Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/244—Interleukins [IL]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
  • A61P31/06—Antibacterial agents for tuberculosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Definitions

• the present invention relates generally to the identification and isolation of antagonists to IL-17A and IL-17F and IL-23 (via pl9) and methods of using the same.
• Cytokines are soluble, small proteins that mediate a variety of biological effects, including the induction of immune cell proliferation, development, differentiation, and/or migration, as well as the regulation of the growth and differentiation of many cell types (see, for example, Arai et al., Annu. Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol. 3:311 (1991); Paul and Seder, Cell 76:241 (1994)). Cytokine-induced immune functions can also include an inflammatory response, characterized by a systemic or local accumulation of immune cells.
• IL-17A, IL-17F and IL-23 are cytokines involved in inflammation.
• Human inter leukin-17A (also known as “IL-17A”) is a cytokine which stimulates the expression of interleukin-6 (IL-6), intracellular adhesion molecule 1 (ICAM-I), interleukin-8 (IL-8), granulocyte macrophage colony-stimulating factor (GM-CSF), and prostaglandin E2 expression, and plays a role in the preferential maturation of CD34+ hematopoietic precursors into neutrophils (Yao et al, J. Immunol 755:5483 (1995); Fossiez et al, J. Exp. Med. 183:2593 (1996)).
• Human interleukin-23 (also known as "IL-23”) is a cytokine which has been reported to promote the proliferation of T cells, in particular memory T cells.
• IL-17A and IL-17F share 55% identity (Kolls and Linden, 2004). In addition to their sequence similarity, both of these cytokines seem are produced by similar cell types, most notably activated, memory CD4+ T cells. See e.g. Agarwal et al., " Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17" J. Biol. Chem. 278:1910-191 (2003); see also Langrish et al. " IL-23 drives a pathogenic T cell population that induces autoimmune inflammation” J. Exp. Med. 201: 233-240 (2005); and Starnes et al.
• IL- 17F shares sequence homology with IL- 17 A, there are key difference between these moelucles.
• IL-17F mRNA is detected in many different tissues (such as, liver, lung, ovary, fetal liver, mast cells and basophils) while IL-17A expression is mostly restricted to T cells.
• T cell IL-17 induces stromal cells to produce pro-inflammatory and hematopoietic cytokines", J. Exp. Med. 183(6):2593-2603, (1996); Toy, D. et al., "Cutting edge: IL-17 signals througha heteodimeric receptor complex", J. Immunol. 177(l):36-39(2006).
• IL- 17F binds IL- 17RA with a much lower affinity than IL- 17 A.
• IL-17A and IL-17F have been implicated as major effector cytokines that trigger inflammatory responses and thereby contribute to a number of autoinflammatory diseases including multiple sclerosis, rheumatoid arthritis, and inflammatory bowel diseases.
• antibodies that bind to IL-23 and to IL-17A or IL- 17F that inhibit the immunological activities of both IL-17A and IL-17F would possess such novel therapeutic qualities.
• the present invention serves this need by providing antagonist molecules, inlcduing antibodies and antibody fragments that bind IL-23 and IL- 17A or IL- 17F, including antagonists that are comprised on one molecule.
• the present invention addresses these needs by providing antagonists to proinflammatory cytokines IL- 17 A, IL- 17F, and IL-23.
• Antagonists provided by the invention are antibodies, or antibody fragments that bind IL-17A and IL-17F, including anibodies that cross-react with IL-17A and IL- 17F, and antibodies or antibody fragments that bind IL-23, including antibodies that bind the pl9 subunit of IL-23.
• the polynucleotide sequence of the human IL- 17A is shown in SEQ ID NO:1 and the corresponding polypeptide sequence is shown in SEQ ID NO:2.
• the polynucleotide sequence of the human pl9 subunit of IL-23 is shown in SEQ ID NO: 3 and the corresponding polypeptide sequence is shown in SEQ ID NO:4.
• the polynucleotide sequence of the human IL-17F is shown in SEQ ID NO:5 and the corresponding polypeptide sequence is shown in SEQ ID NO:6.
• IL-17A is a cytokine which stimulates the expression of IL-6, ICAM-I, IL-8, GM- CSF, and prostaglandin E2 expression, and plays a role in the preferential maturation of CD34+ hematopoietic precursors into neutrophils (Yao et al., J. Immunol. 755:5483 (1995); Fossiez et al., J. Exp. Med. 183:2593 (1996)).
• the pro-inflammatory cytokines IL- 17A and IL- 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 multiple sclerosis, inflammatory bowel disease, 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. IL- 17A mediates its effects through interaction with its cognate receptor, the IL-17 receptor (IL-17RA), and for IL-17F, IL-17RA.
• IL-17RA IL-17 receptor
• the present invention contemplates that a cross-reactive, also called herein cross-binding, antibody may be useful as an antagonist to both IL-17A and IL- 17F, and thus to block both IL-17A and IL- 17F. Accordingly, the present invention addresses this need by providing therapeutic molecules (e.g. antibodies) which may block, inhibit, reduce, antagonize or neutralize the activity of both IL-17A and IL-17F.
• therapeutic molecules e.g. antibodies
• the present invention is directed to bispecific antibodies, with one antibody portion comprising a cross-reactive antibody, or antibody fragment that binds IL- 17A or IL- 17F and one antibody portion comprising an antibody or antibody fragment that binds the pl9 subunit of IL- 23, such as the antibodies described herein.
• the invention further provides uses therefor in inflammatory disease, as well as related compositions and methods.
• IL-23 is a heterodimeric cytokine composed of a unique subunit, pl9 (herein referred to interchangeably as “IL-23", “pl9” and IL23/pl9”), and the p40 subunit, which is shared with interleukin-12 (IL-12) (Oppmann, Immunity 13:115 (2000)).
• IL-23 has been found to stimulate the production and/or maintenence of IL-17 from activated CD4+ T cells in what has now been termed as a "new" T-helper (Th) subset, designated Thl7.
• Th new T-helper
• Thl7 cells have most likely evolved to provide adaptive immunity to specific classes of pathogens, such as extracellular bacteria.
• pathogens such as extracellular bacteria.
• inappropriate Th 17 responses have been strongly implicated in a growing list of autoimmune disorders, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, and psoriasis.
• IL-17 and IL-23 have also been reported to play important roles in many autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, Crohn's disease, and psoriasis.
• Both IL-23 and IL-17 are overexpressed in the central nervous system of humans with multiple sclerosis and in mice undergoing an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE).
• EAE experimental autoimmune encephalomyelitis
• MOG myelin oligodendrocyte glycoprotein
• PGP proteolipid peptide
• IL-17 and Thl7 cells can be produced from IL-23- independent sources, and the in vivo development of an IL-17 effector response has been shown to be IL23 -independent (Mangan et al, Nature 441:231 (2006)). Neutralization of IL-23 would theoretically eliminate existing IL-17 producing cells, but would not prevent the development of new Thl7 cells.
• the present invention concerns the inhibition of proinflammatory cytokines, IL-17A, IL- 17F and IL-23/pl9.
• This inhibition can be by administration of one or more molecules that inhibit IL- 17A or IL- 17F, such as a cross- reactive antibody, and one or more molecules that inhibit IL-23, such as an antibody or antibody fragment that binds the pl9 subunit of IL-23.
• This inhibition can be by administration of one molecule that comprises a binding entity that binds IL- 17A or IL- 17F and that also binds the pl9 subunit of IL-23.
• the present invention concerns the inhibition or neutralization of IL- 17A or IL- 17F and IL-23 (via pi 9) with a single antagonistic molecule or neutralizing entity. Since the portion of the single antagonistic molecule or neutralizing entity that binds IL- 17A or IL- 17F can bind either IL- 17A or IL- 17F, administration of this molecule or entity will inhibit or neutralize IL- 17A and IL- 17F. As such, this portion of the molecule or entity will inhibit or neutralize IL-17A homodimers, IL-17F homodimers, and IL-17A/F heteterodimers.
• the single antagonisitic molecule or neutralizing entity can be used to reduce, limit, neutralize, or block the proinflammatory effects of the IL-17A homodimer, the IL- 17F homodimer, or the IL- 17 A/F heterodimer.
• the single antagonisitic molecule or neutralizing entity can be used to reduce, limit, neutralize, or block the pro-cancerous effects of the IL-17A homodimer, the IL-17F homodimer, or the IL- 17 A/F heterodimer.
• the anti-IL-23pl9 portion of the single antagonistic molecule or neutralizing entity is used to reduce, limit, neutralize, or block production of new T cells that would produce IL-17A and/or IL-17F, including homodimers and heterodimers.
• the antagonistic molecules or neutralizing entities described herein can be used to treat autoimmune diseases, such as multiple sclerosis, inflammatory bowel disease, and psoriasis.
• the antagonistic molecules or neutralizing entities described herein can also be used to treat cancer, including angiogenesis.
• the present invention is based on the surprising discovery that antagonizing both IL- 23 (via p 19) and IL-17A is more effective therapeutically than neutralization of IL-23 alone (either via pl9 or p40) or IL-17A alone and thus, necessary for the effective treatment of inflammatory diseases (including cancers). See co-owned US Patent Application Serial Number 11/762,738, filed June 13, 2007 and WIPO Publication Number 2007/147019, published December 21, 2007.
• the antagonistic molecule or neutralizing entity inhibits the activity of IL-17A or IL- 17F and IL-23 (via the pl9 subunit), and thus, inhibits the production, maintenance, and activity of new and existing IL-17A and IL-17F and IL-17-producing T cells (Thl7).
• the invention further concerns the use of antagonists or neutralizing entities of IL-17A, IL-17F,and IL-23/pl9 in the treatment of inflammatory diseases characterized by the presence of elevated levels of IL- 17 A, IL- 17F, and/or IL-23.
• the invention also concerns the use of antagonists to IL- 17 A, IL- 17F, and IL- 23/pl9 in the treatment of cancers characterized by the presence of elevated levels of IL- 17 A, IL- 17F, and/or IL-23.
• the present invention is directed to antagonizing IL- 17 A, IL- 17F, and IL-23/pl9.
• Antagonists, including antibodies and antibody fragments of the present invention which may block, inhibit, reduce, antagonize or neutralize the activity of IL- 17 A, IL- 17F, (including homdimers and heterodimers), and IL-23/pl9 will have advantages over therapies that target only one of these three cytokines.
• the invention further provides uses therefor in inflammatory disease and cancer, as well as related compositions and methods.
• One method to treat immune related diseases is to suppress the immune response.
• Using the antagonists of the present invention i.e. anti-IL-17A or anti-IL-17F and anti-IL-23/pl9 antibodies
• 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.
• IL-17 is a disulfide- linked homodimeric 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]).
• IL-17 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.
• IL- 17 is disclosed in WO9518826 (1995), WO9715320 (1997) and WO9704097 (1997), as well as US Patent No. 6,063,372.
• IL- 17 exhibits pleitropic biological activities on various types of cells.
• IL-17 has been found to stimulate the production of many cytokines. It induces the secretion of IL-6, IL-8, IL-12, leukemia inhibitory factor (LIF), prostaglandin E2, MCP-I and G-CSF by adherent cells like fibroblasts, keratinocytes, epithelial and endothelial cells.
• IL-17 also has the ability to induce ICAM-I surface expression, proliferation of T cells, and growth and differentiation of CD34.sup.+ human progenitors into neutrophils.
• IL-17 is also believed to play a key role in certain other autoimmune disorders such as multiple sclerosis (Matusevicius et al., Mult. Scler. 5:101 (1999); Park et al, Nat Immunol. 6:1133 (2005)). IL-17 has further been shown, by intracellular signalling, to stimulate Ca 2+ influx and a reduction in [cAMP], in human macrophages (Jovanovic et al, J. Immunol. 160:3513 (1998)).
• Fibroblasts treated with IL-17 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)).
• IL-17A exhibits pleitropic biological activities on various types of cells.
• IL-17A has been found to stimulate the production of many cytokines. It induces the secretion of IL-6, IL-8, IL-12, leukemia inhibitory factor (LIF), prostaglandin E2, MCP-I and G-CSF by adherent cells like fibroblasts, keratinocytes, epithelial and endothelial cells.
• LIF leukemia inhibitory factor
• IL-17A also has the ability to induce ICAM-I surface expression, proliferation of T cells, and growth and differentiation of CD34 + human progenitors into neutrophils.
• IL-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]).
• IL-17A has been shown to induce the expression of osteoclast differentiation factor (ODF) mRNA in osteoblasts (Kotake et al., J. Clin. Invest., 103: 1345-1352 [1999]). ODF stimulates differentiation of progenitor cells into osteoclasts, the cells involved in bone resorption.
• ODF osteoclast differentiation factor
• IL-17A 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. IL- 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., J. Immunol., 160:3513 [1998]).
• Fibroblasts treated with IL-17A 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]).
• IL-17A also shares sequence similarity with mammalian cytokine-like factor 7 that is involved in bone and cartilage growth.
• 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.
• IL-17F The expression pattern of IL-17F appears to be similar to that of IL-17A, 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]).
• IL-23 a cytokine produced by dendritic cell, can mediate the production of both IL-17A and IL- 17F, primarily in memory T cells (Aggarwal et al. J. Biol. Chem. 278:1910-1914 [2003]).
• 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.
• IL- 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 collagens (Cai et al. Cytokine 16:10-21 [2001]; Attur et al Arthritis Rheum 44:2078-2083 [2001]).
• IL- 17F Similar to IL- 17 A, overexpression of IL- 17F in mice has also been shown to increase lung neutrophil recruitment and result in increased expression of ThI -associated cytokines in the lung, including IL-6, IFN-gamma, IP-10 and MIG (Starnes et al. J. Immunol. 167: 4137-4140 [2001]). IL- 17F was also upregulated in T cells from allergen-challenged asthmatics (Kawaguchi et al J. Immunol 167:4430-4435 [2001]), and found to induce IL-6 and IL-8 production in NHBE. In contrast to IL- 17 A, IL- 17F appears to inhibit angiogenesis in vitro (Starnes et al. J.
• IL-17F 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-17A, the expression of IL-17F was alos found to be upregulated by IL-23 in mouse.
• the present invention also provides antibodies that bind both IL- 17F and IL-23pl9 and methods for using such antibodies.
• the antibodies may act as antagonists or agonists, and find utility for, among other things, in vitro, in situ, or in vivo diagnosis or treatment of mammalian cells or pathological conditions associated with the presence (or absence) of IL-17F and/or IL-23pl9. In this embodiment, the antibody would bind IL- 17F, but not IL- 17 A.
• Antagonists to IL- 17 A, IL- 17F, and IL-23 activity are useful in therapeutic treatment of inflammatory diseases, particularly as antagonists to IL-17 A, IL-17F, and IL-23/pl9, in the treatment of inflammatory diseases, particularly in the treatment of multiple sclerosis, inflammatory bowel disease, and cancer.
• Theese antagonists are capable of binding, blocking, inhibiting, reducing, antagonizing or neutralizing IL- 17 A, IL- 17F, their homodimers and heterodimers, and IL-23 (via pl9) (either individually or together) in the treatment of atopic and contact dermatitis, multiple sclerosis, 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, psoriasis, eczema, IBS and inflammatory bowel disease (IBD) such as ulcerative colitis and Crohn's disease, Helicobacter pylori infection, intraabdominal adhesions and/or abscesses as results of peritoneal inflammation (i.e.
• systemic lupus erythematosus SLE
• multiple sclerosis systemic 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 graft vs. host disease
• GVHD graft vs. host disease
• SCW streptococcal cell wall
• SCW streptococcal cell wall
• IL-17 and/or IL-23 expression including but not limited to prostate, renal, colon, ovarian and cervical cancer, and leukemias (Tartour et al, Cancer Res.
• the present invention provides novel antagonists of IL- 17F and IL-23/pl9 and their uses in the treatment of inflammatory diseases and autoimmune diseases.
• the IL-17F and IL-23/pl9 antagonists of the present invention can be used to block, inhibit, reduce, antagonize or neutralize the activity of either IL- 17F or IL-23 (via pi 9), or both IL- 17F and IL-23 (via pi 9) in the treatment of inflammation and inflammatory dieases such as multiple sclerosis, cancer (as characterized by the expression of IL- 17F and/or IL-23), psoriasis, psoriatic arthritis, rheumatoid arthritis, endotoxemia, IBS, and inflammatory bowel disease (IBD), colitis, asthma, allograft rejection, immune mediated renal diseases, hepatobiliary diseases, atherosclerosis, promotion
• IBD inflammatory bowel disease
• the present invention provides isolated polypeptides that bind IL-17F (e.g., human IL-17F polypeptide sequence as shown in SEQ ID NO:6).
• the present invention also provides isolated polypeptides as disclosed above that bind IL-23 (e.g., human IL-23 polypeptide sequence as shown in SEQ ID NO:4). More specifically, the present invention provides polypeptides that bind to the pl9 subunit of IL-23 (e.g. human pl9 polypeptide sequence as shown in SEQ ID NO:4).
• the present invention also provides isolated polypeptides and epitopes comprising at least 15 contiguous amino acid residues of an amino acid sequence of SEQ ID NO:2 or 4.
• Illustrative polypeptides include polypeptides that either comprise, or consist of SEQ ID NO:2 or 4, an antigenic epitope thereof.
• the present invention also provides isolated polypeptides as disclosed above that bind to, block, inhibit, reduce, antagonize or neutralize the activity of IL- 17F or IL-23.
• Preferred embodiments of the invention include binding peptides, antibodies, and any fragments or permutations thereof that bind to IL- 17F or IL-23/pl9 (herein refered to interchangeably as “IL-17F/IL-23 antagonists”, “IL-17F antagonists”, “IL-23 antagonists”, “pl9 antagonists” "IL- 17F/IL-23 antibodies”, “IL-17F/pl9 antibodies”, “IL-17F antibodies”, “IL-23 antibodies”, “pl9 antibodies” "IL-17F/IL-23 antibodies”, “IL-17F/pl9 antibodies”, “IL-17F/IL-23/pl9 antibodies” etc.).
• binding peptides or antibodies are capable of specifically binding to both human IL- 17F and IL-23 (via pi 9) and/or are capable of modulating biological activities associated with either or both IL- 17F and IL-23, and thus are useful in the treatment of various diseases and pathological conditions such as inflammation and immune-related diseases.
• the invention include antibodies, and any fragments or permutations thereof, that cross-reacts with IL-17A and IL-17F (herein refereed to interchangeably as “cross-reactive antibodies”, “cross-binding antibodies”, “A/F antibodies”, “IL-17A/F antibodies” etc.) as well as antibodies, including any fragments or permutations thereof, that bind IL-23pl9.
• cross-reactive antibodies cross-binding antibodies
• A/F antibodies antibodies
• IL-17A/F antibodies etc.
• antibodies are capable of specifically binding to both human IL- 17A and IL- 17F and/or are capable of modulating biological activities associated with either or both IL- 17A and IL- 17F and/or their receptors, IL-17RA and IL-17RC, and thus are useful in the treatment of various diseases and pathological conditions such as immune related diseases.
• the antibody is a monoclonal antibody.
• the present invention provides antibodies and antibody fragments that specifically bind with IL- 17 and/or IL-23 (via pi 9).
• Exemplary antibodies include neutralizing antibodies, polyclonal antibodies, murine monoclonal antibodies, chimeric antibodies, humanized antibodies derived from murine monoclonal antibodies, and human monoclonal antibodies.
• Illustrative antibody fragments including F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, scFv, bispecific antibodies or antibody fragments, and minimal recognition units.
• Neutralizing antibodies preferably bind IL-17A, IL- 17F, their homodimers or heterodimers, or IL-23/pl9 such that the interaction of these ligands with their respective receptors is blocked, inhibited, reduced, antagonized or neutralized. That is, the neutralizing antibodies of the present invention can either either bind, block, inhibit, reduce, antagonize or neutralize each of IL-17A, IL-17F, their homodimers or heterodimers, or IL-23 singly, or bind, block, inhibit, reduce, antagonize or neutralize IL-17A, IL-17F, their homodimers and heterodimers and IL-23 together.
• the present invention further includes compositions comprising a carrier and a peptide, polypeptide, or antibody described herein.
• the present invention further includes pharmaceutical compositions, comprising a pharmaceutically acceptable carrier and a polypeptide or antibody described herein.
• the present invention also provides fusion proteins, comprising an antagonist of the present invention and an immunoglobulin moiety.
• the immunoglobulin moiety may be an immunoglobulin heavy chain constant region, such as a human F c fragment.
• the present invention further includes isolated nucleic acid molecules that encode such fusion proteins.
• the antibodies are linked to one or more non-proteinaceous polymers selected from the group consisting of polyethylene glycol, polypropylene glycol, and polyoxyalkylene, or to a cytotoxic agent or enzyme, or to a radioisotope, fluorescent compound or chemiluminescent compound.
• the present invention provides bispecific antibodies or binding proteins that bind both IL-17A, or IL-17F, and IL-23.
• Bispecific antibodies are antibodies that have two different antigen binding sites, such that the antibody specifically binds to two different antigens.
• Antibodies having higher valencies i.e., the ability to bind to more than two antigens can also be prepared; they are referred to as multispecific antibodies.
• the bispecific antibody can be a monoclonal antibody (MAb).
• the antibody is chimeric, or humanized, or fully human.
• Fully human antibodies may be generated by procedures that involve immunizing transgenic mice, wherein human immunoglobulin genes have been introduced into the mice, as discussed below.
• the hybridoma cell line which produces monoclonal antibodies of the present invention.
• the IL-17/IL-23 antibodies are linked to one or more non-proteinaceous polymers selected from the group consisting of polyethylene glycol, polypropylene glycol, and polyoxyalkylene, or to a cytotoxic agent or enzyme, or to a radioisotope, fluorescent compound or chemiluminescent compound.
• compositions of the invention may include pharmaceutically acceptable carriers or diluents.
• the compositions will include one or more antibodies in an amount which is therapeutically effective to treat a pathological condition or disease.
• antagonists of the present invention are also useful to prepare medicines and medicaments for the treatment of immune-related and inflammatory diseases, including for example, systemic lupus erythematosis, arthritis, rheumatoid arthritis, osteoarthritis, demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, inflammatory bowel disease, colitis, ulcerative colitis, Crohn's disease, gluten-sensitive enteropathy, cancer, neoplastic diseases, and angiogenesis.
• such medicines and medicaments comprise a therapeutically effective amount of an anti-IL- 17A/F cross-binding antibody/IL-23 antibody with a pharmaceutically acceptable carrier.
• the admixture is sterile.
• the IL-17A/F cross-binding antibodies bind to an epitope on both IL- 17A and IL-17F, wherein said epitope comprises residues Ile(23), Lys (25), Gly(27), Thr (29) and Pro(34) of the following sequences of human IL- 17F and the equivalent sequence found in human IL- 17A shown below.
• Residues 23, 25, 27, 29, and 34 are predicted to be on the surface of both IL-17A and IL- 17F and therefore are accessible to the binding of an antibody of the present invention or an equivalent protein binding antagonist.
• the IL-17 A/F antibodies bind to another epitope on both IL-17A and IL- 17F, wherein said epitope comprises residues Arg(67), Ser(68), Thr(69), Ser(70), Pro(71), Trp(72), Asn(73) of the following sequences of human IL- 17F and the equivalent sequence found in human IL-17A, as shown below.
• Residues 69, 71 and 73 are predicted to be on the surface of the bioactive cytokine and therefore are accessible to the binding of an antibody of the present invention or equivalent protein binding antagonist.
• the IL- 17 A/F antibodies bind to another epitope on both IL-17A and IL- 17F, wherein said epitope comprises residues Asp(79), Pro(80), Asn(81), Arg(82), Tyr(83), Pro(84) and Ser(85) of the following sequences of human IL-17F and the equivalent sequence found in human IL- 17 A, as shown below. All residues of this epitope are predicted to be on the surface of the bioactive cytokine and therefore are accessible to the binding of an antibody of the present invention or equivalent protein binding antagonist.
• hIL-17A (Asp81-Ser87 of SEQ ID NO:2) DPERYPS
• the IL-17A/F antibodies bind to another epitope on both IL-17A and IL- 17F, wherein said epitope comprises residues Thr(146), Pro(147), Val(148), Ile(149), His(150), His(151), VaI(152) of the following sequences of human IL- 17F and the corresponding sequence found in human IL- 17 A, as hown below. These residues are predicted to be on the surface of the bioactive cytokine and therefore to be accessible to the binding of an antibody of the present invention or equivalent protein binding antagonist.
• the IL-17A/F antibodies bind to another epitope on both IL-17A and IL- 17F, wherein said epitope is a discontinuous epitope comprising residues from two separate peptide chains of human IL-17F, as shown below; or the equivalent sequence found in human IL-17A, as shown below.
• residues 105-109, 147-152 of hIL-17F and 107-111, 148-154 of hIL-17A are predicted to be on the surface of the bioactive cytokine and therefore are accessible to the binding of an antibody of the present invention or equivalent protein binding antagonist.
• the IL-17 A/F antibodies bind to another epitope on both IL-17A and IL- 17F, wherein said epitope is a discontinuous epitope comprising residues of two or three separate peptide chains of human IL- 17F, as shown below; or the equivalent sequence found in human IL- 17A.
• residues 81, 82, 121, 132, 134 of hIL-17F and 83, 84, 123, 134, 136 of hIL-17A are predicted to be on the surface of the bioactive cytokine and therefore to be accessible to the binding of an antibody of the present invention or equivalent protein binding antagonist.
• an epitope of IL-17A or to IL- 17F to which neutralizing cross-reacting antibodies of the present invention may bind may be from residues 34 to residue 41 of SEQ ID NO: 2 (i.e., PNSEDKNF) or from residues 52 to 64 of SEQ ID NO: 2 (i.e., HNRNTNTNPKRSS).
• the an epitope of IL-17A to which non-neutralizing antibodies of the present invention may bind may be from residues 77 to 85 of SEQ ID NO: 2 (i.e., HRNEDPERY).
• epitopes of IL-23pl9 to which antibodies of the present invention may bind may be from residues 55 to 66 of SEQ ID NO: 4 (i.e., DLREEGDEETTN), from residues 74 to 85 (i.e., GDGCDPQGLRDN); from residues 137 to 146 (i.e., PEGHHWETQQ) and from residues 155 to 164 (i.e, PWQRLLLRFK).
• the present invention provides bispecific antibodies with one binding entity that is cross-reactive for IL- 17A and IL- 17F and one binding entity that binds IL- 23pl9.
• Bispecific antibodies are antibodies that have two different antigen binding sites, such that the antibody specifically binds to two different antigens.
• Antibodies having higher valencies i.e., the ability to bind to more than two antigens can also be prepared; they are referred to as multispecific antibodies.
• the hybridoma cell line which produces monoclonal antibodies of the present invention.
• the antibodies that bind to IL-17A or IL- 17F are linked to one or more non-proteinaceous polymers selected from the group consisting of polyethylene glycol, polypropylene glycol, and polyoxyalkylene, or to a cytotoxic agent or enzyme, or to a radioisotope, fluorescent compound or chemiluminescent compound.
• Typical methods of the invention include methods to treat pathological conditions or diseases in mammals associated with or resulting from increased or enhanced IL- 17F expression and/or activity.
• IL- 17F antibodies may be administered which preferably block or reduce the respective receptor binding or activation to their receptor(s).
• compositions which comprise IL-17F antibodies are provided.
• the compositions of the invention will include pharmaceutically acceptable carriers or diluents.
• the compositions will include one or more IL-17F antibodies in an amount which is therapeutically effective to treat a pathological condition or disease.
• the present invention concerns compositions and methods useful for the diagnosis and treatment of immune related disease in mammals, including humans.
• the present invention is based on the identification of antibodies that bind to IL- 17F (including agonist and antagonist antibodies) which either stimulate or inhibit the immune response in mammals.
• Immune related diseases can be treated by suppressing or enhancing the immune response.
• Antibodies that enhance the immune response stimulate or potentiate the immune response to an antigen.
• Antibodies which stimulate the immune response can be used therapeutically where enhancement of the immune response would be beneficial.
• antibodies that suppress the immune response attenuate or reduce the immune response to an antigen e.g., neutralizing antibodies
• attenuation of the immune response would be beneficial e.g., inflammation.
• antibodies that bind IL- 17F of the present invention and are also useful to prepare medicines and medicaments for the treatment of immune-related and inflammatory diseases including for example, systemic lupus erythematosis, arthritis, psoriatic arthritis, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis, idiopathic inflammatory myopathies, Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, diabetes mellitus, immune-mediated renal disease, demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, and chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases such as infectious, autoimmune chronic active hepatitis, primary biliary cirratives, and
• Models to test the effects of an antibody that binds IL- 17A or IL- 17F and binds IL- 23pl9 on cancer are known in the art.
• One model is the RENCA model in which tumor growth is measured in groups of mice that are injected s.c with the RENCA tumor on Day 0. Mice are then injected with 50-200ug control reagent or IL-17/IL-23 antagonist lX-3X/week for 3 weeks. Tumor volume is monitored 3X/week for 5 weeks. Significantly smaller tumors compared to control reagent injected mice would suggest neutralization or inhibits tumor growth.
• Ten-week old female BALB/c mice (Charles River Laboratories) are injected s.c.
• mice on the right flank with 0.1 x 106 RENCA cells on Day 0.
• Tumor growth is monitored 3X/week for 5 weeks using caliper measurements.
• Tumor volume is calculated using the formula V2*(B)2*L (mm3).
• Another model is the B 16 Melanoma model.
• mice are injected s.c with the B 16 tumor on Day 0.
• Mice are then injected with 50-200ug control reagent or IL-17/IL-23 antagonist lX-3X/week for 3 weeks. Tumor volume is monitored 3X/week for 5 weeks. Significantly smaller tumors compared to control reagent injected mice would suggest neutralization or inhibits tumor growth.
• Tumor growth is monitored 3X/week for 5 weeks using caliper measurements. Tumor volume is calculated using the formula 1 ⁇ 2 *(B)2*L (mm3).
• Another model is the LL/2 Lung Carcinoma model: to test if an IL-17/IL-23 antagonist has effects on tumor growth in mice, groups of mice are injected s.c with the LL/2 tumor on Day 0. Mice are then injected with 50-200ug control reagent or IL-17/IL-23 antagonist IX- 3X/week for 3 weeks. Tumor volume is monitored 3X/week for 5 weeks. Significantly smaller tumors compared to control reagent injected mice would suggest neutralization or inhibits tumor growth.
• Ten-week old female C57BL/6 mice (Charles River Laboratories) are injected s.c. on the right flank with 0.1 x 106 LL/2 cells on Day 0.
• mice injected i.p. with 50-200ug of either control reagent or IL-17/IL-23 antagonist lX-3X/week for 3 weeks
• Tumor growth is monitored 3X/week for 5 weeks using caliper measurements.
• Tumor volume is calculated using the formula 1 ⁇ 2 *(B)2*L (mm3).
• Another model measures tumor growth in the Ct-26 Colon Carcinoma model: to test if the the IL-17/IL-23 antagonist has effects on tumor growth in mice, groups of mice are injected s.c with the CT-26 tumor on Day 0. Mice are then injected with 50-200ug control reagent or IL-17/IL-23 antagonist lX-3X/week for 3 weeks. Tumor volume is monitored 3X/week for 5 weeks. Significantly smaller tumors compared to control reagent injected mice would suggest neutralization or inhibits tumor growth.
• Ten-week old female BALB/c mice (Charles River Laboratories) are injected s.c. on the right flank with 0.1 x 106 CT-26 cells on Day 0.
• mice injected i.p. with 50-200ug of either control reagent or IL-17/IL-23 antagonist lX-3X/week for 3 weeks
• Tumor growth is monitored 3X/week for 5 weeks using caliper measurements.
• Tumor volume is calculated using the formula 1 ⁇ 2 *(B)2*L (mm3).
• mice are injected s.c with the 4Tl tumor on Day 0. Mice are then injected with 50-200ug control reagent or IL-17/IL-23 antagonist lX-3X/week for 3 weeks. Tumor volume is monitored 3X/week for 5 weeks. Significantly smaller tumors compared to control reagent injected mice would suggest neutralization or inhibits tumor growth.
• Ten-week old female BALB/c mice (Charles River Laboratories) are injected s.c. on the right flank with 0.1 x 106 4Tl cells on Day 0.
• mice injected i.p. with 50-200ug of either control reagent or IL-17/IL-23 antagonist lX-3X/week for 3 weeks
• Tumor growth is monitored 3X/week for 5 weeks using caliper measurements.
• Tumor volume is calculated using the formula ! /2*(B)2*L (mm3).
• such medicines and medicaments comprise a therapeutically effective amount of an IL- 17F/IL-23 antibody with a pharmaceutically acceptable carrier.
• a pharmaceutically acceptable carrier Preferably, the admixture is sterile.
• the present invention concerns an isolated antibody which binds to IL- 17F.
• the antibody mimics the activity of IL-17F (an agonist antibody) or conversely the antibody inhibits or neutralizes the activity of IL-17F (an antagonist antibody).
• the antibody is a monoclonal antibody, which preferably has nonhuman complementarity determining region (CDR) residues and human framework region (FR) residues.
• the invention concerns a method of identifying antagonist antibodies of IL-17F and IL-23/pl9, said method comprising contacting both IL-17F and pl9 with a candidate molecule and monitoring a biological activity mediated by IL- 17F and/or IL-23.
• the invention concerns a composition of matter comprising an IL-17F/IL-23 antagonist antibody which binds both IL- 17F and IL-23 in admixture with a carrier or excipient.
• the composition comprises a therapeutically effective amount of the IL-17F/IL-23 antibody.
• antagonistic IL-17A/F antibodies are useful for: (a) decreasing infiltration of inflammatory cells into a tissue of a mammal in need thereof, (b) inhibiting or reducing an immune response in a mammal in need thereof, (c) decreasing the activity of T-lymphocytes or (d) decreasing the proliferation of T-lymphocytes in a mammal in need thereof in response to an antigen.
• the composition comprises a further active ingredient, which may, for example, be a further antibody or a cytotoxic or chemotherapeutic agent.
• the composition is sterile.
• the invention concerns a method of treating an immune related disorder in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of an IL-17A/F-IL-23 antagonist.
• the invention concerns an isolated polynucleotide that encodes a polypeptide of the present invention, wherein said polypeptide is capable of binding to IL- 17A or IL- 17F and IL-23pl9.
• the polypeptide inhibits the activity of IL-17A or IL- 17F and IL-23.
• the invention concerns an isolated polypeptide of the present invention, wherein said polypeptide is capable of binding to IL-17A or IL- 17F and IL- 23pl9.
• the polypeptide inhibits the activity of IL-17A or IL- 17F and IL-23.
• the invention concerns a method for inhibiting IL- 17 production by T cells comprising treating the T cells with an antagonist of IL-23/pl9 (IL-23).
• Processes for producing the polypeptide of the invnetion are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of said antibody and recovering said antibody from the cell culture.
• the invention concerns an article of manufacture, comprising: (a) a composition of matter comprising an antibody described herein; (b) a container containing said composition; and (c) a label affixed to said container, or a package insert included in said container referring to the use of said antibody in the treatment of an immune related disease.
• the invention also provides articles of manufacture and kits which include one or more antibodies described herein.
• Antibodies are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
• antibody or “antibody peptide(s)” refers to an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding and includes chimeric, humanized, fully human, and bispecific antibodies. In certain embodiments, binding fragments are produced by recombinant DNA techniques.
• binding fragments are produced by enzymatic or chemical cleavage of intact antibodies. Binding fragments include, but are not limited to, Fab, Fab', F(ab) 2 , F(ab') 2 , Fv, and single-chain antibodies.
• isolated antibody refers to an antibody that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
• the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lo wry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
• Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
• a “variant" antibody refers herein to a molecule which differs in amino acid sequence from a "parent" antibody amino acid sequence by virtue of addition, deletion and/or substitution of one or more amino acid residue(s) in the parent antibody sequence.
• the variant comprises one or more amino acid substitution(s) in one or more hypervariable region(s) of the parent antibody.
• the variant may comprise at least one, e.g. from about one to about ten, and preferably from about two to about five, substitutions in one or more hypervariable regions of the parent antibody.
• the variant will have an amino acid sequence having at least 75% amino acid sequence identity with the parent antibody heavy or light chain variable domain sequences, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.
• Identity or homology with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the parent antibody residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence shall be construed as affecting sequence identity or homology.
• the variant retains the ability to bind human IL- 17A or IL-IyF and IL-23 (via pi 9) and preferably has properties which are superior to those of the parent antibody.
• the variant may have a stronger binding affinity, enhanced ability to inhibit IL-17A or IL- 17F and IL-23-induced inflammation.
• a Fab form of the variant to a Fab form of the parent antibody or a full length form of the variant to a full length form of the parent antibody, for example.
• the variant antibody of particular interest herein is one which displays at least about 10 fold, preferably at least about 20 fold, and most preferably at least about 50 fold, enhancement in biological activity when compared to the parent antibody.
• parent antibody refers to an antibody which is encoded by an amino acid sequence used for the preparation of the variant.
• the parent antibody has a human framework region and, if present, has human antibody constant region(s).
• the parent antibody may be a humanized or human antibody.
• agonist refers to any compound including a protein, polypeptide, peptide, antibody, antibody fragment, large molecule, or small molecule (less than 10 kD), that increases the activity, activation or function of another molecule.
• the term "antagonist” refers to any compound including a protein, polypeptide, peptide, antibody, antibody fragment, large molecule, or small molecule (less than 10 kD), that decreases the activity, activation or function of another molecule.
• binding(ing) of a polypeptide includes, but is not limited to, the binding of a ligand polypeptide of the present invention to a receptor; the binding of a receptor polypeptide of the present invention to a ligand; the binding of an antibody of the present invention to an antigen or epitope; the binding of an antigen or epitope of the present invention to an antibody; the binding of an antibody of the present invention to an anti-idiotypic antibody; the binding of an anti-idiotypic antibody of the present invention to a ligand; the biding of an anti-idiotypic antibody of the present invention to a receptor; the binding of an anti-anti-idiotypic antibody of the present invention to a ligand, receptor or antibody, etc.
• a "bispecific” or “bifunctional” antibody is a hybrid antibody having two different heavy/light chain pairs and two different binding sites.
• Bispecific antibodies may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann (1990), Clin. Exp. Immunol. 79:315-321; Kostelny et al. (1992), /. Immunol. 148: 1547-1553.
• chimeric antibody refers to antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species.
• variable segments of the genes from a mouse monoclonal antibody may be joined to human constant segments, such as gamma 1 and gamma 3.
• a typical therapeutic chimeric antibody is thus a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant domain from a human antibody, although other mammalian species may be used.
• a chimeric antibody is produced by recombinant DNA technology in which all or part of the hinge and constant regions of an immunoglobulin light chain, heavy chain, or both, have been substituted for the corresponding regions from another animal's immunoglobulin light chain or heavy chain.
• the antigen-binding portion of the parent monoclonal antibody is grafted onto the backbone of another species' antibody.
• One approach described in EP 0239400 to Winter et al. describes the substitution of one species' complementarity determining regions (CDRs) for those of another species, such as substituting the CDRs from human heavy and light chain immunoglobulin variable region domains with CDRs from mouse variable region domains.
• the term "effective neutralizing titer" as used herein refers to the amount of antibody which corresponds to the amount present in the serum of animals (human or cotton rat) that has been shown to be either clinically efficacious (in humans) or to reduce virus by 99% in, for example, cotton rats.
• the 99% reduction is defined by a specific challenge of, e.g., 10 3 pfu, 10 4 pfu, 10 5 pfu, 10 6 pfu, 10 7 pfu, 10 8 pfu, or 10 9 pfu) of RSV.
• epitopic determinants refers to the portion of an antigen to which an antibody specifically binds.
• epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
• IL-17 epitope refers to a portion of the corresponding polypeptide having antigenic or immunogenic activity in an animal, preferably in a mammal, and most preferably in a mouse or a human.
• An epitope having immunogenic activity is a portion of an IL-17A or IL- 17F or IL-23/pl9 polypeptide that elicits an antibody response in an animal.
• An epitope having antigenic activity is a portion of an IL-17A or IL- 17F or IL-23/pl9 polypeptide to which an antibody immunospecifically binds as determined by any method well known in the art, for example, by immunoassays.
• Antigenic epitopes need not necessarily be immunogenic.
• Such epitopes can be linear in nature or can be a discontinuous epitope.
• the term "conformational epitope" refers to a discontinuous epitope formed by a spatial relationship between amino acids of an antigen other than an unbroken series of amino acids.
• epitope tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of antibodies of the present invention.
• the epitope tag preferably is sufficiently unique so that the antibody thereagainst does not substantially cross-react with other epitopes.
• Suitable tag polypeptides generally have at least 6 amino acid residues and usually between about 8- 50 amino acid residues (preferably between about 9-30 residues).
• the epitope tag is a "salvage receptor binding epitope".
• the term "salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgGi, IgG 2 , IgG 3 , or IgG 4 ) that is responsible for increasing the in vivo serum half -life of the IgG molecule.
• fragment refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues, at least contiguous 90 amino acid residues, at least contiguous 100 amino acid residues, at least contiguous 125 amino acid residues, at least 150 contiguous amino acid residues, at least contiguous 175 amino acid residues, at least contiguous 200 amino acid residues, or at least contiguous 250 amino acid residues of the amino acid sequence of a IL- 17 or IL-23/pl9 polypeptide or an antibody that immunospecifically binds to
• immunoglobulin refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes.
• One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.
• Full-length immunoglobulin "light chains” (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus.
• Full-length immunoglobulin "heavy chains” (about 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes (about 330 amino acids).
• Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively.
• variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids.
• An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions.
• hypervariable region refers to the amino acid residues of an antibody which are responsible for antigen binding.
• the hypervariable region comprises amino acid residues from a "Complementarity Determining Region” or "CDR" (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (Chothia and Lesk, 1987, J. MoI. Biol.
• Framework Region or "FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.
• the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
• a "human framework region” is a framework region that is substantially identical (about 85% or more, usually 90-95% or more) to the framework region of a naturally occurring human immunoglobulin.
• the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDR's. The CDR's are primarily responsible for binding to an epitope of an antigen.
• the term "humanized” immunoglobulin refers to an immunoglobulin comprising a human framework region and one or more CDR's from a non-human (usually a mouse or rat) immunoglobulin.
• the non-human immunoglobulin providing the CDR's is called the "donor” and the human immunoglobulin providing the framework is called the "acceptor”.
• Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical.
• all parts of a humanized immunoglobulin, except possibly the CDR's are substantially identical to corresponding parts of natural human immunoglobulin sequences.
• a “humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin.
• a humanized antibody would not encompass a typical chimeric antibody as defined above, e.g., because the entire variable region of a chimeric antibody is non-human.
• human antibody includes and antibody that has an amino acid sequence of a human immunoglobulin and includes antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described, for example, by Kucherlapati et al. in U.S. Patent No. 5,939,598.
• the term "genetically altered antibodies” means antibodies wherein the amino acid sequence has been varied from that of a native antibody. Because of the relevance of recombinant DNA techniques in the generation of antibodies, one need not be confined to the sequences of amino acids found in natural antibodies; antibodies can be redesigned to obtain desired characteristics. The possible variations are many and range from the changing of just one or a few amino acids to the complete redesign of, for example, the variable or constant region. Changes in the constant region will, in general, be made in order to improve or alter characteristics, such as complement fixation, interaction with membranes and other effector functions. Changes in the variable region will be made in order to improve the antigen binding characteristics.
• immunoglobulins may exist in a variety of other forms including, for example, single-chain or Fv, Fab, and (Fab') 2 , as well as diabodies, linear antibodies, multivalent or multispecific hybrid antibodies (as described above and in detail in: Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), which are incorporated herein by reference).
• Hood et al. "Immunology", Benjamin, N. Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporated herein by reference).
• single-chain Fv refers to antibody fragments that comprises the variable regions from both the heavy and light chains, but lacks the constant regions, but within a single polypeptide chain.
• a single-chain antibody further comprises a polypeptide linker between the VH and VL domains which enables it to form the desired structure which would allow for antigen binding.
• Single chain antibodies are discussed in detail by Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
• single-chain antibodies can also be bi-specific and/or humanized.
• a "Fab fragment” is comprised of one light chain and the C H i and variable regions of one heavy chain.
• the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
• a "Fab' fragment” contains one light chain and one heavy chain that contains more of the constant region, between the C H1 and C H2 domains, such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab') 2 molecule.
• a "F(ab') 2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the C H1 and C H2 domains, such that an interchain disulfide bond is formed between two heavy chains.
• diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) in the same polypeptide chain (V H - V L ).
• V H heavy chain variable domain
• V L light chain variable domain
• the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
• Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. ScL USA 90:6444-6448 (1993).
• linear antibodies refers to the antibodies described in Zapata et al. Protein Eng. 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (V H -C HI -V H -C H1 ) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
• immunoglobulin fragment refers to a polypeptide fragment that contains at least the variable domains of the immunoglobulin heavy and light chains.
• An immunologically functional immunoglobulin fragment of the invention is capable of binding to a ligand, preventing binding of the ligand to its receptor, interrupting the biological response resulting from ligand binding to the receptor, or any combination thereof.
• an immunologically functional immunoglobulin fragment of the invention binds specifically to either IL-17A or IL-17F and IL-23/pl9.
• the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
• the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
• 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 DNA and RNA), or analogs of naturally- occurring nucleotides (e.g., ⁇ -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.
• the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
• 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.
• 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.
• nucleic acid molecule refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence.
• 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).
• structural gene refers to a nucleic acid molecule that is transcribed into messenger RNA (inRNA), which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
• An "isolated nucleic acid molecule” is a nucleic acid molecule that is not integrated in the genomic DNA of an organism.
• a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA 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.
• nucleic acid molecule construct 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.
• 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.
• cDNA complementary DNA
• cDNA complementary DNA
• cDNA double-stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand.
• cDNA also refers to a clone of a cDNA molecule synthesized from an RNA template.
• a "promoter” is a nucleotide sequence that directs the transcription of a structural gene.
• 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 al., MoI. Endocrinol. 7:551 (1993)), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman, Seminars in Cancer Biol.
• DSEs differentiation-specific elements
• CREs cyclic AMP response elements
• SREs serum response elements
• GREs glucocorticoid response elements
• binding sites for other transcription factors such as CRE/ATF (O'Reilly et al, J. Biol. Chem. 267:19938 (1992)), AP2 (Ye et 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 et al., eds., Molecular Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), and Lemaigre and Rousseau, Biochem.
• 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.
• Repressible promoters are also known.
• 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.
• a “regulatory element” is a nucleotide sequence that modulates the activity of a core promoter.
• 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.
• An “enhancer” is a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.
• Heterologous DNA refers to a DNA molecule, or a population of DNA molecules, that does not exist naturally within a given host cell.
• DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species (i.e., endogenous DNA) so long as that host DNA is combined with non-host DNA (i.e., exogenous DNA).
• 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.
• a heterologous DNA molecule can comprise an endogenous gene operably linked with an exogenous promoter.
• 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.
• 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.”
• a "protein” is a macromolecule comprising one or more polypeptide chains.
• a protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates 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.
• a peptide or polypeptide encoded by a non-host DNA molecule is a "heterologous" peptide or polypeptide.
• 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 cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
• an "expression vector” is a nucleic acid molecule encoding a gene that is expressed in a host cell.
• 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.
• a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.
• a "recombinant host” is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or expression vector.
• a recombinant host is a cell that produces an antagonist of the present inventionfrom an expression vector.
• an antagonist can be produced by a cell that is a "natural source" of said antagonist, and that lacks an expression vector.
• a "fusion protein” is a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes.
• 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 IL-17RA using affinity chromatography.
• 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 IL-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.
• 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.
• secretory signal sequence denotes a DNA 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.
• secretory peptide a DNA sequence that encodes a 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.
• 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.
• 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.
• isolated polypeptide does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
• 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 carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
• expression refers to the biosynthesis of a gene product.
• expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
• immunomodulator includes cytokines, stem cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic factors, an dthe like, and synthetic analogs of these molecules.
• complement/anti-complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
• biotin and avidin are prototypical members of a complement/anti-complement pair.
• Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like.
• the complement/anti-complement pair preferably has a binding affinity of less than 10 9 M "1 .
• a "therapeutic agent” is a molecule or atom which is conjugated to an antibody moiety to produce a conjugate which is useful for therapy.
• therapeutic agents include drugs, toxins, immunomodulators, chelators, boron compounds, photoactive agents or dyes, and radioisotopes.
• a "detectable label” is a molecule or atom which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis.
• detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties.
• 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.
• affinity tag 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. 4:1075 (1985); Nilsson et al, Methods Enzymol.
• 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.
• the antibodies of the invention comprise a first antibody portion that binds to IL- 17A or IL- 17F and and second antibody portion that binds to IL-23 (via pi 9).
• the antibodies of the invention specifically bind a monomeric form of both IL- 17A or IL- 17F .
• the antibodies of the invention bind a homodimeric form of either IL-17A or IL- 17F.
• the antibodies of the invention bind a heterodimeric form of IL-17A/F.
• the antibodies of the invention specifically bind a multimeric form of IL- 17 (e.g., a heterodimeric form).
• IL-17 can form a heterodimer with any other member of the IL-17 family of ligands, such as IL-17B, IL-17C, or IL-17F.
• the antibodies of the invention bind a homodimeric or heterodimeric form of IL-23 (via binding to the pl9 subunit)
• Preferred antibodies of the invention block the biological activities of IL- 17A or IL- 17F and IL-23, either singly or together.
• the antibodies of the invention include portions of intact antibodies that retain antigen-binding specificity, for example, Fab fragments, Fab' fragments, F(ab') 2 fragments, F(v) fragments, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, and the like.
• antigen binding fragments, as well as full-length dimeric or trimeric polypeptides derived from the above-described antibodies are themselves useful.
• Humanized antibodies are produced by recombinant DNA technology, in which at least one of the amino acids of a human immunoglobulin light or heavy chain that is not required for antigen binding has been substituted for the corresponding amino acid from a nonhuman mammalian immunoglobulin light or heavy chain.
• the immunoglobulin is a mouse monoclonal antibody
• at least one amino acid that is not required for antigen binding is substituted using the amino acid that is present on a corresponding human antibody in that position.
• the "humanization" of the monoclonal antibody inhibits human immunological reactivity against the foreign immunoglobulin molecule.
• a method of performing complementarity determining region (CDR) grafting may be performed by sequencing the mouse heavy and light chains of the antibody of interest that binds to the target antigen (e.g., IL-17 and/or IL-23/pl9) and genetically engineering the CDR DNA sequences and imposing these amino acid sequences to corresponding human V regions by site directed mutagenesis.
• Human constant region gene segments of the desired isotype are added, and the "humanized" heavy and light chain genes are co-expressed in mammalian cells to produce soluble humanized antibody.
• a typical expression cell is a Chinese Hamster Ovary (CHO) cell. Suitable methods for creating the chimeric antibodies may be found, for example, in Jones et al.
• the antibodies of the invention may be used alone or as immunoconjugates with a cytotoxic agent.
• the agent is a chemotherapeutic agent.
• the agent is a radioisotope, including, but not limited to Lead-212, Bismuth-212, Astatine-211, Iodine-131, Scandium-47, Rhenium-186, Rhenium-188, Yttrium-90, Iodine-123, Iodine-125, Bromine-77, Indium-I l l, and fissionable nuclides such as Boron-10 or an Actinide.
• the agent is a toxin or cytotoxic drug, including but not limited to ricin, modified Pseudomonas enterotoxin A, calicheamicin, adriamycin, 5-fluorouracil, and the like. Methods of conjugation of antibodies and antibody fragments to such agents are known in the literature.
• the antibodies of the invention include derivatives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to its epitope.
• suitable derivatives include, but are not limited to fucosylated antibodies and fragments, glycosylated antibodies and fragments, acetylated antibodies and fragments, pegylated antibodies and fragments, phosphorylated antibodies and fragments, and amidated antibodies and fragments.
• the antibodies and derivatives thereof of the invention may themselves by derivatized by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other proteins, and the like.
• At least one heavy chain of the antibody is fucosylated.
• the fucosylation is ⁇ - linked.
• at least one heavy chain of the antibody comprises a fucosylated, ⁇ -linked oligosaccharide.
• the antibodies of the invention include variants having single or multiple amino acid substitutions, deletions, additions, or replacements that retain the biological properties (e.g., block the binding of IL-17A or IL-17F and/or IL-23 to their respective receptors, block the biological activity of IL-17A or IL- 17F and IL-23, binding affinity) of the antibodies of the invention.
• the skilled person can produce variants having single or multiple amino acid substitutions, deletions, additions or replacements.
• variants may include, inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or nonconservative amino acids, (b) variants in which one or more amino acids are added to or deleted from the polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like.
• Antibodies of the invention may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or nonconserved positions. In another embodiment, amino acid residues at nonconserved positions are substituted with conservative or nonconservative residues.
• the techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to the person having ordinary skill in the art.
• Antibodies of the invention also include antibody fragments.
• a “fragment” refers to polypeptide sequences which are preferably at least about 40, more preferably at least to about 50, more preferably at least about 60, more preferably at least about 70, more preferably at least about 80, more preferably at least about 90, and more preferably at least about 100 amino acids in length, and which retain some biological activity or immunological activity of the full-length sequence, for example, the ability to block the binding of IL-17 and/or IL-23 to their respective receptors, block the biological activity of IL-17 and IL-23, binding affinity.
• the invention also encompasses fully human antibodies such as those derived from peripheral blood mononuclear cells of ovarian, breast, renal, colorectal, lung, endometrial, or brain cancer patients. Such cells may be fused with myeloma cells, for example, to form hybridoma cells producing fully human antibodies against both IL-17A or IL-17F and IL-23/pl9.
• the invention also encompasses bispecific antibodies that bind to both IL- 17A or IL- 17F and IL-23 (via pi 9).
• the antibodies of the invention are preferably nontoxic as demonstrated, for example, in in vivo toxicology studies.
• the antibodies and derivatives thereof of the invention have binding affinities that include a dissociation constant (K d ) of less than 1 X 10 -2 .
• the K d is less than 1 X 10 -3 .
• the K d is less than 1 X 10 -4 .
• the K d is less than 1 X 10 -5 .
• the K d is less than 1 X 10 -6 .
• the K d is less than 1 X 10 -7 .
• the K d is less than 1 X 10. -8 .
• the v is less than 1 X 10 -9 .
• the v is less than 1 X 10 -10 . In still other embodiments, the K d is less than 1 X 10 -11 . In some embodiments, the K d is less than 1 X 10 -12 . In other embodiments, the K d is less than 1 X 10 -13 . In other embodiments, the K d is less than 1 X 10 -14 . In still other embodiments, the K d is less than 1 X 10 -15 .
• the invention also includes nucleic acids encoding the heavy chain and/or light chain of the antibodies of the invention.
• Nucleic acids of the invention include nucleic acids having at least 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98% homology to nucleic acids of the invention. The terms "percent similarity", “percent identity” and “percent homology” when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program.
• Nucleic acids of the invention also include complementary nucleic acids. In some instances, the sequences will be fully complementary (no mismatches) when aligned. In other instances, there may be up to about a 20% mismatch in the sequences.
• nucleic acids encoding both a heavy chain and a light chain of an antibody of the invention are provided.
• Nucleic acids of the invention can be cloned into a vector, such as a plasmid, cosmid, bacmid, phage, artificial chromosome (BAC, YAC) or virus, into which another genetic sequence or element (either DNA or RNA) may be inserted so as to bring about the replication of the attached sequence or element.
• the expression vector contains a constitutively active promoter segment (such as but not limited to CMV, SV40, Elongation Factor or LTR sequences) or an inducible promoter sequence such as the steroid inducible pIND vector (Invitrogen), where the expression of the nucleic acid can be regulated.
• Expression vectors of the invention may further comprise regulatory sequences, for example, an internal ribosomal entry site.
• the expression vector can be introduced into a cell by transfection, for example.
• the invention also provides methods of producing antibodies, including monoclonal antibodies that specifically bind to IL-17A or IL-17F and IL-23pl9, either singly or together.
• Antibodies of the invention may be produced in vivo or in vitro. Standard methods are known for creating monoclonal antibodies including, but are not limited to, the hybridoma technique (see Kohler & Milstein, (1975) Nature 256:495-497); the trioma technique; the human B-cell hybridoma technique (see Kozbor et al. (1983) Immunol. Today 4:72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al. in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., 1985, pp. 77-96).
• Both IL-17F and IL-23 may be purified from cells or from recombinant systems using a variety of well-known techniques for isolating and purifying proteins.
• both IL- 17F and IL-23 may be isolated based on the apparent molecular weight of the protein by running the protein on an SDS-PAGE gel and blotting the proteins onto a membrane. Thereafter, the appropriate size band corresponding to either protein may be cut from the membrane and used as an immunogen in animals directly, or by first extracting or eluting the protein from the membrane.
• the protein may be isolated by size-exclusion chromatography alone or in combination with other means of isolation and purification.
• the invention also provides methods of producing monoclonal antibodies that specifically bind to homodimeric, heterodimeric, and/or multimeric forms of both IL-17F and IL- 23/pl9.
• These different forms may be purified from cells or from recombinant systems using a variety of well-known techniques for isolating and purifying proteins.
• both IL- 17F and IL-23/pl9 may be isolated based on the apparent molecular weight of the protein by running the protein on an SDS-PAGE gel and blotting the proteins onto a membrane.
• the appropriate size band corresponding to each may be cut from the membrane and used as an immunogen in animals directly, or by first extracting or eluting the protein from the membrane.
• the protein may be isolated by size-exclusion chromatography alone or in combination with other means of isolation and purification.
• animals are generally immunized with either IL- 17 A, IL- 17F, or IL-23 or an immunogenic portion of either.
• the antigen is generally combined with an adjuvant to promote immunogenicity.
• Adjuvants vary according to the species used for immunization.
• adjuvants include, but are not limited to: Freund's complete adjuvant (“FCA”), Freund's incomplete adjuvant (“FIA”), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions), peptides, oil emulsions, keyhole limpet hemocyanin (“KLH”), dinitrophenol (“DNP”), and potentially useful human adjuvants such as Bacille Calmette-Guerin (“BCG”) and corynebacterium parvum.
• FCA Freund's complete adjuvant
• FFA Freund's incomplete adjuvant
• mineral gels e.g., aluminum hydroxide
• surface active substances e.g., lysolecithin, pluronic polyols, polyanions
• peptides e.g., oil emulsions
• KLH keyhole limpet hemocyanin
• DNP dinitrophenol
• BCG
• the dose and immunization regimen will depend on the species of mammal immunized, its immune status, body weight, and/or calculated surface area, etc.
• blood serum is sampled from the immunized mammals and assayed for anti-IL-17 and IL-23/pl9 antibodies using appropriate screening assays as described below, for example.
• a common method for producing humanized antibodies is to graft CDR sequences from a MAb (produced by immunizing a rodent host) onto a human Ig backbone, and transfection of the chimeric genes into Chinese Hamster Ovary (CHO) cells which in turn produce a functional Ab that is secreted by the CHO cells (Shields, R. L., et al. (1995) Anti-IgE monoclonal antibodies that inhibit allergen-specific histamine release. Int Arch. Allergy Immunol. 707:412-413).
• the methods described within this application are also useful for generating genetic alterations within Ig genes or chimeric Igs transfected within host cells such as rodent cell lines, plants, yeast and prokaryotes (Frigerio L, et al. (2000) Assembly, secretion, and vacuolar delivery of a hybrid immunoglobulin in plants. Plant Physiol. 723:1483-1494).
• Splenocytes from immunized animals may be immortalized by fusing the splenocytes (containing the antibody-producing B cells) with an immortal cell line such as a myeloma line.
• myeloma cell line is from the same species as the splenocyte donor.
• the immortal cell line is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
• HAT medium culture medium containing hypoxanthine, aminopterin and thymidine
• the myeloma cells are negative for Epstein-Barr virus (EBV) infection.
• EBV Epstein-Barr virus
• the myeloma cells are HAT-sensitive, EBV negative and Ig expression negative. Any suitable myeloma may be used.
• Murine hybridomas may be generated using mouse myeloma cell lines (e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0- Agl4 myeloma lines). These murine myeloma lines are available from the ATCC. These myeloma cells are fused to the donor splenocytes polyethylene glycol ("PEG”), preferably 1500 molecular weight polyethylene glycol (“PEG 1500"). Hybridoma cells resulting from the fusion are selected in HAT medium which kills unfused and unproductively fused myeloma cells. Unfused splenocytes die over a short period of time in culture. In some embodiments, the myeloma cells do not express immunoglobulin genes.
• PEG polyethylene glycol
• PEG 1500 1500 molecular weight polyethylene glycol
• HAT medium kills unfused and unproductively fused myeloma cells.
• Hybridomas producing a desired antibody which are detected by screening assays such as those described below may be used to produce antibodies in culture or in animals.
• the hybridoma cells may be cultured in a nutrient medium under conditions and for a time sufficient to allow the hybridoma cells to secrete the monoclonal antibodies into the culture medium. These techniques and culture media are well known by those skilled in the art.
• the hybridoma cells may be injected into the peritoneum of an unimmunized animal. The cells proliferate in the peritoneal cavity and secrete the antibody, which accumulates as ascites fluid. The ascites fluid may be withdrawn from the peritoneal cavity with a syringe as a rich source of the monoclonal antibody.
• Hybridomas expressing mouse monoclonal antibodies that cross bind to human IL- 17A and and human IL- 17F were produced using methods similar to those described above were deposited with the American Type Tissue Culture Collection (ATCC; Manassas VA) patent depository as original deposits under the Budapest Treaty and were given the following ATCC Accession No.s: clone 339.15.5.3 (ATCC Patent Deposit Designation PTA-7987, deposited on November 7, 2006); clone 339.15.3.6 (ATCC Patent Deposit Designation PTA-7988, deposited on November 7, 2006); and clone 339.15.6.16 (ATCC Patent Deposit Designation PTA-7989, deposited on November 7, 2006.
• ATCC American Type Tissue Culture Collection
• the resulting transgenic animals are capable of functionally rearranging the transgenic immunoglobulin sequences and producing a repertoire of antibodies of various isotypes encoded by human immunoglobulin genes.
• the B-cells from the transgenic animals are subsequently immortalized by any of a variety of methods, including fusion with an immortalizing cell line (e.g., a myeloma cell).
• the antibodies of the present invention may also be prepared in vitro using a variety of techniques known in the art. For example, but not by way of limitation, fully human monoclonal antibodies against IL-17A or IL- 17F and IL-23/pl9 may be prepared by using in vitro-primed human splenocytes (Boerner et al. (1991) /. Immunol. 147:86-95).
• the antibodies of the invention may be prepared by "repertoire cloning" (Persson et al. (1991) Proc. Nat. Acad. ScL USA 88:2432-2436; and Huang and Stollar (1991) J. Immunol. Methods 141:227-236).
• U.S. Pat. No. 5,798,230 describes preparation of human monoclonal antibodies from human B antibody-producing B cells that are immortalized by infection with an Epstein-Barr virus that expresses Epstein-Barr virus nuclear antigen 2 (EBNA2).
• EBNA2 Epstein-Barr virus nuclear antigen 2
• antibodies of the invention are formed by in vitro immunization of peripheral blood mononuclear cells ("PBMCs"). This may be accomplished by any means known in the art, such as, for example, using methods described in the literature (Zafiropoulos et al. (1997) /. Immunological Methods 200:181-190).
• PBMCs peripheral blood mononuclear cells
• the invention provides a method for inhibiting inflammation in a mammal comprising administering an antagonist of IL-23 and an antagonist of IL- 17A or IL- 17F to the mammal, wherein the antagonist of IL- 17A or IL- 17F can bind IL- 17A or IL- 17F.
• the antagonist of IL-17A or IL- 17F is an antibody or antibody fragment.
• the antibody or antibody fragment is a cross-reactive antibody to IL-17A and IL- 17F.
• the antagonist of IL-23 binds the pl9 subunit.
• the antagonist of IL-23 is an antibody or antibody fragment.
• the invention provides a method for inhibiting inflammation in a mammal comprising administering an antagonist of IL2-3 and an antagonist of IL-17A or IL- 17F, wherein the antagonist of IL- 17A or IL- 17F comprises an antibody or antibody fragment comprising a HCDRl amino acid sequence, a HCDR2 amino acid sequence, and a HCDR3 amino acid sequence of the variable heavy region selected from the group consisting of: the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7987; the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7988; and the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7989 and wherein the variable light region comprises a LCDRl amino acid sequence, a LCDR2 amino acid sequence, and a LCDR3 amino acid sequence of the variable light region selected from the group consisting of: the variable light region of the hybridoma of ATCC Patent Deposit Designation PTA-7987; the variable light
• the antibody or antibody fragment that binds IL-17A or IL- 17F comprises an amino acid sequence of the variable heavy region selected from the group consisting of: a) the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7987; the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7988; and the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7989.
• the antibody or antibody fragment that binds IL- 17A or IL- 17F comprises an amino acid sequence of the variable light region selected from the group consisting of: the variable light region of the hybridoma of ATCC Patent Deposit Designation PTA-7987; the variable light region of the hybridoma of ATCC Patent Deposit Designation PTA-7988; and the variable light region of the hybridoma of ATCC Patent Deposit Designation PTA-7989.
• the antagonist of IL2-3 and the antagonist of IL-17A or IL- 17F are contained on one molecule.
• the invention provides a method for inhibiting inflammation in a mammal comprising administering an antagonist of IL2-3 and an antagonist of IL-17A or IL- 17F, wherein the antagonist of IL- 17A or IL- 17F comprises an antibody or antibody fragment comprising an amino acid sequence of the variable heavy region and an amino acid sequence of the variable light region and wherein the amino acid sequence of the variable heavy region is selected from the group consisting of: the amino acid sequence of the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7987; the amino acid sequence of the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7988; and the amino acid sequence of the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7989 and wherein the variable light region comprises the amino acid sequence of the variable light region selected from the group consisting of: the amino acid sequence of the variable light region of the hybridoma of ATCC Patent Deposit Designation PTA-7987; the amino acid sequence of the variable light region of the
• the antibody or antibody fragment is humanized. Within other aspects the antibody is chimeric. Within other aspects the antibody fragment is selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2.
• the invention provides a method for inhibiting inflammation in a mammal comprising administering an antagonist of IL2-3 and an antagonist of IL-17A or IL- 17F, wherein the antagonist of IL-23 comprises an antibody or antibody fragment that binds the pl9 subunit of IL-23 and wherein the antibody or antibody fragment comprises a HCDRl amino acid sequence, a HCDR2 amino acid sequence, and a HCDR3 amino acid sequence of the amino acid sequence of variable heavy region as shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO
• variable light region of the antibody or antibody fragment that binds the pl9 subunit of IL-23 comprises an amino acid sequence as shown in SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO:
• the invention provides a method of inhibiting inflammation comprising administering an antagonist to a mammal wherein the antagonist is an antibody or antibody fragment that binds the pl9 subunit of IL-23 comprises a variable heavy region and a variable light region and wherein the amino acid sequence of the variable heavy region is shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
• the invention provides a method for inhibiting inflammation in a mammal comprising administering an antagonist of IL-23 and an antagonist of IL- 17A or IL- 17F to the mammal, wherein the antagonist of IL- 17A or IL- 17F can bind IL- 17A or IL- 17F, and wherein the inflammation is associated with a disease selected from the group consisting of multiple sclerosis (MS), chronic inflammation, autoimmune diabetes, rheumatoid arthritis (RA) and other arthritic conditions, asthma, systhemic lupus erythrematosus, psoriasis, Crohn's Disease, ulcerative colitis, irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD).
• MS multiple sclerosis
• RA rheumatoid arthritis
• asthma systhemic lupus erythrematosus
• psoriasis Crohn's Disease
• ulcerative colitis irri
• the invention provides an antibody or antibody fragment that is a single chain antibody.
• the invention provides an antibody or antibody fragment that is a bispecific antibody.
• the invention provides an antibody that is a tascFv, a biscFv, or a BiAb antibody.
• the invention provides a method for inhibiting inflammation in a mammal comprising administering an antagonist of IL-23 and an antagonist of IL- 17A or IL- 17F to the mammal, wherein the antagonist of IL- 17A or IL- 17F can bind IL- 17A or IL- 17F, and wherein the antibody or antibody fragment binds an IL- 17F epitope, wherein said epitope is selected from the group consisting of: a) an epitope comprising amino acid residues 23, 25, 27, 29 and 34 of SEQ ID NO:6; b) an epitope comprising amino acid residues 23-34 of SEQ ID NO:6; c) an epitope comprising amino acid residues 67-73 of SEQ ID NO:6; d) an epitope comprising amino acid residues 79- 85 of SEQ ID NO:6; e) an epitope comprising amino acid residues 146-152 of SEQ ID NO:4; f) an epitope comprising at least
• the invention provides a method for inhibiting inflammation in a mammal comprising administering an antagonist of IL-23 and an antagonist of IL- 17A or IL- 17F to the mammal, wherein the antagonist of IL- 17A or IL- 17F can bind IL- 17A or IL- 17F, and wherein the antibody or antibody fragment binds an IL-17A epitope, wherein said epitope is selected from the group consisting of: a) an epitope comprising amino acid residues 23, 25, 27, 29 and 34 of SEQ ID NO:2; b) an epitope comprising amino acid residues 20-31 of SEQ ID NO:2; c) an epitope comprising amino acid residues 69-75 of SEQ ID NO:2; d) an epitope comprising amino acid residues 81-87 of SEQ ID NO:2; e) an epitope comprising amino acid residues 148- 154 of SEQ ID NO:2; f) an epitope comprising at least
• the invention provides a method for inhibiting inflammation in a mammal comprising administering an antagonist of IL-23 and an antagonist of IL- 17A or IL- 17F to the mammal, wherein the antagonist of IL- 17A or IL- 17F can bind IL- 17A or IL- 17F, and wherein the antibody or antibody fragment binds an IL-1723 epitope, wherein said epitope is selected from the group consisting of: a) an epitope comprising amino adic residues 55 to 66 of SEQ ID NO: 4; b) an epitope comprising amino adic residues 74 to 85 of SEQ ID NO: 4; and c) an epitope comprising amino adic residues 155 to 164 of SEQ ID NO: 4.
• the antibody or antibody fragment also comprises a PEG moiety.
• the antibody or antibody fragment also comprises an Fc moiety.
• the antibody or antibody fragment is bivalent, trivalent, or tetravalent.
• the invention provides a method for treating disease characterized by elevated expression of IL-17A, IL-17F or IL-23 in a mammalian subject, comprising administering to said subject an effective amount of an antagonist of IL-17A, IL-17F and IL-23.
• the invention provides an antagonist of IL-23 and of IL-17A or IL- 17F compising a an antibody or antibody fragment that binds the pl9 subunit of IL-23 as shown in SEQ ID NO: 4 and that comprises an antibody or antibody fragment that binds IL-17A as shown in SEQ ID NO: 2 or that binds IL-17F as shown in SEQ ID NO: 6.
• the antibody or antibody fragment that binds IL-17A is a cross-reactive antibody that binds IL- 17F.
• the invention provides an antagonist of IL-23 and of IL- 17 A or IL- 17F compising an antibody or antibody fragment that binds IL- 17A or IL- 17F wherein the antibody or antibody fragment comprises a HCDRl amino acid sequence, a HCDR2 amino acid sequence, and a HCDR3 amino acid sequence of the variable heavy region selected from the group consisting of: a) the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7987; b) the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7988; and c) the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7989 and wherein the variable light region comprises a LCDRl amino acid sequence, a LCDR2 amino acid sequence, and a LCDR3 amino acid sequence of the variable light region selected from the group consisting of: d) the variable light region of the hybridoma of ATCC Patent Deposit Designation PTA-7987; e)
• the antibody or antibody fragment that binds IL- 17A or IL- 17F comprises an amino acid sequence of the variable heavy region selected from the group consisting of: a. the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7987; b.the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7988; and c. the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7989.
• the antibody or antibody fragment that binds IL-17A or IL- 17F comprises an amino acid sequence of the variable light region selected from the group consisting of: a.
• the antagonist of IL2-3 and the antagonist of IL-17A or IL-17F are contained on one molecule.
• the invention provides an antagonist of IL-23 and of IL-17A or IL-17F, wherin the antibody or antibody fragment that binds IL- 17A or IL- 17F comprises an amino acid sequence of the variable heavy region and an amino acid sequence of the variable light region and wherein the amino acid sequence of the variable heavy region is selected from the group consisting of: the amino acid sequence of the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA- 7987; the amino acid sequence of the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7988; and the amino acid sequence of the variable heavy region of the hybridoma of ATCC Patent Deposit Designation PTA-7989 and wherein the variable light region comprises the amino acid sequence of the variable light region selected from the group consisting of: the amino acid sequence of the variable light region of the hybridoma of ATCC Patent Deposit Designation PTA-7987; the amino acid sequence of the variable light region of the hybridoma of ATCC Patent Deposit Designation PTA-7988; and
• the antibody or antibody fragment is humanized. Within an aspect the antibody or antibody fragment is chimeric. Within an aspect the antibody fragment is selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2.
• the invention provides an antagonist of IL-23 and of IL-17A or IL- 17F, wherein the antagonist comprises an antibody or antibody fragment that binds the pl9 subunit of IL-23 and wherein the antibody or antibody fragment comprises an amino acid sequence of a variable heavy region as shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 8
• the invention provides an antagonist of IL-23 and of IL-17A or IL- 17F, wherein the antagonist comprises an antibody or antibody fragment that binds the pl9 subunit of IL-23 wherein the antibody or antibody fragment comprises an amino acid sequence of the variable light region as shown in SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:
• the invention provides an antagonist of IL-23 and of IL-17A or IL- 17F, wherein the antagonist comprises an antibody or antibody fragment that binds the pl9 subunit of IL-23 and comprises an amino acid sequence of aa variable heavy region and an amino acid sequence of a variable light region and wherein the amino acid sequence of the variable heavy region is shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO:
• the invention provides an antagonist of IL-23 and of IL-17A or IL- 17F, wherein antagonist can be used to treat inflammation is associated with a disease selected from the group consisting of multiple sclerosis (MS), chronic inflammation, autoimmune diabetes, rheumatoid arthritis (RA) and other arthritic conditions, asthma, systhemic lupus erythrematosus, psoriasis, Crohn's Disease, ulcerative colitis, irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD).
• MS multiple sclerosis
• RA rheumatoid arthritis
• asthma systhemic lupus erythrematosus
• psoriasis psoriasis
• Crohn's Disease ulcerative colitis
• IBS irritable bowel syndrome
• IBD inflammatory bowel disease
• the invention provides an antagonist of IL-23 and of IL-17A or IL- 17F, wherein the antagonist comprises an antibody or antibody fragment that binds an IL- 17F epitope, wherein said epitope is selected from the group consisting of: a) an epitope comprising amino acid residues 23, 25, 27, 29 and 34 of SEQ ID NO:6; b) an epitope comprising g amino acid residues 23-34 of SEQ ID NO:6; c) an epitope comprising amino acid residues 67-73 of SEQ ID NO:6; d) an epitope comprising amino acid residues 79- 85 of SEQ ID NO:6; e) an epitope comprising amino acid residues 146-152 of SEQ ID NO:6; f) an epitope comprising at least one amino acid residue from residues 105-109 and at least one amino acid residue from residues 147-152 of SEQ ID NO:6; g) an epitope comprising at least one amino acid residue from residues,
• the invention provides an antagonist of IL-23 and of IL-17A or IL- 17F, wherein the antagonist comprises an antibody or antibody fragment that binds an IL- 17A epitope, wherein said epitope is selected from the group consisting of: a) an epitope comprising amino acid residues 23, 25, 27, 29 and 34 of SEQ ID NO:2; b) an epitope comprising amino acid residues 20-31 of SEQ ID NO:2; c) an epitope comprising amino acid residues 69-75 of SEQ ID NO:2; d) an epitope comprising amino acid residues 81-87 of SEQ ID NO:2; e) an epitope comprising amino acid residues 148- 154 of SEQ ID NO:2; f) an epitope comprising at least one amino acid residue from residues 107-111 and at least one amino acid residue from residues 149-154 of SEQ ID NO:2; g) an epitope comprising at least one amino acid residue from residues
• the invention provides an antagonist of IL-23 and of IL-17A or IL- 17F, wherein the antagonist comprises an antibody or antibody fragment that binds an IL-23pl9 epitope selected from the group consisting of: a) an epitope comprising amino adic residues 55 to 66 of SEQ ID NO: 4; b) an epitope comprising amino adic residues 74 to 85 of SEQ ID NO: 4; and c) an epitope comprising amino adic residues 155 to 164 of SEQ ID NO: 4.
• the invention provides an antagonist of IL-23 and of IL-17A or IL- 17F, wherein the antagonist comprises an antibody or antibody fragment comprises a PEG moiety.
• the invention provides an antagonist of IL-23 and of IL- 17A or IL- 17F, wherein the antagonist comprises an Fc moiety.
• the invention provides an antagonist of IL-23 and of IL-17A or IL- 17F, wherein the antagonist is bivalent, trivalent, or tetravalent.
• bispecific and single chain antibodies that bind both IL- 17A or IL-17F and IL-23 are made.
• One method comprises fusing hybridoma cells that secrete a monoclonal antibody that cross binds with IL-17A and IL- 17F, with hybridoma cells that secrete a monoclonal antibody that binds IL-23/pl9, thereby preparing a hybrid hybridoma that secretes a bispecific monoclonal antibody that cross binds with IL- 17A or IL- 17F and also binds IL-23pl9 monclonal antibody.
• the method comprises fusing hybridoma cells that secrete an antagonistic (or agonistic) IL- 17A or IL- 17F MAb, with hybridoma cells that secrete an antagonistic (or agonistic) IL-23/pl9 MAb.
• Conventional techniques for conducting such a fusion, and for isolating the desired hybrid hybridoma include those described elsewhere herein, and those illustrated in the examples below.
• U.S. Pat. No. 6,060,285 discloses a process for the production of bispecific antibodies, in which at least the genes for the light chain and the variable portion of the heavy chain of an antibody having a first specificity are transfected into a hybridoma cell secreting an antibody having a second specificity.
• bispecific antibodies are produced, and may be isolated by various means known in the art.
• Single chain antibodies may be formed by linking heavy and light chain variable region (Fv region) fragments via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain.
• Fv region heavy and light chain variable region
• Such single-chain Fvs (scFvs) have been prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable region polypeptides (V L and V H )- The resulting antibody fragments can form dimers or higher oligomers, depending on such factors as the length of a flexible linker between the two variable domains (Kortt et al., Protein Engineering 10:423, 1997).
• two or more scFvs are joined by use of a chemical cross- linking agent.
• Single chain antibodies against IL- 17A or IL- 17F and IL-23 may be concatamerized in either order (i.e., anti-IL-17A-anti-IL-23 or anti-IL-23-anti-IL-17A).
• starting materials for preparing a bispecific antibody include an antagonistic (or agonistic) single chain antibody directed against IL- 17A or against IL- 17F and an antagonistic (or agonistic) single chain antibody directed against IL-23/pl9.
• the scFv entities that bind IL-17A or IL- 17F and IL-23pl9 can be oriented with the variable light region either amino terminal to the variable heavy region or carboxylterminal to it.
• tandem scFvs can be prepared in a number of configurations, such that each target, i.e, IL- 17A or IL- 17F and IL-23pl9 can be bound by its respective variable regions.
• the construct for a tandem scFV molecule can be prepared such that the variable light region and variable heavy region of one antibody can be interspersed with the variable light and variable heavy regions of the other antibody as long as the variable regions are able to bind the targets.
• Tandem scFv molecules that bind both targets can be prepared with a linker between the scFv entites, including a GIy- Ser linker comprising a series of glycine and serine residues and can also include additional amino acids.
• U.S. Pat. No. 5,582,996 discloses the use of complementary interactive domains (such as leucine zipper moieties or other lock and key interactive domain structures) to facilitate heterodimer formation in the production of bispecific antibodies.
• the complementary interactive domain(s) may be inserted between an Fab fragment and another portion of a heavy chain (i.e., C.sub.Hl or C.sub.H2 regions of the heavy chain).
• the use of two different Fab fragments and complementary interactive domains that preferentially heterodimerize will result in bispecific antibody molecules. Cysteine residues may be introduced into the complementary interactive domains to allow disulphide bonding between the complementary interactive domains and stabilize the resulting bispecific antibodies.
• Tetravalent, bispecific molecules can be prepared by fusion of DNA encoding the heavy chain of an F(ab') 2 fragment of an antibody with either DNA encoding the heavy chain of a second F(ab') 2 molecule (in which the CHl domain is replaced by a CH3 domain), or with DNA encoding a single chain Fv fragment of an antibody, as described in U.S. Pat. No. 5,959,083. Expression of the resultant fusion genes in mammalian cells, together with the genes for the corresponding light chains, yields tetravalent bispecific molecules having specificity for selected antigens.
• Bispecific antibodies can also be produced as described in U.S. Pat. No. 5,807,706, which is incorporated by reference herein.
• the method involves introducing a protuberance in a first polypeptide and a corresponding cavity in a second polypeptide, polypeptides interface.
• the protuberance and cavity are positioned so as to promote heteromultimer formation and hinder homomultimer formation.
• the protuberance is created by replacing amino acids having small side chains with amino acids having larger side chains.
• the cavity is created by the opposite approach, i.e., replacing amino acids having relatively large side chains with amino acids having smaller side chains.
• the protuberance and cavity can be generated by conventional methods for making amino acid substitutions in polypeptides.
• a nucleic acid encoding a polypeptide may be altered by conventional in vitro mutagenesis techniques.
• a polypeptide incorporating a desired amino acid substitution may be prepared by peptide synthesis. Amino acids chosen for substitution are located at the interface between the first and second polypeptides.
• Screening for antibodies that specifically bind to IL-17A or IL-17F and IL-23/pl9 may be accomplished using an enzyme-linked immunosorbent assay (ELISA) in which microtiter plates are coated with IL-17A or IL-17F and IL-23 (or pl9 alone).
• ELISA enzyme-linked immunosorbent assay
• antibodies that bind both IL- 17A or IL- 17F and IL-23/pl9 from positively reacting clones can be further screened for reactivity in an ELISA-based assay using microtiter plates coated with the other forms IL-17 and IL-23/pl9, or other IL-17 family members.
• Clones that produce antibodies that are reactive to another forms or family members are eliminated, and clones that produce antibodies that are reactive to both IL- 17A or IL- 17F and IL-23/pl9 may be selected for further expansion.
• Confirmation of reactivity of the antibodies to both IL-17A or IL- 17F and IL-23/pl9 may be accomplished, for example, using a Western Blot assay in which protein from ovarian, breast, renal, colorectal, lung, endometrial, or brain cancer cells and purified FR-. alpha, and other folate receptor isoforms are run on an SDS-PAGE gel, and subsequently are blotted onto a membrane. The membrane may then be probed with the putative anti-FR-. alpha, antibodies.
• Reactivity with both IL-17A or IL-17F and IL- 23/pl9 and not another family member confirms specificity of reactivity for IL-17 A/F cross-binding antibodies and IL-23/pl9.
• Antibody-producing cells of the invention include any insect expression cell line known, such as for example, Spodoptera frugiperda cells.
• the expression cell lines may also be yeast cell lines, such as, for example, Saccharomyces cerevisiae and Schizosaccharomyces pombe cells.
• the expression cells may also be mammalian cells such as, for example, hybridoma cells (e.g., NSO cells), Chinese hamster ovary cells, baby hamster kidney cells, human embryonic kidney line 293, normal dog kidney cell lines, normal cat kidney cell lines, monkey kidney cells, African green monkey kidney cells, COS cells, and non-tumorigenic mouse myoblast G8 cells, fibroblast cell lines, myeloma cell lines, mouse NIH/3T3 cells, LMTK31 cells, mouse Sertoli cells, human cervical carcinoma cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cells, TRI cells, MRC 5 cells, and FS4 cells.
• hybridoma cells e.g., NSO cells
• Chinese hamster ovary cells e.g., baby hamster kidney cells
• human embryonic kidney line 293 normal dog kidney cell lines
• normal cat kidney cell lines monkey kidney cells
• African green monkey kidney cells African green monkey kidney cells
• COS cells and non
• the antibody-producing cells of the invention produce antibodies that specifically bind to IL- 17A or IL- 17F and IL-23/pl9 (either singly or together as with a bispecific antibody or scFV).
• the cells preferably are substantially free of IL-17A, IL-17F and IL-23 binding competitors.
• the antibody-producing cells comprise less than about 10%, preferably less than about 5%, more preferably less than about 1%, more preferably less than about 0.5%, more preferably less than about 0.1%, and most preferably 0% by weight IL- 17A, IL-17F, orIL-23 binding competitors.
• the antibodies produced by the antibody-producing cells are substantially free of IL-17A, IL-17F, and IL-23 competitors.
• antibodies produced by the antibody-producing cells comprise less than about 10%, preferably less than about 5%, more preferably less than about 1%, more preferably less than about 0.5%, more preferably less than about 0.1%, and most preferably 0% by weight both IL-17 and IL-23 binding competitors.
• methods for antibody purification include filtration, affinity column chromatography, cation exchange chromatography, anion exchange chromatography, and concentration.
• the filtration step preferably comprises ultrafiltration, and more preferably ultrafiltration and diafiltration. Filtration is preferably performed at least about 5-50 times, more preferably 10 to 30 times, and most preferably 14 to 27 times.
• Affinity column chromatography may be performed using, for example, PROSEP Affinity Chromatography (Millipore, Billerica, Mass.).
• the affinity chromatography step comprises PROSEP-VA column chromatography. Eluate may be washed in a solvent detergent.
• Cation exchange chromatography may include, for example, SP- Sepharose Cation Exchange Chromatography.
• Anion exchange chromatography may include, for example but not limited to, Q-Sepharose Fast Flow Anion Exchange.
• the anion exchange step is preferably non-binding, thereby allowing removal of contaminants including DNA and BSA.
• the antibody product is preferably nanofiltered, for example, using a Pall DV 20 Nanofilter.
• the antibody product may be concentrated, for example, using ultrafiltration and diafiltration.
• the method may further comprise a step of size exclusion chromatography to remove aggregates.
• Antibodies that bind to both IL-17A or IL-17F and IL-23 can be used to modulate the immune system by binding IL- 17A or IL- 17F and IL-23/pl9 (either singly or together as with a bispecific antibody or scFV), and thus, preventing the binding of IL-17A or IL- 17F with either IL- 17RA or IL- 17RC and IL-23 with its receptor or any other receptor that they may bind.
• the antibodies of the invention can also be used to modulate the immune system by inhibiting the binding of both IL-17A or IL- 17F with the endogenous IL- 17RA and/or IL- 17RC receptor and IL-23 with its endogenous receptor.
• the antibodies of the invention can be also used to treat a subject which produces an excess of either IL-17A or IL-17F and/or IL-23.
• suitable subjects include mammals, such as humans.
• the antibodies of the invention are useful in binding, blocking, inhibiting, reducing, antagonizing or neutralizing of both IL-17A or IL- 17F and IL-23 (either singly or together as with a bispecific antibody or scFV), in the treatment of inflammation and inflammatory dieases such as multiple sclerosis, cancer (characterized by IL-17A or IL-17F and IL-23 expression), psoriasis, psoriatic arthritis, atopic dermatitis, inflammatory skin conditions, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's Disease, diverticulosis, asthma, pancreatitis, type I diabetes (IDDM), pancreatic cancer, pancreatitis, Graves Disease, colon and intestinal cancer, autoimmune disease, seps
• the antibodies of the invention bind to, blocks, inhibits, reduces, antagonizes or neutralizes IL-23 (via pl9) and IL-17A IL-17F either singly or together as with a bispecific antibody or scFV), in vivo.
• antibodies of the invention are useful to:
• IL- 17 A or IL- 17F or IL-23 Block, inhibit, reduce, antagonize or neutralize signaling via IL- 17 A or IL- 17F or IL-23 in the treatment of autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), multiple sclerosis (MS), systemic Lupus erythematosus (SLE), myasthenia gravis, rheumatoid arthritis, IBS and IBD to prevent or inhibit signaling in immune cells (e.g. lymphocytes, monocytes, leukocytes) via their receptors (e.g. IL-17RA and IL-17RC).
• IDDM insulin-dependent diabetes mellitus
• MS multiple sclerosis
• SLE systemic Lupus erythematosus
• myasthenia gravis myasthenia gravis
• IBS and IBD to prevent or inhibit signaling in immune cells (e.g. lymphocytes, monocytes, leukocytes) via their receptors (e.g.
• Blocking, inhibiting, reducing, or antagonizing signaling via IL-17RA and IL-17RC, using the antibodies of the present invention may also benefit diseases of the pancreas, kidney, pituitary and neuronal cells.
• IDDM non-insulin dependent diabetes mellitus
• pancreatitis pancreatic carcinoma
• Anti-IL-17A and IL-17F neutralizing and monoclonal antibodies may signal lymphocytes or other immune cells to differentiate, alter proliferation, or change production of cytokines or cell surface proteins that ameliorate autoimmunity. Specifically, modulation of a T- helper cell response to an alternate pattern of cytokine secretion may deviate an autoimmune response to ameliorate disease (Smith JA et al., J. Immunol. 160:4841-4849, 1998).
• agonistic antibodies may be used to signal, deplete and deviate immune cells involved in asthma, allergy and atopoic disease.
• Signaling via IL- 17RA and IL- 17RC may also benefit diseases of the pancreas, kidney, pituitary and neuronal cells.
• IDDM, NIDDM, pancreatitis, and pancreatic carcinoma may benefit.
• the antibodies described herein can be used to bind, block, inhibit, reduce, antagonize or neutralize IL-23 and IL- 17 A or IL- 17F activity, either singly or together as with a bispecific antibody or scFV, in the treatment of multiple sclerosis, cancer, autoimmune disease, atopic disease, NIDDM, pancreatitis and kidney dysfunction as described above.
• the antibodies of the present invention are useful as antagonists of IL- 17 A or IL- 17F or IL-23. Such antagonistic effects can be achieved by direct neutralization or binding of IL- 17 A or IL- 17F and IL-23 (via pi 9).
• Antibodies herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
• antibodies or binding polypeptides which recognize IL-17A or IL-17F or IL-23 can be used to identify or treat tissues or organs that express a corresponding anticomplementary molecule.
• antibodies to IL- 17 or IL-23 or bioactive fragments or portions thereof can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express these cytokines IL-17 or IL-23 -expressing cancers.
• Suitable detectable molecules may be directly or indirectly attached to the antagonists of the present invention, such as "binding polypeptides,” (including binding peptides disclosed above), antibodies, or bioactive fragments or portions thereof.
• Suitable detectable molecules include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
• Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance). Binding polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin.
• the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the binding polypeptide or antibody portion.
• biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
• binding polypeptide-toxin fusion proteins or antibody- toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues).
• a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest.
• the anticomplementary molecule can be conjugated to a detectable or cytotoxic molecule.
• domain- complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue- specific delivery of generic anti-complementary-detectable/ cytotoxic molecule conjugates.
• Inflammation is a protective response by an organism to fend off an invading agent. Inflammation is a cascading event that involves many cellular and humoral mediators. On one hand, suppression of inflammatory responses can leave a host immunocompromised; however, if left unchecked, inflammation can lead to serious complications including chronic inflammatory diseases (e.g., psoriasis, arthritis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and the like), septic shock and multiple organ failure. Importantly, these diverse disease states share common inflammatory mediators. The collective diseases that are characterized by inflammation have a large impact on human morbidity and mortality.
• anti-inflammatory proteins such as antagonists to IL-17A or IL-17F and IL-23/pl9, such as IL-17A or IL-17F and IL-23/pl9 antibodies, could have crucial therapeutic potential for a vast number of human and animal diseases, from asthma and allergy to autoimmunity, cancers, and septic shock.
• rheumatoid arthritis is a systemic disease that affects the entire body and is one of the most common forms of arthritis. It is characterized by the inflammation of the membrane lining the joint, which causes pain, stiffness, warmth, redness and swelling. Inflammatory cells release enzymes that may digest bone and cartilage.
• the inflamed joint lining can invade and damage bone and cartilage leading to joint deterioration and severe pain amongst other physiologic effects.
• the involved joint can lose its shape and alignment, resulting in pain and loss of movement.
• Rheumatoid arthritis is an immune-mediated disease particularly characterized by inflammation and subsequent tissue damage leading to severe disability and increased mortality.
• a variety of cytokines are produced locally in the rheumatoid joints.
• Numerous studies have demonstrated that IL-I and TNF-alpha, two prototypic pro-inflammatory cytokines, play an important role in the mechanisms involved in synovial inflammation and in progressive joint destruction. Indeed, the administration of TNF-alpha and IL-I inhibitors in patients with RA has led to a dramatic improvement of clinical and biological signs of inflammation and a reduction of radiological signs of bone erosion and cartilage destruction.
• IL-17 and IL-23/pl9 are overexpressed in the synovium and synovial fibroblasts of patients with rheumatoid arthritis compared to individuals without rheumatoid arthritis.
• IL-17 and IL-23/pl9 have been demonstrated to promote matrix degradation and enhance the expression of inflammatory, matrix-destructive cytokines when added to synovium/synoviocyte cultures.
• a molecule that binds or inhibits IL-17 or IL-23 activity such as the antagonists of the present invention, could serve as a valuable therapeutic to reduce inflammation in rheumatoid arthritis, and other arthritic diseases.
• CIA collagen-induced arthritis
• mice develop chronic inflammatory arthritis that closely resembles human rheumatoid arthritis. Since CIA shares similar immunological and pathological features with RA, this makes it an ideal model for screening potential human antiinflammatory compounds.
• the CIA model is a well-known model in mice that depends on both an immune response, and an inflammatory response, in order to occur.
• the immune response comprises the interaction of B-cells and CD4+ T-cells in response to collagen, which is given as antigen, and leads to the production of anti-collagen antibodies.
• the inflammatory phase is the result of tissue responses from mediators of inflammation, as a consequence of some of these antibodies cross- reacting to the mouse's native collagen and activating the complement cascade.
• An advantage in using the CIA model is that the basic mechanisms of pathogenesis are known.
• the relevant T-cell and B-cell epitopes on type II collagen have been identified, and various immunological (e.g., delayed-type hypersensitivity and anti-collagen antibody) and inflammatory (e.g., cytokines, chemokines, and matrix-degrading enzymes) parameters relating to immune-mediated arthritis have been determined, and can thus be used to assess test compound efficacy in the CIA model (Wooley, Curr. Opin. Rheum.
• antagonists of the present invention can be used to neutralize IL-17 and/or IL-23 (via pl9) in the treatment of specific human diseases such as arthritis, psoriasis, psoriatic arthritis, endotoxemia, inflammatory bowel disease (IBD), IBS, colitis, and other inflammatory conditions disclosed herein.
• specific human diseases such as arthritis, psoriasis, psoriatic arthritis, endotoxemia, inflammatory bowel disease (IBD), IBS, colitis, and other inflammatory conditions disclosed herein.
• the injection of 10 - 200 ug of an anti-IL-17 and anti-IL-23/pl9 per mouse can significantly reduce the disease score (paw score, incident of inflammation, or disease).
• the disease score paw score, incident of inflammation, or disease.
• antagonists of the present invention can be efficacious in preventing rheumatoid arthritis, as well as preventing its progression.
• Endotoxemia is a severe condition commonly resulting from infectious agents such as bacteria and other infectious disease agents, sepsis, toxic shock syndrome, or in immunocompromised patients subjected to opportunistic infections, and the like.
• Therapeutically useful of anti-inflammatory proteins, such as antibodies of the invention could aid in preventing and treating endotoxemia in humans and animals. Such antibodies could serve as a valuable therapeutic to reduce inflammation and pathological effects in endotoxemia.
• IBD Inflammatory Bowel Disease
• Ulcerative colitis colon and rectum
• Crohn's Disease small and large intestine
• This review summarizes current knowledge regarding the role of immune-inflammatory mediators in the pathogenesis of inflammatory bowel disease.
• Crohn's disease and ulcerative colitis are immunologically distinct entities.
• Crohn's disease is associated with a ThI T cell-mediated response, characterized by enhanced production of interferon- [gamma] and tumor necrosis factor-[alpha].
• Interleukin (IL)-12 and, possibly, IL-23 govern the ThI cell differentiation, but optimal induction and stabilization of polarized ThI cells would require additional cytokines, such as IL-15, IL- 18 and IL-21.
• IL-15 interferon- [gamma]
• IL-23 Interleukin-23
• cytokines such as IL-15, IL- 18 and IL-21.
• ulcerative colitis the local immune response is less polarized, but it is characterized by CDl -reactive natural killer T cell production of IL-13. Beyond these differences, Crohn's disease and ulcerative colitis share important end-stage effector pathways of intestinal injury, which are mediated by an active cross-talk between immune and non-immune mucosal cells.
• IL-17 and IL-23 are both overexpressed in intestines and/or serum from humans with IBD and in mouse models of IBD. Moreover, neutralization of IL-17 and/or IL-23/pl9 can reduced disease symptoms and pathology in animals models of IBD (Nielson et al, Scand J Gastroenterol. 38:180 (2003); Schmidt et al, Inflamm. Bowel Dis. 11:16 (2005); Fuss et al, Inflamm Bowel Dis. 12:9 (2006)). Moreover, neutralization of IL- 17 and/or IL-23/pl9 can reduce disease symptoms and pathology in animals models of IBD (Yen et al, J. Clin. Invest. ii6:1310 (2006); Zhang et al, Inflamm Bowel Dis. 72:382 (2006)).
• Ulcerative colitis is an inflammatory disease of the large intestine, commonly called the colon, characterized by inflammation and ulceration of the mucosa or innermost lining of the colon. This inflammation causes the colon to empty frequently, resulting in diarrhea. Symptoms include loosening of the stool and associated abdominal cramping, fever and weight loss.
• TNBS 2,4,6-trinitrobenesulfonic acid/ethanol
• DSS dextran sulfate sodium
• Another colitis model uses dextran sulfate sodium (DSS), which induces an acute colitis manifested by bloody diarrhea, weight loss, shortening of the colon and mucosal ulceration with neutrophil infiltration.
• DSS-induced colitis is characterized histologically by infiltration of inflammatory cells into the lamina intestinal, with lymphoid hyperplasia, focal crypt damage, and epithelial ulceration. These changes are thought to develop due to a toxic effect of DSS on the epithelium and by phagocytosis of lamina limba cells and production of TNF-alpha and IFN- gamma.
• DSS is regarded as a T cell-independent model because it is observed in T cell-deficient animals such as SCID mice.
• Psoriasis is a chronic skin condition that affects more than seven million Americans. Psoriasis occurs when new skin cells grow abnormally, resulting in inflamed, swollen, and scaly patches of skin where the old skin has not shed quickly enough. Plaque psoriasis, the most common form, is characterized by inflamed patches of skin ("lesions") topped with silvery white scales. Psoriasis may be limited to a few plaques or involve moderate to extensive areas of skin, appearing most commonly on the scalp, knees, elbows and trunk. Although it is highly visible, psoriasis is not a contagious disease. The pathogenesis of the diseases involves chronic inflammation of the affected tissues.
• IL-17 and IL-23 are both overexpressed in psoriatic skin compared to non-psoriatic skin (Li et al, J Huazhong Univ Sci Technolog Med ScL 24:294 (2004); Piskin et al, J Immunol. 776:1908 (2006)). Therefore, antagonists of the present invention could serve as a valuable therapeutic to reduce inflammation and pathological effects in psoriasis, other inflammatory skin diseases, skin and mucosal allergies, and related diseases.
• Psoriasis is a T-cell mediated inflammatory disorder of the skin that can cause considerable discomfort. It is a disease for which there is no cure and affects people of all ages. Psoriasis affects approximately two percent of the populations of European and North America. Although individuals with mild psoriasis can often control their disease with topical agents, more than one million patients worldwide require ultraviolet or systemic immunosuppressive therapy. Unfortunately, the inconvenience and risks of ultraviolet radiation and the toxicities of many therapies limit their long-term use. Moreover, patients usually have recurrence of psoriasis, and in some cases rebound, shortly after stopping immunosuppressive therapy.
• Antibodies that bind IL-17A and IL- 17F may also be used within diagnostic systems for the detection of circulating levels of IL- 17F or IL- 17 A, and in the detection of IL- 17A and/or IL- 17F associated with acute phase inflammatory response. Elevated or depressed levels of ligand or receptor polypeptides may be indicative of pathological conditions, including inflammation or cancer. IL-17A and IL-17F are known to induce associated acute phase inflammatory response. Moreover, detection of acute phase proteins or molecules such as IL-17A or IL- 17F can be indicative of a chronic inflammatory condition in certain disease states (e.g., asthma, psoriasis, rheumatoid arthritis, colitis, IBD). Detection of such conditions serves to aid in disease diagnosis as well as help a physician in choosing proper therapy.
• a chronic inflammatory condition in certain disease states (e.g., asthma, psoriasis, rheumatoid arthritis, colitis, I
• the activity of antagonists of the present invention on inflammatory tissue derived from human psoriatic lesions can be measured in vivo using a severe combined immune deficient (SCID) mouse model.
• SCID severe combined immune deficient
• xenograft models Several mouse models have been developed in which human cells are implanted into immunodeficient mice (collectively referred to as xenograft models); see, for example, Cattan AR, Douglas E, Leuk. Res. 18:513-22 (1994) and Flavell, DJ, Hematological Oncology 74:67-82 (1996).
• xenograft models see, for example, Cattan AR, Douglas E, Leuk. Res. 18:513-22 (1994) and Flavell, DJ, Hematological Oncology 74:67-82 (1996).
• human psoriatic skin tissue is implanted into the SCID mouse model, and challenged with an appropriate antagonist.
• psoriasis animal models in ther art may be used to evaluate the present antagonists, such as human psoriatic skin grafts implanted into AGR129 mouse model, and challenged with an appropriate antagonist (e.g., see, Boyman, O. et al., J. Exp. Med. Online publication #20031482, 2004, incorporated hereing by reference).
• IL-17/IL-23 antibodies or binding peptides that bind, block, inhibit, reduce, antagonize or neutralize the activity of IL-17, IL-23 or both IL-17 and IL-23 are preferred antagonists.
• tissues or cells derived from human colitis, IBD, arthritis, or other inflammatory lestions can be used in the SCID model to assess the antiinflammatory properties of the IL-17 and IL-23 antagonists described herein.
• Inflammation may also be monitored over time using well-known methods such as flow cytometry (or PCR) to quantitate the number of inflammatory or lesional cells present in a sample, score (weight loss, diarrhea, rectal bleeding, colon length) for IBD.
• flow cytometry or PCR
• therapeutic strategies appropriate for testing in such a model include direct treatment using IL- 17 and IL-23 antagonists (singly or together), or related conjugates or antagonists based on the disrupting interaction of IL- 17 and IL-23 with their receptors.
• Psoriasis is a chronic inflammatory skin disease that is associated with hyperplastic epidermal keratinocytes and infiltrating mononuclear cells, including CD4+ memory T cells, neutrophils and macrophages (Christophers, Int. Arch. Allergy Immunol., 110:199, 1996). It is currently believed that environmental antigens play a significant role in initiating and contributing to the pathology of the disease. However, it is the loss of tolerance to self-antigens that is thought to mediate the pathology of psoriasis. Dendritic cells and CD4+ T cells are thought to play an important role in antigen presentation and recognition that mediate the immune response leading to the pathology.
• AD is a common chronic inflammatory disease that is characterized by hyperactivated cytokines of the helper T cell subset 2 (Th2). Although the exact etiology of AD is unknown, multiple factors have been implicated, including hyperactive Th2 immune responses, autoimmunity, infection, allergens, and genetic predisposition. Key features of the disease include xerosis (dryness of the skin), pruritus (itchiness of the skin), conjunctivitis, inflammatory skin lesions, Staphylococcus aureus infection, elevated blood eosinophilia, elevation of serum IgE and IgGl, and chronic dermatitis with T cell, mast cell, macrophage and eosinophil infiltration. Colonization or infection with S. aureus has been recognized to exacerbate AD and perpetuate chronicity of this skin disease.
• Th2 helper T cell subset 2
• AD Alzheimer's disease
• IL- 17 and IL-23 via pi 9
• specific human diseases such as atoptic dermatitis, inflammatory skin conditions, and other inflammatory conditions disclosed herein.
• IL- 17 plays an important role in allergen-induced T cell activation and neutrophilic influx in the airways.
• the receptor for IL-17 is expressed in the airways (Yao, et al. Immunity 3:811 (1995)) and IL-17 mediated neutrophil recruitment in allergic asthma is largely induced by the chemoattractant IL-8, GRO-D and macrophage inflammatory protein-2 (MIP-2) produced by IL-17 stimulated human bronchial epithelial cells (HBECs) and human bronichial fibroblasts ( Yao, et al. / Immunol 155:5483 (1995)); Molet, et al. / Allergy Clin Immunol 108:430 (2001)).
• MIP-2 macrophage inflammatory protein-2
• IL-17 also stimulates HBECs to release IL-6, a neutrophil-activating factor ( Fossiez, et al, / Exp Med 183:2593 (1996), and Linden, et al. Int Arch Allergy Immunol 126:179 (2001)) and has been shown to synergize with TNF-alpha to prolong the survival of human neutrophils in vitro (Laan, et al. Eur Respir J 21:387 (2003)).
• IL-6 a neutrophil-activating factor
• IL-17 is capable of amplifying the inflammatory responses in asthma by its ability to enhance the secretion of cytokines implicated in airway remodeling such as the profibrotic cytokines, IL-6 and IL-I l and inflammatory mediators granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) (Molet, et al. J Allergy Clin Immunol 108:430 (2001)).
• G-CSF granulocyte colony-stimulating factor
• GM-CSF granulocyte macrophage colony-stimulating factor
• IL-17 is likely to play a significant role in asthma.
• IL-23 is important in the maintenance and differentiation of IL-17 producing cells (e.g. Thl7 cells), IL-23 is also likely to play a role in asthma.
• Patients with mild asthma display a detectable increase in the local concentration of free, soluble IL-17 protein (Molet, et al.
• Anti-IL-17 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- 17 may be involved in determining the balance between neutrophil and eosinophil accumulation following antigen insult Id..
• IL-17F is most closely related to IL-17A.
• the biological activities mediated by IL- 17F are similar to those of IL- 17 A, 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 IL-2, transforming growth factor (TGF)- ⁇ , and monocyte chemoattractant protein (MCP) in endothelial cells Starnes, et al. J Immunol 167:4137 (2001).
• allergen challenge can increase local IL-17F in patients with allergic asthma Kawaguchi, et al.
• antagonists to IL- 17 and/or IL-23 activity to inhibit IL-17 or and/or IL-23 signalling to induce 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-17 and/or IL-23 stimulation. If the addition of antagonists to IL-17 and/or IL-23 activity markedly reduces the production and expression of inflammatory mediators, it would be expected to be efficacious in inflammatory aspects associated with chronic airway inflammation.
• Multiple sclerosis is a relatively commonly occurring autoimmune disease characterized by demyelination and chronic inflammation of the central nervous system (CNS). Although the mechanisms underlying disease initiation are not clearly understood, the disease processes that contribute to clinical progression of multiple sclerosis are inflammation, demyelination, and axonal loss, or neurodegeneration. Macrophages and microglia are the main immune cells of the CNS. These cells, as well as T cells, neutrophils, astrocytes, and microglia, can contribute to the immune-related pathology of, e.g., multiple sclerosis.
• T cell reactivity/autoimmunity to several myelin proteins have been implicated in the induction and perpetuation of disease state and pathology of multiple sclerosis.
• myelin basic protein MBP
• proteolipid protein PBP
• myelin oligodendrocyte protein MAG
• This interaction of autoreactive T cells and myelin proteins can result in the release of proinflammatory cytokines, including TNF-a, IFN-g, and IL-17, among others.
• Additional consequences are the proliferation of T cells, activation of B cells and macrophages, upregulation of chemokines and adhesion molecules, and the disruption of the blood-brain barrier.
• the ensuing pathology is a loss of oligodendrocytes and axons, and the formation of a demyelinated "plaque".
• the plaque consists of a lesion in which the myelin sheath is now absent and the demyelinated axons are embedded within glial scar tissue. Demyelination can also occur as the result of specific recognition and opsinization of myelin antigens by autoantibodies, followed by complement- and/or activated macrophage-mediated destruction. It is this axonal loss and neurodegeneration that is thought to be primarily responsible for the irreversible neurological impairment that is observed in progressive multiple sclerosis.
• This worsening of disease in these cases is classified as secondary-progressive multiple sclerosis, and occurs in approximately 10 - 15% of multiple sclerosis patients. Another 10 - 15% of patients are diagnosed with primary-progressive multiple sclerosis, in which disease symptoms and physical impairment progress at a steady rate throughout the disease process.
• IL-23 and IL-17 are overexpressed in the central nervous system of humans with multiple sclerosis and in mice undergoing an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE).
• EAE experimental autoimmune encephalomyelitis
• MOG myelin oligodendrocyte glycoprotein
• PGP proteolipid peptide
• IL-23 and IL-17 have been published to be upregulated in several human tumors and cancers, including but not limited to those of the colon, breast, ovarian, cervical, prostate, lung, and stomach, as well as melanoma and T cell lymphoma (Tartour et al, Cancer Res. 59:3698 (1999); Kato et al, Biochem. Biophys. Re.s Commun. 282:135 (2001); Steiner et al, Prostate. 56:171 (2003); Langowksi et al, Nature. May 10 [Epub ahead of print], (2006)).
• neutralization of both IL-17 and a key upstream regulator of IL-17, IL-23 is a potent and effective means of treating cancer and other neoplastic diseases. Therefore, neutralizing both IL-17 and IL-23 with antagonists of the present invention (i.e. a single neutralizing entity or antibody to IL- 17 and IL-23 or an antagonistic molecule that will neutralize both together, such as a bispecific antibody or bispecific scFV) will have better efficacy in these diseases than antagonists directed toward either of IL-17 or IL-23 alone.
• antagonists of the present invention i.e. a single neutralizing entity or antibody to IL- 17 and IL-23 or an antagonistic molecule that will neutralize both together, such as a bispecific antibody or bispecific scFV
• Angiogenesis refers to the formation of new capillaries from preexisting vessels. There are several reports that angiogenesis plays important roles in hematological malignancies and solid tumors. The initiation of angiogenesis and the switch to the angiogenic phenotype requires a change between proangiogenic factors and angiogenic inhibitors (Folkman, Na?. Med.1:27 (1995)). IL-17 acts as a stimulatory hematopoietic cytokine by initiating proliferation of mature neutrophils and by expanding myeloid progenitors.
• IL-17 has pro-angiogenic activities and stimulates the migration of vascular endothelial cells, which are associated with tumor promotion (Numasaki et al, Blood, 101:2620 (2003); Yang et al, J. Biol. Chem., 278:33232 (2003); Fujino et al, Gut, 52:65 (2003)).
• In vitro angiogenic activity can be suppressed by neutralizing IL- 17 with a neutralizing anti-IL-17 monoclonal antibody, further supporting the role of IL- 17 in this action.
• IL- 17 may also promote bFGF-, HGF- and VEGF-mediated angiogenesis via bFGF-, HGF- and VEGF-induced growth of vascular endothelial cells (Takahashi et al, Immunol Lett. 98:189 (2005)).
• IL- 17 has been reported to augment the secretion of several angiogenic CXC chemokines (e.g. CXCLl, CXCL5, CXCL6, and CXCL8) in non-small cell lung cancer (NSCLC) lines.
• CXC chemokines e.g. CXCLl, CXCL5, CXCL6, and CXCL8
• Endothelial cell chemotactic activity (a measure of net angiogenic potential) is increased in response to conditioned medium from NSCLC stimulated with recombinant IL-17.
• NSCLC lines transfected with IL- 17 grew more rapidly versus controls when transplanted in SCID mice (Numasaki et al, J Immunol. 175:6177 (2005)).
• IL-17 has been reported to be associated with increased IL-6 at the site of tumors and is well reported to increase MMP-9 expression.
• MMP-9 is an important modulator in diseases of inflammation, autoimmunity, and cancer.
• antagonists of the present invention i.e. a single neutralizing entity or antibody to IL-17 and IL-23 or an antagonistic molecule that will neutralize both together, such as a bispecific antibody or bispecific scFV
• a bispecific antibody or bispecific scFV will have better efficacy than antagonists directed toward either of IL-17 or IL-23 alone.
• IL-23 Similar to IL-17, IL-23 promotes inflammatory responses including upregulation of MMP9, and is also reported to increase angiogenesis and reduce CD8+ T-cell infiltration. Taken together, these actions can lead to enhanced initiation, progression, and/or maintenance of tumors, cancers, and other transformed growths. That IL-23 plays an important role in cancerous diseases is supported by the observation that neutralization of IL-23 with a monoclonal antibody or with genetic deletion in mice reduces tumor growth in several murine tumor models (Langowksi et al. Nature. May 10 (2006) [Epub ahead of print]).
• Efficacy is associated with reduced IL-17 expression and reductions in IL-17-related tumorogenic biomarkers, such as granulocyte infiltration, G-CSF and MMP-9. Therefore, neutralizing both IL-17 and IL23 with antagonists of the present invention (i.e. a single neutralizing entity or antibody to IL-17 and IL-23 or an antagonistic molecule that will neutralize both together, such as a bispecific antibody or bispecific scFV) will have better efficacy in these diseases than antagonists directed toward either of IL-17 or IL-23 alone.
• antagonists of the present invention i.e. a single neutralizing entity or antibody to IL-17 and IL-23 or an antagonistic molecule that will neutralize both together, such as a bispecific antibody or bispecific scFV
• IBS Irritable Bowel Syndrome
• Irritable bowel syndrome represents a disease characterized by abdominal pain or discomfort and an erratic bowel habit.
• IBS patients can be characterized into three main groups based on bowel habits: those with predominantly loose or frequent stools, those with predominantly hard or infrequent stools, and those with variable or normal stools (Talley et al., 2002).
• Altered intestinal motility, abnormalities in epithelial function, abnormal transit of stool and gas, and stress, may contribute to symptoms, while visceral hypersensitivity is a key feature in most patients. Genetic factors affecting pain-signaling and disturbances in central processing of afferent signals are postulated to predispose individuals to IBS following specific environmental exposures.
• IBS gastroesophageal reflux disease
• Converging disease models have been proposed that postulate an enhanced responsiveness of neural, immune or neuroimmune circuits in the central nervous system (CNS) or in the gut to central (psychosocial) or peripheral (tissue irritation, inflammation, infection) perturbations of normal homeostasis (Talley et al., 2002).
• This enhanced responsiveness results in dysregulation of gut motility, epithelial function (immune, permeability), and visceral hypersensitivity, which in turn results in IBS symptoms.
• IBS IGF-like neurotrophic factor
• a number of our in-house molecules are known to be linked to possible activity on neurons due to their direct expression by neurons or expression of their receptors on neurons, including IL-17D, IL-17B and IL-31.
• IL-17 family members and related molecules have been associated with inflammation in the gut, including IL- 17 A, IL- 17F, IL- 23 and IL-31.
• Efficacy of inhibitors of these molecules could be tested in vivo in animal models of disease.
• Several animal models have been proposed that mimic key features of IBS and involve centrally targeted stimuli (stress) or peripherally targeted stimuli (infection, inflammation).
• Stress models Two examples of in vivo animal models that can be used to determine the effectiveness of inhibitors in the treatment of IBS are (i) models focusing on primary CNS-directed pathogeneisis of IBS (stress models), and (ii) models focusing on gut-directed inducers of stress (i.e. gut inflammation, infection or physical stress).
• stress models models focusing on primary CNS-directed pathogeneisis of IBS
• gut-directed inducers of stress i.e. gut inflammation, infection or physical stress.
• IL- 17 and IL-23 there are several molecules and pathogenic pathways that are shared by IL- 17 and IL-23 which play important roles in the devopment, progression, and maintenance of both autoimmune diseases and cancerous diseases. These include the pro-angiogenic roles of IL- 17 and IL-23; enhanced MMP-9 levels and activity by IL- 17 and IL-23; IL-23, TGF-b and IL-6-mediated production and/or maintenece of Th 17 cells; roles of TGF-b and IL-6 in the generation of Foxp3+ regulatory T cells; and additional pathways and molecules.
• the IL-17/IL-23 axis represents an important link to the inappropriate and pathogenic T cell responses associated with autoimmune diseases, tumour-promoting pro-inflammatory processes, and the failure of the adaptive immune surveillance to infiltrate tumours. Therefore, neutralizing both IL-17 and IL23 with antagonists of the present invention (i.e. a single neutralizing entity or antibody to IL-17 and IL-23 or an antagonistic molecule that will neutralize both together, such as a bispecific antibody or bispecific scFV) will have better efficacy in these diseases than antagonists directed toward either of IL-17 or IL-23 alone.
• antagonists of the present invention i.e. a single neutralizing entity or antibody to IL-17 and IL-23 or an antagonistic molecule that will neutralize both together, such as a bispecific antibody or bispecific scFV
• the 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.
• 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.
• the cytokines 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.
• a therapeutically effective amount of 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).
• the antibodies of the present invention can also be administered in combination with other cytokines such as IL-3, -6 and - 11; stem cell factor; erythropoietin; G-CSF and GM-CSF.
• cytokines such as IL-3, -6 and - 11; stem cell factor; erythropoietin; G-CSF and GM-CSF.
• the dosage of administered 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 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.
• Administration of antibodies of the invention to a subject can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection.
• the administration may be by continuous infusion or by single or multiple boluses.
• 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, Adv. Drug Deliv. Rev. 35:199 (1999)).
• Dry or liquid particles comprising antibodies of the invention can be prepared and inhaled with the aid of dry-powder dispersers, liquid aerosol generators, or nebulizers (e.g., Pettit and Gombotz, TIBTECH 76:343 (1998); Patton et al, Adv. Drug Deliv. Rev. 35:235 (1999)).
• dry-powder dispersers liquid aerosol generators
• nebulizers e.g., Pettit and Gombotz, TIBTECH 76:343 (1998); Patton et al, Adv. Drug Deliv. Rev. 35:235 (1999)
• AERX diabetes management system which is a hand-held electronic inhaler that delivers aerosolized insulin into the lungs.
• 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 et al, Science 269:850 (1995)
• a pharmaceutical composition comprising an antibodies of the invention 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).
• antibodies of the invention 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.
• an agent used to treat inflammation is physiologically significant if its presence alleviates the inflammatory response.
• Effective treatment may be assessed in a variety of ways. In one embodiment, effective treatment is determined by reduced inflammation. In other embodiments, effective treatment is marked by inhibition of inflammation. In still other embodiments, effective therapy is measured by increased well-being of the patient including such signs as weight gain, regained strength, decreased pain, thriving, and subjective indications from the patient of better health.
• a pharmaceutical composition comprising antibodies of the invention 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- release forms. The latter form is illustrated by miniosmotic pumps and implants (Bremer et al., Pharm. Biotechnol.
• 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.
• 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 ⁇ m to greater than 10 ⁇ 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., Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and Ostro et al., American J. Hosp. Pharm. 46:1576 (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.
• Liposomes can adsorb to virtually any type of cell and then slowly release the encapsulated agent.
• 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 et al., Ann. N.Y. Acad. Sci. 446:368 (1985)).
• small liposomes 0.1 to 1.0 ⁇ m
• 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)).
• 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 1068:133 (1991); Allen et al., Biochim. Biophys. Acta 1150:9 (1993)).
• Liposomes can also be prepared to target particular cells or organs by varying phospholipid composition or by inserting receptors or ligands into the liposomes.
• 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. 16:960 (1993)).
• These formulations were prepared by mixing soybean phospatidylcholine, ⁇ -tocopherol, and ethoxylated hydrogenated castor oil (HCO-60) in methanol, concentrating the mixture under vacuum, and then reconstituting the mixture with water.
• DPPC dipalmitoylphosphatidylcholine
• SG soybean-derived sterylglucoside mixture
• Cho cholesterol
• 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. 74:287 (1997); Murahashi et al, Biol. Pharm. Bull.20:259 (1997)).
• galactose asialoglycoprotein
• Polyaconitylated human serum albumin liposomes provide another approach for targeting liposomes to liver cells (Kamps et al., Proc. Nat'l Acad. ScL USA 94:11681 (1997)).
• Geho, et 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.
• 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)).
• Antibodies can be encapsulated within liposomes using standard techniques of protein microencapsulation (see, for example, Anderson et al., Infect. Immun. 3i:1099 (1981), Anderson et al, Cancer Res. 50:1853 (1990), and Cohen et al, Biochim. Biophys. 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. 149:124 (1987)).
• therapeutically useful liposomes may contain a variety of components.
• liposomes may comprise lipid derivatives of poly(ethylene glycol) (Allen et al, Biochim. Biophys. Acta 1150:9 (1993)).
• 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.
• the present invention also contemplates chemically modified polypeptides having binding IL-17 and IL-23 activity such as anti-IL-17A and IL-23/pl9 antibodies, which a polypeptide is linked with a polymer, as discussed above.
• compositions may be supplied as a kit comprising a container that comprises an antibody of the invention.
• Antibodies of the invention can be provided in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection.
• a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a therapeutic polypeptide.
• Such a kit may further comprise written information on indications and usage of the pharmaceutical composition.
• such information may include a statement that the antibody composition is contraindicated in patients with known hypersensitivity to IL-17 and IL-23.
• a pharmaceutical composition comprising antibodies of the invention can be furnished in liquid form, in an aerosol, or in solid form.
• Liquid forms are illustrated by injectable solutions, aerosols, droplets, topological solutions and oral suspensions.
• Exemplary solid forms include capsules, tablets, and controlled-release forms. The latter form is illustrated by miniosmotic pumps and implants (Bremer et al., Pharm. Biotechnol.
• 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 ai, Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl.
• 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 ⁇ m to greater than 10 ⁇ 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., Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and Ostro et al., American J. Hosp. Pharm. 46:1576 (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.
• 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.
• Hybridomas expressing monoclonal antibodies to IL- 17A and IL- 17F were described in co-pending and co-owned US Patent Publication No. 2007-0218065, published September 20, 2007 and in US. Patent Application Serial No. 11/741,189, filed April 27, 2007, herein incorporated by reference.
• hybridomas were deposited with the American Type Tissue Culture Collection (ATCC; Manassas VA) patent depository as original deposits under the Budapest Treaty and were given the following ATCC Accession No.s: clone 339.15.5.3 (ATCC Patent Deposit Designation PTA-7987, deposited on November 7, 2006); clone 339.15.3.6 (ATCC Patent Deposit Designation PTA-7988, deposited on November 7, 2006); and clone 339.15.6.16 (ATCC Patent Deposit Designation PTA-7989, deposited on November 7, 2006.
• the variable heavy regions and variable light regions of the antibodies expressed by these hybridomas can be determined by amino acid sequencing.
• the polypeptides comprising the variable heavy regions or variable light regions can also be separated by conventional protein isolation techniques.
• CDRs complementarity determining regions
• heavy chain and light chain complementarity determining regions and variable heavy and light regions can be expressed in cell culture and purified or produced synthetically.
• Antibodies and antibody fragments that bind to IL-23pl9 were identified by screening a phage display library designed so that the antibody light-chain variable region and a portion of the heavy-chain variable region are combined with synthetic DNA encoding human antibody sequences, which are then displayed on phage and phagemid libraries as Fab antibody fragments (Dyax® Human Antibody Libraries, Dyax Corp., Cambridge, MA.). These antibodies and antibody fragments and are described in co-pending and co-owned US Patent Application Serial Number 11/762,738, filed June 13, 2007 and WIPO Publication Number 2007/147019, published December 21, 2007, herein incorpprated by reference. The amino acid sequences of the variable heavy and variable light regions of these sequences are shown in Table 1 , below.
• variable light and heavy chain fragments of antibodies can be isolated in a Fab format. These variable regions can then be manipulated to generate antibodies, including antigen- binding fragments, such as scFvs, to IL-23pl9. Using this technology the variable regions of Fabs have been identified for their charactaristics of binding and or neutralizing IL-23pl9 in plate-based assays described in WIPO Publication Number 2007/147019. EXAMPLE 3 Bispecific antibodies that bind IL-17A or IL-17F and the pl9 subunit of IL-23
• Bispecific antibodies can be constructed from the variable heavy and variable light regions of antibodies that cross-bind to IL- 17A and IL- 17F with variable heavy and variable light regions of antibodies that bind the pl9 subunit of IL-23.
• the antibodies that bind to IL-17A or to IL-17F, which are produced by the ATCC deposited hybridomas described in Example 1 can be joined with the variable heavy and variable light regions of the anti-IL-23pl9 antbodies described in Example 2.
• Such bispecfic antibodies can be joined in a variety of different configurations and can comprise tandem scFv molecules (herein referred to as "tascFv”), and scFv molecules that are not tandem (herein referred to as "biscFv" and "BiAb”).
• tascFv Fc For the tascFv molecule, two scFv molecules are constructed such that one scFv is amino terminal to the other one in a tandem configuration. This can be done in each orientation, and use a tether (e.g., a lambda stump tether or a CHl stump tether, both of which are derived from the native sequence just after the V region in the Fab, or a Gly-Ser tether).
• the tascFv can be further constructed as fusion protein to contain a Fc component ("tascFv Fc").
• anti-IL-17A or anti- IL- 17F binding entitiy can be the scFv entity that is either proximal or distal to a Fc component.
• an anti-IL-23 bidning entity can be the scFv entity that is either proximal or distal to a Fc component.
• the biscFv molecule is not a tandem configuration. Rather, it has a scFv at the N terminus and another at the C terminus of an Fc ("biscFv Fc"). These molecules can be made with the N terminal scFv directly fused to the Fc hinge and with either a short or a long linker at the C terminus connecting to the second scFv. These linkers are Gly-Ser.
• an anti-IL-17A or anti-IL- 17F binding entitiy can be the scFv entity that is either at the N terminus or at the C terminus to a Fc component.
• an anti-IL-23 bidning entity can be the scFv entity that is either at the N terminus or at the C terminus to a Fc component.
• the Biab molecule is also not a tandem format. It comprises of a monoclonal antibody with a scFv fused to the C terminus of the heavy chain. These molecules can be made by converting one scFv back to a light chain (kappa or lambda) and a gammal heavy chain with the second scFv connected by either a short or long Gly-Ser linker.
• an anti-IL-17A or anti-IL-17F binding entity can be either scFv that is converted back to a light chain (kappa or lambda) and a gammal heavy chain or the second scFv fused to the C terminus.
• an anti-IL-23 binding entity can be either scFv that is converted back to a light chain (kappa or lambda) and a gammal heavy chain or the second scFv fused to the C terminus.
• a Fab either anti-IL-17A Fab, anti-IL- 17F Fab or anti-IL-23 binding Fab
• Fab can be fused to the Fc portion rather than converting a scFv back to a light chain (kappa or lambda) and a gamma 1 heavy chain.
• Additional bispecific molecules are known in the art and include single variable domain antibodies, camelid antibodies, variable domains fused to human serum albumin (See Muller, D, et al., J. Biol. Chem. 282 (Issue 17): 12650-12660, 2007), and dual variable domain immunoglobin molecules (See WIPO patent publication number WO/2007/024715, published March 1, 2007, by Wu, Chengbin, et al.), "Knob-into-hole” configurations described by Carter, et al. (1996); "IgG-C- terminal scFv” configuration described by Morrison, et al. (1997); “Tandem scFv-Fc” configuration described by Kanner, et al. (1998); "Diabody-Fc” configuration described by Kontermann, et al. (1999); “scFv-Fc- scFv” configuration described by Barbas, et al. (2003).
• Biotinylated ligands are individually pre-mixed at lug/ml with titrating amounts of antibody in staining media (HBSS + 1%BSA + 0.1% NaAzide + 1OmM HEPES) in lOOul volumes and incubated at RT for 15 minutes.
• staining media HBSS + 1%BSA + 0.1% NaAzide + 1OmM HEPES
• a BHK cell line stably transfected with full length IL17RC is prepared for ligand staining by resuspension with Versene (Invitrogen cat.15040-066), equilibrating to 2 x 10e5 cells/lOOul, pelleting, and resuspension in the ligand/antibody pre-mix.
• Stained cells are incubated at 4° for 30 minutes, washed Ix in staining media, and stained with Streptavidin-PE (BD Pharmingen cat. 554061) at a 1:100 ratio. Cells are incubated at 4° in the dark for 30 minutes, washed 2x in staining media, and re-suspended in a 1:1 ratio of staining media and Cytofix (BD Bioscience 554655).
• the BD LSRII Flow Cytometer or similar instrument is used for data collection and analysis. The software calculates the IC50 for each curve.
• Antibodies having an IC50 value simlar to those of the ATCC Patent Deposit Designation PTA-7987 (clone 339.15.5.3), ATCC Patent Deposit Designation PTA-7988 (clone 339.15.3.6), and ATCC Patent Deposit Designation PTA-7989 (clone 339.15.6.16), will be effective at inhibiting, reducing, or neutralizing the effects of IL-17A or IL- 17F and thus be useful in methods for inhibiting inflammation in a mammal comprising administering an antagonist of IL-23 and an antagonist of IL-17A or IL- 17F to the mammal, wherein the antagonist of IL- 17A or IL- 17F can bind IL-17A or IL- 17F.
• the IC50 (ug/ml) against IL-17A was between 28 and 38 (i.e., 28 ug/ml, 35ug/ml, and 38 ug/ml).
• the IC50 against IL-17F was between 3.5 and 3.6 ug/ml.
• IC50 values against IL-17A and IL-17F between 2 ug/ml and 380ug/ml are contemplated by this invention.
• a murine nih3t3 cell line was stably transfected with the kzl70 (nfkb) reporter construct, containing a neomycin-selectable marker. See U.S. Patent Application Serial Number 11/762,738, filed June 13, 2007. This cell line, or a similar one, can be used to determine the EC50 levels of the antibodies that bind to IL-17A or IL- 17F.
• Antibodies to human IL- 17A are used as antagonists of human IL-17A or human IL- 17AF activation of nfkb elements in a lucif erase assay.
• EC50 levels of human IL- 17 A- or IL-17AF-mediated nfkb activation in the murine nih3t3/kzl70 assay cell line is measured.
• the antibody when used at approx. lO ⁇ g/mL concentration, the antibody can completely neutralize activity induced by human IL- 17A or IL- 17AF, with the inhibition of activity decreasing in a dose dependent fashion at the lower concentrations.
• An isotype-matched negative control mAb tests at the concentrations described above, provided no inhibition of activity.
• SAEC IL-12 PHA Bioassay SAEC IL-12 PHA Bioassay
• SAEC small airway epithelial cells
• rhIL-17 induces the production of cytokines G-CSF, IL-6, and IL-8, which in turn, play a role in the pathology associated with the diseases for which a bispecific neutralizing antibody comprising an antibody or antibodyfragment that cross-binds IL-17A and IL- 17F and an antibody or antibody fragment that binds IL-23pl9 would be efficacious.
• a bispecific neutralizing antibody comprising an antibody or antibodyfragment that cross-binds IL-17A and IL- 17F and an antibody or antibody fragment that binds IL-23pl9 would be efficacious.
• the ability of any of the neutralizing entities described herein to inhibit IL-17-mediated production of these cytokines is measured in this bioassay, thus being predictive of in vivo efficacy against these cytokines as well.
• SAEC cells and growth media purchased from Cambrex, Inc.
• SAEC are plated at 8,000 cells/well in 96-well flat bottom tissue culture multi-well plates, and placed in a 370C, 5% CO2 incubator. The following day, cells are treated with a dose range of the neutralizing entity in combination with 10 - 20 ng/mL rhIL-17. The ligand and neutralizing entity are incubated together for 30 minutes at 370C before adding to the cells. Duplicate or triplicate wells are set up for each dose. After 24 - 48 hours, supernatants are collected, and stored at -800C if not used directly. Before taking supernatants, wells are scanned by inverted microscope to make note of which wells had considerable cell death.
• rhIL-17A treatment induces the production of cytokine IL-6 in human glioblastoma cells U373MG, and of cytokines G-CSF and IL-6 in human glioblastoma cells U87MG.
• the cell lines are available from commercial vendors such as ATCC (Manassas, VA). These cytokines, in turn, play a role in the pathology associated with the diseases for which antagonists of IL- 17A and IL23pl9 would be efficacious.
• the ability of any of the antagonists described herein to inhibit rhlL- 17A-mediated production of G-CSF and IL-6 is measured in this bioassay, thus being predictive of in vivo efficacy against this cytokine as well.
• Method Cells are plated in media (MEM w/Earle's salts, 10% FCS, 2 mM L- glutamine, 1 mM sodium pyruvate, 100 uM NEAA) at 3,000-7,000 cells/well in 96-well flat bottom tissue culture multi-well plates and placed in a 37°C, 5% CO2 incubator from lhr to overnight. Doses of the neutralizing entities are prepared in culture media with FCS concentration reduced to 2%. The cells are treated with a dose range of the neutralizing entity in combination with 0.20-0.5 nM rhIL-17A. The ligand and neutralizing entity are incubated together for 30 minutes at 37°C before adding to the cells. Supernatants are collected after 24 hours and assayed for huG-CSF and huIL-6 using a bead-based assay system (Bio-Rad Laboratories), and IC50 determined.
• the anti-IL-17A antagonists are efficacious at reducing huIL-6 cytokine production by U373MG cells, with IC50 values ranging from 2.3-45 nM.
• IC50 values ranging from 0.17- 0.52 nM.
• EXAMPLE 8 IL- 12 Bioassy
• Leukopheresis PBMC To obtain a consistent pool of PBMCs, normal human donors are voluntarily apheresed. The leukopheresis PBMC are poured into a sterile 500 ml plastic bottle, diluted to 400 ml with room temperature PBS + 1 mM EDTA and transferred to 250 ml conical tubes. The 250 ml tubes are centrifuged at 1500 rpm for 10 minutes to pellet the cells. The cell supernatant is then removed and discarded. The cell pellets are then combined and suspended in 400 ml PBS + 1 mM EDTA.
• the cell suspension (25 ml/tube) is overlaid onto Ficoll (20 ml/tube) in 50 ml conical tubes.
• the tubes are centrifuged at 2000 rpm for 20 minutes at room temperature.
• the interface layer ("buffy coat") containing the white blood cells and residual platelets is collected, pooled and washed repeatedly with PBS + 1 mM EDTA until the majority of the platelets are removed.
• the white blood cells are suspended in 100 ml of ice-cold Cryopreservation medium (70% RPMI + 20% FCS + 10% DMSO) and distributed into sterile cryovials (1 ml cells/vial).
• the cryovials are placed in a -80o C freezer for 24 hours before transfer to a liquid-nitrogen freezer.
• the white blood-cell yield from a typical apheresis is 0.5 - 1.0 x 1010 cells.
• Apheresis cells processed in this manner contain T cells, B cells, NK cells, mon
• T cells must be activated in order to express the IL- 12 receptor and be able to respond to IL- 12 and IL-23.
• Cryopreserved leukopheresis PBMC are thawed, transferred to a sterile 50 ml conical tube, washed once with 50 ml of warm RPMI + 10% heat- inactivated FBS + 1 ug/ml DNAse I (Calbiochem), resuspended in 50 ml of fresh RPMI/FBS/DNAse medium and incubated in a 37° C water bath for at least 1 hour to allow the cells to recover from being thawed.
• the cells are then centrifuged and the cell-supernatant discarded.
• the cell pellet is resuspended in RPMI + 10% FBS and distributed into sterile 75 cm2 tissue culture flasks (1 x 107 cells/flask in 40 ml/flask).
• PHA-L (5 mg/ml stock in PBS) is added to the cells at a final concentration of 5 ug/ml.
• the cells are then cultured at 37 0 C in a humidified incubator for a total of 5 days.
• the cells are "rested" for some experiments by harvesting the cells on the afternoon of day 4, replacing the culture medium with fresh RPMI + 10% FBS without PHA-L (40 ml/flask) and returning the cells to their flasks and incubating at 37 0 C the cells in a humidified incubator for the remainder of the 5 day culture period.
• IL-12 and IL1-23 bioassays Three in vitro assays for detection of human IL-12 and 11-23 bioactivity on normal human T cells have been established: 1) IFN-gamma and MIP-I alpha production, 2) proliferation ([3H] -incorporation) and 3) STAT3 activation. Human PHA blasts (activated T cells) are harvested on day 5 of culture, suspended in fresh RPMI + 10% FBS and plated at the desired cell number per well in 96 well plates. [314] The inclusion of an IL- 12 assay is used to determine specificity of the neutralizing entities described herein for IL-23pl9 and not IL- 12.
• the cells are plated at 1 x 106/well in flat- bottom 96-well plates.
• the cells are cultured at 37o C in a final volume of 200 ul/well with either medium alone, human IL-2 alone (10 ng/ml; R & D Systems), human IL-12 alone (graded doses; Invitrogen), human IL-23 alone (graded doses; made in-house; CHO-derived), anti-human CD28 mAb alone (graded doses; clone 28.2, e-Biosciences), or each cytokine in combination with anti- human CD28 mAb.
• Triplicate wells are set up for each culture condition.
• IFN-gamma production assay cell supernatants (120 ul/well) are harvested after 24 - 48 hours of culturing the cells at 37 0 C in a humidified incubator. Human IFN-gamma and MIP-lalpha concentrations in these supernatants (pooled for each triplicate) are measured using a commercial Luminex bead-based ELISA kit (Invitrogen) following the manufacturer's instructions.
• [317] For the [3H] -incorporation assay the cells are plated at 2 x 105 cells/well in U- bottom 96-well plates. The cells are cultured at 37 degrees C for 72 hours. The cells are pulsed with 1 uCi/well of [3H] -Thymidine (Amersham) for the last 8 hours of this culture period. The cells are then harvested onto glass-fiber filters and the CPMs of [3H] incorporated are quantitated using a beta counter (Topcount NXT, Packard).
• STAT3 Bioassay For the STAT3 Bioassay the cells are plated at 2 x 105 cells/well in U-bottom 96-well plates. Serial dilutions of human IL-12 (R&D) or recombinant human IL-23 (in- house CHO-derived material or eBioscience's Insect heterodimer material) are prepared in assay media (RPMI 1640 with L-Glutamine plus 10% fetal bovine serum), added to the plates containing the cells and incubated together at 37°C for 15 minutes.
• R&D human IL-12
• recombinant human IL-23 in- house CHO-derived material or eBioscience's Insect heterodimer material
• the assay is also used to measure neutralization of IL-12 and IL-23 activity using either commercially- available neutralizing reagents (as "controls") or the anti-IL-23pl9-containing neutralizing entities described herein.
• a half-maximal concentration (EC50, effective concentration at 50 percent) of IL-12 or IL-23 are combined with serial dilutions of anti-human IL- 12 p40 monoclonal antibody (Pharmingen), anti- human IL-23 pl9 polyclonal antibody (R&D, AF1716), human IL-23R-Fc Soluble Receptor, or any of the neutralizing entities described herein, and incubated together at 37°C for 30 minutes in assay media prior to addition to cells. Following pre-incubation, treatments are added to the plates containing the cells and incubated together at 37°C for 15 minutes.
• Capture beads (BIO-PLEX Phospho-STAT3 Assay, BIO-RAD Laboratories) are combined with 50 ul of 1:1 diluted lysates and added to a 96-well filter plate according to manufacture's instructions (BIO-PLEX Phosphoprotein Detection Kit, BIO-RAD Laboratories). The aluminum foil-covered plate is incubated overnight at room temperature, with shaking at 300 rpm. The plate is transferred to a microtiter vacuum apparatus and washed three times with wash buffer. After addition of 25 ⁇ L/well detection antibody, the foil-covered plate is incubated at room temperature for 30 minutes with shaking at 300 rpm. The plate is filtered and washed three times with wash buffer.
• Streptavidin-PE 50 ul/well was added, and the foil-covered plate is incubated at room temperature for 15 minutes with shaking at 300 rpm. The plate is filtered and washed two times with bead resuspension buffer. After the final wash, beads are resuspended in 125 ul/well of bead suspension buffer, shaken for 30 seconds, and read on an array reader (BIO-PLEX, BIO-RAD Laboratories) according to the manufacture's instructions. Data are analyzed using analytical software (BIO-PLEX MANAGER 3.0, BIO-RAD Laboratories).
• [322] Increases in the level of the phosphorylated STAT3 transcription factor present in the lysates are indicative of an IL- 12 or IL-23 receptor-ligand interaction.
• decreases in the level of the phosphorylated STAT3 transcription factor present in the lysates are indicative of neutralization of the IL- 12 or IL-23 receptor-ligand interaction.
• IC50 (inhibitory concentration at 50 percent) values are calculated using GraphPad Prism®4 software (GraphPad Software, Inc., San Diego CA) and expressed as molar ratios for each reagent and/or neutralizing entity in the neutralization assay.
• Recombinant human IL-23 (rhIL-23) induces the production of IL- 17A and IL- 17F in murine splenocytes.
• antagonists to IL-23 neutralization of IL- 17A and IL- 17F production in rhIL-23 treated murine splenocytes is examined.
• Antagonists to rhIL-23 are compared to the commercial neutralizing antibody anti-IL-12p40 (Pharmingen, Franklin Lakes, NJ).
• IL-23 at a concentration of 10 pM is pre-incubated for 30 - 90 minutes at 37°C with 3-fold serial dilutions of the antagonists. Concentrations of the antagonists range from 0 - 343 nM.
• the IL-23 ligand plus antagonists are then added to the splenocytes and incubated at 37° C, 5% CO2 for 24-72 hours. The supernatants are collected and frozen at -80°C until ready to process.
• the levels of IL- 17A and IL- 17F protein in the supernatants are measured using bead-based sandwich ELISAs.
• a commercial kit (Upstate, Charlottesville, VA) is used to measure IL- 17A protein.
• IC50 values for each antagonist are calculated as the amount of antagonist needed to neutralize 50% of the activity of rhIL-23.
• the anti-IL-23pl9 antibodies are efficacious at reducing IL-17A and IL-17F production with IC50 values in the range of of 0.27 - 100.0 nM.
• EAE Mouse Experimental Allergic Encephalomyelitis
• EAE Mouse Allergic Encephalomyelitis
• the extent of disease is evaluated daily in the mice by taking their body weights and assigning a clinical score (0-8) to each mouse, as detailed below.
• the typical pattern of disease symptoms in immunized, but otherwise untreated mice is one of weight loss and paralysis, followed by a period of disease symptom remission, and a subsequent relapse of disease symptoms.
• a pattern of relapses and remissions of disease symptoms ensues, which is also found in humans with this type of multiple sclerosis, known as relapsing-remitting disease.
• Chronic progressive and secondary progressive multiple sclerosis are also targeted indications for this therapeutic combination of an antibody that binds IL- 17 A or IL- 17F and IL-23/pl9 such as a bispecific antibody or scFV as described in this invention.
• an antibody that binds IL- 17 A or IL- 17F and IL-23/pl9 such as a bispecific antibody or scFV as described in this invention.
• Neutralizing monoclonal antibodies to mouse IL- 17 A and IL-23pl9 are administered separately or as a therapeutic combination, during remission from the first peak of EAE disease symptoms.
• the antibodies are delivered as intraperitoneal injections every other day, or as a similar dosing regimen. Groups receive either 25, 50 or 100 ug of each antibody, alone or as a therapeutic combination, per animal per dose, and control groups receive the vehicle control, PBS (Life Technologies, Rockville, MD) or antibody isotype control.
• Blood is collected throughout the experiment to monitor serum levels of cytokine and levels of other mediators of disease. At the time of euthanasia, blood is collected for serum, and brain and spinal cord collected in 10% NBF for histology. In separate animals, tissues (including lymph nodes, brain, spinal cord, spleen, and others) are harvested for the quantification of mRNA by TaqMan quantitative real-time PCR.
• the therapeutic combination of an antibody that binds IL- 17A or IL- 17F and IL23/pl9 may be more efficacious in the treatment of EAE as a model of human multiple sclerosis.
• the therapeutic combination can reduce clinical disease symptoms and works at the molecular level to reduce inflammation, inflammatory infiltrates, inflammatory cytokines/chemokines, and other mechanisms known to be affected in this manner.
• IL-23 and IL-17 are important players in murine colitis and human IBD, via the actions of Th 17 cells.
• IL-23 and IL-17 are upregulated in colitis and IBD, and neutralization of either cytokine alone is efficacious in several animal models of colitis (Fujino et al, Gut, 2003, 52:65-70; Schmidt et al, Inflamm Bowel Dis. 2005, 11:16-23; Yen et al, J Clin Invest. 2006, 116:1310-1316 ; Zhang et al, Inflamm Bowel Dis. 2006, 12:382-388; Kullberg et al, J Exp Med. 2006, 203:2485-94.). Since IL-23 is important for the maintenance, differentiation, and/or induction of Thl7 cells, neutralization of both cytokines would be more efficacious at reducing disease than either cytokine alone.
• mice obtained from Harlan.
• mice are treated topically with 200ul of 3.0% (w/v) oxazalone in 100% ethanol ("sensitization") on the abdomen.
• all mice receive intrarectal injections (120 uL each) of 2.0% (w/v) oxazalone in 50% ethanol while under light isoflurane gas anesthesia ("challenge”).
• Mice are monitored for disease using a Disease Activity Index (DAI) score, which includes stool consistency, body weight, and blood in stool.
• DAI Disease Activity Index
• mice are euthanized on day 2. Serum is collected and stored for later analysis; colons are removed and observed for any gross signs of colitis (lesion, colon shortening, and colon wall thickening). Colons are then cut longitudinally and processed for histology and for 24 h colon cultures.

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