoreactive lymphocytes, antigen presenting cells, or inflammatory cells; induction of a non-reactive state of an immune cell (ie anergy); increasing, decreasing or altering the activity or function of immune cells or the ability to do so, including but not limited to altering the pattern of proteins expressed in these cells. Examples include altered production and / or secretion of certain classes of molecules, such as cytokines, chemokines, growth factors, transcription factors, kinases, co-stimulatory molecules or other cell surface receptors; or any other combination of these modulating events.
For example, administration of an immunomodulatory complex or a polynucleotide encoding an immunomodulatory complex may modulate an immune response by eliminating, sequestrating or inactivating immune cells that mediate or are capable of mediating an unwanted immune response; to induce, generate or activate immune cells that mediate or are capable of mediating a protective immune response; to alter the physical or functional properties of immune cells; or a combination of these effects. Examples of measurements of the modulation of an immune response include, but are not limited to, the examination of the presence or absence of immune cell populations (using fate cytometry, immunohistochemistry, histology, electron microscopy, polymerase chain reaction (PCR)); measurement of the functional capacity of immune cells includes the ability or resistance to proliferation or to divide in response to a signal (such as using T cell proliferation assay based on SH thymidine incorporation after stimulation with anti-CD3 antibody, anti-T cell receptor antibody, anti -CD28 antibody, calcium ionophores, PMA, antigen presenting cells loaded with a peptide or a protein antigen; B cell proliferation assay); measuring the ability to kill or lyse other cells (such as cytotoxic T cell assays); measurements of cytokines, chemokines, cell surface molecules, antibodies and other products of the cells (eg by fate cytometry, enzyme-linked immunosorbent assay, Western blot assay, protein microarray assay, immunoprecipitation assay); measurement of biochemical markers for activation of immune cells or signaling pathways within immune cells (eg Western blot and immunoprecipitation analysis of tyrosine, serine or threonine phosphorylation, polypeptide cleavage and the formation or dissociation of protein complexes; protein array analysis by DNA transcription); DNA arrays or subtractive hybridization); measurements of cell death by apoptosis, necrosis or other mechanisms (eg annexin V staining, TUNEL assays, gel electrophoresis to measure the presence of DNA ladders, histology; ororogenic caspase assays, Western blot assay of caspase substrates); measurements of the genes, proteins and other molecules produced by the immune cells (eg Northern blotting assay, polymerase chain reaction, DNA microarrays, protein microarrays, 2-dimensional gel electrophoresis, Western blotting assay, enzyme-linked immunosorbent assay, fl fate cytometry); and measurements of clinical symptoms or outcomes such as improvements in autoimmune, neurodegenerative and other diseases involving body proteins and body polypeptides (clinical scores, requirements for use in additional therapies, functional status, imaging studies) for example by measuring recurrence rate and disease severity (with use of clinical scores as is known to those skilled in the art) in the case of multiple sclerosis, to measure blood glucose levels in the case of type 1 diabetes or ledin fl ammation in the case of rheumatoid arthritis.
"Individuals" is intended to mean any animal, such as a human, non-human primate, horse, cow, dog, cat, mouse, rat, guinea pig or rabbit.
"Treating", "treating", or "therapy" of a disease or disorder is intended to mean slowing down, stopping or reversing the progression of the disease, as evidenced by the reduction, cessation or elimination of either clinical or diagnostic symptoms, by the administration of a an immunomodulatory complex or a polynucleotide encoding an immunomodulatory complex, either alone or in combination with another compound as described herein. "Treat", "treatment", or "therapy" also means a reduction in the severity of symptoms of an acute or chronic disease or disorder or a reduction in the recurrence intensity, such as in the case of a recurrent or remitting course of autoimmune disease or a reduction of inflammation in the case of an inflammatory aspect of an autoimmune disease. In the preferred embodiment, treating a disease is intended to reverse or stop or limit disease progression, ideally to the point where the disease is completely eliminated. As used herein, improving an illness and treating an illness are equivalent concepts.
"Preventing", "prophylaxis of", or "preventing" a disease or disorder, as used in the context of the present invention, refers to the administration of an immunomodulatory complex or a polynucleotide encoding an immunomodulatory complex, either alone or in isolation. in combination with another compound as described herein, to prevent the onset or onset of a disease or disorder or some or all of the symptoms of a disease or disorder or to reduce the likelihood of a disease or disorder occurring.
A "therapeutically or prophylactically effective amount" of an immunomodulatory complex refers to any amount of the immunomodulatory complex that is sufficient to treat or prevent the disease, for example, by ameliorating or eliminating the symptoms and / or cause of the disease. Therapeutically effective amounts fall, for example, within wide ranges and are determined by clinical trials and are determined for specific patients based on factors known to one skilled in the art, such as the severity of the disease, the patient's weight, age and other factors.
DESCRIPTION OF THE DRAWINGS Figure 1. DNA construct encoding the immunomodulatory complex K-CTA1-R7K / C187A-COL-DD The pCTA1-DD plasmid contains the cholera toxin A1 gene (amino acid 1-194) cloned at HindIII-BamHI and two D fragments from the staphylococcal protein A gene under the control of the trp promoter. Collagen peptide was inserted between the CTA1 and DD fragments to give pCTA1-COL-DD.
R7K and C187A mutations were generated using in vitro mutagenesis, resulting in pK-CTAI-R7K / Cl 87A-COL-DD. Ptrc = Ptrc promoter. COL2A1 = collagen peptide, D = Ig-binding element of protein A from S. aureus.
Figure 2. Comparison of the therapeutic effects of CTA1-R7K-COL-DD and K-CTA1-R7K / Cl 87A-COL-DD in the mouse CIA model.
A. Arthritis Index. B. Arthritis rate.
- I- CTAl-R7K-COL-DD, - Ö - K-CTAl-R7K / Cl87A-COL-DD, - Ü - PBS, control.
Figure 3. Comparison of the therapeutic effects of CTA1-R7K-COL-DD and K-CTA1-R7K / C187A-COL-DD in the mouse CAIA model.
Arthritis index. - I- CTA1-R7K-COL-DD, - O - K-CTA1-R7IUCl87A-COL-DD, - Ü - PBS, control.
DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods and compositions for the prophylaxis, prevention and / or treatment of a disease in an individual, which is associated with one or more of its own proteins, polypeptides, or peptides present in the individual and involved in a non-physiological condition. The invention is more specifically related to methods and compositions for the prophylaxis, prevention and / or treatment of autoimmune diseases associated with one or more of the body's own polypeptides present in an individual in a non-physiological condition such as multiple sclerosis, rheumatoid arthritis. , insulin-dependent diabetes mellitus, autoimmune uveitis, primary biliary cirrhosis, myasthenia gravis, Sjögren's syndrome, pem fi gus vulgaris, scleroderma, pemic anemia, systemic lupus erythematosus and Graves' disease. The present invention provides improved methods for the prophylaxis, prevention and / or treatment of an autoimmune disease, comprising administering an immunomodulatory complex comprising one or more autoantigenic epitopes associated with the disease to an individual. Administration of a therapeutically or prophylactically effective amount of the immunomodulatory complex comprising one or more autoantigenic epitopes to an individual triggers inhibition of an immune response to the autoantigen associated with an autoimmune disease, thereby treating or preventing the disease.
Autoimmune Diseases Several examples of autoimmune diseases associated with autoantigen are presented in Table 1 and specific examples are described in more detail below.
Table 1. Examples of autoimmune diseases and associated autoantigen Autoimmune disease Affected tissue Autoantigen (s) associated with the autoimmune disease Rheumatoid arthritis synovial joints Immunoglobulin, fi brine, fi laggrin, type I, -II, -III, -IV, collagen V, -IX, and XI, GP-39, hnRNP Multiple Sclerosis Central Nervous System Myelin Basal Protein, Proteolipid Protein, Myelin-Associated Glycoprotein, Cyclic Nucleotide Phosphodiesterase, Myelin-Associated Glycoprotein, Myelin-Associated Oligodendrocytin Crystalline Alpha Basal Protein, -IA2, -IA-Zß; dí fl beï fl S mellifllS cells in glutamic acid decarboxylase (65 and 67 kDa- pancreas forms), carboxypeptidase-H, insulin, proinsulin, pre-proinsulin, stress proteins (heat shock proteins), glima-38, öcellsantingen (69 KDa), pglu, 2 23 Sjögren's syndrome exocrine glands HSP60 stress protein, fodrin, ribonucleoproteins: Ro6O (SSA), Ro52 (SSA), and La (SSB), poly (ADP-ribose) polymerase, lipocalin, alpha-amylase Guillain-Barrés peripheral nervous system peripheral myelin protein -In and other syndromes Autoimmune Uveitis Eye, uvea S-antigen, interphotoreceptor retinoid binding protein (IRBP), rhodopsin, recoverin Primary biliary cirrhosis of the bile ducts pyruvate dehydrogenase complex (2-oxo acid dehydrogenase) Autoimmune hepatitis liver 1, -3, and other Myasthenia Gravis nerve-muscle connections acetylcholine receptor Autoimmune gastritis stomach / parietal cell HVKïATPase, internal factor Pemic anemia gastric internal factor Polymyositis muscle Histi dyl-tRNA synthetase, other synthetases, other nuclear antigens Autoimmune thyroiditis thyroid thyroglobulin, thyroid peroxidase Graves' disease thyroid thyroid stimulating thyroid receptor Psoriasis skin unknown Vitiligo skin tyrosinase, tyrosinase-nucleotinous system transglutaminase Rheumatoid Arthritis. Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory synovitis that affects 0.8% of the world's population. It is characterized by chronic inflammatory synovitis which causes erosive joint breakdown. RA is mediated by T cells, B cells and macrophages.
Evidence that T cells play a crucial role in RA includes (l) the dominance of CD4-positive T cells that integrate the joint, (2) clinical improvement associated with inhibition of T cell function by drugs such as cyclosporine (3) association between RA and some HLA-DR alleles. HLA-DR alleles associated with RA contain a similar sequence of amino acids at positions 67-74 in the third hypervariable region of the β-chain involved in peptide binding and presentation to T cells. RA is mediated by autoreactive T cells that recognize a body-specific protein or a modified body-specific protein, which is present in synovial joints. Autoantigens targeting RA include, for example, epitopes from type II collagen; hnRNP; A2 / RA33; Sa; fi laggrin; keratin; eitmllin; cartilage proteins, including gp39; collagen of type-I5 III, -IV, -V, -IX, -XI; HSP-65/60; IgM 24 (rheumatoid factor); RNA polymerase; hnRNP-Bl; hnRNP-D; cardiolipin; aldolase-A; citrulline-modified fi laggrin and fl brin. Autoantibodies that recognize laggrin peptides and that contain a modified arginine residue (diminished to form citrulline) have been identified in the serum of a high proportion of RA patients. Autoreactive T and B cell responses are both directed against the same immunodominant type II collagen peptide (CII peptide) 257-270 in some patients.
Multiple sclerosis. Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system and affects 350,000 Americans and millions of people worldwide. Symptom onset usually occurs between the ages of 20 and 40 and is manifested as an acute or subacute attack of unilateral visual impairment, muscle weakness, paresthesia, ataxia, dizziness, urinary incontinence, dysarthria or mental disorder (in descending order of frequency). Such symptoms result from focal lesions of demyelination, which cause both negative transmission abnormalities due to slower axonal transmission and positive transmission abnormalities due to ectopic impulse generation (eg Lherinitis symptoms).
Diagnosis of MS is based on a history including at least two distinct attacks of neurological dysfunction that are separated in time, the patient presents objective clinical evidence of neurological dysfunction and involves different parts of the white matter in the CNS. Laboratory studies provide additional objective evidence to support the diagnosis of MS, including magnetic resonance imaging (MRI) imaging of white mass lesions in the CNS, cerebrospinal fluid, oligoclonal banding of IgG, and abnormal stimulus responses. Although most patients experience a gradual progressive course of the disease, the clinical course of MS varies widely between individuals and can range from being limited to mild lifelong attacks to fulminant chronic progressive disease. A quantitative increase in the number of myelin autoreactive T cells with the ability to secrete IFN-gamma is associated with the pathogenesis of MS and EAE.
The autoantigenic targets for the autoimmune response in autoimmune demyelinating diseases, such as multiple sclerosis and experimental autoimmune encephalitis (EAE), may include epitopes from proteolipid protein (PLP); myelin basal protein (MBP); myelin oligodendrocyte glycoprotein (MOG); cyclic nucleotide phosphodiesterase (CNPase); myelin-associated glycoprotein (MAG) 5 and myelin-associated oligodendrocytic basal protein (MBOP); alpha-B crystalline (a stress protein); viral and bacterial curing peptides, e.g., indigenous, herpes virus, hepatitis B virus, etc .; OSP (oligodendrocyte specific protein); citrulline-modified MBP (the CS isoform of MBP in which 6 arginines have been deiminated to citrulline), etc. The integral membrane protein PLP is a dominant autoantigen of myelin. Detentinants of PLP antigenicity have been identified in your mouse strains and include residues 139451, 103-116, 215-232, 43-64 and 178-191. At least 26 MBP epitopes have been reported (Meinl et al, J Clin Invest 92, 2633-43, 1993). Residues 1-11, 59-76 and 87-99 are worth noting. Immunodominant MOG epitopes identified in your mouse strains include residues 1-22, 35-55 and 64-96. In human MS patients, the following myelin proteins and epitopes were identified as targets for the autoimmune T and B cell responses. Antibodies eluted from MS plaques in the brain recognized myelin basic protein peptide (MBP peptide) 83-97 (Wucherpfennig et al. J Clin Invest 100: 1 14-1122, 1997). Another study found that approximately 50% of MS patients had T cell reactivity among peripheral blood lymphocytes to myelin oligodendrocyte glycoprotein (MOG) (6-10% in the control), 20% were reactive in MBP (8-12% in the control), 8% were reactive to PLP (0% in the control), 0% were reactive to MAG (0% in the control). In this study, 7 out of 10 MOG-reactive patients had a proliferative T cell response focused on one of the 3 peptide epitopes, including MOG 1-22, MOG 34-56, MOG 64-96 (Kerlero de Rosbo et al. Eur J Immunol 27: 3059-69, 1997). T and B cell responses (antibody eluted from brain lesion) were focused on MBP 87-99 (Oksenberg et al. Nature 362: 68-70, 1993). In MBP 87-99, the amino acid sequence HFFK is a dominant target for both the T cell and B cell responses (Wucherpfennig et al. J Clin Invest 100: 1114-22, 1997). Another study observed lymphocyte reactivity to myelin-associated basal protein (MOBP), including residues MOBP 21-39 and MOBP 37-60 (Holz et al. J Immunol 164: 1103-9, 2000). Using immunogold conjugates of MOG and MBP peptides to stain MS and control brains, both MBP and MOG peptides were recognized by antibodies bound to MS plaques (Genain and Hauser, Methods 10: 420-34, 1996).
Insulin-dependent diabetes mellitus. Human type I or insulin-dependent diabetes mellitus (IDDM) is characterized by autoimmune destruction of the ß cells in the pancreatic Langerhansö cells.
The depletion of ß-cells results in the inability to regulate the levels of glucose in the blood. Obvious diabetes occurs when blood glucose levels rise above a specific level, usually around 250 mg / dl. In humans, the onset of diabetes is preceded by a long presymptomatic period.
During this period, there is a gradual loss of pancreatic beta cell function. The development of the disease is implicated by the presence of autoantibodies to insulin, glutamic acid decarboxylase and tyrosine phosphatase IA2 (IA2).
Markers that can be evaluated during the presymptomatic period are the presence of pancreatic insulin, the level and frequency of antibodies to islet cells, antibodies to islet cell surface, aberrant expression of MHC class II molecules on pancreatic beta cells, blood glucose concentration and plasma concentrations of insulin. An increase in the number of T lymphocytes in the pancreas, antibodies to islet cells and blood glucose is indicative of the disease, which is also a decrease in insulin concentration.
The Non-Obese Diabetic (NOD) mice are an animal model with many clinical immunological and histopathological features common to human IDDM. NOD mice develop 26 inhalation in the islets and destruction of the beta cells spontaneously, leading to hyperglycemia and open diabetes. Both CD4-positive and CD8-positive T cells are required for the development of diabetes, although the roles of each remain unclear. Administration of insulin or GAD5 to NOD mice, in the form of proteins, under tolerating conditions, has been shown to prevent disease and downregulate responses to other autoantigens.
The presence of combinations of autoantibodies with different specificities in serum shows very high sensitivity and specificity for human diabetes mellitus type 1. The presence of autoantibodies to GAD and / or IA-2 shows, for example, approximately 98% sensitivity and 99% specificity for identifying diabetes. type I mellitus from control serum. The presence of autoantibodies specific for two of the three autoantigens including GAD, insulin and IA-2, in healthy first-degree relatives of type 1 diabetes patients provides a positive predictive value of> 90% for the development of type IDM within 5 years. .
Autoantigens targeting human insulin-dependent diabetes mellitus may include, for example, tyrosine phosphatase IA-2; IA-2 [beta]; glutamic acid decarboxylase (GAD), both the 65 kDa and 67 kDa forms; carboxypeptidase H; insulin; proinsulin; stress proteins (HSP); glima 38; islet cell antigen (69 kDa) (ICA69); p52; two ganglioside antigens (GT3 and GM2-1); non-specific glucose-β-phosphatase-related protein (IGRP); and island cell glucose transporter (GLUT 2).
Human IDDM is currently being treated by monitoring blood glucose levels to control injection or pump-based administration of recombinant insulin. Diet and exercise treatments help to achieve adequate blood glucose control.
Autoimmune uveit. Autoimmune uveitis is an autoimmune disease of the eye that is estimated to affect 400,000 people, with an incidence of 43,000 new cases annually in the United States. Autoimmune uveitis is currently treated with steroids, immunosuppressive agents such as methotrexate and cyclosporine, intravenous immunoglobulin and TNF-α antagonists.
Experimental autoimmune uveitis (EAU) is a T-cell-mediated autoimmune disease that targets the neural retina, uvea and related tissues of the eye. EAU shares many clinical and immunological features with human autoimmune uveitis and is induced by peripheral administration of uveitogenic peptide emulsified in Freund's complete adjuvant (CFA).
Autoantigens targeting human autoimmune responses may include S antigen, interphotoreceptor retinoid binding protein (IRBP), rhodopsin and recoverin. 27 Primary biliary cirrhosis. Primary biliary cirrhosis (PBC) is an organ-specific, autoimmune disease that mainly affects women aged 40-60 years. The prevalence reported in this group is approaching 1 per 1000. PBC is characterized by a progressive destruction of intrahepatic biliary epithelial cells (IBEC) covering the inside of the small intrahepatic bile ducts. This leads to obstruction and disturbances in the bile secretion, which eventually leads to cirrhosis. Associations with other autoimmune diseases characterized by damage to the epithelium of the lining / excretory system have been reported, including Sjögren's syndrome, CREST syndrome, autoimmune thyroid disease and rheumatoid arthritis. Attention to the driving antigens has focused on the mitochondria for 50 years, which has led to the discovery of the antimitochondrial antibody (AMA) (Gershwin et al.
Immunol Rev 1741210-225, 2000; Mackay et al. Immunol Rev 174: 226-237, 2000). AMA soon became a cornerstone of laboratory diagnosis of PBC and is present in serum in 90-95% of patients long before clinical symptoms appear. Autoantigenic reactivities in the mitochondria were designated M1 and M2. M2 reactivity is directed at a family of 48-74 kDa components. M2 represents multiple autoantigenic subunits of enzymes from the 2-oxo acid dehydrogenase complex (2-OADC) and is another example of the native protein, polypeptide or peptide of the present invention. Studies that have identified the role of pyruvate dehydrogenase complex antigens (PDC antigens) in the etiopathogenesis of PBC support the concept that PDV plays a central role in the induction of the disease (Gershwin et al. Immunol Rev l74: 210-225, 2000; Mackay et al. Immunol Rev 1742226-23 7, 2000). The most common reactivity in 95% of PBC cases is the E2 74 kDa subunit, which belongs to PDC-E2. There are related but distinct complexes which include: 2- oxoglutarate dehydrogenase complexes (OGDC) and (BC) 2- OADC with branched chain. Three constituent enzymes (E1, 2,3) contribute to the catalytic function, which is to convert the Z-oxo acid substrate to acyl coenzyme A (CoA), with reduction of NADJ 'to NADH. Mammalian PDC contains another component, which is termed protein-X or E-3-binding protein: (E3BP). In PBC patients, the main antigenic response is directed against PDC-E2 and EBBP. The E2 polypeptide contains two tandem repeating lipoyl domains, while E3BP has a single lipoyl domain. The lipoil domain is found in a number of autoantigenic targets in PBC and is referred to herein as the “PBC lipoil domain”. PBC is treated with glucocorticoids and immunosuppressive agents, including methotrexate and cyclosporin A.
Sjögren's syndrome. Sjögren's syndrome (SS) is a chronic autoimmune disease that mainly affects the salivary glands and lacrimal glands, leading to dry eyes (keratoconjunctivitis sicca) and dry mouth (xerostomy). Other organs that may be involved include the bronchial tree, kidneys, liver, blood vessels, peripheral nerves and the pancreas. The dual nature of SS is of particular interest: either alone as a primary disease in women in their 40s and 50s (primary SS) or in conjunction with other autoimmune diseases (secondary SS); glandular (sicca symptoms) and systemic (extraglandular) where clinical manifestations may be present. Characteristic of SS is the presence of rheumatoid factors, antinuclear and precipitating autoantibodies. The 28 cytoplasmic / nuclear ribonucleoprotein particles (Ro / SSA and La / SSB) have a prominent role in the autoimmune response of SS. Other antigens involved in the positive nuclear pattern of immunoresurgency include the following: Ku, NOR-90 (nuclear organizing region), p-80 coilin, HMG-17 (high-mobility group), Ki / SL. Furthermore, organ-specific autoantibodies are also recognized, this includes antityroglobulin, anti-erythrocyte and anti-salivary gland epithelial antibodies. (Overview of Clio et al. Int Arch Allergy Immunol 123: 46-57, 200). A 120-kD, organ-specific autoantigen, has been identified as the cytoskeletal protein oc-fodrin (Haneji et al. Science 2762604-607, 1997). HSP60 is another autoantigen that has been suggested to be involved in SS. Immunization with HSP60 or an HSP60-derived peptide (amino acid residues 437-460) has been shown to reduce SS-related histopathological properties in an animal model of SS (Dalaleu et al. Aithritis Rheum 5822318-2328, 2008). The dominant target antigens Ro / SSA, La / SSB and their corresponding antibodies have been detailed in detail at the molecular level. Ro / SSA is a ribonucleoprotein containing small, cytoplasmic RNA molecules.
The protein component of the Ro / SSA antigen, a 60 kD protein (60-kD Ro / SSA, Ro60) is bound to one of your small cytoplasmic RNA molecules. A 52 kD peptide is another component of Ro / SSA antigens (52-kD Ro / SSA; Ro52). La / SSB antigen is composed of a polypeptide consisting of 408 amino acids. Both 60-kD Ro / SSA and La / SSB proteins are members of a family of RNA-binding proteins that contain a sequence of 80 amino acids known as the RNA recognition motif (RNP). B-cell epitope mapping of 60-kD Ro / SSA, 52-kD Ro / SSA and La / SSB molecules using fl your strategies has revealed specific epitopes in your studies. B-cell epitopes of 60-kD Ro / SSA autoantigen appear to be located in the central region and the carboxy-terminal part of the molecule. Two disease-specific epitopes: TKYKQRNGWSHKDLLRSHLKP (169-190) (SEQ ID NO: 6) and the ELYKEKALSVETEKLLKYLEAV region (211-232) (SEQ 1D NO: 7) have been identified (Routsias et al. Eur J Clin Invest 26). : 514-521, 1996). The antigenic determinants of the 52-kD Ro / SSA protein are substantially linear and are found in the central part of the molecule. Four peptides (amino acids 2-11, 107-126, 277-292 and 365-382) have been reported to be recognizable by anti-Ro / SSA sera (Ricchiuti et al. Clin Exp Immunol 95: 397-407, 1994). Four highly reactive peptides, with purified IgG, spanning regions 145-164, 289-308, 301-320 and 349-368 of the La / SSB protein, have been reported (Tzioufas et al. Clin Exp Immunol 108: 191-198 , 1997).
Other autoimmune diseases and associated autoantigens. Autoantigens related to myasthenia gravis may include epitopes within the acetylcholine receptor. Autoantigens targeting pem fi gus vulgaris could include desmoglein-3. The dominant autoantigen for pem fi gus vulgaris could include desmoglein-3. Myositis-related panels could include tRNA synthetases (eg, threonyl, histidyl, alanyl, isoleucyl, and grycyl); Ku; ScI; SSA; Ul Sn ribonuclear protein; Mi-I; Mi-I; Jo-I; Ku; and SRP. Panels related to scleroderma could include Scl-70; centromer; Ul-ribonuclear proteins; and fi brillarin. Panels related to pemic anemia could 29 include internal factor; and glycoprotein beta subunit of gastric H / K ATPase. Epitope antigens related to systemic lupus erythematosus (SLE) could include DNA; phospholipids; nuclear antigens; Ro; La; Ul-ribonucleoprotein; Ro60 (SS-A); Ro52 (SS-A); La (SS-B); calreticulin; Grp78; Scl-70; histone; Sm protein; and chromatin, etc. For Graves' disease, epitopes could include Na + / I symports; thyrotropin receptor; Tg; and TPO.
Translantate-versus-host disease. One of the biggest limitations of tissue and organ transplants in humans is the rejection of the tissue graft by the recipient's immune system. It is well known that the better the match between MHC class I and II alleles (HLA-A, HLA-B and HLA-DR) between donor and recipient, the better the survival of the transplant.
Graft-versus-host disease (GVHD) causes significant morbidity and mortality in patients receiving grafts containing allogeneic hematopoietic cells. Hematopoietic cells are present in bone marrow transplants, stem cell transplants and other grafts. Approximately 50% of patients receiving a graft from an HLA-matched sibling will develop moderate to severe graft-versus-host disease and the incidence is much higher for non-HLA-matched grafts. One third of patients who develop moderate to severe graft-versus-host disease will die as a result. T lymphocytes and other immune cells of the donor graft will attack the recipient's cells expressing polypeptide variations in their amino acid sequences, in particular variations of proteins encoding the histocompatibility complex genes on chromosome 6 in humans. The most influential graft-versus-host disease proteins in grafts that affect allogeneic hematopoietic cells are the highly polymorphic (large amino acid variation between humans) class I proteins (HLA-A, -B and -C) and class II proteins (DRB1, DQB1 and DPB1) (Appelbaum, Nature 411, 385-389, 2001). Even when the MHC class I alleles are serologically “matched” between donor and recipient, DNA sequencing reveals that there are allele differences in 30% of cases, providing a basis for class I-driven graft-versus-host disease even in matched pairs of donor and recipient (Appelbaum, Nature 411, 385-389, 2001). The smaller histocompatibility antigens, the spontaneous graft-versus-host disease, often cause damage to the skin, intestines, liver, lungs, and pancreas. Graft-versus-host disease is treated with glucocorticoids, cyclosporine, methotrexate, fl udarabine and OKT3.
Tissue transplant rejection. Immunological rejection of tissue grafts, including lung, heart, liver, pancreas, and other organs and tissues, is mediated by immune responses of the transplant recipient, which are directed against the transplanted organ. Allogeneically transplanted organs contain proteins with variations in their amino acid sequences compared to the amino acid sequences of the transplant recipient. Because the amino acid sequence of the transplanted organ is different from that of the transplant recipient, they do not trigger an immune response of the recipient to the transplanted organ. Rejection of transplanted organs is a serious complication and limitation of tissue transplantation and can cause failure of the transplanted organ of the recipient. The chronic inflammation that results from the rejection often leads to dysfunction of the transplanted organ. Graft recipients are currently receiving treatments with a variety of immunosuppressive agents to prevent and inhibit rejection. These substances include glucocorticoids, cyclosporin A, Cellcept, FK-506 and OKT3.
Compositions and Methods of Treatment The present invention provides improved methods and compositions for the treatment, prophylaxis and / or prevention of autoimmune or allergic diseases, comprising an immunomodulatory complex comprising one or more epitopes associated with the disease.
The immunomodulatory complex of the present invention comprises one or more epitopes associated with the autoimmune or allergic disease. The improved method of the present invention includes the administration of an immunomodulatory complex comprising one or more epitopes associated with the disease.
In certain embodiments, the present invention provides improved methods of treating and / or preventing the autoimmune disease diabetes mellitus (IDDM), which comprises administering to an individual an immunomodulatory complex comprising one or more autoantigens associated with diabetes mellitus (IDDM).
The immunomodulatory complex administered to treat or prevent diabetes mellitus (1DDM) could include autoimmune epitopes derived from one or more of your own proteins, such as preproinsulin, proinsulin, glutamic acid decarboxylase-65 and -67 (GAD-65 and GAD-67). ); tyrosine phosphatase-IA-Z; islet cell-specific glucose-6-phosphatase-related protein (IGRP); and / or islet cell antigen 69 kD. The immunomodulatory complex administered to treat or prevent IDDM could alternatively include auto your autoimmune epitopes derived from the same or different body proteins, polypeptides or peptides. In a preferred embodiment, the immunomodulatory complex administered to treat or prevent IDMM could include autoimmune epitopes derived from the body's own polypeptide preproinsulin or proinsulin.
Other embodiments of the present invention provide improved methods for the treatment, prophylaxis and / or prevention of multiple sclerosis (MS), comprising administering an immunomodulatory complex comprising one or more autoantigenic epitopes associated with MS to an individual. The immunomodulatory complex administered to treat MS could include an autoantigenic epitope derived from one or more of 31 body proteins, including but not limited to: myelin basic protein (MBP), myelin oligodendrocytic glycoprotein (MOG), proteolipid protein (PLP) myelinase oligodendrocytic basal protein (MOBP), myelin oligodendrocyte glycoprotein (MOG), and / or myelin-associated glycoprotein (MAG). An immunomodulatory complex could alternatively comprise multiple autoantigenic epitopes derived from the same or different body proteins, polypeptides or peptides associated with the disease.
In other embodiments of the present invention, there are provided improved methods of treating, prophylaxis and / or preventing rheumatoid arthritis (RA), comprising administering an immunomodulatory complex of the invention comprising one or more autoantigenic epitopes associated with RA to a subject. In some embodiments, the autoantigenic epitope is an epitope derived from collagen type I, II, III, IV, V, IX, and XI, GP-39, filaggrin and fibrin. In a preferred embodiment, the epitope is derived from type II collagen, the epitope is preferably the shared immunodominant epitope collagen II peptide, which comprises amino acids 260-273 (CII260-273), SEK. ID. NO.:5.
Alternatively, multiple immunomodulatory complexes comprising autoantigenic epitopes derived from different body polypeptides may be administered.
In a further embodiment, the present invention provides nucleic acid sequences, including DNA and RNA sequences, which encode the immunomodulatory complexes of the invention, as well as plasmids, vectors and expression systems comprising such nucleic acid sequences.
The immunomodulatory complexes of the invention can be produced by recombinant DNA technology.
Techniques for constructing plasmids, vectors and expression systems and cell transfection are well known in the art, and one skilled in the art is well aware of the standard reference materials that describe specific conditions and procedures.
Construction of the plasmids, vectors, and expression systems of the invention utilizes standard ligation and restriction cleavage techniques well known in the art (for a general description, see, e.g., Ausubel, et al., Current Protocols in Molecular Biology, Wiley Interscience, 1989; Sambrook and Russell, Molecular Cloning, A Laboratory Manual 3rd ed. 2001).
Isolated plasmids, DNA sequences or synthesized oligonucleotides are cleaved, tailored and religated to the desired form. Sequences of DNA constructs can be ensured by using e.g. standard DNA sequencing procedures (see, e.g., Sanger et al. (1977) Proc. Natl. Acad. Sci., 74, 5463-5467).
Yet another suitable method for isolating specific molecules is by polymerase chain reaction (PCR) (Mullis et al. Methods Enzymol 1551335-350, 1987) or reverse transcription PCR (RT-PCR). Specific nucleic acid sequences can be isolated from RNA by RT-PCR.
RNA is isolated, for example, from cells, tissues or whole organisms by techniques known to those skilled in the art. Complementary DNA (cDNA) then generates using polyddT or random hexamer primers, deoxynucleotides and an appropriate reverse transcriptase enzyme.
The desired polynucleotide can then be amplified from the generated cDNA by PCR.
The polynucleotide of interest can alternatively be amplified directly from a suitable cDNA library.
Primers that hybridize to both the 5 'and 3' ends of the polynucleotide sequence of interest are synthesized and used for the PCR reaction. The primers may also contain specific restriction enzyme sequences at the 5 'end for easy cleavage and lingering of amplified sequences into similar restriction cleaved plasmid vectors.
Administration of Immunomodulatory Complexes Therapeutically and prophylactically effective amounts of an immunomodulatory complex range from about 1 pg to about 10 mg. A preferred therapeutically or prophylactically effective amount is in the range of about 5 pg to about 1 mg. A most preferred therapeutic amount of immunomodulatory complexes is in the range of 10 pg to 100 pg. In some embodiments, the immunomodulatory complex is administered monthly for 6-12 months and then every three to twelve months as a maintenance dose. Alternative treatment regimens may be developed and may range from daily, to weekly, to biennial, to annual, to a single administration, depending on the severity of the disease, the age of the patient, the immunomodulatory complex being administered and such other factors to be considered by the attending physician. .
In one embodiment, the immunomodulatory complex is administered intranasally. In other variations, the immunomodulatory complex is administered orally, sublingually, subcutaneously, transcutaneously, intradermally, via the mucosa or intramuscularly.
Formulation The immunomodulatory complex may be administered in combination with other substances, such as pharmacological substances, adjuvants, cytokines or immunostimulatory complexes (ISCOMS). EXAMPLES The following examples are specific embodiments of carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
Example 1. Immunomodulatory complex K-CT A 1-R 7K / C18 7A-COL-DD Construction of CTA1-DD mutants, expression and purification of fusion proteins was performed essentially as described by Ågren (J Immunol 1999, 162: 2432-2440) . The pCTA1 -DD plasmid contains the cholera toxin A1 gene (amino acids 1-194) cloned at HindIII-BamHI and DNA encoding two D-fragments from the staphylococcal protein A gene under the control of the trp promoter. DNA encoding a collagen peptide, the shared immunodominant collagen II peptide (CII260-273) was inserted between DNA encoding the CTA1 and the DD moieties, resulting in the pCTA1-COL-DD plasmid. The R7K and C187A mutations were constructed by in vitro mutagenesis, resulting in the plasmid pK-CTA1-R71UC187A-COL-DD (Figure 1).
Example 2. Comparison of the therapeutic effects of CTA1-R7K-COL-DD and K-CTA1-R7K / C187A-COL-DD in the mouse CIA model.
The mouse CIA model for RA was used to compare intranasal treatments with CTA1-R7K-COL-DD and K-CTA1-R7K / C187A-COL-DD tolerogen. The CIA model shares a number of clinical, histological and immunological features with RA and is therefore the most widely used model for testing potential therapeutic agents against RA. DBA1 mice received a primary immunization with chicken type II collagen in Freund's complete adjuvant (FCA) followed by a booster dose with F reund's incomplete 5-adjuvant (IFA) on day 21. The mice were treated intranasally with PBS, CTA1-R7K-COL DD or K-CTA1-R7K / C187A-COL-DD before and / or after the booster immunization. The mice were then monitored for the incidence and severity of arthritis using an arthritis scoring system.
The therapeutic effect of K-CTA1-R7K / C187A-COL-DD was significantly better than the therapeutic effect of CTA1-R7K-COL-DD, which is seen as a decrease in the severity (Figure 2A) and the incidence (Figure 2B) in CIA compared to control group (PBS). The arthritis index for the PBS control group increased dramatically three weeks after the collagen immunizations and peaked at 6 weeks. In the CTA1-R7K-COL-DD group, a slight increase in the arthritis index could be observed. In contrast, the K-CTA1-R7K / Cl87A-COL-DD group had significantly lower arthritis indices and many animals had no symptoms at all.
Example 3 Comparison of the therapeutic effects of CTA1-R7K-COL-DD and K-CTA1-R7K / C1 87A-COL-DD in the CAIA mouse model.
Collagen antibody-induced arthritis (CAIA) was induced in Balb / c mice (Taconic, Denmark) on day 0 by an intravenous injection of a mixture of monoclonal antibodies to collagen II (ArthritoMab cocktail: D1, F10, A2 and D8; MD Biosciences, Zurich, Switzerland ) at a dosage level of 2 mg / mouse. On day 3, lipopolysaccharide (LPS) (ArthritoMab kit, MD Biosciences) was injected intraperitoneally to enhance the incidence and severity of the disease (50 ug / mouse).
Mice were treated intranasally day -2, 0, +3 with 5 pg K-CTA1-R7K / C187A-COL-DD or CTA1-R7K-COL-DD in 20 μl PBS.
On day 4, all mice began to show signs of disease (no data are shown). In the PBS control group and in the group treated with CTA1-R7K-COL-DD, the arthritis index rose dramatically 7 days after immunization. However, in the group treated with K-CTA1-R7K / C187A-COL-DD, the increase in arthritis index was significantly lower throughout the course of the experiment. 35

权利要求:
Claims (1)
[1]
An immunomodulatory complex, which is a fusion protein comprising: (a) a mutated subunit of the ADP-ribosylating Al subunit of the cholera toxin (CTA1) (b) a peptide capable of binding to a specific cellular receptor and (c) a or fl your epitopes associated with an autoimmune or allergic disease, wherein the amino acids corresponding to amino acid 7 arginine and amino acid 187 cysteine in the naturally occurring CTA1 have been replaced in the mutated CTA1 subunit. The immunomodulatory complex of claim 1, wherein the mutated CTA1 subunit is the CTA1 - R7K / C187A mutant, SEQ. ID. NO: 1. The immunomodulatory complex of claim 1, wherein the amino acid lysine has been further inserted at the N-terminus of the mutated CTA1 subunit. The immunomodulatory complex of claim 3, wherein the mutated CTA1 subunit is the K-CTA1-R7IUCl87 mutant, SEQ. ID. NO: 2 Immunomodulatory complex according to any one of claims 1 to 4, wherein one or more of the epitopes are autoimmune epitopes associated with an autoimmune disease. The immunomodulatory complex of claim 5, wherein the autoimmune disease is selected from insulin dependent diabetes mellitus, multiple sclerosis, rheumatoid arthritis, autoimmune uveitis, primary biliary cirrhosis, myasthenia gravis, Sjögren's syndrome, pemous vulgaris, scleroderma, pemic anemia, systemic lupus erythematosus . An immunomodulatory complex according to any one of claims 1 to 4, wherein one or more of the epitopes are allergenic epitopes associated with an allergic disease. The immunomodulatory complex of claim 7, wherein the allergic disease is selected from allergic asthma, allergic rhinitis, atopic dermatitis and food hypersensitivity. An immunomodulatory complex according to any one of claims 1 to 8, wherein the peptide is a peptide that specifically binds to a receptor expressed on a cell capable of antigen presentation. 36 10. ll. The immunomodulatory complex of claim 9, wherein the fusion protein comprises a peptide which specifically binds to a receptor expressed on a cell expressing MHC class I or MHC class II. molecules. The immunomodulatory complex of claim 10, wherein the fusion protein comprises a peptide which specifically binds to a receptor expressed on a cell selected from the group consisting of lymphocytes, such as B lymphocytes, T cells, monocytes, macrophages, dendritic cells, Langerhans cells , epithelial cells and endothelial cells. The immunomodulatory complex of claim 11, wherein said peptide is protein A or a fragment thereof in single or multiple copies, such as one or more D subunits thereof. Immunomodulatory complex K-CTA1-R7IUCl87A-COL-DD SEC. ID. NO: 4. An isolated nucleic acid encoding an immunomodulatory complex according to any one of claims 1 to 13. An expression system comprising a nucleic acid according to claim 14. A transfected cell comprising an expression system according to claim 15. A pharmaceutical composition comprising an immunomodulatory complex according to any one of claims 1 to 13. A pharmaceutical composition according to claim 17 for use in the prophylaxis, prevention and / or treatment of an autoimmune or allergic disease. Pharmaceutical composition according to claim 18, for use in the prophylaxis, prevention and / or treatment of an autoimmune disease selected from insulin-dependent diabetes mellitus, multiple sclerosis, rheumatoid arthritis, autoimmune uveitis, primary biliary cirrhosis, myasthenia gravis, Sjögren's syndrome, pemous vulgaris, scleroderma. , pemic anemia, systemic lupus erythematosus and Graves' disease. A pharmaceutical composition according to claim 18, for use in the prophylaxis, prevention and / or treatment of an allergic disease selected from allergic asthma, allergic rhinitis, atopic dermatitis and food hypersensitivity. Use of an immunomodulatory complex according to any one of claims 1 to 13 for the manufacture of a medicinal product for the prophylaxis, prevention and / or treatment of an autoimmune or allergic disease. Use according to claim 21, wherein the autoimmune disease is selected from insulin dependent diabetes mellitus, multiple sclerosis, rheumatoid arthritis, autoimmune uveitis, primary biliary cirrhosis, myasthenia gravis, Sjögren's syndrome, pemous vulgaris, scleroderma, pemic anemia, systemic lupus erythematosus. Use according to claim 21, wherein the allergic disease is selected from allergic asthma, allergic rhinitis, atopic dermatitis and food hypersensitivity. A method of prophylaxis, prevention and / or treatment of an autoimmune or allergic disease in an individual wherein the method comprises: administering an effective amount of an immunomodulatory complex according to any one of claims 1 to 13 to an individual. The method of claim 24, wherein the autoimmune disease is selected from insulin dependent diabetes mellitus, multiple sclerosis, rheumatoid arthritis, autoimmune uveitis, primary biliary cirrhosis, myasthenia gravis, Sjögren's syndrome, pemous vulgaris, scleroderma, pemic anemia, systemic lupus erythematosus. The method of claim 24, wherein the allergic disease is selected from allergic asthma, allergic rhinitis, atopic dermatitis and food hypersensitivity. 38

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法律状态:
2019-05-28| NUG| Patent has lapsed|

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申请号 | 申请日 | 专利标题

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SE1051122A|SE535625C2|2010-10-28|2010-10-28|New compositions and procedures for the treatment of autoimmune and allergic diseases|SE1051122A| SE535625C2|2010-10-28|2010-10-28|New compositions and procedures for the treatment of autoimmune and allergic diseases|

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CA2795577A| CA2795577A1|2010-10-28|2011-10-28|New compositions and methods for treatment of autoimmune and allergic diseases|

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AU2011321051A| AU2011321051B2|2010-10-28|2011-10-28|New compositions and methods for treatment of autoimmune and allergic diseases|

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EP11836713.5A| EP2633054B1|2010-10-28|2011-10-28|New compositions and methods for treatment of autoimmune and allergic diseases|

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CN201180030427.0A| CN102959082B|2010-10-28|2011-10-28|Be used for the treatment of novel composition and the method for autoimmunity and allergic disease|

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JP2013509020A| JP5346414B2|2010-10-28|2011-10-28|Novel compositions and methods for treating autoimmune and allergic diseases|

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PCT/SE2011/000191| WO2012057671A1|2010-10-28|2011-10-28|New compositions and methods for treatment of autoimmune and allergic diseases|