Modulating gammac-cytokine activity

Abstract

Embodiments relate to peptide antagonists of gamma c-family cytokines associated with important human diseases, such as leukemia, autoimmune diseases, collagenous diseases, diabetes, skin diseases, degenerative neurological diseases, and graft versus host disease (GvHD). Thus, inhibitors of gamma c-cytokine activity are valuable therapeutic and cosmetic agents as well as research tools. Traditional methods of inhibiting gamma c-cytokine activity involve the production of neutralizing antibodies for each individual gamma c-cytokine family member / receptor subunit. However, success is limited, and generally multiple [gamma] c-cytokine family members cooperate to elicit a disease state. The combined use of neutralizing antibodies generated for each factor is impractical and the risk of causing adverse immune responses is increased. The present embodiments overcome these disadvantages by inhibiting gamma c-cytokine activity using peptide antagonists based on consensus gamma c-subunit binding sites. Such methods allow for flexibility in the design of antagonists. In several embodiments, the peptides exhibit simultaneous-blocking (Simull-Block) activity, thereby inhibiting the activity of a plurality of [gamma] c-cytokine family members.

Description

[0001] This application is a divisional application of Chinese patent application No. 201680058779.X. The application date of Chinese patent application No. 201680058779.X is October 6, 2016, and all the contents are incorporated herein.

[0002] Cross-reference to priority and related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 239,761, filed October 9, 2015, which is hereby incorporated by reference in its entirety.

[0004] Sequence Listing in Electronic Format

[0005] This application is submitted with a sequence listing as an ASCII text file via EFS-Web. The sequence listing is provided as a file named BION006WOSEQLIST.txt, created and last saved on October 6, 2016, and is 5,981 bytes in size. Pursuant to 35 U.S.C. §1.52(e), the information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0006] field

[0007] Some embodiments relate to peptide antagonists of γc-family cytokines, which are a group of mammalian cytokines that are primarily produced by epithelial cells, stromal cells, and immune cells and control the normal and pathological activation of various lymphocytes. Some embodiments also relate to therapeutic uses of such peptides for treating certain human diseases. The present embodiment also relates to cosmeceutical applications of such peptides. The target diseases, cosmeceutical applications, and methods of administration, production, and commercialization of the peptides are disclosed.

[0008] background

[0009] Cytokines are a diverse group of soluble factors that mediate various cellular functions, such as growth, functional differentiation, and promotion or prevention of programmed cell death (apoptotic cell death). Unlike hormones, cytokines are not produced by specialized glandular tissues, but can be produced by a variety of cell types, such as epithelial cells, stromal cells, or immune cells.

[0010] The γc-family cytokines are a group of mammalian cytokines that are produced primarily by epithelial cells, stromal cells, and immune cells and control the normal and pathological activation of various lymphocytes. These cytokines are essential for the early development of T cells in the thymus and their homeostasis in the periphery.

[0011] Overview

[0012] In some embodiments, a composite peptide is provided. In some embodiments, the composite peptide comprises at least two amino acid sequences of the interleukin (IL) protein γc-box D-helix region, wherein the composite peptide comprises the amino acid sequence PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3).

[0013] In some embodiments, the composite peptide of claim 1, wherein the composite peptide inhibits the activity of two or more γc-cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In some embodiments, the composite peptide inhibits the activity of at least IL-15 and IL-21.

[0014] In some embodiments, the composite peptide further comprises a signal peptide. In some embodiments, the composite peptide is further conjugated to one or more additional parts at the N-terminus, C-terminus or side chain residues of the composite peptide. In some embodiments of the composite peptide, one or more additional parts are selected from: bovine serum albumin (BSA), albumin, keyhole limpet hemocyanin (Keyhold Limpet Hemocyanin, KLH), the Fc region of IgG, biological proteins used as scaffolds, antibodies against cell-specific antigens, receptors, ligands, metal ions and polyethylene glycol (PEG).

[0015] In some embodiments, the composite peptide consists of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the composite peptide consists of an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3.

[0016] In some embodiments, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a peptide conjugate or a derivative thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or a combination thereof, wherein the peptide conjugate or a derivative thereof modulates the activity of two or more γc-cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, wherein the peptide conjugate comprises the amino acid sequence PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3).

[0017] In some embodiments of the pharmaceutical composition, the peptide conjugate or derivative thereof inhibits the activity of two or more γc-cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.

[0018] In some embodiments of the pharmaceutical composition, the peptide conjugate or its derivative further comprises an additional conjugate located at its N-terminus, C-terminus or side chain residue. In some embodiments of the pharmaceutical composition, the peptide conjugate or its derivative further comprises a signal peptide.

[0019] In some embodiments, the pharmaceutical composition further comprises a protein that stabilizes the structure of the peptide conjugate or its derivative and improves its biological activity, wherein the protein is selected from the group consisting of bovine serum albumin (BSA), albumin, the Fc region of immunoglobulin G (IgG), biological proteins used as scaffolds, polyethylene glycol (PEG) and their derivatives.

[0020] In some embodiments of the pharmaceutical composition, the derivative thereof comprises a peptide sequence that shares at least 90% identity with the amino acid sequence of SEQ ID NO: 3. In some embodiments of the pharmaceutical composition, the derivative thereof comprises a peptide sequence that shares at least 95% identity with the amino acid sequence of SEQ ID NO: 3.

[0021] In some embodiments, methods for blocking signal transduction through two or more γc-cytokine family members are provided. In some embodiments, the method comprises contacting a cell with a composite peptide and / or pharmaceutical composition provided herein.

[0022] In some embodiments, a method of inhibiting γc-cytokine binding to a γc-subunit is provided. In some embodiments, the method comprises contacting a γc-subunit of a cell with a composite peptide and / or a pharmaceutical composition provided herein.

[0023] In some embodiments, a method for treating a γc-cytokine-mediated disease is provided. In some embodiments, the method comprises administering a composite peptide and / or pharmaceutical composition provided herein to a subject in need thereof, wherein the γc-cytokine-mediated disease is selected from the group consisting of: CD4 leukemia, CD8 leukemia, LGL leukemia, systemic lupus erythematosus, Sjögren's syndrome ( syndrome, Wegener's granulomatosis, celiac disease, Hashimoto's thyroiditis, rheumatoid arthritis, diabetes, psoriasis, multiple sclerosis, uvietis, eye inflammation, and graft-versus-host disease (GvHD).

[0024] In some embodiments, a method for treating a disease associated with HTLV-1-related myelopathy (HAM) / tropical spastic paraparesis (TSP) is provided. In some embodiments, the method comprises administering a composite peptide and / or pharmaceutical composition provided herein to a subject in need thereof, wherein the disease associated with HAM / TSP is selected from the group consisting of: adult T-cell leukemia (ATL), HTLV-related myelopathy / tropical spastic paraparesis (HAM / TSP), and other non-tumor inflammatory diseases associated with HTLV (such as uveitis (HU), arthropathy, pulmonary disease, dermatitis, exocrinopathy, and myositis).

[0025] In some embodiments, a method for treating an inflammatory respiratory disease is provided. In some embodiments, the method comprises administering a composite peptide and / or pharmaceutical composition provided herein to a subject in need thereof, wherein the inflammatory respiratory disease is selected from the group consisting of asthma, sinusitis, hay fever, bronchitis, chronic obstructive pulmonary disease (COPD), allergic rhinitis, acute and chronic otitis media, and pulmonary fibrosis.

[0026] In some embodiments, a method for treating a cosmetic disease is provided. In some embodiments, the method comprises administering a composite peptide and / or pharmaceutical composition provided herein to a subject in need thereof, wherein the cosmetic disease is selected from the group consisting of: acne, hair loss, sun spots, nail care, and appearance of aging.

[0027] In some embodiments, a pharmaceutical composition for treating a condition in a patient is provided. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a peptide conjugate or a derivative thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or a combination thereof, wherein the peptide conjugate or a derivative thereof modulates the activity of two or more γc-cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, wherein the peptide conjugate comprises the amino acid sequence PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3).

[0028] In some embodiments of the pharmaceutical composition, the derivative thereof comprises a peptide sequence that shares at least 90% identity with the amino acid sequence of SEQ ID NO: 3. In some embodiments of the pharmaceutical composition, the derivative thereof comprises a peptide sequence that shares at least 95% identity with the amino acid sequence of SEQ ID NO: 3.

[0029] In some embodiments, a pharmaceutical composition is provided for treating a γc-cytokine mediated disease, wherein the γc-cytokine mediated disease is selected from: CD4 leukemia, CD8 leukemia, LGL leukemia, systemic lupus erythematosus, Sjögren's syndrome, Wegener's granulomatosis, celiac disease, Hashimoto's thyroiditis, rheumatoid arthritis, diabetes, psoriasis, multiple sclerosis, uveitis, ocular inflammation and graft-versus-host disease (GvHD).

[0030] In some embodiments, a pharmaceutical composition is provided for treating HTLV-1-associated myelopathy (HAM) / tropical spastic paraparesis (TSP)-associated diseases, wherein the HAM / TSP-associated diseases are selected from: adult T-cell leukemia (ATL), HTLV-associated myelopathy / tropical spastic paraparesis (HAM / TSP), and other HTLV-associated non-tumor inflammatory diseases (such as uveitis (HU), arthropathy, pulmonary disease, dermatitis, exocrinopathy, and myositis).

[0031] In some embodiments, a pharmaceutical composition is provided for treating an inflammatory respiratory disease, wherein the inflammatory respiratory disease is selected from the group consisting of asthma, sinusitis, hay fever, bronchitis, chronic obstructive pulmonary disease (COPD), allergic rhinitis, acute and chronic otitis media, and pulmonary fibrosis.

[0032] In some embodiments, pharmaceutical compositions are provided for use in treating a cosmetic condition, wherein the cosmetic condition is selected from the group consisting of: acne, hair loss, sun spots, nail care, and the appearance of aging.

[0033] In some embodiments, a kit for treating a condition in a patient is provided. In some embodiments of the kit, the condition is a γc-cytokine mediated disease, a HTLV-1 associated myelopathy (HAM) / tropical spastic paraparesis (TSP) associated disease, an inflammatory respiratory disease, a cosmetic disease, or a combination thereof, and the kit comprises a pharmaceutical composition provided herein.

[0034] In some embodiments of the kit, the pharmaceutical composition comprises a therapeutically effective amount of a peptide conjugate or a derivative thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or a combination thereof, wherein the peptide conjugate or a derivative thereof modulates the activity of two or more γc-cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, wherein the peptide conjugate comprises the amino acid sequence PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3).

[0035] In some embodiments of the kit, the derivative thereof comprises a peptide sequence that shares at least 90% identity with the amino acid sequence of SEQ ID NO: 3. In some embodiments of the kit, the derivative thereof comprises a peptide sequence that shares at least 95% identity with the amino acid sequence of SEQ ID NO:3.

[0036] In some embodiments of the kit, the condition is one or more of: CD4 leukemia, CD8 leukemia, LGL leukemia, systemic lupus erythematosus, Sjögren's syndrome ( Sjoegren's syndrome, Wegener's granulomatosis, celiac disease, Hashimoto's thyroiditis, rheumatoid arthritis, diabetes, psoriasis, multiple sclerosis, uveitis, eye inflammation, graft-versus-host disease (GvHD), adult T-cell leukemia (ATL), HTLV-associated myelopathy / tropical spastic paraparesis (HAM / TSP) and other HTLV-associated non-neoplastic inflammatory diseases (such as uveitis (HU), arthropathy, pneumonopathy, dermatitis, exocrinopathy, myositis), asthma, sinusitis, hay fever, bronchitis, chronic obstructive pulmonary disease (COPD), allergic rhinitis, acute and chronic otitis media and pulmonary fibrosis, acne, hair loss, sunburn, nail maintenance or appearance of aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1A An alignment of the D-helical regions of the human γc-cytokine family members is shown.

[0039] Figure 1B The γc-box (SEQ ID NO: 10) motif and the IL-2 / IL-15 box (SEQ ID NO: 11) motif that generate a consensus sequence near the D-helical region of the γc-cytokine are depicted.

[0040] Figure 2 A diagrammatic representation depicting the biochemical properties of amino acids.

[0041] Figure 3A Inhibition of IL-15 and IL-9 activity by BNZ-γ in the PT-18 proliferation assay is shown.

[0042] Figure 3B Shown is a proliferation assay of CTTL2 cells grown in the presence of IL-2 or IL-15 and 0, 0.1, 1 or 10 uM BNZ-γ.

[0043] Figure 3C Inhibition of IL-15-mediated tyrosine phosphorylation of STAT5 by BNZ-γ was shown.

[0044] Figure 4AEx vivo T cell proliferation assay using HAM / TSP peripheral blood is shown. T cell proliferation is inhibited by addition of BNZ-γ.

[0045] Figure 4B The CD4+CD25+ cell population in an ex vivo T cell proliferation assay using HAM / TSP peripheral blood was shown to be reduced after addition of BNZ-γ to the culture.

[0046] Figure 4C The CD4+Ki67 cell population in an ex vivo T cell proliferation assay using HAM / TSP peripheral blood was shown to be reduced after addition of BNZ-γ to the culture.

[0047] Figure 4D It was shown that the percentage of viable cells by Guava staining in an ex vivo T cell proliferation assay using HAM / TSP peripheral blood was not affected after addition of BNZ-γ to the culture.

[0048] Figure 5 The alignment of the sequence of SEQ ID NO:3 with the D-helical regions of different human γc-cytokine family members is shown. The shaded regions represent the amino acid sequences of human γc-cytokine family members that are identical to their corresponding amino acids in the sequence of SEQ ID NO:3.

[0049] Details

[0050] Overview

[0051] More than 100 cytokines have been identified and are thought to have evolved by gene duplication from the original gene pool (see Bazan, JF 1990, Immunol. Today 11: 350-354). To support this view, it is common for a group of cytokines to share components in their multi-subunit receptor systems. The most well-documented shared cytokine subunit in T cells is the common γ subunit (γc-subunit).

[0052] The γc-subunit is shared by 6 known cytokines (interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-15 (IL-15) and interleukin-21 (IL-21), collectively referred to as "γc-cytokines" or "γc-family cytokines") and plays an integral role in transducing the cell activation signals of all of these cytokines. In addition, for each γc-cytokine, there are one or two private cytokine-specific receptor subunits that, when complexed with the γc-subunit, produce a fully functional receptor. (See Rochman et al., 2009, Nat Rev Immunol. 9:480-90.)

[0053] γc-family cytokines are a group of mammalian cytokines that are mainly produced by epithelial cells, stromal cells and immune cells and control the normal and pathological activation of various lymphocytes. These cytokines are crucial for the early development of T cells in the thymus and their homeostasis in the periphery. For example, in the absence of γc-subunits, T cells, B cells and NK cells will not develop in mice. (See Sugamura et al., 1996, Annu. Rev. Immunol. 14: 179-205).

[0054] γc-cytokines are important players in the development of lymphocytes (particularly T cells, B cells and NK cells) that make up the immune system. In addition, γc-cytokines have been implicated in various human diseases. Therefore, factors that inhibit the activity of γc-cytokines will provide useful tools for clarifying the developmental mechanisms of lymphocyte subsets and for treating immune disorders and γc-cytokine-mediated diseases.

[0055] It is known that the germline deletion of the gene encoding the γc-subunit in mice or the mutation of the γc-subunit in humans causes severe combined immunodeficiency (SCID) by destroying the normal appearance or function of NK cells, T cells and B cells. The study of the response of lymphocytes from these mice and human patients to γc-cytokines shows the importance of γc-subunits in the signal transduction of γc-cytokines (IL-2, -4, -7, -9, -15, -21) (reviewed in Sugamura et al., 1995 Adv. Immunol. 59: 225-277). This shows that the destruction of the interaction between γc-subunits and γc-cytokines will effectively block the intracellular signal transduction events performed by γc-cytokine family members. Therefore, it is expected that the antagonist peptide according to the embodiment of the present invention effectively blocks the pathogenic changes in humans suffering from diseases mediated by misregulation of γc-cytokine family members.

[0056] As an alternative to antibody-mediated approaches for modulating the activity of individual γc-cytokines, applicants have designed novel low molecular weight compounds, referred to herein as "simul-blocks," that inhibit the activity of multiple γc-cytokines. These low molecular weight compounds (including both chemicals and peptides) are less immunogenic than antibodies. These properties distinguish simultaneous-blocks as a more effective strategy for mediating γc-cytokine activity in clinical interventions.

[0057] Pathologies associated with γc-cytokines

[0058] Recent studies have shown that dysregulated expression and dysfunction of γc-cytokines may lead to a variety of human immune and hematopoietic diseases.

[0059] IL-2

[0060] Although IL-2 is historically considered a prototypical T cell growth factor, the generation of knockout mice lacking IL-2 expression reveals that IL-2 is not critical for the growth or development of conventional T cells in vivo. However, overexpression of IL-2 leads to preferential expansion of T cell subsets; regulatory T cells (T-reg). (See Antony et al., 2006, J. Immunol. 176: 5255-66.) T-reg suppresses the immune response of other cells, and thus plays a role in maintaining peripheral tolerance (reviewed in Sakaguchi et al., 2008, Cell 133: 775-87). The weakening of peripheral tolerance is believed to cause autoimmune diseases in humans.

[0061] Therefore, the immunosuppressive function of T-regs is thought to prevent the development of autoimmune diseases (see Sakaguchi et al., 2008, Cell 133:775-87). T-regs have also been implicated in cancer, where solid tumors and hematological malignancies have been associated with elevated numbers of T-regs (see De Rezende et al., 2010, Arch. Immunol. Ther. Exp. 58:179-190).

[0062] IL-4

[0063] IL-4 is a non-redundant cytokine involved in the differentiation of T helper cells into the Th2 (T-helper type 2) subset, which promotes the differentiation of immature B cells into plasma cells that produce IgE. IgE levels are elevated in allergic asthma. Therefore, IL-4 is involved in the development of allergic asthma. Antibodies targeting IL-4 can be used to treat or even prevent the onset of allergic asthma. (See LeBuanec et al., 2007, Vaccine 25:7206-16.)

[0064] IL-7

[0065] IL-7 is necessary for the early development of B cells and T cells in the thymus. In mice, abnormal expression of IL-7 causes T cell-related leukemia. (See Fisher et al., 1993, Leukemia 2: S66-68.) However, in humans, misregulation of IL-7 does not seem to cause T cell-related leukemia. In humans, the upregulation of IL-7 alone or in combination with another γc-cytokine family member IL-15 has been involved in large granular lymphocyte (LGL) leukemia.

[0066] IL-9

[0067] Compared with other γc-cytokine family members, the role of IL-9 is still quite unclear. Mice depleted of the IL-9 gene appear normal and do not lack any cell subsets in the lymphoid and hematopoietic compartments. However, recent studies have revealed the in vivo role of IL-9 in the production of Th17 (interleukin-17-induced T-helper) cells (see Littman et al., 2010, Cell 140 (6): 845-58; and Nowak et al., 2009, J. Exp. Med. 206: 1653-60).

[0068] IL-15

[0069] IL-15 is critically involved in the development of NK cells, NK-T cells, some subsets of intraepithelial lymphocytes (IEL), γδ-T cells, and memory-phenotype CD8 T-cells (see Waldmann, 2007, J. Clin. Immunol. 27: 1-18; and Tagaya et al., 1996, EMBO J. 15: 4928-39.) Overexpression of IL-15 in mice leads to the development of NK-T cells and CD8 cell type T cell leukemia (see Fehniger et al., 2001, J. Exp. Med. 193: 219-31; Sato et al., 2011 Blood, in press). These experimentally induced leukemias appear similar to LGL (large granular lymphocyte) leukemias in humans because in both cases the leukemic cells express the CD8 antigen.

[0070] It is also suspected that IL-15-mediated autocrine mechanisms may be involved in the leukemic transformation of CD4 T lymphocytes. (See Azimi et al., 1998, Proc. Natl. Acad. Sci. 95: 2452-7; Azimi et al., 1999, J. Immunol. 163: 4064-72; Azimi et al., 2000, AIDS Res. Hum. Retroviruses 16: 1717-22; and Azimi et al., 2001, Proc. Natl. Acad. Sci. 98: 14559-64). For example, the CD4-tropic HTLV-I that causes adult T-cell leukemia in humans induces autocrine growth of virus-transformed T cells through the production of IL-15 and IL-15Rα (Azimi et al., 1998, Proc. Natl. Acad. Sci. 95: 2452-7).

[0071] In addition to leukemic transformation, recent studies implicate IL-15 in the pathological development of celiac disease (CD), an autoimmune disease. IL-15 is known to stimulate NK cells, CD8 cells, and intestinal intraepithelial lymphocytes (IEL) cells to differentiate into lymphokine-activated killer (LAK) cells by inducing the expression of lytic enzymes (i.e., granzymes and perforins) and interferon-γ. Celiac disease (represented herein as CD) is an immune-mediated enteropathy that is triggered by the consumption of gluten-containing foods by individuals expressing specific HLA-DQ alleles.

[0072] The disease has an incidence of 1% in Western populations. Currently the only treatment for CD is the complete removal of gluten from the patient's diet. The pathology of CD is mainly caused by extensive damage to the intestinal mucosa, which is caused by activated CD8 T cells that have infiltrated the intestinal lamina propria. These CD8 T cells appear to be activated through a mechanism involving IL-15. A recent publication demonstrated in mice that ectopic overexpression of IL-15 by intestinal epithelial cells leads to the development of enteropathy that is very similar to the lesions in CD patients. Neutralization of IL-15 activity significantly reduced the pathological changes. Therefore, interventions that block IL-15 activation of CD8 T cells appear to provide an alternative strategy in enabling CD to cope with a conventional gluten-free diet.

[0073] IL-21

[0074] IL-21 is a recently discovered member of the γc-family. Unlike other family members, IL-21 does not seem to have an effective growth-promoting effect. On the contrary, IL-21 is considered to be more used as a differentiation factor than a factor for controlling cell proliferation (see Tagaya et al., 2010, J. Leuk. Biol. 87: 13-15).

[0075] Current strategies for treating γc-cytokine-mediated disorders

[0076] Since γc-cytokines are believed to be involved in many human diseases, several approaches have been proposed to treat diseases involving γc-cytokines by inhibiting the activity of the γc-cytokine family. These approaches include the use of cytokine-specific monoclonal antibodies to neutralize targeted cytokine activity in vivo; the use of monoclonal antibodies targeting private cytokine-specific receptor subunits (subunits other than the common γc-subunit) to selectively inhibit cytokine activity; and the use of chemical inhibitors that block downstream intracellular cytokine signal transduction pathways.

[0077] Although cytokine-specific antibodies are often the first choice for designing therapeutic agents, cytokines that share receptor components show overlapping functions (see Paul, WE, 1989, Cell 57: 521-24), and more than one cytokine can cooperate to cause disease (see Examples below). Therefore, approaches involving neutralization of a single cytokine may not be effective in treating human diseases involving cytokines.

[0078] Strategies have also been proposed for designing therapeutic agents that inhibit the function of multiple cytokines through antibodies that recognize shared receptor components. However, the multi-subunit nature of the cytokine receptor system and the fact that the functional receptors for a single cytokine can adopt different configurations make this approach difficult.

[0079] For example, a functional IL-15 receptor can be IL-15Rβ / γc or IL-15Rα / β / γc. (See Dubois et al., 2002, Immunity 17:537-47.) An antibody against the IL-15Rβ receptor (TMβ1) is an effective inhibitor of IL-15 function, but only when the IL-15Rα molecule is not present in the receptor complex. (See Tanaka et al., 1991, J. Immunol. 147:2222–28.) Therefore, the effectiveness of monoclonal anti-receptor antibodies, whether generated against shared or private subunits, can be context-dependent and unpredictable in vivo.

[0080] Although it is customary to use monoclonal antibodies against bioactive factors or receptors associated with the pathogenesis of the disease in the clinic, there is little evidence of successful results. In addition, establishing clinically applicable monoclonal antibody therapy is a long and difficult process, and the successful production of neutralizing antibodies is mainly luck. For example, because the γc-subunit is crucial in signal transduction mediated by γc-family cytokines, many attempts have been made to produce polyclonal and monoclonal antibodies against the γc-subunit, and there are many commercial antibodies that recognize the γc-subunit in mice and humans. However, surprisingly, none of these anti-γc-subunit antibodies blocked the function of γc-cytokines.

[0081] Another problem with the therapeutic use of monoclonal antibodies is that they are usually produced by immunizing rodents with human proteins, so the antibodies produced are foreign proteins and are therefore highly immunogenic. To avoid this problem, the amino acid sequence of the monoclonal antibody is molecularly modified so that the antibody molecule is recognized as a human immunoglobulin (a process known as humanization), but this process requires time and expense.

[0082] Targeting JAK3 as an existing alternative for inhibiting multiple γc-cytokines

[0083] The interaction between the γc-subunit and the γc-cytokine leads to the activation of a protein tyrosine kinase in the cell called Janus kinase 3 (Jak3). In turn, Jak3 phosphorylates a variety of signal transduction molecules (including STAT5 and PI3 kinase). The interaction between the γc-subunit and Jak3 is very specific. In fact, no other receptor molecules recruit Jak3 for signal transduction. (See O'Shea, 2004, Ann. Rheum. Dis. 63: (Supplement II): ii67-7.) Therefore, inhibition of cytokine signal transduction through the γc-subunit can be achieved by preventing the activity of Jak3 kinase. Therefore, a variety of chemical inhibitors targeting the kinase activity of Jak3 have been introduced into the market. (See Pesu et al., 2008, Immunol. Rev. 223: 132-142.) One such example is CP690,550.

[0084] The main disadvantage of these protein kinase inhibitors is the lack of specificity for Jak3 kinase. These drugs intercept the binding of ATP (adenosine triphosphate) molecules to Jak3 kinase, a common biochemical reaction of many protein kinases, and therefore tend to block the actions of multiple intracellular protein kinases unrelated to Jak3 kinase, which are very necessary for the health of normal cells in various tissues. Therefore, more specific inhibitors of signal transduction through the γc-subunit are needed.

[0085] Alternative strategies for treating diseases involving γc-cytokines are therefore highly desirable.

[0086] Discovery of the γC-box

[0087] The C-terminus (D-helix) of γc-cytokines contains proposed sites for interaction with common γc-subunits of multiunit cytokine receptors. (Bernard et al., 2004 J. Biol. Chem. 279: 24313-21.) Comparison of the biochemical properties of the amino acids of all γc-cytokines identified in mice and humans revealed that the chemical properties of the amino acids (e.g., hydrophobicity, hydrophilicity, basic / acidic properties), if not identical, are conserved at many positions in the D-helix across members of the γc-cytokine family.

[0088] In contrast, the sequence of IL-13, which is related to the γc-cytokine IL-4 but does not bind to the γc-subunit, does not show significant homology with γc-cytokines in the D-helical region, suggesting that sequence homology in the D-helical region is associated with binding to the γc-subunit. Figure 1AAs shown, alignment of the amino acid sequences of the D-helical regions of the members of the γc-cytokine family in humans revealed a motif of moderate sequence homology among these cytokines, which is referred to herein as the "γc-box".

[0089] The γc-box (SEQ ID NO: 10) comprises 19 amino acids, of which positions 4, 5, and 13 are completely conserved as phenylalanine, leucine, and glutamine, respectively, among the 19 positions. Less conservation is observed at positions 6, 7, and 11 of the γc-box, where the amino acid is one of two or three related amino acids that share physicochemical properties: position 6 can be occupied by the polar amino acids glutamic acid, asparagine, or glutamine; position 7 can be occupied by the non-polar amino acids serine or arginine; and position 11 is occupied by the non-polar aliphatic amino acids leucine or isoleucine. Positions 9 and 16 can be occupied by the non-polar amino acid isoleucine or the polar amino acid lysine. See Figure 1B Some differences in the amino acid composition of the γc-box were observed at position 9 and position 16 among the γc-cytokine subfamily. Comparison of γc-cytokines across species indicated that isoleucine is commonly present at position 9 and position 16 of the IL-2 / 15 subfamily, while other γc-family members typically have lysine at these positions. Without wishing to be bound by a particular theory, isoleucine and lysine are biochemically distinct and may therefore confer specific conformational differences between the IL-2 / 15 subfamily and other γc-cytokines.

[0090] The conservation of the γc-box motif among γc-cytokines is supported by the finding that a glutamine (Gln, Q) residue located in the D-helical region is essential for the binding of γc-cytokines to the γc-subunit. (Bernard et al., 2004 J. Biol. Chem. 279: 24313-21.)

[0091] Peptide inhibitors of γc-cytokine activity

[0092] The activity of γc-family cytokines can be blocked by disrupting the interaction between γc-cytokines and γc-subunits (e.g., by introducing competitive inhibitors that can interact with γc-subunits without stimulating signal transduction through multi-subunit cytokine receptors). Without being bound by a particular theory, the conserved γc-box motif involved in the binding of γc-family cytokines to γc-subunits presents a core base amino acid sequence that can be used to design peptide inhibitors of γc-cytokine signal transduction.

[0093] The core γc-box amino acid sequence comprises: D / EFLE / Q / NS / RXI / KXL / IXQ (SEQ ID NO: 2) (wherein X represents any amino acid). At least some embodiments described herein relate to customized peptide derivatives of the core γc-box amino acid sequence that can inhibit the activity of one or more γc-cytokines. Customized peptide derivatives include any peptide whose partial amino acid sequence shows about 50%, 50-60%, 60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99% or 99.8% identity with the core γc-box amino acid sequence. Customized peptide derivatives also include any peptide, wherein the partial amino acid sequence of this peptide derivative comprises amino acids having similar physicochemical properties to the amino acids of the core γc-box. For example, amino acids having similar physicochemical properties would include phenylalanine, tyrosine, tryptophan and histidine, which are aromatic amino acids. Figure 2 A diagrammatic representation of amino acids with similar physicochemical properties that can be substituted for the amino acids comprising the core γ c-box is shown. The length of the peptide derivatives of the core γ c-box can be 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25-30, 30-35, 35-40, 40-45, 45-50, or more than 50 amino acids. In some embodiments, the custom peptide derivatives can be conjugated to the N-terminus, C-terminus, and / or side chain residues of existing biological proteins / peptides.

[0094] Based on the identification of the conserved γc-box motif in cytokines that bind to the γc-subunit, the applicant has designed a novel 19-mer custom-derived peptide that is an artificial composite peptide that combines the amino acid sequences of the human IL-2 and IL-15 γc-boxes. The 19-mer peptide referred to herein as BNZ-γ consists of the following amino acid sequence: IK- E -FLQ- R -FIHI- V -QS- I -I- N -TS (SEQ ID NO: 1), wherein the amino acids indicated by bold characters are conserved between IL-2 and IL-15, and the underlined amino acids represent positions where the physicochemical properties of the amino acids are conserved.

[0095] Applicants have found that 19-mer BNZ-γ inhibits cell proliferation induced by IL-15 and IL-9, but not induced by IL-3 or IL-4. Figure 3A and Example 2. The applicant further demonstrated that BNZ-γ inhibits the phosphorylation of the intracellular cytokine signal transduction molecule STAT-5 mediated by IL-15. Figure 3C and Example 5. These results demonstrate that tailored peptide derivatives of the conserved γc-box motif can inhibit the activity of multiple γc-cytokines.

[0096] Several embodiments relate to a customized derivative peptide of the 19-mer BNZ-γ amino acid sequence: IKEFLQRFIHIVQSIINTS (SEQ ID NO: 1), which can inhibit the activity of one or more γc-cytokines. The customized peptide derivative of the 19-mer BNZ-γ amino acid sequence includes any peptide whose partial amino acid sequence shows about 50%, 50-60%, 60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99% or 99.8% identity with the amino acid sequence: IKEFLQRFIHIVQSIINTS (SEQ ID NO: 1). The customized peptide derivative also includes any peptide, wherein the partial amino acid sequence of this peptide derivative contains amino acids having physicochemical properties similar to those of the amino acids of the sequence: IKEFLQRFIHIVQSIINTS (SEQ ID NO: 1). In several embodiments, the amino acid residues of the custom derivative peptide retain similar physicochemical properties to those of BNZ-γ, but exhibit different biological inhibitory specificity to the six γc-cytokine family members than the original 19-mer peptide. The length of the peptide derivative of BNZ-γ can be 19, 20, 21, 22, 24, 25-30, 30-35, 35-40, 40-45, 45-50 or more than 50 amino acids. In some embodiments, the custom peptide derivative can be conjugated to the N-terminus, C-terminus and / or side chain residues of an existing biological protein / peptide.

[0097] Several embodiments relate to custom peptide derivatives of the γc-box motif of IL-15, IL-2, IL-21, IL-4, IL-9 or IL-7, which are depicted in Figure 1AOther embodiments relate to custom-derived peptides of artificial composite peptides that combine the amino acid sequences of two or more of the γc-box motifs of human IL-15, IL-2, IL-21, IL-4, IL-9, and IL-7. Several embodiments relate to custom peptide derivatives of the γc-box motif of IL-15, IL-2, IL-21, IL-4, IL-9, or IL-7, wherein the custom peptide derivatives have a partial amino acid sequence that shows about 50%, 50-60%, 60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99%, or 99.8% identity with the amino acid sequence of the γc-box motif of IL-15, IL-2, IL-21, IL-4, IL-9, or IL-7. Customized peptide derivatives of the γc-box motif of IL-15, IL-2, IL-21, IL-4, IL-9 or IL-7 also include any peptide, wherein the partial amino acid sequence of this peptide derivative contains amino acids having physicochemical properties similar to those of the amino acids in the sequence of the γc-box motif of IL-15, IL-2, IL-21, IL-4, IL-9 or IL-7.

[0098] Several embodiments relate to custom peptide derivatives that will inhibit the function of one, all, or selective members of a γc-cytokine. In some embodiments, the custom peptide derivative selectively targets a single γc-cytokine family member. For example, the custom peptide derivative can selectively inhibit the function of IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21. In other embodiments, the custom peptide derivative can inhibit 2 or more γc-cytokine family members.

[0099] For example, the customized peptide derivatives of the embodiments of the present invention can selectively inhibit the function of the following combinations: IL-2 and one or more of IL-4, IL-7, IL-9, IL-15 and IL-21; IL-4 and one or more of IL-7, IL-9, IL-15 and IL-21; IL-7 and one or more of IL-9, IL-15 and IL-21; IL-9 and one or more of IL-2, IL-4, IL-7, IL-15 and IL-21; IL-15 and one or more of IL-2, IL-4, IL-7, IL-9 and IL-21; or IL-21 and one or more of IL-2, IL-4, IL-7, IL-9 and IL-15. In other embodiments, the customized peptide derivatives can comprehensively target all γc-cytokine family members.

[0100] Without wishing to be bound by a particular theory, the customized peptide derivatives may inhibit the function of all or selective members of the γc-cytokine by reducing the binding of the γc-cytokine to the γc-subunit (e.g. as competitive inhibitors). Such customized peptide derivatives may be used in different applications, including as clinical drugs.

[0101] Several embodiments relate to custom peptide derivatives that will regulate (including enhancing or reducing) the function of one, two or more selective members of γc-cytokines. In some embodiments, custom peptide derivatives selectively target individual γc-cytokine family members. For example, custom peptide derivatives can selectively enhance or inhibit the function of IL-2, IL-4, IL-7, IL-9, IL-15 or IL-21. In other embodiments, custom peptide derivatives can enhance or inhibit two or more γc-cytokine family members. In certain embodiments, custom peptide derivatives can include PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3), which can enhance or inhibit the activity of one, two or more of γc-cytokines. In certain embodiments, custom peptide derivatives can include PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3), which can inhibit the activity of at least IL-15 and IL-21.

[0102] In some embodiments, the customized peptide derivative may include any peptide whose partial amino acid sequence shows about 50%, 50-60%, 60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99% or 99.8% identity with the amino acid sequence: PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3). The customized peptide derivative also includes any peptide, wherein the partial amino acid sequence of this peptide derivative contains amino acids having physicochemical properties similar to those of the amino acids of the sequence: PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3).

[0103] In several embodiments, the amino acid residues of the custom-derived peptide retain similar physicochemical properties to those of PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3), but exhibit different biological inhibitory specificity to six gamma c-cytokine family members (i.e., IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21) than the original peptide of PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3). The length of the peptide derivative of the sequence of PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3) can be 19, 20, 21, 22, 24, 25-30, 30-35, 35-40, 40-45, 45-50, or more than 50 amino acids.

[0104] In some embodiments, the custom peptide derivative can be conjugated to the N-terminus, C-terminus and / or side chain residues of existing biological proteins / peptides. In some embodiments, the composite peptide of SEQID NO:3 can be conjugated to other parts through the N-terminus, C-terminus or side chain of the composite peptide. Other parts can include proteins or peptides that stabilize the composite peptide, or include but are not limited to the following other parts: bovine serum albumin (BSA), albumin, keyhole limpet hemocyanin (KLH), Fc region of IgG, biological proteins used as scaffolds, antibodies to cell-specific antigens, receptors, ligands, metal ions and polyethylene glycol (PEG).

[0105] The terms "oligopeptide", "polypeptide", "peptide" and "protein" can be used interchangeably when referring to custom peptide derivatives provided according to embodiments of the present invention, and can be used to specify a series of amino acid residues of any length. Peptides according to embodiments of the present invention can also contain non-natural amino acids. The linker element can be connected to the peptide of embodiments of the present invention by a peptide bond or via a chemical bond. Peptides of embodiments of the present invention can be linear or cyclic and can include (D) as well as (L) amino acids.

[0106] The peptides of the present embodiments may also contain one or more rare amino acids (such as 4-hydroxyproline or hydroxylysine), organic acids or amides and / or derivatives of common amino acids (such as amino acids having a C-terminal carboxylate that is esterified (e.g., benzyl ester, methyl ester or ethyl ester) or amidated and / or having an N-terminal amino group modified (e.g., acetylated or alkoxycarbonylamino), with or without any of a wide variety of side chain modifications and / or substitutions (e.g., methylated, benzylated, tert-butylated, tosylated, alkoxycarbonylamino, etc.)).

[0107] Residues that may be present in addition to the common amino acids include, but are not limited to, penicillamine, tetramethylenecysteine, pentamethylenecysteine, mercaptopropionic acid, pentamethylenemercaptopropionic acid, 2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, ornithine, diaminobutyric acid, aminoadipic acid, m-aminomethylbenzoic acid, and diaminopropionic acid.

[0108] The peptides of the present invention can be produced and obtained by various methods known to those skilled in the art. For example, the peptides can be produced and obtained by genetic engineering based on the nucleotide sequence encoding the peptides of the present invention, or by chemical synthesis such as solid phase synthesis of peptides, or by a combination thereof.

[0109] Those skilled in the art can identify the conserved γc-box motifs disclosed herein and the Figure 2 The invention relates to a method for synthesizing custom peptide derivatives based on the knowledge of the biochemical properties of the amino acids described in the invention. Some embodiments also relate to polynucleotides comprising a nucleotide sequence encoding the peptides of the invention. "Nucleotide sequence", "polynucleotide" or "nucleic acid" can be used interchangeably and are understood to mean double-stranded DNA, single-stranded DNA or a transcription product (e.g., RNA molecule) of the DNA. Rather than administering the peptide itself, the polynucleotide can be administered to a cell or object and expressed by the cell or object. Several embodiments also relate to a gene construct comprising a polynucleotide sequence encoding the peptides of the invention. The gene construct can also include additional regulatory elements (such as promoters and enhancers) and optionally selectable markers.

[0110] Methods for treating gamma c-cytokine mediated diseases

[0111] Several embodiments relate to the use of γc-antagonist peptides in the treatment of γc-cytokine mediated diseases. The use of customized peptide derivatives according to embodiments of the present invention allows flexibility in the design of therapeutic agents (custom design of peptides) and enables more comprehensive results that cannot be achieved by conventional strategies using anti-cytokine or anti-cytokine receptor antibodies.

[0112] Described herein is a novel method for blocking the action of γc-family cytokines. Such operations can produce effective clinical intervention methods to treat diseases related to abnormal regulation or dysfunction of γc-cytokines. Examples of diseases that can be treated by destroying the interaction between γc-cytokines and γc-subunits include autoimmune diseases such as systemic lupus erythematosus, Sjögren's syndrome, Wegener's granulomatosis, celiac disease, Hashimoto's or autoimmune thyroiditis; collagen diseases, including rheumatoid arthritis, inflammatory bowel disease, diabetes, skin autoimmune diseases such as psoriasis; degenerative neurological diseases, such as multiple sclerosis, uveitis or ocular inflammation and sympathetic ophthalmia, graft versus host disease (GvHD) and myasthenia gravis.

[0113] In some embodiments, the γc-antagonist peptides described herein can be used to treat 1-human T-cell lymphotropic type I and II (HTLV-I and HTLV-II) related diseases, including adult T-cell leukemia (ATL), HTLV-related myelopathy / tropical spastic paraparesis (HAM / TSP), and other non-tumor inflammatory diseases associated with HTLV (such as uveitis (HU), arthropathy, lung disease, dermatitis, exocrinopathy, and myositis). In some embodiments, the γc-antagonist peptides described herein can be used to treat other viral diseases (such as influenza, AIDS, HBV, and herpes) or parasitic diseases.

[0114] In several embodiments, the gamma c-antagonist peptides can be administered as immunosuppressants before, during, and after transplantation of various organs.

[0115] In some embodiments, the γc-antagonist peptides described herein can be used to treat immune-mediated diseases such as asthma and other inflammatory respiratory diseases, such as but not limited to sinusitis, hay fever, bronchitis, chronic obstructive pulmonary disease (COPD), allergic rhinitis, acute and chronic otitis media, pulmonary fibrosis. In some embodiments, the γc-antagonist peptides can be administered to treat or prevent allergic reactions caused by exposure to allergens, chemicals, or other common causes of acute respiratory diseases. In some embodiments, the γc-antagonist peptides can be administered to treat or prevent inflammatory responses caused by viruses, bacteria, chemicals, and biochemical agents.

[0116] In several embodiments, the gamma c-antagonist peptides can be administered to treat certain types of malignancies, such as LGL-leukemia, intraepithelial lymphoma and leukemia in refractory celiac disease, NK leukemia / lymphoma, and NK-T leukemia / lymphoma.

[0117] In some embodiments, customized peptide derivatives according to the embodiments described herein can be used for cosmetic purposes such as treating acne, hair loss, sun spots, and nail care, and included in ointments as anti-aging components due to their anti-inflammatory properties.

[0118] Several embodiments relate to therapeutic antagonist peptides that will inhibit the function of all or selective members of γc-cytokines. In some embodiments, the therapeutic antagonist peptide selectively inhibits a single γc-cytokine family member (custom peptide). In other embodiments, the therapeutic antagonist peptide can comprehensively inhibit all γc-cytokine family members (simultaneous-blocking). In some embodiments, the therapeutic antagonist peptide selectively inhibits a subgroup of γc-cytokines. Without wishing to be bound by a particular theory, peptide antagonists can inhibit the function of all or selective members of γc-cytokines by reducing the binding of γc-cytokines to γc-subunits (e.g., as competitive inhibitors).

[0119] In experimental mouse models, several members of the γc-cytokine family, IL-2, IL-7, and IL-15, but not IL-4, have been implicated in graft-versus-host disease (GvHD). (Miyagawa et al., 2008 J. Immunol. 181: 1109-19.) One embodiment relates to the use of therapeutic antagonist peptides that selectively inhibit the activity of IL-2, IL-7, and IL-15 for the treatment of GvHD in humans, thereby allowing the survival of transplanted tissue or bone marrow cells. Other embodiments relate to the use of therapeutic antagonist peptides that selectively inhibit the combination of IL-2 and IL-7, IL-2 and IL-15, or IL-7 and IL-15 for the treatment of GvHD. Other embodiments relate to the use of combinations of therapeutic antagonist peptides that selectively inhibit IL-2, IL-7, or IL-15.

[0120] Some embodiments relate to the use of therapeutic antagonist peptides that selectively inhibit IL-2 function for the treatment of autoimmune disorders in which T-regs have been implicated. In some embodiments, peptide-mediated inhibition of T-regs can enhance innate anti-cancer immunity in humans, thereby providing a new means of anti-cancer therapy.

[0121] Several embodiments relate to the use of therapeutic antagonist peptides that selectively inhibit IL-4 to treat asthma.

[0122] Some embodiments relate to the use of therapeutic antagonist peptides that selectively inhibit IL-7, alone or in combination with therapeutic antagonist peptides that selectively inhibit IL-15, a member of the γc-cytokine family, as therapeutic agents for LGL leukemia. In some embodiments, therapeutic antagonist peptides that selectively inhibit the activity of both IL-7 and IL-15 can be used to treat LGL leukemia. Several embodiments relate to the use of BNZ-γ to treat LGL leukemia. In some embodiments, specific γc-antagonist peptides that selectively inhibit IL-15 alone or specific γc-antagonist peptides that selectively inhibit IL-15 and IL-7 are used as therapeutic agents for CD4 / CD8 T lymphocyte-associated leukemias (including those caused by HTLV I).

[0123] Several embodiments relate to the use of γc-antagonist peptides that selectively inhibit the activity of IL-9 alone or in combination with other γc-cytokine family members as therapeutic agents for human diseases involving abnormal development of Th17 cells.

[0124] Several embodiments relate to the use of therapeutic antagonist peptides that selectively inhibit IL-15 activity as therapeutic agents for treating CD. A recent publication shows that, in addition to IL-15, IL-21 may play a role in the pathogenesis of CD. (See Bodd et al., 2010, Mucosal Immunol. 3: 594-601.) This suggests that the optimal treatment of CD by conventional anti-cytokine or cytokine receptor antibodies will benefit from a combination of at least two antibodies that recognize components belonging to the IL-15 and IL-21 systems. In some embodiments, a custom-derived antagonist peptide that selectively inhibits the activity of both IL-15 and IL-21 is used as a therapeutic agent for treating CD.

[0125] In addition to having therapeutic applications, γc-antagonist peptides also have applications in consumer products. Several embodiments relate to the use of γc-antagonist peptides in skin care products (such as anti-aging, anti-inflammatory, anti-acne and other related applications). Some embodiments relate to the use of γc-antagonist peptides in hair products as anti-hair loss ingredients to treat hair loss caused by autoimmune disorders.

[0126] Another embodiment relates to the development of chemical compounds (non-peptide, non-protein), which have a spatial structure similar to the 19-mer amino acid sequence IKEFLQRFIHIVQSIINTS (SEQ ID NO: 1) and can fit into the pocket of the γc-subunit to structurally hinder the γc-cytokine from approaching the γc-subunit for binding. Some embodiments relate to the use of chemical compounds with similar structures as inhibitors of γc-cytokine activity. Such molecular simulation strategies for further improving the development of synthetic compounds similar to existing biological peptides / protein structures are described in Orzaez et al., 2009 Chem. Med. Chem. 4: 146-160. Another embodiment relates to the use of chemical compounds (non-peptide, non-protein) with a 3D structure similar to the 19-mer amino acid sequence IKEFLQRFIHIVQSIINTS (SEQ ID NO: 1) to treat γc-cytokine-mediated diseases.

[0127] Several embodiments relate to administering a peptide of the amino acid sequence IKEFLQRFIHIVQSIINTS (SEQ ID NO: 1) to treat a γc-cytokine mediated disease. Another embodiment relates to administering a derivative peptide of the amino acid sequence IKEFLQRFIHIVQSIINTS (SEQ ID NO: 1) to treat a γc-cytokine mediated disease, wherein the amino acid sequence of the derivative peptide has similar physicochemical properties to the peptide of the amino acid sequence IKEFLQRFIHIVQSIINTS (SEQ ID NO: 1) but has a different biological activity. Another embodiment relates to administering a peptide of the amino acid sequence IKEFLQRFIHIVQSIINTS (SEQ ID NO: 1) conjugated to the N-terminus and C-terminus or side chain residues of an existing biological protein / peptide to a patient to treat a γc-cytokine mediated disease.

[0128] Several embodiments relate to administering polyclonal antibodies and monoclonal antibodies generated against a peptide consisting of the amino acid sequence IKEFLQRFIHIVQSIINTS (SEQ ID NO: 1) to a patient as an immunogen to treat a γc-cytokine-mediated disease. Another embodiment relates to administering polyclonal antibodies and monoclonal antibodies generated against a derivative peptide of the amino acid sequence IKEFLQRFIHIVQSIINTS (SEQ ID NO: 1) to a patient as an immunogen to treat a γc-cytokine-mediated disease, wherein the amino acid sequence of the derivative peptide has similar physicochemical properties to the peptide of the amino acid sequence IKEFLQRFIHIVQSIINTS (SEQ ID NO: 1) but has a different biological activity.

[0129] Administration of γc-antagonist peptides

[0130] Embodiments of the present invention also include the use of γc-antagonist peptides for the manufacture of drugs for treating diseases. Embodiments of the present invention also include pharmaceutical compositions, which include a combination of γc-antagonist peptides and a pharmaceutically acceptable carrier. The pharmaceutical composition may include a pharmaceutically acceptable carrier and a non-toxic therapeutically effective amount of a γc-antagonist peptide or other compositions of embodiments of the present invention.

[0131] Embodiments of the present invention provide methods of using pharmaceutical compositions of antagonists of γc-cytokines in an effective amount contained in a suitable diluent or carrier. The γc-antagonists of embodiments of the present invention can be formulated according to known methods for preparing pharmaceutically useful compositions. The γc-antagonists can be mixed into a blend as the sole active substance or with other known active substances, together with pharmaceutically suitable diluents (e.g., phosphates, acetates, Tris-HCl), preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifying compounds, solubilizers, adjuvants and / or carriers (such as bovine serum albumin).

[0132] Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 16th edition. 1980 Mack Publishing Co. In addition, such compositions may contain γc-antagonists complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymers (such as polyacetic acid, polyglycolic acid, hydrogels, etc.) or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroplasts. Such compositions will affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate of the γc-antagonist. The γc-antagonist may be conjugated to antibodies, receptors, ligands directed against cell-specific antigens, or coupled to ligands directed against tissue-specific receptors.

[0133] The method of administering the γc-antagonist of the present embodiment can be appropriately selected according to factors such as the type of disease, the condition of the subject and / or the site to be targeted. The γc-antagonist can be administered topically, orally, parenterally, rectally or by inhalation. The term "parenteral" includes subcutaneous injection, intravenous, intramuscular, intraperitoneal, intracisternal injection or infusion techniques. These compositions generally include an effective amount of a γc-antagonist alone or in combination with an effective amount of any other active substance.

[0134] The amount of peptide contained in the pharmaceutical composition of the present invention, the dosage form of the pharmaceutical composition, the frequency of administration, etc. can be appropriately selected according to factors such as the type of disease, the condition of the subject, and / or the site to be targeted. Such dosages and the desired drug concentrations contained in the composition may be affected by many parameters, including the intended use, the patient's weight and age, and the route of administration. Pilot studies will be conducted using animal studies first, and scaling for human administration will be performed according to accepted practices in the field.

[0135] In one embodiment, a host cell that has been genetically modified with a polynucleotide encoding at least one γc-antagonist peptide is administered to a subject to treat a proliferative disorder and / or reduce the growth of malignant cells. The polynucleotide is expressed by the host cell, thereby producing the peptide in the subject. Preferably, the host cell is allogeneic or autologous to the subject.

[0136] On the other hand, the γc-antagonist peptide can be used in combination with other therapies (e.g., therapies that inhibit cancer cell proliferation and growth). The phrase "combination therapy" includes the administration of a γc-antagonist peptide and an additional therapeutic agent as part of a specific treatment regimen designed to provide a beneficial effect from the combined action of these therapeutic agents. The combined administration of these therapeutic agents is usually performed within a defined period of time (usually minutes, hours, days or weeks, depending on the selected combination).

[0137] Combination therapy is intended to include administering these therapeutic agents in a sequential manner (i.e., wherein each therapeutic agent is administered at different times), and administering these therapeutic agents or at least two therapeutic agents in a substantially simultaneous manner. Substantially simultaneous administration can be achieved, for example, by administering to the subject a single capsule of each therapeutic agent with a fixed ratio or with a plurality of single capsules for each therapeutic agent. Sequential or substantially simultaneous administration of each therapeutic agent can be achieved by appropriate routes, including but not limited to oral routes, intravenous routes, intramuscular routes, and direct absorption by mucosal tissues. These therapeutic agents can be administered by the same route or by different routes. The order in which the therapeutic agents are administered is not very critical.

[0138] Combination therapy may also include administration of a therapeutic agent as described above in combination with other biologically active ingredients (such as, but not limited to, a second therapeutic agent and a different therapeutic agent) and additional combinations of non-drug therapies (such as, but not limited to, surgery or radiation therapy). Where the combination therapy also includes radiation therapy, the radiation therapy may be administered at any suitable time, as long as the beneficial effects from the combined effects of the therapeutic agent and the radiation therapy are achieved. For example, in appropriate circumstances, when radiation therapy is temporarily removed from the administration of the therapeutic agent, perhaps for several days or even weeks, a beneficial effect may still be obtained.

[0139] In certain embodiments, the gamma c-antagonist peptide may be administered in combination with at least one antiproliferative agent selected from the group consisting of chemotherapeutic agents, antimetabolites, and anti-neoplastic agents, and anti-mitotic agents, and anti-viral agents, and anti-tumor agents, immunotherapeutic agents, and radiotherapeutic agents.

[0140] In certain embodiments, the gamma c -antagonist peptide may be administered in combination with at least one anti-inflammatory agent selected from the group consisting of steroids, corticosteroids, and non-steroidal anti-inflammatory drugs.

[0141] Also provided are kits for performing any of the methods provided herein. In some embodiments, the kit may include one or more γc-antagonists according to any of the embodiments provided herein. In some embodiments, the kit may include instructions. The instructions may be in written form or in the form of statistical charts, or may be on a recorded medium (), including audio tapes, audio CDs, video tapes, DVDs, CD-ROMs, etc. The kit may include packaging.

[0142] definition

[0143] As used herein, the term "patient" refers to a recipient of therapeutic treatment and includes all organisms within the kingdom Animalia. In preferred embodiments, the animal is within the family of mammals, such as humans, bovines, ovines, porcines, felines, buffalo, canines, goats, equines, donkeys, deer, and primates. The most preferred animal is a human.

[0144] As used herein, the term "treat" or any variation thereof (e.g., treatment, treating, etc.) refers to any treatment of a patient diagnosed with a biological condition such as CD4-, CD8- and LGL-leukemia, autoimmune diseases, systemic lupus erythematosus, Sjögren's syndrome, Wegener's granulomatosis, celiac disease, Hashimoto's thyroiditis, collagen disease, rheumatoid arthritis, inflammatory bowel disease, diabetes, psoriasis, degenerative neurological disease, multiple sclerosis, uveitis, ocular inflammation, graft versus host disease (GvHD), severe myasthenia, 1-human T-cell lymphotropic type I and II (HTLV-I and HTLV-II) related diseases, adult T-cell leukemia (ATL), HTLV-related myelopathy / tropical spastic paraparesis (HAM / TSP), uveitis (HU), arthropathy, lung disease, dermatitis, exocrinopathy, myositis, influenza, AIDS, HBV, herpes, asthma, sinusitis, hay fever, bronchitis, chronic obstructive pulmonary disease (COPD), allergic rhinitis, acute and chronic otitis media, pulmonary fibrosis, NK leukemia / lymphoma and NK-T leukemia / lymphoma.

[0145] As used herein, the term treatment includes: (i) preventing or delaying the presentation of symptoms associated with a target biological condition in a patient at risk who has not yet exhibited symptoms associated with the biological condition; (ii) ameliorating symptoms associated with a target biological condition in a patient diagnosed with a biological condition; (iii) preventing, delaying or ameliorating the presentation of symptoms associated with a complication, condition or disease associated with the target biological condition in a patient at risk or diagnosed with a biological condition; (iv) slowing, delaying or halting the progression of a biological condition; and / or (v) preventing, delaying, slowing, halting or ameliorating the cellular events of inflammation.

[0146] As used herein, the term "symptoms" refers to common signs or indications that a patient suffers from a particular condition or disease.

[0147] As used herein, the term "effective amount" refers to the amount necessary to elicit a desired biological response. According to embodiments of the present invention, an effective amount of a γc-antagonist is the amount necessary to provide an observable effect in at least one biological factor for treating a biological condition.

[0148] "Recombinant DNA technology" or "recombinant" refers to techniques and methods that enable the biosynthesis of heterologous peptides using production of specific polypeptides from microorganisms (e.g., bacteria, yeast), invertebrates (insects), mammalian cells, or organisms (e.g., transgenic animals or plants) that have been transformed or transfected with cloned or synthetic DNA sequences. The native glycosylation pattern can only be achieved with mammalian cell expression systems. Prokaryotic expression systems lack the ability to add glycosylation to synthetic proteins. Yeast and insect cells provide unique glycosylation patterns that may differ from the natural pattern.

[0149] "Nucleotide sequence" refers to a polynucleotide, either as an individual fragment or as a component of a larger DNA construct, derived from DNA or RNA that has been isolated at least once in substantially pure form, free of contaminating endogenous material and in quantities or concentrations that permit identification, manipulation, and recovery of its component nucleotide sequences by standard molecular biology methods (such as those outlined in laboratory manuals in molecular biology).

[0150] "Recombinant expression vector" refers to a plasmid containing a transcription unit comprising the following set: (1) a genetic element or elements that has a regulatory role in gene expression, including a promoter and an enhancer, (2) a structural or coding sequence encoding a polypeptide according to an embodiment of the present invention, and (3) appropriate transcription and translation initiation sequences, and, if necessary, termination sequences. Structural elements intended for use in yeast and mammalian systems preferably include a signal sequence that enables extracellular secretion of the translated polypeptide by yeast or mammalian host cells.

[0151] A "recombinant microbial expression system" refers to a substantially homogenous monoculture of a suitable hotspot microorganism (e.g., a bacterium such as E. coli or a yeast such as S. cerevisiae) that has stably integrated a recombinant transcription unit into the chromosomal DNA or carries the recombinant transcription unit as a component of a residual plasmid. Typically, the host cells that comprise the recombinant microbial expression system are descendants of a single ancestral transformed cell. Once the regulatory elements linked to the structural nucleotide sequence to be expressed are induced, the recombinant microbial expression system will express a heterologous polypeptide.

[0152] As used herein, section headings are only used for organizational purposes and should not be construed as limiting the described subject matter in any way. For any purpose, all documents and similar materials (including but not limited to patents, patent applications, articles, books, papers and internet web pages) cited in this application are expressly incorporated by reference as a whole. When the definition of the term in the incorporated reference seems to be different from the definition provided in this teaching, the definition provided in this teaching will control the term. It should be understood that there is an implied "about" before the temperature, concentration, time, etc. discussed in this teaching, so that slight and insubstantial deviations are within the scope of the teaching herein.

[0153] Although the present invention has been disclosed in the context of certain embodiments and examples, it will be appreciated by those skilled in the art that the present invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of the present invention and to obvious modifications and equivalents thereof. In addition, although several variations of the present invention have been shown and described in detail, other modifications within the scope of the present invention will be apparent to those skilled in the art based on this disclosure.

[0154] It is also contemplated that various combinations or sub-combinations may be made to the specific features and aspects of the embodiments and still fall within the scope of the invention. It should be understood that the various features and aspects of the disclosed embodiments may be combined or substituted with each other to form different modes or embodiments of the disclosed invention. Therefore, it is intended that the scope of the invention disclosed herein is not limited by the above-mentioned specifically disclosed embodiments.

[0155] It should be understood, however, that this detailed description, while indicating preferred embodiments of the invention, is given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art. Example

[0156] The following examples are presented for illustrative purposes and should not be construed as limiting.

[0157] Example 1 - Method for assessing the inhibitory activity of γc-antagonist peptides

[0158] The ability of any custom derived peptide prepared according to embodiments of the present invention to inhibit the action of a gamma c-cytokine family member is determined using a mammalian cell assay to measure their proliferative response to a gamma c-cytokine family member.

[0159] For each of the six γc-cytokines, the indicator cell lines: CTLL-2 (a murine CD8 T cell line available from the American Type Culture Collection), and PT-18 (a murine mast cell line) and its subclone PT-18β were transfected with the human IL-2Rβ gene to render the cells responsive to IL-2 and IL-15 (Tagaya et al., 1996, EMBO J. 15:4928-39), and were used to quantify the growth-promoting activity of the γc-cytokines (see Laboratory Guide in Immunology from Wiley and Sons for method references). The indicator cells exhibited a semi-linear dose-dependent response when measured over a range of concentrations by the colorimetric WST-1 assay (see Clontech PT3946-1 and associated user manuals, incorporated herein by reference, for a detailed description of reagents and methods).

[0160] Once the appropriate dose of the cytokine that produces 50% and 95% of the maximum response from the indicator cell line is determined, various concentrations (ranging from 1 pM to 10 μM) of purified or synthesized custom-derived peptides are added to each well containing the cytokine and the indicator cells. The reduction in light absorption at 450 nm is used as an indicator of inhibition of cytokine-stimulated cell proliferation. Typically, cells are stimulated by cytokines so that the absorbance of the well containing the indicator cell line and cytokine is 2.0 to 3.0, which is reduced to a range of 0.1 to 0.5 by adding inhibitory peptides.

[0161] Example 2 - BNZ-γ peptide specifically inhibits the growth-promoting activity of IL-9 and IL-15

[0162] The ability of the BNZ-γ peptide to specifically inhibit the growth-promoting activity of selected γc-cytokines was determined using PT-18β cells as described above. Figure 3A IL-3 (a non-γc-cytokine that supports the growth of PT-18β cells) was used as a negative control. Briefly, PT-18β cells were incubated with two different dilutions of BNZ-γ peptide produced by HEK293T cells (1:20 or 1:50 dilutions of the original supernatant of HEK293T cells transfected with the BNZ-γ expression construct) or without BNZ-γ peptide in the presence of IL-3, IL-9, IL-15 or IL-4 (1 nM of each cytokine in culture).

[0163] The growth response of the cells was determined using the WST-1 assay 2 days after the introduction of the BNZ-γ peptide and cytokines. The growth promoting activity of IL-3 (a non-γc-cytokine) was not inhibited by BNZ-γ. In contrast, the activity of IL-15 and IL-9 was significantly reduced by the BNZ-γ peptide (p<0.01, Student T test). Cell proliferation stimulated by another γc-cytokine IL-4 was not affected by the addition of the BNZ-γ peptide. Figure 3A Results are shown for IL-3, IL-9, IL-15, and IL-4.

[0164] In a similar assay, the murine cell line CTTL2 was used. In this assay, cells were cultured with 0.5 nM recombinant IL-2 in RPMI 10% fetal bovine serum. To set up the proliferation assay, cells were washed 3 times from cytokines. Cells were seeded at 1x10(5) cells per well of a 96-well plate with a final concentration of 50 pM of IL-2 or IL-15. Various concentrations of BNZ-γ peptide (0.1, 1 and 10 ug / ml) were added to each well. Cells were cultured for 20 hours and in the last 4 hours, 10 μg / ml was added to the plate. 3 H-thymidine. Cells were collected using a plate reader. Data are shown in Figure 3B middle.

[0165] Example 3 - Measurement of the inhibition of γc-cell proliferation by measuring 3H-thymidine incorporation as a marker of cell proliferation Factor activity method

[0166] Antagonist custom-derived peptides were measured by 3H-thymidine incorporation assay to inhibit the proliferation of indicator cell groups induced by γc-cytokines. In short, radiolabeled thymidine (1 microCi) was given to 20-50,000 cells undergoing proliferation in the presence of cytokines. The radioactivity bound to cells was captured on a glass fiber filter using a conventional harvesting machine (e.g., Filtermate universal harvester from Perkin-Elmer), and then the radioactivity was measured using a b-counter (e.g., 1450Trilux microplate scintillation counter) to measure the radioactivity incorporated into the cells.

[0167] Example 4 - For measuring inhibition by determining the incorporation of cell tracking dyes as cell proliferation markers Methods for the activity of γc-cytokines

[0168] Indicator cells are incubated in the presence of selected γc-cytokines or in the presence of selected γc-cytokines and selected custom derived peptides. Cell populations are then labeled in vitro using cell tracking dyes (e.g., CMFDA, C2925 from Invitrogen), and flow cytometry (e.g., Beckton-Dickinson FACScalibur) is used to monitor the decay of green fluorescence of cells during each cell division. Typically, in response to γc-cytokine stimulation, 7 to 10 different peaks corresponding to the number of divisions that the cells have experienced will appear on the green fluorescence channel. Depending on the degree of inhibition, cells are incubated with selected γc-cytokines and antagonist custom derived peptides to reduce the number of peaks to only 1 to 3.

[0169] Example 5 - BNZ-γ and its derived antagonists inhibit intracellular signal transduction

[0170] In addition to stimulating cell proliferation, the binding of γc-cytokines to their receptors causes various intracellular events. (Rochman et al., 2009 Nat. Rev. Immunol. 9: 480-90, Pesu et al., 2005 Immunol. Rev. 203: 127-142.). Immediately after the cytokine binds to its receptor, a tyrosine kinase called Jak3 (Janus kinase 3) is recruited to the receptor at the plasma membrane. This kinase phosphorylates tyrosine residues of various proteins (including γc-subunits, STAT5 (signal transducer and activator of transcription 5) and PI3 (phosphatidylinositol 3) kinase subunits). Among them, the phosphorylation of STAT5 has been implicated in many studies in relation to cell proliferation triggered by γc-cytokines. (Reviewed in Hennighausen and Robinson, 2008 Genes Dev. 22: 711-21.). Based on these published data, we examined whether BNZ-γ peptide inhibited tyrosine phosphorylation of STAT5 in PT-18β cells stimulated by IL-15 (the results are shown in Figure 3C middle).

[0171] PT-18β cells were stimulated with IL-15 in the presence or absence of BNZ-γ peptide. Cytoplasmic proteins were extracted from cells according to conventional methods as described in Tagaya et al., 1996 EMBO J. 15: 4928-39. The extracted cytoplasmic proteins were separated using standard SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the phosphorylation status was confirmed using immunoblotting with anti-phospho-STAT5 antibody (Cell Signaling Technology, catalog #9354, Danvers MA) (see Figure 3C, upper panel). To confirm that each lane exhibited similar total protein loading, the membrane was then stripped and reprobed with anti-STAT5 antibody (Cell Signaling Technology, catalog #9358) (see Figure 3C , figure below).

[0172] These results indicate that tyrosine phosphorylation of STAT5, a marker of signal transduction, is induced by IL-15 in PT-18β cells and that tyrosine phosphorylation of STAT5 is significantly reduced by BNZ-γ peptide.

[0173] Example 6 - Rational design of BNZ-γ derived antagonistic peptides

[0174] Based on the core sequence D / EFLE / Q / NS / RXI / KXL / IXQ (SEQ ID NO: 2) (wherein X represents any amino acid) by using Figure 2 Derivative peptides are prepared by substituting designated amino acids having the same physicochemical properties for defined amino acids of the core sequence.

[0175] Alternatively, custom peptides or their derivatives can be prepared based on sequence alignments of the D-helical regions of different γc-cytokine family members. Figure 5 As shown, one or more sequences conserved among members of the γc-cytokine family (SEQ ID NO:4 - SEQ ID NO:9) can be combined to form a peptide such as SEQ ID NO:3.

[0176] Example 7 - Methods for Identifying Inhibitory Specificity of Antagonistic Custom-Derived Peptides

[0177] The γc-cytokine inhibition specificity of antagonistic customized derived peptides is determined by measuring the ability of customized derived peptides to suppress the proliferation response of cytokine response cell lines to each of 6 γc-cytokines. For example, mouse cell line CTLL-2 is used to determine whether candidate peptides suppress the function of IL-2 and IL-15. PT-18 (β) cells are used to determine whether candidate peptides suppress the function of IL-4 and IL-9. PT-18 (7α) cells are used to determine whether candidate peptides suppress the function of IL-7, and PT-18 (21α) cells are used to determine whether candidate peptides suppress the function of IL-21. PT-18 (β) represents a subclone of PT-18 cells expressing human IL-2Rβ exogenously by gene transfection (see Tagaya et al., 1996), PT-18 (7α) represents a subclone expressing human IL-7Rα by gene transfection, and PT-18 (21Rα) cells express human IL-21Rα.

[0178] Another alternative is to use other cell lines that respond to a range of cytokines. An example of this cell line is human NK cell line NK92, which can be purchased from ATCC (Catalog No. CRL-2407). This cell line is an IL-2 dependent cell line that responds to other cytokines (including IL-9, IL-7, IL-15, IL-12, IL-18, IL-21) (Gong et al., 1994 Leukemia 8:652-658, Kingemann et al., 1996, Biol Blood Marrow Transplant 2:68; 75, Hodge DL et al., 2002 J. Immunol. 168:9090-8).

[0179] Example 8 - Preparation of γc-antagonist peptides

[0180] Custom-derived γc-antagonist peptides were chemically synthesized by manual and automated processes.

[0181] Artificial synthesis: using classical liquid phase synthesis, which involves coupling the carboxyl group or C-terminus of one amino acid to the amino group or N-terminus of another. Alternatively, solid phase peptide synthesis (SPPS) is used.

[0182] Automated synthesis: A number of commercial companies offer automated peptide synthesis at a cost. These companies use a variety of commercial peptide synthesizers, including those provided by Applied Biosystems (ABI). Custom derivatized γc-antagonist peptides were synthesized by automated peptide synthesizers.

[0183] Example 9 - Bioproduction of custom-derived γc-antagonist peptides using recombinant technology

[0184] The custom-derived γc-antagonist peptides are biosynthesized as propeptides consisting of an appropriate tag peptide, a signal peptide, or a peptide derived from a known human protein that enhances or stabilizes the structure of the BNZ-γ peptide or a peptide comprising the sequence of SEQ ID NO: 3 or its derivatives and improves their biological activity. If necessary, an appropriate enzyme cleavage sequence into the N-terminus of the peptide should be designed to remove the tag or any portion of the peptide from the final protein.

[0185] The nucleotide sequence encoding the custom derivative peptide with a stop codon at the 3' end is inserted into a commercial vector having a tag portion derived from thioredoxin of Escherichia coli and a specific peptide sequence between the tag portion and the nucleotide sequence encoding the custom derivative peptide and the stop codon that is recognized and digested by an appropriate proteolytic enzyme (e.g., enterokinase). An example of a suitable vector is the pThioHis plasmid available from Invitrogen, CA. Other expression vectors may be used.

[0186] Example 10 - Customized peptides and derivatives and carrier proteins for immunization purposes and production of antibodies against the customized peptides Conjugation

[0187] BNZ-γ and other customized derivative peptides (such as peptides or derivatives thereof comprising the sequence of SEQ ID NO:3) are used to immunize animals to obtain polyclonal antibodies and monoclonal antibodies. The peptide is conjugated to the N-terminus or C-terminus of a suitable carrier protein (e.g., bovine serum albumin, keyhole limpet hemocyanin (KLH), etc.) by conventional methods using glutaraldehyde or m-maleimidobenzoyl-N-hydroxysuccinimide ester. The conjugated peptide is then used together with a suitable adjuvant to immunize animals (such as rabbits, rodents or donkeys). The specificity of the resulting antibodies is detected using conventional methods. If the resulting antibodies react with the immunogenic peptides, then the ability to inhibit the activity of a single γc-cytokine is tested according to the cell proliferation assay described in Examples 1-3. Due to the composite nature of the derived peptides, it is possible to produce a single antibody (due to the composite nature of these peptides) that simultaneously recognizes two different cytokines.

[0188] Example 11 - Methods for large-scale production of custom-derived γc-antagonist peptides

[0189] As described elsewhere, recombinant proteins were produced on a large scale using a cell-free system. (See Takai et al., 2010 Curr. Pharm. Biotechnol. 11 (3): 272-8.) In brief, cDNA encoding γc-antagonist peptides and tags were subcloned into appropriate vectors (see Takai et al., 2010 Curr. Pharm. Biotechnol. 11 (3): 272-8), which were transcribed in vitro and then immediately translated in vitro to produce labeled peptides. The pre-polypeptide was then purified using an immobilized antibody that recognized the labeled epitope, treated with a proteolytic enzyme, and the eluate (which mainly contained the target custom-derived peptide) was tested for purity using conventional 18% trimethylglycine-SDS-PAGE (Invitrogen) and conventional Coomassie staining. If the required purity of the peptide was not met (> 98%), the mixture was subjected to conventional HPLC (high performance liquid chromatography) for further purification.

[0190] Example 12 - Blocking cytokine function in HAM / TSP using custom derived γc-antagonist peptides

[0191] HTLV-1 associated myelopathy (HAM) / tropical spastic paraparesis (TSP) is a chronic progressive myelopathy seen in some people infected with human T-lymphotropic virus type 1 (HTLV-I). Infiltration of lymphocytes in the spinal cord is associated with the immune response to HTLV-I and leads to the release of certain cytokines. Some of these cytokines may also damage nerves.

[0192] Patients with HAM / TSP exhibit an elevated immune system state similar to that observed in autoimmune diseases (Oh et al. 2008 Neurol Clin. 26: 781-785). This elevated state is demonstrated by the ability of T cells from HAM / TSP patients to undergo spontaneous proliferation for about a week in ex vivo culture in the absence of exogenously added cytokines. The spontaneous proliferation of T cells in HAM / TSP patients is at least partially attributed to the autocrine / paracrine cycle of IL-2, IL-9, and IL-15. Addition of blocking antibodies to IL-2 or IL-15 receptors has been shown to inhibit spontaneous T cell proliferation in HAM / TSP ex vivo culture systems.

[0193] These observations, along with other data from ex vivo studies, have provided the rationale for moving two monoclonal antibodies (anti-IL-2 receptor alpha or anti-Tac and anti-IL-15 receptor beta chain) into the clinic for the treatment of HAM / TSP (Azimi et al., 2001 Proc. Natl. Acad. Sci. 98: 14559-64., Azimi et al., 1999 J. Immunol 163: 4064-72).

[0194] The anti-cytokine receptor antagonists according to the embodiments described herein are valuable not only as therapeutic immunomodulators for treating HAM / TSP, but also the modulation of immune responses in HAM / TSP by the anti-cytokine receptor antagonists according to embodiments of the present invention serves as a proof of concept for the use of anti-cytokine receptor antagonists according to embodiments of the present invention to treat other autoimmune diseases.

[0195] To demonstrate the efficacy of custom-derived γc-antagonist peptides according to the embodiments described herein, we tested the ability of BNZ-γ peptides to block immune responses to HTLV-I in spontaneous T cell proliferation assays using a HAM / TSP ex vivo culture system. Proliferation assays were performed on HAM / TSP patient blood samples with or without the addition of BNZ-γ. These assays evaluated the ability of BNZ-γ to block the function of cytokines (such as IL-2 and IL-15) present in ex vivo HAM / TSP patient blood cultures and to prevent spontaneous T cell proliferation in these samples.

[0196] In an ex vivo spontaneous T cell proliferation assay, PBMCs from HAM / TSP patients were cultured in RPMI-10% FCS at 1x10(6) cells per well of a 96-well plate. Increasing concentrations of BNZ-γ peptide were added to each well. As a control, an irrelevant peptide was used in a similar manner. The cells were incubated in a 37°C CO2 incubator for 3, 4, and 6 days. 1uCi of 3H-thymidine was added to the cells. After an additional 6 hours of incubation, the cells were harvested and their proliferation rates were measured. Figure 4A – Figure 4D Data from a representative HAM / TSP patient are shown in Figure 4A-4D As shown in , BNZ-γ peptide inhibited the spontaneous proliferation of T cells in HAM / TSP cultures at a concentration of approximately 1 ug / ml.

[0197] Other immune markers were additionally measured in this assay. The percentage of virus-specific CD8 cells was measured during ex vivo culture using viral protein tetramers. CD4+CD25+ cell populations (markers of T cell activation) and Ki67 staining (marker of T cell proliferation) were monitored in flow cytometry assays.

[0198] Other forms of conjugated BNZ-γ peptide derivatives or custom peptides comprising the sequence of SEQ ID NO: 3 and their derivatives may be used in similar future assays. They include albumin, BSA, PEG, which can be conjugated to the peptide after chemical synthesis. Other biological forms of custom peptides (such as BNZ-γ peptide conjugates or custom peptides comprising the sequence of SEQ ID NO: 3 and their derivatives) may include regions of known protein entities fused to the custom peptide (including but not limited to the Fc region of human IgG).

[0199] Example 13 - Treatment of Adult T Cell Leukemia in Human Patients by Administration of Custom-Derived γc-Antagonist Peptides Methods of disease (ATL)

[0200] A human patient with adult T-cell leukemia is identified. An effective dose of a custom-derived γc-antagonist peptide (e.g., BNZ-γ, a custom peptide comprising the sequence of SEQ ID NO: 3, or a derivative thereof) as determined by a physician is administered to the patient for a period of time determined by the physician. If the patient enters remission, the treatment is determined to be effective.

[0201] Example 14 - Methods of treating HAM / TSP in human patients by administering custom-derived γc-antagonist peptides

[0202] A human patient with HAM / TSP is identified. An effective dose of a custom-derived γc-antagonist peptide (e.g., BNZ-γ, a custom peptide comprising the sequence of SEQ ID NO: 3, or a derivative thereof) as determined by a physician is administered to the patient for a period of time determined by the physician. The treatment is determined to be effective if the patient's symptoms improve or if disease progression has been stopped or slowed.

[0203] Example 15 - Blocking cytokine function using custom derived γc-antagonist peptides

[0204] A human patient with a need to reduce at least IL-15 and IL-21 function is identified. An effective dose of a custom-derived γc-antagonist peptide (e.g., a composite peptide comprising the sequence of SEQ ID NO: 3 or a derivative thereof) as determined by a physician is administered to the patient for a period of time determined by the physician. If the patient's symptoms improve or if disease progression has been stopped or slowed, the treatment is determined to be effective.

[0205] Example 16 - Methods of treating celiac disease in human patients by administering custom-derived γc-antagonist peptides

[0206] A human patient suffering from celiac disease is identified. An effective dose of a custom-derived γc-antagonist peptide (e.g., a composite peptide comprising the sequence of SEQ ID NO: 3 or a derivative thereof) as determined by a physician is administered to the patient for a period of time determined by the physician. The treatment is determined to be effective if the patient's symptoms improve or if disease progression has been stopped or slowed.

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Claims

1. A composite peptide or a derivative thereof, comprising at least two amino acid sequences of the D-helix region of the γc-box of an interleukin (IL) protein, wherein the composite peptide comprises the amino acid sequence PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3), wherein the derivative comprises an amino acid sequence that shares at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 3, wherein the composite peptide or a derivative thereof inhibits the activity of IL-15 and IL-21.

2. The composite peptide or derivative thereof according to claim 1, further comprising a signal peptide and / or a linker.

3. The composite peptide or derivative thereof as claimed in claim 1, wherein the derivative comprises amino acid residues that retain physicochemical properties similar to those of the amino acid of SEQ ID NO:

3.

4. The composite peptide or derivative thereof as described in any one of claims 1 to 3, wherein the composite peptide or derivative thereof is further conjugated to one or more additional moieties at the N-terminus, C-terminus or side chain residues of the composite peptide.

5. The composite peptide or derivative thereof as described in claim 4, wherein the one or more additional parts are selected from: bovine serum albumin (BSA), albumin, keyhole limpet hemocyanin (KLH), the Fc region of IgG, biological proteins used as scaffolds, antibodies against cell-specific antigens, receptors, ligands, metal ions and polyethylene glycol (PEG).

6. A pharmaceutical composition comprising: A peptide conjugate or a derivative thereof; and A pharmaceutically acceptable carrier, diluent, excipient or a combination thereof; wherein the peptide conjugate or its derivative modulates the activity of IL-15 and IL-21; Wherein the peptide conjugate or derivative thereof comprises an amino acid sequence that shares at least 70% sequence identity with PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3).

7. The pharmaceutical composition of claim 6, wherein the peptide conjugate or its derivative further comprises a signal peptide and / or a linker.

8. The pharmaceutical composition of claim 6, wherein the derivative comprises amino acid residues that retain physicochemical properties similar to those of the amino acid of SEQ ID NO:

3.

9. The pharmaceutical composition according to any one of claims 6 to 8, wherein the peptide conjugate or derivative thereof is further conjugated to one or more additional moieties at its N-terminus, C-terminus or side chain residues.

10. The pharmaceutical composition of claim 9, wherein the one or more additional moieties are selected from the group consisting of bovine serum albumin (BSA), albumin, keyhole limpet hemocyanin (KLH), the Fc region of IgG, biological proteins used as scaffolds, antibodies against cell-specific antigens, receptors, ligands, metal ions, and polyethylene glycol (PEG).

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