Gene expression markers and treatment of multiple sclerosis

Abstract

To provide markers of multiple sclerosis, their use, and treatment with IL-17 antagonists, including IL-17 antibodies, of subjects with increased levels of such markers.SOLUTION: A method of identifying a mammalian subject with multiple sclerosis (MS) or at risk of developing MS, comprising: measuring an expression level of at least one gene selected from the group consisting of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF in a biological sample taken from the subject, wherein an increased level of expression compared to a control indicates that the subject has MS or is at risk of developing MS.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 108,914, filed Jan. 28, 2015, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on January 13, 2016, is named P32496WO_PCTSequenceListing.txt and is 34,331 bytes in size.

[0003] The present invention relates to markers for multiple sclerosis and the use of the markers in identifying patients having MS, at risk of developing MS, and / or for treatment with IL-17 antagonists, such as IL-17 antibodies. [Background technology]

[0004] Multiple sclerosis Multiple sclerosis (MS) is a demyelinating disease affecting the central nervous system where the onset of inflammation results in a highly variable course and progression of symptoms (Compston, A. et al., Lancet 372:1502-1517 (2008)). Onset is usually between the ages of 30 and 50, with a higher incidence in women and high geographic variability (Rosati, G., Neurol Sci 22:117-139 (2001)). There is a wide range of symptoms reflecting the diverse anatomical targets of demyelination, but typical syndromes include weakness, fatigue, vision loss, cognitive impairment, and balance and coordination problems (Compston, supra). The onset of MS is also irregular in timing, leading to the general principle that MS lesions are generalized in both space (location) and time (Adams, R et al., Principles of Neurology.sixth edn, (McGraw-Hill, 1997)). These symptoms are often sufficient to make a diagnosis of MS, but magnetic resonance imaging (MRI) is also informative, in addition to cerebrospinal fluid analysis and neural evoked potential measurements. MS usually begins with a reversible neurological disorder (relapsing-remitting, or RRMS) that eventually progresses to fixed disability (secondary progressive) later in life (Adams et al., supra). As with other aspects of the disease, the pattern, severity, and timing of this progression can vary widely between patients, with some experiencing profound disability with rapid progression from the beginning (primary progressive MS or PPMS), while a minority of other patients have isolated, relatively mild symptoms.

[0005] Currently, there is no cure for MS, and although there have been some recent advances in novel treatments, the disease remains difficult to treat (Kieseier, B.C., et al., Curr Opin Neurol 20:286-293 (2007)). Standard treatments include corticosteroids aimed at suppressing the inflammatory response during acute relapses, sometimes accompanied by plasmaphoresis to remove circulating antibodies from the bloodstream (Giovannoni, G. et al., Curr Opin Neurol 20:261-268 (2007)). Glatiramer acetate and interferon beta 1a are also used in RRMS, but are not particularly effective in PPMS, nor are they effective in altering the eventual course of MS, even in early intervention (Compston et al., supra; Kieseier et al., supra). More specific immunotherapies use monoclonal antibodies to target specific surface molecules involved in MS. Natalizumab binds to α4 integrins on white blood cells, thereby reducing their numbers, but due to the side effects of progressive multifocal leukoencephalopathy (PML), this drug is only used if other treatments have failed (Kieseier et al., supra). Other monoclonal antibodies, rituximab (an anti-CD20 antibody) and daclizumab (which targets CD-25), have a similar rationale, but they too are not curative and have other immunological side effects.

[0006] IL-17 Interleukin-17A (IL-17A, often referred to in the art as IL-17) is a T cell-derived proinflammatory molecule that stimulates epithelial, endothelial, and fibroblast cells to produce other inflammatory cytokines and chemokines, including IL-6, IL-8, G-CSF, and MCP-1 (Yao, Z. et al., J. Immunol. 122(12):5483-5486(1995); Yao, Z. et al., Immunity, 3(6):811-821(1995); Fossiez, F., et al., J. Exp. Med., 183(6):2593-2603(1996); Kennedy, J., et al., J. Interferon Cytokine Res., 16(8):611-7(1996); Cai, XY, et al. al.,Immunol.Lett,62(1):51-8(1998), Jovanovic,DV,et al.,J.Immunol.160(7):3513-21(1998),Laan,M.,et al.,J.Immunol.162(4):2347-52(1999),Linden,A.,et al.,Eur See Respir J, 15(5):973-7(2000) and Aggarwal, S. and Gurney, AL, J Leukoc Biol. 71(1):1-8(2002)). IL-17 also cooperates with other cytokines, including TNF-α and IL-1β, to further induce chemokine expression (Chabaud, M., et al., J. Immunol. 161(1):409-14 (1998)). IL-17A exhibits pleiotropic biological activities on various cell types. IL-17A also inhibits ICAM-1 surface expression, T cell proliferation, and CD34 + It also has the ability to induce the growth and differentiation of human progenitor cells into neutrophils.IL-17A has also been implicated in bone metabolism and has been shown to play an important role in pathologies characterized by the presence of activated T cells and TNF-α production, such as rheumatoid arthritis and bone implant loosening (Van Bezooijen et al., J. Bone Miner. Res., 14:1513-1521 (1999)).

[0007] Interleukin-17A is recognized as the prototype member of a newly emerging family of cytokines. Large-scale sequencing of the genomes of humans and other vertebrates has revealed the presence of additional genes encoding proteins clearly related to IL-17A, thereby defining a new family of cytokines. At least six members of the IL-17 family exist in humans and mice, including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F (see WO01 / 46420 published June 28, 2001). Both IL-17A and IL-17F are secreted as disulfide-linked homodimers ("IL-17AA" and "IL-17FF", respectively). In addition, a heterodimeric species consisting of disulfide-linked IL-17A and IL-17F has also been identified ("IL-17A / F"). Initial characterization indicates that, similar to IL-17A, some of these newly identified IL-17 molecules have the ability to modulate immune function. The potent inflammatory activity identified for some of these elements and their association with major emerging human diseases indicate that these proteins have important roles in the inflammatory process and may provide opportunities for therapeutic intervention.

[0008] The gene encoding human IL-17F is located adjacent to IL-17A (Hymowitz, SG, et al., Embo J, 20(19):5332-41 (2001)). IL-17A and IL-17F share approximately 44% amino acid homology, whereas other members of the IL-17 family share a more limited 15-27% amino acid homology, indicating that IL-17A and IL-17F form a distinct subgroup within the IL-17 family (Starnes, T., et al., J Immunol. 167(8):4137-40 (2001); Aggarwal, S. and Gurney, AL, J. Leukoc Biol, 71(1):1-8 (2002)). IL-17F appears to have similar biological activity as IL-17A and can promote the production of IL-6, IL-8, and G-CSF from a wide variety of cells. Like IL-17A, it can induce cartilage matrix release and inhibit new cartilage matrix synthesis (see, e.g., US 2002-0177188-A1 published Nov. 28, 2002). Thus, like IL-17A, IL-17F can potentially contribute to the pathology of inflammatory diseases. Both IL-17A and IL-17F have been reported to be induced in T cells by the activity of interleukin 23 (IL-23) (Aggarwal, S., et al., J. Biol. Chem., 278(3):1910-4(2003)). More specifically, both IL-17A and IL-17F have been implicated as agents contributing to the progression and pathology of a variety of inflammatory and autoimmune diseases in humans and mouse models of human disease. Indeed, IL-17A, and to a lesser extent IL-17F, have been implicated as effector cytokines that initiate inflammatory responses and thereby contribute to many autoinflammatory (autoimmune) diseases, including multiple sclerosis (MS) (Matusevicius et al., Mult. Scler., 5:101-104 (1999); Kurasawa, K., et al., Arthritis Rheu 43(11):2455-63 (2000)).

[0009] The amino acid sequence of the human receptor for IL-17A, IL-17RA, is available under NCBI GenBank accession number NP_055154.3. To date, at least four additional receptors (IL-17RB, IL-17RC, IL-17RD, and IL-17RE) have been identified in the IL-17R family based on sequence homology with IL-17RA, among which IL-17RC has been shown to be physically associated with IL-17RA, indicating that it may be a functional element in the IL-17R complex (Toy, D. et al., J. Immunol. 177:36-39 (2006)). IL-17RC has been reported to be a receptor for both IL-17A and IL-17F (Presnell, et al., J. Immunol. 179(8):5462-73 (2007)). Summary of the Invention

[0010] In one aspect, the present invention provides a method for identifying a mammalian subject having or at risk of developing multiple sclerosis (MS), comprising measuring the expression level of one or more genes selected from the group of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF in a biological sample obtained from the subject, wherein an increased expression level compared to a control indicates that the subject has or is at risk of developing multiple sclerosis.

[0011] In another aspect, the present invention provides a method for identifying or predicting a mammalian subject having multiple sclerosis (MS) or at risk of developing MS who is likely to be responsive to treatment with an IL-17 antagonist, comprising measuring the expression level of one or more genes selected from the group of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF in a biological sample obtained from the subject, and determining that the subject is likely to be responsive to treatment if the expression level of the one or more genes is increased compared to a control.

[0012] In another aspect, the present invention provides a method for identifying or predicting a mammalian subject having multiple sclerosis (MS) or at risk of developing MS who is unlikely to be responsive to treatment with an IL-17 antagonist, comprising measuring the expression level of one or more genes selected from the group of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF in a biological sample obtained from the subject, and determining that the subject is unlikely to be responsive to the treatment if the expression level of the one or more genes is decreased compared to or at the same level as a control.

[0013] In a further aspect, the present invention provides a method of monitoring a mammalian subject having multiple sclerosis (MS) being treated with an IL-17 antagonist, comprising measuring the expression level of one or more genes selected from the group of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF in a biological sample obtained from the subject, and determining whether the expression level of the one or more genes is increased compared to a control. In one embodiment of the present invention, an increased expression level compared to a control indicates that treatment with the IL-17 antagonist should be continued.

[0014] In another aspect, the present invention also provides a method of treating a mammalian subject having or at risk of developing multiple sclerosis (MS), comprising measuring the expression level of one or more genes selected from the group of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF, and administering an effective amount of an IL-17 antagonist to a subject having an increased expression level of the one or more genes compared to a control.

[0015] In a further aspect, the present invention provides a method of detecting the expression level of one or more genes in a mammalian subject suspected of having or at risk of developing relapsing-remitting multiple sclerosis (RRMS), comprising obtaining a biological sample from the subject and measuring in the biological sample the expression level of one or more genes selected from the group of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF. In one embodiment of the present invention, the method further comprises determining that the subject has or is at risk of developing RRMS if the expression level is increased compared to a control.

[0016] In one embodiment of any aspect of the invention, the mammalian subject is a human patient. In another embodiment, the multiple sclerosis is characterized by an increased level of IL-17. In a particular embodiment, the level of IL-17 is elevated in the cerebrospinal fluid (CSF) of the subject. In a further embodiment, the multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS). In yet another embodiment, the IL-17 is IL-17AA. In a further embodiment, the expression level of the RNA transcript of one or more genes is measured, and in another embodiment, the expression level of the protein product of one or more genes is measured.

[0017] In another embodiment of any aspect of the invention, the biological sample is a biological fluid. In one embodiment, the biological fluid is cerebrospinal fluid (CSF). In another embodiment, the biological fluid is serum, and in yet another embodiment, the biological fluid is plasma.

[0018] In yet another embodiment of any aspect of the invention, the expression level of TIMP1 is measured. In another embodiment, the expression level of LRG1 is measured. In yet another embodiment, the expression level of an RNA transcript of NFκBIZ is measured. In one embodiment of the invention, the expression level of G-CSF is measured in serum. In another embodiment, the expression level of one or more genes selected from the group of CXCL1, CXCL5, and CXCL10 is measured in plasma. In a further embodiment, the expression levels of two or more of the genes are measured, and in another embodiment, the expression levels of three or more of the genes are measured.

[0019] In another embodiment of any aspect of the invention, the expression level of TIMP1 and the expression level of at least one or more genes selected from the group of LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF are measured. In a further embodiment, the expression level of LRG1 and the expression level of at least one or more genes selected from the group of TIMP1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF are measured. In a particular embodiment, the expression levels of TIMP1 and LRG1 are measured. In another embodiment, the expression levels of TIMP1 and LRG1 are measured in CSF. In a further embodiment, the expression levels of TIMP1 and / or LRG1, and G-CSF are measured. In yet another embodiment, the expression levels of TIMP1 and / or LRG1 are measured in CSF and the expression level of G-CSF is measured in serum. In another embodiment, the expression levels of TIMP1, LRG1, and G-CSF are measured.

[0020] In another embodiment of any aspect of the invention, the method further comprises administering an effective amount of an IL-17 antagonist to a subject identified as having, predicted to have, or at risk of developing MS. In a particular embodiment of any aspect of the invention, the method further comprises administering an effective amount of an IL-17 antagonist to a subject determined to have an increased expression level of one or more of the disclosed genes compared to a control. In one embodiment of the invention, the IL-17 antagonist is an antibody or an antigen-binding fragment thereof. In a particular embodiment, the antibody is an IL-17 antibody or an IL-17 receptor antibody. In one embodiment, the antibody is at least one antibody selected from the group of brodalumab, secukinumab, ixekizumab, bimekizumab, CNTO6785, ALX-0761, and afasevikumab. In a further embodiment, the antibody is an IL-17 antibody, which binds to an IL-17A homodimer, an IL-17F homodimer, and / or an IL-17AF heterodimer. In one embodiment, the antibody is an IL-17 antibody that binds to an IL-17A homodimer. In another embodiment, the IL-17 antibody binds to an IL-17AA and an IL-17AF. In yet another embodiment, the antibody is an IL-17 antibody that binds to an IL-17F homodimer. In a further embodiment, the antibody is an IL-17 antibody that binds to an IL-17A / F heterodimer. In another embodiment, the antibody is a monoclonal antibody. In yet another embodiment, the antibody is a chimeric, humanized, or human antibody. In still a further embodiment, the antibody is a bispecific, multispecific, or cross-reactive antibody.

[0021] In yet another embodiment of any of the aspects of the invention, the method of the invention further comprises administering an effective amount of a multiple sclerosis (MS) therapeutic agent. In one embodiment of the invention, the MS therapeutic agent is at least one agent selected from the group of dimethyl fumarate, FTY-720, natalizumab, corticosteroids, beta-interferon, glatiramer acetate, teriflunomide, mitoxantrone, and an anti-CD20 antibody.

[0022] In another aspect, the present invention provides a kit for detecting the expression level of one or more genes in a mammalian subject having or at risk of developing multiple sclerosis (MS), the kit comprising at least one container comprising one or more reagents for detecting the expression level of one or more genes selected from the group of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF. In one embodiment of the present invention, the one or more reagents comprise reagents for detecting the expression level of an RNA transcript and / or the expression level of a protein product of the one or more genes. In another embodiment, the reagents comprise one or more gene-specific primers or probes for the one or more genes. In a further embodiment, the one or more reagents comprise reagents for detecting the expression level of a protein product of the one or more genes. In yet another embodiment, the reagents comprise one or more antibodies or antigen-binding fragments thereof that bind to the protein product of the one or more genes. The kit of the present invention may further comprise a container comprising an IL-17 antagonist and a label or instructions for administering the IL-17 antagonist to the subject. In one embodiment, the IL-17 antagonist is an antibody or an antigen-binding fragment thereof. In another embodiment, the antibody is an IL-17 antibody or an IL-17 receptor antibody. In a further embodiment, the antibody is an IL-17 antibody that binds to IL-17A homodimer, IL-17F homodimer, and / or IL-17AF heterodimer. In various embodiments of the invention, the MS is relapsing-remitting MS (RRMS). [Brief description of the drawings]

[0023] [Figure 1A] 1A-1B show (A) the nucleotide sequence of native human IL-17A cDNA (SEQ ID NO:1) and (B) the amino acid sequence of native human IL-17A (SEQ ID NO:2) derived from the coding sequence of SEQ ID NO:1 shown in Figure 1A. The signal peptide is underlined. [Figure 1B]1A-1B show (A) the nucleotide sequence of native human IL-17A cDNA (SEQ ID NO:1) and (B) the amino acid sequence of native human IL-17A (SEQ ID NO:2) derived from the coding sequence of SEQ ID NO:1 shown in Figure 1A. The signal peptide is underlined. [Figure 2A] Figures 2A-B show (A) the nucleotide sequence of native human IL-17F cDNA (SEQ ID NO:3) and (B) the amino acid sequence of native human IL-17F (SEQ ID NO:4) derived from the coding sequence of SEQ ID NO:3 shown in Figure 2A. The signal peptide is underlined. [Figure 2B] Figures 2A-B show (A) the nucleotide sequence of native human IL-17F cDNA (SEQ ID NO:3) and (B) the amino acid sequence of native human IL-17F (SEQ ID NO:4) derived from the coding sequence of SEQ ID NO:3 shown in Figure 2A. The signal peptide is underlined. [Figure 3A] Figures 3A-3E show (A-D) the nucleotide sequence of native human IL-17RA cDNA (SEQ ID NO:5) and (E) the amino acid sequence of native human IL-17RA (SEQ ID NO:6) derived from the coding sequence of SEQ ID NO:4 shown in Figures 3A-3C. [Figure 3B] Figures 3A-3E show (A-D) the nucleotide sequence of native human IL-17RA cDNA (SEQ ID NO:5) and (E) the amino acid sequence of native human IL-17RA (SEQ ID NO:6) derived from the coding sequence of SEQ ID NO:4 shown in Figures 3A-3C. [Figure 3C] Figures 3A-3E show (A-D) the nucleotide sequence of native human IL-17RA cDNA (SEQ ID NO:5) and (E) the amino acid sequence of native human IL-17RA (SEQ ID NO:6) derived from the coding sequence of SEQ ID NO:4 shown in Figures 3A-3C. [Figure 3D] Figures 3A-3E show (A-D) the nucleotide sequence of native human IL-17RA cDNA (SEQ ID NO:5) and (E) the amino acid sequence of native human IL-17RA (SEQ ID NO:6) derived from the coding sequence of SEQ ID NO:4 shown in Figures 3A-3C. [Figure 3E]Figures 3A-3E show (A-D) the nucleotide sequence of native human IL-17RA cDNA (SEQ ID NO:5) and (E) the amino acid sequence of native human IL-17RA (SEQ ID NO:6) derived from the coding sequence of SEQ ID NO:4 shown in Figures 3A-3C. [Figure 4A] 4A-4B show (A) the nucleotide sequence of native human IL-17RC cDNA (SEQ ID NO:7) and (B) the amino acid sequence of native human IL-17RC (SEQ ID NO:8) derived from the coding sequence of SEQ ID NO:8 shown in FIG. 4A. [Figure 4B] 4A-4B show (A) the nucleotide sequence of native human IL-17RC cDNA (SEQ ID NO:7) and (B) the amino acid sequence of native human IL-17RC (SEQ ID NO:8) derived from the coding sequence of SEQ ID NO:8 shown in FIG. 4A. [Diagram 5] 1 shows the possible role of the IL-17 pathway in the pathogenesis of MS. [Figure 6] As described in Example 1 below, multiple inflammatory mediators, eotaxin, CXCL10, TARC, and MCP4, are shown to be elevated in RRMS CSF samples (p<0.03 vs. healthy donors (HD) for all analytes). [Figure 7A] Figures 7A-B show that (A) CSF IL-17AA and IL-17FF were elevated in RRMS patients versus healthy donors (HD) (p<0.0001, Wilcoxon test), and (B) there was no elevation of serum IL-17AA or IL-17FF in RRMS patients versus HD, as described in Example 1 below. [Figure 7B] Figures 7A-B show that (A) CSF IL-17AA and IL-17FF were elevated in RRMS patients versus healthy donors (HD) (p<0.0001, Wilcoxon test), and (B) there was no elevation of serum IL-17AA or IL-17FF in RRMS patients versus HD, as described in Example 1 below. [Figure 8A]8A-8B show upregulation of NFκBIZ, CXCL1, IL-6, and CSF3 in (A) human brain endothelial cells and (B) microglia upon stimulation with IL-17A, as well as further amplification of the presence of TNFα and IL-6+6R (p<0.05 (pairwise t-test) for all comparisons except those in brackets with "FC" for "fold change"). See Example 2 below. [Figure 8B] 8A-8B show upregulation of NFκBIZ, CXCL1, IL-6, and CSF3 in (A) human brain endothelial cells and (B) microglia upon stimulation with IL-17A, as well as further amplification of the presence of TNFα and IL-6+6R (p<0.05 (pairwise t-test) for all comparisons except those in brackets with "FC" for "fold change"). See Example 2 below. [Figure 9] Described below in Example 3 is the method used to assess gene expression in an experimental autoimmune encephalomyelitis (EAE) model for MS, and the effect of an IL-17 antibody on gene expression pathways. [Figure 10A-1] At peak EAE disease as described in Example 3 below, Figures 10A-1-10A-2 show increased lymphocyte (CD4 and CD45) and Figures 10B-1-10B-2 show decreased myelin component (Mobp and Mog) gene expression. [Figure 10A-2] At peak EAE disease as described in Example 3 below, Figures 10A-1-10A-2 show increased lymphocyte (CD4 and CD45) and Figures 10B-1-10B-2 show decreased myelin component (Mobp and Mog) gene expression. [Figure 10B-1] At peak EAE disease as described in Example 3 below, Figures 10A-1-10A-2 show increased lymphocyte (CD4 and CD45) and Figures 10B-1-10B-2 show decreased myelin component (Mobp and Mog) gene expression. [Figure 10B-2]At peak EAE disease as described in Example 3 below, Figures 10A-1-10A-2 show increased lymphocyte (CD4 and CD45) and Figures 10B-1-10B-2 show decreased myelin component (Mobp and Mog) gene expression. [Figure 11] As described in Example 3 below, we show upregulation of CHI3L1, TIMP1, IL-6, CXCL1, LRG1, and NFκB1 in EAE spinal cord at the peak of disease, and blockade of gene expression with anti-IL-17AA / AF + anti-IL-17FF. [Figure 12A] Figures 12A-12F show plots of transcript abundance for the following genes: (A) NFκBIZ, (B) CXCL1, (C) IL-6, (D) YKL40 (CHI3L1), (E) TIMP1, and (F) LRG1 in EAE models receiving various treatments, as described in Example 3 below. [Figure 12B] Figures 12A-12F show plots of transcript abundance for the following genes: (A) NFκBIZ, (B) CXCL1, (C) IL-6, (D) YKL40 (CHI3L1), (E) TIMP1, and (F) LRG1 in EAE models receiving various treatments, as described in Example 3 below. [Figure 12C] Figures 12A-12F show plots of transcript abundance for the following genes: (A) NFκBIZ, (B) CXCL1, (C) IL-6, (D) YKL40 (CHI3L1), (E) TIMP1, and (F) LRG1 in EAE models receiving various treatments, as described in Example 3 below. [Figure 12D] Figures 12A-12F show plots of transcript abundance for the following genes: (A) NFκBIZ, (B) CXCL1, (C) IL-6, (D) YKL40 (CHI3L1), (E) TIMP1, and (F) LRG1 in EAE models receiving various treatments, as described in Example 3 below. [Figure 12E]Figures 12A-12F show plots of transcript abundance for the following genes: (A) NFκBIZ, (B) CXCL1, (C) IL-6, (D) YKL40 (CHI3L1), (E) TIMP1, and (F) LRG1 in EAE models receiving various treatments, as described in Example 3 below. [Figure 12F] Figures 12A-12F show plots of transcript abundance for the following genes: (A) NFκBIZ, (B) CXCL1, (C) IL-6, (D) YKL40 (CHI3L1), (E) TIMP1, and (F) LRG1 in EAE models receiving various treatments, as described in Example 3 below. [Figure 13] 1 shows plots of transcript abundance of CXCL5 in EAE models undergoing various treatments, as described in Example 3 below. [Figure 14] As described in Example 4 below, we show that TIMP1 and LRG1 are elevated in RRMS CSF and correlate with CSF IL-17AA. [Figure 15] Serum G-CSF levels in EAE models undergoing various treatments, as described in Example 5 below, are shown. [Figure 16] As described in Example 5 below, human G-CSF levels in serum from normal and RRMS patients are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] definition Unless otherwise defined, technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994) and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992) provide those of ordinary skill in the art with a general reference for many of the terms used in this application.

[0025] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0026] The term "IL-17" as used herein refers to IL-17A and / or IL-17F, and homodimers and heterodimers thereof, unless otherwise indicated, and refers to any naturally occurring IL-17 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses full-length unprocessed IL-17, as well as any form of IL-17 obtained by processing within a cell, including the precursor form and the mature form of the protein. The term also encompasses naturally occurring variants of IL-17, such as splice variants or allelic variants. In addition to disulfide-linked IL-17A and IL-17F ("IL-17A / F"), both IL-17A and IL-17F are secreted as disulfide-linked homodimers ("IL-17AA" and "IL-17FF", respectively). Thus, the term also encompasses homodimers and heterodimers of IL-17. As used herein, "IL-17AA" refers to IL-17A homodimer and "IL-17FF" refers to IL-17F homodimer. "IL-17A / F" or "IL-17AF" refers to IL-17A and IL-17F heterodimer. As used herein, "IL-17A" refers to IL-17A homodimer (IL-17AA) unless otherwise specified, and "IL-17F" refers to IL-17F homodimer (IL-17FF) unless otherwise specified. In one embodiment of the invention, IL-17 is IL17-AA. In another embodiment of the invention, IL-17 is IL-17FF. In yet another embodiment, IL-17 is IL-17A / F. The full length polypeptide chains of human IL-17A and IL-17F, including their amino acid sequences, are shown in Figures 1B and 2B (SEQ ID NOs: 2 and 4), respectively, and their corresponding nucleic acid sequences are shown in Figures 1A and 2A (SEQ ID NOs: 1 and 3), respectively. The start and stop codons are shown in bold and underlined in the figures.

[0027] The term "IL-17 receptor" is used to generally refer to the IL-17 receptor family, including IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE, and complexes thereof, and refers to any native IL-17 receptor from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses full-length unprocessed IL-17 receptor, as well as any form of IL-17 receptor obtained by processing within a cell, including IL-17 receptor, soluble IL-17 receptor, e.g., IL-17RA and / or soluble IL-17RC. The term also encompasses naturally occurring variants of the IL-17 receptor, e.g., splice variants or allelic variants. The nucleotide sequence of the native sequence IL-17RA polypeptide is shown in Figures 3A-D, and its amino acid sequence is shown in Figure 3E (SEQ ID NOs: 5 and 6), respectively. The nucleotide sequence of a native sequence IL-17RC polypeptide is shown in Figure 4A and its amino acid sequence is shown in Figure 4B (SEQ ID NOs:7 and 8), respectively.

[0028] As used herein, an "IL-17 antagonist" includes any molecule that interferes with the function or binding of IL-17 and blocks and / or neutralizes the associated activity of IL-17. Thus, IL-17 antagonists include anti-IL-17 antibodies, anti-IL-17 receptor antibodies, including anti-IL-17RA antibodies, and anti-IL-17RC antibodies, or soluble IL-17 receptors, including soluble IL-17RA and soluble IL-17RC.

[0029] An "IL-17 antibody," "anti-IL-17 antibody," or "antibody that binds IL-17" refers to an antibody or antigen-binding fragment thereof that can bind to an IL-17A homodimer, an IL-17F homodimer, and / or an IL-17AF heterodimer, or a portion thereof, with sufficient affinity such that the antibody is useful in targeting IL-17 as an agent for detection, analysis, diagnosis, and / or treatment. An IL-17 antibody may further interfere with IL-17 activity. In addition, an IL-17 antibody may interfere with the expression of other genes or proteins. In one embodiment, an IL-17 antibody can bind to IL-17AA, IL-17FF, and / or IL-17AF. In some embodiments, an anti-IL17 antibody can bind to an IL-17A dimer. In some embodiments, an anti-IL17 antibody can bind to an IL-17A homodimer and an IL-17AF heterodimer. In certain embodiments, the anti-IL-17 antibody can bind to IL-17A homodimer and cannot bind to IL-17AF heterodimer. In certain embodiments, the anti-IL-17 antibody can bind to IL-17F homodimer and cannot bind to IL-17AF heterodimer. In some embodiments, the anti-IL-17 antibody can bind to IL-17A homodimer, IL-17F homodimer, and IL-17AF heterodimer. In some such embodiments, an anti-IL-17 antibody that can bind to IL-17A homodimer, IL-17F homodimer, and IL-17AF heterodimer may also be referred to as an IL-17A and F antibody or an IL-17A and IL-17F cross-reactive antibody, or an IL-17A / F cross-reactive antibody. In certain such embodiments, the IL-17A and F cross-reactive antibodies bind to the same or similar epitopes on IL-17A, IL-17F, and / or IL-17AF heterodimers. In certain embodiments, the IL-17A and F cross-reactive antibodies bind to the same or similar epitopes on IL-17A, IL-17F, and / or IL-17AF heterodimers with sufficient affinity. In certain advantageous embodiments, the IL-17A and F cross-reactive antibodies bind to IL-17A, IL-17F, and IL-17AF with high affinity.The structures of IL-17A and IL-17F have been reported. See Hymowitz et al., 2001, Embo J, 20(19):5332-41; Ely et al., 2009, Nature Immunology 10(12):1245-1252; and Liu et al., 2013, Nature Communications DOI:10.1038 / ncomms2880. Similar or identical epitopes involving amino acid residues present in the surface regions of IL-17A and IL-17F can be inferred from the structures.

[0030] An "IL-17 receptor antibody," "anti-IL-17 receptor antibody," or "antibody that binds to IL-17 receptor" refers to an antibody that can bind to IL-17 receptor or a portion thereof with sufficient affinity such that the antibody is useful for targeting IL-17 receptor as a diagnostic and / or therapeutic agent. In addition, IL-17 receptor antibodies can interfere with IL-17 receptor activity, such as downstream signaling. IL-17 receptor antibodies can also interfere with the expression of other genes or proteins. In one embodiment, an IL-17 receptor antibody can bind to IL-17RA and / or IL-17RC. In other embodiments, an IL-17 receptor antibody can bind to IL-17RA. In some embodiments, an IL-17 receptor antibody can bind to IL-17RC. In certain embodiments, an IL-17 receptor antibody can bind to IL-17RA and IL-17RC.

[0031] An antibody, oligopeptide, or other organic molecule that "binds" to an antigen of interest, e.g., an IL-17 antibody that binds to an IL-17A homodimer, an IL-17F homodimer, and / or an IL-17AF heterodimer, is one that binds to the antigen with sufficient affinity such that the antibody, oligopeptide, or other organic molecule does not nonspecifically bind to other unrelated proteins. In one embodiment, the extent of binding of the antibody, oligopeptide, or other organic molecule to "non-target" proteins will be less than about 10% of the binding of the antibody, oligopeptide, or other organic molecule to their specific target proteins, as determined by fluorescence activated cell sorting (FACS) analysis, radioimmunoprecipitation (RIA), or other means. In certain embodiments, an antibody that binds to IL-17 or an IL-17 receptor will bind to an antigen with sufficient affinity such that the antibody, oligopeptide, or other organic molecule does not nonspecifically bind to other unrelated proteins. -4 M, or at least about 10 -5 M, or at least about 10 -6 M, or at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 -9 M, or at least about 10 -10 M, or at least about 10 -11 M, or at least about 10 -12 M or greater.

[0032] The term "antibody" as used herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments, so long as they exhibit the desired biological activity.

[0033] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogenous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind the same epitope(s), except for possible variants of the antibody that may arise during the production of the monoclonal antibody, and such variants are generally present in minor amounts. Such monoclonal antibodies typically include antibodies comprising a polypeptide sequence that binds to a target, the target-binding polypeptide sequence being obtained by a process that includes the selection of a polypeptide sequence that binds to a single target from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It is understood that the selected target-binding sequence can be further modified, for example, to improve affinity for the target, humanize the target-binding sequence, improve its production in cell culture, reduce immunogenicity in vivo, create multispecific antibodies, etc., and that antibodies comprising modified target-binding sequences are also monoclonal antibodies of the invention. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced using, for example, hybridoma techniques (e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681, (Elsevier, NY, 1981), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display methods (see, e.g., Clackson et al., Nature 352:624-628 (1991); Marks et al. J. Mol. Biol. 222:581-597 (1991); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods 101(34):12467-12472 (2004)). 284(1-2):119-132 (2004), and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO1998 / 24893, WO1996 / 34096, WO1996 / 33735, WO1991 / 10741, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993), Jakobovits et al., Nature, 362:255-258 (1993), Bruggemann et al., Year in Immuno., 7:33 (1993), U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669 (all to GenPharm), U.S. Patent No. 5,545,807, WO1997 / 17852, U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, Marks et al., Bio / Technology, 10:779-783 (1992), Lonberg et al. al.,Nature,368:856-859(1994),Morrison,Nature,368:812-813(1994),Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0034] Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences, as well as "humanized" antibodies.

[0035] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulin. Humanized antibodies are largely human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of human immunoglobulin sequences. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0036] As used herein, an "intact antibody" is one that comprises two antigen binding regions and an Fc region. Preferably, the intact antibody has a functional Fc region.

[0037] An "antigen-binding fragment" includes a portion of an intact antibody that contains its antigen-binding region. Examples of antigen-binding fragments include Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragment(s).

[0038] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain contains at one end a variable domain (V H ) followed by several constant domains. Each light chain has a variable domain (V L ) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain.

[0039] The term "variable" refers to the fact that the sequence of certain portions of the variable domains differs widely among antibodies, and is used in the binding and specificity of each particular antibody to its particular antigen. However, variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments, called hypervariable regions, in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FRs with a predominantly β-sheet structure, which are connected by three hypervariable regions, forming loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions in each chain are held in close proximity to the hypervariable regions of the other chain by the FRs, and contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of cells in antibody-dependent cellular cytotoxicity (ADCC).

[0040] When used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable regions generally consist of amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from the "hypervariable loops", e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0041] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment produces an F(ab') fragment that has two antigen-binding sites and is still capable of cross-linking to antigen. 2 Fragments are obtained.

[0042] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight non-covalent association. The three hypervariable regions of each variable domain interact to form the V H -V LIt is in this configuration that an antigen-binding site is defined on the surface of the dimer. The six hypervariable regions together confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0043] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments in that they have a few additional residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear at least one free thiol group. F(ab') 2 Antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0044] The "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0045] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain and includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined as extending from an amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include an antibody population with all K447 residues removed, an antibody population without the K447 residue removed, and an antibody population with a mixture of antibodies with and without the K447 residue.

[0046] Unless otherwise indicated, the numbering of residues in immunoglobulin heavy chains herein is that of the EU index in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), which is expressly incorporated herein by reference. "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0047] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), and the like. Such effector functions generally require that the Fc region is associated with a binding domain (e.g., an antibody variable domain) and can be assessed, for example, using the various assays disclosed herein.

[0048] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0049] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or the Fc region of a parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions in the native sequence Fc region or in the Fc region of the parent polypeptide, and preferably from about 1 to about 5 amino acid substitutions. The variant Fc region herein will preferably retain at least about 80% homology with the native sequence Fc region and / or the Fc region of the parent polypeptide, and most preferably at least about 90% homology therewith, and more preferably at least about 95% homology therewith.

[0050] Depending on the amino acid sequence of the constant domain of the heavy chain, intact antibodies can be assigned to different "classes". There are five major classes of intact antibodies (IgA, IgD, IgE, IgG, and IgM), and several of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0051] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently cause lysis of the target cells. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in an animal model, such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0052] A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector function. Preferably, the cell expresses at least FcγRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred. Erector cells can be isolated from their natural source, e.g., blood or PBMCs as described herein.

[0053] The term "Fc receptor" or "FcR" is used to refer to a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Additionally, a preferred FcR is one that binds IgG antibodies (gamma receptors), which includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor, FcγRIIB, contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those yet to be identified, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al. J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulates immunoglobulin homeostasis.

[0054] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, for example, a CDC assay as described in Gazzano-Santoro et al., J.Immunol.Methods 202:163 (1996) may be performed.

[0055] "Single-chain Fv" or "scFv" antibody fragments are fragments of the V H and V L Preferably, the Fv polypeptide comprises a V domain, and these domains are present in a single polypeptide chain. H and V L It further comprises a polypeptide linker between the domains which enables the scFv to form the desired structure for antigen binding. For a general review of scFvs, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0056] The term "diabody" refers to a small antibody fragment that has two antigen-binding sites, which are located within the same polypeptide chain (V H ~V L ) and the variable light domain (V L ) bound to the variable heavy domain (V H (Diabodies include a diabody having a complementary domain on another chain, which is paired with the complementary domain on another chain to create two antigen-binding sites, by using a linker that is too short to allow pairing between the two domains on the same chain. Diabodies are described in detail, for example, in EP 404,097, WO 93 / 11161, and Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0057] A "naked antibody" is an antibody that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or radiolabel.

[0058] An "isolated antibody" is an antibody that has been identified and separated and / or recovered from a component of its natural environment. The contaminating components of its natural environment are materials that would interfere with the diagnostic and therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is purified (1) to greater than 95% by weight of the antibody as determined by the Lowry method, and most preferably greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue, or preferably silver stain. An isolated antibody includes an antibody in situ in a recombinant cell, since at least one component of the antibody's natural environment will not be present. Often, however, an isolated antibody will be prepared by at least one purification step.

[0059] An "affinity matured" antibody is one that has one or more modifications in one or more hypervariable regions thereof that result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody that does not have that modification(s). Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. Bio / Technology 10:779-783 (1992) describes affinity maturation by shuffling VH and VL domains. Random mutagenesis of CDR and / or framework residues has been described in Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994), Schier et al. Gene 169:147-155 (1995), Yelton et al. J. Immunol. 155:1994-2004 (1995), Jackson et al., J. Immunol. 154(7):3310-9 (1995), and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0060] An "amino acid sequence variant" antibody herein is an antibody that has an amino acid sequence that differs from that of the primary species antibody. In most cases, amino acid sequence variants will possess at least about 70% homology with the primary species antibody, preferably they are at least about 80%, more preferably at least about 90% homologous with the primary species antibody. Amino acid sequence variants possess substitutions, deletions, and / or additions at specific positions within or adjacent to the amino acid sequence of the primary species antibody. Examples of amino acid sequence variants herein include acidic variants (e.g., deamidated antibody variants), basic variants, antibodies with amino-terminal leader extensions (e.g., VHS-) on one or two of their light chains, antibodies with C-terminal lysine residues on one or two of their heavy chains, and the like, including combinations of heavy and / or light chain amino acid sequence variants. Antibody variants of particular interest herein are those antibodies that contain an amino-terminal leader extension on one or two of their light chains, and optionally further contain other amino acid sequence and / or glycosylation differences compared to the primary species antibody.

[0061] A "glycosylation variant" antibody herein is an antibody having one or more carbohydrate moieties attached thereto that differ from the one or more carbohydrate moieties attached to the main species antibody.

[0062] If the antibody has an Fc region, the oligosaccharide structure may be attached to one or both heavy chains of the antibody, for example, at residue 299 (298, EU numbering of residues).

[0063] "Amino-terminal leader extension" herein refers to one or more amino acid residues of an amino-terminal leader sequence present at the amino terminus of any one or more heavy or light chains of an antibody. An exemplary amino-terminal leader extension comprises or consists of three amino acid residues, VHS, present on one or both of the light chains of an antibody variant.

[0064] A "deamidated" antibody is one in which one or more asparagine residues have been derivatized, eg, to an aspartic acid, a succinimide, or an isoaspartic acid.

[0065] As used herein, the term "expression level" as applied to a gene refers to a determined value for the level of a gene product, e.g., the level of an RNA transcript of the gene or the level of a protein product of the gene.

[0066] The terms "downregulated," "decreased," and "reduced" are used interchangeably and mean that the expression, activity, or level of a gene, or an RNA transcript or protein product of a gene, is lower compared to one or more controls, e.g., one or more positive and / or negative controls.

[0067] The terms "upregulated," "increased," and "elevated" are used interchangeably and mean that the expression, activity, or level of a gene, or of the RNA transcript or protein product of a gene, is greater compared to one or more controls, e.g., one or more positive and / or negative controls.

[0068] "Subject" includes mammals and human subjects. Mammals include, but are not limited to, humans, rodents, sport animals, zoo animals, pet animals, and farm or livestock animals, such as dogs, cats, cows, sheep, pigs, horses, and non-human primates, such as monkeys. Preferably, the rodent is a mouse or a rat. Preferably, the mammal is a human, also referred to herein as a patient.

[0069] "Treatment" refers to the management and treatment of a subject for the purpose of combating any of the diseases or conditions targeted according to the present invention, including but not limited to multiple sclerosis, and specifically relapsing-remitting multiple sclerosis (RRMS). Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, reduction in the rate of disease progression, amelioration or remission of disease, and remission or improvement of prognosis. In some embodiments, the IL-17 antagonists of the present invention are used to delay or slow the progression of the disease.

[0070] "Multiple sclerosis" or "MS" is also known as disseminated sclerosis or disseminated encephalomyelitis, in which the insulating covering of nerve cells in the brain and spinal cord is damaged or demyelinated. MS is classified into four common forms, including relapsing-remitting (RRMS), secondary progressive (SPMS), primary progressive (PPMS), and progressive relapsing MS. RRMS is characterized by unpredictable relapses followed by periods of remission lasting from months to years without any new signs of disease activity, and is the initial course in the majority of individuals with MS. Those with RRMS may later develop SPMS, experiencing progressive neurological decline between acute attacks without periods of remission. PPMS is characterized by progression of disability from the time of onset without any remissions or improvements, or only occasional and to a minor extent. Progressive relapsing MS is the rarest form and is characterized by uniform neurological loss from disease onset and distinct aggravating attacks. See Lublin et al., Neurology 83:278-286 (2014).

[0071] "Multiple sclerosis (MS) therapeutic agent" refers to an agent other than an IL-17 antagonist used to treat MS, for example, dimethyl fumarate (TECFIDERA®), FTY-720 (fingolimod, GILENYA®), nataluzimab (TYSABRI®), corticosteroids, beta-interferons, glatiramer acetate (COPAXONE®), teriflunomide (AUBAGIO®), mitoxantrone, and anti-CD20 antibodies such as ocrelizumab, rituximab, and ofatumumab.

[0072] The term "effective amount" refers to an amount of a drug, such as an IL-17 antagonist, effective to treat multiple sclerosis in a patient.

[0073] In the context of the present invention, "at least one," "at least two," "at least three," "at least four," "at least five," etc. of the genes listed in any particular gene set means any one or any and all combinations of the listed genes.

[0074] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry that are within the skill of the art. Such techniques are described in "Molecular Cloning: A Laboratory Manual, 2006." nd edition (Sambrook et al., 1989), "Oligonucleotide Synthesis" (MJ Gait, ed., 1984), "Animal Cell Culture" (RIFreshney), ed., 1987), "Methods in Enzymology" (Academic Press, Inc.), "Handbook of Experimental Immunology", 4 thedition (D.M. Weir & C.C. Blackwell, eds.), Blackwell Science Inc., 1987); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987), "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987), and "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994).

[0075] Multiple sclerosis (MS) is a demyelinating inflammatory disorder of the central nervous system (CNS) that involves an autoimmune response to myelin antigens. Studies of experimental autoimmune encephalomyelitis (EAE), an animal model for MS, have provided evidence that T cells specific for autoantigens mediate pathology in these diseases. Until recently, type 1 helper T (Th1) cells were thought to be the primary effector T cells responsible for autoimmune inflammation. However, more recent studies have demonstrated that a class of IL-17-secreting CD4 T cells, termed Th17, are involved in the pathogenesis of MS. + The role of T cells in pathogenesis is shown. See FIG. 5. Activated myelin-specific T cells are thought to migrate into the bloodstream and into the central nervous system (CNS). Breakdown of the blood-brain barrier (BBB) ​​occurs, allowing recruitment of other inflammatory cells into the CNS. T cells migrating into the CNS encounter their cognate myelin antigens and are reactivated by local APCs. T cells proliferate and release inflammatory mediators that aid in the recruitment of other immune cells to the site of inflammation. Activation of local microglia and infiltrating cells leads to the production of proteases, glutamate, reactive oxygen species, and other cytotoxins that promote inflammation, cell death, direct neuronal injury, and demyelination. Damage to the myelin sheath surrounding the axons leads to axonopathy and neuronal damage.

[0076] The present invention is based at least in part on the discovery that IL-17, specifically IL-17AA, is found to be elevated in the cerebrospinal fluid (CSF) of a subset of patients with multiple sclerosis, particularly those with relapsing-remitting MS (RRMS).Therefore, the expression level of IL-17, specifically IL-17AA, in MS patients can serve as a biomarker to identify patients who have MS, are at risk of developing MS, and are likely to be responsive to treatment with IL-17 antagonists, such as IL-17 antibodies.These patients may also benefit from novel combination treatments of IL-17 antagonists in combination with other multiple sclerosis therapeutic agents.

[0077] Surrogate markers whose expression is coordinately regulated, either positively or negatively, with the expression of IL-17, specifically IL-17AA, are also suitable as biomarkers for MS. Surrogate markers include genes that are positive regulators of the same pathway or downstream pathways as those positively regulated by IL-17, specifically IL-17AA. Increased expression of such genes compared to controls may identify patients who have MS, are at risk of developing MS, and / or are likely to be responsive to treatment with IL-17 antagonists, such as IL-17 antibodies. Surrogate markers also include genes whose expression is inversely correlated with the expression of IL-17, specifically IL-17AA. Decreased expression of such genes compared to controls may also identify patients who have MS, are at risk of developing MS, and / or are likely to be responsive to treatment with IL-17 antagonists, such as IL-17 antibodies. Thus, the present invention explored surrogate biomarkers for IL-17, specifically IL-17AA.

[0078] Surrogate markers for IL-17AA in multiple sclerosis According to the present invention, the following genes have been identified as being associated with increased levels of IL-17AA in RRMS patients: TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL8, NFκBIZ, YKL40 (CHI3L1), and G-CSF (CSF3). These genes are also listed in Table 1, which lists selected genes analyzed herein along with their respective NCBI GenBank accession numbers. An increased expression level of the RNA transcripts of one or more genes or their protein products in a biological sample obtained from a subject compared to a control indicates that the subject may have MS, is at risk for developing MS, and / or is more likely to be responsive to treatment with an IL-17 antagonist, such as an IL-17 antibody. A decreased expression level compared to a control, or the same level compared to a control, of one or more of the disclosed genes indicates that the subject is not likely to have MS, is not at risk for developing MS, and / or is less likely to be responsive to treatment with an IL-17 antagonist.

[0079] The control may be, for example, the expression of a gene present in the same cells that is known to be upregulated in patients with multiple sclerosis (positive control). Alternatively, or in addition, the control may be the expression of the same gene as the marker gene in normal cells of the same cell type (negative control). The expression level may also be normalized to the expression level of a housekeeping gene, such as, for example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and / or β-actin, or to the expression level of all genes in the sample being tested. The control value may be a predetermined average value obtained from a relevant population. These and other controls and methods are well known in the art and will be apparent to the skilled artisan.

[0080] The disclosed genes were surprisingly found to be associated with increased levels of IL-17AA in RRMS patients.For example, TIMP1 is a well-known endogenous regulator of the matrix metalloprotease (MMP) family of extracellular proteases, but its role in myelin pathology remains unclear, as TIMP1 appears to exert both positive and negative effects on CNS myelin damage. Some reports suggest that TIMP1 plays a protective role in inhibiting MMP-mediated pathogenesis by promoting remyelination in response to CNS myelin injury (see, e.g., Ichiyama et al., J. Neuroimmunology 172:182-186, 2006; Moore et al., J. Neuroscience 31:6247-6254, 2011), while other reports indicate that TIMP1 secreted by T cells promotes neuropathology (see, e.g., Adamson et al., PLoS ONE 8:e59367.doi:10.1371 / journal.pone.0059367, 2013). LRG1 is a protein that has been shown to be involved in protein-protein interactions, signal transduction, and cell adhesion and development, and is produced by inflammation (see, e.g., Wang et al., Nature 499:306-311, 2013; Miyajima et al., PLoS ONE 8:e74453.doi:10.1371 / journal.pone.0074453, 2013). While LRG1 has been associated with Parkinson's disease with dementia (PDD) and progressive supranuclear palsy (PSP) (see, e.g., Miyajima supra), there are no known reports of an association between LRG1 and multiple sclerosis, specifically RRMS. TIFF2025063066000001.tif228170

[0081] Diagnosis and Prognosis In various aspects, the present invention provides a method for identifying a subject having MS, at risk of developing MS, and / or likely to be responsive to treatment with an IL-17 antagonist. The method comprises measuring the expression level of one or more genes selected from the group of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL8, NFκBIZ, YKL40 (CHI3L1), and G-CSF (CSF3) in a biological sample obtained from the subject. The method may further comprise comparing the expression level of the gene(s) to the expression level of a control gene. An increased expression level compared to the control indicates that the subject has MS, is at risk of developing MS, and / or is likely to be responsive to treatment with an IL-17 antagonist. Alternatively, a decreased or the same expression level compared to the control indicates that the subject does not have MS, is not at risk of developing MS, and / or is unlikely to be responsive to treatment with an IL-17 antagonist.

[0082] The biological sample may be, for example, a biopsy tissue sample or a biological fluid, including, but not limited to, spinal fluid (CSF), blood, urine, saliva, ascites fluid, or derivatives such as serum and plasma. In one embodiment, the expression level measured is the expression level of the RNA transcript of the gene. In another embodiment, the expression level measured is the expression level of the protein product of the gene. The expression levels of the RNA transcript or protein product of one, two, three, four, five, six, seven, eight, or more genes may be measured. In one embodiment of the present invention, the expression level of the RNA transcript of TIMP1 or its protein product may be measured. In a particular embodiment, the expression level of the RNA transcript of TIMP1 or its protein product, as well as the expression level of at least one or more genes selected from the group of LRG1, CXCL1, CXCL5, CXCL10, IL8, NFκBIZ, YKL40, and G-CSF may be measured. In another embodiment, the expression level of the RNA transcript of LRG1 or its protein product may be measured. In another particular embodiment, the expression level of the RNA transcript of LRG1 or its protein product, and the expression level of at least one or more genes selected from the group of TIMP1, CXCL1, CXCL5, CXCL10, IL8, NFκBIZ, YKL40, and G-CSF may be measured. In yet another embodiment, the expression levels of the RNA transcript of TIMP1 and LRG1 or their protein products are measured. In a further embodiment, the expression levels of the RNA transcript of TIMP1 and / or LRG1, and G-CSF or their protein products are measured. In a particular embodiment, the expression levels of the RNA transcript of TIMP1, LRG1, and G-CSF or their protein products are measured. In a further embodiment, when the method comprises measuring the expression level of any one of the genes selected from the group of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL8, YKL40, and G-CSF, its protein product is measured. When the method comprises measuring the expression level of NFκBIZ, its RNA transcript is measured.

[0083] In one embodiment, the expression level of one or more of the genes selected from the group TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL8, NFκBIZ, YKL40, and G-CSF may be measured from CSF obtained from an MS patient. In a particular embodiment, when the method comprises measuring the expression level of one or more genes selected from CXCL1, CXCL5, and CXCL10, the RNA transcript or protein product may be measured from plasma obtained from an MS patient. In another embodiment, when the method comprises measuring the expression level of G-CSF, the RNA transcript or protein product may be measured from serum obtained from an MS patient. In a particular embodiment, the expression level of the protein product of G-CSF is measured from serum.

[0084] In any of the methods and embodiments, the MS may be RRMS.

[0085] Methods for measuring expression levels Various methods for determining the expression levels of RNA transcripts (e.g., mRNA) or proteins include, but are not limited to, gene expression profiling, polymerase chain reaction (PCR), including quantitative real-time PCR (qRT-PCR), microarray analysis, which can be performed using commercially available equipment following the manufacturer's protocols, for example, Affymetrix GenChip technology, serial analysis of gene expression (SAGE) (Velculescu et al., Science 270:484-487 (1995), and Velculescu et al., Cell 88:243-51 (1997)), MassARRAY, gene expression analysis by Massively Parallel Signature Sequencing (MPSS) method (Brenner et al., Nature Biotechnology 18:630-634 (2000)), proteomics, immunohistochemistry (IHC), etc. In one embodiment of the present invention, mRNA is quantified. In further embodiments, such mRNA analysis is performed using the technique of polymerase chain reaction (PCR) or by microarray analysis. If PCR is utilized, a preferred form of PCR is quantitative real-time PCR (qRT-PCR).

[0086] General methods for mRNA extraction are known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). RNA isolation may be performed using purification kits, buffer sets, and proteases from commercial manufacturers such as Qiagen, following the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Other commercially available RNA isolation kits include MasterPure™ Complete DNA and RNA Purification Kit (EPICENTRE™, Madison, WI), and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA may also be isolated, for example, by cesium chloride density gradient centrifugation. After analysis of RNA concentration, an RNA repair and / or amplification step may be included as necessary, where RNA is reverse transcribed using a gene-specific promoter followed by PCR. Preferably, real-time PCR is used, which is compatible with both quantitative competitive PCR, in which an internal competitor of each target sequence is used for normalization, and quantitative comparative PCR, in which a normalization gene contained in the sample or a housekeeping gene in RT-PCR is used. For further details, see, for example, "PCR: The Polymerase Chain Reaction", Mullis et al., eds., 1994, and Held et al., Genome Research 6:986-994 (1996).

[0087] Expression levels can also be determined at the protein level, for example, using various types of immunoassays or proteomic techniques.

[0088] In an immunoassay, the target diagnostic protein marker is detected using an antibody that specifically binds to the marker. The antibody will typically be labeled with a detectable moiety. Many labels are available, which can be broadly grouped into the following categories: 1) Radioisotopes, e.g. 35 S, 14 C. 125 I, 3 H, and 131 I. The antibody may be labeled with a radioisotope, for example, using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al. (1991) Ed. Wiley-Interscience, New York, New York, Pub., and radioactivity can be measured using scintillation counting. 2) Fluorescent labels, such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, and Texas Red, can be used. Fluorescent labels can be conjugated to antibodies using techniques disclosed in, for example, Current Protocols in Immunology (supra). Fluorescent labels can be quantified using a fluorometer. 3) A variety of enzyme-substrate labels are available, and U.S. Patent No. 4,275,149 provides a review of some of these. Enzymes generally catalyze a chemical modification of a chromogenic substrate that can be measured using a variety of techniques. For example, enzymes can catalyze a color change in the substrate, which can be measured spectrophotometrically. Alternatively, enzymes can modify the fluorescence or chemiluminescence of the substrate. Techniques for quantifying the modification of fluorescence are described above. Chemiluminescent substrates become electronically excited by a chemical reaction and can then emit light, which can be measured (e.g., using a chemiluminometer) or which donate energy to a fluorescent acceptor. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al. (1981) Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic press, New York 73:147-166.

[0089] Examples of enzyme-substrate combinations include, for example: a) horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, in which hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB)); b) alkaline phosphatase (AP), which uses para-nitrophenyl phosphate as a chromogenic substrate; c) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or with the fluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

[0090] Many other enzyme-substrate combinations are available to those of skill in the art, for reviews of which see U.S. Patent Nos. 4,275,149 and 4,318,980.

[0091] The label may be indirectly conjugated to the antibody. Those skilled in the art will know various techniques to achieve this. For example, the antibody may be conjugated with biotin, and any of the three broad categories of labels mentioned above may be conjugated with avidin or streptavidin, or vice versa. Since biotin selectively binds to avidin, the label may be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label to the antibody, the antibody is conjugated with a small hapten (e.g., digoxin), and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody). In this way, indirect conjugation of the label to the antibody can be achieved.

[0092] In other forms of immunoassay techniques, the antibody need not be labeled, and its presence can be detected using a labeled antibody which binds to the antibody.

[0093] The immunoassays described herein may be in any assay format, including, for example, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

[0094] Competitive binding assays rely on the ability of a labeled standard to compete with the measurement sample analyte for binding to a limited amount of antibody. The amount of antigen in the measurement sample is inversely proportional to the amount of standard bound to the antibody. To facilitate the determination of the amount of bound standard, the antibody is generally insolubilized before or after the competition so that the standard and analyte bound to the antibody can be easily separated from the standard and analyte that remain unbound.

[0095] Sandwich assay involves the use of two antibodies, each capable of binding to a different immunogenic portion or epitope of the protein to be detected. In a sandwich assay, the sample analyte to be measured is bound by a first antibody immobilized on a solid support, and then a second antibody binds to the analyte, thereby forming an insoluble ternary complex. See, for example, U.S. Pat. No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assay) or may be measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is the ELISA assay, in which the detectable moiety is an enzyme.

[0096] Protein levels can also be detected using proteomics techniques. The term "proteome" is defined as the completeness of proteins in a sample (e.g., tissue, period, or cell culture) at a particular time. Proteomics includes, among other things, the study of the global variation of protein expression in a sample (also referred to as "expression proteomics"). Proteomics typically involves the following steps: (1) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE), (2) identification of the individual proteins recovered from the gel, for example, by mass spectrometry or N-terminal sequencing, and (3) analysis of the data using bioinformatics. Proteomics methods are a valuable alternative or complement to other methods of gene expression profiling, and can be used alone or in combination with other methods to detect the products of the tumor resistance markers of the present invention.

[0097] The measurement of biomarker expression levels may be performed using a software program executed by a suitable processor. Suitable software and processors are known in the art and commercially available. The program may be embodied in software stored on a tangible medium, such as a CD-ROM, floppy disk, hard drive, DVD, or memory associated with the processor, although those skilled in the art will readily understand that the entire program or parts thereof may alternatively be executed by devices other than a processor and / or embodied in firmware and / or dedicated hardware, in a known manner.

[0098] Following measurement of the expression levels of one or more of the RNA transcripts of the genes identified herein or their protein products, assay results, findings, diagnoses, predictions, prognoses and / or treatment recommendations are typically recorded and communicated, for example, to specialists, physicians, and / or patients. In certain embodiments, a computer will be used to communicate such information to interested parties, such as the patient and / or the attending physician. In some embodiments, the assays will be performed or the assay results will be analyzed in a country or jurisdiction different from the country or jurisdiction to which the results or diagnoses are communicated.

[0099] In a preferred embodiment, a diagnosis, prediction, prognosis and / or treatment recommendation based on the expression level in a test subject of one or more of the biomarkers disclosed herein is communicated to the subject as soon as possible after the assay is completed and the diagnosis and / or prediction is generated. The results and / or related information may be communicated to the subject by the subject's treating physician. Alternatively, the results may be communicated directly to the test subject by any means of communication, including written communication, electronic form of communication such as email, or telephone. The communication may be facilitated by the use of a computer, as in the case of email communication. In certain embodiments, a communication including the results of a diagnostic test, and / or conclusions drawn from the test and / or treatment recommendations based on the test, may be automatically generated and delivered to the subject using a combination of computer hardware and software, as would be known to one skilled in the art of telecommunications. An example of a communication system in the healthcare field is described in U.S. Pat. No. 6,283,761. However, the present invention is not limited to methods utilizing this particular communication system. In certain embodiments of the methods of the invention, all or some of the method steps, including assaying the sample, diagnosing the disease, and communicating the assay results or diagnosis, may be performed in a different (e.g., foreign) jurisdiction.

[0100] To facilitate diagnosis and / or treatment recommendations, the reference and / or subject biomarker profile or expression levels of one or more of the biomarkers presented herein of the present invention may be displayed on a display device, contained electronically, or in a machine-readable medium, such as, but not limited to, an analog tape, such as one readable by a VCR, CD-ROM, DVD-ROM, USB flash media, etc. Such machine-readable media may also include additional test results, such as, but not limited to, clinical parameters and risk factors in conventional laboratories. Alternatively, or in addition, the machine-readable medium may also include subject information, such as medical history and any relevant family medical history.

[0101] Treatment and methods for monitoring treatment Subjects identified according to the present invention as expressing biomarkers that correlate with IL-17, specifically IL-17AA, are likely to be responsive to treatment with an IL-17 antagonist. Thus, the present invention provides a method of treating a subject with MS or at risk of developing MS by measuring the expression level of an RNA transcript or its protein product of one or more genes selected from the group of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL8, NFκBIZ, YKL40 (CHI3L1), and G-CSF (CSF3) in a biological sample obtained from the subject, as described above. The method may further comprise comparing the expression level of the gene(s) with the expression level of a control gene. Subjects having increased expression levels of one or more genes compared to the subject are treated with an effective amount of an IL-17 antagonist.

[0102] In addition, the present invention provides a method for monitoring a subject having MS treated with an IL-17 antagonist, comprising measuring the expression level of an RNA transcript or its protein product of one or more genes selected from the group of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL8, NFκBIZ, YKL40 (CHI3L1), and G-CSF (CSF3) in a biological sample obtained from the subject, as described above, and determining whether the expression level of the one or more genes is increased compared to a control. In one embodiment of the present invention, an increased expression level compared to a control indicates that treatment with an IL-17 antagonist in the subject should be continued. One embodiment of the present invention provides for further administration of an IL-17 antagonist to the subject.

[0103] In these methods of the invention, the biological sample used and / or obtained may be a biological fluid, including but not limited to, for example, cerebrospinal fluid (CSF), blood, urine, saliva, ascites fluid, or derivatives such as serum and plasma. In one aspect of the invention, the expression level measured is the expression level of the RNA transcript of the gene. In another aspect, the expression level measured is the expression level of the protein product of the gene. The expression levels of the RNA transcript or protein product of one, two, three, four, five, six, seven, eight, or more genes may be measured. In one embodiment of the invention, the expression level of the RNA transcript of TIMP1 or its protein product may be measured. In a particular embodiment, the expression level of the RNA transcript of TIMP1 or its protein product, as well as the expression level of at least one or more genes selected from the group of LRG1, CXCL1, CXCL5, CXCL10, IL8, NFκBIZ, YKL40, and G-CSF may be measured. In another embodiment, the expression level of the RNA transcript of LRG1 or its protein product may be measured. In another particular embodiment, the expression level of the RNA transcript of LRG1 or its protein product, and the expression level of at least one or more genes selected from the group of TIMP1, CXCL1, CXCL5, CXCL10, IL8, NFκBIZ, YKL40, and G-CSF may be measured. In yet another embodiment, the expression levels of the RNA transcript of TIMP1 and LRG1 or their protein products are measured. In a further embodiment, the expression levels of the RNA transcript of TIMP1 and / or LRG1, and G-CSF or their protein products are measured. In a particular embodiment, the expression levels of the RNA transcript of TIMP1, LRG1, and G-CSF or their protein products are measured. In a further embodiment, when the method comprises measuring the expression level of any one of the genes selected from the group of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL8, YKL40, and G-CSF, its protein product is measured. When the method comprises measuring the expression level of NFκBIZ, its RNA transcript is measured.

[0104] In one embodiment, the expression level of one or more of the genes selected from the group TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL8, NFκBIZ, YKL40, and G-CSF may be measured from CSF obtained from an MS patient. In a particular embodiment, when the method comprises measuring the expression level of one or more genes selected from CXCL1, CXCL5, and CXCL10, the RNA transcript or protein product may be measured from plasma obtained from an MS patient. In another embodiment, when the method comprises measuring the expression level of G-CSF, the RNA transcript or protein product may be measured from serum obtained from an MS patient. In a particular embodiment, the expression level of the protein product of G-CSF is measured from serum.

[0105] In any of the above methods and embodiments, the MS may be RRMS.

[0106] The effect of administration of IL-17 antagonists in multiple sclerosis can be measured by a variety of assays or tests known in the art. Many measures can be used to assess the effectiveness of a product in treating MS, including clinical measures, those based on MRI scans, and those based on quality of life. The Expanded Disability Status Scale (EDSS) is a widely used measure to track the course of disability in MS. It classifies the most common MS-related neurological disorders into disability levels ranging from 0 to 10, with each successive step indicating a worsening of the disease. In the lower ranges of the EDSS scale, disease progression is defined primarily by increased levels of disability in specific functional systems measured on neurological examination. A score of 1.0 to 3.5 indicates mild to moderate impairment in a functional system. Higher scores, ranging from 4.0 and above, indicate more severe impairment affecting walking, including the need for assistive devices such as a cane (EDSS of 6.0), a walker (EDSS of 6.5), or a wheelchair (EDSS of 7.0). A score above 7.0 classifies the patient as bedridden.

[0107] The MS Functional Scale (MSFC) (Whitaker et al., Multiple Sclerosis 1:37-47 (1995)) is also used to assess efficacy. Unlike traditional MS clinical outcome measures derived from standard neurological examinations, the MSFC is based on quantitative tests of lower limb function / gait (25-foot walk test), upper limb function (nine-hole peg test), and cognitive function (PASAT 3), which extend the measurements of the EDSS and assess efficacy on clinical aspects not adequately captured by this scale.

[0108] MRI is another tool to assess efficacy in the treatment of MS and can be used alone or to support clinical data to evaluate the effect of treatment on relapse and disability endpoints. MRI is a highly sensitive instrument for monitoring disease activity, detecting approximately 5-10 times more disease activity than is clinically evident in both relapsing-remitting and secondary progressive MS patients) (Isaac et al.,.Neurology 38:1511-1515(1988); Willoughby et al., Ann.Neurol.25:43-44(1989); Khoury et al.,Neurology 44:2120-2124(1994); Thompson et al., Ann.Neurol.9:53-62(1991); Thompson et al.,Neurology 42:60-63(1992)). T2-weighted sequences in MS patients detect new areas of acute demyelination, as well as more chronic areas of demyelination and gliosis. For this reason, T2-weighted MRI is a good technique for monitoring the accumulation of lesions over time, either as the number of active lesions or as a change in the total volume of such lesions.

[0109] Injection of gadolinium diethylenetriaminepentaacetic acid (Gd-DPTA) during the acquisition of T1-weighted sequences allows visualization of blood-brain barrier breakdown secondary to the inflammatory properties of acute MS lesions. Recent evidence indicates that gadolinium (Gd) enhancement is a useful marker of disease activity that correlates with clinical relapses (Molyneux et al., Ann. Neurol. 43:332-339 (1998); Kappos et al., Lancet 353:964-969 (1999); McFarland et al., Multiple Sclerosis 8:40-51 (2002)).

[0110] De novo hypointense lesions on T1-weighted sequences in MS patients correspond either to inflammatory Gd-enhancing lesions (including edema, demyelination, axonal loss, or a combination of these pathologies) (Bruck et al., Ann. Neurol. 42:783-793 (1997)) or to chronic lesions with substantial axonal loss. Approximately half of the acute T1 hypointense lesions on MRI evolve into chronic "T1 black holes," which correlate with disability progression (Simon et al., Neurology 55:185-192 (2000)).

[0111] IL-17 antagonists An IL-17 antagonist includes any molecule that inhibits, reduces, or interferes with the function or binding of IL-17 to a receptor, such as IL-17AA, IL-17FF, and / or IL-17A / F, blocks and / or neutralizes the associated activity of IL-17. An IL-17 antagonist may prevent the interaction between IL-17 (including IL-17AA, IL-17FF, and / or IL-17A / F) and one or more of its receptors. Such antagonists achieve this effect, for example, by binding to IL-17 or a receptor for IL-17 with sufficient affinity and specificity to interfere with IL-17 activity. The term "IL-17 antagonist" is used to refer to any and all antagonists of IL-17AA, IL-17FF, IL-17A / F, IL-17RA, and IL-17RC. In certain embodiments, the inhibition, reduction, interference, blocking, or abolishment may be partial or complete inhibition, reduction, interference, blocking, or abolishment of IL-17AA, IL-17FF, and / or IL-17AF activity. In certain other embodiments, the inhibition, reduction, interference, blocking, or abolishment may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% inhibition, reduction, interference, blocking, or abolishment of IL-17AA, IL-17FF, and / or IL-17AF activity.

[0112] This group of antagonists includes, for example, antibodies against IL-17 or a portion thereof, antibodies reactive with IL-17, or IL-17 receptors or portions thereof, including antibodies that bind IL-17AA, IL-17FF, IL-17A / F, IL-17RA, and / or IL-17RC, as well as soluble molecules of the IL-17 receptors, e.g., soluble IL-17RA and soluble IL-17RC. The term antagonist also includes any agent that interferes with the production of IL-17AA, IL-17FF, and / or IL-17A / F, or antagonizes an IL-17 receptor, such as IL-17RA and / or IL-17RC. Such antagonists may be in chimeric hybrid form, useful for combining the function of the agent with a carrier protein to increase serum half-life of the therapeutic agent or to confer cross-species tolerance. Thus, examples of such antagonists include bio-organic molecules (e.g., peptidomimetics), antibodies, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, drugs and their metabolites, transcriptional and translational control sequences, etc. In one embodiment, the antagonist is an IL-17 antibody capable of binding to IL-17AA, IL-17FF, and / or IL-17A / F, or an IL-17 receptor antibody capable of binding to IL-17RA and / or IL-17RC.

[0113] Exemplary IL-17 antibodies include secukinumab (e.g., U.S. Patent No. 7,807,155), ixekizumab (e.g., U.S. Patent No. 7,838,638), bimekizumab (e.g., U.S. Patent No. 8,580,265), ALX-0761 (e.g., U.S. Publication No. 20140314743), CNTO 6785 (e.g., U.S. Patent No. 8,519,107), and afasevikumab (anti-IL-17 CDR sequences shown in U.S. Patent No. 8,715,669). Exemplary IL-17 receptor antibodies include brodalumab (e.g., U.S. Patent No. 7,833,527).

[0114] Antibody production Exemplary techniques for producing antibodies used in accordance with the present invention are provided below. The antigens used to produce antibodies may be full length or portions thereof containing the desired epitope. Alternatively, antibodies may be produced using cells that express the desired antigen or epitope on their cell surface. Other forms of antigens useful for producing antibodies will be apparent to those skilled in the art.

[0115] Polyclonal antibodies Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Bifunctional or derivatizing agents, such as maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl 2 , or R 1 N=C=NR, where R and R 1 are different alkyl groups) and conjugating the relevant antigen to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.

[0116] For example, animals are immunized against the antigen, immunogenic conjugate, or derivative by combining 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of complete Freund's adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted by subcutaneously injecting 1 / 5 to 1 / 10 of the original amount of peptide or conjugate in complete Freund's adjuvant at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. The animals are boosted until the titer reaches a plateau. Preferably, the animals are boosted with a conjugate of the same antigen, but conjugated to a different protein and / or with a different cross-linking reagent. Conjugates can also be made as protein fusions in recombinant cell culture. Agglutinating agents such as alum are also suitably used to improve the immune response.

[0117] Monoclonal antibodies Various methods for producing the monoclonal antibody herein are available in the art.For example, monoclonal antibody can be produced by recombinant DNA methods (U.S. Patent No. 4,816,567) using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975).

[0118] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized ex vivo. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0119] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas usually contains hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0120] Preferred myeloma cells are those that fuse efficiently, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to media such as HAT medium. Among these, preferred myeloma cell lines are mouse myeloma lines derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001 (1984), and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)).

[0121] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0122] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0123] After hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in animals.

[0124] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0125] DNA encoding a monoclonal antibody is easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of a mouse antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into host cells, such as E. coli cells, Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of the monoclonal antibody in the recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993), and Pluckthun, Immunol. Revs., 130:151-188 (1992).

[0126] In further embodiments, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications have described the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), as well as recombinant infection in vivo as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

[0127] The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567, and Morrison et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0128] Typically, such a non-immunoglobulin polypeptide is substituted for the constant domains of an antibody or for the variable domains of one antigen-binding site of an antibody to create a chimeric bivalent antibody comprising one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0129] Humanized antibodies Methods for humanizing non-human antibodies have been described in the art. Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, typically taken from an "import" variable domain. Humanization can be essentially performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). Such "humanized" antibodies are thus chimeric antibodies (U.S. Pat. No. 4,816,567), in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0130] The selection of both light and heavy human variable domains used to generate humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).

[0131] It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To this end, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are known to those skilled in the art. Computer programs are available which depict and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues which influence the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected and combined from the recipient and import sequences to achieve the desired antibody characteristic, such as increased affinity for the target antigen(s). In general, the variable region residues are directly and most substantially involved in influencing antigen binding.

[0132] Various forms of humanized or affinity matured antibodies are contemplated. For example, the humanized or affinity matured antibody may be an antibody fragment, such as a Fab, which is optionally conjugated with one or more cytotoxins(s) to generate an immunoconjugate. Alternatively, the humanized or affinity matured antibody may be an intact antibody, such as an intact IgG1 antibody.

[0133] Human antibodies As an alternative to humanization, human antibodies may be produced. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, expression of the antibody heavy chain joining region (J) in chimeric and germ-line mutant mice has been demonstrated. HIt has been described that homozygous deletion of the IL-1 gene results in complete inhibition of endogenous antibody production. Introduction of the human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Patent Nos. 5,591,669, 5,589,369, and 5,545,807. Alternatively, human antibodies and antibody fragments can be produced in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors using phage display technology (McCafferty et al., Nature 348:552-553 (1990)). According to this technique, antibody V domain genes are cloned in frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of genes encoding antibodies exhibiting these properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of forms, for reviews of which see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice.Repertoires of V genes from unimmunized human donors can be constructed and antibodies to different array intersections (including self-antigens) can be isolated substantially according to the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0134] As described above, human antibodies can also be generated by in vivo activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

[0135] antigen binding fragment Various techniques have been developed for the production of antibody fragments that contain one or more antigen-binding regions. Traditionally, these fragments were obtained via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992), and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, antigen-binding fragments can be isolated from the antibody phage libraries mentioned above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to produce F(ab') 2 (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab') 2The fragments can be directly isolated from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO93 / 16185, U.S. Pat. No. 5,571,894, and U.S. Pat. No. 5,587,458. The antibody fragment may also be a linear antibody, e.g., as described in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

[0136] multispecific antibodies Multispecific antibodies have binding specificities for at least two different epitopes, which are usually derived from different antigens. Such molecules will usually only bind two different epitopes (i.e., bispecific antibodies, BsAbs), but as used herein, antibodies with additional specificities, such as trispecific antibodies, are encompassed in this expression. Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually performed by an affinity chromatography step, is somewhat cumbersome and results in low product yields. Similar procedures are disclosed in WO93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991). According to a different approach, an antibody variable domain (antibody-antigen combining site) with the desired binding specificity is fused to an immunoglobulin constant domain sequence. The fusion is preferably with an immunoglobulin heavy chain constant domain, including at least a part of the hinge, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CH1), including the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construct provide optimal yields. However, when expression of at least two polypeptide chains in equal ratios results in higher yields, or when the ratio is not particularly significant, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector.

[0137] In a preferred embodiment of this approach, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It has been discovered that this asymmetric structure facilitates separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides an easy method of separation. This approach is disclosed in WO94 / 04690. For further details of the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0138] According to another approach described in WO96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. A preferred interface comprises at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). A compensatory "cavity" of identical or similar size to the larger side chain(s) is created on the interface of a second antibody molecule by replacing the large amino acid side chain with a smaller side chain (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers compared to other unwanted end products such as homodimers.

[0139] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Such antibodies have been proposed, for example, to target immune cells to unwanted cells (U.S. Pat. No. 4,676,980) and for treating HIV infection (WO 91 / 00360, WO 92 / 200373). Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents, along with a number of cross-linking techniques, are known in the art and are disclosed in U.S. Pat. No. 4,676,980. In certain embodiments, the IL-17 antibody is a bispecific or multispecific antibody that binds IL-17AA, IL-17FF, and / or IL-17AF. In certain other embodiments, the IL-17 antibody is a bispecific or multispecific antibody in which one antigen-binding site binds IL-17AA and IL-17AF and the other antigen-binding site binds IL-17FF. Meanwhile, in yet another embodiment, one antigen binding site binds IL-17AA and the other antigen binding site binds IL-17FF and IL-17AF. In certain other embodiments, one binding site binds IL-17AF and IL-17FF and the other antigen binding site binds IL-17AA, IL-17FF, and IL-17AF. In certain other embodiments, one antigen binding site of the bispecific or multispecific antibody binds IL-17AA, IL-17AF, and / or IL-17FF and the other antigen binding site binds a second antigen. In some embodiments, the second antigen is an inflammatory mediator. In some embodiments, the second antigen is TNFα or IL-1.

[0140] Techniques for producing bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) report that intact antibodies are proteolytically cleaved to produce F(ab') fragments. 2A procedure is described to generate Fab' fragments that are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0141] Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) described a fully humanized bispecific antibody F(ab') 2 The production of the molecules is described. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.

[0142] Various techniques have also been described for producing and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form the antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described in Hollinger et al., Proc. Nat. Acad. Sci. USA, 90:6444-6448 (1993) provides an alternative mechanism for making bispecific antibody fragments. These fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are paired with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol, 152:5368 (1994).

[0143] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147:60 (1991).

[0144] Other Amino Acid Sequence Modifications Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletion of, and / or insertion of, and / or substitution of, residues from the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties. Amino acid changes can also alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites.

[0145] One useful method for identifying particular antibody residues or regions that are preferred positions for mutagenesis is called "alanine scanning mutagenesis" described in Cunningham and Wells Science, 244:1081-1085 (1989). Here, target residues or groups are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Those amino acid positions that show functional sensitivity to the substitution are then improved by introducing further or other variants at or for the substitution positions. Thus, while the sites for introducing amino acid sequence diversity are predetermined, the nature of the mutations themselves need not be predetermined. For example, to analyze the performance of a variant at a given site, ala scanning or random mutagenesis is performed at the target codon or region, and the expressed antibody variants are screened for the desired activity.

[0146] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or an antibody fused to a cytotoxic polypeptide. Other insertional variants of antibody molecules include the fusion of the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0147] Another type of variant is amino acid substitution variant. These variants have at least one amino acid residue replaced by a different residue in the antibody molecule. The most targeted positions for mutagenesis include the antibody variable region, but FR modification is also contemplated. Conservative substitutions are shown under the heading of "preferred substitutions" in Table 2. If such substitutions cause changes in biological activity, they are named "exemplary substitutions" in the following table, or further substantial changes as described below for amino acid classes may be introduced, and the products screened. TIFF2025063066000002.tif150170

[0148] Substantial modifications in the biological properties of antibodies are achieved by selecting substitutions which differ significantly in their effect on (a) the structure of the polypeptide backbone in the region of the substitution, e.g., sheet or helix structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Amino acids can be grouped by similarities in the properties of their side chains (in A Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): Non-polar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M) Uncharged polar: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q) Acidic: Asp(D), Glu(E) Basic: Lys(K), Arg(R), His(H) Alternatively, the residues present in the transfer can be grouped based on common side chain properties: Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; Acidic: Asp, Glu; Basic: His, Lys, Arg; Residues affecting chain orientation: Gly, Pro; Aromatic: Trp, Tyr, Phe.

[0149] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0150] Any cysteine ​​residue not involved in maintaining the proper conformation of the antibody may also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) may be added to the antibody to improve its stability, particularly where the antibody is an Fv fragment or other such antibody.

[0151] A particularly preferred type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they were generated. A convenient means of generating such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. Antibody variants thus generated are displayed from filamentous phage particles in a monovalent fashion as fusions to the gene III product of M13 housed within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning methods can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be advantageous to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants is subjected to screening as described herein, and antibodies with superior properties in one or more relevant assays are selected for further development.

[0152] Another type of amino acid variant of an antibody alters the original glycosylation pattern of the antibody, by deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites that are not present in the antibody.

[0153] Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0154] Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). The alteration may also be accomplished by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0155] If the antibody comprises an Fc region, the carbohydrate attached thereto may be modified. For example, antibodies having a mature carbohydrate structure lacking fructose attached to the Fc region of the antibody are described in US Patent Application Publication No. 2003 / 0157108A1 (Presta, L.). See also US Patent Application Publication No. 2004 / 0093621A1 (Kyowa Hakko Kogyo Co., Ltd.). Antibodies having bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the Fc region of the antibody are referenced in WO03 / 011878, Jean-Mairet et al. and U.S. Patent No. 6,602,684, Umana et al. Antibodies having at least one galactose residue in the oligosaccharide attached to the Fc region of the antibody are reported in WO97 / 30087, Patel et al. See also WO98 / 58964 (Raju, S.) and WO99 / 22764 (Raju, S.) concerning antibodies with engineered carbohydrate attached to the Fc region thereof.

[0156] It may be desirable to modify the antibodies of the present invention with respect to effector function, for example to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively, or in addition, cysteine ​​residue(s) may be introduced in the Fc region to allow interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992), and Shopes, BJ Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

[0157] WO00 / 42072 (Presta, L.) describes antibodies with improved ADCC function in the presence of human effector cells, when the antibodies have amino acid substitutions in their Fc region. Preferably, the antibodies with improved ADCC comprise substitutions at positions 298, 333, and / or 334 (Eu numbering of residues) of the Fc region. Preferably, the modified Fc region is a human IgG1 Fc region comprising or consisting of substitutions at one, two, or three of these positions. Such substitutions are optionally combined with substitution(s) that increase C1q binding and / or CDC.

[0158] Antibodies with altered C1q binding and / or complement dependent cytotoxicity (CDC) are described in WO 99 / 51642, U.S. Patent No. 6,194,551B1, U.S. Patent No. 6,242,195B1, U.S. Patent No. 6,528,624B1, and U.S. Patent No. 6,538,124 (Idusogie et al.). The antibodies comprise amino acid substitutions at one or more of amino acid positions 270, 322, 326, 327, 329, 313, 333, and / or 334 (Eu numbering of residues) of their Fc region.

[0159] To increase the serum half-life of an antibody, a salvage receptor binding epitope may be incorporated into the antibody (particularly an antibody fragment), for example as described in U.S. Pat. No. 5,739,277. As used herein, a "salvage receptor binding epitope" refers to an epitope that is present in an IgG molecule (e.g., an IgG 1 , IgG 2 , IgG 3 , or IgG 4 ) epitope in the Fc region of

[0160] Antibodies with improved binding to neonatal Fc receptor (FcRn) and increased half-life are described in WO00 / 42072 (Presta, L.) and US2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region therein with one or more substitutions that improve binding of the Fc region to FcRn. For example, the Fc region may have a substitution at one or more of positions 238, 250, 256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, 428, or 434 (Eu numbering of residues). Preferred Fc region-containing antibody variants with improved FcRn binding comprise amino acid substitutions at one, two, or three of positions 307, 380, and 434 (Eu numbering of residues) of the Fc region.

[0161] Engineered antibodies with three or more (preferably four) functional antigen binding sites are also contemplated (US Application No. US2002 / 0004587A1, Miller et al.).

[0162] Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art, including, but not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated mutagenesis (or site-directed), PCR mutagenesis, and cassette mutagenesis of pre-prepared mutated or non-mutated versions of the antibody.

[0163] Administration and Formulation The IL-17 antagonist may be administered by any suitable route, including, but not limited to, parenteral routes of administration, such as intravenous (IV), intramuscular (IM), subcutaneous (SC), and intraperitoneal (IP), as well as transdermal, buccal, sublingual, rectal, intranasal, and inhalation routes. IV, IM, SC, and IP administration may be by bolus or infusion, or in the case of SC, by slow release implantation devices, such as, but not limited to, pumps, slow release formulations, and medical devices. In one embodiment of the invention, administration is systemic.

[0164] In certain embodiments, the method of administration of the IL-17 antagonist is by subcutaneous infusion, particularly using a metered dose infusion device such as a pump. Such pumps can be reusable, disposable, and implantable or external. Medical infusion pumps useful for this purpose include, for example, the pumps disclosed in U.S. Patent Nos. 5,637,095, 5,569,186, and 5,527,307. The composition can be administered intermittently or intermittently from such a device.

[0165] Therapeutic formulations of IL-17 antagonists suitable for storage include lyophilized formulations or aqueous solutions of the antagonist having the desired degree of purity in admixture with pharma- ceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers, such as phosphate, citrate, histidine, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride, benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as Examples of suitable surfactants include serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEENT™, PLURONICS™, or polyethylene glycol (PEG). Examples of lyophilized anti-IL-17 antibody formulations are described in WO 97 / 04801. These compositions contain an antagonist to IL-17, preferably in soluble form, containing about 0.1-90% by weight, more typically about 10-30% active antagonist.

[0166] The active ingredient may also be incorporated into microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

[0167] IL-17 antagonists, such as anti-IL-17 antibodies disclosed herein, may also be formulated as immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985), Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980), U.S. Patent Nos. 4,485,045 and 4,544,545, and WO 97 / 38731 published October 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

[0168] Particularly useful liposomes can be generated by the reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to liposomes via a disulfide interconversion reaction as described in Martin et al., J.Biol.Chem., 257:286-288 (1982).

[0169] Sustained release preparations may be prepared.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which matrices are in the form of shaped articles, such as films, or microcapsules.Examples of sustained release matrices include polyesters, hydrogels (such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and L-glutamic acid y-ethyl, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres made of lactic acid-glycolic acid copolymers and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0170] Any specific antagonist can be added to the carrier protein to increase the serum half-life of the therapeutic antagonist. For example, soluble immunoglobulin chimeras, such as those described herein, can be obtained for each specific IL-17 antagonist or its antagonist portion, as described in U.S. Pat. No. 5,116,964. The immunoglobulin chimeras are easily purified via IgG-binding protein A-Sepharose chromatography. The chimeras have the ability to form immunoglobulin-like dimers, which have higher avidity and serum half-life at the same time.

[0171] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0172] The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other, and such active compounds can be administered separately to the mammal being treated.

[0173] For example, it may be desirable to further provide a multiple sclerosis therapeutic that is not an IL-17 antagonist. Multiple sclerosis (MS) therapeutics include, but are not limited to, dimethyl fumarate (TECFIDERA®), FTY-720 (fingolimod, GILENYA®), nataluzimab (TYSABRI®), corticosteroids, beta-interferons, glatiramer acetate (COPAXONE®), teriflunomide (AUBAGIO®), mitoxantrone, and anti-CD20 antibodies such as ocrelizumab, rituximab, and ofatumumab. In certain embodiments, the MS therapeutic is dimethyl fumarate. In another embodiment, the MS therapeutic is FTY-720.

[0174] The multiple sclerosis therapeutic agent may be administered in combination with an IL-17 antagonist or separately. Such multiple sclerosis therapeutic agents are suitably present or administered in combination in an amount effective for the intended purpose, typically less than that used when administered alone without an IL-17 antagonist. When these are formulated together, they may be formulated in an amount determined by, for example, the type of indication, the subject, the subject's age and weight, the current clinical condition, the administration time, the dosage form, the administration method, etc. For example, the combination drug is used in a ratio of about 0.0001 to 10,000 parts by weight to 1 part by weight of the IL-17 antagonist.

[0175] The dosage of the antagonist administered to a subject with multiple sclerosis will be determined by a physician taking into consideration the relevant circumstances, including the subject's condition, the type of antagonist, the type of indication, and the selected route of administration. The dosage ranges presented herein are not intended to limit the scope of the invention in any way. The "effective amount" for multiple sclerosis for purposes of this specification is determined by the factors described above, but is generally about 0.01-100 mg / kg body weight / day. In one embodiment, the dose is about 0.1-50 mg / kg / day, more specifically about 0.1-25 mg / kg / day. Furthermore, when the IL-17 antagonist is administered once a day, the intravenous or intramuscular dose in humans may be about 0.3-10 mg / kg body weight, more specifically about 0.5-5 mg / kg per day. For subcutaneous administration, the dose may be greater than the therapeutic equivalent of the intravenous or intramuscular dose. In one embodiment, the once daily subcutaneous dose for a human is about 0.3 to 20 mg / kg, more specifically about 0.5 to 5 mg / kg.

[0176] The present invention contemplates a variety of administration schedules. The present invention encompasses continuous administration schedules, in which the IL-17 antagonist is administered regularly (once a day, once a week, once a month, depending on the dose and dosage form) without substantial interruption. In one embodiment, continuous administration schedules include continuous infusion once a day, in which the IL-17 antagonist is injected every day, and continuous bolus administration schedules, in which the IL-17 antagonist is administered at least once a day by bolus injection or by inhalation or intranasal route. The present invention also encompasses discontinuous administration schedules. The exact parameters of discontinuous administration schedules will vary depending on the formulation, delivery method, and clinical needs of the subject being treated. For example, if the IL-17 antagonist is administered by infusion, the administration schedule may include a first phase of administration followed by a second phase that is longer, the same, or shorter than the first phase, in which the IL-17 antagonist is not administered.

[0177] When administration is by bolus injection, particularly a bolus injection of a slow release formulation, the administration schedule may also be continuous, i.e., the IL-17 antagonist is administered daily, or may be discontinuous, having a first and second phase as described above.

[0178] Continuous and discontinuous dosing schedules in any manner also include dosing schedules in which dosing is adjusted over the first phase, for example, such that the dose is low at the beginning of the first phase and increases towards the end of the first phase, or the dose is high initially and decreases during the first phase, or the dose is low initially, increases to a peak level and then is reduced towards the end of the first phase, and any combination thereof.

[0179] kit Materials for use in the methods of the present invention are suitable for preparing kits. The kits of the present invention may include one or more gene-specific or gene-selective probes and / or primers for quantifying the expression levels of RNA transcripts of genes disclosed herein. Such kits may optionally contain one or more reagents for the extraction of RNA from biological samples. The kits may include one or more containers each having one or more of the various materials or reagents utilized in the methods, including, for example, chromatographic columns, prefabricated microarrays, buffers, suitable nucleotide triphospages, reverse transcriptases, DNA polymerases, RNA polymerases, and one or more probes and primers for quantifying the expression levels of RNA transcripts disclosed herein.

[0180] Alternatively, or in addition, the kits of the invention may include one or more reagents for quantifying the expression levels of the protein products of the genes disclosed herein, including antibodies specific for the protein products, which may optionally be labeled.

[0181] The kits of the invention may additionally or separately comprise one or more containers of one or more IL-17 antagonists, such as IL-17 antibodies. The kits may further comprise a set of instructions, typically written instructions, regarding the use and dosage of the IL-17 antagonist(s) for the treatment of multiple sclerosis. The instructions included in the kit will typically include information regarding dosage, administration schedule, and route of administration for the treatment of multiple sclerosis. The IL-17 antagonist(s) may be provided in unit doses, bulk packages (e.g., multi-dose packages), or subunit doses.

[0182] Further details of the invention are described in the following non-limiting examples.

[0183] Example 1 CSF IL17 levels and other inflammatory mediators are elevated in RRMS patients IL-17AA, IL-17FF, and various other proteins were measured in cerebrospinal fluid (CSF) and serum from 50 RRMS patients receiving standard of care therapy and 20 healthy donors (HD) using immunoassays based on the Erenna® immunoassay platform (Singulex, Inc.) in the case of IL-17A and IL-17F, or standard sandwich immunoassays. The IL-17A immunoassay clearly measured the IL-17AA homodimer cytokine, which has approximately 30% cross-reactivity with the IL-17AF heterodimer, while the IL-17F assay measured only the IL-17FF homodimer, which has no detectable cross-reactivity with IL-17AF or AA. All immunoassays used monoclonal capture and detection antibodies.

[0184] As shown in FIG. 6, multiple inflammatory mediators were elevated in several RRMS CSF samples, including eotaxin, CXCL10, TARC, and MCP4 (p<0.03 vs. healthy donors for all analytes).

[0185] IL-17AA and IL-17FF levels were also analyzed in CSF samples of RRMS patients. The results in Figure 7 show that IL-17AA levels were higher in the CSF of RRMS patients than in control subjects. In particular, IL-17AA was detectable in 76% of RRMS patients compared to 50% of control subjects, and the median CSF IL-17AA level was 0.4 pg / mL in RRMS patients compared to 0.07 pg / mL in control subjects (dynamic range approximately 10 fold). See the left panel of Figure 7A (*p<0.0001, Wilcoxon test). IL-17FF levels were detectable in the CSF in 12% of RRMS patients compared to 0% of control subjects. See the right panel of Figure 7A. In contrast, no elevation of serum IL-17AA and IL-17FF levels was observed in RRMS patients. See Figure 7B.

[0186] However, the levels of IL-17AA and IL-17FF in CSF were still considered to be insufficient to be useful biomarkers for in vivo diagnosis. Therefore, surrogate biomarkers were sought in the subset of patients with RRMS who presented with high IL-17AA levels.

[0187] Example 2 Upregulation of IL-17-responsive genes in human brain endothelial cells and microglia in vivo hCMEC / D3 immortalized human brain endothelial cell line and SV40 transformed human microglial cell line were grown using standard cell culture protocols and then stimulated with TNFα, IL-6 plus sIL-6R, IL-17AA, or combinations of these cytokines for 6 hours. RNA was then isolated from the cells and microarray analysis was performed to compare gene expression induced by IL-17 with or without additional cytokines.

[0188] Human brain endothelial cells (see FIG. 8A) and microglia (see FIG. 8B) were stimulated with IL-17A in the presence or absence of TNFα and IL-6+6R. Figures 8A and 8B show that amplification of gene expression related to STAT and NF-κB signaling, e.g., NFκBIZ, CXCL1, IL-6, and CSF3 (G-CSF), was increased by IL-17A but was further amplified in the presence of TNFα or IL6+IL6R in both cell types (p<0.05 (pairwise t-test) for all comparisons except those in brackets with "FC" for "fold change").

[0189] Example 3 Identification of in vivo RRMS biomarkers in EAE models Experimental autoimmune encephalomyelitis (EAE) in mice is a disease caused by CD4 + T cell-mediated autoimmune disease, which is characterized by perivascular CD4 + EAE is characterized by T cell and mononuclear cell inflammation followed by primary demyelination of axonal pathways within the central nervous system (CNS), which results in hind limb paralysis. + T H 1 / T H It provides a powerful model for the study of the pathogenesis and immune regulation of 17-mediated tissue damage and is generally regarded as a relevant model for the human immune-mediated demyelinating disease multiple sclerosis. 35-55 EAE can be induced by immunization with a peptide corresponding to IL-17AA. The EAE model is useful for identifying biomarkers that correlate with IL-17AA by determining the modulation of the biomarkers by various IL-17 blocking antibodies.

[0190] Whole genome expression analysis was performed on RNA isolated from spinal cord tissue from mice with active EAE versus controls 48 hours after treatment with anti-IL-17 antibody, see Figure 9. In particular, 300 μg of MOG in complete Freund's adjuvant of heat-inactivated Mycobacterium tuberculosis (Mtb) was administered.35-55 C57BL / 6 mice were immunized with 100 mg of 10 ... TIFF2025063066000003.tif131170

[0191] The effect of anti-IL-17 antibodies was compared with small molecule drugs approved for the treatment of multiple sclerosis. Dimethyl fumarate (TECFIDERA®) is an oral treatment approved in the United States for the treatment of relapsing forms of multiple sclerosis, including RRMS. Dimethyl fumarate is known not to affect the IL-17 pathway. FTY-720 (fingolimod, GILENYA®) is a sphingosine-1-phosphate (S1P) modulator approved for the treatment of relapsing forms of MS, including RRMS.

[0192] Antibody treatment groups (anti-IL-17 and control anti-gp120) were treated on day 14 at the doses shown in Table 3 and harvested 48 hours later on day 16 (1 dose total). DMF and vehicle treatment groups were dosed bid (twice a day) starting on day 14 through day 15 (pm administration, 4 doses total) and harvested on day 16. FTY-120 treatment groups were dosed QD (once a day) from day 14 through day 15 (2 doses total) and harvested on day 16.

[0193] Spinal cords and serum were collected at harvest on day 16, and gene expression analysis was performed on spinal cord tissue RNA.

[0194] Figure 10A shows that lymphocyte levels, measured by markers such as CD4 and CD45, were increased in peak EAE disease, while Figure 10B shows that transcript abundance of myelin components, such as myelin-associated oligodendrocyte basic protein (Mobp) and myelin oligodendrocyte glycoprotein (Mog), was decreased in peak EAE disease versus naive controls with p<0.0007 for all genes (Wilcoxon test on each pair). The changes in expression levels of inflammatory and myelination-related genes shown in Figure 10 correlated with the induction of disease, thereby validating the EAE animal model of MS. However, the changes in gene expression were not modulated by anti-IL-17 treatment.

[0195] FIG. 11A shows a heat map of selected additional genes upregulated in EAE spinal cord at the peak of disease and the reduction in expression of these genes by anti-IL-17AA / AF+anti-IL-17FF antibodies. The goal of this study was to identify genes not previously identified as associated with IL-17 or associated with RRMS or other neurodegenerative diseases. FIG. 11 shows that RNA levels of CHI3L1, TIMP1, IL-6, CXCL1, LRG1, and NFκBIZ increased at the peak of disease (compared to before disease control) and decreased upon treatment with anti-IL-17AA / AF and anti-IL-17FF antibodies compared to control treatment (anti-ragweed and CTLA-4-Ig immunoadhesin). FIG. 12A-F show plots of the respective transcript abundance of each gene with each treatment. FIG. 13 shows that treatment with anti-IL-17 antibodies also reduced gene expression of CXCL5. Blockade of IL-17 target genes in the EAE spinal cord was in some cases better achieved with treatments including anti-IL-17AA / AF antibodies compared to treatments with anti-IL-17AA or anti-IL-FF antibodies alone (see Figures 12 and 13).

[0196] Example 4 RRMS biomarkers in human samples IL-17A, IL-17F, and various target proteins were measured in cerebrospinal fluid (CSF) and serum from 50 RRMS patients and 20 healthy donors (HD) using the Erenna® immunoassay platform (Singulex, Inc.) or a sandwich assay as described in Example 1.

[0197] Table 4 below shows the protein levels of selected genes tested in human CSF samples, including LRG1, TIMP1, YKL40 (CHI3L1), CXCL1, CXCL10, IL-6, and IL-8. The protein levels of all genes showed significant elevation in RRMS versus healthy controls (see the "Differential Levels by Diagnosis" column in Table 4). Except for IL-6, the protein levels of all genes in Table 4 showed good correlation with the protein levels of IL-17AA in human RRMS CSF samples (see the "Spearman Correlation with IL17 CSF" column). TIFF2025063066000004.tif73170

[0198] In particular, the correlation of TIMP1 and LRG1 with IL-17AA is shown in FIG. 14. FIG. 14 shows that the protein levels of TIMP1 and LRG1 are elevated in CSF samples of human RRMS patients, and their protein levels in CSF are also correlated with the protein levels of IL-17AA in CSF of human RRMS patients, confirming the results from the mouse EAE model. In addition, the levels of IL17A, TIMP1, and LRG1 in CSF correlated with the CSF / serum albumin ratio, which is a measure of blood-brain barrier permeability. Serum and CSF albumin were measured using a Sigma albumin ELISA kit (catalog number RAB0603) according to the manufacturer's instructions. For the assay, serum was diluted 1 / 1,000,000 and CSF was diluted 1 / 5,000. The results are shown in Table 5 below. TIFF2025063066000005.tif37170

[0199] Example 5 G-CSF as a serum marker for RRMS G-CSF (CSF3) is a cytokine regulated by IL-17 in multiple cell lineages and is a key driver of neutrophil proliferation. G-CSF was not detectable in CSF but was detectable in serum. Therefore, the correlation of G-CSF serum levels with IL-17AA was investigated in both EAE models and human serum samples.

[0200] Serum was obtained from EAE mice as described in Example 3 above at the time points indicated and indicated in Figure 9. G-CSF was measured using a sandwich ELISA with a monoclonal antibody specific for G-CSF.

[0201] As shown in Figure 15, peak levels of G-CSF were observed prior to clinical disease onset in the EAE model (day 8 when the clinical score was 0). Serum G-CSF was decreased following treatment with anti-IL-17 antibodies, including aIL-17AA / AF+aIL-17FF (combination of aIL-17AA / AF and aIL-17FF) described in Table 3 above, but not following treatment with DMF or FTY-720.

[0202] In human serum samples, G-CSF was elevated in high IL-17AA RRMS patients and correlated with CSF IL-17AA levels. See FIG. 16.

[0203] Thus, serum G-CSF levels could be used as a serum marker to identify IL-17AA high RRMS patients, particularly those who would benefit from therapies targeting IL-17AA, IL-17FF, and / or IL-17AF.

[0204] Although the present invention has been described in the foregoing specification with reference to specific embodiments, it is not limited thereto. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and are intended to fall within the scope of the appended claims.

[0205] All references cited throughout this specification, and the references cited therein, are expressly incorporated herein by reference in their entireties.

Claims

1. 1. A method for identifying a mammalian subject having or at risk of developing multiple sclerosis (MS), comprising: measuring the expression level of one or more genes selected from the group consisting of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF in a biological sample obtained from the subject; The method, wherein an increased level of expression relative to a control indicates that the subject has or is at risk of developing multiple sclerosis.

2. The method of claim 1 , wherein the mammalian subject is a human patient.

3. The method of claim 1 or 2, wherein the multiple sclerosis is characterized by increased levels of IL-17.

4. The method of claim 3, wherein the level of IL-17 is elevated in the cerebrospinal fluid (CSF) of the subject.

5. The method according to any one of claims 1 to 4, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS).

6. The method according to any one of claims 1 to 5, wherein the IL-17 is IL-17AA.

7. The method of any one of claims 1 to 6, wherein the expression level of an RNA transcript of one or more genes is measured.

8. The method of any one of claims 1 to 6, wherein the expression level of the protein product of one or more genes is measured.

9. The method according to any one of claims 1 to 8, wherein the biological sample is a biological fluid.

10. 10. The method of claim 9, wherein the biological fluid is cerebrospinal fluid (CSF).

11. The method of claim 9 , wherein the biological fluid is serum.

12. The method of claim 9 , wherein the biological fluid is plasma.

13. The method according to any one of claims 1 to 12, wherein the expression level of TIMP1 is measured.

14. The method according to any one of claims 1 to 12, wherein the expression level of LRG1 is measured.

15. The method according to any one of claims 1 to 7 and 9 to 12, wherein the expression level of an RNA transcript of NFκBIZ is measured.

16. The method of claim 11, wherein the expression level of G-CSF is measured in the serum.

17. The method of claim 12, wherein the expression level of one or more genes selected from the group of CXCL1, CXCL5, and CXCL10 is measured in the plasma.

18. The method of any one of claims 1 to 17, wherein the expression levels of two or more of said genes are measured.

19. The method of any one of claims 1 to 17, wherein the expression levels of three or more of said genes are measured.

20. The method according to any one of claims 1 to 19, wherein the expression level of TIMP1 and the expression level of at least one or more genes selected from the group consisting of LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF are measured.

21. The method according to any one of claims 1 to 19, wherein the expression level of LRG1 and the expression level of at least one or more genes selected from the group consisting of TIMP1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF are measured.

22. The method according to any one of claims 1 to 14, wherein the expression levels of TIMP1 and LRG1 are measured.

23. The method of claim 22, wherein the expression levels of TIMP1 and LRG1 are measured in the CSF.

24. The method according to any one of claims 1 to 14, wherein the expression levels of TIMP1 and / or LRG1, and G-CSF are measured.

25. The method of claim 24, wherein the expression level of TIMP1 and / or LRG1 is measured in the CSF and the expression level of G-CSF is measured in serum.

26. The method of claim 24 or 25, wherein the expression levels of TIMP1, LRG1, and G-CSF are measured.

27. 27. The method of any one of claims 1 to 26, further comprising administering to the subject identified as having or at risk of developing MS an effective amount of an IL-17 antagonist.

28. 28. The method of claim 27, wherein the IL-17 antagonist is an antibody or an antigen-binding fragment thereof.

29. The method of claim 28, wherein the antibody is an IL-17 antibody or an IL-17 receptor antibody.

30. 30. The method of claim 29, wherein the antibody is an IL-17 antibody, and the IL-17 antibody binds to an IL-17A homodimer, an IL-17F homodimer, and / or an IL-17AF heterodimer.

31. 1. A method for identifying or predicting a mammalian subject having or at risk of developing multiple sclerosis (MS) that is likely to be responsive to treatment with an IL-17 antagonist, comprising: Measuring the expression level of one or more genes selected from the group consisting of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF in a biological sample obtained from the subject; and determining that the subject is likely to be responsive to the treatment if the expression level of the one or more genes is increased compared to a control.

32. 1. A method for identifying or predicting a mammalian subject having or at risk of developing multiple sclerosis (MS) that is unlikely to be responsive to treatment with an IL-17 antagonist, comprising: Measuring the expression level of one or more genes selected from the group consisting of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF in a biological sample obtained from the subject; and determining that the subject is unlikely to be responsive to the treatment if the expression level of the one or more genes is decreased or at the same level as compared to a control.

33. 1. A method for monitoring a mammalian subject having multiple sclerosis (MS) being treated with an IL-17 antagonist, comprising: Measuring the expression level of one or more genes selected from the group consisting of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF in a biological sample obtained from the subject; determining whether the expression level of the one or more genes is increased compared to a control.

34. 34. The method of claim 33, wherein an increased level of expression compared to a control indicates that treatment with the IL-17 antagonist should be continued.

35. The method of any one of claims 31 to 34, wherein the mammalian subject is a human patient.

36. The method of any one of claims 31 to 35, wherein the multiple sclerosis is characterized by increased levels of IL-17.

37. 37. The method of claim 36, wherein the level of IL-17 is elevated in the cerebrospinal fluid (CSF) of the subject.

38. The method of any one of claims 31 to 37, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS).

39. The method of any one of claims 36 to 37, wherein the IL-17 is IL-17AA.

40. The method of any one of claims 31 to 39, wherein the expression level of an RNA transcript of one or more genes is measured.

41. 40. The method of any one of claims 31 to 39, wherein the expression level of the protein product of one or more genes is measured.

42. The method according to any one of claims 31 to 41, wherein the biological sample is a biological fluid.

43. 43. The method of claim 42, wherein the biological fluid is cerebrospinal fluid (CSF).

44. 43. The method of claim 42, wherein the biological fluid is serum.

45. 43. The method of claim 42, wherein the biological fluid is plasma.

46. The method according to any one of claims 31 to 45, wherein the expression level of TIMP1 is measured.

47. The method of any one of claims 31 to 45, wherein the expression level of LRG1 is measured.

48. The method according to any one of claims 31 to 40 and 42 to 45, wherein the expression level of an RNA transcript of NFκBIZ is measured.

49. The method of claim 44, wherein the expression level of G-CSF is measured in the serum.

50. 46. ​​The method of claim 45, wherein the expression level of one or more genes selected from the group of CXCL1, CXCL5, and CXCL10 is measured in the plasma.

51. The method of any one of claims 31 to 50, wherein the expression levels of two or more of said genes are measured.

52. The method of any one of claims 31 to 50, wherein the expression levels of three or more of said genes are measured.

53. The method according to any one of claims 31 to 52, wherein the expression level of TIMP1 and the expression level of at least one or more genes selected from the group consisting of LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF are measured.

54. The method according to any one of claims 31 to 52, wherein the expression level of LRG1 and the expression level of at least one or more genes selected from the group consisting of TIMP1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF are measured.

55. The method of any one of claims 31 to 47, wherein the expression levels of TIMP1 and LRG1 are measured.

56. 56. The method of claim 55, wherein the expression levels of TIMP1 and LRG1 are measured in the CSF.

57. The method according to any one of claims 31 to 47, wherein the expression levels of TIMP1 and / or LRG1, and G-CSF are measured.

58. 58. The method of claim 57, wherein the expression level of TIMP1 and / or LRG1 is measured in the CSF and the expression level of G-CSF is measured in serum.

59. The method of claim 57 or 58, wherein the expression levels of TIMP1, LRG1, and G-CSF are measured.

60. 60. The method of any one of claims 31 and 33-59, further comprising administering to said subject identified as having an increased expression level of said one or more genes an effective amount of an IL-17 antagonist.

61. 61. The method of claim 60, wherein the IL-17 antagonist is an antibody or an antigen-binding fragment thereof.

62. The method of claim 61 , wherein the antibody is an IL-17 antibody or an IL-17 receptor antibody.

63. 63. The method of claim 62, wherein the antibody is an IL-17 antibody, and the IL-17 antibody binds to an IL-17A homodimer, an IL-17F homodimer, and / or an IL-17AF heterodimer.

64. 1. A method of treating a mammalian subject having or at risk of developing multiple sclerosis (MS), comprising: Measuring the expression level of one or more genes selected from the group consisting of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF in a biological sample obtained from the subject; administering to said subject having an increased expression level of said one or more genes as compared to a control an effective amount of an IL-17 antagonist.

65. 65. The method of claim 64, wherein the mammalian subject is a human patient.

66. 66. The method of claim 64 or 65, wherein the multiple sclerosis is characterized by increased levels of IL-17.

67. 67. The method of claim 66, wherein the IL-17 is elevated in cerebrospinal fluid (CSF).

68. 68. The method of any one of claims 64 to 67, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS).

69. The method of any one of claims 66 to 68, wherein the increased level of IL-17 is an increased level of IL-17AA.

70. 70. The method of any one of claims 64 to 69, wherein the expression level of an RNA transcript of one or more genes is measured.

71. 70. The method of any one of claims 64 to 69, wherein the expression level of the protein product of one or more genes is measured.

72. The method of any one of claims 64 to 71, wherein the biological sample is a biological fluid.

73. 73. The method of claim 72, wherein the biological fluid is cerebrospinal fluid (CSF).

74. 73. The method of claim 72, wherein the biological fluid is serum.

75. 73. The method of claim 72, wherein the biological fluid is plasma.

76. The method according to any one of claims 64 to 75, wherein the expression level of TIMP1 is measured.

77. The method of any one of claims 64 to 75, wherein the expression level of LRG1 is measured.

78. The method of any one of claims 64 to 70 and 72 to 75, wherein the expression level of an RNA transcript of NFκBIZ is measured.

79. 75. The method of claim 74, wherein the expression level of G-CSF is measured in the serum.

80. 76. The method of claim 75, wherein the expression level of one or more genes selected from the group of CXCL1, CXCL5, and CXCL10 is measured in the plasma.

81. The method of any one of claims 64 to 80, wherein the expression levels of two or more of said genes are measured.

82. The method of any one of claims 64 to 80, wherein the expression levels of three or more of said genes are measured.

83. The method of any one of claims 64 to 82, wherein the IL-17 antagonist is an antibody or an antigen-binding fragment thereof.

84. 84. The method of claim 83, wherein the antibody is an IL-17 antibody or an IL-17 receptor antibody, or an antibody-binding fragment thereof.

85. 85. The method of claim 84, wherein the antibody is at least one antibody selected from the group consisting of brodalumab, secukinumab, ixekizumab, bimekizumab, CNTO 6785, ALX-0761, and afasevicumab.

86. 85. The method of claim 84, wherein the antibody is an IL-17 antibody, wherein the IL-17 antibody is an IL-17A homodimer, an IL-17F homodimer, and / or an IL-17AF heterodimer.

87. The method of claim 84, wherein the antibody is an IL-17 antibody that binds to an IL-17A homodimer.

88. 88. The method of claim 87, wherein the anti-IL-17 antibody binds to IL-17AA and IL-17AF.

89. 85. The method of claim 84, wherein the antibody is an IL-17 antibody that binds to an IL-17F homodimer.

90. The method of claim 84, wherein the antibody is an IL-17 antibody that binds to an IL-17AF heterodimer.

91. The method of any one of claims 83 to 90, wherein the antibody is a monoclonal antibody.

92. 92. The method of claim 91, wherein the antibody is a chimeric, humanized, or human antibody.

93. 92. The method of claim 91, wherein the antibody is a bispecific, multispecific, or cross-reactive antibody.

94. 94. The method of any one of claims 64-93, further comprising administering an effective amount of a multiple sclerosis (MS) therapeutic agent.

95. 95. The method of claim 94, wherein the MS therapeutic agent is at least one agent selected from the group consisting of dimethyl fumarate, FTY-720, nataluzimab, corticosteroids, beta-interferon, glatiramer acetate, teriflunomide, mitoxantrone, and anti-CD20 antibodies.

96. 1. A method for detecting the expression level of one or more genes in a mammalian subject suspected of having or at risk of developing relapsing-remitting multiple sclerosis (RRMS), comprising: obtaining a biological sample from the subject; measuring in the biological sample the expression level of one or more genes selected from the group consisting of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF.

97. 97. The method of claim 96, further comprising determining that the subject has or is at risk for developing RRMS if the expression level is increased relative to a control.

98. 98. The method of claim 96 or 97, wherein the mammalian subject is a human patient.

99. The method of any one of claims 96-98, wherein the RRMS is characterized by increased levels of IL-17.

100. 100. The method of claim 99, wherein the level of IL-17 is elevated in the cerebrospinal fluid (CSF) of the subject.

101. The method of claim 99 or 100, wherein the IL-17 is IL-17AA.

102. The method of any one of claims 96 to 101, wherein the expression level of an RNA transcript of one or more genes is measured.

103. 102. The method of any one of claims 96 to 101, wherein the expression level of the protein product of one or more genes is measured.

104. The method of any one of claims 96 to 103, wherein the biological sample is a biological fluid.

105. 105. The method of claim 104, wherein the biological fluid is cerebrospinal fluid (CSF).

106. 105. The method of claim 104, wherein the biological fluid is serum.

107. 105. The method of claim 104, wherein the biological fluid is plasma.

108. The method of any one of claims 96 to 107, wherein the expression level of TIMP1 is measured.

109. The method of any one of claims 96 to 107, wherein the expression level of LRG1 is measured.

110. The method of any one of claims 96 to 102 and 104 to 107, wherein the expression level of an RNA transcript of NFκBIZ is measured.

111. The method of claim 106, wherein the expression level of G-CSF is measured in the serum.

112. 108. The method of claim 107, wherein the expression level of one or more genes selected from the group of CXCL1, CXCL5, and CXCL10 is measured in the plasma.

113. The method of any one of claims 96 to 112, wherein the expression levels of two or more of said genes are measured.

114. The method of any one of claims 96 to 112, wherein the expression levels of three or more of said genes are measured.

115. The method according to any one of claims 96 to 107, wherein the expression level of TIMP1 and the expression level of at least one or more genes selected from the group consisting of LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF are measured.

116. The method according to any one of claims 96 to 107, wherein the expression level of LRG1 and the expression level of at least one or more genes selected from the group consisting of TIMP1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF are measured.

117. The method of any one of claims 96 to 109, wherein the expression levels of TIMP1 and LRG1 are measured.

118. The method of claim 117, wherein the expression levels of TIMP1 and LRG1 are measured in the CSF.

119. The method according to any one of claims 96 to 109, wherein the expression levels of TIMP1 and / or LRG1, and G-CSF are measured.

120. The method of claim 119, wherein the expression level of TIMP1 and / or LRG1 is measured in the CSF and the expression level of G-CSF is measured in serum.

121. The method of claim 119 or 120, wherein the expression levels of TIMP1, LRG1, and G-CSF are measured.

122. 122. The method of any one of claims 97-121, further comprising administering to said subject identified as having or at risk of developing MS an effective amount of an IL-17 antagonist.

123. 123. The method of claim 122, wherein the IL-17 antagonist is an antibody or an antigen-binding fragment thereof.

124. The method of claim 123, wherein the antibody is an IL-17 antibody or an IL-17 receptor antibody.

125. 125. The method of claim 124, wherein the antibody is at least one antibody selected from the group consisting of brodalumab, secukinumab, ixekizumab, bimekizumab, CNTO 6785, ALX-0761, and afasevicumab.

126. The method of claim 124, wherein the antibody is an IL-17 antibody that binds to an IL-17A homodimer, an IL-17F homodimer, and / or an IL-17AF heterodimer.

127. The method of claim 124, wherein the antibody is an IL-17 antibody that binds to an IL-17A homodimer.

128. The method of claim 124, wherein the antibody is an IL-17 antibody that binds to IL-17AA and IL-17AF.

129. The method of claim 124, wherein the antibody is an IL-17 antibody that binds to an IL-17F homodimer.

130. The method of claim 124, wherein the antibody is an IL-17 antibody that binds to IL-17A / F.

131. The method of any one of claims 123 to 130, wherein the antibody is a monoclonal antibody.

132. The method of claim 131 , wherein the antibody is a chimeric, humanized, or human antibody.

133. 132. The method of claim 131, wherein the antibody is a bispecific, multispecific, or cross-reactive antibody.

134. 134. The method of any one of claims 122-133, further comprising administering an effective amount of a multiple sclerosis (MS) therapeutic agent.

135. 135. The method of claim 134, wherein the MS therapeutic agent is at least one agent selected from the group consisting of dimethyl fumarate, FTY-720, nataluzimab, corticosteroids, beta-interferon, glatiramer acetate, teriflunomide, mitoxantrone, and anti-CD20 antibodies.

136. 1. A kit for detecting the expression level of one or more genes in a mammalian subject having or at risk of developing multiple sclerosis (MS), comprising: The kit comprises at least one container comprising one or more reagents for detecting the expression level of one or more genes selected from the group consisting of TIMP1, LRG1, CXCL1, CXCL5, CXCL10, IL-8, NFκBIZ, YKL40, and G-CSF.

137. 137. The kit of claim 136, wherein the one or more reagents comprise reagents for detecting the expression level of an RNA transcript and / or the expression level of a protein product of the one or more genes.

138. 138. The kit of claim 137, wherein the reagents comprise one or more gene-specific primers or probes for the one or more genes.

139. 137. The kit of claim 136, wherein the one or more reagents comprise reagents for detecting the expression level of a protein product of the one or more genes.

140. 140. The kit of claim 139, wherein the reagent comprises one or more antibodies or antigen-binding fragments thereof that bind to the protein product of the one or more genes.

141. 141. The kit of any one of claims 136-140, further comprising a container containing an IL-17 antagonist and a label or instructions for administering said IL-17 antagonist to said subject.

142. The kit of claim 141, wherein the IL-17 antagonist is an antibody or an antigen-binding fragment thereof.

143. The kit of claim 142, wherein the antibody is an IL-17 antibody or an IL-17 receptor antibody.

144. The kit of claim 143, wherein the antibody is an IL-17 antibody that binds to an IL-17A homodimer, an IL-17F homodimer, and / or an IL-17AF heterodimer.

145. The kit of any one of claims 136 to 144, wherein the MS is relapsing-remitting MS (RRMS).

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