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HK1152053B - Il-17ra-il-17rb antagonists and uses thereof - Google Patents

Il-17ra-il-17rb antagonists and uses thereof Download PDF

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Publication number
HK1152053B
HK1152053B HK11106212.6A HK11106212A HK1152053B HK 1152053 B HK1152053 B HK 1152053B HK 11106212 A HK11106212 A HK 11106212A HK 1152053 B HK1152053 B HK 1152053B
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Hong Kong
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antagonist
antibody
treatment
receptor complex
antibodies
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HK11106212.6A
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Chinese (zh)
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HK1152053A1 (en
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Alison L. Budelsky
Michael R. Comeau
Joel E. Tocker
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Amgen K-A, Inc.
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Priority claimed from PCT/US2009/001085 external-priority patent/WO2009136976A2/en
Publication of HK1152053A1 publication Critical patent/HK1152053A1/en
Publication of HK1152053B publication Critical patent/HK1152053B/en

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Description

IL-17RA-IL-17RB antagonists and uses thereof
Cross Reference to Related Applications
The present application claims the benefit of U.S. provisional application No. 61/145,901, filed on month 20, 2009, and 35u.s.c. § 119, U.S. provisional application No. 61/066,538, filed on month 21, 2008, which are incorporated herein by reference.
Technical Field
The present invention relates to the discovery that interleukin-17 ligand and receptor family members, and IL-17 receptor a and IL-17 receptor B form a heteromeric complex that is biologically active. Antagonists of the IL-17RA-IL-17RB heteromeric receptor complex and methods of use are disclosed.
Background
The interleukin-17 family is a group of 6 structurally related cytokines, designated IL-17A through IL-17F, which are important in the regulation of immune responses. At the primary structure level, the most similar appears to be a C-terminal region that contains 4 conserved cysteine residues (reviewed in Kawaguchi et al, J.Allergy Clin Immunol 114: 1265, 2004; Kolls and Linden, Immunity 21: 467, 2004). The crystal structure of IL-17F has been determined and found to share structural features with the cystine knot family of growth factors (Hymowitz et al, 2001, EMBO J.20: 5532-.
The IL-17 receptor (IL-17R) also forms a family of related type I transmembrane proteins. 5 different members of this family (IL-1RA through IL-1RE) have been identified, several of which also exhibit alternative splicing, including soluble forms that can act as decoy receptors (Kolls and Linden, supra; Moseley et al, Cytokine Growth Factor Rev.14: 155, 2003). Although IL-17RA multimerizes independently of ligand and has been shown to form a biologically active heteromeric receptor complex with IL-17RC (Toy et al, JImmunol.177: 36; 2007), the possibility of forming other heteromeric IL-17R complexes (transmembrane or soluble type) and the resulting biological activity, if any, has not been previously known. This and other aspects of various embodiments of the present invention are provided.
Brief Description of Drawings
FIG. 1 is a graph illustrating the effect of IL-17RA-IL-17RB antagonists on airway hyperresponsiveness. Open circles indicate results obtained in mice given MSA and MuFc (N ═ 4); open squares represent results obtained in mice given IL-25 and MuFc (N ═ 3); the filled triangles represent results obtained in mice given IL-25 and M751 (N ═ 3).
Figure 2 presents a Western blot prepared essentially as described in example 11. Lanes 1 and 4 contain molecular weight markers. Plate A was blotted with anti-IL-17 RA and plate B was blotted with anti-HIS. Lane 2 presents a positive control for IL-17RA HIS, and lane 3 shows the result of IL-17RA HIS precipitation with IL-17RB Fc. Lane 5 presents an IL-17RD HIS positive control, and lane 6 shows that IL-17RD HIS cannot be precipitated with IL-17RB Fc.
Figure 3 is a graph illustrating Airway Hyperresponsiveness (AHR) in mice in the OVA asthma model from experiment 1 of example 14. Mice were challenged with increasing concentrations of methacholine and changes in PENH above baseline ± SEM were calculated.
Figure 4 illustrates lung Resistance (RL) in mice in an OVA asthma model as described in example 14. Mean airway resistance (R) Area Under Curve (AUC) is shown for each treatment group ± SEM. Figure 4a presents the results from experiment 2 and 4b from experiment 3.
Figure 5 presents an analysis of bronchoalveolar lavage fluid (BALF) cell content as described in example 14, experiment 1. The results are shown as total BALF (4a) leukocytes, (4b) eosinophils, (4c) neutrophils, (4d) lymphocytes and (4e) macrophages. Each filled circle represents BALF cell content from one mouse. Statistical analysis was compared using a non-parametric one-way ANOVA with Dunn's multiple comparison test (. p < 0.05).
Fig. 6 is similar to fig. 5, but presents the results from example 14, experiment 2. The results are shown as total BALF (4a) leukocytes, (4b) eosinophils, (4c) neutrophils, (4d) lymphocytes and (4e) macrophages. Statistical analysis comparisons were performed using one-way ANOVA with Bonferroni's multiple comparison test (. p < 0.05).
Figure 7 presents the results from example 14, experiment 3. The results are shown as total BALF (4a) leukocytes, (4b) eosinophils, (4c) neutrophils, (4d) lymphocytes and (4e) macrophages. Each filled circle represents BALF cell content from one mouse. Statistical analysis was compared using a non-parametric one-way ANOVA with Dunn's multiple comparison test (. p < 0.05).
FIG. 8 illustrates BALF IL-13 concentrations from mice in OVA asthma model. From example 14: (a) experiment 1, (b) experiment 2, (c) BALF samples from each mouse of the 3 independent experiments described in experiment 3 were assayed for IL-13 concentration by ELISA. Each filled circle represents the value from one mouse. The horizontal line represents the group mean. Comparisons between groups were performed using one-way ANOVA. P < 0.05.
Figure 9 presents BALF IL-5 concentrations from mice in the OVA asthma model. From example 14: (a) experiment 1, (b) experiment 2, (c) BALF samples from each mouse in 3 independent experiments described in experiment 3 were assayed for IL-5 concentration by ELISA. Each filled circle represents the value from one mouse. The horizontal line represents the group mean. Comparisons between groups were performed using one-way ANOVA. P < 0.05.
Figure 10 illustrates the assay by ELISA as in example 14: (a) experiment 1, (b) experiment 2, (c) serum IgE concentration in individual mice in the OVA asthma model described in experiment 3. Serum IgE concentrations of each mouse are shown as filled circles. The horizontal line represents the group mean. Comparisons between groups were performed using one-way ANOVA. P < 0.05.
Figure 11 presents lung histology scores from a population of mice in the OVA asthma model as described in experiment 3 of example 14. Statistical comparisons used unpaired t-test × p < 0.0001.
Detailed Description
Section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, and the like. Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly practiced in the art or as described herein. The following methods and techniques may be performed in accordance with conventional methods well known in the art and generally as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al, 2001, Molecular Cloning: a Laboratory Manual, third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the terms and experimental methods and techniques used in the analytical chemistry, organic chemistry and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques may be used for chemical synthesis, chemical analysis, preparation, formulation and delivery of drugs, and treatment of patients.
The identification, cloning and preparation of IL-17RA has been described by way of example in USPN 6,072,033, granted at 6.6.2000, which is hereby incorporated by reference in its entirety. The amino acid sequence of human IL-17RA is shown in SEQ ID NO of USPN 6,072,033: 10(GenBank accession No. NM _ 014339). Human IL-17RA has an N-terminal signal peptide with a predicted cleavage site between about amino acids 27 and 28. This signal peptide is followed by a 293 amino acid extracellular domain, a 21 amino acid transmembrane domain and a 525 amino acid cytoplasmic tail. Soluble forms of human IL-17RA (huIL-17RA) useful in the methods of the invention include the extracellular domain (residues 1-320 or residues 28-320 excluding the signal peptide) or fragments of the extracellular domain that retain the ability to bind IL-17A. Other forms of IL-17RA useful in the methods of the invention include muteins and variations of amino acid identity of at least 70% to 99% with native IL-17RA that retains the ability to bind IL-17A, as described in more detail in USPN 6,072,033.
IL-17 receptor B (IL-17RB) and many isoforms thereof are known in the art, e.g., Tian et al, Oncogene 19: 2098(2000) to those disclosed and described in (a). Additional examples include sequences available from public databases such as, but not limited to, GenBank accession No. NM _ 018725. In addition, as described below, IL-17RB can also include biologically active fragments and/or variants.
IL-17RA associates with IL-17RB to form a heteromeric receptor complex that is biologically active (i.e., upon binding of a ligand, the receptor complex is activated and signals into cells expressing it, resulting in the induction of biological activities such as mRNA, secretion of cytokines, changes in cell morphology or activation state, etc.). Each member of the heteromeric receptor complex is referred to as its "component" or "subunit". As used herein, "IL-17 RA-IL-17RB heteromeric receptor complex" (or "heteromeric receptor complex") refers to a complex comprising at least IL-17RA and IL-17 RB; additional subunits or components may also form part of the heteromeric receptor complex.
Accordingly, certain aspects of the invention relate to agents (e.g., antigen binding proteins as described below) and methods for blocking the association of IL-17RA with IL-17RB (and/or with additional receptor subunits), thereby preventing the formation of a functional receptor complex (a receptor complex that can be activated). Other aspects of the invention relate to antagonists that bind to the IL-17RA-IL-17RB heteromeric receptor complex, or subunits or components thereof, and inhibit binding of a ligand (i.e., IL-25) and subsequent activation of the receptor complex. Further aspects of the invention relate to antagonists that bind to the IL-17RA-IL-17RB heteromeric receptor complex or subunits thereof and prevent activation from occurring. Prevention of functional complex formation and/or activation will reduce or prevent signal transduction and reduce downstream pro-inflammatory effects of IL-17RA/IL-17RB activation. Such methods and antagonists would be useful in the treatment of various inflammatory and autoimmune disorders affected by the IL-17/IL-17R pathway. Embodiments of the invention are useful in vitro assays to screen antagonists or agonists of the IL-17RA-IL-17RB heteromeric receptor complex and/or to identify cells expressing the IL-17RA-IL-17RB heteromeric receptor complex.
Furthermore, the recognition that both IL-17RA and IL-17RB are essential for the formation of a functional IL-25 receptor complex has led to additional useful agents (e.g., antigen binding proteins) for inhibiting or antagonizing IL-25 biological activity. For example, an antigen binding protein that binds to one or more subunits of the receptor complex (e.g., an antibody that binds IL-17RA or an antibody that binds IL-17RB) and inhibits IL-25 binding or activates the receptor complex would be a potent IL-25 antagonist.
In certain embodiments, an antagonist of the invention is an "isolated" or "substantially pure" (or "substantially homologous") molecule. The term "isolated molecule" (where the molecule is, for example, a polypeptide, peptide, or antibody) is a molecule that, depending on its source or derivative source, (1) does not accompany a naturally associated component with which it is associated in its native state, (2) is substantially free of other molecules from the same species, (3) is expressed from a cell of a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components.
Molecules may also be rendered substantially free of naturally associated components (i.e., "purified" proteins) by isolation using purification techniques well known in the art. Molecular purity or homogeneity can be determined by a number of methods well known in the art. For example, the purity of a polypeptide sample can be determined using polyacrylamide gel electrophoresis and staining the gel to present the polypeptide using techniques well known in the art. For some purposes, higher resolution may be provided by using HPLC or other methods for purification well known in the art.
An "isolated" antagonist (i.e., a protein, polypeptide, peptide, or antibody) is not associated with at least some of the substances with which it is normally associated in its native state, and in one embodiment constitutes at least about 5%, and in another embodiment at least about 50%, by weight of the total protein administered to a sample. A "substantially pure" protein comprises at least about 75%, specifically at least about 80%, and particularly at least about 90%, by weight of total protein. The definition includes the production of a protein from one organism in a different organism or host cell. Alternatively, the protein can be produced at significantly higher concentrations than are commonly seen by using inducible promoters or high expression promoters, such that the protein is produced at increased concentration levels.
These are but a few of the many aspects of the various embodiments of the invention described herein.
IL-17RA-IL-17RB antagonists
IL-17RA associates with IL-17RB to form a heteromeric receptor complex that is biologically active (i.e., when activated by binding a ligand, transduces a signal into a cell, resulting in a change in the biological activity of the cell, such as induction of mRNA, secretion of cytokines, change in cell morphology or activation state, etc.). The IL-17RA-IL-17RB heteromeric receptor complex is defined as the association of at least IL-17RA and IL-17RB proteins (such as, but not limited to, inter-protein interactions), which appear as a heteromeric receptor complex on the outer membrane of a cell. The heteromeric receptor complex is necessary for at least IL-25 signaling (i.e., activation of IL-17RA and/or IL-17 RB). It is understood that the IL-17RA-IL-17RB heteromeric receptor complex may further comprise additional proteins (i.e., "helper" proteins). For example, the signaling molecule known as Act-1 is part of the IL-17A signaling cascade, and recent evidence suggests that it may also be involved in IL-25 signaling (Claudio et al, J.Immunol.182: 1617, 2009; Swaidani et al, J.Immunol.182: 1631, 2009). Activation of the IL-17RA-IL-17RB heteromeric receptor complex is achieved by binding to an IL-17 ligand family member, such as, but not limited to, IL-25 (IL-17E). Activation of the IL-17RA-IL-17RB heteromeric receptor complex includes, but is not limited to, initiation of intracellular signaling cascades and downstream events (e.g., gene transcription and translation).
Embodiments relate to antagonists (including antigen binding proteins) that inhibit association of subunits (i.e., IL-17RA and IL-17RB and/or accessory proteins) to form IL-17RA-IL-17RB heteromeric receptor complexes, and antagonists (i.e., antigen binding proteins) that inhibit binding of a ligand (i.e., IL-25) to an IL-17RA-IL-17RB heteromeric receptor complex or subunit thereof. Additional embodiments relate to antagonists (including antigen binding proteins) that bind to one or more subunits of the IL-17RA-IL-17RB heteromeric receptor complex and cause a conformational change that prevents association of the complex subunits, ligand binding thereto, or activation thereof.
An "antigen binding protein" as used herein is a protein that specifically binds to an identified target protein (e.g., a subunit of the 17RA-IL-17RB heteromeric receptor complex or the heteromeric receptor complex itself). By "specifically binds" is meant that the antigen binding protein has a higher affinity for the identified target protein than for other proteins. Typically, "specific binding" refers to an equilibrium dissociation constant of < 10-7-10-11M, or < 10-8-＜10-10M, or < 10-9-＜10-10M。
Antigen binding proteins include antibodies or fragments thereof that specifically bind to an identified target protein (as defined elsewhere herein), as well as peptides or polypeptides that specifically bind to an identified target protein. Antigen binding proteins that inhibit the formation of the IL-17RA-IL-17RB heteromeric receptor complex, or inhibit ligand binding to or signaling therefrom, are referred to herein as IL-17RA-IL-17RB antagonists. Thus, embodiments of IL-17RA-IL-17RB antagonists can bind to any portion of the IL-17RA-IL-17RB heteromeric receptor complex (i.e., to the complex itself or a subunit thereof) and inhibit receptor activation. Subgenera of the IL-17RA-IL-17RB antagonist genus include antibodies (as defined herein, supra), as well as peptides and polypeptides.
Activation or activation of a receptor is defined herein as the participation of one or more intracellular signaling pathways and transduction of intracellular signals (i.e., signal transduction) in response to molecules that bind to membrane-bound receptors, such as, but not limited to, receptors: the ligands interact. As used herein, signal transduction is the transmission of a signal by a transformation from one physical or chemical form to another; for example, in cell biology, the process by which a cell converts an extracellular signal into a response (e.g., secretion of cytokines, proliferation of the cell, or a change in activation state).
"inhibition" can be measured as a decrease in the activity of the IL-17RA-IL-17RB heteromeric receptor complex, e.g., the formation of the heteromeric receptor complex, the binding of a ligand (i.e., IL-17AIL-17F and/or IL-25) to the heteromeric receptor complex (or at least one subunit thereof), or a decrease in biological activity (i.e., stimulation of cytokine secretion, alteration in cell number or activity state, or other biological effect) in response to a ligand (e.g., IL-17A, IL-17F and/or IL-25) of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In one embodiment, the antagonists of the invention reduce the activity of the IL-17RA-IL-17RB heteromeric receptor complex by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%; in another embodiment, an antagonist of the invention inhibits activity by at least 35%, 45%, 55%, 65%, 75%, 85%, 95% or more.
Inhibition of heteromeric receptor complex formation can be measured by any method known in the art, such as, but not limited to, the co-immunoprecipitation method described herein. Other examples include Forster Resonance Energy Transfer (FRET) assays and other methods known in the art and useful for quantitative or qualitative analysis of ligand/receptor interactions. Inhibition of ligand binding can also be measured by any method known in the art, e.g., FACS, EIA, RIA, the above assays, and methods known in the art for evaluating interactions between two or more molecules, including those described herein and USSN 11/906,094.
Furthermore, "inhibition" can be measured as a loss of IL-17RA-IL-17RB heteromeric receptor complex IL-25 activation measured in a biologically relevant readout, such as, but not limited to, upregulated gene transcription (e.g., IL-5, IL-13, eotaxin, MCP-1, and/or IL-17RBmRNA level elevation) and/or gene translation, and/or release of various factors associated with IL-17RA-IL-17RB heteromeric receptor complex activation (including IL-5 and/or IL-13), as well as any other pro-inflammatory mediators known in the art to be released by any cell expressing IL-17RA and/or IL-17RB. Additional biologically relevant reads include changes in the number and/or appearance of cells in the biological sample (e.g., increased cell content in a bronchoalveolar lavage sample, goblet cell hyperplasia and/or vascular/perivascular inflammation in a lung tissue sample).
IL-17RA-IL-17RB antagonists additional embodiments relate to IL-17RA-IL-17RB antagonists that bind to IL-17RA. In one embodiment, the antagonist partially inhibits or completely inhibits the association of IL-17RA-IL-17RB heteromeric receptor complex subunits, thereby preventing heteromeric receptor complex formation. In certain embodiments, the IL-17RA-IL-17RB antagonist does not block IL-25 binding to the IL-17RA-IL-17RB heteromeric receptor complex. In alternative embodiments, the IL-17RA-IL-17RB antagonist can block IL-25 binding to the IL-17RA-IL-17RB heteromeric receptor complex (or subunit thereof).
IL-17RA-IL-17RB antagonists additional embodiments relate to IL-17RA-IL-17RB antagonists that bind to IL-17RB. In one embodiment, the antagonist partially inhibits or completely inhibits the association of IL-17RA-IL-17RB heteromeric receptor complex subunits, thereby preventing heteromeric receptor complex formation. In certain embodiments, the IL-17RA-IL-17RB antagonist does not block IL-25 binding to the IL-17RA-IL-17RB heteromeric receptor complex. In alternative embodiments, the IL-17RA-IL-17RB antagonist can block IL-25 binding to the IL-17RA-IL-17RB heteromeric receptor complex (or subunit thereof).
IL-17RA-IL-17RB antagonists additional embodiments relate to IL-17RA-IL-17RB antagonists that bind to both IL-17RA and IL-17RB, including those that bind to heteromeric receptor complexes. In one embodiment, the antagonist partially inhibits or completely inhibits the association of IL-17RA-IL-17RB heteromeric receptor complex subunits, thereby preventing heteromeric receptor complex formation. In certain embodiments, the IL-17RA-IL-17RB antagonist does not block IL-25 binding to the IL-17RA-IL-17RB heteromeric receptor complex. In alternative embodiments, the IL-17RA-IL-17RB antagonist can block IL-25 binding to the IL-17RA-IL-17RB heteromeric receptor complex (or subunit thereof).
Various embodiments of the above IL-17RA-IL-17RB antagonists include IL-17RA-IL-17RB antagonists that bind to IL-17RA, or IL-17RB, or the heteromeric receptor complex, and sterically inhibit or block the association of subunits of the heteromeric receptor complex, thereby preventing formation of the IL-17RA-IL-17RB heteromeric receptor complex. In one example of steric hindrance of the heteromeric receptor complex subunit association, binding of the antagonist to the subunit occurs at a site that is necessary for, or sufficiently proximal to, association of that subunit with other subunits of the receptor complex such that the spatial arrangement of the antagonist prevents association of the heteromeric receptor complex subunits. Alternatively, various embodiments of the above IL-17RA-IL-17RB antagonists include IL-17RA-IL-17RB antagonists that bind to IL-17RA, or IL-17RB, or the heteromeric receptor complex, and induce (or prevent) a conformational change in one or more subunits of the heteromeric receptor complex, thereby inhibiting the formation of the IL-17RA-IL-17RB heteromeric receptor complex. In one example of a conformational change preventing association of subunits of the heteromeric receptor complex, binding of the antagonist to a subunit occurs at a site that can be remote from the site necessary for association of that subunit with other subunits of the receptor complex, causing a conformational change in that subunit that prevents its association with other subunits, or a conformational change necessary for subunit association. Similarly, various embodiments of the above IL-17RA-IL-17RB antagonists include IL-17RA-IL-17RB antagonists that bind to IL-17RA, or IL-17RB, or the heteromeric receptor complex, and induce (or prevent) a conformational change that inhibits signaling or sterically hinders signaling of the heteromeric receptor complex.
In another alternative embodiment, the various IL-17RA-IL-17RB antagonists described above include IL-17RA-IL-17RB antagonists that bind to IL-17RA, or IL-17RB, or the heteromeric receptor complex, and induce a conformational change in the heteromeric receptor complex (or a subunit thereof), thereby inhibiting IL-25 (or another ligand) binding to the IL-17RA-IL-17RB heteromeric receptor complex. The embodiments also include IL-17RA-IL-17RB antagonists that bind to IL-17RA, or IL-17RB, or both IL-17RA and IL-17RB, and sterically hinder or inhibit binding of a ligand (e.g., IL-25) to the IL-17RA-IL-17RB heteromeric receptor complex. Embodiments of the invention also include antagonists that bind to IL-17RA, or IL-17RB, or both IL-17RA and IL-17RB, and inhibit (partially or completely) the signaling pathway of the receptor complex, thereby inhibiting signaling through the IL-17RA-IL-17RB heteromeric receptor complex.
In another alternative embodiment, the IL-17RA-IL-17RB antagonist binds to a ligand (i.e., IL-17A, IL-25, etc.) and inhibits signaling through the IL-17RA-IL-17RB heteromeric receptor complex. Such antagonists may act by inhibiting binding to one or more than one subunit of the IL-17RA-IL-17RB heteromeric receptor complex. Thus, for example, an antagonist may allow a ligand to bind to a first receptor subunit, but prevent interaction of a second receptor subunit with the ligand or the first receptor subunit. Such inhibition may occur as described above, e.g., by steric hindrance of binding, induction of conformational change, etc., so as to inhibit (partially or completely) signaling through the IL-17RA-IL-17RB heteromeric receptor complex.
1.1IL-17RA-IL-17RB antagonists: antibodies
Embodiments of IL-17RA-IL-17RB antagonists include antibodies or fragments thereof, as broadly defined herein. Thus, the IL-17RA-IL-17RB antagonist includes polyclonal antibodies, monoclonal antibodies, bispecific antibodies, diabodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, fully human antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments thereof.
IL-17RA-IL-17RB antagonist antibodies can also include single domain antibodies that comprise a dimer of two heavy chains and no light chain, such as those found in camels and llamas (see, e.g., Muldermans et al, 2001, J.Biotechnol.74: 277-302; Desmyter et al, 2001, J.biol.Chem.276: 26285-26290).
The IL-17RA-IL-17RB antagonist antibody can comprise a tetramer or fragment thereof. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). The amino-terminal portion of each chain includes a variable region primarily responsible for antigen recognition. The carboxy-terminal portion of each strand defines a constant region that primarily serves a effector function. Human light chains are divided into kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha or epsilon heavy chains, and define the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG 4. There are subclasses of IgM, including but not limited to IgM1 and IgM 2. IL-17RA-IL-17RB antagonist antibodies include all such isoforms. For exemplary purposes, antibody fragments include, but are not limited to, F (ab), F (ab') 2, Fv and single chain Fv fragments (scfv), as well as single chain antibodies. IL-17RA-IL-17RB antagonist antibodies can include any of the foregoing examples.
The structure of antibodies is well known in the art and need not be repeated here, but as an example, the variable regions of heavy and light chains typically exhibit the same general structure, with relatively conserved Framework Regions (FRs) connected by three hypervariable regions (also called complementarity determining regions or CDRs). The CDRs are the hypervariable regions of an antibody (or antigen-binding protein, as outlined herein) which are responsible for antigen recognition and binding. The CDRs from each pair of two chains are positioned by the framework regions, enabling binding to a specific epitope. From N-terminus to C-terminus, both light and heavy chains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. In certain embodiments, the amino acid assignments to each domain may be consistent with those defined in Kabat Sequences of Proteins of Immunological Interest. See, Chothia et al, 1987, j.mol.biol.196: 901-; chothia et al, 1989, Nature 342: 878-883.
As used herein, "complementarity determining regions" or "CDRs" refer to binding protein regions that constitute the primary surface contact points for antigen binding. A binding protein of the invention can have six CDRs, for example, one heavy chain CDR1 ("CDRH 1"), one heavy chain CDR2 ("CDRH 2"), one heavy chain CDR3 ("CDRH 3"), one light chain CDR1 ("CDRL 1"), one light chain CDR2 ("CDRL 2"), one light chain CDR3 ("CDRL 3"). CDRH1 typically comprises from about 5 to about 7 amino acids, CDRH2 typically comprises from about 16 to about 19 amino acids, and CDRH3 typically comprises from about 3 to about 25 amino acids. CDRL1 typically comprises about 10 to about 17 amino acids, CDRL2 typically comprises about 7 amino acids, and CDRL3 typically comprises about 7 to about 10 amino acids.
The IL-17RA-IL-17RB antagonist antibody includes at least all or part of a light chain or heavy chain variable region, or all or part of both light and heavy chain variable regions, which specifically bind to IL-17RA, or IL-17RB, or both IL-17RA and IL-17RB. Examples of fragments (i.e., "portions") of the variable regions include CDRs. In other words, the IL-17RA-IL-17RB antagonist antibody comprises at least one CDR of the variable region, wherein the CDR specifically binds IL-17RA, or IL-17RB, or both IL-17RA and IL-17RB. In alternative embodiments, the IL-17RA-IL-17RB antagonist antibody comprises at least two, or at least three, or at least four, or at least five, or at least all six CDRs of the variable region, wherein at least one CDR specifically binds IL-17RA, or IL-17RB, or both IL-17RA and IL-17RB. The CDRs may be from either the heavy or light chain and may be any of the three CDRs in each chain, i.e. each of the CDRs is independently selected from CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL 3.
Embodiments of the IL-17RA-IL-17RB antagonist antibodies can include scaffolds into which useful CDRs are grafted. Certain embodiments include a human scaffold component for a humanized antibody. In one embodiment, the scaffold structure is a traditional, tetrameric antibody structure. Thus, embodiments of the IL-17RA-IL-17RB antagonist antibodies can include additional components such as frameworks, J and D regions, constant regions, and the like, that make up the heavy or light chain. Embodiments of the IL-17RA-IL-17RB antagonist antibodies can include antibodies with modified Fc domains (referred to as Fc variants). An "Fc variant" refers to a molecule or sequence that is modified from a native Fc but still contains a binding site for a salvage receptor (FcRn). Additional examples of "Fc variants" include molecules or sequences humanized from non-human native Fc. In addition, native Fc contains sites that can be removed, as the structural features or biological activity provided by these sites are not essential to the fusion molecules of the invention. Thus, the term "Fc variant" encompasses molecules or sequences that lack one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).
Embodiments of IL-17RA-IL-17RB antagonist antibodies include human monoclonal antibodies. Human monoclonal antibodies directed against human IL-17RA, or IL-17RB, or both IL-17RA and IL-17RB can be made by any method known in the art, such as, but not limited toXenoMouseTMTechniques (see, for example, U.S. Pat. Nos. 6,114,598, 6,162,963, 6,833,268, 7,049,426, 7,064,244; Green et al, 1994, Nature Genetics 7: 13-21; Mendez et al, 1997, Nature Genetics 15: 146-. Another example of making fully Human antibodies includes the Ultimab Human Antibody Development SystemTMAnd Trans-phase TechnologyTM(Metarex Corp., Princeton, NJ), phage display Technology, ribosome display Technology (see, e.g., Cambridge Antibody Technology, Cambridge, UK), and any other method known in the art.
Certain embodiments of IL-17RA-IL-17RB antagonist antibodies include chimeric and humanized antibodies or fragments thereof. Generally, both chimeric and humanized antibodies refer to antibodies that bind to regions from more than one species. For example, chimeric antibodies typically comprise a variable region from a non-human and a constant region from a human. Humanized antibodies generally refer to non-human antibodies in which the variable domain framework regions have been exchanged for sequences found in human antibodies. Typically, in humanized antibodies, the entire antibody is encoded by a human polynucleotide except for the CDRs, or is identical to such an antibody except for within its CDRs. The CDRs, some or all of which are encoded by nucleic acids derived from non-human organisms, are grafted into the β -sheet framework of a human antibody variable region to produce an antibody, the specificity of which is determined by the grafted CDRs. The manufacture of such antibodies is well known in the art (see, e.g., Jones, 1986, Nature 321: 522-525; Verhoeyen et al, 1988, Science 239: 1534-1536). Humanized antibodies can also be generated using mice with genetically engineered immune systems or using any other method or technique known in the art (see, e.g., Roque et al, 2004, Biotechnol. prog.20: 639-654). In certain embodiments, the CDRs are human, and thus both humanized and chimeric antibodies herein can comprise some non-human CDRs; for example, humanized antibodies can be generated that comprise CDRH3 and CDRL3 regions, with one or more of the other CDR regions being of a different particular origin.
In one embodiment, the IL-17RA-IL-17RB antagonist antibody comprises a multispecific antibody. These are antibodies that bind to two (or more) different antigens. An example of a bispecific antibody known in the art is a "diabody". Diabodies can be produced by various methods known in the art, for example, chemically or from hybrid hybridomas (Holliger and Winter, 1993, Current Opinion Biotechnol.4: 446-. A specific embodiment of the multispecific IL-17RA-IL-17RB antagonist antibody is an antibody that has the ability to bind both IL-17RA and IL-17RB.
In alternative embodiments, the IL-17RA-IL-17RB antagonist antibody comprises a minibody. Minibodies are miniaturised antibody-like proteins comprising a single chain Fv (scFv; described below) linked to a CH3 domain (see, for example, Hu et al, 1996, Cancer Res.56: 3055-3061).
In alternative embodiments, the IL-17RA-IL-17RB antagonist antibody comprises a domain antibody; such as those described in U.S. patent No. 6,248,516. Domain antibodies (dAbs) are functional binding domains of antibodies, corresponding to the variable regions of the heavy (VH) or light (VL) chains of human antibodies. dAbs have a molecular weight of about 13kDa, or 1/10 less than the size of an intact antibody. dAbs are well expressed in a variety of hosts including bacterial, yeast and mammalian cell systems. Furthermore, dAbs are highly stable and retain activity even after being subjected to harsh conditions (e.g. freeze drying or heat denaturation). See, for example, U.S. patent 6,291,158; 6,582,915, respectively; 6,593,081, respectively; 6,172,197, respectively; U.S. serial No. 2004/0110941; european patent 0368684; U.S. Pat. No. 6,696,245, WO04/058821, WO04/003019 and WO 03/002609.
As previously described, the IL-17RA-IL-17RB antagonist antibody can include an antibody fragment, i.e., a fragment of any of the antibodies described herein, that retains specificity of binding to IL-17RA, or IL-17RB, or both IL-17RA and IL-17RB. Specific antibody fragments include, but are not limited to, (i) Fab fragments consisting of the VL, VH, CL and CH1 domains, (ii) Fd fragments consisting of the VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody, (iv) single variable fragmentsdAb fragments consisting of variable domains (see, e.g., Ward et al, 1989, Nature 341: 544-546), (v) isolated CDR regions, (vi) F (ab')2Fragments, bivalent fragments comprising two linked Fab fragments, (vii) single chain Fv molecules (scFv), wherein the VH domain and the VL domain are linked by a peptide linker that allows association of the two domains to form an antigen binding site (see, e.g., Bird et al, 1988Science 242: 423. SP 426; Huston et al, 1988, Proc. Natl. Acad. Sci.85: 5879; viii) bispecific single chain Fv dimers and (ix) "diabodies" or "triabodies", multivalent or multispecific fragments constructed by gene fusion (see, e.g., Tomlinson et al, 2000, Methods enzymol.326: 461. SP 479; WO 94/13804; Holliger et al, 1993, Proc. Natl. Acad. Sci.90: 6444. SP 6448). The antibody fragment may be modified. For example, the molecule may be stabilized by incorporating a disulfide bridge connecting the VH and VL domains (see, e.g., Reiter et al, 1996, Nature Biotech.14: 1239-1245). Again, as outlined herein, the non-CDR components of these fragments are preferably human sequences.
In additional embodiments, the IL-17RA-IL-17RB antagonist antibody comprises an antibody fusion protein (sometimes referred to herein as an "antibody conjugate"). The conjugation partner may be proteinaceous or non-proteinaceous; the latter are typically generated using functional groups on the antigen binding protein (see discussion regarding covalent modification of antigen binding proteins) and the conjugation partner. Such as linkers known in the art; such as homobifunctional or heterobifunctional linkers, as are well known in the art (see, for example, the Pierce Chemical Company catalog of 1994, incorporated herein by reference, for technical section on cross-linkers, page number 155-. Suitable conjugates include, but are not limited to, labels, drugs, or cytotoxic agents (including, but not limited to, cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or active fragments of such toxins) as described below. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, and the like. Cytotoxic agents also include radiochemicals, made by conjugating a radioisotope to an antigen binding protein, or binding a radionuclide to a chelator that has been covalently attached to an antigen binding protein. Additional embodiments utilize calicheamicin, auristatin, geldanamycin, and maytansine.
In one embodiment, the IL-17RA-IL-17RB antagonist antibody comprises an antibody analog, sometimes referred to as a "synthetic antibody". For example, various alternative protein scaffolds or artificial scaffolds can be grafted with CDRs from IL-17RA-IL-17RB antagonist antibodies. Such scaffolds include, but are not limited to, mutations introduced to stabilize the three-dimensional structure of the binding protein, as well as fully synthetic scaffolds composed of, for example, biocompatible polymers. See, for example, Korndorfer et al, 2003, Proteins: structure, Function, and Bioinformatics, volume 53, phase 1: 121-129; roque et al, 2004, biotechnol.prog.20: 639-654. In alternative embodiments, the IL-17RA-IL-17RB antagonist antibodies may include peptide antibody mimetics or "PAMs," as well as antibody mimetics that utilize a fibronectin component as a scaffold.
1.2IL-17RA-IL-17RB antagonists: peptides/polypeptides
Embodiments of IL-17RA-IL-17RB antagonists include proteins in the form of peptides and polypeptides that specifically bind to IL-17RA, or IL-17RB, or both IL-17RA and IL-17RB, and inhibit the activity of IL-17A, IL-17F and/or IL-25. In certain embodiments, an IL-17RA-IL-17RB antagonist inhibits association of subunits of the IL-17RA-IL-17RB heteromeric receptor complex; inducing (or preventing) a conformational change in the receptor subunits, thereby inhibiting their interaction; inhibiting the binding of a ligand (i.e., IL-25) to the heteromeric receptor complex (or subunit thereof), or inducing a conformational change in the heteromeric receptor complex (or subunit thereof) that inhibits the binding of a ligand thereto.
Embodiments include recombinant IL-17RA-IL-17RB antagonists. A "recombinant protein" is a protein made using recombinant techniques, i.e., by expressing a recombinant nucleic acid using methods known in the art.
As used herein, "peptide" refers to a molecule of 1-100 amino acids. Exemplary peptides can include those generated from a random pool that bind to IL-17RA, or IL-17RB, or both IL-17RA and IL-17RB, inhibit the association of L-17RA and IL-17RB to form an IL-17RA-IL-17RB heteromeric receptor complex, or inhibit IL-17RA-IL-17RB heteromeric receptor complex signaling. Such as peptide sequences derived from completely random sequences (e.g., selected by phage display or RNA-peptide screening), and sequences in which one or more residues of a naturally occurring molecule are replaced with an amino acid residue not present at that position in the naturally occurring molecule. Exemplary methods for identifying peptide sequences include phage display, E.coli display, ribosome display, RNA-peptide screening, chemical screening, and the like.
As used herein, "protein" refers to at least two covalently linked amino acids, and includes proteins, polypeptides, oligopeptides, and peptides. In certain embodiments, the two or more covalently linked amino acids are linked by a peptide bond. The protein may be composed of naturally occurring amino acids and peptide bonds, for example when the protein is produced recombinantly using expression systems and host cells as outlined below. Alternatively, in certain embodiments (e.g., when screening protein candidates for the ability to inhibit the association of IL-17RA and IL-17RB), the protein may comprise synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and norleucine) or peptidomimetic structures, i.e., "peptide or protein analogs," such as peptoids (see, Simon et al, 1992, Proc. Natl. Acad. Sci. U.S.A.89: 9367, incorporated herein by reference), which are resistant to proteases or other physiological and/or storage conditions. Such synthetic amino acids can be incorporated, particularly when the protein is synthesized in vitro by conventional methods well known in the art. In addition, any combination of peptidomimetics, synthetic and naturally occurring residues/structures may be used. "amino acid" also includes imino acid residues such as proline and hydroxyproline. The amino acid "R group" or "side chain" may be in the (L) -or (S) -configuration. In a particular embodiment, the amino acid is in the (L) -or (S) -configuration.
An example of an antagonist protein is the soluble IL-17RA-IL-17RB heteromeric receptor. Methods for preparing such soluble heteromeric receptors are known in the art, for example, as described in U.S. patent 6,589,764, issued to 7/8/2003, which is incorporated herein by reference. The IL-17A-IL-17B receptor complex includes IL-17RA and IL-17RB (and/or additional subunits) as proteins that are co-expressed in the same cell, or as receptor subunits that are linked to each other (e.g., covalently linked by any suitable means, such as by a cross-linking agent or polypeptide linker). In one embodiment, the heteromeric receptor is formed from a fusion protein of each receptor component with a portion of an antibody molecule (e.g., an Fc region). Alternatively, the heteromeric IL-17A-IL-17B receptor may be formed by a non-covalent interaction, such as the non-covalent interaction of biotin with avidin.
2.0IL-17RA-IL-17RB antagonists
As noted above, IL-17RA-IL-17RB antagonists include IL-17RA-IL-17RB antigen binding proteins, including but not limited to antibodies, peptides, and polypeptides, as well as other antagonists (including other polypeptides or proteins). An alternative embodiment of an IL-17RA-IL-17RB antagonist (e.g., an IL-17RA-IL-17RB antigen binding protein) includes covalent modification of the IL-17RA-IL-17RB antagonist. Such modification may be done post-translationally. For example, several covalent modifications of the IL-17RA-IL-17RB antagonist are introduced into the molecule by reacting specific amino acid residues of the antagonist with an organic derivatizing agent that is capable of reacting with selected side chains or N-or C-terminal residues. Examples of such modifications to IL-17RA-IL-17RB antagonists are shown below.
Most commonly, the cysteinyl residue is reacted with an α -haloacetate (and corresponding amine), such as chloroacetic acid or chloroacetamide, to give a carboxymethyl or carboxyamidomethyl derivative. Also by reacting with bromotrifluoroacetone, α -bromo- β - (5-imidazolyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoic acid, 2-chloromercuriyl-4-nitrophenol or chloro-7-nitrobenzo-2--1, 3-diazole reaction to derivatize cysteinyl residues. Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0, since this reagent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably carried out at a pH of 6.00.1M sodium cacodylate. Lysyl and the amino terminal residue are reacted with succinic anhydride or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysyl residue. Other suitable reagents for derivatizing alpha amino-containing residues include imidoesters, such as methyl picoliniminate; pyridoxal phosphate; pyridoxal; chloroborohydhdide; trinitrobenzenesulfonic acid; o-methyl isourea; 2, 4-pentanedione; and transaminase-catalyzed reactions with glyoxylate. Arginyl residues are modified by reaction with one or several conventional reagents, such as benzoylformaldehyde, 2, 3-butanedione, 1, 2-cyclohexanedione and ninhydrin. Because of the high pK of the guanidine functionaThe derivatization of arginine residues requires that the reaction be carried out under basic conditions. In addition, these reagents can react with the groups of lysine as well as the arginine epsilon amino group.
Specific modification of tyrosyl residues, of particular interest for introducing spectroscopic tags into tyrosyl residues, can be obtained by reaction with aromatic diazo compounds or tetranitromethane. Most commonly, N-acetylimidazole (N-acetylimidizole) and tetranitromethane are used to form O-acetyl tyrosines and 3-nitro derivatives, respectively. Use of125I or131I iodination of tyrosyl residues to produce labeled proteins for use in IL-17RA radioimmunoassays, the chloramine-T method described above is suitable. Pendant carboxyl groups (aspartyl or glutamyl) are selectively modified by reaction with a carbodiimide (R ' -N ═ C ═ N — R '), where R and R ' are optionally different alkyl groups, for example 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azocation-4, 4-dimethylpentyl) carbodiimide. In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
The derivatization of the bifunctional agent is useful for crosslinking the IL-17RA-IL-17RB antagonist to a matrix or surface of a water-insoluble carrier for use in various methods. Commonly used crosslinking agents include, for example, 1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide ester, such as esters with 4-azidosalicylic acid, homobifunctional imidoesters (including bis-succinimidyl esters (e.g., 3' -dithiobis (succinimidyl propionate)), and bifunctional maleimides (e.g., bis-N-maleimidyl-1, 8 octane), such as methyl-3- [ (p-azidophenyl) dithio ] imido ester, produce photoactivatable intermediates, alternatively, a water-insoluble reactive matrix such as cyanogen bromide activated carbohydrates and reactive substrates described in U.S. Pat. Nos. 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537 and 4,330,440 are used for protein immobilization.
Glutaminyl and asparaginyl residues are often deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Any form of these residues falls within the scope of the present invention. Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the alpha amino groups of the lysine, arginine and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp.79-86[1983]), acetylation of the N-terminal amino group and amidation of any C-terminal carboxyl group.
Another covalent modification of the IL-17RA-IL-17RB antagonist within the scope of the present invention includes altering the glycosylation pattern of the protein. As is known in the art, the glycosylation pattern can depend on the sequence of the protein (e.g., the presence or absence of particular glycosylated amino acid residues, discussed below), or the host cell or organism in which the protein is produced. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a 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 any of these tripeptide sequences in a polypeptide creates potential glycosylation sites. 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. The addition of glycosylation sites to the IL-17RA-IL-17RB antagonist is readily accomplished by altering the amino acid sequence such that it comprises one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also result from the addition of one or more serine or threonine residues or substitution of the starting sequence (for an O-linked glycosylation site). Briefly, the antigen binding protein amino acid sequence is preferably altered by a change at the DNA level, particularly by mutation of the DNA encoding the polypeptide of interest at preselected bases, such that codons are generated that will translate into the desired amino acids.
Another method of increasing the number of carbohydrate moieties on the IL-17RA-IL-17RB antagonist is by chemically or enzymatically coupling the glycoside to the protein. These methods have the advantage that they do not require the production of the protein in a host cell with glycosylation capability for N-or O-linked glycosylation. Depending on the coupling means used, the saccharide may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups (e.g. those of cysteine), (d) free hydroxyl groups (e.g. those of serine, threonine or hydroxyproline), (e) aromatic residues (e.g. those of phenylalanine, tyrosine or tryptophan) or (f) the amide group of glutamine. These methods are described in WO 87/05330 and Aplin and Wriston, 1981, CRC Crit. Rev. biochem., pp.259-306, published on 9/11 1987.
Removal of the carbohydrate moiety present in the starting IL-17RA-IL-17RB antagonist may be achieved chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid or equivalent compound. This treatment results in the cleavage of most or all of the sugars except the linked sugar (N-acetylglucosamine or N-acetylgalactosamine), leaving the polypeptide intact. Chemical deglycosylation was performed by hakimudin et al, 1987, arch.biochem.biophysis.259: 52 and Edge et al, 1981, anal. biochem.118: 131. Enzymatic cleavage of the carbohydrate moiety on a polypeptide can be achieved by using various endo-and exo-glycosidases, such as Thotakura et al, 1987, meth.enzymol.138: 350. Glycosylation at potential glycosylation sites can be prevented by using the compound tunicamycin, as described by Duskin et al, 1982, J.biol.chem.257: 3105. Tunicamycin blocks the formation of protein-N-glycoside linkages.
Another covalent modification of the IL-17RA-IL-17RB antagonist involves attaching antigen binding proteins to various non-proteinaceous polymers, including but not limited to various polyols, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene, in the manner set forth in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337. In addition, as is known in the art, amino acid substitutions can be made at various positions within the antigen binding protein to facilitate the addition of polymers such as PEG.
Covalent modification of IL-17RA-IL-17RB antagonists is included within the scope of the present invention, and is typically, but not always, achieved post-translationally. For example, several covalent modifications of the IL-17RA-IL-17RB antagonist are introduced into the molecule by reacting specific amino acid residues of the IL-17RA-IL-17RB antagonist with an organic derivatizing agent capable of reacting with selected side chains or N-or C-terminal residues.
In certain embodiments, the covalent modification of the antigen binding proteins of the present invention comprises the addition of one or more labels. Generally, depending on the method of detecting the label, the labels fall into a variety of categories a) isotopic labels, which can be radioisotopes or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) a redox active moiety; d) an optical dye; enzymatic groups (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase); e) a biotinylated group; and f) a predetermined polypeptide epitope recognized by the secondary reporter (e.g., leucine zipper sequence pair, binding site of a second antibody, metal binding domain, epitope tag, etc.). In certain embodiments, the labeling group is coupled to the antigen binding protein through spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used in the practice of the present invention.
Specific labels include optical dyes including, but not limited to, chromophores, phosphors, and fluorophores, the latter of which will be embodied in many instances. The fluorophore may be a "small molecule" fluorescent agent or a proteinaceous fluorescent agent. "fluorescent label" refers to any molecule that can be detected by its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, phycoerythrin, coumarin, methylcoumarin, pyrene, malachite green, stilbene, lucifer Yellow, Cascade BlueJ, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy5, Cy5.5, LC Red 705, Oregon green (Oregon green), Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow and R-Phycoerythrin (PE) (Molecular Probes, Eugene, OR), rhodamine, and Texas (Cys, Yellow), Cyxanth Yellow 355, Cys 5, Cys Ha green (Rockha green, Iregon green). Suitable optical dyes, including fluorophores, are described in Molecular Probes Handbook, Richard p.
Suitable proteinaceous fluorescent markers also include, but are not limited to, green fluorescent protein (including GFP (Chalfie et al, 1994, Science 263: 802-plus 805) of the species Renilla, Ptilosarcus or Aequorea), EGFP (Clontech Laboratories, Inc., Genbank accession No. U55762), blue fluorescent protein (BFP, Quantum Biotechnology, Inc.1801de Maison neve.West, 8th Floor, Montreal, Quebec, Canada H3H J9; Stauber, 1998, Biotechiques 24: 462-plus 471; Heim et al, 1996, Curr.6: 178-plus 182), enhanced yellow fluorescent protein (EImYFP, Clontech Laboratories, Laborie 6342), luciferase (Achiki et al, J54545442-plus 5442, WO 2607, WO 4685, WO 967: 967/11, WO 14611: 5876995 7, WO 4685, WO 14611: 3676, WO 9-plus) and WO 14611/11-plus. All references cited above are expressly incorporated herein by reference.
All of the above modifications of the antigen binding protein may also be made to any other protein class IL-17RA-IL-17RB antagonist, such as a heteromeric IL-17RA-IL-17RB complex or a polypeptide or peptide antagonist as described herein.
3.0 methods of use
The invention also provides methods of using IL-17RA-IL-17RB antagonists, including, for example, using IL-17RA-IL-17RB antagonists for diagnostic purposes or for therapeutic purposes. It will be appreciated that for treatment, the use of IL-17RA-IL-17RB antagonists is generally useful for reducing or ameliorating the disease and/or symptoms of the disease or condition for which treatment is administered. The present invention provides IL-17RA-IL-17RB antagonists as described throughout this specification, which antagonists may be used in the preparation or manufacture of medicaments for the treatment of various conditions or diseases as described herein. Furthermore, an effective amount of an IL-17RA-IL-17RB antagonist and a therapeutically effective amount of one or more additional active substances as described herein can be used in the preparation or manufacture of a medicament useful in such treatment. Certain embodiments include kits comprising, in part, an IL-17RA-IL-17RB antagonist; optionally, such a kit may comprise at least one additional active ingredient for separate, simultaneous or subsequent administration to a subject in need thereof.
Additional embodiments include methods of inhibiting IL-17RA and/or IL-17RB activation in IL-17 RA-and IL-17 RB-expressing cells using one or more IL-17RA-IL-17RB antagonists described herein. For example, a method of inhibiting IL-17RA and/or IL-17RB activation in a cell expressing IL-17RA and IL-17RB, comprising exposing the cell to an IL-17RA-IL-17RB antagonist, wherein the IL-17RA-IL-17RB antagonist binds to at least one subunit or component of the heteromeric receptor complex and partially inhibits or completely inhibits its association (by steric hindrance or conformational change) with another subunit or component of the heteromeric receptor complex, thereby preventing IL-17RA-IL-17RB heteromeric receptor complex formation. In certain embodiments, the IL-17RA-IL-17RB antagonist binds to a subunit of the heteromeric receptor complex. In alternative embodiments, the IL-17RA-IL-17RB antagonist binds to more than one subunit of the heteromeric receptor complex or to the heteromeric receptor complex itself. In certain embodiments, the IL-17RA-IL-17RB antagonist need not inhibit binding of a ligand (e.g., IL-25) to one or more components of the heteromeric receptor complex to inhibit IL-17RA and/or IL-17RB activation. In alternative embodiments, the IL-17RA-IL-17RB antagonist inhibits binding of a ligand (i.e., IL-25) to IL-17RA and/or IL-17RB, and inhibits IL-17RA and/or IL-17RB activation. Additional embodiments include a method wherein the IL-17RA-IL-17RB antagonist is an antigen binding protein as defined herein; the antigen binding protein is optionally in the form of a pharmaceutical composition.
Additional embodiments include methods of inhibiting IL-17RA and/or IL-17RB activation in cells expressing at least IL-17RA and IL-17RB in vivo, using one or more IL-17RA-IL-17RB antagonists described herein. For example, a method of inhibiting IL-17RA and/or IL-17RB activation in a cell expressing IL-17RA and IL-17RB in vivo comprises exposing the cell to an IL-17RA-IL-17RB antagonist, wherein the IL-17RA-IL-17RB antagonist binds to at least one subunit or component of the heteromeric receptor complex and partially inhibits or completely inhibits its association (by steric hindrance or conformational change) with another subunit or component of the heteromeric receptor complex, thereby inhibiting IL-17RA-IL-17RB heteromeric receptor complex activation. In certain embodiments, the IL-17RA-IL-17RB antagonist binds to a subunit of the heteromeric receptor complex. In alternative embodiments, the IL-17RA-IL-17RB antagonist binds to more than one subunit of the heteromeric receptor complex, or to the heteromeric receptor complex itself. In certain embodiments, the IL-17RA-IL-17RB antagonist need not block binding of a ligand (e.g., IL-25) to one or more components of the heteromeric receptor complex to inhibit IL-17RA and/or IL-17RB activation. In alternative embodiments, the IL-17RA-IL-17RB antagonist inhibits binding of a ligand (i.e., IL-25) to IL-17RA and/or IL-17RB, and inhibits IL-17RA and/or IL-17RB activation. Additional embodiments include a method wherein the IL-17RA-IL-17RB antagonist is an antigen binding protein as defined herein; the antigen binding protein is optionally in the form of a pharmaceutical composition.
Additional embodiments include methods of reducing proinflammatory mediators released upon activation of the IL-17RA-IL-17RB heteromeric receptor complex in cells expressing the complex in vivo using one or more IL-17RA-IL-17RB antagonists described herein. For example, a method of reducing the release of a proinflammatory mediator following activation of an IL-17RA-IL-17RB heteromeric receptor complex in a cell expressing the complex in vivo, comprising exposing the cell to an IL-17RA-IL-17RB antagonist, wherein the IL-17RA-IL-17RB antagonist binds to at least one subunit or component of the heteromeric receptor complex and partially inhibits or completely inhibits the formation or activation (via steric hindrance or conformational change) of the IL-17RA-IL-17RB heteromeric receptor complex, thereby reducing the release of the proinflammatory mediator. In certain embodiments, the IL-17RA-IL-17RB antagonist binds to a subunit of the heteromeric receptor complex. In alternative embodiments, the IL-17RA-IL-17RB antagonist binds to more than one subunit of the heteromeric receptor complex, or to the heteromeric receptor complex itself. In certain embodiments, the IL-17RA-IL-17RB antagonist need not inhibit the binding of a ligand (e.g., IL-25) to one or more components of the heteromeric receptor complex to reduce the release of pro-inflammatory mediators. In alternative embodiments, the IL-17RA-IL-17RB antagonist inhibits the binding of a ligand (i.e., IL-25) to IL-17RA and/or IL-17RB, and reduces the release of pro-inflammatory mediators. Additional embodiments include a method wherein the IL-17RA-IL-17RB antagonist is an antigen binding protein as defined herein; the antigen binding protein is optionally in the form of a pharmaceutical composition.
Additional embodiments include the method as described above, wherein the pro-inflammatory mediator is at least one of: IL-5, IL-6, IL-8, IL-12, IL-13, CXCL1, CXCL2, GM-CSF, G-CSF, M-CSF, IL-1 β, TNF α, RANK-L, LIF, PGE2, MMP3, MMP9, GRO α, NO, eotaxin, MCP-1 and IL-17RB, and any other pro-inflammatory mediator known in the art that is released from any cell by activation of IL-17RA and/or IL-17RB.
Additional embodiments include methods as described above for treating disorders associated with IL-17 family members (such as, but not limited to, inflammatory and autoimmune disorders) with said IL-17RA-IL-17RB antagonists.
Additional embodiments include methods of treating inflammation, wherein the IL-17RA-IL-17RB heteromeric receptor complex is partially or completely blocked from activation by administering one or more IL-17RA-IL-17RB antagonists described herein. For example, a method of treating inflammation in a patient in need thereof comprises administering an IL-17RA-IL-17RB antagonist to the patient, wherein the IL-17RA-IL-17RB antagonist binds to at least one subunit or component of the heteromeric receptor complex and partially inhibits or completely inhibits formation or activation (via steric hindrance or conformational change) of the heteromeric receptor complex, thereby facilitating treatment of inflammation. In certain embodiments, the IL-17RA-IL-17RB antagonist binds to a subunit of the heteromeric receptor complex. In alternative embodiments, the IL-17RA-IL-17RB antagonist binds to more than one subunit of the heteromeric receptor complex, or to the heteromeric receptor complex itself. In certain embodiments, the IL-17RA-IL-17RB antagonist need not block the binding of a ligand (e.g., IL-25) to one or more components of the heteromeric receptor complex, to be useful for treating inflammation. In alternative embodiments, the IL-17RA-IL-17RB antagonist inhibits the binding of a ligand (i.e., IL-25) to IL-17RA and/or IL-17RB, and is useful for treating inflammation. Additional embodiments include a method wherein the IL-17RA-IL-17RB antagonist is an antibody as defined herein; the antibody is optionally in the form of a pharmaceutical composition.
Additional embodiments include methods of treating autoimmune disorders, wherein the IL-17RA-IL-17RB heteromeric receptor complex is partially or completely blocked from activation by administering one or more IL-17RA-IL-17RB antagonists described herein. For example, a method of treating an autoimmune disorder in a patient in need thereof comprises administering an IL-17RA-IL-17RB antagonist to the patient, wherein the IL-17RA-IL-17RB antagonist binds to at least one subunit or component of the heteromeric receptor complex and partially inhibits or completely inhibits formation or activation of the heteromeric receptor complex, thereby facilitating treatment of the autoimmune disorder. In certain embodiments, the IL-17RA-IL-17RB antagonist binds to a subunit of the heteromeric receptor complex. In alternative embodiments, the IL-17RA-IL-17RB antagonist binds to more than one subunit of the heteromeric receptor complex, or to the heteromeric receptor complex itself. In certain embodiments, the IL-17RA-IL-17RB antagonist need not block binding of a ligand (e.g., IL-25) to one or more components of the heteromeric receptor complex, to be useful for treating an autoimmune disorder. In alternative embodiments, the IL-17RA-IL-17RB antagonist inhibits the binding of a ligand (i.e., IL-25) to IL-17RA and/or IL-17RB, and is useful for treating an autoimmune disorder. Additional embodiments include a method wherein the IL-17RA-IL-17RB antagonist is an antibody as defined herein; the antibody is optionally in the form of a pharmaceutical composition.
Additional embodiments include methods of treating inflammatory and/or autoimmune disorders as described above, wherein the disorders include, but are not limited to, cartilage inflammation and/or bone degeneration, arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis of the oligoarticular type, juvenile rheumatoid arthritis of the polyarticular type, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Latt's Syndrome, SEA Syndrome (seronegative, onset, arthropathy Syndrome), juvenile dermatomyositis, juvenile psoriasis arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, juvenile rheumatoid arthritis of the oligoarticular type, rheumatoid arthritis of the type I, rheumatoid arthritis of the type II, and/or, Polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, reiter's syndrome, SEA syndrome (seronegative, onset and arrest disease, arthrosis syndrome), dermatomyositis, psoriatic arthritis, scleroderma, systemic lupus erythematosus, vasculitis, myositis, polymyositis, dermatomyositis, osteoarthritis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, primary biliary fibrosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, psoriasis dropsicca, dermatosis, bullous psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus erythematosus, inflammatory bowel disease, inflammatory, Stutterian disease (Still's disease), Systemic Lupus Erythematosus (SLE), myasthenia gravis, Inflammatory Bowel Disease (IBD), Crohn's disease (Crohn's disease), ulcerative colitis, celiac disease, Multiple Sclerosis (MS), asthma (including extrinsic and intrinsic asthma and associated chronic inflammatory conditions, or airway hyperreactivity), chronic obstructive pulmonary disease (COPD, i.e. chronic bronchitis, emphysema), Acute Respiratory Disorder Syndrome (ARDS), respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, acute lung injury, allergic bronchopulmonary aspergillosis, allergic pneumonia, eosinophilic pneumonia, bronchitis, allergic bronchitis bronchiectasis, tuberculosis, allergic pneumonia, occupational asthma, asthma-like disorders, sarcoidosis, reactive airway disease (or dysfunction) syndrome, Gossypium, interstitial lung disease, hypereosinophilic syndrome, rhinitis, sinusitis and parasitic diseases of the lung, airway hyperreactivity associated with conditions induced by viruses such as Respiratory Syncytial Virus (RSV), parainfluenza virus (PIV), Rhinovirus (RV) and adenovirus, Guillain-Barre disease, type I diabetes, Graves ' disease, Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD and the like.
Additional embodiments include pharmaceutical compositions comprising a therapeutically effective amount of one or more IL-17RA-IL-17RB antagonists and pharmaceutically acceptable diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants. Furthermore, the invention provides methods of treating a patient by administering such pharmaceutical compositions, and methods of making or producing medicaments for treating the above conditions.
Acceptable formulation materials are non-toxic to the subject at the dosages and concentrations employed. In certain embodiments, the pharmaceutical composition may comprise formulation materials for altering, maintaining or preserving the composition, such as pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); an antibacterial agent; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffering agents (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); a filler; a monosaccharide; a disaccharide; and other carbohydrates (e.g., glucose, mannose, or dextrins); proteins (e.g., serum albumin, gelatin, or immunoglobulins); coloring agents, flavoring agents, and diluents; an emulsifier; hydrophilic polymers (e.g., polyvinylpyrrolidone); a low molecular weight polypeptide; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); a suspending agent; surfactants or wetting agents (e.g., poloxamers, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancing agents (e.g., alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); a delivery vehicle; a diluent; excipients and/or pharmaceutical adjuvants. See, REMINGTON' S PHARMACEUTICAL SCIENCES, 18 "Edition, (A.R. Genrmo, ed.), 1990, Mack Publishing Company.
In certain embodiments, the optimal pharmaceutical composition will be determined by one of skill in the art based on, for example, the intended route of administration, the form of delivery, and the desired dosage. See, for example, REMINGTON's breathing medicine science, supra. In certain embodiments, such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the IL-17RA-IL-17RB antagonist. In certain embodiments, the primary vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, saline or artificial cerebrospinal fluid, to which other substances commonly used in compositions for parenteral administration may be added. Neutral buffered saline or saline mixed with serum albumin are additional exemplary carriers. In particular embodiments, the pharmaceutical composition comprises Tris buffer at a pH of about 7.0-8.5 or acetate buffer at a pH of about 4.0-5.5, and may further comprise sorbitol or a suitable substitute. In certain embodiments, the IL-17RA-IL-17RB antagonist compositions can be prepared for storage as lyophilized cakes or aqueous solutions by combining a selected composition having the desired purity with an optional formulation (REMINGTON's SPHARMACEMENT SCIENCES, supra). Furthermore, in certain embodiments, the IL-17RA-IL-17RB antagonist preparation can be formulated as a lyophilizate using a suitable excipient, such as sucrose.
The pharmaceutical compositions of the present invention may be selected for parenteral delivery. Alternatively, the composition may be selected for inhalation or delivery through the digestive tract (e.g., oral). The preparation of such pharmaceutically acceptable compositions is within the skill of the art. The formulation components are preferably present within acceptable concentrations at the site of administration. In certain embodiments, a buffer is used to maintain the composition at physiological pH or at a slightly lower pH, typically in the range of about pH 5 to about pH 8.
When intended for parenteral administration, the IL-17RA-IL-17RB antagonist can be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired IL-17 receptor antigen binding protein in a pharmaceutically acceptable carrier. A particularly suitable carrier for parenteral injection is sterile distilled water, wherein the IL-17RA-IL-17RB antagonist is formulated as a sterile, isotonic solution and is well-preserved. In certain embodiments, the preparation may involve the formulation of the desired molecule with a substance, such as an injectable microsphere, a bioerodible particle, a polymer (e.g., polylactic acid or polyglycolic acid), a bead, or a liposome, which formulation may result in controlled or sustained release of the product that can be delivered by depot injection. In certain embodiments, hyaluronic acid may be used, having the effect of promoting sustained release duration in the circulation. In certain embodiments, an implantable drug delivery device can be used to introduce a desired antigen binding protein.
The pharmaceutical compositions of the present invention may be formulated for inhalation. In these embodiments, the IL-17RA-IL-17RB antagonist can be formulated as a dry, inhalable powder. Inhalation solutions can also be formulated with a propellant for aerosol delivery. In certain embodiments, the solution may be prepared for spraying. Pulmonary administration and formulation methods therefor are further described in international patent application No. PCT/US94/001875, which is incorporated by reference and describes pulmonary administration of chemically-modified proteins.
It is also contemplated that the formulation may be administered orally. The IL-17RA-IL-17RB antagonist administered in this manner may be formulated with or without carriers conventionally used in solid dosage (e.g., tablet and capsule) formulations. In certain embodiments, the capsule may be designed to release the active portion of the formulation at a point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. Additional substances may be included to facilitate uptake of the IL-17RA-IL-17RB antagonist. Diluents, flavoring agents, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binding agents may also be used.
Preferably, pharmaceutical compositions of the invention are provided comprising an effective amount of one or more IL-17RA-IL-17RB antagonists in admixture with non-toxic excipients suitable for the manufacture of tablets. The solution may be prepared in unit dosage form by dissolving the tablet in sterile water or other suitable vehicle. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or a binder, such as starch, gelatin or acacia; or a lubricant, such as magnesium stearate, stearic acid or talc.
Additional pharmaceutical compositions will be apparent to those skilled in the art, which compositions include formulations that allow the IL-17RA-IL-17RB antagonist to be included in a sustained or controlled release dosage formulation. Techniques for formulating various other sustained or controlled release delivery vehicles (e.g., liposome carriers, bioerodible microparticles or porous beads, and depot injections) are also known to those skilled in the art. See, for example, international patent application No. PCT/US93/00829, which is incorporated by reference and describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions. Sustained release preparations may comprise semipermeable polymeric matrices in the form of shaped articles, for example, films, or microcapsules. The sustained release matrix may comprise a polyester, a hydrogel, a polylactide (as disclosed in U.S. Pat. No. 3,773,919 and European patent application publication No. EP 058481, each incorporated by reference), a copolymer of L-glutamic acid and L-glutamic acid gamma-ethyl ester (Sidman et al, 1983, Biopolymers 2: 547-556), poly (2-hydroxyethyl methacrylate) (Langer et al, 1981, J.biomed.Mater.Res.15: 167-277 and Langer, 1982, chem.Tech.12: 98-105), ethylene vinyl acetate (Langer et al, 1981, supra) or poly-D (-) -3-hydroxybutyric acid (European patent application publication No. EP 133,988). Sustained release compositions may also comprise liposomes, which may be prepared by any of several methods known in the art. See, for example, Eppstein et al, 1985, proc.natl.acad.sci.u.s.a.82: 3688-; european patent application publication nos. EP 036,676, EP 088,046 and EP 143,949, which are incorporated by reference.
Pharmaceutical compositions for in vivo administration are typically provided as sterile formulations. Sterilization can be achieved by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization can be performed using this method either before or after lyophilization and reconstitution. Compositions for parenteral administration may be stored in lyophilized form or in aqueous solution. Parenteral compositions are typically placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
Once formulated into the pharmaceutical composition, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations may be stored in a ready-to-use form or in a form that is reconstituted prior to administration (e.g., as a lyophile). The invention also provides kits for preparing single dosage administration units. The kits of the invention may each comprise a first container having a dry protein and a second container having a liquid formulation. In certain embodiments of the invention, kits are provided comprising single and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes).
The therapeutically effective amount of a pharmaceutical composition comprising an IL-17RA-IL-17RB antagonist used will depend, for example, on the treatment setting and the subject. One skilled in the art will recognize that the appropriate dosage level for treatment will vary depending, in part, on the molecule being administered, the indication for which the IL-17RA-IL-17RB antagonist is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (age and general health) of the patient. In certain embodiments, the clinician may titrate the dosage and alter the route of administration to obtain the optimal therapeutic effect. Typical dosages may vary from about 0.1 μ g/kg up to about 30mg/kg or more, depending on the factors discussed above. In particular embodiments, the dosage may vary from 0.1 μ g/kg up to about 30mg/kg, optionally from 1 μ g/kg up to about 30mg/kg or from 10 μ g/kg up to about 5 mg/kg. Of course, it should be understood that this is at the discretion of a qualified physician and that these dosages are exemplary only. The frequency of administration will depend on the pharmacokinetic parameters of the particular IL-17RA-IL-17RB antagonist in the formulation used. Typically, the clinician administers the composition until a dosage is reached that achieves the desired effect. Thus, the composition may be administered over time in a single dose, or in two or more doses (which may comprise the same or different amounts of the desired molecule), or by continuous infusion through an implanted device or catheter. Further refinement of appropriate dosages is routinely made by those of ordinary skill in the art and is within the scope of their routinely performed tasks. Suitable dosages can be determined by using suitable dose response data. In certain embodiments, the IL-17RA-IL-17RB antagonist can be administered to the patient over an extended period of time. Chronic administration of IL-17RA-IL-17RB antagonists, e.g., anti-human antigen antibodies produced in non-human animals, e.g., non-fully human antibodies or non-human antibodies produced in non-human species, minimizes deleterious immune or allergic responses that are typically associated with non-fully human IL-17RA-IL-17RB antagonists.
The route of administration of the pharmaceutical composition is in accordance with known methods, e.g., oral, by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional injection; by slow release systems or by implanted devices. In certain embodiments, the composition can be administered by bolus injection or continuous infusion or by an implanted device.
The compositions may also be administered topically by implantation of a film, sponge, or other suitable substance that has absorbed or encapsulated the desired molecule. In certain embodiments using an implanted device, the device may be implanted in any suitable tissue or organ and may deliver the desired molecule by diffusion, timed release bolus injection, or continuous administration.
The IL-17RA-IL-17RB antagonists described herein can be used in combination (pre-treatment, post-treatment, or concurrent treatment) with pharmaceutical agents useful for the treatment of the diseases and conditions described herein. In one embodiment, the IL-17RA-IL-17RB antagonists described herein can be used with any one or more of those for treatment or prognosisTNF inhibitors for use in combination (pre-treatment, post-treatment, or concurrent treatment) to protect against the diseases and conditions described herein, such as, but not limited to, all types of soluble TNF receptors, including etanercept (e.g.) And all types of monomeric or polymeric p75 and/or p55TNF receptor molecules and fragments thereof; anti-human TNF antibodies, such as but not limited to infliximab (e.g.) And D2E7 (e.g.) And the like. Such TNF inhibitors include compounds and proteins that block the in vivo synthesis or extracellular release of TNF. In particular embodiments, the invention relates to the use of an IL-17RA-IL-17RB antagonist in combination (pre-treatment, post-treatment, or concurrent treatment) with any one or more of the following TNF inhibitors: TNF binding proteins (soluble TNF receptors type I and soluble TNF receptors type II ("sTNFRs") as defined herein), anti-TNF antibodies, granulocyte colony stimulating factor, thalidomide, BN 50730, tenidap, E5531, tiapafant PCA 4248, nimesulide, panavir, rolipram, RP 73401, peptides T, MDL 201, 449A, (1R, 3S) -cis-1- [9- (2, 6-diaminopurine) hydrochloride]-3-hydroxy-4-cyclopentene, (1R, 3R) -trans-1- [9- (2, 6-diamino) purine]-3-acetoxycyclopentane, (1R, 3R) -trans-1- (9-adenine) -3-azidocyclopentane hydrochloride and (1R, 3R) -trans-1- (6-hydroxy-purin-9-yl) -3-azidocyclopentane. TNF binding proteins are disclosed in the art (EP 308378, EP 422339, GB 2218101, EP 393438, WO 90/13575, EP 398327, EP 412486, WO91/03553, EP 418014, JP 127,800/1991, EP 433900, U.S. Pat. No. 5,136,021, GB 2246569, EP 464533, WO92/01002, WO 92/13095, WO92/16221, EP 512528, EP 526905, WO 93/07863, EP 568928, WO 93/21946, WO 93/19777, EP 417563, WO 94/06476 and PCT International application No. PCT/US 97/12244). For example, EP 393438 and EP 422339 indicate the amino acid and nucleic acid sequences of type I soluble TNF receptors (also known as "sTNFR-I" or "30 kDa TNF inhibitors") and type II soluble TNF receptors (also known as "sTNFR-II" or "40 kDa TNF inhibitors") (collectively "sTNFRs") as well as modifications thereof (e.g., fragments, functional derivatives and variants). EP 393438 and EP 422339 also disclose methods for isolating the gene responsible for encoding the inhibitor, cloning the gene into suitable vectors and cell types and expressing the gene for the production of the inhibitor. In addition, multivalent forms of sTNFR-I and sTNFR-II (i.e., molecules comprising more than one active moiety) have also been disclosed. In one embodiment, the multivalent form may be constructed by chemically coupling the at least one TNF inhibitor and the further moiety to any clinically acceptable linker, such as polyethylene glycol (WO92/16221 and WO 95/34326), by peptide linker (Neve et al (1996), Cytokine, 8 (5): 365-. anti-TNF antibodies include the MAK 195F Fab antibody (Holler et al (1993), 1st International Symposium on Cytokines in Bone Marrow Transplantation, 147), CDP 571 anti-TNF monoclonal antibody (Rankin et al (1995), British Journal of Rheumatology, 34: 334-.
The IL-17RA-IL-17RB antagonists described herein can be used with all types of IL-1 inhibitors, such as, but not limited to, kiniret (e.g.) In combination (pre-treatment, post-treatment or concurrent treatment). Interleukin-1 receptor antagonists (IL-1ra) are human proteins that are natural inhibitors of interleukin-1. Interleukin-1 receptor antagonists and methods of making and using sameProcesses are described in U.S. Pat. No. 5,075,222, WO 9I/08285, WO 91/17184, AU 9173636, WO92/16221, WO 93/21946, WO 94/06457, WO 94/21275, FR 2706772, WO 94/21235, DE 4219626, WO 94/20517, WO 96/22793 and WO 97/28828. The proteins include glycosylated as well as non-glycosylated IL-1 receptor antagonists. Specifically, three types of IL-1ra (IL-1ra α, IL-1ra β and IL-1rax), each encoded by the same DNA coding sequence and variants thereof, are disclosed and described in U.S. Pat. No. 5,075,222. Methods for preparing IL-1 inhibitors, particularly IL-1ras, are also disclosed in patent 5,075,222. Additional classes of interleukin-1 inhibitors include compounds that specifically prevent the activation of IL-1 cell receptors. Such compounds include IL-1 binding proteins, such as soluble receptors and monoclonal antibodies. Such compounds also include monoclonal antibodies to the receptor. Another class of interleukin-1 inhibitors includes compounds and proteins that block the in vivo synthesis and/or extracellular release of IL-1. Such compounds include substances that affect IL-1 gene transcription or IL-1 proprotein processing.
The IL-17RA-IL-17RB antagonists described herein may be used with all types of CD28 inhibitors, such as, but not limited to, Abirapep (abatacept) (e.g., as) In combination (pre-treatment, post-treatment or concurrent treatment). The IL-17RA-IL-17RB antagonist may be used in combination (pre-treatment, post-treatment, or concurrent treatment) with one or more cytokines, lymphokines, hematopoietic factors, and/or anti-inflammatory agents.
Treatment of the diseases and conditions described herein may include the use of a first-line drug to control pain and/or inflammation in combination with (pre-treatment, post-treatment, or concurrent treatment with) treatment with one or more IL-17RA-IL-17RB antagonists provided herein. These drugs are classified as non-steroidal anti-inflammatory drugs (NSAIDs). The second class of therapies includes corticosteroid long-acting antirheumatic drugs (SAARDs) or Disease Modifying (DM) drugs. Information on The following compounds can be found in The Merck Manual of Diagnosis and Therapy, eighteenth edition, Merck, Sharp & Dohme Research Laboratories, Merck & Co., Rahway, N.J. (2006) and Pharmaprojects, PJB Publications Ltd.
In particular embodiments, the invention relates to the use of an IL-17RA-IL-17RB antagonist and any one or more NSAIDs for the treatment of the diseases and conditions described herein (pre-treatment, post-treatment or concurrent treatment). The anti-inflammatory effects of NSAIDs are at least partially attributed to The inhibition of prostaglandin synthesis (Goodman and Gilman, "The pharmaceutical Basis of Therapeutics," MacMillan 7th edition (1985)). NSAIDs can be characterized by at least nine groups: (1) salicylic acid derivatives, (2) propionic acid derivatives, (3) acetic acid derivatives, (4) fenamic acid derivatives, (5) carboxylic acid derivatives, (6) butyric acid derivatives, (7) oxicams, (8) pyrazoles and (9) pyrazolones.
In another embodiment, the invention relates to the use of an IL-17RA-IL-17RB antagonist in combination (pre-treatment, post-treatment, or concurrent treatment) with any one or more of a salicylate derivative, prodrug ester, or pharmaceutically acceptable salt thereof. Such salicylic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof include: acexamine, alopridine, aspirin, benorilate, bromosaligenin, calcium acetylsalicylate, choline magnesium trisalicylate, magnesium salicylate, choline salicylate, diflusinal, etosalate, fendulac, gentisic acid, ethylene glycol monosalicylate, imidazole salicylate, lysine acetylsalicylic acid, 5-aminosalicylic acid, salicylic morpholine, 1-naphthyl salicylate, olsalazine, pasamide, phenyl acetylsalicylate, phenyl salicylate, salicylamide, acetylsalicylamide, O-acetic acid, salsalate, sodium salicylate, and sulfasalazine. This group is also intended to cover structurally related salicylic acid derivatives with similar analgesic and anti-inflammatory properties.
In yet another embodiment, the invention relates to the use of an IL-17RA-IL-17RB antagonist in combination (pre-treatment, post-treatment, or concurrent treatment) with any one or more of the propionic acid derivatives, prodrug esters, or pharmaceutically acceptable salts thereof. The propionic acid derivative and a prodrug thereofEsters and pharmaceutically acceptable salts include: alminoprofen and benzeneLoxfen, bucloxic acid, carprofen, dexindoprofen, fenoprofen, flurnoprofen, flurbiprofen, ibuprofen aluminum, ibuspron, indoprofen, isoprofen, ketoprofen, loxoprofen, miroprofen, naproxen sodium, oxaprozin, pioprofen, pimeloprofen, pimelaprofen, pirprofen, pranoprofen, protiazic acid, piridoprofen, suprofen, tiaprofenic acid, and tioxateRofen. This group is also intended to cover structurally related propionic acid derivatives with similar analgesic and anti-inflammatory properties.
In yet another embodiment, the invention relates to the use of an IL-17RA-IL-17RB antagonist in combination (pre-treatment, post-treatment, or concurrent treatment) with any one or more of an acetic acid derivative, prodrug ester, or pharmaceutically acceptable salt thereof. The acetic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof include: acemetacin, alclofenac, amfenac, bufexamic acid, cinnamyl, clopidogenic acid, demeclocin, diclofenac potassium, diclofenac sodium, etodolac, felbinac, fenclofenac, fenclorac, fentiazac, furofenac, dimeglumacin, ibufenac, indomethacin, triazolic acid, isofenac, clonazole acid, difenoxacin, oxapinacin, oxpinac, pimetacin, proglumic, sulindac, tamicin, tiamidt, thiofenac, tolmetin sodium, zidometacin, and zomepirac. This group is also intended to cover structurally related acetic acid derivatives with similar analgesic and anti-inflammatory properties.
In another embodiment, the invention relates to the use of an IL-17RA-IL-17RB antagonist in combination (pre-treatment, post-treatment, or concurrent treatment) with any one or more fenamic acid derivatives, prodrug esters, or pharmaceutically acceptable salts thereof. The fenamic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof include: phenylethylanthranilic acid, etofenamate, flufenamic acid, isonicotin, meclofenamic acid, mefenamic acid, niflumic acid, talniflumic ester, terrofenamate, tolfenamic acid, and ufenamate. This group is also intended to cover structurally related fenamic acid derivatives with similar analgesic and anti-inflammatory properties.
In another embodiment, the invention relates to the use of an IL-17RA-IL-17RB antagonist in combination (pre-treatment, post-treatment, or concurrent treatment) with any one or more carboxylic acid derivatives, prodrug esters, or pharmaceutically acceptable salts thereof. The carboxylic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof that can be used include: clidanac, diflunisal, flunixin, inoridine, ketorolac, and tenolidine. This group is also intended to cover structurally related carboxylic acid derivatives with similar analgesic and anti-inflammatory properties.
In yet another embodiment, the invention relates to the use of an IL-17RA-IL-17RB antagonist in combination (pre-treatment, post-treatment, or concurrent treatment) with any one or more of the butyric acid derivatives, prodrug esters, or pharmaceutically acceptable salts thereof. The butyric acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof include: butapropiophenone hydrazine, butenafine, fenbufen and biphenyl butyric acid. This group is also intended to cover structurally related butyric acid derivatives with similar analgesic and anti-inflammatory properties.
In another embodiment, the invention relates to the use of an IL-17RA-IL-17RB antagonist in combination (pre-treatment, post-treatment, or concurrent treatment) with any one or more of the oxicams, prodrug esters, or pharmaceutically acceptable salts thereof. The oxicams, prodrug esters and pharmaceutically acceptable salts thereof include: flexion typeOxicams, enoxicam, isoxicam, piroxicam, sudoxicam, tenoxicam and 4-hydroxy-1, 2-benzothiazine 1, 1-dioxido 4- (N-phenyl) -carboxamide. This group is also intended to cover structurally related oxicams with similar analgesic and anti-inflammatory properties.
In yet another embodiment, the invention relates to the use of an IL-17RA-IL-17RB antagonist in combination (pre-treatment, post-treatment, or concurrent treatment) with any one or more pyrazoles, prodrug esters, or pharmaceutically acceptable salts thereof. The pyrazoles, prodrug esters and pharmaceutically acceptable salts thereof that can be used include: diphenyl oxazole and epiprazole. This group is also intended to cover structurally related pyrazoles with similar analgesic and anti-inflammatory properties.
In another embodiment, the invention relates to the use of an IL-17RA-IL-17RB antagonist in combination (pre-treatment, post-treatment, or concurrent treatment) with any one or more pyrazolones, prodrug esters, or pharmaceutically acceptable salts thereof. The pyrazolones, prodrug esters and pharmaceutically acceptable salts thereof that can be used include: azapropazone (apazone), azapropazone (azapropazone), benproperazone, fiprone, mofebuzone, molbupone, oxybutyzone, phenylbutazone, pipobuzone, isopropanyline, raminones, succinbuperazone, and thiazolidinoketone (thiazolidinobutazone). This group is also intended to cover structurally related pyrazolones having similar analgesic and anti-inflammatory properties.
In another specific embodiment, the invention relates to the use of an IL-17RA-IL-17RB antagonist in combination (pre-treatment, post-treatment, or concurrent treatment) with any one or more of the following NSAIDs: epsilon-acetamido hexanoic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amicin, anilazine, antrofinin, bendazac acid, bendazac lysine, benzydamine, beprozin, bromopimol, bucolone, butylbenzazole, ciprofloxacin, clotrimipramine ester, dacoziamine, diprosamide, detoxamine, dipyridamide, difenimide, difeniramide, difeniramine, ditisalamide, ditrazazole, emofazone, mefanazine (fanetilate), fenflumizole, flofenin, flunimide, flunixin, fluquinazone, fopirtine, salicylic acid, guanymetrix, guaiazolene, isonixi, hydranilide hydrochloride, lenide, clemizole, clonixine, cloquinazine, lysine, oxsulosin, oxsulindac, olne, olsalazine, olsala, Renitorine, pirimozole, perisoxazole citrate, piprofen, piproxen, pirazolic acid, pirfenidone, Proquinol, Prosxazole, thielavin B, telflumidazole, timegadine, tolmetin, tolado, trpitamid and those named with the company code, for example 480156S, AA861, AD1590, AFP802, AFP860, AI77B, AP504, AU8001, BPPC, BW540C, CHINOIN 127, CN100, EB382, EL508, F1044, FK-506, GV3658, ITF182, KCNTEI6090, KME4, LA2851, MR714, MR897, MY309, ONO3144, PR823, PV102, PV108, R830, RS2131, SCR152, SPASH 440, SIR, S510, SQ 133, ST281, ST 1, ST 4139, ST 6001-539 770, Wbenzoyl 6026, WX 770-5, WX 770, W6026 and WX 5. This group is also intended to cover structurally related NSAIDs having similar analgesic and anti-inflammatory properties to said NSAIDs.
In yet another embodiment, the invention relates to the combination (pre-treatment, post-treatment, or concurrent treatment) of an IL-17RA-IL-17RB antagonist with any one or more corticosteroids, prodrug esters or pharmaceutically acceptable salts thereof for the treatment of the diseases and disorders described herein. Corticosteroids, prodrug esters and pharmaceutically acceptable salts thereof include hydrocortisone and compounds derived from hydrocortisone such as 21-acetoxypregnenolone, alclomerase, algestrone, amcinonide, beclomethasone, betamethasone valerate, budesonide, prednisone, clobetasol propionate, clobetasol, clocortolone, prednole, corticosterone, cortisone, clobazole, deflazacon, desonide, desoximerasone, dexamethasone, diflorasone, diflorolone, difluprednate, glycyrrhetinic acid, fluzacort, fluorodichloropine, flumethasone pivalate, flucolone acetate, flunisolone, fluneolone ester, fluocinolone acetonide, flucoconide, flucolone butyrate, fluocinolone caproate, fluxolone acetate, fluxolone acetonide, flupredone acetate, fluprednide, and the like, Flupredlone, flurandrenolide, formocortat, halcinonide, halomethasone, haloprednisolone acetate, hydrocortisone ester, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone phosphate, hydrocortisone 21-sodium succinate, hydrocortisone tertbutylate, methylprednisolone, mometasone furoate, paramethasone, prednisolone 21-diedryaminoacetate, prednisolone sodium phosphate, prednisolone sodium succinate, prednisolone 21-sodium sulfobenzoate, prednisolone 21-sodium stearoylethanolaminate, prednisolone tert-butyl ethyl ester, prednisolone 21-trimethyl acetate, prednisone, prednisolone valerate, prednisolone 21-diethylaminoethyl ester, ticortisone, triamcinolone acetonide and triamcinolone acetonide. This group is also intended to cover structurally related corticosteroids that have similar analgesic and anti-inflammatory properties.
In another embodiment, the invention relates to IL-17RA-IL-17RB antagonists in combination (pre-treatment, post-treatment, or concurrent treatment) with any one or more of long acting antirheumatic drugs (SAARDs) or disease modifying antirheumatic drugs (DMARDS), prodrug esters, or pharmaceutically acceptable salts thereof for use in treating the diseases and disorders described herein. SAARDs or DMARDS, prodrug esters and pharmaceutically acceptable salts thereof include: sodium allose, auranofin, chlorthioglucose, thioacetamide, azathioprine, brequinar sodium, buclizine, 3-gold sulfide-2-propanol-1-sulfonic acid calcium, chlorambucil, chloroquine, chlorobuzali, cuproxyline, cyclophosphamide, cyclosporine, dapsone, 15-deoxyspergualin, diacerein, glucosamine, gold salts (e.g., cycloquinate, disodium aurothiodine, sodium thioaurothiosulfate), hydroxychloroquine sulfate, hydroxyurea, kebuperazone, levamisole, clobenzaprine, melittin, 6-mercaptopurine, methotrexate, mizoribine, mycophenolate mofetil, calcium thioacetate, nitrogen mustard, D-penicillamine, pyridinidazole (pyrinolidazole) (e.g., SKNF86002 and SB203580), rapamycin, thiols, thymopoietin, and vincristine. This group is also intended to cover structurally related SAARDs or DMARDs with similar analgesic and anti-inflammatory properties.
In another embodiment, the invention relates to the use of an IL-17RA-IL-17RB antagonist (pre-treatment, post-treatment, or concurrent treatment) with any one or more COX2 inhibitor, prodrug ester, or pharmaceutically acceptable salt thereof for the treatment of the diseases and conditions described herein. Examples of COX2 inhibitors, prodrug esters, or pharmaceutically acceptable salts thereof include, for example, celecoxib. This group is also intended to cover structurally related COX2 inhibitors with similar analgesic and anti-inflammatory properties. Examples of COX-2 selective inhibitors include, but are not limited to, etoricoxib, valdecoxib, celecoxib, lincomone, lumiracoxib, rofecoxib, and the like.
Treatment of the diseases and disorders described herein includes the use of first-line drugs that control inflammatory responses (e.g., hyperresponsiveness in the airways of an affected individual) in combination (pre-treatment, post-treatment, or concurrent treatment) with the treatment of one or more IL-17RA-IL-17RB antagonists provided herein. Drugs commonly used to treat such diseases or conditions include β 2-agonists, leukotriene inhibitors, methylxanthines, anti-inflammatory agents, anticholinergics, bronchodilators, corticosteroids, and combinations of these agents. Information on The following compounds can be found in The Merck Manual of Diagnosis and Therapy, eighteenth edition, Merck, Sharp & Dohme Research Laboratories, Merck & Co., Rahway, N.J. (2006) and Pharmaprojects, PJB Publications Ltd.
In another embodiment, the invention relates to IL-17RA-IL-17RB antagonists (pre-treatment, post-treatment, or concurrent treatment) and any one or more beta 2-agonists, prodrug esters, or pharmaceutically acceptable salts thereof, for use in treating the diseases and disorders described herein. Examples of beta-2 agonists, prodrug esters or pharmaceutically acceptable salts thereof include, for example, salbutamol (RProair HFA、Ventolin ) Oxetalin (b), andthe solution is absorbed,Syrup), pirbuterol acetate (Maxair)) And terbutaline sulfate (f)). Long-acting beta-2 agonists (some of which are associated with other substances (e.g. pharmaceutical compositions)And) Combinations) are known and useful in combination with IL-17RA-IL-17RB antagonists.
Another embodiment of the invention relates to the use of an IL-17RA-IL-17RB antagonist (pre-treatment, post-treatment, or concurrent treatment) with any one or more leukotriene inhibitors, prodrug esters or pharmaceutically acceptable salts thereof for the treatment of the diseases and disorders described herein. Examples of leukotriene inhibitors, prodrug esters or pharmaceutically acceptable salts thereof include, for example, zileutonZafirlukastAnd montelukast
In yet another embodiment, the invention relates to the use of an IL-17RA-IL-17RB antagonist (pre-treatment, post-treatment, or concurrent treatment) with any one or more methylxanthines, prodrug esters, or pharmaceutically acceptable salts thereof, for the treatment of the diseases and disorders described herein. Examples of methylxanthines, prodrug esters or pharmaceutically acceptable salts thereof include, for example, theophylline (e.g., theophylline)Slo- Slo- Theo- Theo- ) And aminophylline (e.g. aminophylline)。
In another embodiment, the invention relates to IL-17RA-IL-17RB antagonists (pre-treatment, post-treatment, or concurrent treatment) with any one or more anti-inflammatory agents, prodrug esters, or pharmaceutically acceptable salts thereof for use in the treatment of the diseases and disorders described herein. Examples of such anti-inflammatory agents include, but are not limited to, Cromolyn (Cromolyn)And nedocromil
Another embodiment of the invention relates to the use of an IL-17RA-IL-17RB antagonist (pre-treatment, post-treatment, or concurrent treatment) with any one or more anticholinergics, prodrug esters, or pharmaceutically acceptable salts thereof for the treatment of the diseases and disorders described herein. Examples of such anticholinergics, prodrug esters, or pharmaceutically acceptable salts thereof include, but are not limited to, ipratropium bromideAnd tiotropium bromide (tiotropium)
In another embodiment, the invention relates to IL-17RA-IL-17RB antagonists (pre-treatment, post-treatment, or concurrent treatment) in combination with any one or more corticosteroids, prodrug esters or pharmaceutically acceptable salts thereof for use in the treatment of the diseases and disorders described herein. Examples of inhaled corticosteroids include beclomethasone dipropionate (I)And) Triamcinolone acetonide (b)Tri- ) And flunisolideExamples of other corticosteroids useful in the present invention include Prednisone (Prednisone)) And prednisolone
Yet another embodiment of the invention relates to the use of an IL-17RA-IL-17RB antagonist (pre-treatment, post-treatment, or concurrent treatment) with any one or more inhaled beta-2 agonists, prodrug esters, or pharmaceutically acceptable salts thereof, for the treatment of the diseases and disorders described herein. Examples of corticosteroids, prodrug esters or pharmaceutically acceptable salts thereof include, for example, salbutamolOcinalinPibuterol acetateTerbutalineIsotadineAnd levosalbutamol (levalbuterol)
In yet another embodiment, the invention relates to the use of an IL-17RA-IL-17RB antagonist (pre-treatment, post-treatment, or concurrent treatment) with any one or more bronchodilators (or anticholinergics), prodrug esters, or pharmaceutically acceptable salts thereof for the treatment of the diseases and disorders described herein. Examples of bronchodilators include ipratropiumAnd tiotropium bromide
Treatment of the diseases and conditions described herein may include the use of a first-line drug to treat or control infectious disease in combination (pre-treatment, post-treatment, or concurrent treatment) with the treatment of one or more IL-17RA-IL-17RB antagonists provided herein. Drugs commonly used to treat such diseases or conditions include antibiotics, antimicrobials, antivirals, and combinations thereof. Information on The following compounds can be found in The Merck Manual of Diagnosis and Therapy, eighteenth edition, Merck, Sharp & Dohme Research Laboratories, Merck & Co., Rahway, N.J. (2006) and Pharmaprojects, PJB Publications Ltd.
In yet another embodiment, the invention relates to the use of an IL-17RA-IL-17RB antagonist in combination (pre-treatment, post-treatment, or concurrent treatment) with any one or more of an antibacterial agent, prodrug ester, or pharmaceutically acceptable salt thereof for the treatment of the diseases and conditions described herein. Antibacterial agents include, for example, the broad spectrum of penicillins, cephalosporins and other beta-lactams, aminoglycosides, pyrroles, quinolones, macrolides, rifamycins, tetracyclines, sulfonamides, lincosamides, and polymyxins. The penicillins include, but are not limited to, penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, ampicillin/sulbactam, amoxicillin/clavulanate (clavulanate), natacillin, cyclopenicillin, bacampicillin, carbenicillin, cairinin, ticarcillin/clavulanate, azlocillin, mezlocillin, piperacillin (peperacili), and mecillin. The cephalosporins and other beta-lactams include, but are not limited to, cephalothin, cefapirin, cephalexin, cephradine, cefazolin, cefadroxil, cefaclor, cefamandole, cefotetan, cefetametCetin, ceruroxime, cefonicid, cefradine, cefixime, cefotaxime, moxalactam, ceftizoxime, ceftriaxone, cephaperazone, ceftazidime, imipenem, and aztreonam. The aminoglycosides include, but are not limited to, streptomycin, gentamicin, tobramycin, amikacin, netilmicin, kanamycin, and neomycin. The azoles include, but are not limited to, fluconazole. The quinolones include, but are not limited to, nalidixic acid, norfloxacin, enoxacin, ciprofloxacin, ofloxacin, sparfloxacin, and temafloxacin. Macrolides include, but are not limited to, erythromycin, spiramycin, and azithromycin. The rifamycins include, but are not limited to rifampin. The tetracyclines include, but are not limited to, spicycline, chlortetracycline, chlormoxycline, demeclocycline, doxycycline, guanmecycline, lymecycline, meclocycline, methacycline oxide, minocycline, oxytetracycline, pipecolin, pipcycline, rolicycline, sancycline, succinicin, pirimicycline, and tetracycline. The sulfanilamide medicines include, but are not limited to, sulfanilamide, sulfamethoxazoleAzole, sulfacetamide, sulfadiazine and sulfadiazineAzole and Compound sulfamethoxazole (trimethoprim/Sulfamethoxamine)Oxazole). The lincosamide antibiotics include, but are not limited to, clindamycin and lincomycin. Such polymyxins (polypeptides) include, but are not limited to, polymyxin B and polymyxin E.
4.0 screening assay
Additional embodiments include methods of screening for antagonists of the IL-17RA-IL-17RB heteromeric receptor complex. Screening assay formats known in the art and suitable for identifying antagonists of the IL-17RA-IL-17RB heteromeric receptor complex are contemplated. For example: a method of screening for an antagonist of an IL-17RA-IL-17RB heteromeric receptor complex, the method comprising providing IL-17RA and IL-17RB in an IL-17RA-IL-17RB heteromeric receptor complex; exposing a candidate substance to the receptor complex; and determining the amount of receptor complex formation relative to that not exposed to the candidate substance. The step of exposing the candidate substance to the receptor complex may be before, during or after IL-17RA and IL-17RB form an IL-17RA-IL-17RB heteromeric receptor complex.
Additional embodiments include methods of screening for antagonists of IL-17RA-IL-17RB heteromeric receptor complex activation, the method comprising providing IL-17RA and IL-17RB in an IL-17RA-IL-17RB heteromeric receptor complex; exposing a candidate substance to the receptor complex; adding one or more IL-17 ligands; and determining the amount of activation of the IL-17RA-IL-17RB heteromeric receptor complex relative to that which is not exposed to the candidate substance. Candidate agents that reduce IL-17RA-IL-17RB heteromeric receptor complex activation in the presence of one or more IL-17 ligands are considered positive as determined by a biologically relevant readout (see below). The IL-17 ligand may be IL-17A, IL-17F, IL-25 or any other IL-17 ligand that binds to and activates IL-17RA, IL-17RB, or IL-17RA-IL-17RB heteromeric receptor complex. Activation is defined elsewhere in the specification. Relevant biological readouts include IL-5, IL-6, IL-8, IL-13, CXCL1, CXCL2, GM-CSF, G-CSF, M-CSF, IL-1 β, TNF α, RANK-L, LIF, PGE2, IL-12, MMP3, MMP9, GRO α, NO, and any other molecule known in the art that is released from any cell expressing IL-17RA and/or IL-17RB. The step of exposing the candidate substance to the receptor complex may be before, during or after IL-17RA and IL-17RB form an IL-17RA-IL-17RB heteromeric receptor complex. It is understood that the candidate substance may partially inhibit the IL-17RA-IL-17RB heteromeric receptor complex, i.e., less than 100% inhibition. Under certain assay conditions, the candidate substance may completely inhibit the IL-17RA-IL-17RB heteromeric receptor complex.
In one aspect, the invention provides cell-based assays to detect the effect of a candidate substance on the association of IL-17RA and IL-17RB, the 17RA-IL-17RB heteromeric receptor complex, and the activation of the 17RA-IL-17RB heteromeric receptor complex. Accordingly, the present invention provides for the addition of candidate substances to cells to screen 17RA-IL-17RB heteromeric receptor complex antagonists.
"candidate substance" or "drug candidate" as used herein describes any molecule that can be screened for activity as outlined herein, such as, but not limited to, peptides, fusion proteins of peptides (e.g., peptides that bind IL-17RA, IL-17RB, or the 17RA-IL-17RB heteromeric receptor complex covalently or non-covalently bound to other proteins, such as antibody fragments or protein-based scaffolds known in the art), proteins, antibodies, small organic molecules (including known drugs and drug candidates), polysaccharides, fatty acids, vaccines, nucleic acids, and the like.
Candidate substances include a wide variety of chemical species. In one embodiment, the candidate substance is an organic molecule, preferably a small organic compound having a molecular weight greater than 100 and less than about 2500 daltons. Including small organic compounds having a molecular weight greater than 100 but less than about 2000 daltons, more preferably less than about 1500 daltons, more preferably less than about 1000 daltons, more preferably less than 500 daltons. Candidate substances comprise functional groups necessary for structural interaction with proteins, especially hydrogen bonding, and typically comprise at least one amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of said chemical functional groups. The candidate materials often comprise cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate substances are also found in biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.
Candidate substances are obtained from a variety of sources, including libraries of synthetic or natural compounds. For example, numerous methods exist for the random and directed synthesis of a variety of organic compounds and biomolecules, including the expression and/or synthesis of random oligonucleotides and peptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily available. In addition, natural or synthetically prepared libraries and compounds are readily modified by conventional chemical, physical and biochemical means. Known pharmacological substances may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidation (amidification) to produce structural analogs.
In alternative embodiments, the candidate bioactive substance can be a protein or protein fragment. Thus, for example, protein-containing cell extracts, or random or directed digests of proteinaceous cell extracts, may be used. This allows the production of libraries of prokaryotic and eukaryotic proteins for screening in the system described herein. This embodiment includes bacterial protein libraries, fungal protein libraries, viral protein libraries, and mammalian protein (including human protein) libraries.
In certain embodiments, the candidate substance is a peptide. In this embodiment, it may be useful to use a peptide construct comprising a display structure. "display structure" or grammatical equivalents refer herein to a sequence that, when fused to a candidate biologically active substance, causes the candidate substance to assume a conformationally constrained form. Proteins interact with each other primarily through conformational restriction domains. Although small peptides with freely rotating amino and carboxyl termini, as known in the art, can have robust functions, the structural conversion of such peptides into pharmacological agents is difficult due to the inability to predict the side chain positions for peptidomimetic synthesis. Thus, display of the peptide in a conformationally constrained structure would be beneficial for next generation drugs and would likely also result in higher affinity interaction of the peptide with the target protein. This fact has been recognized in combinatorial library generation systems that use short peptides produced biologically in bacterial phage systems. Many workers have constructed small domain molecules in which random peptide structures can be displayed. Specific display structures maximize accessibility to the peptide by displaying the peptide in the outer loop. Thus, suitable display structures include, but are not limited to, minibody structures, loop and coiled-coil stem structures at the beta-sheet turn (where residues not critical to the structure are random), zinc finger domains, cysteine-linked (disulfide) structures, transglutaminase linked structures, cyclic peptides, B-loop structures, barrels or bundles of helices, leucine zipper motifs, and the like. See U.S. patent No. 6,153,380, which is incorporated by reference.
Phage display libraries are of particular use in screening assays; see, for example, U.S. Pat. Nos. 5,223,409, 5,403,484, 5,571,698, and 5,837,500, all of which are expressly incorporated by reference in their entirety for purposes of phage display methods and constructs. Typically, phage display libraries may utilize synthetic protein (e.g., peptide) inserts, or may utilize digests of genomes, cdnas, and the like.
Depending on the assay and the desired results, a variety of cell types can be used, including eukaryotic and prokaryotic cells; mammalian cells and human cells find particular use in the present invention. In one embodiment, the cell may be genetically engineered, e.g., it may comprise exogenous nucleic acids, such as those encoding IL-17RA and IL-17RB. In certain instances, the IL-17RA and IL-17RB proteins of the invention are engineered to comprise a marker (e.g., an epitope marker), such as, but not limited to, those for immunoprecipitation testing or other uses.
The candidate substance is added to the cells and allowed to incubate for a suitable period. The step of exposing the candidate substance to the receptor complex may be before, during or after IL-17RA and IL-17RB form an IL-17RA-IL-17RB heteromeric receptor complex. In one embodiment, the association of IL-17RA and IL-17RB is assessed in the presence and absence of the candidate substance. Immunoprecipitation assays can be performed, for example, by using labeled constructs and antibodies. Then, for interference with IL-17RA and IL-17RB associated candidate substance, IL-17 ligand family members (such as IL-17A and IL-17F) signal transduction activity, for example by testing the IL-17 ligand family member activated gene expression.
In certain embodiments, the IL-17RA and/or IL-17RB proteins are fusion proteins. For example, a receptor protein may be modified in a manner to form a chimeric molecule comprising an apoprotein (i.e., the protein portion of the chimeric molecule or complex) fused to another heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of one or more receptors with a marker polypeptide that provides an epitope to which an anti-marker antibody can selectively bind. The epitope tag is typically located at the amino or carboxy terminus of the receptor protein. Antibodies against the marker polypeptide can be used to detect the presence of this epitope-tagged form of the receptor. In addition, the provision of such epitope tags enables the receptor polypeptides to be readily purified by affinity purification methods using anti-tag antibodies or other types of affinity matrices that bind the epitope tags. These epitope tags can be used to immobilize to a solid support as outlined herein.
Various marker polypeptides and their respective antibodies are well known in the art. Examples include polyhistidine (poly his) or polyhistidine-glycine (poly his-gly) tags; influenza HA marker polypeptide and its antibody 12CA5[ Field et al, mol.cell.biol., 8: 2159-2165(1988) ]; the C-myc marker and its 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies [ Evan et al, Molecular and Cellular Biology, 5: 3610-3616(1985) ]; and the herpes simplex virus glycoprotein D (gD) marker and antibodies thereto [ Paborsky et al, Protein Engineering, 3 (6): 547-553(1990)]. Other marker polypeptides include FLAGGTM-peptide [ Hopp et al, Biotechnology, 6: 1204-1210(1988) ]; KT3 epitope peptide [ Martin et al, Science, 255: 192-; tubulin epitope peptides [ Skinner et al, j.biol.chem., 266: 15163-15166(1991) ]; and the T7 gene 10 protein peptide marker [ Lutz-Freyermuth et al, proc.natl.acad.sci.usa, 87: 6393-6397(1990)].
Various expression vectors can be prepared. The expression vector may be a self-replicating extrachromosomal vector or a vector that integrates into the host genome. Typically, these expression vectors include transcription and translation regulatory nucleic acids operably linked to a nucleic acid encoding a metalloprotein. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, promoters, optional operator sequences and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
In certain embodiments, the assay for inhibiting binding to the IL-17RA-IL-17RB heteromeric receptor complex is performed in vitro. For example, the components of the assay mixture (candidate substance, IL-17RA and IL-17RB) are immobilized on a surface, and the other components (one of which is labeled in certain embodiments) are added. For example, IL-17RA or IL-17RB may be attached to a surface, and a candidate substance and labeled IL-17RA and/or IL-17RB may be added. After washing, the presence of the marker was assessed. In this embodiment, the IL-17RA and IL-17RB proteins are isolated as known in the art.
In general, the linkage is generally effected as is known in the art, and depends on the composition of the two species to be linked. In general, a linker is used by utilizing functional groups available for attachment on each component. Functional groups for attachment are amino, carboxyl, oxo, hydroxyl and thiol groups. These functional groups may then be directly or indirectly attached by using a linker. Linkers are well known in the art; for example, homobifunctional or heterobifunctional linkers are well known (see the 1994Pierce Chemical Company catalog, incorporated herein by reference, for technical sections on crosslinkers, page 155-200). Linking linkers include, but are not limited to, alkyl groups (including substituted alkyl groups and alkyl groups containing heteroatom moieties), including short alkyl groups, esters, amides, amines, epoxy groups, and glycols and derivatives. Alternatively, a fusion partner is used; suitable fusion partners include other immobilization components such as histidine tags for attachment to surfaces with nickel, functional components for attachment of linkers and tags, and proteinaceous tags.
In one embodiment, particularly when the candidate substance is immobilized on a solid support, a suitable fusion partner is an auto-fluorescent protein label. Suitable fluorescent markers for proteins also include, but are not limited to, Green Fluorescent Protein (GFP) (including GFP from the species Renilla, Ptilosarcus or Aequorea (Chalfie et al, 1994, Science 263: 802-containing 805)), EGFP (Clontech Laboratories, Inc., Genbank accession No. U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc.1801de Maison neuve blvd. West, 8th Floor, Montreal, Quebec, Canada H3H J9, Stauber, 1998, Biotechniques 24: 462-471; Heim et al, 1996, Curr. biol. 6: 178-containing 182), enhanced yellow fluorescent protein (ECloroop, ntech Laboratories, Inc.: 6342-containing), luciferase (Ochiki et al, Acmu. J. 1993, 5454150: 545445, WO 1467/5472, WO 9611, WO 9/32-containing 94), WO 19/32-S.7, WO 14611-containing No. 7,4685, WO 19, WO9,977, WO 19, WO-S. 11/No. 7,4685, WO-S. 36637,72, WO-S. No. 11,977/11, WO 19, WO 19,72, WO 11,32,32, WO 7,32,32,32,32,32,32,32,32,32,76, US). All references cited above are expressly incorporated herein by reference.
The insoluble support may be comprised of any component to which the composition can be bound, which is readily separated from soluble material and is otherwise compatible with all screening methods. The surface of such a support may be solid or porous and of any suitable shape. Examples of suitable supports include microtiter plates, arrays, membranes and beads, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other substances, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene, and the like), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, ceramics and various other polymers. In certain embodiments, the solid support allows optical detection and does not significantly fluoresce itself. Furthermore, as is known in the art, the solid support may be coated with a number of substances, including polymers, such as dextran, acrylamide, gelatin, agarose, and the like. Exemplary solid supports include silicon, glass, polystyrene and other plastics and acrylics. Microtiter plates and arrays are particularly convenient because large numbers of assays can be performed simultaneously using small amounts of reagents and samples. The particular manner in which the compositions are combined is not critical so long as it is compatible with the agents and all methods of the invention, retains the activity of the compositions, and is non-diffusible.
The candidate substance is contacted with the other components of the assay under reaction conditions that favor a targeted interaction of the substance. Typically, the condition is a physiological condition. The incubation may be carried out at any temperature that favours optimal activity, typically between 4 and 40 ℃. The incubation period for optimal activity was chosen, but could also be optimized to facilitate rapid high throughput screening. Typically 0.1-1 hours is sufficient. In the case of solid phase assays, excess reagents are typically disposed of or washed away. Assay formats are discussed below.
Various other reagents may be included in the assay. These include reagents such as salts, neutral proteins, e.g. albumin, detergents, etc., which can be used to promote optimal apoprotein-substance binding and/or reduce non-specific or background interactions. In addition, reagents that additionally improve assay efficiency, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like, can also be used. The mixing of the ingredients may be added in any order that provides the necessary combination.
In one embodiment, any of the assays outlined herein can utilize a robotic system for high throughput screening. Many systems typically involve the use of 96 (or more) well microtiter plates, but as those skilled in the art will recognize, any number of different plates or configurations may be used. Further, any or all of the steps outlined herein may be automated; thus, for example, the system may be fully or partially automated.
As those skilled in the art will recognize, there are a variety of components that may be used, including but not limited to one or more robotic arms; a plate handler (plate handler) for position control of the microplate; an automated lid handler (lid handler) for removing and replacing lids for holes in a non-cross-contamination plate; tip assemblies (tip assemblies) with disposable tips for sample distribution; a washable tip assembly for sample distribution; a 96-well loading block; cooling the reagent rack; microtiter plate pipette position (arbitrary cooling); flat and top stacker towers (racking tower); and a computer system.
Fully robotic or microfluidic systems include automated liquid handling, particle handling, cell handling, and organism handling, including high throughput pipetting to perform all steps of screening applications. This includes liquid, particle, cell and organism manipulations such as pumping, dispensing, mixing, diluting, washing, precision volume transfer; recovering and discarding the pipette tip; and equal volume repeat pipetting for multiple deliveries from a single sample draw. These operations are the transfer of non-cross-contaminating liquids, particles, cells and organisms. The apparatus performs automated replication of microplate samples to filters, membranes and/or daughter plates, high density transfer, full plate serial dilution, and high volume operations.
In one embodiment, chemically derivatized particles, plates, tubes, magnetic particles or other solid state matrices specific for the assay components may be used. Binding surfaces of microplates, tubes, or any solid phase matrix include non-polar surfaces, highly polar surfaces, modified dextran coatings to facilitate covalent binding, antibody coatings, affinity media to bind fusion proteins or peptides, surface immobilized proteins (e.g., recombinant protein a or G), nucleotide resins or coatings, and other affinity matrices useful in the present invention.
In one embodiment, platforms or platforms with multiple volumes for multi-well plates, multi-tubes, small tubes, deep well plates, microcentrifuge tubes, cryovials, square well plates, filters, chips, optical fibers, beads and other solid phase matrices are housed in a scalable modular platform for additional capacity. The combined platform comprises a variable-speed fixed-track oscillator, an electroporator, a multi-station working plate for diluting a source sample, a sample and a reagent, a measuring plate, a sample and reagent storage, a pipette tip and a movable washing station.
In one embodiment, a cyclovariable temperature and temperature regulation system is used to stabilize the temperature of a heat exchanger (e.g., a controlled block or platform) to provide accurate temperature control of the incubated sample from between 4 ℃ to 100 ℃.
In certain embodiments, the instrumentation will include a detector, which may be a variety of different detectors depending on labeling and assay. In one embodiment, useful detectors include microscopes with multichannel fluorescence; a plate reader with single and dual wavelength end points and kinetic properties, Fluorescence Resonance Energy Transfer (FRET), SPR systems, luminescence, quenching, two photon excitation and intensity redistribution providing fluorescence, ultraviolet and visible spectrophotometric detection; a CCD camera that captures and transforms data and images into a quantifiable form; and a computer workstation. These will enable the following monitoring: size, growth and phenotypic expression of specific markers in cells, tissues and organisms; the target is determined; optimizing a guide object; integration of data analysis, mining, organization, and high throughput screening with public and proprietary databases.
The 17RA-IL-17RB heteromeric receptor complex is a biologically active form of the receptor, and ligand-specific activation has been shown herein to be responsive to the release of pro-inflammatory mediators. It is known in the art that various disease states (as exemplified herein) are associated with increased physiological levels of members of the IL-17 ligand family. In one embodiment, the IL-17RA-IL-17RB antigen binding proteins are useful for detecting the IL-17RA-IL-17RB heteromeric receptor complex in a biological sample, and identifying cells or tissues that express the complex. This has important value to the research community.
The antigen binding proteins of the invention may be used for diagnostic purposes to detect, diagnose or monitor diseases and/or conditions associated with IL-17 or the IL-17RA or L-17RB receptor. The present invention provides for the detection of the presence of IL-17 receptors in a sample using classical immunohistological methods known to those skilled in the art (e.g., Tijssen, 1993, Practice and Theory of Enzyme Immunoassays, vol 15(EdsR. H. Burdon and P. H. van Knipponberg, Elsevier, Amsterdam); Zola, 1987, Monoclonal antibodies: A Manual Tec of electrons, pp.147-158(CRC Press, Inc.; Jalkanen et al, 1985, J.cell. biol. 101: 976-985; Jalkanen et al, 1987, J.CellBiol. 105: 3087-3096). The detection of the IL-17 receptor can be performed in vivo or in vitro.
The diagnostic applications provided herein include the use of the antigen binding proteins to detect the expression of the IL-17IL-17RA and IL-17RB proteins, and the binding of ligands to the IL-17 receptor. Examples of methods useful for detecting the presence of the IL-17 receptor include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and Radioimmunoassays (RIAs). As outlined above, the use of co-immunoprecipitation is very useful for detecting the IL-17RA-IL-17RB heteromeric receptor complex. For diagnostic applications, the antigen binding protein may typically be labeled with a detectable labeling group as defined herein.
One aspect of the invention provides for the identification of a single or a plurality of cells expressing the IL-17RA-IL-17RB heteromeric receptor complex. In a specific embodiment, the antigen binding protein is labeled with a labeling group and the binding of the labeled antigen binding protein to the IL-17 receptor is detected. In another specific embodiment, the binding of the antigen binding protein to the IL-17 receptor is detected in vivo. In another specific embodiment, the antigen binding protein-IL-17 receptor is isolated, using the known in the art of technology measurement. See, for example, Harlow and Lane, 1988, Antibodies: ALaborory Manual, New York: cold Spring Harbor (1991 edition, periodical supplement); john e.coligan, 1993 edition, Current Protocols In Immunology New York: john Wiley & Sons.
5.0 preparation of IL-17RA-IL-17RB antagonists
Suitable host cells for expression of IL-17RA-IL-17RB antagonists include prokaryote, yeast, or higher eukaryote cells. Suitable Cloning and expression Vectors for use with hosts of bacterial, fungal, yeast and mammalian cells are described, for example, in Pouwels et al Cloning Vectors: the ALaborory Manual, Elsevier, New York, (1985). Using RNA from the DNA constructs disclosed herein, cell-free translation systems can also be used to produce the LDCAM polypeptides.
Prokaryotes include gram-negative or gram-positive organisms, such as E.coli or Bacillus (Bacillus). Suitable prokaryotic host cells for transformation include, for example, Escherichia coli, Bacillus subtilis, Salmonella typhimurium and various other species within the genera Pseudomonas, Streptomyces and Staphylococcus. In prokaryotic host cells such as E.coli, the IL-17RA-IL-17RB antagonist may comprise an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant IL-17RA-IL-17RB antagonist.
The IL-17RA-IL-17RB antagonist can be expressed in a yeast host cell, preferably from Saccharomyces (Saccharomyces) (e.g., Saccharomyces cerevisiae). Other genera of yeast may also be used, such as the genera Pichia (Pichia), Kluyveromyces lactis (K.lactis) or Kluyveromyces (Kluyveromyces). Yeast vectors often contain replication initiation sequences, Autonomous Replication Sequences (ARS), promoter regions, polyadenylation sequences, transcription termination sequences, and selectable marker genes from a 2. mu.yeast plasmid. Suitable promoter sequences for yeast vectors include, inter alia, the following promoters: metallothionein, 3-phosphoglycerate kinase (Hitzeman et al, J.biol. chem.255: 2073, 1980) or other glycolytic enzymes (Hess et al, J.adv. enzyme Reg.7: 149, 1968; and Holland et al, biochem.17: 4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other suitable vectors and promoters for expression in yeast are further described in Hitzeman, EPA-73, 657 or Fleer et al, Gene, 107: 285-195 (1991); and van den Berg et al, Bio/Technology, 8: 135, 139 (1990). Another alternative promoter is the glucose-repressible ADH2 promoter, described by Russell et al (J.biol.chem.258: 2674, 1982) and Beier et al (Nature 300: 724, 1982). Shuttle vectors, which are replicable in both yeast and E.coli, can be constructed by inserting the DNA sequence from pBR322, which is used in the large intestine rod, into the above-mentioned yeast vectorsSelection and replication in bacteria (Amp)rGenes and origins of replication).
The yeast alpha factor leader sequence can be used to direct secretion of the IL-17RA-IL-17RB antagonist. The alpha factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, for example, Kurjan et al, Cell 30: 933, 1982; bitter et al, proc.natl.acad.sci.usa 81: 5330, 1984; U.S. patent No. 4,546,082; and EP 324,274. Other leader sequences suitable for promoting secretion of recombinant polypeptides by yeast hosts are known to those skilled in the art. The leader sequence may be modified near its 3' end to include one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
Methods for transformation of yeast are known to those skilled in the art. One such method is described by hinen et al, proc.natl.acad.sci.usa 75: 1929, 1978. Hinnen et al selected Trp + transformants in a selective medium consisting of 0.67% yeast nitrogen source, 0.5% casamino acids, 2% glucose, 10. mu.g/ml adenine and 20. mu.g/ml uracil. Yeast host cells transformed with a vector comprising the ADH2 promoter sequence can be cultured for inducible expression in "rich" medium. An example of a rich medium is a medium consisting of 1% yeast extract, 2% peptone and 1% glucose, with the addition of 80. mu.g/ml adenine and 80. mu.g/ml uracil. Derepression of the ADH2 promoter occurs when glucose is depleted in the culture medium.
Recombinant IL-17RA-IL-17RB antagonists may also be expressed using mammalian or insect host cell culture systems. Luckow and Summers, Bio/Technology 6: 47(1988) review baculovirus systems for the production of heterologous proteins in insect cells. Established cell lines of mammalian origin may also be used. Examples of suitable mammalian host Cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al, Cell 23: 175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese Hamster Ovary (CHO) cells, HeLa cells and BHK (ATCC CRL 10) Cell lines and CV-1/EBNA-1 Cell lines derived from the African green monkey kidney Cell line CVI (ATCC CCL 70) as described by McMahan et al (EMBO J.10: 2821, 1991).
Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from the viral genome. Commonly used promoter and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40(SV40) and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, such as the SV40 start, early and late promoters, enhancers, splicing and polyadenylation sites, can be used to provide additional genetic components for expression of structural gene sequences in mammalian host cells. The early and late promoters of the virus are particularly useful because both are readily available from the viral genome as fragments which may also contain the viral origin of replication (Fiers et al, Nature 273: 113, 1978). Smaller or larger SV40 fragments may also be used, provided that the approximately 250bp sequence extending from the Hind III site to the Bgl I site at the SV40 viral origin of replication is included.
Exemplary expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (mol. cell. biol. 3: 280, 1983). A practical system for stable high level expression of mammalian cDNA in C127 murine mammary epithelial cells can be constructed essentially as described by Cosman et al (mol. Immunol.23: 935, 1986). By Cosman et al, Nature 312: 768, 1984, and PMLSV N1/N4, which has been deposited as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A-0367566, incorporated herein by reference, and U.S. patent application Ser. No. 07/701,415, filed 5, 16, 1991. The vector may be derived from a retrovirus. In place of the native signal sequence, and in addition to the start codon methionine, a heterologous signal sequence may be added, such as the signal sequence for IL-7 described in U.S. Pat. No. 4,965,195, Cosman et al, Nature 312: 768(1984) the signal sequence of the IL-2 receptor, the IL-4 signal peptide described in EP 367,566, the type I IL-1 receptor signal peptide described in U.S. Pat. No. 4,968,607 and the type II IL-1 receptor signal peptide described in EP 460,846.
The IL-17RA-IL-17RB antagonists of the invention, which are isolated, purified or homologous proteins, can be produced or purified from naturally occurring cells by recombinant expression systems as described above.
A method of producing an IL-17RA-IL-17RB antagonist comprising culturing a host cell transformed with an expression vector comprising a DNA sequence encoding at least one IL-17RA-IL-17RB antagonist under conditions sufficient to drive expression of said IL-17RA-IL-17RB antagonist. The IL-17RA-IL-17RB antagonist is then recovered from the culture medium or cell extract, depending on the expression system employed. As known to the skilled person, the method of purifying the recombinant protein will vary depending on factors such as the type of host cell used and whether the recombinant protein is secreted into the culture medium. For example, when using an expression system that secretes a recombinant protein, the culture medium can first be concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration units. After the concentration step, the concentrate can be applied to a purification matrix, such as a gel filtration medium. Alternatively, ion exchange resins, such as matrices or matrices having pendant Diethylaminoethyl (DEAE) groups, may be used. The matrix may be acrylamide, agarose, dextran, cellulose or other species commonly used for protein purification. Alternatively, a cation exchange step may be employed. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, one or more reverse phase high performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium, such as silica gel having methyl or other aliphatic group pendant groups, may be employed to further purify the IL-17RA-IL-17RB antagonist. Some or all (in various combinations) of the foregoing purification steps are well known and can be used to provide substantially homogeneous recombinant proteins.
It is possible to affinity purify the expressed IL-17RA-IL-17RB antagonist using an affinity column comprising IL-17RA, or IL-17RB, or both IL-17RA and IL-17RB, or IL-17RA-IL-17RB heteromeric receptor complex proteins. The IL-17RA-IL-17RB antagonist can be isolated from the affinity column using conventional techniques, such as in a high salt elution buffer, followed by dialysis against a lower salt buffer, or by changing the pH or other components (depending on the affinity matrix used). Alternatively, the affinity column may comprise an antibody that binds to an antagonist of IL-17RA-IL-17 RB.
Recombinant proteins produced in bacterial culture can be isolated by first disrupting the host cells, centrifuging, extracting from the cell mass (if insoluble polypeptides) or from the supernatant (if soluble polypeptides), followed by one or more steps of concentration, salting out, ion exchange, affinity purification or size exclusion chromatography. Finally, RP-HPLC can be used in the final purification step. Microbial cells may be disrupted by any conventional method, such as freeze-thaw cycling, sonication, mechanical disruption, or use of a cell lysing substance.
The IL-17RA-IL-17RB antagonist can be expressed as a secreted polypeptide using transformed yeast host cells to simplify purification. Secreted recombinant polypeptides from yeast host cell fermentation can be produced by methods similar to Urdal et al 1984, j.chromanog.296: 171. Urdal et al describe two sequential reverse phase HPLC steps for purification of recombinant human IL-2 in a preparative HPLC column.
All references cited within the body of this specification are expressly incorporated herein by reference in their entirety. The following practical and prophetic examples are provided for the purpose of illustrating particular embodiments or features of the invention and are not to be construed as limiting the scope thereof.
Examples
His, goat anti-hIL-17 RA polyclonal antibody, goat anti-hIL-17 RB polyclonal antibody, goat anti-hIL-17 RC polyclonal antibody and all ELISA kits were obtained from R & D Systems (Minneapolis, MN) and used according to the manufacturer's instructions. Murine IL-13 was obtained from Invitrogen Biosource (Carlsbad, CA). Murine Serum Albumin (MSA) was obtained from Sigma-Aldrich (st. louis, MO). Monoclonal antibodies against human and mouse IL-25, IL-17RA and IL-17RB were prepared essentially as described by Yao et al (Yao et al, 1995, Immunity 3: 811-54821; Yao et al, 1995, J.Immunol.155: 5483-5486; Yao, 1997, Cytokine 9: 794-800). cDNAs encoding human and mouse IL-17RA have been previously described (see the three Yao references above). The open reading frames encoded by human and mouse IL-17RB are the same as previously described (Tian et al, 2000, Oncogene 19 (17): 2098-. cDNA encoding murine IL-25 has been previously described (Hurst et al, 2002, J Immunol.169 (1): 443-. Murine IL-25 was expressed and purified in E.coli as described (Hurst et al, supra). The extracellular region of human IL-17RA was fused to either poly HIS or human Fc IgG1(IL-17RA: HIS or IL-17RA: Fc, respectively), essentially as described by Yao et al, 1995, Immunity (supra); the extracellular region of human IL-17RB was fused to either poly HIS (IL-17RB. HIS) or human Fc IgG1(IL-17RB. Fc). In some experiments, commercially available murine and human IL-25, IL-17RAFc and IL-17RB Fc (R & D Systems) were used.
Example 1
This example demonstrates that IL-17RB is essential for response to IL-25in vivo. IL-17 RB-/-mice were generated using methods known in the art. Briefly, gene targeting vectors were constructed by replacing the genomic sequence containing murine IL-17RB exon 3 with the PGKneo cassette. The thymidine kinase cassette (MC-TK) was inserted into the 5' end of the vector. 129-source Embryonic Stem (ES) cells were electroporated with a targeting vector and selected in the presence of G418 and ganciclovir as described (Kolls, J et al, 1994.Proc. Natl. Acad. Sci. USA.91: 215-219). ES clones carrying the desired mutation in IL-17RB were identified by a combination of PCR and genomic Southern blot analysis and injected into Swiss Black blastocysts. Male chimeras were crossed to female Swiss Black to generate IL-17RB mutated heterozygous mice that were then crossed to generate IL-17 RB-deficient mice. Marker Assisted accelerated Backcrossing (MAX-BAX) is usedSM) The technique (Charles River Laboratories, Wilmington, Mass.) the mice were migrated to a C57BL/6 background by 5 consecutive backcrosses to C57BL/6 mice. Mice identified as 99.5% C57BL/6 were used to establish breeding populations to generate experimentally used mice.
Control C57BL/6 mice (WT) or IL-17 RB-/-mice (KO) were administered 50 microliters of MSA (Sigma-Aldrich, St. Louis MO; 10 micrograms/mL) or mouse IL-25 (Amgen; 10 micrograms/mL) Intranasally (IN) once daily for four days, essentially as described by Hurst et al (J.Immunol.169: 443, 2002). On day 5, bronchoalveolar lavage fluid (BALF) and lung tissue were collected from the mice and analyzed.
Bronchoalveolar lavage (BAL) was performed by inserting tubes into mice anesthetized with 300 microliters of IP injection of 2.5% Avermectin (2-2-2-tribromoethanol, Sigma), followed by rinsing the lungs with two 600 microliter volumes of ice-cold Dulbecco's PBS (Gibco). The BAL liquid cells were spheronized by centrifugation at 1000rpm for 10 minutes, followed by resuspension with PBS + 5% fetal bovine serum (FBS; Hyclone; Logan, UT) for dilution with120 blood machines (bench-top analyzers for processing and analyzing blood samples; Siemens Diagnostics, Tarrytown, N.Y.) calculate and analyze the total leukocyte content and the changes in the number of various cell types. Also by ELISA (R)&D Systems; detection limit: IL-531 pg/mL; IL-1362 pg/mL) the IL-5 and IL-13 protein concentrations of the BALF were determined.
Essentially as described previously (Hartel, C. et al, 1999Scand. J. Immunol.49 (6): 649-654), use was made of assay-On-DemandPrimers (Applied Biosystems, Foster City, Calif.) were introduced through(a rapid real-time fluorophore-based polymerase chain reaction method) to determine mRNA levels of various inflammatory mediators in lung tissue. The procedure was performed on an ABI Prism 7900HT FastRT-PCR System (Applied Biosystems)And (6) analyzing. Phase of each gene in each treatment group on beta-actin, HPRT or GAPDH gene expressionExpression was determined using the Sequence Detection System 2.2.3(Applied Biosystems). The results of two independent experiments are shown in tables 1-4 below.
Table 1: analysis of BALF cell content, IL-5 concentration and IL-13 concentration in IL-17RB KO and WT mice intranasally administered IL-25
N is 5/group; values shown are (mean ± SD); the value assigned to samples below the detection range of the IL-5ELISA was 31 pg/mL.
Table 2: analysis of IL-5, IL-13 and IL-17RA mRNA IN the lungs of IL-17RB KO and WT mice IN response to IN IL-25 challenge
N is 5/group; values shown are (mean ± SD); the IL-5 and IL-13 values shown are relative to beta-actin gene expression (2E-. DELTA.Ct) (mean. + -. SD). The IL-17RA values shown are gene expression relative to HPRT (2E-. DELTA.Ct) (mean. + -. SD). N/A ═ unanalyzed
Experiments IN which IN IL-25 was challenged to this IL-17RB KO mouse were repeated IN essentially the same manner: addition of mouse Interleukin-13 attack arm (IL-13; Invitrogen Biosource)TMCarlsbad, CA; once daily dosing of 50 microliters (10 micrograms/mL) per time for four days; the results are shown in tables 3-4 below.
Table 3: analysis of BALF cell content IN IL-17RB KO and WT mice IN response to IN IL-13 or IL-25 challenge
N is 5/group; the indicated values are (mean. + -. SD)
Table 4: analysis of IL-5, IL-13, eotaxin, MCP-1, IL-9, IL-10, IL-17A and IL-17RA mRNA IN the lungs of IL-17RB KO and WT mice IN response to IN IL-25 challenge
N-4 lungs from individual mice; values shown are gene expression relative to HPRT (2E-. DELTA.Ct) (mean. + -. SD)
For lung pathology experiments, mice were treated with CO2Suffocation is painless and lethal. Lungs were harvested, fixed in 10% Neutral Buffered Formalin (NBF), processed, sectioned at 6 microns and treated with hematoxylin and eosin (H.A. J.R., and J.A. Hotchkiss, am.J. Pathol.141: 307; 1992) essentially as described (Harkema, J.R., and J.A. Hotchkiss, am.J. Pathol.141: 307; 1992)&E) Staining or Periodic Acid Schiff (PAS) staining; the graded scale for analyzing tissue sections is shown as the following four different categories. The mean total inflammation score for each group is reported in table 5.
Table 5: histological analysis of lung tissue inflammation and goblet cell proliferation IN IL-17RB KO and WT mice challenged with IN IL-25
Reported mean scores ± SD.
Goblet cell proliferation (PAS staining)
0 is normal
1-microscopic, goblet cells proliferate in large bronchioles
2 mild, goblet cell proliferation in bronchioles of the large and medium tracts
3-intermediate, goblet cells proliferate in large, medium and some small bronchioles
4-significant, goblet cells proliferate throughout the airways
Inflammation of the peribronchial region
0 is normal
Very minimal eosinophil/macrophage/lymphocyte mantle (discontinuous to monolayer) without edema
2-mild eosinophil/macrophage/lymphocyte mantle (2-5 cells); minimal edema, fibrous tissue formation
3-intermediate eosinophils/macrophages/lymphocytes casing (5-10 cells); presence of edema and fibrous tissue formation
4 ═ significant eosinophil/macrophage/lymphocyte commitment (> 10 cells); marked edema and fibrous tissue formation
Bronchopneumonia
0 is normal
Local accumulation of 1 ═ microscopic macrophages/neutrophils/eosinophils/MNGC
2-slight local accumulation of macrophages/neutrophils/eosinophils/MNGC
Multiple local accumulation of 3 ═ moderate macrophages/neutrophils/eosinophils/MNGC
Multiple local accumulation of 4 ═ significant macrophages/neutrophils/eosinophils/MNGC
Pulmonary perivasculitis/vasculitis
0 is normal
Very minimal eosinophil/lymphocyte/macrophage mantle (discontinuous to monolayer) with no intimal infiltration/hyperplasia
2-mild eosinophils/lymphocytes/macrophages casing (2-5 cells); local eosinophilic intimal infiltration and endothelial hyperplasia
3-intermediate eosinophils/lymphocytes/macrophages casing (5-10 cells); extensive intimal eosinophil infiltration and endothelial hyperplasia with few MNGCs
4 ═ significant eosinophil/lymphocyte/macrophage repopulation (> 10 cells); catheter walls sometimes disappeared and MNGC was significant; presence of sparse (discrete) vasculitis
In wild type C57BL/6 mice, the effects of intranasal administration of IL-25 included (1) an increase in total BALF leukocyte counts, including an increase in the number of BALF eosinophils, neutrophils, lymphocytes and macrophages, and an increase in BALF IL-5 and IL-13 concentrations (tables 1 and 3); (2) increased lung mRNA levels of IL-5, IL-13, eotaxin and MCP-1 (tables 2 and 4); and (3) goblet cell proliferation in large and medium airways, and strong perivascular/vascular inflammation involving arteries and veins, with the exception of alveolar capillaries (table 5). These effects were not observed when IL-25 was intranasally administered to IL-17RB KO mice (tables 1-5). IL-17RA mRNA was present in IL-17RB KO mice (tables 2 and 4). These data indicate that IL-17RB is essential for all IL-25 activities that have been measured in the lung to date.
Example 2
This example demonstrates in vivo that IL-17RA is required for response to IL-25. The generation of C57BL/6IL-17 RA-/-mice has been described previously (Ye, P. et al, 2001J.exp. Med.194: 519-) (527). Control C57BL/6 mice (WT) or IL-17 RA-/-mice (KO) were treated essentially as described in example 1 for IL-17 RB-/-mice; the results are shown in tables 6 and 7 below.
Table 6: comparative analysis of BALF in IL-17RA KO and C57BL/6WT mice: cell content and proteins
N is 5; values shown are (mean ± SD). N/a is not tested. Samples below the detection range of the IL-5ELISA were assigned the lower limit of detection, i.e., 31 pg/mL.
Table 7: comparative analysis of lung tissue in IL-17RA KO and C57BL/6WT mice: mRNA level
N is 4; values shown are relative to gene expression (2E- Δ Ct) for β -actin (mean ± SD). Lung tissue from IL-13 treated mice was not analyzed in this experiment.
Lung tissue was sectioned, prepared for histological analysis, staining and analysis essentially as described in example 1. The average total inflammation score for each group is reported in table 8.
Table 8: histological analysis comparison of lung tissue in IL-17RA KO and WT mice
KO，IL-25 KO，MSA WT，IL-25 WT，MSA
Goblet cell proliferation 0.6±0.5 0 2.8±0.4 0
Inflammation of the peribronchial region 0.8±0.4 0.8±0.9 3.2±.5 0
Bronchopneumonia 1.0±0 1.0±0 1.2±0.5 0.6±0.5
Perivasculitis/vasculitis of the lung 1.6±0.5 1.2±0.5 3.4±0.5 0.8±0.4
Total score 4.0±1 3.0±1.4 10.6±1.3 1.4±0.9
All groups except MSA-treated IL-17RA KO mice had N ═ 5 and MSA-treated IL-17RA KO mice had N ═ 4. Reported mean score ± SD.
The experiment was repeated in essentially the same manner; the results are shown in tables 9 and 10 below. No histological analysis of the lungs was performed in this experiment.
Table 9: comparative analysis of BALF in KO and WT mice
N is 5; the indicated values are (mean. + -. SD)
Table 10: comparative analysis of lung tissue in KO and WT mice: mRNA level
N is 4; the values shown are the gene expression (2E-. DELTA.Ct) (mean. + -. SD) relative to GAPDH
IN wild type C57BL/6 mice, the effects of IN administration on IL-25 included: (1) increased total BALF leukocyte numbers, increased numbers of BALF eosinophils, neutrophils, lymphocytes and macrophages, and increased BALF IL-5 and IL-13 concentrations (tables 1 and 4); (2) goblet cell proliferation in large and medium airways, and strong perivascular/vascular inflammation involving arteries and veins, with the exception of alveolar capillaries (table 3); and (3) increased levels of lung mRNA for IL-5, IL-13, eotaxin, MCP-1, and IL-17RB (tables 2 and 5). Although IL-17RB mRNA was present in L-17RA KO mice, these effects were not observed when IL-25 was intranasally administered to IL-17RA KO mice (tables 1-5). These data indicate that IL-17RA is essential for IL-25 activity in the lung.
Example 3
This example demonstrates that IL-17RA and IL-17RB are required to respond to IL-25in vitro. The production of splenocytes has been described previously (Hamilton et al, 1978, J Clin invest.62 (6): 1303-12). Briefly, individual spleens from C57BL/6WT, C57BL/6IL-17RB KO, and C57BL/6IL-17RA KO mice were aseptically removed and treated with 0.4mg/mL collagenase D (Roche Applied Science, Indianapolis, IN) and 0.1% DNAse-I (Roche Applied Science) IN RPMI 1640(Gibco-Invitrogen, Carlsbad, Calif.) to produce single cell suspensions. Splenocytes were cultured in complete DMEM medium (Gibco-Invitrogen) alone, or with the addition of 1 microgram/mL concanavalin A (Con A; Sigma-Aldrich) or complete DMEM medium (Gibco-Invitrogen) with the addition of IL-25(Amgen) at the final concentrations indicated, at 2.0X 107Individual cells/ml culture. At 5% CO2The cells were cultured in a humidified incubator at 37 ℃ for 72 hours. By ELISA (R)&D Systems) the IL-5 and IL-13 concentrations of the supernatants were determined. Spleen cell assays were repeated twice for each genotype in IL-17RA KO, IL-17RB KO and WT animals using different litters; data from two independent experiments are presented below (tables 11-14).
Table 11: IL-25 stimulated IL-17RA KO and WT splenocyte produced IL-5 and IL-13
N ═ 2 individual spleens; values shown are (mean ± SD). The value assigned to samples below the detection range of the IL-5ELISA was 31 pg/mL. The value assigned to samples below the detection range of the IL-13ELISA was 62 pg/ml.
Table 12: IL-25 stimulated production of IL-5 and IL-13 by WT and IL-17RA KO splenocytes
Table 13: IL-25 stimulated IL-17RB KO and WT spleen cell produced IL-5 and IL-13
N-3 individual spleens; values shown are (mean ± SD); the value assigned to samples below the detection range of the IL-5ELISA was 31 pg/mL. The value assigned to samples below the detection range of the IL-13ELISA was 62 pg/ml.
Table 14: IL-25 stimulated IL-17RB KO and WT spleen cell produced IL-5 and IL-13
N-3 individual spleens; values shown are (mean ± SD); the value assigned to samples below the detection range of the IL-5ELISA was 31 pg/mL. The value assigned to samples below the detection range of the IL-13ELISA was 62 pg/ml.
Stimulation with IL-25 induced IL-5 and IL-13 production by wild type C57BL/6 spleen cells in culture. This cytokine production was not induced by IL-25 stimulation of IL-17RB KO or IL-17RA KO splenocytes (tables 11-14). Con A, a positive control for splenocyte activation, induced IL-17RB KO splenocytes production of IL-13, IL-17RA KO splenocytes production of IL-5 and IL-13. Con A stimulation did not induce IL-5 production by IL-17RB KO splenocytes in one experiment, but induced IL-5 production by IL-17RB KO splenocytes in a second experiment. These in vitro cell culture data provide further support for: both IL-17RB and IL-17RA are essential for IL-25 signaling.
Example 4
This example characterizes the ability of anti-IL-17 RB-M735 and anti-IL-25-M819 antibodies to inhibit IL-25 responses in vitro. Spleen cells were prepared from spleens from naive BALB/C miceSingle cell suspension of cells, subsequently diluted to 4X 10 in complete DMEM medium (Gibco-Invitrogen, Carlsbad, Calif.)7Individual cells/mL. Cells (100. mu.l) were added to a 96-well plate and brought to a final concentration of 4X 10 using the following conditions6Individual cells/well:
culture medium only
10ng/mL muIL-25 (stimulated control)
MuIL-25+100ng/mL muIL-17RB. muFc (blocking control)
10ng/mL muIL-25+463, 154, 51, 17, 5.7, 1.9, 0.64, 0.21, 0.07, 0.023, 0.007, 0.003ng/mL anti-muIL-17 RB M735
10ng/mL muIL-25+1000, 100, 10, 1.0, or 0.1ng/mL anti-muIL-25M 819.
Each of the conditions listed above was tested with three independent biological samples (each sample consisting of splenocytes from the spleens of two BALB/c mice used for the first time in the experiment), and this test was repeated three times in three independent experiments. Cultures were incubated at 37 ℃ and 10% CO2The following incubations were carried out for 72 hours, and supernatants were collected at 72 hours and IL-5 concentrations were determined by ELISA. Both M735 and M819 inhibit IL-25-induced secretion of IL-5 by mouse splenocytes; the IC50 values calculated for each antibody in 3 independent splenocyte experiments are shown in tables 15-16 below, and the IC50 value is the inhibition of IL-25 induced IL-5 production by cultured BALB/c splenocytes.
Table 15: IC50 value for anti-IL-17 RB M735
Description of the antibodies IC50
mAb M735, experiment 1 1.32ng/mL
mAb M735, experiment 2 0.166ng/mL
mAb M735, experiment 3 2.15ng/mL
Table 16: IC50 value for anti-IL-25M 819
Description of the antibodies IC50
mAb M819, experiment 1 0.24ng/mL
mAb M819, Exp 2 4.2ng/mL
mAb M819, Exp 2 1.12ng/mL
Both anti-IL-17 RB M735 and anti-IL-25-M819 inhibit IL-25-induced IL-5 production. These data provide further support for: IL-17RB is essential for IL-25 signaling in splenocytes.
Example 5
This example characterizes the ability of various anti-IL-17 RA antibodies to inhibit the IL-25 response in vitro. Single cell suspensions of splenocytes were prepared essentially as described in example 4 above. Cells (100. mu.l) were added to a 96-well plate and brought to a final concentration of 4X 10 using the following conditions6Individual cells/well:
culture medium only
10ng/mL muIL-25 (stimulated control)
MuIL-25+100ng/mL muIL-17RB. muFc (blocking control)
10ng/mL muIL-25+1000, 100, 10, 1.0 or 0.1ng/mL anti-muIL-17 RA monoclonal antibody.
Each condition was tested with three independent biological samples, each consisting of splenocytes from two mice. Cultures were incubated at 37 ℃ and 10% CO2The following incubations were carried out for 72 hours, and supernatants were collected at 72 hours and IL-5 concentrations were determined by ELISA. A panel of eight different rat anti-mouse IL-17RA monoclonal antibodies was tested. None of them significantly inhibited IL-25-induced secretion of IL-5 by mouse splenocytes.
In addition to these rat anti-mouse antibodies, a mouse anti-mouse IL-17RA monoclonal antibody M751 was evaluated twice in the splenocyte assay. M751 inhibits IL-25-induced secretion of IL-5 from mouse splenocytes. The calculated IC50 for anti-mIL-17 RA M751 in 2 independent splenocyte experiments is shown in table 17 below. Thus, anti-IL-17 RA-M751 was the best anti-IL-17 RA inhibitor of IL-25-induced IL-5 production in this splenocyte assay, but was not as potent as anti-IL-17 RB-M735 as compared to anti-IL-17 RB-M735 (Table 15).
Table 17: IC50 values for anti-IL-17 RA-M751
Description of the antibodies IC50
mAb M751, experiment 1 4.03ng/mL
mAb M751, experiment 2 2.79ng/mL
Example 6
This example demonstrates the inhibition of the IL-25 response in vivo with an anti-IL-17 RA antibody M751, which inhibits IL-25 activity in an in vitro bioassay (previously described). Murine serum albumin (MSA; Sigma, 10. mu.g/mL) or murine IL-25(Amgen, TO; 10. mu.g/mL) was intranasally administered TO BALB/c mice once daily for four days. On days 1-4, mice were injected intraperitoneally with 200 micrograms of neutralizing anti-IL-17 RA antibody (M751), neutralizing anti-IL-17A antibody (M210), or isotype control antibody (murine Fc; Amgen) four hours prior to intranasal instillation of MSA or IL-25. On day 5, bronchoalveolar lavage fluid (BALF) and lung tissue were collected and analyzed as previously described. The results of two independent experiments are shown in tables 18-21 below.
Table 18: analysis of BALF cell content, IL-5 and IL-13 concentrations IN BALB/c mice treated with IN IL-25IN the Presence or absence of mouse IL-17RA blocking antibody M751-experiment 1
N is 5; the indicated values are (mean. + -. SD)
The value assigned to samples below the detection range of the IL-5ELISA was 31 pg/mL. The value assigned to samples below the detection range of the IL-13ELISA was 62 pg/mL.
Table 19: analysis of BALF cell content, IL-5 and IL-13 concentrations IN BALB/c mice treated with IN IL-25IN the Presence or absence of mouse IL-17RA blocking antibody M751-experiment 2
N is 5; the indicated values are (mean. + -. SD)
Table 20: analysis of IL-13, IL-5, IL-17RB, eotaxin and MCP-1mRNA IN Lung tissue from IN IL-25 challenged mice IN the absence or presence of mouse IL-17RA blocking antibody M751-experiment 1
N is 4; the values shown are the gene expression (2E-. DELTA.Ct) (mean. + -. SD) relative to GAPDH
Table 21: analysis of IL-13, IL-5, IL-17RB, eotaxin and MCP-1mRNA IN Lung tissue from IN IL-25 challenged mice IN the absence or presence of mouse IL-17RA blocking antibody M751-experiment 2
N is 4; values shown are gene expression (2E- Δ Ct) relative to GAPDH (mean ± SD); not determined N/D
Treatment with anti-IL-17 RA monoclonal antibody M751 inhibited IL-25-induced BALF cell content, as well as IL-25-induced BALF IL-5 and IL-13 concentrations and pulmonary transcription induction. In contrast, treatment with anti-IL-17A monoclonal antibody M210 did not significantly affect IL-25-induced BALF cell content (although the data show that this antibody may affect IL-25-induced BALF neutrophil levels). These data, together with those previously described in IL-17RA KO mice, indicate that IL-17RA is essential for IL-25-induced increases in BALF cell content and IL-5 and IL-13 concentrations. As shown by anti-IL-17A treatment significantly reduced IL-25-induced neutrophil influx into BALF, the effect of IL-25in vivo appears not to be mediated by IL-17A, in addition to IL-25-induced neutrophil recruitment.
Example 7
This example demonstrates the effect on Airway Hyperresponsiveness (AHR) induced by IL-25, as well as anti-IL-17 RA-M751 and anti-IL-17A-M210. BALB/c mice were administered MSA or mouse IL-25IN daily over a four day period, essentially as described previously. On day 5, Airway Hyperresponsiveness (AHR) to acetylcholine (MCh) challenge in conscious, unbound mice was first measured non-invasively using a whole-body plethysmograph (Buxco Electronics, Troy, NY). Based on the pressure waveform in the plethysmograph in response to increasing concentrations of MCh attacks, enhancement Pauses (PENH) were measured and reported as a percentage change relative to the baseline reading displayed prior to the MChS exposure. PC200 is the MCh concentration required to induce a PENH of 200% above baseline and is reported herein below in tables 22 and 23.
Table 22: AHR of MCh challenge IN BALB/c mice treated with IN IL-25IN the presence or absence of mouse IL-17RA or IL-17A blocking antibodies
Treatment of PC200，MCh，mg/mL
muFc，MSA 19.3±5.3
M751，MSA 20.7±7.6
M210，MSA 25.8±10.6
muFc，IL-25 4±4.7
M751，IL-25 15.9±1.5
M210，IL-25， 2±2.2
N is 5/group; the indicated values are (mean. + -. SD)
Table 23: BALB/c mice treated with IN IL-25IN the presence or absence of mouse IL-17RA blocking antibody M751, AHR of acetylcholine challenge
Treatment of PC200，MCh，mg/mL
muFc，MSA 12.5±2.6
M751，MSA 17.5±3.4
M210，MSA 21.5±3.4
MuFc，IL-25 4.3±0.5
M751，IL-25 9.0±1.7
M210，IL-25 5.3±1.3
N is 4/group; the indicated values are (mean. + -. SD)
Airway hyperreactivity was also measured in anesthetized and mechanically ventilated mice (treated with intranasal IL-25 and anti-IL-17 RA-M751). BALB/c mice were administered MSA or mouse IL-25IN daily over a four day period, essentially as described previously. On day 5, mice were sedated with xylazine hydrochloride (20mg/kg, given intraperitoneally) and anesthetized with sodium pentobarbital (100mg/kg, given intraperitoneally). The catheter was inserted into the trachea with a metal needle, and the mice were then connected to a small animal respirator (flexiVent, SCIRES: Scientific Respiratory apparatus, Montreal, Canada). Each mouse was ventilated with sinusoidal inspiration and passive expiration at a rate of 150 breaths/min and an amplitude of 10mL/kg mouse weight. A Positive End Expiratory Pressure (PEEP) of 3.0cmH2O was established by connecting the mice to the water column.
After one minute of ventilation of the mice, the lungs were expanded twice to total lung volume (TLC, 30cmH2O amplitude). An aerosol of saline or increasing concentrations of acetyl- β -methylcholine (MCh, Sigma-Aldrich) was delivered to the lungs for 15s followed by ventilation for 15 s. Followed by gasAerosol and ventilation, 2.5Hz Volume Drive (VD) oscillations were applied to the airway orifice. Each 10-2.5Hz VD oscillation had an amplitude of 0.20mL and lasted for 1.25 s. Lungs were expanded twice to TLC before the next dose of MCh. Recording pressure and volume measurements over time in the respiratory system with a small animal ventilator and calculating respiratory system resistance (R) by fitting the data to a single chamber model of the respiratory system in which P istr＝RV+EV+PO(PtrPressure, volume, time, elasticity, volume, POBaseline pressure). The lung resistance measured at different concentrations of MCh is shown in figure 2.
These results indicate that, in addition to inhibiting IL-25 activity in vitro and IL-25-induced increases in BALF cell content and IL-5 and IL-13 concentrations in vivo, M751 also inhibits IL-25-induced AHR, indicating that antibodies that bind IL-17RA and inhibit IL-25 activity are useful for treating or ameliorating IL-25 mediated conditions involving AHR.
Example 8
This example demonstrates the inhibition of the IL-25 response in vivo with either an anti-IL-17 RB antibody (M735) or an anti-IL-25 antibody (M819), both of which inhibit IL-25 activity in an in vitro bioassay (described above). PBS or mouse IL-25 was administered intranasally to BALB/c mice followed by intraperitoneal injection of 250 micrograms of neutralizing mouse anti-mouse IL-17RB antibody (M735), neutralizing rat anti-mouse IL-25 antibody (M819), irrelevant control mouse IgG1 antibody (muIgG 1; Amgen), murine Fc protein (muFc; Amgen), or whole rat IgG (Pierce, Rockford IL). On day 5, bronchoalveolar lavage fluid (BALF) was collected and analyzed as previously described. Carrying out independent repeated experiments; in the second, BALF IL-5 and IL-13 protein concentrations were not determined. The results are shown in tables 24 to 26 below.
Table 24: analysis of BALF cell content, IL-5 concentration and IL-13 concentration from IN IL-25 challenged mice IN the absence or presence of IL-17RB blocking antibody (M735)
N is 5; the indicated values are (mean. + -. SD)
Table 25: analysis of BALF cell content, IL-5 concentration and IL-13 concentration from IN IL-25 challenged mice IN the absence or presence of IL-17RB blocking antibody (M735) or IL-25 blocking antibody (M819)
N is 5; the indicated values are (mean. + -. SD)
Table 26: analysis of BALF cell content, IL-5 concentration and IL-13 concentration from IN IL-25 challenged mice IN the absence or presence of IL-17RB blocking antibody (M735) or IL-25 blocking antibody (M819)
N is 5; the indicated values are (mean. + -. SD)
Example 9
This example demonstrates the induction of Airway Hypersensitive Response (AHR) by IL-25 and the effect thereof by anti-IL-17 RB antibody (M735) or anti-IL-25 antibody (M819). Performing a sequence of experiments substantially as described previously; AHR of conscious, unbound mice was measured non-invasively using a whole-body plethysmograph. The results of three independent experiments are shown in tables 27-29 below.
Table 27: AHR values from IN IL-25 challenged mice IN the absence or presence of anti-IL-17 RB blocking antibody (M735)
Treatment of PC200，MCh，mg/mL
PBS 37.6±15
muIgG1，IL-25 5.5±3.2
M735，IL-25， 16.5±5.7
N is 5; the indicated values are (mean. + -. SD)
Table 28: AHR values from IN IL-25 challenged mice IN the absence or presence of anti-IL-17 RB blocking antibody (M735) or IL-25 blocking antibody (M819)
Treatment of PC200，MCh，mg/mL
PBS 42±13
muFc，IL-25 0.5±0.8
M735，IL-25 19.7±6.2
rIgG，IL-25 6.1±4.3
M819，IL-25 18.4±2.8
N is 5; the indicated values are (mean. + -. SD)
Table 29: AHR values from IN IL-25 challenged mice IN the absence or presence of anti-IL-17 RB blocking antibody (M735) or IL-25 blocking antibody (M819)
Treatment of PC200，MCh，mg/mL
PBS 29.9±3.3
muIgG1，IL-25 6.38±1.2
M735，IL-25 16.8±1.6
rIgG，IL-25 10.9±1.2
M819，IL-25 15.6±1.9
These results indicate that IL-25 increases AHR, and this effect can be mitigated by either anti-IL-17 RB or anti-IL-25.
Example 10
This example provides histological confirmation of the blocking of IL-25in vivo responses by treatment with an anti-IL-17 RB antibody (M735), an anti-IL-25 antibody (M819), or an anti-IL-17 RA antibody (M751). PBS or mouse IL-25 was intranasally administered to BALB/c mice intraperitoneally with 200 micrograms of neutralizing anti-IL-17 RB antibody (M735), 200 micrograms of neutralizing anti-IL-25 antibody (M819), 200 micrograms of neutralizing anti-IL-17 RA antibody (M751), 200 micrograms of neutralizing anti-IL 17A antibody (M210), or isotype control antibody essentially as described previously. On study day 5, by CO2Asphyxiation caused the mouse to die painlessly. Lungs were collected, fixed, processed, sectioned, stained and evaluated as described. A summary of the histopathological results is shown in table 30 below.
Table 30: histological analysis of lung inflammation and goblet cell proliferation IN mice challenged with IN IL-25 and treated with anti-IL-17 RA, anti-IL-17A, anti-IL-25, anti-IL-17 RB or control
N is 5/group; reported mean scores ± SD.
The mice had an average score of 1.8 ± 0.8 compared to mice challenged with MSA and treated with an isotype control; mice challenged with IL-25 and treated with isotype control had the deepest lesions with an average score of 7.6. + -. 2.2. As shown by the mean score of 6.8. + -. 1.3, treatment of mice with anti-IL-17A antibody had essentially no effect on lung injury. In contrast, anti-IL-17 RA (score 1.0. + -. 0.7), anti-IL-25 (score 1.4. + -. 1.1) or anti-IL-17 RB (score 1.8. + -. 1.5) treatments were all effective in inhibiting IL-25-induced inflammation to background levels, indicating that blocking of IL-25 or any of the proteins involved in the receptor complex is an equally effective treatment.
Example 11
This example demonstrates the association between IL-17RA and IL-17RB. A series of immunoprecipitations were performed using the extracellular domains of human IL-17RA and IL-17RB fused to the Fc region of human IgG (R & D Systems, Minneapolis, MN) or a polyhistidine tag (Amgen). 50 microliter of protein G slurry was added to the Eppendorf tube, washed with Phosphate Buffered Saline (PBS), followed by rotary incubation with 2 micrograms of IL-17RA. Fc or IL-17RB. Fc protein for 1 hour at 4 ℃. At the end of this incubation, 2. mu.g of the opposite soluble receptor protein was added (i.e., IL-17RA-HIS was added to IL-17RB: Fc and IL-17RB-HIS was added to IL-17RA: Fc), and this final combination was incubated overnight at 4 ℃ with rotation.
The next morning, the tubes were centrifuged at 12,000rpm for 1 minute, followed by washing of the protein G beads with PBS, followed by RIPA buffer (Sigma-Aldrich, st. The beads were resuspended in 60 microliters of 2 × Tris-glycine SDS sample buffer (Invitrogen, Carlsbad CA) containing 10% β -mercaptoethanol and subsequently stored on ice or at-20 ℃. In 4-20% Tris-glycine 10-well mini-acrylamide gel (Invitrogen, Carlsbad CA) followed by transfer to a nitrocellulose membrane (Invitrogen, Carlsbad CA). With gentle shaking, at room temperature for 1 hour or at 4 ℃ overnightThe blocking buffer solution is used for blocking the membrane, and the blocking buffer solution is used for infrared determination (Li-Biosciences, Lincoln, NE) optimizedWestern blot blocking buffer. The membrane was then incubated with primary antibody (in the presence of 0.1% Tween-20)Diluted 1: 1000 to 1: 5000 in blocking buffer) were incubated at 4 ℃ for 60 minutes with gentle shaking. The membrane was washed 4 times in PBS + 0.1% Tween-20, and then the membrane was washed with a second antibody (in the presence of 0.1% Tween-20)Diluted 1: 10,000 in blocking buffer) was incubated for 60 minutes at 4 ℃ with gentle shaking. The membranes were washed four times in PBS + 0.1% Tween-20, followed by Li-The infrared imaging system presents the protein. The following antibodies were used:
a first antibody: second antibody:
goat anti-hIL-17 RA, affinity purified polypeptides800CW donkey anti-goat
Cloning antibody (R)&D Systems, IgG (H + L), high adsorption (Li-
Minneapolis，MN) Biosciences，Lincoln，NE；
Infrared dye: US06027709)
Goat anti-hIL-17 RB, affinity purified polypeptidesMonoclonal antibodies (directed anti)
Cloning of antibodies (R & D Systems, mouse monoclonal antibodies with His Tag sequences
Minneapolis，MN) (IgG1)；Novagen，EMD Chemicals，
Inc.，San Diego，CA)
Goat anti-hIL-17 RC, affinity purified polypeptides
The cloning of antibodies (R & D Systems,
Minneapolis，MN)
Alexa 680 rabbit anti-mouse
IgG(H+L)(Invitrogen，Carlsbad，
CA; alexa Fluor 680: berlier JE, et al,
J Histochem Cytochem 51，
1699-712(2003))
a representative blot is shown in figure 2. In several experiments, IL-17RB.Fc was able to immunoprecipitate IL-17 RA.HIS. In this experimental system, IL-17ra.fc was also able to immunoprecipitate IL-17rc.his, indicating that this system was able to reproduce biochemical interactions between proteins that had been previously shown in other systems (Toy, d. et al, JI, 2006, 177: 36). IL-17RA.Fc or IL-17RB.Fc were not able to immunoprecipitate IL-17RD.HIS (R & D Systems, Minneapolis, MN), indicating that the IL-17RA and IL-17RB interactions are characteristic of these proteins and not inherent to all IL-17R family members. This is the first description of biochemical interactions between IL-17RA and IL-17RB.
Example 12
As described in USSN 11/906,094 (incorporated herein by reference), using Abgenix (now Amgen Fremont Inc.)Techniques (U.S. Pat. Nos. 6,114,598, 6,162,963, 6,833,268, 7,049,426, 7,064,244, which are incorporated herein by reference in their entirety; Green et al, 1994, Nature Genetics 7: 13-21; Mendez et al, 1997, Nature Genetics 15: 146-. As described therein, fully human anti-IL-17 RA antibodies are screened for their ability to inhibit human IL-17A binding to human IL-17RA (and to cynomolgus IL-17 RA). A panel of antibodies was identified and selected for further spreading and analysis; the amino acid sequences of the variable heavy and light chains are shown in the sequence listing, while a table summarizing the various sequences is shown below. One antibody 3.454.1 shows evidence of both forms of variable light chains.
Table 31: summary of anti-huIL-17A antibodies
The antibodies were further characterized with respect to: its ability to inhibit IL-17A and/or IL-17F bioactivity, and which domains of IL-17RA are important for antibody binding.
Determination of IL-17A/IL-17F induced cytokine/chemokine secretion
The assay uses a Human Foreskin Fibroblast (HFF) cell line. anti-IL-17 RA antibody at 36 degrees C and HFF cells (96 hole plate 5000 cells/hole) temperature in 30 minutes; the cultures were then stimulated overnight with IL-17A (5ng/ml) alone or IL-17F (20ng/ml) and TNF-. alpha. (5 ng/ml). Fibroblast culture supernatants were analyzed for the presence of IL-6 or GRO-alpha by ELISA. The antibodies inhibit the biological activity of IL-17A and IL-17F as indicated by a decrease in the amount of IL-6 and/or GRO- α produced in the assay.
Cross-competition assay
Cross-competition studies were performed to determine the IL-17RA binding properties of certain antibodies as described in USSN 11/906,094. The multiplex binding method described by modified Jia et al (see Jia et al, J.Immun. meth., 2004, 288: 91-98) was used, in which a Bio-Plex workstation and software (BioRad, Hercules, Calif.) andcompany's reagent (Austin, TX). Generally following the basic manufacturer's protocol. Testing the antibodies in a paired combination; if two antibodies cross-compete with each other, they are grouped or "boxed" together. In general, antibodies that are boxed differently bind to different sites of IL-17RA, while antibodies that are boxed the same bind to similar sites of IL-17RA.
Evaluation of neutralization determinants: Hu/Mu chimeras
Using a large number of chimeric human/mouse IL-17RA, studies were conducted to determine where various IL-17RA antagonists (in the form of human antibodies) bind to human IL-17RA. The method utilizes the non-cross-reactivity of various IL-17RA antibodies with mouse IL-17RA. For each chimera, one or both regions of the extracellular domain of human IL-17RA were replaced with the corresponding region of mouse IL-17RA. Chimeras of 6 single regions and 8 double regions are constructed; multiplex analysis using Bio-Plex workstation and software (BioRad, Hercules, Calif.) was performed to determine the neutralizing determinant of human IL-17RA by analyzing the differential binding of exemplary human IL-17RA mAbs to chimeric and wild-type IL-17RA proteins.
Evaluation of neutralization determinants: arginine scan
Further studies were conducted using a number of mutant IL-17RA proteins having arginine substitutions at selected amino acid residues of human IL-17RA. Arginine scanning is an art-recognized method of assessing where an antibody or other protein binds to another protein, see, e.g., Nanevicz, t. et al, 1995, j.biol.chem., 270: 37, 21619-: 29, 20464-20473. In general, arginine side chains are positively charged and relatively bulky compared to other amino acids, which would disrupt the mutation-introducing region of antibody binding to antigen. Arginine scanning is a method of determining whether a residue is part of a neutralization determinant and/or epitope. The 95 amino acids distributed throughout the extracellular domain of human IL-17RA were selected for mutation to arginine. The selection is biased towards charged or polar amino acids, so that the residues are most likely on the surface and the probability of mutations leading to protein misfolding is reduced.
Stratagene based using standard techniques known in the artII protocol kits (Stratagene/Agilent, Santa Clara, Calif.) provided standard designs of sense and antisense oligonucleotides containing mutated residues. Use ofII kit (Stratagene) mutagenesis of Wild Type (WT) HuIL-17RA-Flag-pHis was performed. All chimeric constructs were constructed to encode a FLAG-histidine tag (6 histidines) on the carboxy terminus of the extracellular domain to facilitate purification by means of a poly-His tag. Multiplex analysis using Bio-Plex workstation and software (BioRad, Hercules, Calif.) was performed to determine the neutralizing determinant of human IL-17RA by analyzing the differential binding of certain human IL-17RA mAbs to arginine mutants and wild-type IL-17RA protein.
The results of these studies are summarized in table 32 below.
Table 32: summary of the Properties of certain IIL-17RA antibodies
Example 13
This example describes an IL-25 re-stimulation assay useful for assessing the effect of L-17RA-IL-17RB antagonists on IL-25 bioactivity. Human Peripheral Blood Mononuclear Cells (PBMC) were isolated from normal donors at 5X 106Individual cells/ml in thymus stromal lymphopoietin (TSLP (Quentmeier et al, Leukemia.2001Aug; 15 (8): 1286); 100 ng/ml; purchased from R&D Systems, Minneapolis, MN) for 24 hours. The PBMCs are then collected and placed in the presence of IL-2(10 ng/ml, R) in the presence or absence of the substance whose inhibitory activity is being tested&D Systems, Minneapolis, MN) and IL-25(10 nanograms/ml; r&D Systems, Minneapolis, MN). Restimulated cultures were prepared as single cell suspensions and diluted to 4X 107Individual cells/ml; 100 microliter of cells were added to 48-well plates to a final concentration of 4X 106Individual cells/well. After 3 days, the supernatant was collected and used for ELISA (R)&D Systems, Minneapolis, MN) for IL-5. The substances tested included the soluble form of IL-17RB (previously described) and the following sets of polyclonal and monoclonal antibodies summarized below:
MAB 1771: anti-HuIL-17 RA MuIgG2b (R & D Systems)
MAB 1207: anti-HuIL-17 RB MuIgG2b (R & D Systems)
AF 177: anti-HuIL-17 RA goat polyclonal IgG (R & D Systems)
Several fully human anti-HuIL-17 RA HuIgG2 (described in example 12)
The results of testing various substances in several different re-stimulation assays using PBMCs from different donors are shown in table 33 below.
Table 33: IL-5 production by IL-2+ IL-25 stimulated human PBMCs (TSLP treated) in the presence or absence of various IL-17RB and IL-17RA inhibitors
Description of inhibitors [ inhibitor of]microgram/mL [IL-5]，pg/mL % inhibition
Is free of Is free of 93.9±9.7 0％
HuIL-17RB:Fc 10 42.0±10.8 55％
HuIL-17RB:HIS 10 31.2±11.5 67％
3.1404 10 35.9±53 62％
3.1404 1.0 42.9±5.1 54％
3.1404 0.1 83.4±4.6 12％
Is free of Is free of 576±6.8 0％
HuIL-17RB:Fc 10 72.7±3.8 87％
MAB1771 10 437.7±7.8 24％
MAB1207 10 499.4±6.3 13％
AF177 10 105.8±4.0 82％
3.1404 10 100.5±5.1 83％
Is free of Is free of 191.6+4.9 0％
HuIL-17RB:Fc 10 24.5±4.2 88％
MAB1771 10 127.9±3.6 33％
AF177 10 26.0±4.0 86％
3.1404 10 19.2±3.4 90％
4.16 10 22.2±3.4 88％
3.381 10 0.0±0.0 100％
A panel of human antibodies that bind IL-17RA was tested in 3 independent restimulation assays using different PBMC donors on different days and with different preparations of antibody. The results are shown in Table 34 below.
Table 34: IL-5 production by IL-2+ IL-25 stimulated human PBMC (TSLP treatment) in the presence or absence of various IL-17RA antibodies
The results of the block analysis of these antibodies were ambiguous.
Substantially similar results were obtained with additional preparations of these antibodies. This result indicates that certain antibodies that bind IL-17RA and inhibit IL-17A also inhibit IL-25.
Example 14
This example describes a mouse model of asthma. Mice were sensitized with an antigen (e.g., BALB/c) by intraperitoneal injection of the antigen (e.g., ovalbumin [ OVA ]) in alum or another adjuvant. Several sensitization schemes are known in the art; one regimen was three injections of 10 micrograms of OVA (in alum) at weekly intervals (i.e., day-21, day-14, and day-7). The mice were then challenged with antigen by aerosol exposure (5% OVA) or intranasal administration (0.1mg OVA). The attack schedule may be selected from shorter periods (i.e., daily attacks on days 1, 2, and 3) or longer periods (i.e., weekly attacks for two to three weeks). Endpoints to be measured may include AHR, BAL fluid cell number and composition, in vitro draining lung lymph node cytokine levels, serum IgE levels, and histopathological assessment of lung tissue. Other animal models of asthma are known, including the use of other animals (e.g., C57BL/6 mice), sensitization protocols (e.g., intranasal vaccination, use of other adjuvants or no adjuvants, etc.), and/or antigens (including peptides (e.g., peptides derived from OVA or other proteinaceous antigens), cockroach extracts, ragweed extracts, or other extracts (e.g., extracts for desensitization therapy, etc.)). The effect of the antibodies on IL-17RA, IL-17RB, IL-17 and IL-25 was evaluated in this model using a group of mice as shown below.
Female BALB/c mice were immunized with OVA IP in alum on days-21, -14 and-7 and exposed to aerosol challenge with OVA (in PBS) on days 1-3. In experiments 1 and 2, mice were injected IV with antibody one day before OVA aerosol challenge (day-1); or in experiment 3, mice were IP injected with antibody on the day of the first OVA aerosol challenge (day 1), 30 minutes prior to OVA challenge; or in experiments 1 and 3, mice were IP injected with dexamethasone (Dex) (positive control) or Phosphate Buffered Saline (PBS) (negative control) 30 minutes prior to each aerosol exposure to OVA (days 1-3). Age-matched, antigen OVA-only exposed groups were included for comparison. Airway Hyperresponsiveness (AHR) to MCh challenge was measured 48 hours after the last OVA challenge. 72 hours after the last OVA challenge, mice were sacrificed and serum, BAL fluid, draining lung lymph nodes and lungs were collected for analysis. A series of three experiments were performed.
Experiment 1 included the following treatment groups:
group 1: naive mice exposed to antigen, n-10
Group 2: PBS, IP, n ═ 10
Group 3: 1mg/kg Dex, IP, n ═ 10
Group 4: 500 microgram mIgG1 isotype control ab, IV, n ═ 10
Group 5: 500 microgram of anti-IL-17 RB M735mAb, IV, n ═ 10
Group 6: 500 microgram of chimeric anti-mIL-17 RA mAb M751, IV, n-10
Group 7: 500 microgram rat IgG control ab, IV, n ═ 10
Group 8: 500 micrograms of anti-mIL-25M 819, IV, n ═ 10
Group 9: 500 microgram of anti-mIL-17 mAb M210, IV, n-10
Experiment 2 included the following treatment groups:
group 1: naive mice exposed to antigen, n-10
Group 2: 500 microgram mIgG1 isotype control ab, IV, n ═ 10
Group 3: 500 microgram of chimeric anti-mIL-17 RA mAb M751, IV, n-10
Experiment 3 included the following treatment groups:
group 1: naive mice exposed to antigen, n-10
Group 2: PBS, IP, n ═ 10
Group 3: 1mg/kg Dex, IP, n ═ 10
Group 4: 500 microgram mIgG1 isotype control ab, IP, n ═ 10
Group 5: 500 microgram of anti-IL-17 RB M735mAb, IP, n ═ 10
Group 6: 500 microgram of chimeric anti-mIL-17 RA mAb M751, IP, n-10
Group 7: 500 microgram rat IgG control ab, IP, n ═ 10
Group 8: 500 microgram anti-mIL-25M 819, IP, n ═ 10
Group 9: 500 microgram of anti-mIL-17 mAb M210, IP, n ═ 10
Group 10: 500 microgram of anti-mIL-17F mAb M850, IP, n ═ 10
Neutralizing antibodies to IL-17RB, IL-17RA, or IL-25, but not IL-17A, reduced AHR in a mouse OVA asthma model; the results are shown in FIGS. 1 to 3. Percent change in the mean value of PENH from baseline was reported for each treatment group ± SE from experiment 1 (figure 1). Airway hyperresponsiveness was measured essentially as previously described in example 7. The degree of bronchoconstriction is expressed as percent change in PENH from baseline. Treatment with neutralizing antibodies to IL-17RB, IL-17RA, or IL-25, but not IL-17A, reduced AHR in response to MCh challenge compared to mice treated with PBS or control antibodies (FIG. 3).
In experiments 2 and 3, the pulmonary resistance (R) of mechanically ventilated mice in response to methacholine challenge was measuredL). Measurements of pressure and volume in the respiratory system over time were recorded with a small animal ventilator and respiratory system resistance (R ═ cmH) was calculated by fitting the data to a single chamber model of the respiratory system2O/mL), P in the single chamber modeltr＝RV+EV+PO(PtrPressure, volume, time, elasticity, volume, POBaseline pressure). The area of airway resistance (R) under the curve (AUC) was calculated by summing all R measurements for each mouse at each concentration of methacholine. In experiment 2, treatment with IL-17RA neutralizing antibody reduced lung resistance in response to methacholine challenge compared to mice treated with control antibody (figure 4 a). In experiment 3, treatment with neutralizing antibodies to IL-17RB, IL-17RA or IL-25, but not IL-17A, reduced the pulmonary resistance to methacholine challenge compared to control antibody treated mice (FIG. 4 b).
The effect of the antibody on BALF cell number and composition was also determined; the results are shown in FIGS. 5 to 7. In experiment 1, neutralizing antibodies to IL-17RB, IL-17RA or IL-25, but not IL-17A, significantly reduced the total leukocyte (FIG. 5a), eosinophil (FIG. 5b) and lymphocyte (FIG. 5d) numbers of BALF in this mouse OVA asthma model compared to treatment with the appropriate control antibodies. Neutralizing antibodies to IL-17RB or IL-17RA but not IL-17A significantly reduced the total BALF neutrophil count (FIG. 5 c). Neutralizing antibodies to IL-25 reduced BALF total neutrophil counts but were not significant (fig. 5 c).
In experiment 2, neutralizing antibodies to IL-25, IL-17RB and IL-17RA significantly reduced the total leukocyte (FIG. 6a), eosinophil (FIG. 6b) and lymphocyte (FIG. 6d) numbers of BALF in this mouse OVA asthma model compared to treatment with the appropriate control antibodies. These antibodies had no significant effect on total BALF neutrophil (fig. 6c) or macrophage (fig. 6e) numbers.
In experiment 3, neutralizing antibodies to IL-17RB, IL-17RA or IL-25 but not IL-17A or IL-17F significantly reduced the total leukocyte (FIG. 7A), eosinophil (FIG. 7b) and lymphocyte (FIG. 7d) counts of BALF in this mouse OVA asthma model. Neutralizing antibodies to IL-17RB, IL-17RA, or IL-25 but not IL-17A or IL-17F reduced the total neutrophil count of BALF (FIG. 7c), but only IL-17RB and IL-25 antibodies had a significant effect. Neutralizing antibodies to IL-17RB or IL-17RA but not IL-25, IL-17A or IL-17F reduced BALF total macrophage numbers (FIG. 7e), but only IL-17RA antibody had a significant effect.
As shown in FIG. 8a (experiment 1) and FIG. 8c (experiment 3), neutralizing antibodies to IL-17RB, IL-17RA or IL-25, but not IL-17A or IL-17F, significantly reduced BALF IL-13 concentration. In experiment 2, the concentrations of BALLFIL-13 in mice treated with neutralizing antibodies to IL-17RB, IL-17RA or IL-25 were lower but not significant, probably due to overall lower levels of IL-13 induction (compared to that typically observed in the present mouse model) (FIG. 8 b).
Neutralizing antibodies to IL-17RB, IL-17RA or IL-25 but not IL-17A or IL-17F also reduced BALF IL-5 concentration in this mouse OVA asthma model, but the group treated with anti-IL-25 mAb alone was significantly lower in experiment 1 (FIG. 9a) compared to isotype control antibody treated mice, whereas the groups treated with anti-IL-17 RB, anti-IL-17 RA and anti-IL-25 mAb were all significantly lower in experiment 3 (FIG. 9 c). Furthermore, in experiment 2, the BALF IL-5 concentration was significantly reduced by treatment with neutralizing antibodies to IL-17RB, IL-17RA and IL-25 (FIG. 9 b).
Similarly, neutralizing antibodies to IL-17RB, IL-17RA, or IL-25, but not IL-17A or IL-17F, reduced total serum IgE concentrations in the present mouse OVA asthma model. In experiment 1, neutralizing antibodies to IL-17RB, IL-17RA, or IL-25 reduced total serum IgE concentrations compared to the appropriate isotype control antibody treated group, but only the IL-25 neutralizing antibody treated group significantly reduced total serum IgE concentrations (FIG. 10 a). In experiment 2, neutralizing antibodies to IL-25, IL-17RB or IL-17RA reduced total serum IgE concentrations compared to the control antibody treated group, but not significantly (FIG. 10 b). In experiment 3, neutralizing antibodies to IL-17RB, IL-17RA or IL-25, but not IL-17A or IL-17F, significantly reduced total serum IgE concentrations compared to the respective appropriate control antibody-treated groups (FIG. 10 c).
Lungs from 8 mice from each treatment group in experiment 3 were analyzed histologically. Lung tissue sections were stained with H & E or PAS and then scored by a pathologist as described in example 1 previously. Treatment with neutralizing antibodies to IL-17RB, IL-17RA, or IL-25, but not IL-17A or IL-17F, significantly reduced the inflammatory score in this mouse OVA asthma model (FIG. 11).
These results indicate that treatment with anti-IL-17 RB mAb M735, anti-IL-17 RA mAb M751, or anti-IL-25 mAb M819 significantly reduced multiple parameters of inflammation in this mouse OVA-induced asthma model, which is considered a model of human lung inflammatory conditions (e.g., asthma). In contrast, treatment with anti-IL-17A mAb or anti-IL-17F mAb did not significantly reduce inflammation in this model. Therefore, IL-25 and its receptors IL-17RB and IL-17RA play a role in mediating inflammation in this mouse OVA asthma model.

Claims (6)

  1. Use of an IL-17RA-IL-17RB antagonist in the manufacture of a medicament for inhibiting a biological activity of IL-25, wherein the IL-17RA-IL-17RB antagonist is a polypeptide comprising the amino acid sequence set forth in seq id NO: 40 and a light chain variable domain consisting of SEQ ID NO: 14, or a heavy chain variable domain.
  2. 2. The use of claim 1, wherein the release of at least one pro-inflammatory mediator is inhibited, wherein the pro-inflammatory mediator is IL-5.
  3. Use of an IL-17RA-IL-17RB antagonist in the manufacture of a medicament for inhibiting a biological activity of IL-25in vivo, wherein the IL-17RA-IL-17RB antagonist is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 40 and a light chain variable domain consisting of SEQ ID NO: 14, or a heavy chain variable domain.
  4. 4. The use of claim 3, wherein the release of at least one pro-inflammatory mediator is inhibited, wherein the pro-inflammatory mediator is IL-5.
  5. Use of an IL-17RA-IL-17RB antagonist in the manufacture of a medicament for inhibiting a biological activity of IL-25in vitro, wherein the IL-17RA-IL-17RB antagonist is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 40 and a light chain variable domain consisting of SEQ ID NO: 14, or a heavy chain variable domain.
  6. 6. The use of claim 5, wherein the release of at least one pro-inflammatory mediator is inhibited, wherein the pro-inflammatory mediator is IL-5.
HK11106212.6A 2008-02-21 2009-02-20 Il-17ra-il-17rb antagonists and uses thereof HK1152053B (en)

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