Specification
TRUNCATED IL-17RA SOLUBLE RECEPTOR AND METHODS OF USING IN INFLAMMATION
BACKGROUND OF THE INVENTION
[1] Cytokines are soluble, small proteins that mediate a variety of biological effects, including the regulation of the growth and differentiation of many cell types (see, for example, Aral et al, Anmi. Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol. 3:311 (1991); Paul and Seder, Cell 75:241 (1994)). Proteins that constitute the cytokine group include interieukins, interferons, colony stimulating factors, tumor necrosis factors, and other regulatory molecules. For example, human interleukin-17 is a cytokine which stimulates the expression of interleukin-6, intracellular adhesion molecule 1, interleukin-8, granulocyte macrophage colony-stimulating factor, and prostaglandin E2 expression, and plays a role in the preferential maturation of CD34+ hematopoietic precursors into neutrophils (Yao el al, ./. Immunol. 755:5483 (1995); Fossiez el al, .1. Exp. Med. 183:2593 (1996)).
[2] Receptors that bind cytokines are typically composed of one or more integral membrane proteins that bind the cytokine with high affinity and transduce this binding event to the cell through the cytoplasmic portions of the certain receptor subunits. Cytokine receptors have been grouped into several classes on the basis of similarities in their extracellular ligand binding domains.
[3] The demonstrated in vivo activities of cytokines and their receptors illustrate the clinical potential of, and need for, other cytokines, cytokine receptors, cytokine agonists, and cytokine antagonists. For example, demonstrated in vivo activities of the pro-inflammatory cytokine family illustrates the enormous clinical potential of, and need for antagonists of pro-inflammatoi7 molecules.
DETAILED DESCRIPTION OF THE INVENTION
[4] The present invention addresses these needs by providing antagonists to pro-inflammatoiy cytokine 1L-17A, and 1L-17F. Specifically, the antagonists of the invention are engineered variants as shown in SEQ ID N0:6 (polynucleotide shown in SEQ ID N0:5). The variant polypeptide shown in SEQ ID N0:6 comprises exons 7-10 of 1L-17RA fused to Fc5 with otPA pre-pro signal sequence. The II-17RA variant can also have a native 1L-17RA signal sequence, and can be fused to an FclO. Specifically, the pro-inflammatory cytokines 1L-17A and 1L-17F have a high degree of sequence similarity, share many biological properties, and are both produced by activated T cells. They have both been implicated as factors that contribute to the progression of various autoimmune and inflammatory diseases including rheumatoid arthritis and asthma. In fact, reagents that negate iL-17A function significantly ameliorate disease incidence and severity in several mouse models of human disease. 1L-17A mediates its effects through interaction with its cognate receptor.
the lL-17 receptor (IL-)7RA), and for IL-17F, IL-17RA. Previously, we had reported that IL-17RA is a receptor for both IL-17A and IL-17F. and binds both with a similar high affinity. (L-17RA on the other hand, binds IL-I7A with high affinity, but binds IL-I7F with very low affinity. Consistent with this, it has been shown that a soluble form of IL-17RA blocks IL-17A binding and signaling in cells expressing either receptor, but does not interfere with binding or function of IL-17F to IL-17RA. Thus, the present invention has determined that a shortened lL-17RA soluble receptor may be used to block IL-17A and possibly 1L-17F. Since 1L-17A intervention has been proposed as an effective therapy for several auto-immune diseases, using such a truncated soluble receptor may address concerns surrounding immungenicity of the longer soluble receptor. Thus, the present invention is directed to IL-I7RA antagonists, such as the truncated, soluble receptor described herein in SEQ ID NOs:5 and 6, which may block, inhibit, reduce, antagonize or neutralize the activity of IL-17A, IL-17F, or both IL-17A and IL-17F.. The invention further provides uses therefor in inflammatory disease, as well as related compositions and methods.
A) Overview
[5] Immune related and inflammatory diseases are the manifestation or consequence of fairly complex, often multiple interconnected biological pathways which in normal physiology are critical to respond to insult or injury, initiate repair from insult or injury, and mount innate and acquired defense against foreign organisms. Disease or pathology occurs when these normal physiological pathways cause additional insult or injury either as directly related to the intensity of the response, as a consequence of abnormal regulation or excessive stimulation, as a reaction to self, or as a combination of these.
[6] Though the genesis of these diseases often involves multi-step pathways and often multiple different biological systems/pathways, intervention at critical points in one or more of these pathways can have an ameliorative or therapeutic effect. Therapeutic intervention can occur by either antagonism of a detrimental process/pathway or stimulation of a beneficial process/pathway.
|7] Many immune related diseases are known and have been extensively studied. Such diseases include immune-mediated inflammatory diseases (such as rheumatoid arthritis, immune mediated renal disease, hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, and asthma), non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, neoplasia, etc.
[8] T lymphocytes (T cells) are an important component of a mammalian immune response. T cells recognize antigens which are associated with a self-molecule encoded by genes within the major histocompatibility complex (MHC). The antigen may be displayed together with MHC molecules on the surface of antigen presenting cells, virus infected cells, cancer cells, grafts.
etc. The T cell system eliminates tliese altered cells which pose a health threat to the host mammal. T cells include helper T cells and cytotoxic T cells. Helper T cells proliferate extensively following ecognition of an antigen-MHC complex on an antigen presenting cell. Helper T cells also secrete a variety of cytokines, i.e., lymphokines, which play a central role in the activation of B cells, cytotoxic cells and a variety of other cells which participate in the immune response.
[9] A central event in both humoral and cell mediated immune responses is the activation and clonal expansion of helper T cells. Helper T cell activation is initiated by the interaction of the T cell receptor (TCR)—CD3 complex with an antigen-MHC on the surface of an antigen presenting cell. This interaction mediates a cascade of biochemical events that induce the resting helper T cell to enter a cell cycle (the GO to Gl transition) and results in the expression of a high affinity receptor for lL-2 and sometimes IL-4. The activated T cell progresses through the cycle proliferating and differentiating into memory cells or effector cells.
[10] In addition to the signals mediated through the TCR, activation of T cells involves additional costimulation induced by cytokines released by the antigen presenting cell or through interactions with membrane bound molecules on the antigen presenting cell and the T cell. The cytokines IL-1 and lL-6 have been shown to provide a costimulatory signal. Also, the interaction between the B7 molecule expressed on the surface of an antigen presenting cell and CD28 and CTLA-4 molecules expressed on the T cell surface effect T cell activation. Activated T cells express an increased number of cellular adhesion molecules, such as ICAM-l, integrins, VLA-4, LFA-I, CD56, etc.
[11] T-cell proliferation in a mixed lymphocyte culture or mixed lymphocyte reaction (MLR) is an established indication of the ability of a compound to stimulate the immune system. In many immune responses, inflammatory cells infiltrate the site of injury or infection. The migrating cells may be neutrophilic, eosinophilic, monocytic or lymphocytic as can be determined by histologic examination of the affected tissues. Current Protocols in Immunology, ed. .lohn E. Coligan, 1994, .lohn Wiley & Sons, Inc.
[12] Immune related diseases could be treated by suppressing the immune response. Using soluble receptors and/or neutralizing antibodies that inhibit molecules having immune stimulatory activity would be beneficial in the treatment of immune-mediated and inflammatory diseases. Molecules which inhibit the immune response can be utilized (proteins directly or via the use of antibody agonists) to inhibit the immune response and thus ameliorate immune related disease.
[13] Interleukin-17 (IL-17A) has been identified as a cellular ortholog of a protein encoded by the T lymphotropic Herpes virus Saimiri (HSV) [see, Rouvier et al., .1. Immunol,. 150(12): 5445-5456 (19993); Yao et al., J. Immunol., 122(12):5483-5486 (1995) and Yao et al.. Immunity, 3(6):811-821 (1995)]. Subsequent characterization has shown that this protein is a potent
cytokine that acts to induce proinflammatory responses in a wide variety of peripiieral tissues. IL-17A is a disuifide-iinked Jiomodimeric cytokine of about 32 kDa which is synthesized and secreted only by CD4+activated memory T cells (reviewed in Fossiez et al.. Int. Rev. Immunol.. 16: 541-551 [1998]). Specifically, IL-I7 is synthesized as a precursor polypeptide of 155 amino acids with an N-terminal signal sequence of 19-23 residues and is secreted as a disulfide-linked homodimeric glycoprotein. II-17A is disclosed in W09518826 (1995), WO9715320 (1997) and WO9704097 (1997), as well as US Patent No. 6,063,372.
|14] Despite its restricted tissue distribution, IL-17A exhibits pieitropic biological activities on various types of cells. IL-17A has been found to stimulate the production of many cytokines. It induces the secretion of lL-6, lL-8, lL-12, leukemia inhibitory factor (LIF), prostaglandin E2, MCP-1 and G-CSF by adherent cells like fibroblasts, keratinocytes, epithelial and endothelial cells. IL-17A also has the ability to induce ICAM-1 surface expression, proliferation of T cells, and growth and differentiation of CD34.sup.-i- human progenitors into neutrophils. 1L-17A has also been implicated in bone metabolism, and has been suggested to play an important role in pathological conditions characterized by the presence of activated T cells and TNF-.alpha. production such as rheumatoid arthritis and loosening of bone implants (Van Bezooijen et al., J. Bone Miner. Res. 14: 1513-1521 [1999]). Activated T cells of synovial tissue derived from rheumatoid arthritis patients were found to secrete higher amounts of iL-17A than those derived from normal individuals or osteoarthritis patients (Chabaud et al., Arthritis Rheum. 42: 963-970 [1999]). It was suggested that this proinflammatory cytokine actively contributes to synovial inflammation in rheumatoid arthritis. Apart from its proinflammatory role, 1L-17A seems to contribute to the pathology of rheumatoid arthritis by yet another mechanism. For example, IL-17A has been shown to induce the expression of osteoclast differentiation factor (ODF) mRNA in osteoblasts (Kotake et al., J. Clin. Invest., 103; I345-I352 [1999]). ODF stimulates differentiation of progenitor cells into osteoclasts, the cells involved in bone resorption.
[15] Since the level of IL-17A is significantly increased in synovial fluid of rheumatoid arthritis patients, it appears that IL-17A induced osteoclast formation plays a crucial role in bone resorption in rheumatoid arthritis. 1L-17A is also believed to play a key role in certain other autoimmune disorders such as multiple sclerosis (Matusevicius et al.. Mult. Scler., 5: 101-104 [1999]). IL-17A has further been shown, by intracellular signalling, to stimulate Ca.sup.2+ influx and a reduction in [cAMP], in human macrophages (Jovanovic et al., .1. Immunol., 160:3513 [1998]). Fibroblasts treated with IL-J7A induce the activation of NF-.kappa.B, [Yao et al.. Immunity, 3:811 (1995), Jovanovic et al., supra], while macrophages treated with it activate NF-.kappa.B and mitogen-activated protein kinases (Shalom-Barek et al., J. Biol. Chem., 273:27467 [1998]).
[16] Additionally, IL-17A also sliares sequence similarity with mammalian cytokine-like factor 7 that is involved in bone and cartilage growth. Other proteins with which IL-17A polypeptides share sequence similarity are human embryo-derived interleukin-related factor (EDIRF) and interleukin-20.
[17] Consistent with IL-17A's wide-range of effects, the cell surface receptor for IL-17A has been found to be widely expressed in many tissues and cell types (Yao et al.. Cytokine, 9:794 [1997]). While the amino acid sequence of the human IL-17A receptor (IL-17RA) (866 amino acids) predicts a protein with a single transmembrane domain and a long, 525 amino acid intracellular domain, the receptor sequence is unique and is not similar to that of any of the receptors from the cytokine/growth factor receptor family. This coupled with the lack of similarity of 1L-17A itself to other known proteins indicates that IL-17A and its receptor may be part of a novel family of signalling proteins and receptors. It has been demonstrated that 1L-17A activity is mediated through binding to its unique cell surface receptor, wherein previous studies have shown that contacting T cells with a soluble form of the 1L-17A receptor polypeptide inhibited T cell proliferation and IL-2 production induced by PHA, concanavalin A and anti-TCR monoclonal antibody (Yao et al., J. Immunol., 155:5483-5486 [1995]). As such, there is significant interest in identifying and characterizing novel polypeptides having homology to the known cytokine receptors, specifically IL-17A receptors.
|18] The expression pattern of lL-17F appears to be similar to that of IL-1 7A, such that it includes only activated CD4+ T cells and monocytes (Starnes et al. J. Immunol. 167: 4137-4140 [2001]). IL-17F has been demonstrated to induce G-CSF, IL-6, and IL-8 in fibroblasts (Hymowitz et al, EMBO J. 20:5322-5341 [2001]) and TGF-b in endothelial cells (Starnes et al. J. Immunol. 167: 4137-4140 [2001]). It has recently been reported that IL-23, a cytokine produced by dendritic cell, can mediate the production of both 1L-17A and 1L-17F, primarily in memory T cells (Aggarwal et al. .1. Biol. Chem. 278:1910-1914 [2003]).
[19] Moreover, over expression or upregulation of both IL-17A and IL-17F have been shown in arthritic and asthmatic individuals (reviewed in Moseley et al. CytokineGrowth Factor Rev 14:155-174 [2003]). With regards to arthritis, these cytokines act in a manner characteristic to the cartilage and joint destruction that is associated with rheumatoid- and osteo-arthritis. For example, 1L-17A and IL-17F have been demonstrated to enhance matrix degradation in articular cartilage explants via release of cartilage proteoglycan glycosaminoglycans and collagen fragments, while inhibiting the synthesis of new proteoglycans and coUagens (Cai et al. Cytokine 16:10-21 [2001]; Attur et al Arthritis Rheum 44:2078-2083 [2001 ]).
[20] Similar to IL-17A, overexpression of IL-17F in mice has also been shown to increase lung neutrophil recruitment and result in increased expression of Thl-associated cytokines in the
lung, including lL-6, IFN-gamma, iP-10 and MIG (Starnes et al. J. Immunol. 167: 4137-4140 [2001]). 1L-17F was also upregulated in T cells from allergen-challenged asthmatics (Kawaguchi et al ,1. Immunol 167:4430-4435 [2001]), and found to induce IL-6 and lL-8 production in NHBE. In contrast to 1L-17A, IL-I7F appears to inhibit angiogenesis in vitro (Starnes et al. .1. Immunol. 167: 4137-4140 [2001]).
[21] IL-I7F mRNA was not detected by northern blot in various human tissues but was dramatically induced upon activation of CD4+ T cells and monocytes. Id. In mice, Th2 cells and mastr cells were found to express IL-17F upon activation. See Dumont, Expert Opin. Ther. Patents 13(3) (2003). Like IL-I7A, the expression of 1L-17F was alos found to be upregulated by IL-23 in mouse.
|22] The IL-17 cytokine/receptor families appear to represent a unique signaling system within the cytokine network that will offer innovative approaches to the manipulation of immune and inflammatory responses. Accordingly, the present invention is based on the discovery of a new IL-17 family receptor, IL-17RA and its ability to bind both lL-17A and IL-17F.
[23] As such, antagonists to IL-I7F and IL-17A activity, such as 1L-17RA soluble receptors of the present invention, are useful in therapeutic treatment of inflammatory diseases, particularly as antagonists to both IL-17F and 1L-17A singly or together in the treatment of inflammation. Moreover, antagonists to IL-17F activity, such as 1L-17RA soluble receptors of the present invention, are useful in therapeutic treatment of other inflammatoiy diseases for example as bind, block, inhibit, reduce, antagonize or neutralize 1L-17F and IL-17A (either individually or together) in the treatment of psoriasis, atopic and contact dermatitis, IBD, colitis, endotoxemia, arthritis, rheumatoid arthritis, psoriatic arthritis, adult respiratory disease (ARD), septic shock, multiple organ failure, inflammatory lung injury such as asthma, chronic obstructive pulmonary disease (COPD), airway hyper-responsiveness, chronic bronchitis, allergic asthma, bacterial pneumonia, psoriasis, eczema, , and inflammatory bowel disease such as ulcerative colitis and Crohn's disease, helicobacter pylori infection, intraabdominal adhesions and/or abscesses as results of peritoneal inflammation (i.e. from infection, injury, etc.), systemic lupus erythematosus (SLE), multiple sclerosis, systemic sclerosis, nephrotic syndrome, organ allograft rejection, graft vs. host disease (GVHD), kidney, lung, heart, etc. transplant rejection, streptococcal cell wall (SCW)-induced arthritis, osteoarthritis, gingivitis/periodontitis, herpetic stromal keratitis, cancers including prostate, renal, colon, ovarian, cervical, leukemia, angiogenesis, restenosis and kawasaki disease.
[24] Cytokine receptors subunits are characterized by a multi-domain structure comprising a ligand-binding domain and an effector domain that is typically involved in signal transduction. Multimeric cytokine receptors include monomers, homodimers (e.g., PDGF receptor aa and f3p isoforms, erythropoietin receptor, MPL [thrombopoietin receptor], and G-CSF receptor).
heterodimers whose subunits each have ligand-binding and effector domains (e.g., PDGF receptor a(3 isoform), and multiniers having component subunits with disparate functions (e.g., IL-2, iL-3, iL-4, IL-5, lL-6, IL-7, and GM-CSF receptors). Some receptor subunits are common to a plurality of receptors. For example, the AIC2B subunit. which cannot bind ligand on its own but includes an intracellular signal transduction domain, is a component of lL-3 and GM-CSF receptors. Many cytokine receptors can be placed into one of four related families on the basis of their structures and functions. Class I hematopoietic receptors, for example, are characterized by the presence of a domain containing conserved cysteine residues and the WSXWS motif. Additional domains, including protein kinase domains; fibronectin type III domains; and immunoglobulin domains, which are characterized by disulfide-bonded loops, are present in certain hematopoietic receptors. Cytokine receptor structure has been reviewed by Urdal, Ann. Reports Med. Chem. 26:221-228, 1991 and Cosman, Cytokine 5.:95-106, 1993. It is generally believed that under selective pressure for organisms to acquire new biological functions, new receptor family members arose from duplication of existing receptor genes leading to the existence of multi-gene families. Family members thus contain vestiges of the ancestral gene, and these characteristic features can be exploited in the isolation and identification of additional family members.
[25] Amongst other inventions, the present invention provides novel uses for a soluble receptor, designated "IL-17RA" or "soluble IL-17RA" or "slL-17RA", all of which may be used herein interchangeably, or and neutralizing antibodies to 1L-17RA cytokine receptors. The present invention also provides soluble IL-17RA polypeptide fragments and fusion proteins, for use in human inflammatory and autoimmune diseases. The anti- IL-17RA antibodies, and soluble IL-17RA receptors of the present invention, including the neutralizing anti-IL-17RA antibodies of the present invention, can be used to block, inhibit, reduce, antagonize or neutralize the activity of either IL-1 7F or IL-17A, or both IL-I7A and IL-17F in the treatment of inflammation and inflammatory dieases such as psoriasis, psoriatic arthritis, rheumatoid arthritis, endotoxemia, inflammatory bowel disease (IBD), colitis, asthma, allograft rejection, immune mediated renal diseases, hepatobiliary diseases, multiple sclerosis, atherosclerosis, promotion of tumor growth, or degenerative Joint disease and other inflammatory conditions disclosed herein.
[26] An illustrative nucleotide sequence that encodes a truncated 1L-17RA soluble receptor is shown in SEQ ID "NOs'.S and 6. Specifcally, this truncated variant comprises exons 7-10 of IL-1 7RA fused to Fc5 with otPA pre-pro signal sequence. Variant can also have a native lL-17RA signal sequence, and can be fused to an FclO.
[27] These and other aspects of the invention will become evident upon reference to the following detailed description. In addition, various references are identified below and are incorporated by reference in their entirety.
B) Definitions
[28] In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the invention.
[29] As used herein, "nucleic acid" or "nucleic acid molecule" refers to polynucleotides,
such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments
generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation,
scission, endonuclease action, and exonuclease action. "Nucleic acid molecules can be composed of
monomers that are naturally-occurring nucleotides (such as D"NA and RNA), or analogs of naturally-
occurring nucleotides (e.g., a-enantiomeric forms of naturally-occurring nucleotides), or a
combination of both. Modified nucleotides can have alterations in sugar moieties and/or in
pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or
more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be
functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically
and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of
modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or
pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by
phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include
phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term "'nucleic acid molecule" also includes so-called "peptide nucleic acids," which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.
|30] The term "complement of a nucleic acid molecule" refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
[31] The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that includes one or more degenerate codons as compared to a reference nucleic acid molecule that encodes a polypeptide. Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
[32] The term "structural gene" refers to a nucleic acid molecule that is transcribed into messenger RNA (mRNA), which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
[33] An "isolated nucleic acid molecule" is a nucleic acid molecule that is not integrated in the genomic DNA of an organism. For example, a DNA molecule that encodes a growth factor that has been separated from the genomic DIM A of a cell is an isolated DIM A molecule. Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism. A nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species.
[34] A "nucleic acid molecule construcf is a nucleic acid molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature.
135] "Linear DNA" denotes non-circular DNA molecules having free 5' and 3' ends. Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption.
[36] "Complementary DNA (cDNA)" is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription. Those skilled in the art also use the term "cDNA" to refer to a double-stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand. The term "cDNA" also refers to a clone of a cDNA molecule synthesized from an RN A template.
[37] A "promoter" is a nucleotide sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee et ai, Mol. Endocrinol. 7:55] (1993)), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman, Seminars in Cancer Biol. 1:41 (1990)), glucocorticoid response elements (GREs), and binding sites for other transcription factors, such as CRE/ATF (O'Reilly et al, J. Biol. Chem. 2(57:19938 (1992)), AP2 (Ye el al, J. Biol Chem. 269:25728 (1994)), SPl, cAMP response element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamer factors (see, in general, Watson ei al., eds., Molecular Biologx' of the Gene. 4th ed. (The Benjamin/Cummings Publishing Company, inc. 1987), and Lemaigre and Rousseau, Biochem. ./. 303:1 (1994)). If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter. Repressibie promoters are also known.
[38] A "core promoter" contains essential nucleotide sequences for promoter function, including the TATA box and start of transcription. By this definition, a core promoter may or may
not have detectable activity in the absence of specific sequences that may enhance the activity or confer tissue specific activity.
|39] A "regulatory element" is a nucleotide sequence that modulates the activity of a core promoter. For example, a regulatory element may contain a nucleotide sequence that binds with cellular factors enabling transcription exclusively or preferentially in particular cells, tissues, or organelles. These types of regulatory elements are normally associated with genes that are expressed in a "cell-specific," "tissue-specific," or "organelle-specific" manner.
[40] An "enhancer" is a type of regulatoiy element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.
|41] "Heterologous DMA" refers to a DMA molecule, or a population of DNA molecules, that does not exist naturally within a given host cell. DMA molecules heterologous to a particular host cell may contain D"NA derived from the host cell species (i.e., endogenous DNA) so long as that host DMA is combined with non-host DMA (i.e., exogenous DTsIA). For example, a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a transcription promoter is considered to be a heterologous DNA molecule. Conversely, a heterologous DNA molecule can comprise an endogenous gene operably linked with an exogenous promoter. As another illustration, a DNA molecule comprising a gene derived from a wild-type cell is considered to be heterologous DNA if that DNA molecule is introduced into a mutant cell that lacks the wild-type gene.
|42] A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides."
[43] A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carboliydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
[44] A peptide or polypeptide encoded by a non-host DNA molecule is a "heterologous" peptide or polypeptide.
[45] A "cloning vector" is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage, that has the capability of replicating autonomously in a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites that allow insertion of a nucleic acid molecule in a determinable fashion without loss of an essential biological function of
the vector, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of ceils transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance orampicillin resistance.
[46] An "'expression vector" is a nucleic acid molecule encoding a gene tiiat is expressed in a host cell. Typically, an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be "operably linked to" the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.
|47] A "recombinant host" is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or expression vector. In the present context, an example of a recombinant host is a cell that produces IL-17RA from an expression vector, hi contrast, IL-17RA can be produced by a cell that is a "natural source" of IL-I7RA, and that lacks an expression vector.
[48] "hitegrative transformants" are recombinant host cells, in which heterologous DNA has become integrated into the genomic DNA of the cells.
[49] A "fusion protein" is a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes. For example, a fusion protein can comprise at least part of a IL-17RA polypeptide fused with a polypeptide that binds an affinity matrix. Such a fusion protein provides a means to isolate large quantities of 1L-17RA using affinity chromatography.
[50] The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule termed a "ligand." This interaction mediates the effect of the ligand on the cell. Receptors can be membrane bound, cytosolic or nuclear; monomeric {e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and lL-6 receptor). Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. In certain membrane-bound receptors, the extracellular ligand-binding domain and the intracellular effector domain are located in separate polypeptides that comprise the complete functional receptor.
[51] In general, the binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell, which in turn leads to an alteration in the metabolism of the cell. Metabolic events that are often linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation. increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.
[52] A "soluble receptor" is a receptor polypeptide that is not bound to a cell membrane. Soluble receptors are most commonly ligand-binding receptor polypeptides that lack transmembrane and cytoplasmic domains, and other linkage to the cell membrane such as via glycophosphoinositol (gpi). Soluble receptors can comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to a substrate, or immunoglobulin constant region sequences. Many cell-surface receptors have naturally occurring, soluble counterparts that are produced by proteolysis or translated from alternatively spliced mRNAs. Soluble receptors can be monomeric, homodimeric, heterodimeric, or multimeric, with multimeric receptors generally not comprising more than 9 subunits, preferably not comprising more than 6 subunits, and most preferably not comprising more than 3 subunits. Receptor polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively. Soluble receptors of cytokine receptors generally comprise the extracellular cytokine binding domain free of a transmsmbrane domain and intracellular domain. For example, representative soluble receptors include soluble receptors for IL-17RA. It is well within the level of one of skill in the art to delineate what sequences of a known cytokine receptor sequence comprise the extracellular cytokine binding domain free of a transmsmbrane domain and intracellular domain. Moreover, one of skill in the art using the genetic code can readily determine polynucleotides that encode such soluble receptor polyptides.
[53] The term "secretory signal sequence" denotes a D"NA sequence that encodes a peptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
[54] An "isolated polypeptide" is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature. Typically, a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95%) pure, such as 96%, 97%, or 98%) or more pure, or greater than 99% pure. One way to show that a particular protein preparation contains an isolated polypeptide is by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining of the gel. However, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
[55] The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a
particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyi-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at tiie carboxyl terminus of the complete polypeptide.
[56] The term "expression" refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression involves transcription of the structural gene into niRNA and the translation of mRN A into one or more polypeptides.
[57] The term "splice varianf is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a polypeptide encoded by a splice variant of an mRN A transcribed from a gene.
|58] As used herein, the term "immunomodulator" includes cytokines, stem cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic factors, an dthe like, and synthetic analogs of these molecules.
|59] The term "complement/anti-complement pair" denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair. Other exemplary complement/anti-complement pairs include receptor/1 igand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of less than 10'^ M"'.
[60] An "anti-idiotype antibody" is an antibody that binds with the variable region domain of an immunoglobulin, hi the present context, an anti-idiotype antibody binds with the variable region of an anti-IL-i7RA antibody, and thus, an anti-idiotype antibody mimics an epitope of IL-17RA.
[61] An "antibody fragment" is a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab. and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, an anti-IL-17RA monoclonal antibody fragment binds with an epitope of IL-17RA.
[62] The term "antibody fragment" also includes a synthetic or a genetically engineered polypeptide that binds to a specific antigen, such as polypeptides consisting of the light chain variable region. "Fv" fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide
linker ("scFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
163] A "chimeric antibody" is a recombinant protein that contains the variable domains and complementary determining regions derived from a rodent antibody, while the remainder of the antibody molecule is derived from a human antibody.
[64] "Humanized antibodies" are recombinant proteins in which murine complementarity determining regions of a monoclonal antibody have been transferred from heavy and light variable chains of the murine immunoglobulin into a human variable domain. Construction of humanized antibodies for therapeutic use in humans that are derived from murine antibodies, such as those that bind to or neutralize a human protein, is within the skill of one in the art.
[65] As used herein, a "therapeutic agenf is a molecule or atom which is conjugated to an antibody moiety to produce a conjugate which is useful for therapy. Examples of therapeutic agents include drugs, toxins, immunomodulators, chelators, boron compounds, photoactive agents or dyes, and radioisotopes.
[66] A "detectable label" is a molecule or atom which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis. Examples of detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties.
[67] The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A (Nilsson et al, EMBO J. 4:\Q15 (1985); Nilsson el ah. Methods Enzymol. 198-3 (1991)), glutathione S transferase (Smith and Johnson. Geue 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer ei al, Proc. Natl. Acad. Sci. USA 82:7952 (1985)), substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)), streptavidin binding, peptide, or other antigenic epitope or binding domain. See, in general. Ford el a!.. Protein Expression and Purification 2:95 (1991). DNA molecules encoding affinity tags are available from commercial suppliers {e.g., Pharmacia Biotech, Piscataway, NJ).
[68] A "naked antibody" is an entire antibody, as opposed to an antibody fragment, which is not conjugated with a therapeutic agent. Naked antibodies include both polyclonal and monoclonal antibodies, as well as certain recombinant antibodies, such as chimeric and humanized antibodies.
[69] As used herein, the term "antibody component" includes both an entire antibody and an antibody fragment.
[70] An "immunoconjugate" is a conjugate of an antibody component with a therapeutic agent or a detectable label.
[71] As used herein, the term "antibody fusion protein" refers to a recombinant molecule that comprises an antibody component and a iL-17RA polypeptide component. Examples of an antibody fusion protein include a protein that comprises a IL-17RA extracellular domain, and either an Fc domain or an antigen-binding region.
[72] A "target polypeptide" or a "target peptide" is an amino acid sequence that comprises at least one epitope, and that is expressed on a target cell, such as a tumor cell, or a cell that carries an infectious agent antigen. T cells recognize peptide epitopes presented by a major histocompatibility complex molecule to a target polypeptide or target peptide and typically lyse the target cell or recruit other immune cells to the site of the target cell, thereby killing the target cell.
[73] An "antigenic peptide" is a peptide which will bind a major histocompatibility complex molecule to form an MHC-peptide complex which is recognized by a T cell, thereby inducing a cytotoxic lymphocyte response upon presentation to the T cell. Thus, antigenic peptides are capable of binding to an appropriate major histocompatibility complex molecule and inducing a cytotoxic T cells response, such as cell lysis or specific cytokine release against the target cell which binds or expresses the antigen. The antigenic peptide can be bound in the context of a class I or class n major histocompatibility complex molecule, on an antigen presenting cell or on a target cell.
[74[ In eukaryotes, RNA polymerase II catalyzes the transcription of a structural gene to produce mRNA. A nucleic acid molecule can be designed to contain an RNA polymerase II template in which the RNA transcript has a sequence that is complementary to that of a specific mRNA. The RNA transcript is termed an "anti-sense RNA" and a nucleic acid molecule that encodes the anti-sense RNA is termed an "anti-sense gene." Anti-sense RNA molecules are capable of binding to mRNA molecules, resulting in an inhibition of mRNA translation.
[75] An "anti-sense oligonucleotide specific for IL-17RA" or a "IL-17RA anti-sense oligonucleotide" is an oligonucleotide having a sequence (a) capable of forming a stable triplex with a portion of the IL-17RA gene, or (b) capable of forming a stable duplex with a portion of an mRNA transcript of the IL-17RA gene.
[76] A "ribozyme" is a nucleic acid molecule that contains a catalytic center. The term includes RNA enzymes, self-splicing RNAs, self-cleaving RNAs, and nucleic acid molecules that perform these catalytic functions. A nucleic acid molecule that encodes a ribozyme is termed a "ribozyme gene."
[77] An "external guide sequence" is a nucleic acid molecule that directs the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acid molecule that encodes an external guide sequence is termed an "external guide sequence gene."
[78] The term "variant IL-17RA gene" refers to nucleic acid molecules that encode a polypeptide having an amino acid sequence that is a modification of the known iL-17RA amino acid sequence.
[79] Alternatively, variant IL-17RA genes can be identified by sequence comparison. Two amino acid sequences have "100% amino acid sequence identity" if the amino acid residues of the two amino acid sequences are the same when aligned for maximal correspondence. Similarly, two nucleotide sequences have "100% nucleotide sequence identity" if the nucleotide residues of the two nucleotide sequences are the same when aligned for maximal correspondence. Sequence comparisons can be performed using standard software programs such as those included in the LASERGENE bioinformatics computing suite, which is produced by DNASTAR (Madison, Wisconsin). Other methods for comparing two nucleotide or amino acid sequences by determining optimal alignment are well-known to those of skill in the art (see, for example, Peruski and Peruski, The Internet and the New Biology: Tools for Genomic and Molecular Research (ASM Press, Inc. 1997), Wu et al. (eds.), "Information Superhighway and Computer Databases of Nucleic Acids and Proteins," in Methods in Gene Biotechuolog)\ pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.). Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc. 1998)). Particular methods for determining sequence identity are described below.
[80[ Regardless of the particular method used to identify a variant IL-17RA gene or variant 1L-17RA polypeptide, a variant gene or polypeptide encoded by a variant gene may be functionally characterized the ability to bind specifically to an anti-IL-17RA antibody. A variant IL-17RA gene or variant 1L-I7RA polypeptide may also be functionally characterized the ability to bind to its ligand, for example, IL-17A and/or IL-17F, using a biological or biochemical assay described herein.
[81] The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
[82] The term "ortholog" denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
[83] "Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, a-globin, P-globin, and myoglobin are paralogs of each other.
[84] The present invention includes functional fragments of/Z-777L4 genes. Within the context of this invention, a "functional fragment"' of a IL-17RA gene refers to a nucleic acid molecule that encodes a portion of a IL-17RA polypeptide which is a domain described herein or at least specifically binds with an anti-IL-17RA antibody.
[85] Due to the imprecision of standard analytical methods, molecular weights and lengths of polymers are understood to be approximate values. When such a value is expressed as "about'" X or "approximately" X, the stated value of X will be understood to be accurate to ±10%.
C) Production of IL-17RA Polvnucleotides or Genes
[86] Nucleic acid molecules encoding a human IL-17RA gene can be obtained by screening a human cDNA or genomic library using polynucleotide probes based upon the sequence of IL-17RA. These techniques are standard and well-established, and may be accomplished using cloning kits available by commercial suppliers. See, for example, Ausubel et al. (eds.). Short Protocols in Molecular Biology, 3''' Edition, John Wiley & Sons 1995; Wu el al. Methods in Gene Biotechnology, CRC Press, Inc. 1997; Aviv and Leder, Proc. Nat'I Acad Sci. USA 69.-1408 (1972); Huynh el al., "Constructing and Screening cDNA Libraries in XgtlO and Xgtl 1," in DNA Cloning: A Practical Approach Vol I, Glover(ed.), page 49 (IRL Press, 1985); Wu (1997) at pages 47-52.
[87] Nucleic acid molecules that encode a human IL-J7RA gene can also be obtained using the polymerase chain reaction (PCR) with oligonucleotide primers having nucleotide sequences that are based upon the nucleotide sequences of the IL-17RA gene or cDNA. General methods for screening libraries with PCR are provided by, for example, Yu et al, "Use of the Polymerase Chain Reaction to Screen Phage Libraries," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), Humana Press, hie, 1993. Moreover, techniques for using PCR to isolate related genes are described by, for example, Preston, "Use of Degenerate Oligonucleotide Primers and the Polymerase Chain Reaction to Clone Gene Family Members," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, Wliite (ed.), Humana Press, Inc. 1993. As an alternative, a IL-17RA gene can be obtained by synthesizing nucleic acid molecules using mutually priming long oligonucleotides and the nucleotide sequences described herein (see, for example, Ausubel (1995)). Established techniques using the polymerase chain reaction provide the ability to synthesize DNA molecules at least two kilobases in length (Adang et al. Plant Molec. Biol 27:1131 (1993), Bambot et al, PCR Methods and Applications 2:266 (1993), Dillon et al, "Use of the Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 263-268, (Humana Press, Inc. 1993), and Holowachuk et al, PCR Methods Appl. 4:299 (1995)). For reviews on polynucleotide synthesis, see, for example. Click and
J
Pasternak. Molecular Biotechiolog}>, Principles and Applications of RecomhinanI DNA (ASM Press 1994). Itakura et al. Amru. Rev. Biochem. 5i;323 (1984). and Ciimie el al.. Proc. Nail Acad Sci. USA 87:633 i]990).
D) Production of IL-l 7RA Gene Variants
[88] The present invention provides a variety of nucleic acid molecules, including DNA and RNA molecules, that encode the !L-17RA polypeptides disclosed herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules.
[89] Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules.
[90] Table 1 sets forth the one-letter codes to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a code letter. "Complement" indicates the code for the complementaiy nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.
Table 1
|91| The degenerate codons, encompassing all possible codons for a given amino acid, are set forth in Table 2.
[92] One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon. representative of all possible codons encoding an amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine
(AGR). and the degenerate codon for argiiiiiie (MGN) can. in some circumstances, encode serine (AGY). A similar relationsliip exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degene?"ate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of lL-17RA. Variant sequences can be readily tested for functionality as described herein.
[93] Different species can exhibit "preferential codon usage." In general, see, Grantham el a!., Nml. Acids Res. 8:1893 (1980), Haas et al. Curr. Biol. 6:315 (1996), Wain-Hobson et ai, Gene J3:355 (1981), Grosjean and Fiers, Gene 75:199 (1982), Holm, Nuc. Acids Res. ]4:3015 (1986). Ikemura, ./. Mol. Biol. 158:513 (1982), Sharp and Matassi, Curr. Opin. Genet. Dev. 4:S5\ (1994). Kane, Cwr. Opin. Biotechnol. 6:494 (1995), and Makrides, Microbiol. Rev. 60:5)2 (1996). As used herein, the term "preferential codon usage" or "preferential codons" is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2). For example, the amino acid threonine (Tlir) may be encoded by ACA, ACC, ACG, or ACT. but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential. Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequences disclosed herein serve as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
[94] A IL-17RA-encoding cDNA can be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction with primers designed from the representative human IL-17RA sequences disclosed herein. In addition, a cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to IL-17RA polypeptide.
[95] Those skilled in the art will recognize that the 1L-17RA sequence represents a single allele of human 1L-17RA, and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the nucleotide sequences disclosed herein, including those containing silent mutations and those in which mutations result in amino acid
sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of the amino acid sequences disclosed herein. cDNA molecules generated from alternatively spliced niRNAs, which retain the properties of the IL-17RA polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and niRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
[96] Using the methods discussed above, one of ordinary skill in the art can prepare a variety of polypeptides that comprise a soluble IL-17RA receptor subunit that is substantially homologous to the sequence of IL-17RA, or allelic variants thereof and retain the ligand-binding properties of the wild-type IL-17RA receptor. Such polypeptides may also include additional polypeptide segments as generally disclosed herein.
|97] Within certain embodiments of the invention, the isolated nucleic acid molecules can hybridize under stringent conditions to nucleic acid molecules comprising nucleotide sequences disclosed herein. For example, such nucleic acid molecules can hybridize under stringent conditions to nucleic acid molecules comprising the nucleotide sequence of IL-17RA.
[98] In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (T^^) for the specific sequence at a defined ionic strength and pH. The T,-,-, is the
temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Following hybridization, the nucleic acid molecules can be washed to remove non-hybridized nucleic acid molecules under stringent conditions, or under highly stringent conditions. See, for example, Sambrook el al. Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989); Ausubel et al, (eds.). Current Protocols in Molecular Biolog)-' fiohn Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Cril. Rev. Biochem. Mol. Biol. 26:211 (1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake, MN) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, CA), as well as sites on the Internet, are available tools for analyzing a given sequence and calculating T,-,^ based on user-defined criteria. It is well within
the abilities of one skilled in the art to adapthybridization and wash conditions for use with a particular polynucleotide hybrid.
[99] The present invention also provides isolated 1L-I7RA polypeptides that have a substantially similar sequence identity to the polypeptides of the sequence of 1L-17RA or their orthologs. The term "substantially similar sequence identity" is used herein to denote polypeptides having at least 70%, at least 80%, at least 90%, at least 95%, such as 96%, 97%. 98%, or greater than 95% sequence identity to the sequences shown in the sequence of IL-17RA, or their orthologs. For example, variant and orthoiogous IL-17RA receptors can be used to generate an immune response
and raise cross-reactive antibodies to human 1L-17RA. SUCIT antibodies can be iiiimanized, and modified as described herein, and used therauputically to treat psoriasis, psoriatic arthritis, !BD. colitis, endotoxemia as well as in other therapeutic appHcations described herein.
[100] Percent sequence identity is determined by conventional methods. See, for example, Altschul el al. Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff, Proc. Natl. Acad Sci. USA 89:]09]5 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one-letter codes). The percent identity is then calculated as: ([Total number of identical matches]/ [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences])(l 00).
and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative IL-17RA variant. The FASTA algorithm is described by Pearson and Lipman, Proc. Natl Acad. Sci. USA (S'J:2444 (1988). and by Pearson, Melh. Enzymol 183:63 (1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., the sequence of IL-17RA) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, ./. Afo/. Biol. 48:444 (1970); Sellers, SIAMJ. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions. Illustrative parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).
[102] FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as described above.
[103] The present invention includes nucleic acid molecules that encode a polypeptide having a conservative amino acid change, compared with an amino acid sequence disclosed herein. For example, variants can be obtained that contain one or more amino acid substitutions of the sequence of 1L-17RA, in which an alkyl amino acid is substituted for an alkyl amino acid in a IL-17RA amino acid sequence, an aromatic amino acid is substituted for an aromatic amino acid in a IL-17RA amino acid sequence, a sulfur-containing amino acid is substituted for a sulfur-containing amino acid in a 1L-17RA amino acid sequence, a hydroxy-containing amino acid is substituted for a hydroxy-containing amino acid in a IL-17RA amino acid sequence, an acidic amino acid is substituted for an acidic amino acid in a IL-17RA amino acid sequence, a basic amino acid is substituted for a basic amino acid in a IL-17RA amino acid sequence, or a dibasic monocarboxylic amino acid is substituted for a dibasic monocarboxylic amino acid in a IL-17RA amino acid
sequence. Among the common amino acids, for example, a "conservative amino acid substitution" is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine. The BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Natl Acad. Sci. USA ^'9:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed above), the language "conservative amino acid substitution" preferably refers to a substitution represented by a BLOSUM62 value of greater than -I. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2. or 3. According to this system, preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUIV162 value of at least 2 (e.g., 2 or 3).Particular variants of 1L-I7RA are characterized by having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% such as 96%, 97%), 98%), or 99%o or greater sequence identity to the corresponding amino acid sequence (e.g., the sequence of IL-17RA), wherein the variation in amino acid sequence is due to one or more conservative amino acid substitutions.
[104] Conservative amino acid changes in a lL-17RA gene can be introduced, for example, by substituting nucleotides for the nucleotides recited in the sequence of 1L-17RA. Such "conservative amino acid" variants can be obtained by oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like (see Ausubel (1995); and McPherson (ed.). Directed Mutagenesis: A Practical Approach (IRL Press 1991)). A variant 1L-I7RA polypeptide can be identified by the ability to specifically bind anti-IL-17RA antibodies.
(105] The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without h'mitation, trans-3-methylproline, 2,4-methanoproline, c/.v-4-hydroxyproline, /ra/7s-4-hydroxyproline, A^-methylglycine, a//o-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, /er/-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fiuorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be
employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is typically carried out in a ceii-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson el al.. J. Am. Chem. Soc. 113:2122 (1991), Ellman el al. Methods Enzymol. 202:301 (1991), Chung el al., Science 259:806 (1993), and Chung et al., Proc. Nal 7 Acad. Set USA 90:10145 (1993).
|106] In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al, J. Biol. Chem. 277:19991 (1996)). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced {e.g., phenylalanine) and in the presence of the desired non-naturaliy occurring amino acid(s) {e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al, Diochem. 55:7470 (1994). Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2.395 (1993)).
[107] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturaliy occurring amino acids, and unnatural amino acids may be substituted for IL-l 7RA amino acid residues.
[108] Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et al, Proc. Nat'I Acad. Sci. USA 88:4498 (1991), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering," in Proteins: Analysis and Design, Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity to identify' amino acid residues that are critical to the activity of the molecule. See also, Hilton et al, J. Biol. Chem. 277:4699 (1996).
|109] Although sequence analysis can be used to further defme the IL-l 7RA ligand binding region, amino acids that play a role in IL-17RA binding activity (such as binding of IL-17RA to either 11-17A or IL-l 7F, or to an anti-IL-17RA antibody) can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos el al. Science 255:306 (1992), Smith el al, ./. Mol Biol 224:899 (1992). and Wlodaver et al, FEES Lett. 309:59 (1992).
[110] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer (Proc. Nat'1 Acad. Sci. USA 86:2)52 (1989)). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman el al. Biochem. 50:10832 (1991), Ladner et a!., U.S. Patent No. 5,223,409, Huse, international publication No. WO 92/06204, and region-directed mutagenesis (Derbyshire el al.. Gene ¥6:145 (1986), and Ner et al, DNA 7.-127, (1988)). Moreover, IL-17RA labeled with biotin or FITC can be used for expression cloning of IL-17RA ligands.
[Ill] Variants of the disclosed IL-17RA nucleotide and polypeptide sequences can also be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389 (1994), Stemmer, Proc. Nat'I Acad Sci. USA 97:10747 (1994), and international publication No. WO 97/20078. Briefly, variant DNA molecules are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNA molecules, such as allelic variants or DNA molecules from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
[112] Mutagenesis methods as disclosed herein can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode biologically active polypeptides, or polypeptides that bind with anti-IL-17RA antibodies, can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
[113] The present invention also includes "functional fragments" of IL-1 7RA polypeptides and nucleic acid molecules encoding such functional fragments. Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a IL-17RA polypeptide. As an illustration. DNA molecules having the sequence of IL-17RA can be digested with 5^/31 nuclease to obtain a series of nested deletions. The fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for the ability to bind anti-IL-17RA antibodies. One alternative to exonuclease digestion is to use oiigonucleotide-directed mutagenesis to introduce deletions or stop codons to specify
production of a desired fragment. Alternatively, particular fragments of a 1L-17RA gene can be synthesized using the polymerase chain reaction.
jll4] This general approach is exemplified by studies on the truncation at either or both termini of interferons have been summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover, standard techniques for functional analysis of proteins are described by, for example, Treuter et al., Molec. Gen. Genet. 240:\\3 (1993), Content et al.. "Expression and preliminary deletion analysis of the 42 kDa 2-5A synthetase induced by human interferon," in Biological Interferon Systems. Proceedings of ISIR-TNO Meeting on Interferon Systems. Cantell (ed.), pages 65-72 (Nijhoff 1987), Herschman, "The EGF Receptor," in Control of Animal Cell Proliferation, Vol. 7, Boynton et al., (eds.) pages 169-199 (Academic Press 1985), Coumailleau el al.. J. Biol. Chem. 270:29210 (1995); Fukunaga et al.. J. Biol. Chem. 270:2529] (1995); Yamaguchi el al., Biochem. Pharmacol. 50:1295 (1995), and Meisel et al, Plant Molec. Biol. 30:] (1996).
[115] The present invention also contemplates functional fragments of a IL-17RA gene that have amino acid changes, compared with an amino acid sequence disclosed herein. A variant IL-17RA gene can be identified on the basis of structure by determining the level of identity with disclosed nucleotide and amino acid sequences, as discussed above. An alternative approach to identifying a variant gene on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant 1L-17RA gene can hybridize to a nucleic acid molecule comprising the sequence of IL-17RA.
[116] The present invention also includes using functional fragments of IL-17RA polypeptides, antigenic epitopes, epitope-bearing portions of IL-17RA polypeptides, and nucleic acid molecules that encode such functional fragments, antigenic epitopes, epitope-bearing portions of IL-17RA polypeptides. Such fragments are used to generate polypeptides for use in generating antibodies and binding partners that bind, block, inhibit, reduce, antagonize or neutralize activity of 1L-17A or 1L-17F or both 1L-17A and 1L-17F. A "functional" 1L-17RA polypeptide or fragment thereof as defined herein is characterized by its ability to block, inhibit, reduce, antagonize or neutralize 1L-17A or IL-17F inflammatory, proliferative or differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to bind specifically to an anti-IL-17RA antibody, cell, 1L-17A or IL-17F. As previously described herein, 1L-17RA is characterized by a unique cytokine receptor structure and domains as described herein. Thus, the present invention further contemplates using fusion proteins encompassing: (a) polypeptide molecules comprising one or more of the domains described above; and (b) functional fragments comprising one or more of these domains. The other polypeptide portion of the fusion protein may be contributed by another cytokine receptor, such as IL-IOR, 1L-13R, IL-17RA, IL-1 ORB (CRF2-4), or by a non-native and/or an unrelated secretory signal peptide that facilitates secretion of the fusion protein.
[117] The present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a IL-17RA polypeptide described herein. Such fragments or peptides may comprise an '"immunogenic epitope," which is a part of a protein that elicits an antibody response when the entire protein is used as an immunogen. Immunogenic epitope-bearing peptides can be identified using standard methods (see, for example, Geysen el al, Proc. Nail Acad. Sci. USA 57:3998(1983)).
[118] In contrast, polypeptide fragments or peptides may comprise an "antigenic epitope," which is a region of a protein molecule to which an antibody can specifically bind. Certain epitopes consist of a linear or contiguous stretch of amino acids, and the antigenicity of such an epitope is not disrupted by denaturing agents, it is known in the art that relatively short synthetic peptides that can mimic epitopes of a protein can be used to stimulate the production of antibodies against the protein (see, for example, Sutcliffe et al.. Science 2/9:660 (1983)). Accordingly, antigenic epitope-bearing peptides, antigenic peptides, epitopes, and polypeptides of the present invention are useful to raise antibodies that bind with the polypeptides described herein, as well as to identify and screen anti-lL-I7RA monoclonal antibodies that are neutralizing, and that may bind, block, inhibit, reduce, antagonize or neutralize the activity of IL-17F and IL-17A (individually or together). Such neutralizing monoclonal antibodies of the present invention can bind to an 1L-17RA antigenic epitope. Hopp/Woods hydrophilicity profiles can be used to determine regions that have the most antigenic potential within the sequence of 1L-I7RA (Hopp et al., Proc. Natl. Acad. Sci.78:3824-3828. 198!; Hopp, J. Immun. Meth. 88-"l-'8, 1986 and Triquier et al.. Protein Engineering Jl: 153-169, 1998). The profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored. In IL-17RA these regions can be determined by one of skill in the art. Moreover, 1L-17RA antigenic epitopes as predicted by a .lameson-Wolf plot, e.g., using DNASTAR Protean program (DNASTAR, Inc., Madison, WI) serve as preferred antigenic epitpoes, and can be determined by one of skill in the art.
[119] In preferred embodiments, antigenic epitopes to which
neutralizing antibodies of the present invention bind would contain residues of IL-17RA that are important to ligand-receptor binding, for example, with IL-17RA and IL-17A or 1L-17F (individually or together).
[120] Antigenic epitope-bearing peptides and polypeptides can contain at least four to ten amino acids, at least ten to fifteen amino acids, or about 15 to about 30 amino acids of an amino acid sequence disclosed herein. Such epitope-bearing peptides and polypeptides can be produced by fragmenting a IL-17RA polypeptide, or by chemical peptide synthesis, as described herein. Moreover, epitopes can be selected by phage display of random peptide libraries (see, for example. Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993), and Cortese e/ al, Ciirr. Opin. Biotechnol
7:616 (1996)). Standard methods for identifying epitopes and producing antibodies from small peptides that comprise an epitope are described, for example, by Mole, "Epitope Mapping." in Methods in Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (The Humana Press, Inc. 1992), Price, "'Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies: Production, Engineering, and Clinical Application, Ritterand Ladyman (eds.), pages 60-84 (Cambridge University Press 1995), and Coligan et ah (eds.). Current Protocols in Immunology, pages 9.3.1 - 9.3.5 and pages 9.4.1 - 9.4.11 (John Wiley & Sons 1997).
[121] For any IL-17RA polypeptide, including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables I and 2 above. Moreover, those of skill in the art can use standard software to devise IL-17RA variants based upon the nucleotide and amino acid sequences described herein.
E) Production of 1L-17RA Polypeptides
[122] The polypeptides of the present invention, including full-length polypeptides; soluble monomeric, homodimeric, heterodimeric and multimeric receptors; full-length receptors; receptor fragments (e.g. ligand-binding fragments and antigenic epitopes), functional fragments, and fusion proteins, can be produced in recombinant host cells following conventional techniques. To express a lL-17RA gene, a nucleic acid molecule encoding the polypeptide must be operably linked to regulatoiy sequences that control transcriptional expression in an expression vector and then, introduced into a host cell. In addition to transcriptional regulatory sequences, such as promoters and enhancers, expression vectors can include translational regulatory sequences and a marker gene which is suitable for selection of cells that carry the expression vector.
[123] Expression vectors that are suitable for production of a foreign protein in eukaryotic cells typically contain (1) prokaryotic DMA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DMA elements that control the processing of transcripts, such as a transcription termination/polyadenylation sequence. As discussed above, expression vectors can also include nucleotide sequences encoding a secretory sequence that directs the heterologous polypeptide into the secretory pathway of a host cell. For example, an 1L-17RA expression vector may comprise a IL-17RA gene and a secretory sequence derived from any secreted gene.
[124] IL-17RA proteins of the present invention may be expressed in mammalian cells. Examples of suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells
(BHK-21. BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-Kl: ATCC CCL61; CHO DG44 (Chasin el a/.. Som. Cell. Molec. Genet. 72:555, 1986)), rat pituitary cells (GHl; ATCC CCL82), HeLa S3 cells (ATCC CCL2,2). rat hepatoma cells (H-4-n-E; ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).
[125] For a mammalian host, the transcriptional and translational regulatory signals may be derived from mammalian viral sources, for example, adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression. Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, for example, actin, collagen, myosin, and metallothionein genes.
[126] Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis. Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer et al, J. Molec. Appl. Genet. 7:273 (1982)), the TK promoter of Herpes virus (McKnight, Cell 57:355 (1982)), the SV40 early promoter (Benoist et al. Nature 290:304 (1981)), the Rous sarcoma virus promoter (Gorman el al. Proc. Nat'l Acad. Sci. USA 79:6777 (1982)), the cytomegalovirus promoter (Foecking et al., Gene "^5:101 (1980)), and the mouse mammary tumor virus promoter (see, generally, Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture," in Protein Engineering: Principles and Practice, Cleland et al. (eds.), pages 163-181 (John Wiley & Sons, Inc. 1996)).
[127] Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNA polymerase promoter, can be used to control IL-17RA gene expression in mammalian cells if the prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et al., Mol. Cell. Biol. J0:4529 (1990), and Kaufman et al., Nucl. Acids Re.s. 79:4485 (1991)).
[128] In certain embodiments, a DNA sequence encoding a IL-17RA soluble receptor polypeptide, or a fragment of IL-17RA polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers. Multiple components of a soluble receptor complex can be co-transfected on individual expression vectors or be contained in a single expression vector. Such techniques of expressing multiple components of protein complexes are well known in the art.
|129] An expression vector can be introduced into host cells using a variety or sranaaru techniques including calcium phosphate transfection. liposome-niediated transfection. microprojectile-mediated delivery, eiectroporation, and the like. The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome. Techniques for introducing vectors into eukaryotic cells and techniques for selecting such stable transformants using a dominant selectable marker are described, for example, by Ausubel (1995) and by Murray (ed.), Gene Transfer and Expressio)! Protocols (Humana Press 1991).
[130] For example, one suitable selectable marker is a gene that provides resistance to the antibiotic neomycin. In this case, selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A suitable amplifiable selectable marker is dihydrofolate reductase (DHFR), which confers resistance to methotrexate. Other drug resistance genes {e.g., hygroniycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternatively, markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4. CDS, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
[131] 1L-17RA polypeptides can also be produced by cultured mammalian cells using a viral delivery system. Exemplary viruses for this purpose include adenovirus, retroviruses, herpesvirus, vaccinia virus and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see Becker et a/.. Meth. Cell Biol. 43:\6\ (1994), and Douglas and Curiel, Science & Medicine 4:44 (1997)). Advantages of the adenovirus system include the accommodation of relatively large DNA inserts, the ability to grow to high-titer, the ability to infect a broad range of mammalian cell types, and flexibility that allows use with a large number of available vectors containing different promoters.
[132] By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. An option is to delete the essential El gene from the viral vector, which results in the inability to replicate unless the El gene is provided by the host cell. Adenovirus vector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), for example, can be grown as adherent cells or in suspension culture at
relatively high cell density to produce significant amounts of protein (see Gamier ei al, Cylotechnol. /5:145 (1994)).
[133] IL-17RA can also be expressed in other higher eukaryotic cells, such as avian, fungal, insect, yeast, or plant cells. The baculovirus system provides an efficient means to introduce cloned IL-17RA genes into insect cells. Suitable expression vectors are based upon the Aiilographa californica multiple nuclear polyhedrosis virus (AcMNPV), and contain well-known promoters such as Drosophila heal shock protein (hsp) 70 promoter, Aulographa californica nuclear polyhedrosis virus immediate-early gene promoter {ie-1) and the delayed early 39K promoter, baculovirus pIO promoter, and the Drosophila metallothionein promoter. A second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, el al, ./. Virol. 67:4566 (1993)). This system, which utilizes transfer vectors, is sold in the BAC-to-BAC kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, PFASTBAC (Life Technologies) containing a Tn7 transposon to move the D"NA encoding the IL-17RA polypeptide into a baculovirus genome maintained in E. coli as a large piasmid called a "bacmid." See, Hill-Perkins and Possee, J. Gen. Virol. 71:91] (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk, and Rapoport, J. Biol. Chem. 270:]543 (1995). In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed IL-17RA polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al., Proc. Nail Acad. Sci. 82:1952 (1985)). Using a technique known in the art, a transfer vector containing a 1L-17RA gene is transformed into E. coli, and screened for bacmids which contain an interrupted /acZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is then isolated using common techniques.
[134j The illustrative PFASTBAC vector can be modified to a considerable degree. For example, the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins (see. for example, Hill-Perkins and Possee, J. Gen. Virol. 77:971 (1990), Bonning, f/a/., J. Gen. Virol. 75:1551 (1994), and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). In such transfer vector constructs, a short or long version of the basic protein promoter can be used. Moreover, transfer vectors can be constructed which replace the native IL-17RA secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen Corporation; Carlsbad, CA), or baculovirus gp67 (PharMingen: San Diego, CA) can be used in constructs to replace the native IL-17RA secretory signal sequence.
[135] The recombinant virus or bacmid is used to transfect host cells. Suitable insect host cells include cell lines derived from 1PLB-5/-21, a Spodopiera frugiperda pupal ovarian cell line, such as Sfi (ATCC CRL 1711), 5-/21AE, and 5/21 (Invitrogen Corporation; San Diego, CA). as well as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line (Invitrogen) derived from Trichophisia ni (U.S. Patent No. 5,300,435). Commercially available serum-free media can be used to grow and to maintain the cells. Suitable media are Sf900 H™ (Life Technologies) or ESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa, KS) or Express FiveO™ (Life Technologies) for the T. ni cells. When recombinant virus is used, the cells are typically grown up from an inoculation density of approximately 2-5 x 10^ cells to a density of 1-2 x 10^' cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
[136] Established techniques for producing recombinant proteins in baculovirus systems are provided by Bailey et al.. "Manipulation of Baculovirus Vectors," in Methods in Molecular Biology, Volume 7: Gene Transfer and Expression Protocols, Murray (ed.), pages 147-168 (The Humana Press, Inc. 1991), by Patel e/ al, "The baculovirus expression system," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages 205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37 to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (The Humana Press, Inc. 1995), and by Lucknow, "Insect Cell Expression Technology," in Protein Engineering: Principles and Practice, Cleland et al. (eds.), pages 183-218 (John Wiley & Sons, Inc. 1996).
J137] Fungal cells, including yeast cells, can also be used to express the genes described herein. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Suitable promoters for expression in yeast include promoters from GALl (galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOXl (alcohol oxidase), H1S4 (histidinol dehydrogenase), and the like. Many yeast cloning vectors have been designed and are readily available. These vectors include YIp-based vectors, such as Yip5, YRp vectors, such as YRpl7, YEp vectors such as YEpl3 and YCp vectors, such as YCpl9. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311, Kawasaki et al., U.S. Patent No. 4,931,373. Brake, U.S. Patent No. 4,870,008. Welch et al, U.S. Patent No. 5.037,743, and Murray el al., U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A suitable vector system for use in Saccharomyces cerevisiae is the POTl vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Additional suitable promoters and
terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311, Kingsman et al., U.S. Patent No. 4,615,974, and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446, 5.063,154. 5,139.936, and 4,661,454.
[138] Transformation systems for other yeasts, including Hansermla poJymorpha, Schizosaccharomyces pomhe, Kluyveromyces lactis, Kluyveromyce,'; fragills, Ustilugo maydis, Pichia pasloris, Pichia methanolica, Pichia guiUermondii and Candida mahosa are known in the ait. See, for example, Gleeson el al, J. Gen. Microbiol. 732:3459 (1986), and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight el al, U.S. Patent No. 4,935,349. Methods for transforming ,4c7"eOT0777i/m chrysogenum are disclosed by Sumino el al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533.
[139] For example, the use of Pichia methanolica as host for the production of recombinant proteins is disclosed by Raymond, U.S. Patent No. 5,716,808, Raymond, U.S. Patent No. 5,736,383, Raymond et al, YeasI N-.W-li (1998), and in international publication Nos. WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide production in P. methanolica, the promoter and terminator in the plasmid can be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG] or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences. A suitable selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), and which allows ade2 host cells to grow in the absence of adenine. For large-scale, industrial processes where it is desirable to minimize the use of methanol, host cells can be used in which both methanol utilization genes (AUG! and AUG2) are deleted. For production of secreted proteins, host cells can be deficient in vacuolar protease genes (PEP4 and PRBl). Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. P. methanolica cells can be transformed by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
[140] Expression vectors can also be introduced into plant protoplasts, intact plant tissues, or isolated plant cells. Methods for introducing expression vectors into plant tissue include the direct
infection or co-cultivation of plant tissue witii Agrohacteriiim tumefaciens, microprojectile-mediated delivery, DNA injection, electroporation, and the like. See, for example, Horsch el al. Science 227:1229 (1985). Klein et al, Biotechnology 10:268 (1992), and Miki et al, "Procedures for Introducing Foreign DMA into Plants," in Methods in Plant Molecular Biology and Biotechnology, Click et al. (eds.), pages 67-88 (CRC Press, 1993).
[141] Alternatively, IL-17RA genes can be expressed in prokaryotic host cells. Suitable promoters that can be used to express IL-17RA polypeptides in a prokaryotic host are well-known to those of skill in the art and include promoters capable of recognizing the T4, T3, Sp6 and T7 polymerases, the PR and PL promoters of bacteriophage lambda, the trp, recA, heat shock. lacUV5. tac, lpp-lacSpi\ phoA, and lacZ promoters of E. coli, promoters of B. suhtilis, the promoters of the bacteriophages of Bacillus, Streptomyces promoters, the int promoter of bacteriophage lambda, the hla promoter of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene. Prokaryotic promoters have been reviewed by Click,./. Ind. Microbiol. 1:111 (1987), Watson et al., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), and by Ausubel et al. (1995;.
[142] Suitable prokaryotic hosts include £. coli and Bacillus subtilus. Suitable strains of £. coli include BL21(DE3), BL21(DE3)pLysS, BL2!(DE3)pLysE, DHl, DH4I, DH5, DH51, DH51F', DHSIMCR, DHIOB, DH10B/p3, DHl IS, C600, HBlOl, JMlOl, JMI05, JM109, JMl 10, K38, RRl. Y1088, YI089, CSH18, ER1451, and ER1647 (see, for example. Brown (ed.). Molecular Biolog}' Lahfax (Academic Press 1991)). Suitable strains o^Bacillus subtilus include BRl 51, YB886, Ml 119, M1120, and B170 (see, for example. Hardy, "Bacillus Cloning Methods,'" in DNA Cloning: A Practical Approach, Glover (ed.) (IRL Press 1985)).
[143[ When expressing a 1L-17RA polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the ceils (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
[144] Methods for expressing proteins in prokaryotic hosts are well-known to those of skill in the art (see, for example, Williams et al. "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition. Glover et al (eds.). page 15 (Oxford University Press 1995), Ward et al, "Genetic
Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applicalions, page 137 (Wiley-Liss, inc. 1995), and Georgiou, "Expression of Proteins in Bacteria." in Protein Engineering: Principles and Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc. 1996)).
1145] Standard methods for introducing expression vectors into bacterial, yeast, insect, and plant ceils are provided, for example, by Ausubel (1995).
[146] General methods for expressing and recovering foreign protein produced by a mammalian cell system are provided by, for example, Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture," in Protein Engineering: Principles and Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recovering protein produced by a bacterial system is provided by, for example, Grisshammer et al., "Purification of over-produced proteins from E. coli cells," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages 59-92 (Oxford University Press 1995). Established methods for isolating recombinant proteins from a baculovirus system are described by Richardson (ed.), Baculovirus Expression Protocols (The Humana Press, Inc. 1995).
[147] As an alternative, polypeptides of the present invention can be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. These synthesis methods are well-known to those of skill in the art (see, for example, Merrifield, J. Am. Chem. Soc. 55:2149 (1963), Stewart et al. "Solid Phase Peptide Synthesis" (2nd Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept. Prol. J;3 (1986), Atherton et al.. Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989), Fields and Colowick, "Solid-Phase Peptide Synthesis," Methods in Enzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al.. Chemical Approaches to the Synthesis of Peptides and Proteins (CRC Press, Inc. 1997)). Variations in total chemical synthesis strategies, such as "native chemical ligation" and "expressed protein ligation" are also standard (see, for example, Dawson et al., Science 266:116 (1994), Hackeng et al.. Proa. Nat'I Acad Sci. USA 9-/:7845 (1997), Dawson, Methods Enzymol. 287: 34 (1997), Muir et al. Proc. Natl Acad Sci. USA 95:6705 (1998), and Severinov and Muir, J. Biol. Chem. 273:16205 (1998)).
[148] Peptides and polypeptides of the present invention comprise at least six, at least nine, or at least 15 contiguous amino acid residues of the sequence of IL-17RA. As an illustration, polypeptides can comprise at least six, at least nine, or at least 15 contiguous amino acid residues of the sequence of IL-I7RA. Within certain embodiments of the invention, the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of these amino acid sequences. Nucleic acid molecules encoding such peptides and polypeptides are useful as polymerase chain reaction primers and probes.
[149] Moreover, IL-17RA polypeptides and fragments thereof can be expressed as monomers, homodimers, heterodimers, or multimers within higher eukaryotic cells. Such cells can be used to produce 1L-17RA monomeric, homodimeric, heterodimeric and multimeric receptor polypeptides that comprise at least one 1L-17RA polypeptide ("lL-17RA-comprising receptors'" or "IL-17RA-comprising receptor polypeptides"), or can be used as assay cells in screening systems, Within one aspect of the present invention, a polypeptide of the present invention comprising the IL-17RA extracellular domain is produced by a cultured cell, and the ceil is used to screen for ligands for the receptor, including the natural ligand, IL-I7F, as well as IL-17A, or even agonists and antagonists of the natural ligand. To summarize this approach, a cDNA or gene encoding the receptor is combined with other genetic elements required for its expression {e.g.., a transcription promoter), and the resulting expression vector is inserted into a host cell. Cells that express the DMA and produce functional receptor are selected and used within a variety of screening systems. Each component of the monomeric, homodimeric, heterodimeric and multimeric receptor complex can be expressed in the same cell. Moreover, the components of the monomeric, homodimeric, heterodimeric and multimeric receptor complex can also be fused to a transmembrane domain or other membrane fusion moiety to allow complex assembly and screening of transfectants as described above.
[150] To assay the 1L-17A and 1L-17F antagonist polyepeptides and antibodies of the present invention, mammalian cells suitable for use in expressing IL-17RA-comprising receptors or other receptors known to bind IL-17A or IL-17F (e.g., cells expressing IL-17RA) and transducing a receptor-mediated signal include cells that express other receptor subunits that may form a functional complex with IL-I7RA. It is also preferred to use a ceil from the same species as the receptor to be expressed. Within a preferred embodiment, the cell is dependent upon an exogenously supplied hematopoietic growth factor for its proliferation. Preferred cell lines of this type are the human TF-I cell line (ATCC number CRL-2003) and the AML-193 cell line (ATCC number CRL-9589), which are GM-CSF-dependent human leukemic cell lines and BaF3 (Palacios and Steinmetz, Cell 41: 727-734, (1985)) which is an IL-3 dependent murine pre-B cell line. Other cell lines include BHK, COS-1 and CHO cells. Suitable host cells can be engineered to produce the necessaiy receptor subunits or other cellular component needed for the desired cellular response. This approach is advantageous because cell lines can be engineered to express receptor subunits from any species, thereby overcoming potential limitations arising from species specificity. Species orthologs of the human receptor cDNA can be cloned and used within cell lines from the same species, such as a mouse cDNA in the BaF3 cell line. Cell lines that are dependent upon one hematopoietic growth factor, such as GM-CSF or lL-3, can thus be engineered to become dependent upon another cytokine that acts through the 1L-17RA receptor, such as 1L-17F or 1L-17A.
[151] Cells expressing functional receptor are used within screening assays. A variety of suitable assays are known in the art. These assays are based on the detection of a biological response in a target cell. One such assay is a cell proliferation assay. Cells are cultured in the presence or absence of a test compound, and cell proliferation is detected by, for example, measuring incorporation of tritiated thymidine or by colorimetric assay based on the metabolic breakdown of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Mosman, J. Immunol. Melh. 65: 55-63, (1983)). An alternative assay format uses cells that are further engineered to express a reporter gene. The reporter gene is linked to a promoter element that is responsive to the receptor-linked pathway, and the assay detects activation of transcription of the reporter gene. A preferred promoter element in this regard is a serum response element, or SRE. See, e.g., Shaw el a!.. Cell 56:563-572, (1989). A preferred such reporter gene is a luciferase gene (de Wet el al., Mol. Cell. Biol. 7:725, (1987)). Expression of the luciferase gene is detected by luminescence using methods known in the art (e.g., Baumgartner el al, J. Biol. Chem. 269:29094-29101, (1994); Schenborn and Goiffin, PromegaJ^oles 47:11, 1993). Luciferase activity assay kits are commercially available from, for example, Promega Corp., Madison, WI. Target cell lines of this type can be used to screen libraries of chemicals, cell-conditioned culture media, fungal broths, soil samples, water samples, and the like. For example, a bank of cell-conditioned media samples can be assayed on a target cell to identify cells that produce iigand. Positive cells are then used to produce a cDNA library in a mammalian expression vector, which is divided into pools, transfected into host cells, and expressed. Media samples from the transfected cells are then assayed, with subsequent division of pools, re-transfection, subculturing, and re-assay of positive cells to isolate a cloned cDNA encoding the Iigand.
[152] An additional screening approach provided by the present invention includes the use of hybrid receptor polypeptides. These hybrid polypeptides fall into two general classes. Within the first class, the intracellular domain of IL-17RA, is joined to the ligand-binding domain of a second receptor. A second class of hybrid receptor polypeptides comprise the extracellular (ligand-binding) domain of IL-17RA with an intracellular domain of a second receptor, preferably a hematopoietic cytokine receptor, and a transmembrane domain. Hybrid 1L-17RA monomers, homodimers, heterodimers and multimers of the present invention receptors of this second class are expressed in cells known to be capable of responding to signals transduced by the second receptor. Together, these two classes of hybrid receptors enable the identification of a responsive cell type for the development of an assay for detecting IL-17F or 1L-17A. Moreover, such cells can be used in the presence of IL-17F or IL-17A to assay the soluble receptor antagonists of the present invention in a competition-type assay. In such assay, a decrease in the proliferation or signal transduction activity of IL-i7F or IL-17A in the presence of a soluble receptor of the present invention demonstrates
antagonistic activity. Moreover IL-lTRA-soiubie receptor binding assays, an cell-based assays, can also be used to assess whether a soluble receptor binds, blocks, inhibits, reduces, antagonizes or neutralizes IL-17F or IL-I7A activity.
F) Production of IL-I7RA Fusion Proteins and Conjugates
[153] One general class of IL-17RA analogs are variants having an amino acid sequence that is a mutation of the amino acid sequence disclosed herein. Another general class of IL-17RA analogs is provided by anti-idiotype antibodies, and fragments thereof, as described below. Moreover, recombinant antibodies comprising anti-idiotype variable domains can be used as analogs (see, for example, Monfardini et al. Proc. Assoc. Am. Physicians 108:420 (1996)). Since the variable domains of anti-idiotype IL-17RA antibodies mimic IL-17RA, these domains can provide IL-17RA binding activity. Methods of producing anti-idiotypic catalytic antibodies are known to those of skill in the art (see, for example, Joron ef at., Ann. N Y Acad. Sci. 672:216 (1992), Friboulet et a/.. Appl. Biochem. Biotechnol. 47:229 (1994), and Avalle et al, Ann. N YAcad. Sci. 864:\ 18 (1998)).
[154] Another approach to identifying 1L-17RA analogs is provided by the Use of combinatorial libraries. Methods for constructing and screening phage display and other combinatorial libraries are provided, for example, by Kay et al. Phage Display of Peptides and Proteins (Academic Press 1996), Verdine. U.S. Patent No. 5,783,384, Kay, et. al. U.S. Patent No. 5,747,334, and Kauffman et al, U.S. Patent No. 5,723,323.
[155] 1L-17RA polypeptides have both in vivo and in vitro uses. As an illustration, a soluble form of 1L-17RA can be added to cell culture medium to inhibit the effects of the IL-17RA ligand (i.e. 1L-17F, IL-17A or both) produced by the cultured cells.
[156] Fusion proteins of 1L-17RA can be used to express IL-17RA in a recombinant host, and to isolate the produced 1L-17RA. As described below, particular 1L-17RA fusion proteins also have uses in diagnosis and therapy. One type of fusion protein comprises a peptide that guides a IL-17RA polypeptide from a recombinant host cell. To direct a IL-17RA polypeptide into the secretory pathway of a eukaryotic host cell, a secretory signal sequence (also known as a signal peptide, a leader sequence, prepro sequence or pre sequence) is provided in the 1L-17RA expression vector. While the secretory signal sequence may be derived from 1L-17RA, a suitable signal sequence may also be derived from another secreted protein or synthesized de novo. The secretory signal sequence is operably linked to a IL-17RA-encoding sequence such that the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the nucleotide sequence encoding the polypeptide of interest, although certain secretory signal sequences may be
positioned elsewhere m the nucleotide sequence of interest (see, e.g., Welch el a!.. U.S. Patent No. 5.037.743: Holland e/ ciL. U.S. Patent No. 5,143.830).
[157j Although the secretory signal sequence of 1L-17RA or another protein produced by nsammalian cells {e.g.. tissue-type plasminogen activator signal sequence, as described, for example, in U.S. Patent Mo. 5,641,655) is useful for expression of 1L-17RA in recombinant mammalian hosts, a yeast signal sequence is preferred for expression in yeast cells. Examples of suitable yeast signal sequences are those derived from yeast mating phermone a-factor (encoded by the MFal gene), invertase (encoded by the SUC2 gene), or acid phosphatase (encoded by the PH05 gene). See, for example, .Romanos ei a/., "'Expression of Cloned Genes in Yeast," in DNA Cloning 2: A Praclicai Approach, 2'"^ Edition, Glover and Hames (eds). pages 123-167 (Oxford University Press 1995).
jl58] IL-17RA soluble receptor polypeptides can be prepared by expressing a truncated DNA encoding the extracellular domain, for exam.ple. a polypeptide which contains the sequence of iL-17RA. or the corresponding region of a non-human receptor, it is preferred that the extracellular domain polypeptides be prepared in a form substantially free of transmembrane and intracellular polypeptide segirients. To direct the export of the receptor domain from the host cell, the receptor DN.'^i is linked to a second DNA segment encoding a secretory peptide, such as a t-PA secretory peptide. To facilitate purification of the secreted receptor domain, a C-terminal extension, sucli as a poly-histidine tag, substance P. Flag™ peptide (Hopp el at.. Biolechnology 6';!204-!210. (1988); available from Eastman Kodak Co., New Haven, CT) or another polypeptide or protein for vvhich ari antibody or other specific binding agent is available, can be fused to the receptor polypeptide. Moreover, IL-!7RA antigenic epitopes from the extracellular cytokine binding domains are also prepared as de.scribed above.
1159] In an alternative approach, a receptor extracellular domain of iL-i7RA or other cytokine receptor component can be expressec as a fusion with immunoglobulin heavy chain constant regions, typically an F^- fragment, which contains two constant region domains and a hinge region but
lacks the variable region (See, Sledziewski, AZ et al., US Patent No. 6,018,026 and 5,750.375). The soluble iL-17RA polypeptides of the present invention mclude such fusions. One such fusion is shown in shown in Example 8. Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded to each other and two receptor polypeptides are arrayed in closed proximity to each other. Fusions of this type can be used to affinity purify the cognate ligand from solution, as an in vitro assay tool, to block, inhibit or reduce signals in vitro by specifically titrating out ligand, and as antagonists in vivo by administering them parenterally to bind circulating ligand and clear it from the circulation. To purify ligand, a IL-17RA-Ig chimera is added to a sample containing the ligand (e.g., cell-conditioned culture media or tissue extracts) under conditions that facilitate receptor-ligand binding (typically near-physiological temperature, pH, and ionic strength).
The chimera-ligand complex is then separated by the mixture using protein A, which is immobilized on a solid support (e.g., insoluble resin beads). The ligand is then ekited using conventional chemical techniques, such as with a salt or pH gradient. In the alternative, the chimera itself can be bound to a solid support, with binding and elution carried out as above. The chimeras may be used in vivo to regulate inflammatory responses including acute phase responses such as serum amyloid A (SAA), C-reactive protein (CRP), and the like. Chimeras with high binding affinity are administered parenterally (e.g.. by intramuscular, subcutaneous or intravenous injection). Circulating molecules bind ligand and are cleared from circulation by normal physiological processes. For use in assays, the chimeras are bound to a support via the F^, region and used in an ELISA format.
[160] To assist in isolating anti-lL-l7RA and binding partners of the present invention, an assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/ anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, N.I) may be advantageously employed. Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40. 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. A receptor, antibody, member or fragment is covalently attached, using amine or sulfliydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding. Alternatively, ligand/receptor binding can be analyzed using SELDl(TM) technology (Ciphergen, Inc., Palo Alto, CA). Moreover, BIACORE technology, described above, can be used to be used in competition experiments to determine if different momnoclonal antibodies bind the same or different epitopes on the IL-i7RA polypeptide, and as such, be used to aid in epitope mapping of neutralizing antibodies of the present invention that bind, block, inhibit, reduce, antagonize or neutralize IL-17F or both IL-! 7A and IL-17F.
[161] Ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and calorimetric assays (Cunningham et al.. Science 253:545-48, 1991: Cunningham etal.. Science 245:821-25. 1991).
[162] The present invention further provides a variety of other polypeptide fusions and related multimeric proteins comprising one or more polypeptide fusions. For example, a soluble IL-17RA receptor can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos.
5,155,027 and 5,567,584. Preferred dimerizing proteins in this regard include immunoglobulin constant region domains, e.g., IgGyl, and the human K light chain, [mmunoglobulin-soiuble [L-I7RA fusions can be expressed in genetically engineered ceils to produce a variety of multimeric 1L-17RA receptor analogs. Auxiliary domains can be fused to soluble IL-17RA receptor to target them to specific cells, tissues, or macromolecuies (e.g.. collagen, or ceils expressing the IL-I 7RA ligands, IL-17F or IL-17A). A IL-17RA polypeptide can be fused to two or more moieties, such as an affmity tag for purification and a targeting domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al.. Connective Tissue Research 34:1-9. 1996.
[163] In bacterial cells, it is often desirable to express a heterologous protein as a fusion protein to decrease toxicity, increase stability, and to enhance recovery of the expressed protein. For example, IL-17RA can be expressed as a fusion protein comprising a glutathione S-transferase polypeptide. Glutathione S-transferease fusion proteins are typically soluble, and easily purifiable from E. coli lysates on immobilized glutathione columns, in similar approaches, a iL-1 7RA fusion protein comprising a maltose binding protein polypeptide can be isolated with an amylose resin column, while a fusion protein comprising the C-terminal end of a truncated Protein A gene can be purified using IgG-Sepharose. Established techniques for expressing a heterologous polypeptide as a fusion protein in a bacterial cell are described, for example, by Williams et al, "Expression of Foreign Proteins in E. coli Using Plasmid Vectors and Purification of Specific Polyclonal Antibodies," in DNA Cloning 2: A Practical Approach, 2'"' Edition, Glover and Hames (Eds.), pages 15-58 (Oxford University Press 1995). In addition, commercially available expression systems are available. For example, the PINPOINT Xa protein purification system (Promega Corporation; Madison, Wi) provides a method for isolating a fusion protein comprising a polypeptide that becomes biotinylated during expression with a resin that comprises avidin.
[164] Peptide tags that are useful for isolating heterologous polypeptides expressed by either prokaryotic or eukaryotic cells include polyHistidine tags (which have an affinity for nickel-chelating resin), c-myc tags, calmodulin binding protein (isolated with calmodulin affinity chromatography), substance P, the RYIRS tag (wliich binds with anti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which binds with anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem. Biophys. 329:2\5 (1996), Morganti et al, Biotechnol. Appl Biochein. 23:61 (1996), and Zheng el al., Gene 186:55 (1997). Nucleic acid molecules encoding such peptide tags are available, for example, from Sigma-Aldrich Corporation (St. Louis, MO).
[165] Another form of fusion protein comprises a iL-17RA polypeptide and an immunoglobulin heavy chain constant region, typically an F^ fragment, which contains two or three
constant region domains and a hinge region but lacks the variable region. As an illustration, Chang el al., U.S. Patent No. 5,723,125, describe a fusion protein comprising a human interferon and a human
immunoglobulin Fc fragment. The C-terminal of the interferon is linked to the N-terminai of the Fc fragment by a peptide linker moiety. An example of a peptide linker is a peptide comprising primarily a T cell inert sequence, which is immunologically inert. An exemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGG S. In this fusion protein, an illustrative Fc moiety is a human y4 chain, which is stable in solution and has little or no complement activating activity. Accordingly, the present invention contemplates a iL-!7RA fusion protein that comprises a IL-17RA moiety and a human Fc fragment, wherein the C-terminus of the IL-17RA moiety is attached to the N-terminus of the Fc fragment via a peptide linker, such as a peptide comprising the amino acid sequence of the sequence of IL-17RA. The FL-17RA moiety can be a IL-17RA molecule or a fragment thereof.
[166] In another variation, a IL-17RA fusion protein comprises an IgG sequence, a IL-17RA moiety covalently joined to the aminoterminal end of the IgG sequence, and a signal peptide that is covalently joined to the aminoterminal of the 1L-I7RA moiety, wherein the IgG sequence consists of the following elements in the following order: a hinge region, a CH2 domain, and a CH3 domain. Accordingly, the IgG sequence lacks a CHi domain. The IL-I7RA moiety displays a IL-17RA activity, as described herein, such as the ability to bind with a IL-17RA iigand. This general approach to producing fusion proteins that comprise both antibody and nonantibody portions has been described by LaRochelle e/ al, EP 742830 (WO 95/21258).
[167] Fusion proteins comprising a IL-17RA moiety and an Fc moiety can be used, for example, as an in vitro assay tool. For example, the presence of a IL-17RA Iigand in a biological sample can be detected using a lL-l7RA-immunoglobulin fusion protein, in which the IL-17RA moiety is used to bind the Iigand, and a macromolecule, such as Protein A or anti-Fc antibody, is used to bind the fusion protein to a solid support. Such systems can be used to identify agonists and antagonists that interfere with the binding of a IL-17RA ligands, e.g., IL-17F or both IL-I7A and IL-17F, to their receptor.
[168] Other examples of antibody fusion proteins include polypeptides that comprise an antigen-binding domain and a IL-17RA fragment that contains a lL-17RA extracellular domain. Such molecules can be used to target particular tissues for the benefit of IL-17RA binding activity.
[169] The present invention further provides a variety of other polypeptide fusions. For example, part or all of a domain(s) conferring a biological function can be swapped between IL-17RA of the present invention with the functionally equivalent domain(s) from another member of the cytokine receptor family. Polypeptide fusions can be expressed in recombinant host cells to produce a variety of IL-17RA fusion analogs. A JL-17RA polypeptide can be fused to two or more moieties or domains, such as an affinity tag for purification and a targeting domain. Polypeptide
fusions can also comprise one or more cleavage sites, particularly between domains. See, for example, Tuan et al. Connective Tissue Research 34:\ (1996).
[170] Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. General methods for enzymatic and chemical cleavage of fusion proteins are described, for example, by Ausubel (1995) at pages 16-19 to 16-25.
[171] IL-17RA binding domains can be further characterized by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids of lL-17RA ligand agonists. See, for example, de Vos et al. Science 255:306 (1992), Smith et al.,./. Mo/. Biol. 224:899 (1992), and Wlodaver et al, FEES Lett 309:59 (1992).
[172] The present invention also contemplates chemically modified IL-17RA compositions, in which a IL-17RA polypeptide is linked with a polymer. Illustrative 1L-17RA polypeptides are soluble polypeptides that lack a functional transmembrane domain. Typically, the polymer is water soluble so that the IL-17RA conjugate does not precipitate in an aqueous environment, such as a physiological environment. An example of a suitable polymer is one that has been modified to have a single reactive group, such as an active ester for acylation, or an aldehyde for alkylation. In this way, the degree of polymerization can be controlled. An example of a reactive aldehyde is polyethylene glycol propionaldehyde, or mono-(C 1-C10) alkoxy, or aryloxy derivatives thereof (see, for example, Harris, et al, U.S. Patent No. 5,252,714). The polymer may be branched or unbranched. Moreover, a mixture of polymers can be used to produce lL-17RA conjugates.
[173] {L-I7RA conjugates used for therapy can comprise pharmaceutically acceptable water-soluble polymer moieties. Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, mono-(Cl-C10)aIkoxy-PEG, aiyloxy-PEG, poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde, Aw-succinimidyl carbonate PEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols {e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or other carbohydrate-based polymers. Suitable PEG may have a molecular weight from about 600 to about 60,000, including, for example, 5,000, 12,000, 20.000 and 25.000. A IL-17RA conjugate can also comprise a mixture of such water-soluble polymers.
[174] One example of a [L-I7RA conjugate comprises a IL-i7RA moiety and a polyaikyi oxide moiety attached to the /^-terminus of the lL-17RA moiety. PEG is one suitable polyaikyi oxide. As an illustration, IL-17RA can be modified with PEG, a process known as "PEGylation."
PEGylation of IL-17RA can be carried out by any of the PEGylation reactions i Clin Jmiminol 108:430 (2001)).
|224] Clinical evidence shows that acute, severe exacerbations of asthma are associated with recruitment and activation of neutrophils in the airways, thus fL-l? is likely to play a significant role in asthma. Patients with mild asthma display a detectable increase in the local concentration of free, soluble IL-17A protein (Molet, et al. .1 Allergy Clin Immunol 108:430 (2001)) while healthy human volunteers with induced, severe airway inflammation due to the exposure to a swine confinement, display a pronounced increase in the concentration of free, soluble IL-17A protein in the bronchoalveolar space ( Fossiez, et al, J Exp Med 183:2593 (1996), and Linden, et al. Int Arch Allergy Immunol 126:179 (2001)). Furthermore, IL-17 levels in sputum have correlated with individuals who have increased airway hyper-reactivity Barczyk, et al. Respir Med 97:726 (2003).
[225] In animal models of airway hyper-responsiveness, chronic inhalation of ovalbumin by sensitized mice resulted in bronchial eosinophilic inflammation and early induction of IL-17 mRNA expression in inflamed lung tissue, together with a bronchial neutrophilia Hellings, et al. Am J Respir Cell Mol Biol 28:42 (2003). Anti-IL-!7 monoclonal antibodies strongly reduced bronchial neutrophilic influx but significantly enhanced IL-5 levels in both bronchoalveolar lavage fluid and serum, and aggravated allergen-induced bronchial eosinophilic influx, suggesting that IL-17A may be involved in determining the balance between neutrophil and eosinophil accumulation following antigen insult Id..
|226] Among the IL-17 family members, IL-17F is most closely related to IL-17A. The biological activities mediated by IL-17F are similar to those of IL-17A, where IL-17F stimulates production of IL-6, IL-8 and G-CSF Hurst, et al. J Immunol 169:443 (2002). IL-17F also induces production of lL-2, transforming growth factor (TGF)- , and monocyte chemoattractant protein (MCP) in endothelial cells Starnes, et al. J Immunol 167:4137 (2001). Similarly, allergen challenge can increase local IL-17F in patients with allergic asthma Kawaguchi, et al. J Immunol 167:4430 (2001). Gene delivery of IL-17F in murine lung increases neutrophils in the bronchoalveolar space, while mucosal transfer of the IL-! 7F gene enhances the levels of Ag-induced pulmonary neutrophilia and airway responsiveness to methacholine Oda, et al. Am J Respir Crit Care Med 171:12 (2005).
|227] Apart from asthma, several chronic inflammatory airway diseases are characterized by neutrophil recruitment in the airways and IL-17 has been reported to play an important role in the pathogenesis of respiratory conditions such as chronic obstructive pulmonary disease (COPD),
bacterial pneumonia and cystic fibrosis (Linden, et al. Eur Respir J 15:973 (2000), Ye, et ai. Am J Respir Ceil Moi Biol 25:335(2001), Rahman, et al. Clin Immunol 115:268(2005)). An anti-IL-I7A and/or anti-IL-17F therapeutic molecule could be demonstrated to be efficacious for chronic inflammatory airway disease in an in vitro model of inflammation. The ability of antagonists to IL-17F and/or IL-17A activity, such as IL-17RA soluble receptors and antibodies thereto including the anti-human-lL-]7RA monoclonal and neutralizing antibodies of the present invention to inhibit IL-17A or and/or lL-17F-induced cytokine, and chemokine production from cultured HBECs or bronchial fibroblasts could be used as a measure of efficacy for such antagonists in the prevention of the production of inflammatory mediators directly resulting from IL-17A and/or F stimulation. If the addition of antagonists to 1L-17F and/or 1L-17A activity, such as IL-17RA soluble receptors and antibodies thereto including the anti-human-IL-17RA monoclonal and neutralizing antibodies of the present invention markedly reduces the production and expression of inflammatory mediators, it would be expected to be efficacious in inflammatory aspects associated with chronic airway inflammation.
[228] For pharmaceutical use, the soluble IL-17RA or anti-IL-I7RA antibodies of the present invention are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. Intravenous administration will be by bolus injection, controlled release, e.g, using mini-pumps or other appropriate technology, or by infusion over a typical period of one to several hours, hi general, pharmaceutical formulations will include a hematopoietic protein in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to provent protein loss on vial surfaces, etc. When utilizing such a combination therapy, the cytokines may be combined in a single formulation or may be administered in separate formulations. Methods of formulation are well known in the art and are disclosed, for example, in Remington's Pharmaceutical Sciences, Gennaro, ed.. Mack Publishing Co., Easton PA, 1990, which is incorporated herein by reference. Therapeutic doses will generally be in the range of 0.1 to 100 mg/kg of patient weight per day, preferably 0.5-20 mg/kg per day, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. The proteins will commonly be administered over a period of up to
28 days following chemotherapy or bone-marrow transplant or until a platelet count of >20,000/mm-',
preferably >50,000/mm^, is achieved. More commonly, the proteins will be administered over one week or less, often over a period of one to three days. In general, a therapeutically effective amount of soluble iL-17RA or anti-IL-]7RA antibodies of the present invention is an amount sufficient to produce a clinically significant increase in the proliferation and/or differentiation of lymphoid or
myeloid progenitor cells, which will be manifested as an increase in circulating levels of mature cells (e.g. platelets or neutrophils). Treatment of platelet disorders will thus be continued until a platelet
count of at least 20,000/mm-'. preferably 50.000/mm-'. is reached. The soluble IL-!7RA or anti-IL-17RA antibodies of the present invention can also be administered in combination with other cytokines such as lL-3, -6 and -11; stem cell factor; erythropoietin; G-CSF and GM-CSF. Within regimens of combination therapy, daily doses of other cytokines will in general be; EPO, 150 IJ/kg; GM-CSF. 5-15 Ig/kg; IL-3, 1-5 Ig/kg; and G-CSF, 1-25 Ig/kg. Combination therapy with EPO. for example, is indicated in anemic patients with low EPO levels.
|229] Generally, the dosage of administered soluble IL-17RA (or IL-17RA analog or fusion protein) or anti-IL-17RA antibodies will vary depending upon such factors as the patient's age. weight, height, sex, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of soluble IL-17RA or anti-lL-17RA antibodies which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient), although a lower or higher dosage also may be administered as circumstances dictate.
[230] Administration of soluble 1L-)7RA or anti-lL-17RA antibodies to a subject can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection. When administering therapeutic proteins by injection, the administration may be by continuous infusion or by single or multiple boluses.
[231] Additional routes of administration include oral, mucosal-membrane, pulmonary, and transcutaneous. Oral delivery is suitable for polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid-based systems (see, for example, DiBase and Morrel, "Oral Delivery of Microencapsulated Proteins," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). The feasibility of an intranasal delivery is exemplified by such a mode of insulin administration (see, for example, Hinchcliffe and Ilium, ^Jv. Drug Deliv. Rev. 55:199 (1999)). Dry or liquid particles comprising soluble 1L-17RA or anti-lL-!7RA antibodies can be prepared and inhaled with the aid of dry-powder dispersers, liquid aerosol generators, or nebulizers (e.g., Pettit and Gombotz, TIBTECH J6:343 (1998); Patton et al., Adv. Drug Deliv. Rev. 35:235 (1999)). This approach is illustrated by the AERX diabetes management system, which is a hand-held electronic inhaler that delivers aerosolized insulin into the lungs. Studies have shown that proteins as large as 48,000 kDa have been delivered across skin at therapeutic concentrations with the aid of low-frequency ultrasound, which illustrates the feasibility of trascutaneous administration (Mitragotri el al.. Science 269:850 (1995)). Transdermal delivery using electroporation provides another means to administer a molecule having IL-17RA binding activity (Potts et al., Pharm. Biotechnol. 70:213 (1997)).
[232] A pharmaceutical composition comprising a soluble 1L-17RA or anti-lL-]7RA antibody can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic proteins are combined in a mixture with a pharmaceutically acceptable carrier. A composition is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
[233] For purposes of therapy, soluble IL-17RA or anti-IL-!7RA antibody molecules and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount. A combination of a therapeutic molecule of the present invention and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient. For example, an agent used to treat inflammation is physiologically significant if its presence alleviates the inflammatory response.
[234] A pharmaceutical composition comprising 1L-17RA (or IL-17RA analog or fusion protein) or neutralizing anti-lL-17RA antibody can be furnished in liquid form, in an aerosol, or in solid form. Liquid forms, are illustrated by injectable solutions and oral suspensions. Exemplary solid forms include capsules, tablets, and controlled-reiease forms. The latter form is illustrated by miniosmotic pumps and implants (Bremer el al. Pharm. Biotechnol. 10:239 (1997); Ranade, "Implants in Drug Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al., "Protein Delivery with Infusion Pumps," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997); Yewey et al., "Delivery of Proteins from a Controlled Release Injectable Implant," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).
[235] Liposomes provide one means to deliver therapeutic polypeptides to a subject intravenously, intraperitoneally, intrathecally, intramuscularly, subcutaneously, or via oral administration, inhalation, or intranasal administration. Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounding aqueous compartments (see, generally, Bakker-Woudenberg et al, Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. /):S61 (1993), Kim, Drugs 4(y.6\% (1993), and Ranade, "Site-Specific Drug Delivery Using Liposomes as Carriers," in Drug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRC Press 1995)). Liposomes are similar in composition to cellular membranes and as a result, liposomes can be administered safely and are biodegradable. Depending on the method of preparation, liposomes may be unilamellar or multilamellar, and liposomes can vary in size with diameters ranging from 0.02 \x.m to greater than 10
|j,m. A variety of agents can be encapsulated in liposomes: hydrophobic agents partition in the biiayers and hydrophilic agents partition within the inner aqueous space(s) (see, for example, Machy el al. Liposomes In Cell Biology And PharmacoJog)' (John Libbey 1987), and Ostro el uL, American J. Hosp. Pharm. 46A516 (1989)). Moreover, it is possible to control the therapeutic availability of the encapsulated agent by varying liposome size, the number of biiayers, lipid composition, as well as the charge and surface characteristics of the liposomes.
[236] Liposomes can adsorb to virtually any type of cell and then slowly release the encapsulated agent. Alternatively, an absorbed liposome may be endocytosed by cells that are phagocytic. Endocytosis is followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents (Scherphof f/ al.. Aim. N.Y. Acad Sci. 446:36S (1985)). After intravenous administration, small liposomes (0.1 to 1.0 \im) are typically taken up by cells of the reticuloendothelial system, located principally in the liver and spleen, whereas liposomes larger than 3.0 fxm are deposited in the lung. This preferential uptake of smaller liposomes by the cells of the reticuioendotheliai system has been used to deliver chemotherapeutic agents to macrophages and to tumors of the liver.
[237] The reticuloendothelial system can be circumvented by several methods including saturation with large doses of liposome particles, or selective macrophage inactivation by pharmacological means (Claassen el al., Biochim. Biophys. Acta S(92:428 (1984)). in addition, incorporation of glycolipid- or polyethelene glycol-derivatized phospholipids into liposome membranes has been shown to result in a significantly reduced uptake by the reticuloendothelial system (Allen et al, Biochim. Biophys. Acta ]068:\33 (1991); Allen et al., Biochim. Biophys. Acta J J 50:9 0993)}.
[238] Liposomes can also be prepared to target particular cells or organs by varying phospholipid composition or by inserting receptors or ligands into the liposomes. For example, liposomes, prepared with a high content of a nonionic surfactant, have been used to target the liver (Hayakawa et al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull. 76:960 (1993)). These formulations were prepared by mixing soybean phospatidylcholine, a-tocopherol, and ethoxylated hydrogenated castor oil (HCO-60) in methanol, concentrating the mixture under vacuum, and then reconstituting the mixture with water. A liposomal formulation of dipalmitoylphosphatidyicholine (DPPC) with a soybean-derived sterylglucoside mi,xture (SG) and cholesterol (Ch) has also been shown to target the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).
[239] Alternatively, various targeting ligands can be bound to the surface of the liposome,
■ such as antibodies, antibody fragments, carbohydrates, vitamins, and transport proteins. For example.
liposomes can be modified with branched type galactosyllipid derivatives to target asialoglycoprotein
(galactose) receptors, which are exclusively expressed on the surface of liver cells (Kato and
Sugiyama, Crit. Rev. Ther. Drug Carrier Syst. J4:287 (1997); Miirahashi et ciL, Biol. Pharm. Bul!.20:259 (1997)). Similarly. Wu et al., Hepatology 27:112 (1998), have shown that labeling liposomes with asialofetuin led to a shortened liposome plasma half-life and greatly enhanced uptake of asialofetuin-labeled liposome by hepatocytes. On the other hand, hepatic accumulation of liposomes comprising branched type galactosyllipid derivatives can be inhibited by preinjection of asialofetuin (Murahashi et al., Biol Pharm. BuU.2Q:259 (1997)). Polyaconitylated human serum albumin liposomes provide another approach for targeting liposomes to liver cells (Kamps el al.. Proc. Nat'I Acad Sci. USA 94:11681 (1997)). Moreover, Geho, et a/. U.S. Patent No. 4,603,044, describe a hepatocyte-directed liposome vesicle delivery system, which has specificity for hepatobiliary receptors associated with the specialized metabolic cells of the liver.
[240] In a more general approach to tissue targeting, target cells are prelabeled with biotinylated antibodies specific for a ligand expressed by the target cell (Harasym et al. Adv. Drug Deliv. Rev. 32:99 (1998)). After plasma elimination of free antibody, streptavidin-conjugated liposomes are administered. In another approach, targeting antibodies are directly attached to liposomes (Harasym et al. Adv. Drug Deliv. Rev. 32:99 (1998)).
(241] Polypeptides and antibodies can be encapsulated within liposomes using standard techniques of protein microencapsulation (see, for example, Anderson et al. Infect. Inmnm. 37:1099 (1981), Anderson et al, Cancer Res. 50:18.53 (1990), and Cohen et al. Biochim. Biophys. Ada 1063:95 (1991), Alving et al "Preparation and Use of Liposomes in Immunological Studies," in Lipo.some Technology, 2nd Edition, Vol. HI, Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al, Meth. Enzymol 149:\24 (1987)). As noted above, therapeutically useful liposomes may contain a variety of components. For example, liposomes may comprise lipid derivatives of poly(ethylene glycol) (Allen et al, Biochim. Biophys. Acta 1150:9 (1993)).
|242] Degradable polymer microspheres have been designed to maintain high systemic levels of therapeutic proteins. Microspheres are prepared from degradable polymers such as poly(lactide-co-glycolide) (PLC), polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetate polymers, in which proteins are entrapped in the polymer (Gombotz and Pettit, Bioconjugate Chem. 6:332 (1995); Ranade, "Role of Polymers in Drug Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.), pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable Controlled Release Systems Useful for Protein Delivery," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92 (Plenum Press 1997); Bartus et al. Science 2\^ (1993), and Ranade. "Site-Specific Drug Delivery Using Liposomes as Carriers,'" in Drug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRC Press 1995)). Liposomes are similar in composition to cellular membranes and as a result, liposomes can be administered safely and are biodegradable. Depending on the method of preparation, liposomes may be unilamellar or multilamellar, and liposomes can vary in size with diameters ranging from 0.02 |.un to greater than 10 p,m. A variety of agents can be encapsulated in liposomes: hydrophobic agents partition in the bilayers and hydrophilic agents partition within the inner aqueous space(s) (see, for example, Machy et al., Lipo.somes In Cell Biology And Pharmacology {io\\x\ Libbey 1987), and Ostro et al, American J. Hosp. Pharm. 46:\516 (1989)). Moreover, it is possible to control the therapeutic availability of the encapsulated agent by varying liposome size, the number of bilayers, lipid composition, as well as the charge and surface characteristics of the liposomes.
[248] Liposomes can adsorb to virtually any type of cell and then slowly release the encapsulated agent. Alternatively, an absorbed liposome may be endocytosed by cells that are phagocytic. Endocytosis is followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents (Scherphof e/ al, Ann. N.Y. Acad. Sci. 446:16% (1985)). After intravenous administration, small liposomes (0.1 to 1.0 |a,m) are typically taken up by cells of the reticuloendothelial system, located principally in the liver and spleen, whereas liposomes larger than 3.0 |u,m are deposited in the lung. This preferential uptake of smaller liposomes by the cells of the reticuloendothelial system has been used to deliver chemotherapeutic agents to macrophages and to tumors of the liver.
|249] The reticuloendothelial system can be circumvented by several methods including saturation with large doses of liposome particles, or selective macrophage inactivation by pharmacological means (Claassen et al, Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporation of glycolipid- or polyethelene glycol-derivatized phospholipids into liposome membranes has been shown to result in a significantly reduced uptake by the reticuloendothelial system (Allen et al, Biochim. Biophys. Ada J068:]33 (1991); Allen et al, Biochim. Biophys. Acta 7750:9(1993)).
[250] Liposomes can also be prepared to target particular cells or organs by varying phospholipid composition or by inserting receptors or ligands into the liposomes. For example, liposomes, prepared with a high content of a nonionic surfactant, have been used to target the liver (Hayakawa et al. Japanese Patent 04-244,018; Kato et al, Biol Pharm. Bull J6:960 (1993)). These formulations were prepared by mixing soybean phospatidylcholine, a-tocopherol, and ethoxylated hydrogenated castor oil (HCO-60) in methanol, concentrating the mixture under vacuum, and then
reconstituting tiie mixture with water. A liposomal formulation of dipalmitoylpliosphatidylcholine (DPPC) with a soybean-derived sterylglucoside mixture (SG) and cholesterol (Ch) has also been shown to target the liver (Shimizu el al., Biol. Pharm. Bull. 2»:881 (1997)).
[251] Alternatively, various targeting ligands can be bound to the surface of the liposome, such as antibodies, antibody fragments, carbohydrates, vitamins, and transport proteins. For example, liposomes can be modified with branched type galactosyllipid derivatives to target asialoglycoprotein (galactose) receptors, which are exclusively expressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev. Ther. Drug Carrier Svst. 14:287 (1997); Murahashi et al, Biol. Pharm. Bull. 20:259 (1997)). Similarly, Wu el al, Hepatologv 27:772 (1998), have shown that labeling liposomes with asialofetuin led to a shortened liposome plasma half-life and greatly enhanced uptake of asialofetuin-labeled liposome by hepatocytes. On the other hand, hepatic accumulation of liposomes comprising branched type galactosyllipid derivatives can be inhibited by preinjection of asialofetuin (Murahashi et al, Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serum albumin liposomes provide another approach for targeting liposomes to liver cells (Kamps el al, Proc. Nat'I Acad. Sci. USA 94:11681 (1997)). Moreover, Geho, el al U.S. Patent No. 4,603,044, describe a hepatocyte-directed liposome vesicle delivery system, which has specificity for hepatobiliary receptors associated with the specialized metabolic cells of the liver.
|252] In a more general approach to tissue targeting, target cells are prelabeled with biotinylated antibodies specific for a ligand expressed by the target cell (Harasym el al, Adv. Drug Deliv. Rev. 32:99 (1998)). After plasma elimination of free antibody, streptavidin-conjugated liposomes are administered. In another approach, targeting antibodies are directly attached to liposomes (Harasym et al. Adv. Drug Deliv. Rev. 32:99 (1998)).
[253] Anti-IL-I7RA neutralizing antibodies and binding partners with IL-I7F OR IL-I7A binding activity, or 1L-17RA soluble receptor, can be encapsulated within liposomes using standard techniques of protein microencapsulation (see, for example, Anderson et al. Infect. Immun. 31:1099 (1981), Anderson et al, Cancer Res. 50:1853 (1990), and Cohen el al, Biochim. Biophvs. Acta 1063:95 (1991), Alving et al "Preparation and Use of Liposomes in Immunological Studies," in Liposome Technology, 2nd Edition, Vol. Ill, Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al. Meth. Enzymol 149:\2A (1987)). As noted above, therapeutically useful liposomes may contain a variety of components. For example, liposomes may comprise lipid derivatives of poivi'ethylene giycol) (Allen etal. Biochim. Biophvs. Acta 1150:9 (1993)).
[254] Degradable polymer microspheres have been designed to maintain high systemic levels of therapeutic proteins. Microspheres are prepared from degradable polymers such as poly(lactide-co-glycolide) (PLG), polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetate polymers, in which proteins are entrapped in the polymer (Gombotz and Pettit, Bioconjugate
Chem. 6:332 (1995); Ranade, "Role of Polymers in Drug Delivery," in Drug Delivery Systems. Ranade and Hollinger (eds.), pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable Controlled Release Systems Useful for Protein Delivery," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92 (Plenum Press 1997); Bartus et al. Science 281:1161 (1998); Putney and Burke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem. Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres can also provide carriers for intravenous administration of therapeutic proteins (see, for example, Gref tV «/., Pharm. Biotechnol. 10:167(1997)).
[255] The present invention also contemplates chemically modified Anti-lL-17RA antibody or binding partner, for example anti-Anti-Il.-17RA antibodies or IL-17RA soluble receptor, linked with a polymer, as discussed above.
[256] Other dosage forms can be devised by those skilled in the art, as shown, for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5"' Edition (Lea & Febiger 1990), Gennaro (ed.). Remington's Pharmaceutical Sciences, 19* Edition (Mack Publishing Company 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRC Press 1996).
[257] The present invention contemplates compositions of anti-IL-17F antibodies, and methods and therapeutic uses comprising an antibody, peptide or polypeptide described herein. Such compositions can further comprise a carrier. The carrier can be a conventional organic or inorganic carrier. Examples of carriers include water, buffer solution, alcohol, propylene glycol, macrogol, sesame oil, corn oil, and the like.
K) Production of Transgenic Mice
[258] Transgenic mice can be engineered to over-express the either iL-17F, 1L-17A or the !L-17RA gene in all tissues or under the control of a tissue-specific or tissue-preferred regulatory element. These over-producers can be used to characterize the phenotype that results from over-expression, and the transgenic animals can serve as models for human disease caused by excess IL-17F, IL-17A or IL-17RA. Transgenic mice that over-express any of these also provide model bioreactors for production of IL-17RA, such as soluble IL-17RA, in the milk or blood of larger animals. Methods for producing transgenic mice are well-known to those of skill in the art (see, for example, Jacob, "Expression and Knockout of Interferons in Transgenic Mice," in Overe.xpression and Knockout of Cytokines in Transgenic Mice, Jacob (ed.), pages 111-124 (Academic Press, Ltd. 1994), Monastersky and Robl (eds.), Strategies in Transgenic Animal Science (ASM Press 1995), and Abbud and Nilson, "Recombinant Protein Expression in Transgenic Mice," in Gene Expression Systems: Using Nature for the Art of Expression, Fernandez and Hoeffler (eds.), pages 367-397 (Academic Press, Inc. 1999)).
[259] For example, a method for producing a transgenic mouse tliat expresses a iL-!7RA gene can begin witii adult, fertile males (studs) (B6C3fl, 2-8 months of age (laconic Farms, Germantown, NY)), vasectomized males (duds) (B6D2fl, 2-8 months, (Taconic Farms)), prepubescent fertile females (donors) (B6C3fl, 4-5 weeks, (Taconic Farms)) and adult fertile females (recipients) (B6D2fl, 2-4 months. (Taconic Farms)). The donors are acclimated for one week and then injected with approximately 8 lU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company; St. Louis, MO) I.P., and 46-47 hours later. 8 lU/mouse of human Chorionic Gonadotropin (hCG (Sigma)) l.P. to induce superovulation. Donors are mated with studs subsequent to hormone injections. Ovulation generally occurs within 13 hours of hCG injection. Copulation is confirmed by the presence of a vaginal plug the morning following mating.
[260] Fertilized eggs are collected under a surgical scope. The oviducts are collected and eggs are released into urinanalysis slides containing hyaluronidase (Sigma). Eggs are washed once in hyaluronidase, and twice in Whitten's W640 medium (described, for example, by Menino and O'Claray, Biol. Reprod. 77:159 (]986), and Dienhart and Downs, Zygote 4:\29 {\996)) that has been incubated with 5% CO,, 5% O,, and 90% N, at 37°C. The eggs are then stored in a 37°C/5% CO,
incubator until microinjection.
[261] Ten to twenty micrograms of plasmid DNA containing a 1L-17RA encoding sequence is linearized, gel-purified, and resuspended in 10 mM Tris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10 nanograms per microliter for microinjection.
[262] Plasmid DNA is microinjected into harvested eggs contained in a drop of W640 medium overlaid by warm, CO,-equilibrated mineral oil. The DNA is drawn into an injection needle
(pulled from a 0.75mm ID, I mm OD borosilicate glass capillary), and injected into individual eggs. Each egg is penetrated with the injection needle, into one or both of the haploid pronuclei.
[263] Picoliters of DNA are injected into the pronuclei, and the injection needle withdrawn without coming into contact with the nucleoli. The procedure is repeated until all the eggs are injected. Successfully microinjected eggs are transferred into an organ tissue-culture dish with pre-gassed W640 medium for storage overnight in a 37°C/5% CO, incubator.
[264] The following day, two-cell embryos are transferred into pseudopregnant recipients. The recipients are identified by the presence of copulation plugs, after copulating with vasectomized duds. Recipients are anesthetized and shaved on the dorsal left side and transferred to a surgical microscope. A small incision is made in the skin and through the muscle wall in the middle of the abdominal area outlined by the ribcage, the saddle, and the hind leg, midway between knee and spleen. The reproductive organs are exteriorized onto a small surgical drape. The fat pad is stretched
out over the surgical drape, and a baby serrefine (Roboz, Rockville, MD) is attached to the fat pad and left hanging over the back of the mouse, preventing the organs from sliding back in.
[265] With a fine transfer pipette containing mineral oil followed by alternating W640 and air bubbles, 12-17 healthy two-cell embryos from the previous day's injection are transferred into the recipient. The swollen ampulla is located and holding the oviduct between the ampulla and the bursa, a nick in the oviduct is made with a 28 g needle close to the bursa, making sure not to tear the ampulla or the bursa.
[266] The pipette is transferred into the nick in the oviduct, and the embryos are blown in, allowing the first air bubble to escape the pipette. The fat pad is gently pushed into the peritoneum, and the reproductive organs allowed to slide in. The peritoneal wall is closed with one suture and the skin closed with a wound clip. The mice recuperate on a 37°C slide warmer for a minimum of four hours.
[267] The recipients are returned to cages in pairs, and allowed 19-21 days gestation. After birth, 19-21 days postpartum is allowed before weaning. The weanlings are sexed and placed into separate sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off the tail with clean scissors.
[268] Genomic DMA is prepared from the tail snips using, for example, a QIAGEN DNEASY kit following the manufacturer's instructions. Genomic DNA is analyzed by PCR using primers designed to amplify a IL-17RA gene or a selectable marker gene that was introduced in the same plasmid. After animals are confirmed to be transgenic, they are back-crossed into an inbred strain by placing a transgenic female with a wild-type male, or a transgenic male with one or two wild-type feiTiale(s). As pups are born and weaned, the sexes are separated, and their tails snipped for genotyping.
[269] To check for expression of a transgene in a live animal, a partial hepatectomy is performed. A surgical prep is made of the upper abdomen directly below the zyphoid process. Using sterile technique, a small 1.5-2 cm incision is made below the sternum and the left lateral lobe of the liver exteriorized. Using 4-0 silk, a tie is made around the lower lobe securing it outside the body cavity. An atraumatic clamp is used to hold the tie while a second loop of absorbable Dexon (American Cyanamid; Wayne, N.J.) is placed proximal to the first tie. A distal cut is made from the Dexon tie and approximately 100 mg of the excised liver tissue is placed in a sterile petri dish. The excised liver section is transferred to a 14 ml polypropylene round bottom tube and snap frozen in liquid nitrogen and then stored on dry ice. The surgical site is closed with suture and wound clips, and the animal's cage placed on a 37°C heating pad for 24 hours post operatively. The animal is checked daily post operatively and the wound clips removed 7-10 days after surgery. The expression
level of IL-17RA mRNA is examined for each transgenic mouse using an RNA solution hybridization assay or polymerase chain reaction.
[270] in addition to producing transgenic mice that over-express IL-17F, 1L-17A or IL-17RA, it is useful to engineer transgenic mice with either abnormally low or no expression of any of these genes. Such transgenic mice provide useful models for diseases associated with a lack of !L-17F, lL-17A or IL-17RA. As discussed above, IL-17RA gene expression can be inhibited using anti-sense genes, ribozyme genes, or external guide sequence genes. To produce transgenic mice that under-express the IL-17RA gene, such inhibitory sequences are targeted to IL-17RA mRNA. Methods for producing transgenic mice that have abnormally low expression of a particular gene are known to those in the art (see, for example, Wu el al., "Gene Underexpression in Cultured Cells and Animals by Antisense DMA and RNA Strategies," in Methods in Gene Biotechnology, pages 205-224 (CRC Press 1997)).
|271] An alternative approach to producing transgenic mice that have little or no IL-17RA gene expression is to generate mice having at least one normal iL-17RA allele replaced by a nonfunctional IL-I7RA gene. One method of designing a nonfunctional 1L-17RA gene is to insert another gene, such as a selectable marker gene, within a nucleic acid molecule that encodes IL-17RA. Standard methods for producing these so-called "knockout mice" are known to those skilled in the art (see, for example, Jacob, "Expression and Knockout of Interferons in Transgenic Mice," in Overexpression and Knockout of Cytokines in Transgenic Mice, Jacob (ed.), pages 111-124 (Academic Press, Ltd. 1994), and Wu et al, "New Strategies for Gene Knockout," in Methods in Gene Biotechnology, pages 339-365 (CRC Press 1997)).
|272] The invention is further illustrated by the following non-limiting examples.
EXAMPLES
EXAMPLE 1 IL-17F mRNA is Upregulated in a Murine Model of Asthma
[273] lL-17F mRNA levels were measured in a sensitization and airway challenge model in mice. Groups of mice, 8 to 1,0 wks of age, were sensitized by intraperitoneal injection of 10 ug of recombinant Dermatophagoides pteronyssinus allergen 1 (DerPl) (Indoor biotechnologies, Cardiff, UK) in 50 % Imject Alum (Pierce) on days 0 and 7. Seven days later, mice were challenged on 3 consecutive days (days 14, 15 and 16) with 20 ug of DerPl in 50 ul PBS. There were 4 mice representing this group. Negative controls included 5 mice given phosphate buffered saline (PBS) sensitization, followed by PBS challenge. In addition to 3 mice given DerPl sensitization, followed
by PBS challenge. Forty-eight hours following allergen, or control challenge whole lung tissue was harvested and total RNA was isolated.
[274] First strand cDNA was prepared using identical amounts of total RNA from each subject. IL-17F PCR was applied using Qiagen hotstar polymerase (Qiagen, Valencia, CA) and the manufacturer's recommendations. The IL-17F PCR utilized 35 cycles of amplification with sense primer as shown in SEQ ID N0:1 and antisense primer as shown in SEQ ID N0:2. In order to establish that the template quality was uniform amongst all subjects, Beta Actin PCR was applied to the same amount of each template used in the IL-1 7F amplification. B actin PCR included 25 cycles of PCR with sense primer as shown in SEQ ID N0:3 and antisense primer as shown in SEQ ID N0:4.
[275] All 4 mice from the DerPl sensitized, DerPl challenged treatment group (the asthma simulation) showed robust 1L-17F amplification. In contrast, weak IL-17F amplification was seen from the negative controls, including 3 of 3 subjects representing the DerPl sensitized/PBS challenged treatment group and 5 of 5 subjects from the PBS sensitized/PBS challenged treatment group. B actin amplification was at least as robust for the negative controls as for the asthma-simulated subjects, demonstrating that the weak negative control IL-17F amplification was not due to template problems.
EXAMPLE 2 IL-17A Induces Elevated Levels of IFN-gamma and TNF-alpha in Human Peripheral Blood
Mononuclear Cells [276] Human peripheral blood mononuclear cells (PBMC) are purified by ficoll density gradient centrifugation and then incubated overnight at 37"C in media alone, 50 ng/ml anti-human CD3 antibody, or the combination of 50 ng/ml anti-human CD3 antibody plus 1 )J,g/ml anti-human CD28 antibody. Replicate cultures for each of these conditions are set up and are given no cytokine, 25 ng/ml human IL-17A, or 25 ng/ml human IL-I7F. After 24-hour incubations, supernatants from each culture are harvested and assayed for cytokine content using B-D Bioscience's human Thl/Th2 Cytometric Bead Array (CBA). We found that cultures that had been stimulated with either anti-CD3 or anti-CD3 plus anti-CD28 and had been supplemented with IL-! 7A contained significantly elevated levels of IFN-gamma and TNF-alpha (3-5-fold elevation of each) over cultures with no cytokine added or those that received IL-17F. Cultures in which no anti-CD3 stimulation was added did not show significant changes in cytokine levels. In addition, IL-17A addition induced no significant changes in other cytokines assayed for with the CBA including IL-2, IL-4, IL-5, and IL-10. This data indicates that IL-I7A. but not IL-I7F, can augment the production of IFN-gamma and TNF-alpha in PBMC cultures stimulated with anti-CD3 or anti-CD3 plus anti-CD28.
EXAMPLE 3 IL-17RA-FC Decreases Disease Incidence and Progression in Mouse Collagen Induced Arthritis
(CIA) Model
A) Mouse Collagen Induced Arthritis (CIA) Model
[277] Ten week old male DBA/1 J mice (Jackson Labs) are divided into 3 groups of 13 mice/group. On day-21, animals are given an intradermal tail injection of 50-100 |LII of 1 mg/ml chick Type II collagen formulated in Complete Freund's Adjuvant (prepared by Chondrex, Redmond, WA), and three weeks later on Day 0 they are given the same injection except prepared in Incomplete Freund's Adjuvant. IL-17RA-Fc is administered as an intraperitoneal injection 3 times a week for 4 weeks, at different time points ranging from Day 0, to a day in which the majority of mice exhibit moderate symptoms of disease. Groups receive either 10 or 100 |j,g of IL-17RA-Fc per animal per dose, and control groups receive the vehicle control, PBS (Life Technologies, Rockville, MD). Animals begin to show symptoms of arthritis following the second collagen injection, with most animals developing inflammation within 1.5-3 weeks. The extent of disease is evaluated in each paw by using a caliper to measure paw thickness, and by assigning a clinical score (0-3) to each paw: 0=Normal, 0.5=Toe(s) inflamed, l=Mild paw inflammation, 2=Moderate paw inflammation, and 3=Severe paw inflammation as detailed below.
B) Monitoring Disease
[278] Animals can begin to show signs of paw inflammation soon after the second collagen injection, and some animals may even begin to have signs of toe inflammation prior to the second collagen injection. Most animals develop arthritis within 1.5-3 weeks of the boost injection, but some may require a longer period of time. Incidence of disease in this model is typically 95-100%, and 0-2 non-responders (determined after 6 weeks of observation) are typically seen in a study using 40 animals. Note that as inflammation begins, a common transient occurrence of variable low-grade paw or toe inflammation can occur. For this reason, an .animal is not considered to have established disease until marked, persistent paw swelling has developed.
[279] AH animals are observed daily to assess the status of the disease in their paws, which is done by assigning a qualitative clinical score to each of the paws. Every day, each animal has its 4 paws scored according to its state of clinical disease. To determine the clinical score, the paw can be thought of as having 3 zones, the toes, the paw itself (manus or pes), and the wrist or ankle joint. The extent and severity of the inflammation relative to these zones is noted including: observation of each toe for swelling; torn nails or redness of toes; notation of any evidence of edema or redness in any of the paws; notation of any loss of fine anatomic demarcation of tendons or bones; evaluation of the wrist or ankle for any edema or redness; and notation if the inflammation extends proximally up the
leg. A paw score of 1, 2. or 3 is based first on the overall impression of severity, and second on how
many zones are involved. The scale used for clinical scoring is shown below.
C) Clinical Score
0 = Normal
0.5 = One or more toes involved, but only the toes are inflamed
1 = mild inflammation involving the paw (1 zone), and may include a toe or toes
2 = moderate inflammation in the paw and may include some of the toes and/or the wrist/ankle (2 zones)
3 = severe inflammation in the paw, wrist/ankle, and some or ail of the toes (3 zones)
|280] Established disease is defined as a qualitative score of paw inflammation ranking 2 or
more, that persists for two days in a row. Once established disease is present, the date is recorded and designated as that animal's first day with "established disease".
[281] Blood is collected throughout the experiment to monitor serum levels of anti-collagen antibodies, as well as serum immunoglobulin and cytokine levels. Serum anti-collagen antibodies correlate well with severity of disease. Animals are euthanized on Day 21, and blood collected for serum and CBC's. From each animal, one affected paw is collected in 10%NBF for histology and one is frozen in liquid nitrogen and stored at -80°C for mRNA analysis. Also, 1/2 spleen, 1/2 thymus, 1/2 mesenteric lymph node, one liver lobe and the left kidney are collected in RNAIater for RMA analysis, and .1/2 spleen, 1/2 thymus, 1/2 mesenteric lymph node, the remaining liver, and the right kidney are collected in 10% NBF for histology. Serum is collected and frozen at -80°C for immunoglobulin and cytokine assays.
[282] Groups of mice receiving IL-1 7RA-Fc at all time points are characterized by a delay in the onset and/or progression of paw inflammation. These results indicate that IL-17RA can reduce inflammation, as well as disease incidence and progression associated with this model. These results are further supported by the observation that IL-17RA-Fc resulted in decreased levels of serum TNFa. IL-lb, and anti-collagen antibodies.
EXAMPLE 4 Truncated IL-17RA Soluble Receptors Decrease Disease Incidence and Progression in an
Inflammatory Bowel Disease (IBD) Model
[283] This model is designed to show that cultured intestinal tissue from patients with IBD produce higher levels of inflammatory mediators compared to tissue from healthy controls. This enhanced production of inflammatory mediators (including but not limited to IL-lb, IL-4, lL-5, IL-6, IL-8, IL-12, lL-13, IL-15, IL-17 A and F, lL-18, lL-23, TNF-a, IFN-g, MIP family members, MCP-1, G- and GM-CSF, etc.) contributes to the symptoms and pathology associated with IBDs such as
Crohn's disease (CD) and ulcerative colitis (UC) by way of tiieir effect(s) on activating inflammatory pathways and downstream effector cells. These pathways and components then lead to tissue and cell damage/destruction observed in vivo. Therefore, this model can simulate this enhanced inflammatory mediator aspect of IBD. Furthermore, when intestinal tissue from healthy controls or from human intestinal epithelial cell (lEC) lines is cultured in the presence of these inflammatory components, inflammatory pathway signaling can be observed, as well as evidence of tissue and cell damage.
[284] Therapeutics that would be efficacious in human IBD in vivo would work in the above ex vivo or lEC models by inhibiting and/or neutralizing the production and/or presence of inflammatory mediators.
[285] In this model, human intestinal tissue is collected from patients with IBD or from healthy controls undergoing intestinal biopsy, re-sectioning or from post-mortem tissue collection, and processed using a modification of Alexakis et al (Gut 53:85-90; 2004). Under aseptic conditions, samples are gently cleaned with copious amounts of PBS, followed by culturing of minced sections of tissue, in the presence of complete tissue culture media (plus antibiotics to prevent bacterial overgrowth). Samples from the same pool of minced tissue are treated with one of the following: vehicle (PBS); recombinant human (rh) IL-17A; rhlL-17F; or rhlL-17A+rhIL-17F. In addition, these are treated with or without an antagonist of either IL-17A or IL-17F, alone or in combination (such as a soluble IL-17RA). This experimental protocol is followed for studies with human lEC lines, with the exception that cells are passaged from existing stocks. After varying times in culture (from 1 h to several days), supernatants are collected and analyzed for levels of inflammatory mediators, including those listed above. In samples from patients with IBD or in samples treated with rh!L-17A and/or F, levels of inflammatory cytokines and chemokines are elevated compared to untreated healthy control tissue samples. The addition of antagonists to 1L-17F and/or IL-17A activity, such as IL-17RA soluble receptors and antibodies thereto including the anti-human-IL-17RA monoclonal and neutralizing antibodies of the present invention markedly reduces the production of inflammatory mediators, and thus, would expect to be efficacious in human IBD.
EXAMPLE 5 Truncated IL-17RA Soluble Receptors Decrease Disease Incidence and Progression in a
Multiple Sclerosis (MS) Model
[286] Multiple sclerosis (MS) is a complex disease that is thought to be mediated by a
number of factors, including the presence of lymphocytic and mononuclear cell inflammatory
infiltrates and demyelination throughout the CNS. Microglia are macrophage-like cells that populate
the central nervous system (CNS) and become activated upon injury or infection. Microglia have
been implicated as playing critical roles in various CNS diseases including MS, and may be used to study mechanism(s) of initiation, progression, and therapy of the disease (Nagai et al. Neurobiol Dis 8:1057-1068; 2001; Olson et al. J Neurosci Methods 128:33-43; 2003). Immortalized human microglial cell lines and/or established human astroglia cell lines can, therefore, be used to study some of the effects of inflammatory mediators on these cell types and their potential for neutralization. Inflammatory mediators (including but not limited to IL-lb, IL-6, IL-8, iL-12, IL-i3, lL-15. lL-17 A and F. IL-18, IL-23, TNF-a, IFN-g, MIP family members, RANTES, lP-10. MCP-1. G- and GM-CSF, etc.) can contribute to the symptoms and pathology associated with MS by way of their effect(s) on activating inflammatory pathways and downstream effector cells.
[287] in order to evaluate the pro-inflammatory actions of 1L-17A and 1L-17F, and the ability of an antagonist to 1L-17F and/or 1L-17A activity, such as 1L-17RA soluble receptors and antibodies thereto including the anti-human-IL-17RA monoclonal and neutralizing antibodies of the present invention to neutralize or decrease these effects, cultured glial cells are treated with one of the following: vehicle; rhIL-17A; rhIL-17F; rhIL-17A+lL-17F. In addition, these are treated with or without an antagonist of either IL-17A or 1L-17F, alone or in combination (such as a soluble IL-17RA). After varying times in culture (from I h to several days), supernatants and cells are collected and analyzed for levels and/or expression of inflammatory mediators, including those listed above. Levels of inflammatory cytokines and chemokines are elevated in the presence of rhlL-17A and/or IL-I7F compared to cultures treated with vehicle alone. The addition of antagonists to IL-I 7F and/or IL-17A activity, such as lL-17RA soluble receptors and antibodies thereto including the anti-human-1L-17RA monoclonal and neutralizing antibodies of the present invention markedly reduces the production and expression of inflammatory mediators, and thus, would expect to be efficacious in inflammatory aspects associated with human MS.
EXAMPLE 6
Truncated IL-17RA Soluble Receptors Decrease Disease Incidence and Progression in a
Rheumatoid Arthritis (RA) and Osteoarthritis (OA) Model
[288] This model is designed to show that human synovial cultures (including synovial
macrophages, synovial fibroblasts, and articular chondrocytes) and explants from patients with RA
and OA produce higher levels of inflammatory mediators compared to cultures/explants from healthy
controls. This enhanced production of inflammatory mediators (including but not limited to
oncostatin M. IL-lb, IL-6, iL-8, IL-12, IL-I5, IL-17 A and F, IL-18, IL-23, TNF-a, IFN-g, IP-10,
RANTES. RANKL. MIP family members, MCP-1, G- and GM-CSF, nitric oxide, etc.) contributes to
the symptoms and pathology associated with RA and OA by way of their effect(s) on activating
inflammatory pathways and downstream effector cells. These pathways and components then lead to
inflammatory infiltrates, cartilage and matrix loss/destruction, bone loss, and iipregulation of prostaglandins and cyclooxygenases. Therefore, this model can simulate the destructive inflammatory aspects of RA and OA in in vitro and ex vivo experiments. Furthermore, when explants and synovial cultures from healthy controls are cultured in the presence of several of these inflammatory components (e.g. oncostatin M, TNF-a, IL-lb, IL-6, IL-I7A and F, IL-15, etc.), inflammatory pathway signaling can be observed. Therapeutics that would be efficacious in human RA in vivo ^ would work in the above in vitro and ex vivo models by inhibiting and/or neutralizing the production and/or presence of inflammatory mediators.
[289] In this model, human synovial explants are collected from patients with RA, OA, or from healthy controls undergoing joint replacement or from post-mortem tissue collection, and processed using a modification of Wooley and Tetlow (Arthritis Res 2: 65-70; 2000) and van 't Hof at al (Rheumatology 39:1004-1008; 2000). Cultures of synovial fibroblasts, synovial macrophages and articular chondrocytes are also studied. Replicate samples are treated with one of the following: vehicle (PBS); recombinant human (rh) IL-17A; rhIL-17F; or rhIL-17A+rhlL-l 7F, and some samples contain various combinations of oncostatin M, TNF-a, IL-lb, lL-6, 1L-17A, IL-17F, and IL-15. In addition, these are treated with or without an antagonist to IL-17F and/or IL-17A activity, such as IL-17RA soluble receptors and antibodies thereto including the anti-human-lL-17RA monoclonal and neutralizing antibodies of the present invention. After varying time of culture (from 1 h to several days), supernatants are collected and analyzed for levels of inflammatory mediators, including those listed above. In samples from patients with RA or OA, or in samples treated with rhIL-17A and/or F (either alone or in combination with other inflammatory cytokines), levels of inflammatory cytokines and chemokines are elevated compared to untreated healthy control explants or in untreated cell cultures. The addition of antagonists to IL-17F and/or IL-I7A activity, such as IL-17RA soluble receptors and antibodies thereto including the anti-human-IL-17RA monoclonal and neutralizing antibodies of the present invention markedly reduces the production of inflammatory mediators, and thus, would expect to be efficacious in human RA and OA.
EXAMPLE 7 IL-17A, IL-17F and IL-17RA Expression in Murine Disease Models
[290] Four murine models of disease (asthma, DSS colitis, atopic dermatitis and experimental allergic encephalomyelitis) were analyzed using know techniques for the expression of IL-17A. lL-17Fand 1L-17RA.
[291] In the asthma model, IL-17A and IL-17F are expressed at very low to undetectable levels in lung, spleen, lung draining lymph nodes and lung infiltrating cells in diseased and non-diseased mice. IL-17RA message was found to be more highly expressed in lung compared to spleen
and lymph node but was not regulated with disease. IL-17RA was more highly expressed in spleen and lung draining lymph node compared to lung but was also not regulated with disease.
[292] Contrary to the asthma model, IL-17A and IL-17F were highly up-regulated in diseased but not normal mice in the DSS-colitis model in both proximal and distal colon. Neither cytokine was significantly up-regulated in the mesenteric lymph node. Further, it was found that up-regulation of both cytokines in the context of acute DSS-induced colitis and not in chronic DSS-induced colitis. IL-17RA was found to be prominently expressed in mesenteric lymph nodes as compared to proximal and distal colon, but was not regulated with disease. In contrast, IL-17RA was more highly expressed in proximal distal colon tissue compared to mesenteric lymph nodes. IL-17RA expression was also not regulated with disease.
[293] In atopic dermatitis, IL-17.A. niRNA was not detectable. IL-17F was found to be expressed in both skin and skin-draining lymph nodes but did not appear to be significantly regulated with disease. IL-17RA niRNA was more highly expressed in skin-draining lymph nodes as compared to skin but was not regulated with disease. 1L-! 7RA was more highly expressed in skin compared to skin-draining lymph nodes but was also not regulated with disease.
[294] In experimental allergic encephalomyelitis, both 1L-I7A and 1L-17F appeared to up-regulated in spinal chord in diseased but not healthy mice. IL-17F may have been more highly expressed in lymph nodes compared to spinal cord but expression in the lymph nodes was not regulated with disease. However, overall levels of expression in these tissues was quite low. IL-I7RA was more highly expressed in lymph node tissue compared to brain and spinal cord. 1L-17RA was not tested.
[295] In short, IL-17A and IL-17F expression appears to be regulated with disease in the context of the DSS-induced colitis and experimental allergic encephalomyelitis models but apparently not for asthma or atopic dermatitis. IL-17RA and IL-17RA expression does not appear to be regulated with disease but IL-17RA expression appears to be enriched in lymphoid tissues while IL-17RA expression appears to be enriched in non-lymphoid tissues.
EXAMPLE 8 Plate Based Protein binding Assays of IL17RA shortened Variants
[296] The format of the Capture EIA is as follows: Coat the ELISA plate with Goat anti Human IgG at 1 p-g/ml and incubate overnight at 4°C. Wash and block the plate with 200 |il per well 1% BSA for 1 hour at room temperature. Wash, add the IL-17RA engineered receptor variant as described above with the following dilution series (100 |.ig/ml through 0.10 p^g/ml) to the plate and incubate for 1 hour at room temperature. Wash, add biotin labeled ligand (® 10:1 (1L17A) or 6:1
(1L17F) and incubate for 1 hour at room temperature. Wash, add Strept Avidin -Horse Radish Peroxidase (w 0.5 µg/mL and incubate for I hour at room temperature. Wash, add TMB substrate for 4 minutes. Stop the reaction by adding Stop Solution. (Note: All reagents volumes were 50 µ\ per well unless stated otherwise). A positive result would be high OD values, generally above 0.5.
[297] The format of the Neutralization EIA is as follows: Coat the ELISA plate with the engineered IL-17RA variant described above at 1 µg/ml and incubate overnight at 4°C. Wash and block the plate with 200 p.! per well 1% BSA for 1 hour at room temperature. While blocking, in a separate plate incubate the IL-17RA engineered soluble receptor variant with the dilution series (50 µglm\ through 0.05 j-ig/ml) with biotin labeled ligand % 10:1 (IL17A) or 6:1 (ILI7F) in equal volumes for 1 hour at room temperature. Wash the blocked plate, add the receptor-ligand complex to the blocked plate and incubate for 1 hour at room temperature. Wash, add Strept Avidin -Horse Radish Peroxidase @ 0.5 )j,g/mL and incubate for 1 hour at room temperature. Wash, add TMB substrate for 7 minutes. Stop the reaction by adding Stop Solution. (Note: All reagents volumes were 50 10,1 per well unless stated otherwise). A positive result would be low OD values, generally below 0.5. Neutalization indicates that the variant protein is binding the biotinylated ligand.
[298] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.
WE CLAIM
What is claimed is:
1. An isolated soluble receptor polynucleotide comprising SEQ ID N0:5.
2. The isolated soluble receptor polynucleotide of claim 1, wherein the polynucleotide consists of SEQ lD NO:5.
3. An isolated polynucleotide encoding a soluble receptor polypeptide, wherein said polypeptide comprises SEQ ID N0:6.
4. The isolated polynucleotide of claim 3, wherein said soluble receptor polypepyide consists of SEQ ID N0:6.
5. An isolated soluble receptor comprising a first subunit, wherein said first subunit comprises SEQ1DN0:6.
6. The soluble receptor of claim 5, wherein the soluble receptor comprises a second subunit, wherein said subunits are linked together by a polypeptide linker.
7. The soluble receptor of claim 5, wherein said soluble receptor reduces the pro-inflammatory activity of IL-17A or IL-17F.
8. The soluble receptor of claim 5, wherein said soluble receptor reduces the pro-inflammatory activity of both IL-17A and IL-17F.
9. The soluble receptor of claim 6, wherein the polypeptide linker has about 100 to 240 amino acid residues.
10. A method of reducing IL-17A-induced or lL-17F-induced inflammation comprising administering to a mammal with inflammation an amount of a composition of a soluble receptor according to any of claims 1-9. sufficient to reduce inflammation.
11. A method of reducing IL-17A-induced and IL-17F-induced inflammation comprising administering to a mammal with inflammation an amount of a composition of a soluble receptor according to any of claims 1 -9, sufficient to reduce inflammation.
12. A method of treating a mammal afflicted with an inflammatory disease in which lL-17A or 1L-17F plays a role, comprising: administering a soluble receptor according to any of claims 1-9, wherein the inflammatory activity of either IL-I7A or IL-17F is reduced.
13. A method of treating a mammal afflicted with an inflammatory disease in which lL-17A and lL-17F plays a role, comprising: administering a soluble receptor according to any of claims 1 -9, wherein the inflammatory activity of IL-17A and IL-17F is reduced.
14. The method of any of claims 10-13, wherein the disease is asthma.
15. The method of any of claims 10-13, wherein the disease is a chronic inflammatory disease.
16. The method of claim 15, wherein the disease is a chronic inflammatory disease comprising inflammatory bowel disease, ulcerative colitis, Crohn's disease, arthritis, atopic dermatitis, or psoriasis.
17. The method of any of claims 10-13, wherein the disease is an acute inflammatory disease.
18. The method of claim 17, wherein the disease is an acute inflammatory disease comprising endotoxemia, septicemia, toxic shock syndrome or infectious disease.
19. A method of treating a pathological condition in a subject associated with lL-17RA activity comprising administering an effective amount of the soluble receptor of any of claims 1 -9, thereby treating said pathological condition.
20. The method of claim 19, wherein said pathological condition is asthma.
21. The method of claim 20, wherein said pathological condition is a chronic inflammatory condition.
22. The method of claim 21, wherein said chronic inflammatory condition comprising
inflammatory bowel disease, ulcerative colitis, Crohn's disease, arthritis, atopic dermatitis, or
psoriasis.
23. The method of claim 19, wherein said pathological condition is an acute inflammatory
condition.
24. The method of claim 23, wherein said acute inflammatory condition comprises endotoxemia,
septicemia, toxic shock syndrome, or infectious disease.
25. A method of treating a mammal afflicted with an inflammatory disease in which IL-17RA plays a role, comprising: a soluble receptor according to any of claims 1-9 wherein the inflammatory activity is reduced.
26. The method of claim 25, wherein the disease is asthma.
27. The method of claim 25, wherein the disease is a chronic inflammatory disease.
28. The method of claim 27, wherein the disease is a chronic inflammatory disease comprising
inflammatory bowel disease, ulcerative colitis, Crohn's disease, arthritis, atopic dermatitis, or
psoriasis.
29. The method of claim 25, wherein the disease is an acute inflammatory disease.
30. The method of claim 29, wherein the disease is an acute inflammatory disease comprising
endotoxemia, septicemia, toxic shock syndrome or infectious disease.
