HK1188610A - Cross-reactive and bispecific anti-el 17a/f antibodies - Google Patents

Abstract

The present invention relates generally to antibodies cross-reactive with IL-17A and IL-17F, and bispecific anti-IL-17A/F and their uses.

Description

Cross-reactive and bispecific anti-IL-17A/F antibodies

The present application is a divisional application of chinese patent application No.200980133845.5 entitled "cross-reactive and bispecific anti-IL-17A/F antibody" filed on 8/28/2009.

Technical Field

The present invention relates generally to antibodies that cross-react with IL-17A and IL-17F, and bispecific anti-IL-17A/F antibodies, and uses thereof.

Background

Interleukin-17 (IL-17) is a pro-inflammatory molecule from T cells that stimulates epithelial, endothelial, and fibroblasts to produce other inflammatory cytokines and chemokines, including IL-6, IL-8, G-CSF, and MCP-1[ see, Yao, z. et al, j.immunol. (journal of immunology), 122 (12): 5483-5486 (1995); yao, z. et al, Immunity, 3 (6): 811-821 (1995); fossiez, F et al, J.Mep. (journal of experimental medicine), 183 (6): 2593-2603 (1996); kennedy, J et al, J. interferon Cytokine Res, (journal of interferon Cytokine research), 16 (8): 611-7 (1996); cai, x.y et al, immunol.lett (immunological communication), 62 (1): 51-8 (1998); jovanovic, D.V et al, j.immunol. (journal of immunology), 160 (7): 3513-21 (1998); laan, M et al, j. immunol. (journal of immunology), 162 (4): 2347-52 (1999); linden, a et al, Eur Respir J (european journal of respiration), 15 (5): 973-7 (2000); and Aggarwal, s. and Gurney, a.l., JLeukoc Biol. (journal of leukocyte biology) 71 (1): 1-8(2002)]. IL-17 also synergizes with other cytokines, including TNF- α and IL-1 β, to further induce chemokine expression (Chabaud, M et al, J.Immunol. (J.Immunol.) 161 (1): 409-14 (1998)). Interleukin-17 (IL-17) exhibits pleiotropic biological activities on various types of cells. IL-17 also has the ability to induce ICAM-1 surface expression, T cell proliferation and CD34⁺The ability of human progenitor cells to grow and differentiate into neutrophils. IL-17 is also involved in bone metabolism and has been shown 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. (J. bone and mineral research), 14: 1513-]). Activated T cells from synovial tissue of rheumatoid Arthritis patients were found to secrete higher amounts of IL-17 than those from normal individuals or osteoarthritis patients (Chabaud et al, Arthritis Rheum. (Arthritis and rheumatism), 42: 963-]). This proinflammatory cytokine is proposed to actively promote synovial inflammation in rheumatoid arthritis. In addition to its pro-inflammatory effects, IL-17 appears to contribute to the pathology of rheumatoid arthritis by another mechanism. For example, IL-17 has been shown to induce the expression of Osteoclast Differentiation Factor (ODF) mRNA in osteoblasts (Kotake et al, J.Clin.Invest. (J.Clin. Clin.) (103: 1345) -1352[1999 ]]). ODF stimulates progenitor cells to differentiate into osteoclasts (cells involved in bone resorption). Since IL-17 is significantly elevated in synovial fluid of rheumatoid arthritis patients, it seems to be shown that IL-17-induced osteoclast formation plays a role in bone resorption in rheumatoid arthritisHas important function. IL-17 is also thought to play a key role in certain other autoimmune disorders such as multiple sclerosis (Matusevicius et al, Mult. Scler. (multiple sclerosis), 5: 101-104 (1999); Kurasawa, K et al, Arthritis Rheu disease 43 (11): 2455-63(2000)) and psoriasis (Teunissen, M.B et al, J Invest Dermatol (J. Dermatology) 111 (4): 645-9 (1998); Albanesi, C et al, J Invest Dermatol (J. dermatology) 115 (1): 81-7 (2000); and Homey, B et al, J. Immunol. (J. immunology) 164 (12: 6621-32 (2000)).

It has further been shown that stimulation of Ca in human macrophages by intracellular signaling of IL-17²⁺Internal flow and [ cAMP]_iReduction of (Jovanovic et al, J. Immunol. (J. Immunol.), 160: 3513[1998 ] A]). IL-17 treated fibroblasts induced activation of NF- κ B [ Yao et al, Immunity, 3: 811(1995), Jovanovic et al, supra]Whereas its treated macrophages activate NF-. kappa.B and mitogen-activated protein kinases (Shalom-Barek et al, J.biol.chem. (J.Biol.Chem.), 273: 27467[1998 ] A]). In addition, IL-17 also has sequence similarity to mammalian cytokine-like factor 7, which is involved in bone and cartilage growth. Other proteins with sequence similarity to the IL-17 polypeptide are human Embryonic Derived Interleukin Related Factor (EDIRF) and interleukin-20.

Consistent with the widespread effects of IL-17, it has been found that the cell surface receptor for IL-17 is widely expressed in many tissues and cell types (Yao et al, Cytokine, 2: 794[1997 ]). Although the amino acid sequence (866 amino acids) of the human IL-17 receptor (IL-R) predicts a protein with a single transmembrane domain and a long, 525 amino acid intracellular domain, the receptor sequence is unique and dissimilar to any receptor from the cytokine/growth factor receptor family. This, coupled with the lack of similarity of IL-17 itself to other known proteins, suggests that IL-17 and its receptor may be part of a new family of signaling proteins and receptors. IL-17 activity has been demonstrated to be mediated by binding to its unique cell surface receptor (referred to herein as human IL-17R), where previous studies have shown that T cell proliferation and IL-2 production induced by PHA, concanavalin A and anti-TCR monoclonal antibodies are inhibited by contacting T cells with a soluble form of an IL-17 receptor polypeptide (Yao et al, J.Immunol. (J.Immunol., 155: 5483-5486[1995 ]). Therefore, there is considerable interest in identifying and characterizing novel polypeptides having homology to known cytokine receptors, particularly the IL-17 receptor.

Interleukin-17 is now believed to be the prototypical member of the emerging cytokine family. Large scale sequencing of human and other vertebrate genomes has revealed the presence of other genes encoding proteins that are clearly related to IL-17, thus defining a new cytokine family. There are at least six members of the IL-17 family in humans and mice, including IL-17B, IL-17C, IL-17D, IL-17E and IL-17F, as well as the novel receptors IL-17RH1, IL-17RH2, IL-17RH3 and IL-17RH4 (see WO01/46420 published 6/28.2001). One such IL-17 member (referred to as IL-17F) has been shown to bind to the human IL-17 receptor (IL-17R) (Yao et al, Cytokine 9 (11): 794-800 (1997)). Initial characterization showed that several of these newly identified molecules have the ability to modulate immune function, as with IL-17. The strong inflammatory role of several of these factors that have been identified, and the emerging association with major human diseases, suggests that these proteins may play a significant role in the inflammatory process and may provide opportunities for therapeutic intervention.

The gene encoding human IL-17F is located in the vicinity of IL-17 (Hymowitz, S.G et al, Embo J.20 (19): 5332-41 (2001)). IL-17 and IL-17F share 44% amino acid identity while other members of the IL-17 family share a more limited 15-27% amino acid identity, indicating that IL-17 and IL-17F form different subgroups within the IL-17 family (Starnes, T. et al, J. Immunol. 167 (8): 4137-40 (2001); Aggarwal, S. and Gurney, A.L., J.Leukoc Biol (J. Leucocytobiology), 71 (1): 1-8 (2002)). IL-17F appears to have a biological effect similar to that of IL-17 and is capable of promoting the production of IL-6, IL-8 and G-CSF by a variety of cells. Similar to IL-17, it is capable of inducing the release of cartilage matrix and inhibiting the synthesis of new cartilage matrix (see US-2002-0177188-A1, published on 11/28/2002). Thus, like IL-17, IL-17F may potentially contribute to the pathology of inflammatory disorders. Recently, these authors have observed that both IL-17 and IL-17F are induced in T cells by the action of interleukin 23(IL-23) (Aggarwal, S et al, J.biol.chem. (J.Biol.Chem., 278 (3): 1910-4 (2003)). The observation that IL-17 and IL-17F share similar chromosomal localization and significant sequence similarity, as well as the observation that IL-17 and IL-17F appear to be induced in the same cell population in response to specific stimuli, led to the identification of novel human cytokines consisting of IL-17 and IL-17F covalent heterodimers (referred to herein as IL-17A/F).

It is now recognized that IL-17A and IL-17F are the major proinflammatory factors secreted by the Th17T cell subset. They target almost all cell types in vivo and induce inflammation and tissue damage.

Human IL-17A/F is a different novel cytokine that is distinguishable from human IL-17 and IL-17F in both protein structure and cell-based activity assays. By using purified recombinant human IL-17A/F as a standard, a human IL-17A/F-specific ELISA has been developed. By using this specific ELISA, the induced expression of human IL-17A/F was detected, confirming that IL-17A/F is naturally produced from cultured activated human T cells. Thus, IL-17A/F is a different novel cytokine, detectable as a natural product of isolated activated human T cells, the recombinant form of which has been characterized in both protein structure and cell-based assays, which is different and distinguishable from the relevant cytokine.

IL-17A/F is disclosed in U.S. application publication No.20060270003, published on 30/11 2006, and 20070160576, published on 12/7/2007. IL-17A/F has been described as a target for the treatment of a variety of immune-mediated diseases (Chang and Dong, Cell Res. (Cell research) 17 (5): 435-40 (2007)); it has been shown that the IL-17A/F protein produced by mouse Th17 cells induces recruitment of airway neutrophils and thus has in vivo function in airway neutrophilia (Liang et al, J Immunol (J Immunol) 179 (11): 7791-9 (2007)); cytokines that heterodimerize human IL-17A/F have been reported to signal through the IL-17RA/IL-17RC receptor complex (Wright et al, J Immunol (J Immunol) 181 (4): 2799-805 (2008)).

Given the pro-inflammatory properties of both IL-17A and IL-17F, it is desirable to generate cross-reactive and bispecific antibodies that can block both IL-17A and IL-17F with high efficiency. Such cross-reactive and bispecific antibodies provide new therapeutic opportunities in the treatment of inflammatory and immune-related diseases, including autoimmune diseases, and in the treatment of cancer.

Summary of The Invention

The invention is based, at least in part, on the generation and characterization of antibodies that cross-react with both IL-17A and IL-17F. The invention also relates to bispecific antibodies that bind to both IL-17A and IL-17F.

In one aspect, the invention relates to an antibody, or a functional fragment thereof, comprising an antigen binding domain that binds to IL-17A and IL-17F and inhibits a biological function of both IL-17A and IL-17F, wherein the biological function can be, for example, pro-inflammatory activity.

In one embodiment, the antibody comprises a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 regions, wherein at least one of the CDRL1, CDRL2 and CDRL3 regions is selected from the group consisting of:

(a) CDRL1 comprising the sequence DVSTAVA (SEQ ID NO: 24) or SISSYLA (SEQ ID NO: 25),

(b) CDRL2 comprising the sequences SASFLYS (SEQ ID NO: 26) or GASSRAS (SEQ ID NO: 27) and

(c) CDRL3, which comprises the sequence SYTTPPT (SEQ ID NO: 28) or RYSQPIT (SEQ ID NO: 29),

or an affinity matured variant thereof, or a functional fragment of said antibody or of said affinity matured variant.

In another embodiment, each of the CDRL1, CDRL2, and CDRL3 regions is selected from the group consisting of:

(a) CDRL1 comprising the sequence DVSTAVA (SEQ ID NO: 24) or SISSYLA (SEQ ID NO: 25),

(b) CDRL2 comprising the sequences SASFLYS (SEQ ID NO: 26) or GASSRAS (SEQ ID NO: 27) and

(c) CDRL3, which comprises the sequence SYTTPPT (SEQ ID NO: 28) or RYSQPIT (SEQ ID NO: 29).

In a further embodiment of the process of the present invention,

(a) CDRL1 comprises the sequence DVSTAVA (SEQ ID NO: 24),

(b) CDRL2 comprises the sequence SASFLYS (SEQ ID NO: 26), and

(c) CDRL3 comprises the sequence SYTTPPT (SEQ ID NO: 28).

In yet another embodiment of the present invention,

(a) CDRL1 comprises the sequence SISSYLA (SEQ ID NO: 25),

(b) CDRL2 comprises the sequence GASSRAS (SEQ ID NO27), and

(c) CDRL3 comprises the sequence RYSQPIT (SEQ ID NO: 29).

In yet another embodiment, in the affinity matured variant, CDRL3 comprises the sequence SYTAKLT (SEQ ID NO: 30).

In yet another embodiment, in the affinity matured variant,

(a) CDRL1 comprises the sequence DVSTAVA (SEQ ID NO: 24), and

(b) CDRL2 comprises the sequence SASFLYS (SEQ ID NO: 26, and CDRL3 may comprise the sequence YIIYPAT (SEQ ID NO: 31), or a functional fragment thereof.

In yet another embodiment, in the affinity matured variant,

(a) CDRL1 comprises the sequence DVSTAVA (SEQ ID NO: 24), and

(b) CDRL2 comprises the sequence SASFLYS (SEQ ID NO: 26), and CDRL3 may comprise the sequence HNDLPLT (SEQ ID NO: 32).

In another embodiment, in the affinity matured variant,

(a) CDRL1 comprises the sequence VISSLA (SEQ ID NO: 33), and

(b) CDRL2 comprises the sequence GASFLYS (SEQ ID NO: 34).

In another embodiment, in the affinity matured variant, CDRL3 comprises the sequence SYTPRST (SEQ ID NO: 75), or a functional fragment thereof.

In yet another embodiment, in the affinity matured variant,

(a) CDRL1 comprises the sequence DVSTAVA (SEQ ID NO: 24); and

(b) CDR2 comprises the sequence SASFLYS (SEQ IS NO: 26).

In yet another embodiment, in the affinity matured variant, CDRL3 comprises the sequence QYYSTTTT (SEQ ID NO: 76), or a functional fragment thereof.

In yet another embodiment, in the affinity matured variant,

(a) CDRL1 comprises the sequence DVSTAVA (SEQ ID NO: 24); and

(b) CDR2 comprises the sequence SASFLYS (SEQ ID NOT: 26).

In a different embodiment, in the affinity matured variant, CDRL3 comprises sequence QQSQNPQTT (SEQ ID NO: 77), or a functional fragment thereof.

In another embodiment, in the affinity matured variant,

(a) CDRL1 comprises the sequence DVSTAVA (SEQ ID NO: 24); and

(b) CDRL2 comprises the sequence SASFLYS (SEQ ID NO: 26).

In another embodiment, in the affinity matured variant, CDRL3 comprises the sequence RFSQHIT (SEQ ID NO: 78), or a functional fragment thereof.

In yet another embodiment, in the affinity matured variant,

(a) CDRL1 comprises the sequence RIKRYLA (SEQ ID NO: 89); and

(b) CDRL2 comprises the sequence GASSRAS (SEQ ID NO: 27).

In yet another embodiment, in the affinity matured variant, CDRL3 comprises the sequence RYSWHTT (SEQ ID NO: 79), or a functional fragment thereof.

In yet another embodiment, in the affinity matured variant,

(a) CDRL1 comprises the sequence SISSYLA (SEQ ID NO: 25); and

(b) CDRL2 comprises the sequence GASSRAS (SEQ ID NO: 27).

In a different embodiment, in the affinity matured variant, CDRL3 comprises the sequence RYSLPIT (SEQ ID NO: 80), or a functional fragment thereof.

In yet another embodiment, in the affinity matured variant,

(a) CDRL1 comprises the sequence SISSYLA (SEQ ID NO: 25); and

(b) CDRL2 comprises the sequence GASSRAS (SEQ ID NO: 27).

In still other embodiments, the antibody comprises a heavy chain variable domain comprising CDRH1, CDRH2, and CDRH3 regions, wherein at least one of the CDRH1, CDRH2, and CDRH3 regions is selected from the group consisting of:

(a) a CDRH1 comprising a sequence selected from the group consisting of: GFTFTDYDIS (SEQ ID NO: 35), GFSFIDYDIS (SEQ ID NO: 36), GFSFTSYMMS (SEQ ID NO: 81), GFSFPSYFIS (SEQ ID NO: 82); and GFTFYDYDIS (SEQ ID NO: 88);

(b) a CDRH2 comprising a sequence selected from the group consisting of: SDGYTYYADSVKG (SEQ ID NO: 37), PDCYTYYADSVKG (SEQ ID NO: 38), PDAYTYPDCYTYYADSVKG (SEQ ID NO: 39), PDSYTTYYADSVKG (SEQ ID NO: 90), YDGYTYYADSVKG (SEQ ID NO: 41), YEGYTYYADSVKG (SEQ ID NO: 44), ESGATDYADSVKG (SEQ ID NO: 83), VSGATVYADSVKG (SEQ ID NO: 84), YDGYAYYADSVKG (SEQ ID NO: 85); and LDGYSYYADSVKG (SEQ ID NO: 86); and

(c) CDRH3 comprising the sequences YLLYWSYV (SEQ ID NO: 40) and EGYYYSTSIKYYPW (SEQ ID NO: 87),

or a functional fragment thereof.

In still other embodiments, the antibody comprises a heavy chain variable domain comprising CDRH1, CDRH2, and CDRH3 regions, wherein each of the CDRH1, CDRH2, and CDRH3 regions is selected from the group consisting of:

(a) a CDRH1 comprising a sequence selected from the group consisting of: GFTFTDYDIS (SEQ ID NO: 35), GFSFIDYDIS (SEQ ID NO: 36), GFSFTSYMMS (SEQ ID NO: 81) and GFSFPSYFIS (SEQ ID NO: 82),

(b) a CDRH2 comprising a sequence selected from the group consisting of: SDGYTYYADSVKG (SEQ ID NO: 37), PDCYTYYADSVKG (SEQ ID NO: 38), PDAYTYPDCYTYYADSVKG (SEQ ID NO: 39), PDSYTTYYADSVKG (SEQ ID NO: 90), YDGYTYYADSVKG (SEQ ID NO: 41), YEGYTYYADSVKG (SEQ ID NO: 44), ESGATDYADSVKG (SEQ ID NO: 83) and VSGATVYADSVKG (SEQ ID NO: 84); and

(c) CDRH3 comprising the sequence YLLYWSYV (SEQ ID NO: 40) or EGYYYSTSIKYYPW (SEQ ID NO: 87),

or a functional fragment thereof.

In another embodiment, the invention relates to an antibody comprising the CDRH1, CDRH2 and CDRH3 regions, wherein

(a) CDRL1 comprises the sequence DVSTAVA (SEQ ID NO: 24),

(b) CDRL2 comprises the sequence SASFLYS (SEQ ID NO: 26),

(c) CDRL3 comprises the sequence SYTTPPT (SEQ ID NO: 28), and

(d) CDRH1 comprises the sequence GFTFTDYDIS (SEQ ID NO: 35),

(e) CDRH2 comprises sequence SDGYTYYADSVKG (SEQ ID NO: 37), and

(f) CDRH3 comprises the sequence YLLYWSYV (SEQ ID NO: 40),

or a functional fragment thereof.

In yet another embodiment, the invention relates to an antibody comprising the CDRH1, CDRH2 and CDRH3 regions, wherein

(a) CDRL1 comprises the sequence SISSYLA (SEQ ID NO: 25),

(b) CDRL2 comprises the sequence GASSRAS (SEQ ID NO: 27),

(c) CDR3 comprises the sequence RYSQPIT (SEQ ID NO: 29),

(d) CDRH1 comprises the sequence GFTFTDYDIS (SEQ ID NO: 35),

(e) CDRH2 comprises sequence SDGYTYYADSVKG (SEQ ID NO: 37), and

(f) CDRH3 comprises the sequence YLLYWSYV (SEQ ID NO: 40),

or a functional fragment thereof.

In yet another embodiment, the antibody further comprises CDRH1, CDRH2, and CDRH3 regions, wherein

(d) CDRH1 comprises the sequence GFSFIDYDIS (SEQ ID NO: 36),

(e) CDRH2 comprises the sequence PDCYTYYADSVKG (SEQ ID NO: 38), and

(f) CDRH3 comprises the sequence YLLYWSYV (SEQ ID NO: 40).

In yet another embodiment, the antibody further comprises CDRH1, CDRH2, and CDRH3 regions, wherein

(d) CDRH1 comprises the sequence SFSGIDYDIS (SEQ ID NO: 91),

(e) CDRH2 comprises the sequence YDGYTYYADSVKG (SEQ ID NO: 41), and

(f) CDRH3 comprises the sequence YLLYWSYV (SEQ ID NO: 40).

In another embodiment, the antibody further comprises CDRH1, CDRH2, and CDRH3 regions, wherein

(d) CDRH1 comprises the sequence GFTFTDYDIS (SEQ ID NO: 35),

(e) CDRH2 comprises sequence SDGYTYYADSVKG (SEQ ID NO: 37), and

(f) CDRH3 comprises the sequence YLLYWSYV (SEQ ID NO: 40).

In another embodiment, the antibody further comprises CDRH1, CDRH2, and CDRH3 regions, wherein

(d) CDRH1 comprises the sequence GFSFIDYDIS (SEQ ID NO: 36),

(e) CDRH2 comprises the sequence PDAYTYYADSVKG (SEQ ID NO: 42), and

(f) CDRH3 comprises the sequence YLLYWSYV (SEQ ID NO: 40).

In yet another embodiment, the antibody further comprises CDRH1, CDRH2, and CDRH3 regions, wherein

(d) CDRH1 comprises the sequence GFSFIDYDIS (SEQ ID NO: 36),

(e) CDRH2 comprises the sequence PDSYTYYADSVKG (SEQ ID NO: 43), and

(f) CDRH3 comprises the sequence YLLYWSYV (SEQ ID NO: 40).

In yet another embodiment, the antibody further comprises CDRH1, CDRH2, and CDRH3 regions, wherein

(d) CDRH1 comprises the sequence SFSGIDYDIS (SEQ ID NO: 91),

(e) CDRH2 comprises the sequence YEGYTYYADSVKG (SEQ ID NO: 44), and

(f) CDRH3 comprises the sequence YLLYWSYV (SEQ ID NO: 40).

In another embodiment, the antibody binds to substantially the same epitope of IL-17A and IL-17F as a cross-reactive antibody selected from the group consisting of YW241.47, YW278.15, YW279.1, or an affinity matured variant thereof, or a functional fragment of such antibody or such affinity matured variant.

In yet another embodiment, the affinity matured variant is selected from the group consisting of: antibodies YW278.15.18, YW178.15.2, YW278.15.3, YW278.15.18C55A, YW278.15.18C55S and yw278.15.2.d 54e.

In yet another embodiment, the antibody comprises an antigen binding domain, or functional fragment thereof, that binds to IL-17A and IL-17F with substantially the same binding affinity.

In yet another embodiment, the antibody comprises at least about 10^-10To 10^-11The binding affinity of M binds to the antigen binding domains of IL-17A and IL-17F, or functional fragments thereof.

In another embodiment, the invention relates to antibodies, or functional fragments thereof, comprising an antigen binding domain that binds to IL-17A and IL-17F, both as monomers and homodimers or heterodimers.

In yet another embodiment, the invention relates to antibodies, or functional fragments thereof, that bind to human IL-17A/F and non-human primate IL-17A/F. The non-human primate can be, for example, a cynomolgus monkey (cynomolgus (cyno)) or a Rhesus monkey (Rhesus monkey). In another embodiment, the antibody binds to human IL-17A/F and optionally non-human primate IL-17A/F, but does not bind to rodents such as rat or mouse IL-17A/F.

In yet another embodiment, the antibody is capable of reducing demyelination in an inflammatory or immune-related disease.

The antibodies of the invention may be monoclonal, and may be chimeric, humanized or human.

Antibody fragments of the invention include, but are not limited to, Fab ', F (ab')₂And Fv fragments; diabodies (diabodies); a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

In a different aspect, the invention relates to a bispecific antibody comprising a first antigen-binding site that binds to IL-17A and a second antigen-binding site that binds to IL-17F, wherein

(1) The first antigen binding site comprises a first heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 regions, wherein at least one of the CDRH1, CDRH2 and CDRH3 regions is selected from the group consisting of:

(a) CDRH1 comprising the sequence TSYEIS (SEQ ID NO: 45),

(b) CDRH2 comprising the sequence WVGSIYLWGG (SEQ ID NO: 46) and

(c) CDRH3 comprising the sequences ARFGQRYA (SEQ ID NO: 47) and

(2) the second antigen binding site comprises a second heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 regions, wherein at least one of the CDRH1, CDRH2 and CDRH3 regions is selected from the group consisting of:

(a) CDRH1 comprising the sequence TSPYIS (SEQ ID NO: 48),

(b) CDRH2 comprising the sequence WVASIFYYSG (SEQ ID NO: 49) and

(c) CDRH3 comprising sequence ARGGYGYNQWFYSIYQSY (SEQ ID NO: 50),

or an affinity matured variant thereof, or a functional fragment of the antibody or the affinity matured variant.

In one embodiment, the bispecific antibody comprises CDRH1, CDRH2, and CDRH3 regions, wherein

(1) In the first antigen-binding site, the antigen binding site,

(a) CDRH1 comprises the sequence TSYEIS (SEQ ID NO: 45),

(b) CDRH2 comprising the sequence WVGSIYLWGG (SEQ ID NO: 46) and

(c) CDRH3 comprises the sequences ARFGQRYA (SEQ ID NO: 47) and

(2) in the second antigen-binding site,

(a) CDRH1 comprises the sequence TSPYIS (SEQ ID NO: 48),

(b) CDRH2 comprising the sequence WVASIFYYSG (SEQ ID NO: 49) and

(c) CDRH3 comprises the sequence ARGGYGYNQWFYSIYQSY (SEQ ID NO: 50),

or an affinity matured variant thereof, or a functional fragment of the antibody or the affinity matured variant.

In another embodiment, the bispecific antibody further comprises a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 regions, wherein at least one of the CDRL1, CDRL2 and CDRL3 regions is selected from the group consisting of:

(a) CDRL1, which comprises the sequence SISSYLA (SEQ ID NO: 25),

(b) CDRL2 comprising the sequences GASSRAS (SEQ ID NO: 27) and

(c) CDRL3, which comprises the sequence YYSSPLT (residues 91-97 of SEQ ID NO: 21),

or an affinity matured variant thereof, or a functional fragment of the antibody or the affinity matured variant.

In yet another embodiment, in the bispecific antibody or affinity matured variant thereof, or a functional fragment of the antibody or affinity matured variant, the light chain variable domain,

(a) CDRL1 comprises the sequence SISSYLA (SEQ ID NO: 25),

(b) CDRL2 comprises the sequences GASSRAS (SEQ ID NO: 27) and

(c) CDRL3 comprises the sequence YYSSPLT (residues 91-97 of SEQ ID NO: 21).

In another embodiment, the bispecific antibody, or functional fragment thereof, binds to IL-17A and IL-17F with substantially the same binding affinity.

In yet another embodiment, the bispecific antibody, or functional fragment thereof, is administered in an amount of at least about 10^-10To 10^-11M binds to IL-17A and IL-17F with binding affinity.

In another embodiment, the bispecific antibody, or functional fragment thereof, binds to IL-17A and IL-17F both as monomers and as homodimers or heterodimers.

In yet another embodiment, the bispecific antibody, or functional fragment thereof, binds IL-17A/F of both human and non-human primates, wherein the non-human primate can be, for example, a cynomolgus monkey (cyno) or Rhesus monkey (Rhesus monkey). In another embodiment, the antibody binds to human IL-17A/F and optionally non-human primate IL-17A/F but does not bind to rodents, e.g., rat or mouse IL-17A/F.

In various embodiments, the bispecific antibody, or functional fragment thereof, is capable of reducing demyelination in inflammatory or immune-related diseases.

As before, the bispecific antibody may also be monoclonal, chimeric, humanized or human, and the antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibodyAntibody fragments include, but are not limited to, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and a multispecific antibody formed from the antibody fragment.

In various aspects, the invention relates to the cross-reactive and bispecific antibodies described hereinbefore that inhibit the biological function of IL-17A at a molar ratio of antibody to IL-17A of from about 10: 1 to about 1: 10, or from about 5: 1 to about 1: 5, or from about 1: 1 to about 1: 2, wherein IL-17A represents an IL-17A homodimer.

In yet another aspect, the invention relates to the cross-reactive and bispecific antibodies described hereinbefore which inhibit the biological function of IL-17F in a molar ratio of antibody to IL-17F of from about 10: 1 to about 1: 10, or from about 5: 1 to about 1: 5, or from about 1: 1 to about 1: 3, or from about 1: 1 to about 1: 2, wherein IL-17F represents an IL-17F homodimer.

In yet another aspect, the invention relates to the cross-reactive and bispecific antibodies described hereinbefore that inhibit the biological function of heterodimeric forms of IL-17A and IL-17F at a molar ratio of antibody to heterodimer of about 10: 1 to about 1: 10, or about 5: 1 to about 1: 5, or about 1: 1 to about 1: 3, or about 1: 1 to about 1: 2.

In another aspect, the invention relates to an isolated nucleic acid molecule encoding a light chain of a cross-reactive or bispecific antibody or antibody fragment as described hereinbefore.

In a further aspect, the present invention relates to a recombinant host cell comprising a nucleic acid molecule encoding the light chain of a cross-reactive or bispecific antibody or antibody fragment as described hereinbefore. The host cell may be eukaryotic or prokaryotic, including, for example, Chinese Hamster Ovary (CHO) cells and escherichia coli (e.coli) cells.

The invention also relates to a pharmaceutical composition comprising a cross-reactive or bispecific antibody or antibody fragment as described hereinbefore in admixture with a pharmaceutically acceptable excipient.

Furthermore, the present invention relates to a method of treating an inflammatory or immune related disease comprising administering to a mammalian subject in need thereof an effective amount of a cross-reactive or bispecific antibody of the present invention, or a functional fragment thereof. The mammalian subject is preferably a human patient.

The inflammatory or immune-related disorder may, for example, be selected from the group consisting of: systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathy (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Graye's disease), hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes, immune-mediated nephropathy (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous system such as multiple sclerosis, idiopathic demyelinating polyneuropathy or acute polyneuritis (idiopathic-Barre syndrome), And chronic inflammatory demyelinating polyneuropathy, diseases of the liver and gallbladder such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepadnaviral hepatitis), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, Inflammatory Bowel Disease (IBD) including ulcerative colitis, Crohn's disease, gluten-sensitive enteropathy, and Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skin disease, erythema multiforme and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food allergy and urticaria, immunological diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplant-related diseases including graft rejection and graft-versus-host disease, infectious diseases include viral diseases such as AIDS (HIV infection), hepatitis A, hepatitis B, hepatitis C, hepatitis D and E, herpes, etc., bacterial infections, fungal infections, protozoal infections and parasitic infections.

In a preferred embodiment, the inflammatory or immune-related disease is selected from the group consisting of Rheumatoid Arthritis (RA), Inflammatory Bowel Disease (IBD) and asthma.

In yet another aspect, the present invention relates to an article of manufacture comprising (a) a container; (b) a label on the container; and (c) a composition of matter comprising a cross-reactive or bispecific antibody of the invention contained in the container, wherein the label on the container indicates that the composition of matter is useful for treating an inflammatory or immune-related disease.

The invention also relates to the use of an antibody herein for the manufacture of a medicament for the treatment of an inflammatory or immune-related disease.

Brief Description of Drawings

FIG. 1 shows the nucleotide sequence of the native sequence of human IL-17AcDNA (SEQ ID NO: 1).

FIG. 2 shows the sequence represented by SEQ ID NO: 1(SEQ ID NO: 2).

FIG. 3 shows the nucleotide sequence of the native sequence of human IL-17F cDNA (SEQ ID NO: 3).

FIG. 4 shows the sequence represented by SEQ ID NO: 3(SEQ ID NO: 4).

FIG. 5A shows an alignment of light chain sequences (SEQ ID NOs: 5-14) of IL-17A/F cross-reactive antibodies YW248.65, YW240.27, YW241.47, YW271.25, YW278.15, YW278.27, YW278.57, YW278.60, YW279.1, and YW 280.3.

FIG. 5B shows an alignment of the heavy chain sequences (SEQ I NOs: 51-60) of IL-17A/F cross-reactive antibodies YW248.65, YW240.27, YW241.47, YW271.25, YW278.15, YW278.27, YW278.57, YW278.60, YW279.1, and YW 280.3.

FIG. 5C shows an alignment of the light chain sequences (SEQ ID NOS: 63-68) of IL-17A/F cross-reactive antibodies YW278.15.7, YW278.15.8, YW278.15.9, YW279.1.20, YW279.1.21, and YW279.1.22.

FIG. 5D shows an alignment of the heavy chain sequences (SEQ ID NOS: 69-74) of IL-17A/F cross-reactive antibodies YW278.15.7, YW278.15.8, YW278.15.9, YW279.1.20, YW279.1.21, and YW279.1.22.

FIG. 6 shows an alignment of the light chain sequences of IL-17A/F cross-reactive antibody YW278.15(SEQ ID NO: 9) and its affinity matured variant, YW278.15.18(SEQ ID NO: 15), YW278.15.2(SEQ ID NO: 16) and YW278.15.3(SEQ ID NO: 17). CDRL1, CDRL2 and CDRL3 sequences are indicated in boxes.

FIG. 7 shows an alignment of the heavy chain sequences of IL-17A/F cross-reactive antibody YW278.15(SEQ ID NO: 92) and its affinity matured variant, YW278.15.18(SEQ ID NO: 93), YW278.15.2(SEQ ID NO: 94), YW278.15.2.D54E (SEQ ID NO: 95), YW278.15.3(SEQ ID NO: 92), YW278.15.18C55A (SEQ ID NO: 18) and YW278.15.18C55S (SEQ ID NO: 19). CDRL1, CDRL2 and CDRL3 sequences are indicated in boxes. Note: YW278.15.18C55A and YW278.15.18C55S have the same light chain as YW278.15.18.

FIGS. 8A and 8B show the results of BIAcore immunoassays performed with three affinity matured IL-17A/F cross-reactive antibodies. A. The binding affinities (Kd (M)) for recombinant human IL-17A (rhIL-17A), recombinant human IL-17F (rhIL-17F), cynomolgus monkey IL-17F and human IL-17A/F in solution are shown in the last column. B. The binding affinities (Kd (M)) for recombinant human IL-17A (rhIL-17A), recombinant human IL-17F (rhIL-17F), cynomolgus monkey IL-17F, cynomolgus monkey IL-17A (FIG. 8B) and human IL-17A/F in solution are shown in the last column.

FIG. 9 shows the results of BIAcore immunoassays performed by comparing two further affinity matured IL-17A/F cross reactive antibodies to YW278.15.18. The binding affinities (Kd (M)) for recombinant human IL-17A (rhIL-17A), recombinant human IL-17F (rhIL-17F), cynomolgus monkey IL-17F and human IL-17A/F in solution are shown in the last column.

FIG. 10 shows the results of BIAcore immunoassays performed with three affinity matured IL-17A/F cross reactive antibodies. Binding affinity to rhesus IL-17A is shown in the last column.

FIG. 11 shows an alignment of the light chain variable region sequences of IL-17A-specific antibody YW264.21(SEQ ID NO: 20) and IL-17F-specific antibody YW265.01(SEQ ID NO: 21).

FIG. 12 shows an alignment of the heavy chain variable region sequences of IL-17A-specific antibody YW264.21(SEQ ID NO: 22) and IL-17F-specific antibody YW265.01(SEQ ID NO: 23).

FIGS. 13A and 13B show the IC50 and IC90 data for several IL-17A/F cross-reactive antibodies, as well as the molar ratio of antibodies required to inhibit the activity of the target IL-17A/F polypeptide.

FIG. 14 shows IL-17A and IL-17F binding affinity data for IL-17A/F bispecific antibodies compared to anti-IL-17A antibody YW264.1 and anti-IL-17F antibody YW 265.01.

FIG. 15 shows another alignment of the light chain variable region sequences (SEQ ID NOS61 and 20-21) of IL-17A/F bispecific antibodies, including antibody YW264.03(SEQ ID NO: 61).

FIG. 16 shows another alignment of the heavy chain variable region sequences (SEQ ID NOS62 and 22-23) of an IL-17A/F bispecific antibody including YW264.03(SEQ ID NO: 62).

FIG. 17 shows Fab binding to IL-17F dimer.

FIG. 18 shows epitopes for Fab on IL-17F dimer.

Detailed Description

I. Definition of

Unless defined otherwise, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some instances, terms having patch-understood meanings are defined herein for clarity and/or ease of reference, and the inclusion of such definitions herein is not necessarily to be construed as representing a substantial difference from what is commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are commonly employed using conventional methods, such as, for example, the widely used methods of molecular cloning as described in the following references: sambrook et al, Molecular Cloning: a Laboratory Manual (molecular cloning: Laboratory Manual) second edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.. Procedures involving the use of commercially available kits and reagents, if appropriate, are generally carried out according to manufacturer-specified protocols and/or parameters, unless otherwise indicated.

"native sequence IL-17A/F polypeptides" include polypeptides having the same amino acid sequence as a corresponding IL-17A/F polypeptide derived from nature. Such native sequence IL-17A/F polypeptides may be isolated from nature, or may be produced by recombinant and/or synthetic means. The term "native sequence IL-17A/F polypeptide" specifically encompasses naturally occurring truncated or secreted forms of a particular IL-17A/F polypeptide (e.g., an extracellular domain sequence), naturally occurring variant forms of the polypeptide (e.g., alternatively spliced forms), and naturally occurring allelic variants. In various embodiments of the invention, the native sequence IL-17A/F polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acid sequences shown in the figures. In this figure, the start and stop codons are shown in bold and underlined.

An "IL-17A/F polypeptide variant" refers to an active IL-17A/F polypeptide as defined above or below having at least about 80% amino acid sequence identity to a full-length native sequence IL-17A/F polypeptide sequence disclosed herein, an IL-17A/F polypeptide sequence lacking a signal peptide disclosed herein, or any other fragment of a full-length IL-17A/F polypeptide sequence disclosed herein. Such IL-17A/F polypeptide variants include, for example, IL-17A/F polypeptides in which one or more amino acid residues are added or deleted from the N-or C-terminus of the full-length native amino acid sequence. Typically, an IL-17A/F polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, with a full-length native sequence IL-17A/F polypeptide sequence disclosed herein, an IL-17A/F polypeptide sequence disclosed herein lacking a signal peptide, or any other specifically defined fragment of a full-length IL-17A/F polypeptide sequence disclosed herein, or a fragment thereof, Alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity, and alternatively at least about 99% amino acid sequence identity. Typically, an IL-17A/F variant polypeptide is at least about 10 amino acids in length, alternatively at least about 20 amino acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 300 amino acids in length, or longer. The same definition applies to further variants of the variant IL-17A/F polypeptides.

"percent (%) amino acid sequence identity" with respect to an IL-17A/F polypeptide sequence identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in a particular IL-17A/F polypeptide sequence after aligning the candidate sequence with the particular IL-17A/F polypeptide sequence (and introducing gaps, if necessary) to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to obtain maximum alignment over the full length of the sequences being compared. To this end, however, a% amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The author of the ALIGN-2 sequence alignment computer program was Genentech, inc, and the source code was submitted with a user document to the U.S. copyright office (Washington d.c., 20559) with U.S. copyright registration number TXU 510087. The ALIGN-2 program is publicly available through Genentech, Inc. (South SanFrancisco, Calif.) or is compiled from this source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably on the numeral UNIXV4.0D. All sequence alignment parameters were set by the ALIGN-2 program and were unchanged.

In the case of ALIGN-2 as applied to amino acid sequence comparisons, the amino acid sequence identity% (or so: a given amino acid sequence A has or contains some% amino acid sequence identity with respect to, with, or against a given amino acid sequence B) of a given amino acid sequence A with respect to (to), with (with), or against (against) is calculated as follows: the X/Y ratio is multiplied by 100, where X is the number of amino acid residues scored as identical matches in the A and B alignments of the sequence alignment program ALIGN-2, and where Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence a is not equal to the length of amino acid sequence B, the% amino acid sequence identity of a to B will not equal the% amino acid sequence identity of B to a.

Unless specifically stated otherwise, all% amino acid sequence identity values used herein are obtained as described in the preceding paragraph using the ALIGN-2 computer program. However, the% value of amino acid sequence identity can also be obtained using the WU-BLAST-2 computer program as will be described below (Altschul et al, Methods in Enzymology 266: 460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to default values. Those parameters that are not set to default values, such as tunable parameters, may be set with the following values: overlap span (overlap span) is 1, overlap fraction (overlap fraction) is 0.125, word threshold (T) is 11, and scoring matrix (scoring matrix) is BLOSUM 62. Using WU-BLAST-2, the value of% amino acid sequence identity is obtained as follows: the number of amino acid residues (a) that are a perfect match between the amino acid sequence of the polypeptide of interest (which has a sequence derived from the native polypeptide), and the comparative amino acid sequence of interest (i.e., the sequence compared to the polypeptide of interest, which may be an IL-17A/F variant polypeptide), as determined by WU-BLAST-2, is divided by the total number of amino acid residues (b) of the polypeptide of interest. For example, in the description of "a polypeptide comprising an amino acid sequence a having at least 80% amino acid sequence identity to amino acid sequence B", amino acid sequence a is a comparative amino acid sequence of the "comparative protein" of interest, and amino acid sequence B is an amino acid sequence of the polypeptide of interest.

The percent amino acid sequence identity can also be determined using the sequence alignment program NCBI-BLAST2(Altschul et al, Nucleic Acids Res. (Nucleic Acids research) 25: 3389-3402 (1997)). The NCBI-BLAST2 sequence alignment program was downloaded from http:// www.ncbi.nlm.nih.gov or from the National Institute of Health (National institutes of Health), Bethesda, MD.. NCBI-BLAST2 uses several search parameters, all of which are set to default values, including, for example, uncovered (uncovered) is (yes), chain (strand) is all (all), expected number of occurrences is 10, minimum low complexity length 15/5, multiple pass e-value is 0.01, multiple pass constant is 25, missing for final gap alignment is 25, and scoring matrix is BLOSUM 62.

The amino acid sequence identity of a given amino acid sequence A relative to, with, or against a given amino acid sequence B when aligned using NCBI-BLAST2 (or so-called: a given amino acid sequence A has or contains some% amino acid sequence identity relative to, with, or against a given amino acid sequence B) is calculated as follows: the X/Y ratio is multiplied by 100, where X is the number of amino acid residues scored as identical matches in the A and B alignments of the program using the sequence alignment program NCBI-BLAST2, and where Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence a is not equal to the length of amino acid sequence B, the% amino acid sequence identity of a to B will not equal the% amino acid sequence identity of B to a.

An "IL-17A/F variant polynucleotide" or "IL-17A/F variant nucleic acid sequence" refers to a nucleic acid molecule as defined below that encodes an active IL-17A/F polypeptide and that has at least about 80% nucleic acid sequence identity to a nucleotide sequence encoding a full-length native sequence IL-17A/F polypeptide sequence disclosed herein, a full-length native sequence IL-17A/F polypeptide sequence lacking a signal peptide disclosed herein, or any other fragment of a full-length IL-17A/F polypeptide sequence disclosed herein. Typically, an IL-17A/F variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, with a nucleotide sequence encoding the full-length native sequence IL-17A/F polypeptide sequences disclosed herein, the full-length native sequence IL-17A/F polypeptide sequences disclosed herein lacking a signal peptide, or any other fragment of the full-length IL-17A/F polypeptide sequences disclosed herein, Alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity, and alternatively at least about 99% nucleic acid sequence identity. Variants do not include the native nucleotide sequence.

Typically, an IL-17A/F variant polynucleotide is at least about 30 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 900 nucleotides in length, or longer.

"percent (%) nucleic acid sequence identity" with respect to an IL-17A/F encoding nucleic acid sequence identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in the IL-17A/F nucleic acid sequence of interest after aligning the candidate sequence with the IL-17A/F nucleic acid sequence of interest (and introducing gaps, if necessary) to obtain the maximum percentage of sequence identity. Sequence alignments can be performed using various methods in the art to determine percent nucleic acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. To this end, however, the% nucleic acid sequence identity value is generated using the sequence comparison computer program ALIGN-2, the complete source code of which has been submitted with the user profile to the U.S. copyright office (Washington d.c., 20559) under U.S. copyright registration No. TXU 510087. The ALIGN-2 program is publicly available through Genentech, Inc. (South San Francisco, Calif.) or is compiled from this source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably on digital UNIX V4.0D. All sequence alignment parameters were set by the ALIGN-2 program and were unchanged.

In the case of ALIGN-2 as applied to nucleic acid sequence comparison, the percent nucleic acid sequence identity (or in other words: a given nucleic acid sequence C has or contains some percent nucleic acid sequence identity relative to, with, or against a given nucleic acid sequence D) of a given nucleic acid sequence C relative to, with, or against a given nucleic acid sequence D is calculated as follows: the W/Z ratio is multiplied by 100, where W is the number of nucleotides scored as identical matches in the C and D alignments of the sequence alignment program ALIGN-2, and where Z is the total number of nucleotides in D. It will be understood that when the length of nucleic acid sequence C is not equal to that of nucleic acid sequence D, the% nucleic acid sequence identity of C relative to D will not equal the% nucleic acid sequence identity of D relative to C.

Unless specifically stated otherwise, all values of% nucleic acid sequence identity as used herein are obtained as described in the preceding paragraph using the ALIGN-2 computer program. However, the% value for nucleic acid sequence identity can also be obtained using the WU-BLAST-2 computer program as will be described below (Altschul et al, Methods in Enzymology 266: 460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to default values. Those parameters that are not set as default values, i.e. adjustable parameters, can be set with the following values: overlap span (overlap span) is 1, overlap fraction (overlap fraction) is 0.125, word threshold (T) is 11, and scoring matrix (scoring matrix) is BLOSUM 62. Using WU-BLAST-2, the value of% nucleic acid sequence identity is obtained as follows: the number of perfectly matched nucleotides between a nucleic acid sequence encoding a nucleic acid molecule of interest encoding an IL-17A/F polypeptide (which has a sequence derived from a nucleic acid encoding a native sequence IL-17A/F polypeptide), as determined by WU-BLAST-2, and a comparison nucleic acid sequence of interest (i.e., a sequence compared to a nucleic acid molecule of interest encoding an IL-17A/F polypeptide, which may be a variant IL-17A/F polynucleotide), is divided by the total number of nucleotides of a nucleic acid molecule of interest encoding an IL-17A/F polypeptide (a). For example, in the context of "an isolated nucleic acid molecule comprising a nucleic acid sequence A having at least 80% nucleic acid sequence identity to nucleic acid sequence B", nucleic acid sequence A is a comparison nucleic acid molecule of interest and nucleic acid sequence B is the nucleic acid sequence of a nucleic acid molecule of interest encoding an IL-17A/F polypeptide.

Percent Nucleic acid sequence identity can also be determined using the sequence alignment program NCBI-BLAST2(Altschul et al, Nucleic Acids Res. (Nucleic Acids research) 25: 3389-3402 (1997)). The NCBI-BLAST2 sequence alignment program was downloaded from http:// www.ncbi.nim.nih.gov or from the National Institute of Health (National institutes of Health), Bethesda, Md.. NCBI-BLAST2 uses several search parameters, all of which are set to default values, including, for example, uncovered (uncovered) is (yes), chain (strand) is all (all), expected number of occurrences is 10, minimum low complexity length 15/5, multiple pass e-value is 0.01, multiple pass constant is 25, missing for final gap alignment is 25, and scoring matrix is BLOSUM 62.

When comparing nucleic acid sequences using NCBI-BLAST2, the percent nucleic acid sequence identity (or in other words: a given nucleic acid sequence C has or contains some percent nucleic acid sequence identity relative to, with, or against a given nucleic acid sequence D) for a given nucleic acid sequence C relative to, with, or against a given nucleic acid sequence D is calculated as follows: the W/Z ratio is multiplied by 100, where W is the number of nucleotides scored as identical matches in the C and D alignments of the sequence alignment program using the sequence alignment program NCBI-BLAST2, and where Z is the total number of nucleotides in D. It will be understood that when the length of nucleic acid sequence C is not equal to that of nucleic acid sequence D, the% nucleic acid sequence identity of C relative to D will not equal the% nucleic acid sequence identity of D relative to C.

In other embodiments, IL-17A/F variant polynucleotides are nucleic acid molecules that encode active IL-17A/F polypeptides and are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length IL-17A/F polypeptides disclosed herein. The IL-17A/F variant polypeptides may be those encoded by IL-17A/F variant polynucleotides.

As used in describing the various polypeptides disclosed herein, "isolated" refers to a polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment refer to substances that would normally interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the polypeptide is purified (1) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a rotor sequencer, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue (or preferably silver staining). Since at least one component of the natural environment of the IL-17A/F polypeptide is not present, isolated polypeptides include polypeptides in situ within recombinant cells. However, isolated polypeptides are typically prepared by at least one purification step.

Nucleic acids encoding "isolated" IL-17A/F polypeptides or nucleic acids encoding other polypeptides refer to nucleic acid molecules that have been identified and separated from at least one contaminating nucleic acid molecule with which they are normally associated in the natural source of the nucleic acid encoding the polypeptide. Nucleic acid molecules encoding an isolated polypeptide differ from the form or situation in which they are found in nature. Nucleic acid molecules encoding an isolated polypeptide are distinguished from nucleic acid molecules encoding a particular polypeptide when present in a native cell. However, a nucleic acid molecule encoding an isolated polypeptide includes a nucleic acid molecule encoding a polypeptide contained in a cell that normally expresses the polypeptide, where, for example, the nucleic acid molecule is in a different chromosomal location than in the native cell.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is "operably linked" if it is in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence; alternatively, if the ribosome binding site is positioned to facilitate translation, it is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous. Ligation may be achieved by ligation reactions at appropriate restriction sites. If there are no such sites, synthetic oligonucleotide adaptors or linkers can be used in accordance with conventional practice.

The "stringency" of a hybridization reaction can be readily determined by one of ordinary skill in the art, and is generally calculated empirically based on probe length, wash temperature, and salt concentration. Generally, longer probes require higher temperatures for proper renaturation, while shorter probes require lower temperatures. Hybridization generally relies on the ability of denatured DNA to renature when complementary strands are present in an environment below their melting temperature. The higher the degree of homology desired between the probe and hybridizable sequence, the higher the relative temperature that can be used. It is therefore believed that higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures are less stringent. For additional details and explanations of the stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology (protocol in modern Molecular Biology), Wiley Interscience Publishers (1995).

"stringent conditions" or "high stringency conditions" as defined herein can be determined as follows: (1) washing with low ionic strength and high temperature, e.g., 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate, 50 ℃; (2) denaturing agents such as formamide, e.g., 50% (v/v) formamide and 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer pH6.5 with 750mM sodium chloride, 75mM sodium citrate, 42 ℃; or (3) washed at 42 ℃ with 50% formamide, 5XSSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solution, sonicated salmon sperm DNA (50. mu.g/ml), 0.1% SDS, and 10% dextran sulfate (at 42 ℃), washed at 55 ℃ in 0.2XSSC (sodium chloride/sodium citrate) and 50% formamide, followed by a high stringency wash at 55 ℃ in 0.1XSSC with EDTA.

"moderately stringent conditions" can be as defined in Sambrook et al, Molecular Cloning: ALaborory Manual (molecular cloning: laboratory Manual), New York: cold Spring harborPress, 1989, including the use of less stringent wash solutions and hybridization conditions (e.g., temperature, ionic strength, and% SDS) than those described above. An example of moderately stringent conditions is incubation at 37 ℃ overnight in a solution containing 20% formamide, 5XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 XDenhardt's solution, 10% dextran sulfate, and 20mg/ml denatured sheared salmon sperm DNA, followed by washing the filter at about 37-50 ℃ in 1 XSSC. The skilled person will recognize how to adjust the temperature, ionic strength, etc. as required to accommodate factors such as probe length.

The term "epitope tagged" as used herein refers to a chimeric polypeptide comprising an IL-17A/F polypeptide fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, but is short enough so that it does not interfere with the activity of the polypeptide to which it is fused. The tag polypeptide is also preferably quite unique such that the antibody is substantially non-cross-reactive with other epitopes. Suitable tag polypeptides typically have at least 6 amino acid residues and typically between about 8 and about 50 amino acid residues (preferably between about 10 and about 20 amino acid residues).

The term "immunoadhesin" as used herein refers to antibody-like molecules that combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, immunoadhesins comprise fusions of amino acid sequences and immunoglobulin constant domain sequences having a desired binding specificity that is different from the antigen recognition and binding site of the antibody (i.e., is "heterologous"). The adhesin part of an immunoadhesin molecule is typically a contiguous amino acid sequence comprising at least the binding site for a receptor or ligand. The immunoglobulin constant domain sequence in immunoadhesins can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits, or neutralizes a biological activity of a native IL-17A/F polypeptide disclosed herein. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics the biological activity of the native IL-17A/F polypeptides disclosed herein. Suitable agonist or antagonist molecules specifically include agonistic or antagonistic antibodies or antibody fragments, fragments or amino acid sequence variants of the native IL-17A/F polypeptide, peptides, antisense oligonucleotides, small organic molecules, and the like. Methods for identifying agonists or antagonists of an IL-17A/F polypeptide may comprise contacting an IL-17A/F polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the IL-17A/F polypeptide.

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the progression of the targeted pathological state or disorder. Those in need of treatment include those already with the disorder as well as those prone to the disease or those in which the disorder is to be prevented.

"Long-term" administration refers to the administration of one or more agents in a continuous mode, as opposed to a short-term mode, to maintain the initial therapeutic effect (activity) for an extended period of time. By "intermittent" administration is meant treatment that is not continuous, uninterrupted, but rather periodic in nature.

For therapeutic purposes, "mammal" refers to any animal classified as a mammal, including humans, non-human higher primates, livestock and farm animals, and zoo, sports, or pet animals such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, and the like. The mammal preferably refers to a human.

Administration of one or more other therapeutic agents "in combination" includes simultaneous (co-) administration and sequential administration in any order.

As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers, which are non-toxic to the cells or mammal to which they are exposed at the dosages and concentrations employed. Typically, the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN^TMPolyethylene glycol (PEG) and PLURONICS^TM。

The term "antibody" is used in the broadest sense and specifically covers, for example, single anti-IL-17A/F or anti-IL-17A or anti-IL-17F monoclonal antibodies (including agonistic, antagonistic and neutralizing antibodies), corresponding antibody compositions with polyepitopic specificity, polyclonal antibodies, single chain antibodies, and antibody fragments (see below), so long as they exhibit the desired biological or immunological activity. The term "immunoglobulin" (Ig) is used interchangeably herein with antibody.

An "isolated antibody" refers to an antibody that has been identified and separated from and/or recovered from a component of its natural environment. Contaminant components of its natural environment refer to substances that interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified (1) to greater than 95% by weight, most preferably greater than 99% by weight of the antibody as determined by the Lowry method, (2) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by use of a rotor sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue (or preferably silver staining). An isolated antibody includes an antibody in situ within a recombinant cell, since at least one component of the antibody's natural environment will not be present. However, isolated antibodies are typically prepared by at least one purification step.

The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H) (IgM antibodies are composed of 5 basic heterotetrameric units and an additional polypeptide called the J chain, and thus contain 10 antigen-binding sites; secretory IgA antibodies can be polymerized to form multivalent assemblies containing 2-5 basic 4-chain units and the J chain). In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, the number of disulfide bonds depending on the isotype of the heavy chain. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain at the N-terminus (V)_H) Followed by three constant domains (C)_H) (for each of the alpha and gamma chains) And four C_HDomains (for μ and ε isoforms). Each light chain has a variable domain at the N-terminus (V)_L) Followed by a constant domain at its other end (C)_L)。V_LAnd V_HAre arranged side by side, and C_LTo the first constant domain (C) of the heavy chain_H1) And are arranged side by side. Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. Paired V_HAnd V_LTogether forming an antigen binding site. For the structure and properties of different classes of antibodies, see, e.g.Basic and Clinical ImmunologyEighth edition, Daniel p.stites, Abba i.terr and Tristram g.parslow (editors), Appleton&Lange, Norwalk, conn., 1994, page 71 and chapter 6.

Light chains from any vertebrate species can be classified into one of two distinct types called kappa and lambda, depending on their constant domain amino acid sequences. Depending on its heavy chain (c.sub.h) constant domain amino acid sequence, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, have heavy chains called α, δ, ε, γ and μ, respectively. According to C_HWith relatively minor differences in sequence and function, the γ and α classes can be further divided into subclasses, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2.

The term "variable" refers to the case where certain segments in the variable domains differ extensively in sequence in an antibody. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110 amino acid span of the variable domain. In fact, the V region is composed of relatively invariant segments of 15-30 amino acids called Framework Regions (FRs) and shorter regions of 9-12 amino acids each of which is highly variable called "hypervariable regions" separating the framework regions. The variable domains of native heavy and light chains each comprise four FRs, which largely adopt a β -sheet conformation, connected by three hypervariable regions that form loops connecting, and in some cases forming part of, the β -sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and together with the hypervariable regions from the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition Public Health Service, national institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions typically comprise amino acid residues from a "complementarity determining region" or "CDR" (e.g., V)_LAbout residues 24-34(L1), 50-56(L2) and 89-97(L3), and V_HAbout 1-35(H1), 50-65(H2) and 95-102 (H3); kabat et al, Sequences of proteins of Immunological Interest, fifth edition public Health Service, National Institutes of Health (National Institutes of Health), Bethesda, Md. (1991)) and/or those residues from "hypervariable loops" (e.g., V.V._LIn residues 26-32(L1), 50-52(L2) and 91-96(L3), and V_HIn the vicinity of residues 26-32(H1), 53-55(H2) and 96-101 (H3); chothia and Lesk j.mol.biol. (journal of molecular biology) 196: 901-917(1987)).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention can be prepared by the hybridoma method (first described by Kohler et al, Nature (Nature), 256: 495 (1975)), or can be prepared by recombinant DNA methods in bacteria, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No.4,816,567). "monoclonal antibodies" can also be used, for example, in Clackson et al, Nature (Nature), 352: 624-: 581-597(1991) from phage antibody libraries.

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

An "intact" antibody is an antibody comprising an antigen binding site and c.sub.l and at least the heavy chain constant domains CH1, CH2 and CH 3. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. Preferably, the whole antibody has one or more effector functions.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; linear antibodies (see U.S. Pat. No.5,641,870, example 2; Zapata et al, Protein eng. (protein engineering) 8 (10): 1057-1062[1995]) (ii) a A single chain antibody molecule; and multispecific antibodies formed from antibody fragments. In a preferred embodiment, the fragment is "functional", i.e., qualitatively retains the ability of the corresponding intact antibody to bind to the target IL-17A and IL-17F polypeptides, and also qualitatively retains such inhibitory properties if the intact antibody also inhibits the biological activity or function of IL-17A/F. Qualitatively retained means that activity is maintained in type, but the degree of binding affinity and/or activity may vary.

Digestion of an antibody with papain produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. Fab fragments consist of intact light and heavy chain variable domains (V)_H) And, the first constant domain (C) of one heavy chain_H1) And (4) forming. Each Fab fragment is monovalent for antigen binding, i.e. it has one antigen binding site. Pepsin treatment of the antibody produced a larger F (ab')₂A fragment, roughly equivalent to two Fab fragments linked by a disulfide bond, having bivalent antigen binding activity and still capable of cross-linking antigen. Fab' fragment is due to C_H1The carboxy-terminal end of the domain is differentiated from the Fab fragment by the addition of an additional few residues, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residue of the constant domain carries a free thiol group. F (ab')₂Antibody fragments were originally produced as pairs of Fab 'fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of two heavy chains held together by disulfide bonds. The effector function of an antibody is determined by sequences in the Fc region, which is also the portion recognized by Fc receptors (FcR) found on certain types of cells.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in close, non-covalent association. Six hypervariable loops (3 loops each for the heavy and light chains) which contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody are highlighted from the folding of these two domains. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, however with lower affinity than the entire binding site.

"single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments comprising v.sub.h and v.sub.l antibody domains joined into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the v.sub.h and v.sub.l domains such that the sFv forms the desired structure for antigen binding. For reviews on sFv see Pluckthun in The Pharmacology of monoclonal Antibodies (monoclonal antibody Pharmacology), vol.113, Rosenburg and Moore eds, Springer-Verlag, New York, pp.269-315 (1994); borebaeck 1995, see below.

The term "diabodies" refers to small antibody fragments prepared by using short linkers (about 5-10 residues) at V_HAnd V_LsFv fragments (see preceding paragraph) were constructed between domains to obtain inter-chain rather than intra-chain pairing of the V domains to generate bivalent fragments, i.e., fragments with two antigen binding sites. Bispecific diabodies are heterodimers of two "cross" sFv fragments, where the V of the two antibodies_HAnd V_LThe domains are present on different polypeptide chains. Diabodies are described, for example, in EP404,097; WO 93/11161; and Hollinger et al, proc.natl.acad.sci. (journal of the american academy of sciences) USA, 90: 6444- > 6448 (1993).

"humanized" forms of non-human (e.g., rodent) antibodies refer to chimeric antibodies that contain, at a minimum, sequences derived from non-human antibodies. For the most part, humanized antibodies refer to human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired antibody specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibody. In general, a humanized antibody will comprise substantially all of the variable domains of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See Jones et al, Nature 321 for more details: 522-525 (1986); riechmann et al, Nature (Nature) 332: 323-329 (1988); and Presta, curr.op.struct.biol. (new view of structural biology) 2: 593-596(1992).

A "cross-reactive antibody" is an antibody that recognizes the same or similar epitope on more than one antigen. Thus, the cross-reactive antibodies of the invention recognize the same or similar epitopes present on both IL-17A and IL-17F. In a specific embodiment, the cross-reactive antibody binds to both IL-17A and IL-17F using the same or substantially the same paratope. Preferably, the cross-reactive antibodies herein also block the function (activity) of both IL-17A and IL-17F.

The term "paratope" as used herein refers to the portion of an antibody that binds to a target antigen.

A "species-dependent antibody" such as a mammalian anti-IL-17A/F antibody is an antibody that has a stronger binding affinity for an antigen from a first mammalian species than it does for a homolog of the antigen from a second mammalian species. Normally, the species-dependent antibody "specifically" binds to a human antigen (i.e., has no more than about 1x 10)^-7M, preferably not more than about 1x10^-8M and is optimalPreferably no more than about 1x10^-9M, but has an affinity for a homolog of the antigen from a second non-human mammalian species that is at least about 50-fold weaker than its binding affinity for the human antigen, or at least about 500-fold weaker than its binding affinity for the human antigen. The species-dependent antigen may be any of a variety of antibody types as defined above, but is preferably a humanized or human antibody.

An antibody that "binds" an antigen of interest is one that binds the antigen with sufficient affinity for the antibody to be used as a diagnostic and/or therapeutic agent that targets cells or tissues that express the antigen, and does not significantly cross-react with other proteins. In such embodiments, the antibody binds to a "non-target" protein to less than about 10% of the binding of the antibody to its particular target protein, as determined by Fluorescence Activated Cell Sorting (FACS) analysis or Radioimmunoprecipitation (RIA). With respect to binding of an antibody to a target molecule, the term "specifically binds" or "specifically binds to" a particular polypeptide or an epitope on a target of a particular polypeptide means that binding other than non-specific interaction can be measured. Specific binding can be determined, for example, by measuring binding of the molecule and comparing it to binding of a control molecule, which is typically a molecule of similar structure but lacking binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, e.g., an excess of unlabeled target. In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the term "specifically binds" or "specifically binds to" or "is specific for" a particular polypeptide or an epitope on a target of a particular polypeptide can be manifested by the molecule having, for example, a Kd for the target of at least about 10^-4M, alternatively at least about 10^-5M, alternatively at least about 10^-6M, alternatively at least about 10^-7M, alternatively at least about 10^-8M, alternatively at least about 10^-9M, alternatively at least about 10^-10M, alternatively at least about 10^-11M, alternatively at least about10^-12M, or larger molecules. In one embodiment, the term "specifically binds" refers to binding wherein the molecule binds to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. In a preferred embodiment, the specific binding affinity is at least about 10^-10M。

Antibody "effector functions" refer to those biological activities attributable to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region), and which vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and complement dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) cause these cytotoxic effector cells to specifically bind to antigen-bearing target cells, followed by killing of the target cells with cytotoxins. The antibody "arms" the cytotoxic cells and is absolutely necessary for such killing. The main cells mediating ADCC (NK cells) express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravatch and Kinet, annu.rev.immunol. (annual review of immunology) 9: 457-92(1991) page table 3 summarizes FcR expression on hematopoietic cells. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as described in U.S. patent No.5,500,362 or 5,821,337. Effector cells useful in such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of a molecule of interest may be assessed in vivo, for example in animal models such as Clynes et al proc.natl.acad.sci.u.s.a. (journal of the american academy of sciences) 95: 652-.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is one that binds an IgG antibody (gamma receptor) and which includes receptors of the Fc γ RI, Fc γ RII and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA comprises an Immunoreceptor Tyrosine Activation Motif (ITAM) in its cytoplasmic domain. The inhibitory receptor Fc γ RIIB comprises an Immunoreceptor Tyrosine Inhibitory Motif (ITIM) in its cytoplasmic domain (see review M.in Daeron, Annu.Rev.Immunol. (annual review in immunology) 15: 203-234 (1997)). For a review of fcrs see ravatch and Kinet, annu.rev.immunol. (annual review of immunology) 9: 457-492 (1991); capel et al, immunolmethods (immunization methods) 4: 25-34 (1994); and de Haas et al, j.lab.clin.med. (journal of laboratory clinical medicine) 126: 330-41(1995). The term "FcR" encompasses other fcrs herein, including those that will be identified in the future. The term also includes the neonatal receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, j.immunol. (journal of immunology) 117: 587(1976) and Kim et al, j.immunol. (journal of immunology) 24: 249 (1994)).

"human effector cells" refer to leukocytes which express one or more fcrs and perform effector functions. Preferably, the cell expresses at least Fc γ RIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; among them, PBMC and NK cells are preferable. The effector cells may be isolated from a natural source, for example from blood.

"complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that bind to its cognate antigen. To assess complement activation, CDC assays can be performed, such as Gazzano-Santoro et al, immunol. methods (immunological methods) 202: 163 (1996).

The word "label" as used herein refers to a detectable compound or composition that is conjugated, directly or indirectly, to an antibody to produce a "labeled" antibody. The label may be detectable by itself (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzymatic label, may catalyze detectable chemical alteration of a substrate compound or composition.

"solid phase" refers to a non-aqueous matrix to which the antibody of the invention may be attached. Examples of solid phases contemplated herein include those made partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicone. In certain embodiments, depending on the context, the solid phase may comprise the wells of an assay plate; in other embodiments, it refers to a purification column (e.g., an affinity chromatography column). This term also includes discontinuous solid phases of discrete particles, such as those described in U.S. Pat. No.4,275,149.

"liposomes" refers to vesicles composed of various types of lipids, phospholipids, and/or surfactants that can be used to deliver drugs (e.g., IL-17A/F polypeptides or antibodies thereto) to mammals. The components of liposomes are generally arranged in bilayer form, similar to the lipid arrangement of biological membranes.

For purposes herein, "active" or "activity" or "function" when referring to an IL-17A/F polypeptide refers to one or more forms of the IL-17A/F polypeptide that retain the biological and/or immunological activity of the native or naturally-occurring IL-17A/F polypeptide, wherein "biological" activity refers to the biological function (either inhibitory or stimulatory) caused by the native or naturally-occurring IL-17A/F polypeptide in addition to inducing antibody production against an antigenic epitope possessed by the native or naturally-occurring IL-17A/F polypeptide, and "immunological" activity refers to the ability to induce antibody production against an epitope possessed by the native or naturally-occurring IL-17A/F polypeptide. One preferred biological activity includes induction of activation of NF-. kappa.B and stimulation of the production of the pro-inflammatory chemokines IL-8 and IL-6. Another one isOne preferred biological activity includes stimulation of peripheral blood mononuclear cells or CD4⁺A cell. Another preferred biological activity includes stimulation of T lymphocyte proliferation. Another preferred biological activity includes, for example, the release of TNF- α from THP1 cells. Another activity includes enhancing matrix synthesis in articular cartilage. Alternatively, another activity includes promoting articular cartilage matrix degradation and inhibiting matrix synthesis. Another preferred biological activity includes modulating the level of interleukin-17 signaling pathway during the moderate to severe phase of inflammatory bowel disease or during stroke.

With respect to anti-IL-17A/F (or anti-IL-17A or anti-IL-17F) antibodies, the terms "active" or "activity" or "function" and grammatical variations thereof, are used to refer to the ability to inhibit (blocking or antagonistic) or mimic (agonistic) at least one of the foregoing activities. Antibodies and antibody fragments that are said to be "functional" are characterized by such properties.

"immunological" activity refers only to the ability to induce the production of antibodies to epitopes possessed by native or naturally occurring IL-17A/F polypeptides.

Degenerative cartilage disease describes a number of diseases characterized primarily by the destruction of the cartilage matrix. Other pathologies include increased nitric oxide production and proteoglycan degradation. Exemplary conditions encompassed within this definition include, for example, arthritis (e.g., osteoarthritis, rheumatoid arthritis, psoriatic arthritis).

The term "immune-related disorder" refers to a disorder that is caused, mediated, or otherwise contributes to the pathogenesis of a mammal by a component of the mammalian immune system. Also included are diseases in which stimulation or intervention of the immune response has an ameliorating effect on disease progression. The term includes immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, neoplasias, and the like.

The term "T cell mediated disease" refers to a disease in which T cells directly or indirectly mediate or otherwise contribute to pathogenesis in a mammal. T cell mediated diseases may be associated with cell mediated effects, lymphokine mediated effects, and the like, and even effects associated with B cells if stimulated, e.g., by lymphokines secreted by T cells.

Examples of immune-related and inflammatory diseases which may be treated according to the invention, some of which are immune or T cell mediated, include systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (grave's disease), hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes, immune-mediated nephropathy (glomerulonephritis, multiple sclerosis, multiple, Tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous system such as multiple sclerosis, idiopathic demyelinating polyneuropathy or acute idiopathic polyneuritis (Guillain-Barresyndrome), and chronic inflammatory demyelinating polyneuropathy, diseases of the liver and gallbladder such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepadnaviruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, Inflammatory Bowel Diseases (IBD) including ulcerative colitis, Crohn's disease, rhinitis enteropathy, and Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skin disease, erythema multiforme and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic diseases, erythema multiforme, and contact dermatitis, Atopic dermatitis, food allergy and urticaria, immunological diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, graft-related diseases including graft rejection and graft-versus-host disease, infectious diseases including viral diseases such as AIDS (HIV infection), hepatitis a, hepatitis b, hepatitis c, hepatitis d and e, herpes and the like, bacterial infections, fungal infections, protozoal infections and parasitic infections.

The term "effective amount" is the concentration or amount of the IL-17A/F polypeptide and/or agonist/antagonist that results in the particular specification being made. An "effective amount" of an IL-17A/F polypeptide or agonist or antagonist thereof can be determined empirically. In addition, a "therapeutically effective amount" is a concentration or amount of an IL-17A/F polypeptide and/or agonist/antagonist effective to achieve the indicated therapeutic effect. This amount may also be determined empirically.

Detailed description of the invention

The present invention provides for the production of antibodies that cross-react with IL-17A/F and bispecific antibodies that specifically bind to IL-17A and IL-17F. IL-17A and IL-17F are the major proinflammatory cytokines secreted by a subset of Th17T cells. They target almost all cell types in vivo and induce tissue inflammation and tissue damage. Since these cytokines are involved in inflammation, and in particular in immune-related diseases, such as autoimmune diseases, e.g. Rheumatoid Arthritis (RA) and Inflammatory Bowel Disease (IBD), it is desirable to generate antibodies capable of blocking both IL-17A and IL-17F. The invention provides such antibodies. More specifically, the invention provides antibodies that cross-react with IL-17A and IL-17F and bispecific antibodies with binding specificity for both IL-17A and IL-17F.

General methods for recombinant production of antibodies

The antibodies and other recombinant proteins herein can be produced by well-known techniques of recombinant DNA technology. Thus, in addition to the antibodies specifically identified herein, the skilled artisan is able to generate antibodies to the antigen of interest (e.g., by the techniques described below).

Antibodies generated according to the invention are directed against two antigens of interest, IL-17A and IL-17F. The IL-17A/F cross-reactive and bispecific antibodies of the invention have been generated by phage display technology, but other known techniques for making bispecific antibodies and antibodies having antigen-binding regions that bind to two different antigens can also be used.

Cross-reactive antibodies

According to one embodiment, antibodies with specificity for one of IL-17A are diversified such that they produce specificity for IL-17F while retaining specificity for IL-17A. Alternatively, antibodies with specificity for IL-17F are diversified such that they produce specificity for IL-17A while retaining specificity for IL-17F. In general, such methods comprise the step of (1) rendering an antibody light chain variable domain (V)_L) Wherein prior to diversification the antibody comprises a V capable of binding to an epitope on the first IL-17 protein (IL-17A or IL-17F)_LAnd heavy chain variable domain (V)_H) And (2) selecting a diversified antibody that is capable of binding to an epitope on a first IL-17 protein (IL-17A or IL-17F) and an epitope on a second IL-17 protein (IL-17F or IL-17A). A detailed description of this method is provided in co-pending application publication No.20080069820, published 3/20 of 2008, the disclosure of which is expressly incorporated herein by reference in its entirety. A specific generation and selection method for the production of IL-17A/F cross-reactive antibodies is exemplified in example 1.

Bispecific antibodies

According to another embodiment, antibodies specific for two IL-17 polypeptides (IL-17A and IL-17F) are isolated from a non-human mammal (e.g., a mouse or rat) injected with IL-17A and IL-17F.

Methods for making bispecific antibodies are known in the art. The conventional preparation of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al, Nature, 305: 537-539 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) generate a mixture of potentially 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is usually performed by an affinity chromatography step, is rather cumbersome and the product yield is low. WO93/08829 and Traunecker et al, EMBO J. (Lew. European society of molecular biology), 10: 3655-3659(1991) disclose a similar procedure.

According to another method described in W096/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces comprise at least a portion of the C of the antibody constant domain_H3 domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size to one or more large side chains are created at the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism to increase the yield of heterodimers, but not other unwanted end products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one antibody in the heterologous conjugate may be coupled to avidin and the other antibody to biotin. For example, such antibodies have been proposed for targeting immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treating HIV infection (WO91/00360, WO92/200373, and EP 03089). Heteroconjugated antibodies can be prepared using any suitable crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No.4,676,980, along with a number of crosslinking techniques.

For reviews on the preparation of bispecific antibodies see, e.g., Holliger, p. and Winter, g., curr. opin. biotechnol. (biotechnology new view) 4, 446-449 (1993); carter, p. et al, j.hematotherpy 4, 463-470 (1995); and Pluckthun, A. and Pack, P., Immunotechnology 3, 83-105 (1997). Bispecific and/or bivalent properties have been obtained by fusing two scFv molecules via a flexible linker, a leucine zipper motif, heterodimerization, and by association of the scFv molecules to form bivalent monospecific diabodies and related structures.

Thus, for example, to generate a bispecific antibody, one can start with two monospecific antibodies (e.g., an anti-IL-17A and an anti-IL-17F antibody). The two arms of the antibody can then be separated and covalently recombined to form a bispecific antibody. Such bispecific antibodies may comprise a common Fc portion and a Fab portion from each parent molecule. Thus, one Fab portion is specific for one of the receptor subunits and another is specific for a different receptor subunit. Of course, the starting material need not be an intact bivalent antibody. For example, they may be fragments such as Fab fragments, or Fab fragments (e.g. F (ab ═)2 fragments) further comprising one or more heavy chain CH2 and/or CH3 domains.

Bispecific antibodies can also be prepared using chemical bonds. Brennan et al, Science 229: 81(1985) describes a method of proteolytic cleavage of intact antibodies to F (ab')₂And (3) fragment. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab ' -TNB derivatives is then reverted back to Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody. In yet another embodiment, Fab' -SH fragments recovered directly from E.coli can be chemically coupled in vitro to form bispecific antibodies. Shalaby et al, j.exp.med. (journal of experimental medicine) 175: 217-225(1992).

Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one antibody in the heterologous conjugate may be coupled to avidin and the other antibody to biotin. Heteroconjugated antibodies can be prepared using any suitable crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No.4,676,980, along with a number of crosslinking techniques.

Leucine zippers have also been used to generate bispecific antibodies. Kostelny et al, j.immunol. (journal of immunology) 148 (5): 1547-1553(1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' -portions of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. Prepared by Hollinger et al, proc.natl.acad.sci.usa (journal of the american academy of sciences) 90: 6444-. The fragment comprises a light chain variable domain (V) linked by a linker_L) Heavy chain variable domain of (V)_H) The linker is too short to allow pairing between the two domains on the same strand. Thus, V on a segment is forced_HAnd V_LThe complementary V on the Domain and the other fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by using single chain fv (sFv) dimers has also been reported. See Gruber et al, j.immunol. (journal of immunology) 152: 5368(1994). Alternatively, the bispecific antibody may be a human Protein Eng (Protein engineering) such as Zapata et al 8 (10): 1057-1062 (1995).

Any of a variety of conventional methods can be used to chemically couple (crosslink) two polypeptide chains (e.g., antibody moieties). Covalent attachment can be obtained by direct condensation of existing side chains (e.g. formation of a disulfide bond between two cysteine residues) or by incorporation of external bridging molecules. Many bivalent or multivalent reagents are useful in conjugating polypeptides. For a description of some methods that can be used to chemically crosslink antibodies, see, e.g., Cao et al (1988) Bioconjugate Chemistry 9, 635-644; shalaby et al (1992) J.Exp.Med. (journal of Experimental medicine) 175, 217-225; glennie et al (1987) J.Immunol. (J.Immunol) 139, 2367-; jung et al (1991) Eur.J.Immunol. (European journal of immunology) 21, 2431-2435; VanDijk et al (1989) int. J. cancer (J. International cancer) 44, 738-743; pierce immunology catalog & Handbook (Pierce immunology catalog and Handbook) (1991) E8-E39; karpovsky et al (1984) j.exp.med. (journal of experimental medicine) 160, 1686; liu et al (1985) proc.natl.acad.sci.usa (journal of the american academy of sciences) 82, 8648; kranz et al (1981), PNAS 78, 5807; perez et al (1986), j.exp.med. (journal of experimental medicine) 163, 166-; brennan (1986) Biotech.4, 424; and U.S. patent nos.4,676,980, 6,010,902 and 5,959,083.

In addition, recombinant techniques can be used to produce single chain bispecific antibodies, for example, as described below: whitlow et al (1991), Methods: a company to Methods in Enzymology (Methods: guide for Methods in Enzymology), Vol.2, page 97; bird et al (1988), Science 242, 423-; U.S. Pat. Nos.4,946,778; pack et al (1993), Bio/Technology 11, 1271-77; and Sandhu (1992), crit. rev. biotech.12, 437. In particular, methods for producing bispecific single chain antibodies are described in, for example, U.S. patent nos. 5,892,020; gruber et al (1994), j.immunol. (journal of immunology) 152, 5368-74; mahllender et al (1994), Biochemistry 33, 10100-; winter et al (1991) Nature 349, 293-299; schmidt et al (1996). International Journal of Cancer 65, 538-546; and Thirion et al (1996), Eur.J.of Cancer Prevention (J.European Cancer Prevention) 5, 507-511.

Specific methods for making bispecific antibodies are described in example 2 herein.

Recombinant production of antibodies

For recombinant production of the antibody, the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. In another embodiment, the antibody can be produced by homologous recombination, for example as described in U.S. patent No.5,204,244, which is specifically incorporated herein by reference. DNA encoding the monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). Many vectors are available. Carrier ingredients generally include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters and transcription termination sequences, for example, as described in U.S. patent No.5,534,615, issued 7/9/1996, and which is specifically incorporated herein by reference.

Suitable host cells for cloning or expressing DNA encoding an antibody chain include mammalian host cells. There has been much interest in mammalian host cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7, ATCC CRL1651), human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al, J.GenVirol.36: 59(1977)), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, Proc.Natl.Acad.Sci.USA (Proc.Natl.Acad.Acad.77: 4216 1980), mouse sertoli cells (TM4, Mather, biol.Reprod.23: 243-251(1980)), monkey kidney cells (MDCC 1, ATCC CCL70), African green monkey kidney cells (562O-76, ATCC CRL-1587), human hepato cells (HELA, CCL2), canine kidney cells (MDCC 3528), bovine mouse lung cells (ATCC 1443, ATCC 14465), bovine liver mouse (CCL) mouse lung 73138, ATCC 144138, ATCC accession No.3 (MMffRL 369, ATCC accession No. 7, ATCC CCL51), TRI cells (Mather et al, annalsn.y.acad.sci. (new york academy of sciences) 383: 44-68(1982)), MRC5 cells, FS4 cells, and the human hepatoma (Hep G2) line.

Host cells are transformed with expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences.

Mammalian host cells can be cultured in a variety of media. Can be used forCommercially available media such as Ham's F10(Sigma), minimal essential Medium (MEM, Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing the host cells. In addition, any of the media described in the following documents can be used as the medium for the host cells: ham et al, meth.enz.58: 44(1979), Barnes et al, anal. biochem. (Biochemical annual newspaper) 102: 255(1980), U.S. patent nos.4,767,704; 4,657,866, respectively; 4,927,762, respectively; 4,560,655, respectively; or 5,122,469; WO 90/03430; WO 87/00195; or us reissue patent 30,985. Any of these media may be supplemented as needed with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN)^TM) Trace elements (defined as inorganic compounds that are typically present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be included at suitable concentrations. Culture conditions such as temperature, pH, etc. are previously used for the host cell selected for expression, as will be apparent to the ordinarily skilled artisan.

Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a preferred purification technique. The suitability of protein a as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein a can be used to purify antibodies based on human gamma 1, human gamma 2, or human gamma 4 heavy chains (Lindmark et al, j. immunol. meth. (journal of immunological methods) 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3((Guss et al, EMBO J. (Proc. Eur. Biologicals) 5: 15671575(1986)) the matrix to which the affinity ligand is attached is most often agarose, but other matrices can be usedBakerbond ABX^TMPurification was performed on resin (j.t.baker, phillips burg, n.j.). Depending on the antibody to be recovered, other protein purification techniques such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, heparin Sepharose may also be used^TMChromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, hydrophobic interaction chromatography, and ammonium sulfate precipitation.

After any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to additional purification steps to obtain the desired level of purity.

Humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be performed essentially according to the method of Winter and co-workers (Jones et al, Nature (Nature), 321: 522-525 (1986); Riechmann et al, Nature (Nature), 332: 323-327 (1988); Verhoeyen et al, Science, 239: 1534-1536(1988)) by replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No.4,816,567) in which substantially less than the entire human variable domain is replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.

The choice of human variable domains (including light and heavy chains) used to make humanized antibodies is important for reducing antigenicity. The entire library of known human variable domain sequences is screened with the variable domain sequences of rodent antibodies according to the so-called "best-fit" (best-fit) "method. The closest human sequence to rodents is then used as the human FR for the humanized antibody (Sims et al, J.Immunol. (J.Immunol., 151: 2296 (1993)). Another approach uses a particular framework derived from the consensus sequence of all human antibodies of a particular subclass of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al, J. Immunol., 151: 2623 (1993)).

More importantly, the antibodies retain high affinity for the antigen after humanization as well as other favorable biological properties. To achieve this, according to a preferred method, humanized antibodies are prepared by a method of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are also available which are capable of illustrating and displaying the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these display images enables analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the recipient and import sequences and combined to obtain a desired antibody characteristic, such as increased affinity for one or more target antigens. Generally, CDR residues are directly and most substantially involved in the effect on antigen binding.

Alternatively, it is now possible to generate transgenic animals (e.g. mice) which can be immunized to generate a full human antibody profile (reporters) in the absence of endogenous immunoglobulin production. For example, the region of the antibody heavy chain junction (J) has been described in chimeric and germline mutant mice_H) Homozygous deletion of the gene, which results in complete suppression of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays into such germline mutant mice will result in the generation of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, proc.natl.acad.sci.usa (journal of the american academy of sciences), 90: 2551 (1993); jakobovits et al, Nature, 362: 255-258 (1993); bruggermann et al, yearinn immunity, 7: 33 (1993); and Duchosal et al Nature 355: 258(1992). Human antibodies can also be derived from phage display libraries (Hoogenboom et al, j.mol.biol. (r.))Journal of molecular biology), 227: 381 (1991); marks et al, j.mol.biol. (journal of molecular biology), 222: 581-597 (1991); vaughan et al Nature Biotech (Nature: Biotechnology) 14: 309(1996)).

Therapeutic uses

The antibodies of the invention are useful in the treatment of inflammatory and immune-related diseases associated with the production of IL-17A and/or IL-17F. As discussed previously, given the pro-inflammatory properties of both IL-17A and IL-17F, cross-reactive and bispecific antibodies capable of blocking both IL-17A and IL-17F with high efficiency provide a new therapeutic opportunity in the treatment of inflammatory and immune-related diseases, including autoimmune diseases.

Exemplary inflammatory and immune-related diseases targeted by the cross-reactive and bispecific antibodies of the invention may be, but need not be, immune or T cell-mediated, including systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (grave's disease), hashimoto thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes, immune-mediated renal diseases (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous system such as multiple sclerosis, idiopathic demyelinating polyneuropathy or acute idiopathic polyneuritis (Guillain-Barre syndrome), and chronic inflammatory demyelinating polyneuropathy, diseases of the liver and gall bladder such as infectious hepatitis (A, B, C, D, E and other non-hepadnaviral hepatitis), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, Inflammatory Bowel Diseases (IBD) including ulcerative colitis, Crohn's disease, gluten-sensitive bowel disease, and Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skin disease, erythema multiforme and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food allergy and urticaria, immunological diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, graft-related diseases including graft rejection and graft-versus-host disease, infectious diseases including viral diseases such as AIDS (HIV infection), hepatitis a, hepatitis b, hepatitis c, hepatitis d and e, herpes and the like, bacterial infections, fungal infections, protozoal infections and parasitic infections.

In particular embodiments, the antibodies are used to treat Rheumatoid Arthritis (RA), Inflammatory Bowel Disease (IBD) including ulcerative colitis and crohn's disease, asthma, or other allergic or autoimmune diseases.

Dosage and formulation

The antibody or antibody fragment compositions of the invention can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners. Determining a "therapeutically effective amount" of an antibody or antibody fragment to be administered requires consideration of the above factors, which is the minimum amount required to prevent, ameliorate, or treat the target disease (such as any of the diseases and disorders listed above). The antibody or antibody fragment is not required but is optionally formulated with one or more agents currently used to prevent or treat the target disease. The effective amount of such other agents depends on the amount of the antibody or antibody fragment of the invention present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as used above, or about 1-99% of the dosages used so far.

In general, treatment of a target disease involves alleviating one or more symptoms or medical problems associated with the target disease, e.g., if the target disease is an inflammatory disease or condition, the treatment can reduce and/or alleviate one or more symptoms associated with inflammation.

In the case of rheumatoid arthritis, symptoms include, without limitation, muscle and joint pain, stiffness, swelling, joint pain, or tenderness, one or more of which may be alleviated by administration of an antibody of the invention. In addition, the cross-reactive and bispecific anti-IL-17A/F antibodies of the invention can prevent, alleviate or delay the signs of target disease (physical manueffects). For example, if the target disease is rheumatoid arthritis, treatment may prevent, reduce or delay the progression of structural damage (e.g., bone erosion and joint damage characteristic of the disease). For this indication, the cross-reactive and bispecific antibodies of the invention may be administered with other therapeutic modalities used in clinical practice (e.g., alone or in combination with methotrexate)Rituximab) in combination to treat patients with moderate-to-severe active Rheumatoid Arthritis (RA). Other therapeutic options that may be combined with the antibodies of the invention include, without limitation, non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, TNF antagonist therapies such as etanercept (etanercept), infliximab (infliximab), or adalimumab (adalimumab).

If the target disease is Inflammatory Bowel Disease (IBD), including ulcerative colitis and Crohn's disease, treatment may prevent, reduce or delay the development of inflammation, and/or one or more accompanying symptoms including abdominal pain, diarrhea or constipation, fatigue, and fever. Traditional therapies that can treat IBD in combination via treatment with the cross-reactive and bispecific antibodies of the present invention include aminosalicylates, corticosteroids, antibiotics, and other biological or therapeutic options such as TNF antagonist therapies such as etanercept, infliximab, or adalimumab.

Therapeutic formulations can be prepared by combining the active ingredient in the desired purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (20 th edition), editors a.gennaro, 2000, Lippincott, Williams&Wilkins, philiadelphia, Pa.) was prepared using standard methods known in the art for mixing. Acceptable carriers include saline or buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols such as mannitol, or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN^TM、PLURONICS^TMOr PEG.

Optionally, but preferably, the formulation contains a pharmaceutically acceptable salt, preferably sodium chloride, and preferably at about physiological concentrations. Optionally, the formulations of the present invention may contain a pharmaceutically acceptable preservative. In some embodiments, the preservative concentration ranges from 0.1 to 2.0%, typically in v/v. Suitable preservatives include those known in the pharmaceutical arts. Preferred preservatives are benzyl alcohol, phenol, m-cresol, methyl paraben and propyl paraben. Optionally, the formulation of the invention may comprise a pharmaceutically acceptable surfactant at a concentration of 0.005 to 0.02%.

The formulations herein may also contain more than one active compound necessary for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. Suitably, such molecules are present in combination in amounts effective for the intended purpose.

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

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No.3,773,919, copolymers of L-glutamic acid with gamma ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- (-) -3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of releasing molecules for over 100 days, certain hydrogels release proteins for shorter periods of time. When the encapsulated antibodies are maintained in vivo for extended periods of time, they may denature or aggregate by exposure to a humid environment at 37 ℃, resulting in a loss of biological activity and possible changes in immunogenicity. Rational stabilization strategies can be devised based on the relevant mechanisms. For example, if the aggregation mechanism is found to be intermolecular S — S bond formation via sulfur-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling humidity, employing appropriate additives, and developing specific polymer matrix compositions.

The antibodies and antibody fragments described herein are administered to a human subject according to known methods, such as, for example, intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebral, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Topical administration may be particularly desirable if the antagonistic effect of the antibodies herein is associated with a wide range of side effects or toxicity. Ex vivo (ex vivo) strategies may also be used in therapeutic applications. Ex vivo strategies involve transfecting or transducing cells obtained from a subject by a polynucleotide encoding an antibody or antibody fragment. The transfected or transduced cells are then returned to the subject. The cells may be of many types, including but not limited to hematopoietic cells (e.g., bone marrow cells, macrophages, monocytes, dendritic cells, T cells, or B cells), fibroblasts, epithelial cells, endothelial cells, keratinocytes, or muscle cells.

Article and kit

Another embodiment of the invention is an article of manufacture comprising a material useful in the treatment of a disease or disorder targeted by an antibody of the invention. The article of manufacture comprises a container and a label or package insert (package insert) on or with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be made of various materials such as glass or plastic. The container contains a composition that is therapeutically effective for the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a glass vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a cross-reactive or bispecific antibody or antibody fragment of the invention. The label or package insert indicates that the composition is for use in treating a particular condition. The label and package insert also include instructions for administering the antibody composition to a patient. Also contemplated are articles of manufacture and kits comprising the combination therapies described herein.

Package insert refers to an insert conventionally included in commercial packaging of therapeutic products containing information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. In one embodiment, the package insert indicates that the composition is for use in the treatment of an inflammatory or immune-related disorder, such as, for example, any of the disorders listed above, including various forms of arthritis, such as rheumatoid arthritis, Inflammatory Bowel Disease (IBD), other autoimmune diseases, or allergic disorders, such as asthma.

In addition, the article of manufacture may further comprise a second container having a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may further comprise other desirable materials from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

The foregoing written description is considered to be sufficient to enable one skilled in the art to practice the invention. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Unless otherwise indicated, the commercially available reagents mentioned in the examples were used according to the manufacturer's instructions. The source of those cells identified in the examples below and throughout the specification as ATCC accession numbers is the American Type culture Collection (American Type culture Collection, Manassas, VA). Unless otherwise indicated, the present invention uses standard procedures for recombinant DNA technology, such as those described above and in the following texts: sambrook et al, supra; the subject of Ausubel et al,current Protocols in Molecular Biology (Experimental protocol in modern Molecular Biology)(Green Publishing Associates and Wiley Interscience, N.Y., 1989); innis et al, in the name of Innis,PCR Protocols: a Guide to Methods and Applications (PCR protocol: Methods and Applications) Application guide)(Academic Press, Inc.: N.Y., 1990); the result of Harlow et al is that,Antibodies：A laboratory Manual (antibody: Laboratory Manual)(Cold Spring Harbor Press：ColdSpring Harbor，1988)；Gait，Oligonucleotide Synthesis (Oligonucleotide Synthesis)(IRLPress：Oxford，1984)；Freshney，Animal Cell Culture1987; the results of Coligan et al,current Protocols in Immunology (modern enzymology protocol)，1991。

Examples

The following examples are provided for illustrative purposes only and are in no way intended to limit the scope of the present invention.

All patents and references cited in this specification are incorporated herein by reference in their entirety.

Example 1

anti-IL-17A/F cross-reactive antibodies

Materials and methods

Library sorting and screening to identify anti-IL-17A/F antibodies

Human IL-17A (R & D Systems, cat # 317-IL-050/CF) and IL-17F (R & DSsystems, cat #1335-IL-025/CF) were used as antigens for library sorting. For conventional sorting, phage libraries were sorted for four rounds against IL-17A or IL-17F alone. For alternative sorting, phage libraries can be interchangeably sorted in alternate rounds for IL-17A and IL-17F. Nunc 96-well Maxisorp immunoplates were coated with target antigen (10. mu.g/ml) overnight at 4 ℃ and blocked by phage blocking buffer PBST (phosphate buffered saline (PBS) and 1% (w/v) Bovine Serum Albumin (BSA) and 0.05% (v/v) tween-20) for 1 hour at room temperature. Antibody phage libraries VH (see e.g., Lee et al, J.Immunol.meth. (J.Immunol. methods.) 284: 119-132, 2004) and VH/VL (see Liang et al, JM.366: 815-829, 2007) were added separately to antigen plates and incubated overnight at room temperature. The next day the antigen coated plates were washed ten times with PBT (PBS containing 0.05% Tween-20), and the bound phage were eluted for 30 minutes by 50mM HCl and 500mM NaCl and neutralized by an equal volume of 1M Tris base (pH 7.5). The recovered phage were amplified in E.coli (E.coli) XL-1Blue cells. In subsequent selection rounds, the incubation of antibody phage with antigen-coated plates was reduced to 2-3 hours and the stringency of plate washes was gradually increased.

After 4 rounds of panning, significant enrichment was observed. From VH and VH/VL each library sorting in 96 clones were picked to determine if they specifically bound to both human IL-17A and IL-17F. The variable regions of these clones were sequenced by PCR to identify unique sequence clones.

The affinity of the phage antibodies was graded using a spot competition elisa (spot competition elisa). The IC50 values for phage antibodies were further determined using a competitive phage binding ELISA. The unique phage antibody that binds both human IL-17A and IL-17F with the highest IC50 was selected and reformatted (reformatted) into full-length IgG for evaluation in an in vitro cellular assay.

By cloning individual V_LAnd V_HThe regions were cloned into the LPG3 and LPG4 vectors, respectively, transiently expressed in mammalian CHO cells, and the clones of interest were reformatted into iggs by protein a column purification.

Determination of IC50/90 for Cross-reactive antibodies

Human neonatal foreskin fibroblasts (Invitrogen) were seeded on the first day at 2x104 cells/150 μ l medium/well in 96-well plates. The next day the medium was replaced with cytokine/antibody containing medium (150. mu.l). Recombinant human IL-17A was used at 5 ng/ml. Recombinant human IL-17F was used at 50 ng/ml. Recombinant human IL-17A/F heterodimer was purified autonomously (in-house) and used at 25 ng/ml. Supernatants were harvested 24 hours later and subjected to a G-CSF ELISA to measure G-CSF induction. Data were plotted in the PRISM and IC50/90 values were calculated using the same software.

For deriving from V _H Library of constructs with improved affinity for cloning of the library

Phagemid pW0703 (derived from phagemid pV0350-2b (Lee et al)Human, J.mol.biol (journal of molecular biology) 340, 1073-1093(2004)), containing a stop codon (TAA) at all CDR-L3 positions and displaying a monovalent Fab on the surface of the M13 phage was used as a library template for the extraction of a protein from V._HLibrary grafting of heavy chain variable domains (V) of clones of interest_H) For affinity maturation. Both hard and soft randomization strategies are used for affinity maturation. For hard randomization, a light chain library with selected sites of three light chain CDRs was randomized using amino acids designed to mimic natural human antibodies, the degeneracy of the designed DNA being described in Lee et al (j.mol.biol. 340, 1073-. For soft randomization, residues at positions 91-94 and 96 of CDR-L3, 28-31 and 34-35 of CDR-H1, 50, 52 and 53-58 of CDR-H2, 95-99 and 100A of CDR-H3 were targeted; two different CDR loop combinations L3/H1/H2 and L3/H3 were chosen for randomization. To obtain soft randomized conditions, which introduce approximately 50% mutation rate at selected sites, mutant DNA was synthesized by a 70-10-10-10 mixture of bases supporting wild-type nucleotides (Gallop et al, Journal of medicinal chemistry 37: 1233-1251 (1994)).

Generating affinity-improved phage sorting strategies

For affinity-improved selection, phage libraries were subjected to a first round of plate sorting followed by four or five rounds of solution sorting. The library was sorted individually for human IL-17A and IL-17F. For the first round of plate sorting, the library was sorted for 2 hours at room temperature against a target coated plate (NUNC Maxisorp plate) with phage input of approximately 3o.d./ml in 1% BSA and 0.05% Tween 20. After the first round of sorting, solution sorting was performed to increase the stringency of selection. For solution sorting, 1o.d./ml of phage propagated by the first plate sorting were incubated with 100nM biotinylated target protein (concentration based on IC50 values of parental clonal phage) in 100 μ l buffer containing 1% superblock (pierce biotechnology) and 0.05% Tween20 for 30 minutes at room temperature. The mixture was further diluted 10X by 1% Superblock, 100 μ Ι/well was applied to neutravidin-coated wells (5 μ g/ml) with gentle shaking at room temperature for 15 min for biotinylated target to bind phage. The wells were washed ten times with PBS-0.05% Tween 20. To determine background binding, control wells containing phage with non-biotinylated target were captured on neutravidin-coated plates. Bound phage were eluted for 20 min by 0.1N HCl, neutralized by 1/10 volumes of 1M TrispH11, titrated and propagated for the next round. Thereafter, five additional rounds of solution sorting were performed together with increasing selection stringency. The first round was on-rate selection by decreasing the concentration of biotinylated target protein from 10nM to 1nM, and the second round was off-rate selection by adding excess (100-fold more) of non-biotinylated target protein to compete for weaker binders, all at room temperature. In addition, the input of phage was reduced (0.1-0.5 O.D/ml) to reduce binding of background phage.

High throughput affinity screening ELISA (Single spot competition)

Colonies were selected from the fifth or sixth round of screening. Colonies were grown overnight at 37 ℃ in 150. mu.l/well 2YT medium containing 50. mu.g/ml carbenicillin and 1E10/ml KO7 in 96-well plates (Falcon). From the same plate, colonies of XL-1 infected parent phage were picked as controls. 96-well Nunc Maxisorp plates were coated with 100. mu.l/well of human IL-17A and IL-17F (2. mu.g/ml) each in PBS overnight at 4 ℃ or for 2 hours at room temperature. The plate was blocked for 30 minutes by 65. mu.l of 1% BSA and for another 30 minutes by 40. mu.l of 1% Tween 20.

Phage supernatants were diluted 1: 10 in ELISA (enzyme-linked immunosorbent assay) buffer (PBS with 0.5% BSA, 0.05% Tween 20) with or without 10nM target protein in a total volume of 100. mu.l and incubated in F-plates (NUNC) for at least 1 hour at room temperature. 75 μ l of the mixture with or without the target protein was transferred side by side to the target protein coated plate. The plate was warmed and shaken for 15 minutes to allow capture of unbound phage onto target protein coated plates. The plate was washed at least five times with PBS-0.05% Tween 20. KnotConjugation was quantified by adding horseradish peroxidase (HR) -conjugated anti-M13 antibody to ELISA buffer (1: 5000) and incubating for 30 min at room temperature. The plate was washed at least five times with PBS-0.05% Tween 20. Thereafter, 100. mu.l/well of 3, 3 ', 5, 5' -Tetramethylbenzidine (TMB) peroxidase substrate and peroxidase solution B (H) at a 1: 1 ratio₂O₂) (Kirkegaard-Perry Laboratories (Gaithersburg, Md.)) was added to the wells and incubated for 5 minutes at room temperature. By adding 100. mu.l of 1M phosphoric acid (H)₃PO₄) The reaction was stopped by reaching each well and allowing to incubate for 5 minutes at room temperature. The OD (absorbance) of the yellow color in each well was determined at 450nm using a standard ELISA plate reader. The reduction (%) of OD was calculated by the following equation.

OD_450nmReduction (%) - (OD of the wells containing competitor)_450nm) /(OD of Compette-free wells_450nm)]*100

OD of well from parent phage_450nmReduction (%) (100%) selection with OD less than 50% for both human and murine targets_450nmThe clones reduced (%) were subjected to sequence analysis. Unique clones were selected for phage preparations to determine binding affinity for both human IL-17A and IL-17F by comparison to parental clones (phage IC 50). The most affinity improved clones were then reformatted into adult IgG1 for antibody generation and further BIAcore binding kinetic analysis and other in vitro or in vivo assays.

Characterization of anti-IL-17A/F antibodies (Biacore)

Binding affinity against IL-17A/FIG Using BIAcore by Surface plasmon resonance (SRP)^TM3000 instrumental measurements. anti-IL-17A/F human IgG was captured by a mouse anti-human Fc antibody (GE Healthcare, cat # BR-1008-39) coated on a CM5 biosensor chip to obtain approximately 200 Response Units (RU). For kinetic measurements, human IL-17A (R)&D Systems，cat# 317-IL-050/CF)，IL-17F(R&D, cat #1335-IL-025/CF), IL-17A/F (GNE), rhesus IL-17F (GNE), cynomolgus ITwo-fold serial dilutions (0.98nM to 125nM) of L-17A (PUR17200) were injected into PBT buffer (PBS containing 0.05% Tween 20) at 25 ℃ at a flow rate of 30. mu.l/min. Binding rates (k) were calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation software version 3.2))_on) And dissociation rate (k)_off). Equilibrium dissociation constant (K)_D) At a ratio of k_off/k_onAnd (4) calculating.

Results

As discussed previously, IL-17A and IL-17F are the major proinflammatory cytokines secreted by the Th17T cell subset. They target almost all cell types in the body and induce tissue inflammation and tissue damage. The objective of the work described in this example was to generate cross-reactive antibodies that can block both IL-17A and F with high efficiency and cross-species reactivity.

The light chain variable region sequences of the cross-reactive anti-IL-17A/F clones are shown in FIG. 5A (SEQ ID NOs: 5-14), including the CDRL1, CDRL2, and CDRL3 sequences. The heavy chain variable region sequence of the cross-reactive anti-IL-17A/F clones is shown in FIG. 5B (SEQ ID NOs: 51-60). YW241.47, YW278.15 and YW279.1, identified in cross-reactive clones, showed particularly strong binding to both homodimeric and heterodimeric forms of IL-17A and IL-17F. It is noteworthy that the cross-reactive antibodies YW278.15 and 279.1 showed almost equal blocking of IL-17A and F. Of these, the cross-reactive antibody YW278.15 was selected for affinity maturation.

The light chain variable region sequences of further cross-reactive anti-17A/F antibodies, including affinity matured variants of YW278.15 and YW279.1, are shown in FIG. 5C (SEQ ID Nos: 63-68).

The heavy chain variable region sequences of further cross-reactive anti-A/F antibodies, affinity matured variants including YW278.15 and YW279.1 are shown in FIG. 5D (SEQ ID Nos: 69-74).

FIG. 6 shows an alignment of the light chain sequences of IL-17A/F cross-reactive antibody YW278.15(SEQ ID NO: 9) and its affinity matured variant, YW278.15.18(SEQ ID NO: 15), YW278.15.2(SEQ ID NO: 16) and YW278.15.3(SEQ ID NO: 17). CDRL1, CDRL2 and CDRL3 sequences are indicated in boxes.

FIG. 7 shows an alignment of the heavy chain sequences of IL-17A/F cross-reactive antibody YW278.15(SEQ ID NO: 92) and its affinity matured variant, YW278.15.18(SEQ ID NO: 93), 278.15.2(SEQ ID NO: 94), YW278.15.2.D54E (SEQ ID NO: 95), YW278.15.3(SEQ ID NO: 92), YW278.15.18C55A (SEQ ID NO: 18) and YW278.15.18C55S (SEQ ID NO: 19). CDRL1, CDRL2 and CDRL3 sequences are indicated in boxes. YW278.15.18C55A and YW278.15.18C55S have the same light chain as YW278.15.18 and introduce C55A and C55S mutations in the antibody heavy chain to aid in preparation.

FIG. 8A shows BIAcore by three affinity matured IL-17A/F cross-reactive antibodies, YW278.15.2, YW278.15.18, and YW278.15.3^TMResults of the immunoassay. The binding affinities (Kd (M)) for recombinant human IL-17A (rhIL-17A), recombinant human IL-17F (rhIL-17F), cynomolgus monkey IL-17F and human IL-17A/F in solution are shown in the last column.

FIG. 8B shows IL-17A/F cross-reactive antibody, YW278.15.2.D54E, matured by affinity; YW278.15.3, respectively; YW278.15.9, respectively; YW279.1.20, respectively; YW279.1.21, respectively; YW279.1.22 BIAcore^TMResults of the immunoassay. The binding affinities (Kd (M)) for recombinant human IL-17A (rhIL-17A), recombinant human IL-17F (rhIL-17F), cynomolgus monkey IL-17A, cynomolgus monkey IL-17F and human IL-17A/F in solution are shown in the last column.

FIG. 9 shows IL-17A/F cross-reactive antibodies matured by two further affinities, YW278.15.18.C55A and YW278.15.18.C55S, BIAcore in comparison with YW278.15.18^TMResults of the immunoassay. The binding affinities (Kd (M)) for recombinant human IL-17A (rhIL-17A), recombinant human IL-17F (rhIL-17F), cynomolgus monkey IL-17F and human IL-17A/F in solution are shown in the last column.

FIG. 10 shows the performance of IL-17A/F cross-reactive antibodies by three affinity maturationBIAcore^TMAs a result of the immunoassay, rhesus IL-17A was used as a target. Binding affinity to rhesus IL-17A is shown in the last column.

Fig. 13A and 13B show IC50 and IC90 data determined by repeated assays for a panel of cross-reactive antibodies. The molar ratio of antibody to target cytokine is also shown.

The results show that the affinity of YW278.15 and 279.1 is improved by CDR randomization for both IL-17A and IL-17F. Among the cross-reactive antibodies tested, YW278.15.2 and YW278.15.18 showed the best improvements in affinity and cell blocking activity. YW278.15.2 and YW278.15.18 both have similar activity on IL-17A and IL-17F.

Example 2

anti-IL-17A/F bispecific antibodies

Materials and methods

Library sorting and screening to identify anti-IL-17A/F antibodies

Human IL-17A (R & D Systems, cat # 317-IL-050/CF) and IL-17F (R & DSsystems, cat #1335-IL-025/CF) were used as antigens for library sorting. For conventional sorting, phage libraries were sorted for four rounds against IL-17A or IL-17F alone. For alternative sorting, phage libraries can be interchangeably sorted in alternate rounds for IL-17A and IL-17F. Nunc96 well Maxisorp immune plates were coated with target antigen (10. mu.g/ml) overnight at 4 ℃ and blocked by phage blocking buffer PBST (phosphate buffered saline (PBS) and 1% (w/v) Bovine Serum Albumin (BSA) and 0.05% (v/v) tween-20) for 1 hour at room temperature. Antibody phage libraries VH (see e.g., Lee et al, J.Immunol. meth. (J. Immunol. methods.) 284: 119-132, 2004) and VH/VL (see Liang et al, JMB. 366: 815-829, 2007) were added separately to antigen plates and incubated overnight at room temperature. The next day the antigen coated plates were washed ten times with PBT (PBS containing 0.05% Tween-20), and the bound phage were eluted for 30 minutes by 50mM HCl and 500mM NaCl and neutralized by an equal volume of 1M Tris base (pH 7.5). The recovered phage were amplified in E.coli (E.coli) XL-1Blue cells. In subsequent selection rounds, the incubation of antibody phage with antigen-coated plates was reduced to 2-3 hours and the stringency of plate washes was gradually increased.

After 4 rounds of panning, significant enrichment was observed. From VH and VH/VL each library sorting in 96 clones were picked to determine if they specifically bound to both human IL-17A and IL-17F. The variable regions of these clones were sequenced by PCR to identify unique sequence clones.

The affinity of the phage antibodies was graded using a spot competition ELISA. Phage supernatants were diluted 1: 5 in ELISA (enzyme linked immunosorbent assay) buffer (PBS with 0.5% BSA, 0.05% Tween 20) with or without 50nM target protein in a total volume of 100. mu.l and incubated in F-plates (NUNC) for at least 1 hour at room temperature. 75 μ l of the mixture with or without the target protein was transferred side by side to target protein coated plates (1ug/ml IL-17A or IL-17F coated overnight). The plate was warmed and shaken for 15 minutes to allow capture of unbound phage onto target protein coated plates. The plate was washed 10 times with PBS-0.05% Tween 20. Binding was quantified by adding horseradish peroxidase (HR) -conjugated anti-M13 antibody to ELISA buffer (1: 5000) and incubating for 30 min at room temperature. The plate was washed 10 times with PBS-0.05% Tween 20. Thereafter, 100. mu.l/well of 3, 3 ', 5, 5' -Tetramethylbenzidine (TMB) peroxidase substrate and peroxidase solution B (H) at a 1: 1 ratio₂O₂) (Kirkegaard-Perry Laboratories (Gaithersburg, Md.)) was added to the wells and incubated for 5 minutes at room temperature. By adding 100. mu.l of 0.1M phosphoric acid (H)₃PO₄) The reaction was stopped by reaching each well and allowing to incubate for 5 minutes at room temperature. The OD (absorbance) of the yellow color in each well was determined at 450nm using a standard ELISA plate reader. The reduction (%) of OD was calculated by the following equation.

OD_450nmReduction (%) - (OD of the wells containing competitor)_450nm) /(OD of Compette-free wells_450nm)]*100

OD of well from parent phage_450nmReduction (%) (100%) picking a target with less than 60% OD to one of IL-17A or IL-17F_450nmReduced (%) clones and reformatted to full-length human IG as follows: v of Individual clones_LAnd V_HThe regions were cloned into LPG3 and LPG4 vectors, respectively, transiently expressed in mammalian CHO cells, and purified by a protein a column. Bispecific antibodies (which are specific for IL-17A in one arm and IL-17F in the other) were constructed using the knob-and-hole technique (cf. Merchant et al, Nature Biotechnology (Nature: Biotechnology), 16: 677-. These mono-and bispecific antibodies were then used for in vitro cellular assay evaluation and for BIAcore binding kinetics analysis.

Characterization of anti-IL-17A/F antibodies (Biacore)

Binding affinity against IL-17A/FIG Using BIAcore by Surface plasmon resonance (SRP)^TM3000 instrumental measurements. anti-IL-17A/F human IgG was captured by a mouse anti-human Fc antibody (GE Healthcare, cat # BR-1008-39) coated on a CM5 biosensor chip to obtain approximately 200 Response Units (RU). For kinetic measurements, human IL-17A (R)&D Systems, cat # 317-IL-050/CF) or IL-17F (R)&D, cat #1335-IL-025/CF) was injected into PBT buffer (PBS containing 0.05% Tween 20) at 25 ℃ at a flow rate of 30. mu.l/min. Binding rates (k) were calculated using a simple one-to-one Langmuir binding model (BIAcore evaluation software version 3.2 (BIAcore EvaluationSoftware version 3.2))_on) And dissociation rate (k)_off). Equilibrium dissociation constant (K)_D) At a ratio of k_off/k_onAnd (4) calculating.

Results

FIG. 11 shows an alignment of the light chain variable region sequences of IL-17A-specific antibody YW264.21(SEQ ID NO: 20) and IL-17F-specific antibody YW265.01(SEQ ID NO: 21).

FIG. 12 shows an alignment of the heavy chain variable region sequences of IL-17A-specific antibody YW264.21(SEQ ID NO: 22) and IL-17F-specific antibody YW265.01(SEQ ID NO: 23).

FIG. 14 shows binding affinity data for IL-17A and IL-17F for IL-17A/F bispecific antibodies as compared to anti-IL-17A antibody YW264.21 and anti-IL-17F antibody YW 265.01.

FIG. 15 shows an alignment of the light chain variable region sequences of IL-17A-specific antibodies YW264.21(SEQ ID NO: 20) and YW264.03(SEQ ID NO: 61) and IL-17F-specific antibody YW265.01(SEQ ID NO: 21).

FIG. 16 shows an alignment of the heavy chain variable region sequences of IL-17A-specific antibodies YW264.21(SEQ ID NO: 22) and YW264.03(SEQ ID NO: 62) and IL-17F-specific antibody YW265.01(SEQ ID NO: 23).

Example 3

Crystal structure of IL-17F-Fab complex

Materials and methods

Protein expression:

IL-17F such as Hymowitz SG, et al, EMBO J. (Proc. Eur. Mol. Biol., 2001, 20: 5332 expression and purification was performed as described in 5341. Briefly, the DNA encoding IL-17F was subcloned into the pET15b (Novagen) site to introduce an N-terminal His-tag and a thrombin cleavage site. After another PCR step, the coding region was subcloned into the baculovirus transfer vector pAcGP67B (PharMingen), which was then co-transfected with BaculoGold DNA (PharMingen) into Sf9 cells, and the recombinant virus was isolated and amplified in Sf9 cells. For protein production, Hi5 cells were infected by the amplified baculovirus. The medium was harvested by centrifugation and the pH was adjusted to 7.0. The medium was filtered and loaded onto a Ni-NTA column. Fractions containing IL-17F were eluted with imidazole, mixed and dialyzed into PBS (pH6.5) overnight at 4 ℃ with thrombin. The protein sample was then concentrated and the thrombin and His-tag were removed by purification on a size exclusion column (size exclusion column).

Fab fragments of YW278.15.18.C55S (hereinafter "Fab") were expressed in E.coli, purified using Protein G-Sepharose, and eluted with 0.58% acetic acid. The Fab containing fractions were then purified by 20mM MES pH5.5 and NaCl gradient using a SPHiTrap column (GE Healthcare). The final buffer was 25mM Tris, 100mM NaCl, pH 7.5.

And (3) complex purification:

purified fab was mixed with excess purified recombinant IL 17F. The complex was purified by size exclusion column. The fractions containing the complex were mixed and concentrated to 25 mg/mL.

And (3) crystallization:

the IL-17F-fab complex was crystallized using hanging drop vapor diffusion. The hanging drops were formed by mixing 2ul of protein and 2ul of well solution (40% MPD, 5% PEG8000 and 0.1M sodium arsenate pH6.5) at 19 ℃. After 5 days of incubation crystals grew, were washed and frozen directly in the mother liquor in liquid nitrogen.

Crystallographic data and refinement (refinishment):

crystallographic data were collected in a beam line (beamline)11-1 at a Stanford Synchtron Radiation Laboratory. The data sets were processed using the procedures in the HKL package (HKL, Charlottesville, VA). The structure was analyzed as follows: variants of humanized 4D5Fab (PDB code 1FVE) were used as search model by molecular replacement using the program PHASER (CCP4) followed by refinement by REFMAC5(CCP 4). The final model has an excellent geometry in which 99.6% of all residues are in the best or extra-allowed region of the Ramachandran plot (Ramachandran plot) and only 0.6% (6 residues) are in the general allowed or disallowed region.

TABLE 1X-ray data Collection and refinement statistics

Parenthetical values are for the highest-resolution peripheral program (highest-resolution shell).

FIG. 17 shows Fab binding to IL-17F dimer, and FIG. 18 shows epitopes for Fab on IL-17F dimer. This epitope is centered on the cysteine junction and is largely conserved between L17A and IL17F (residues in parentheses are sequence differences between F and a).

The foregoing written description is considered to be sufficient to enable those skilled in the art to practice the invention. The invention is not limited in scope by the deposited constructs, as the deposited embodiments are intended as single illustrations of certain aspects of the invention, and any functionally equivalent constructs fall within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims (37)

1. An antibody or antigen-binding fragment thereof that binds IL-17A, comprising three heavy chain and three light chain Complementarity Determining Regions (CDRs) comprising:

(a) a CDRH1, the CDRH1 comprising: sequence TSYEIS (SEQ ID NO: 45), or SEQ ID NO: 62 of the amino acid residues 30 to 35,

(b) a CDRH2, the CDRH2 comprising: sequence WVGSIYLWGG (SEQ ID NO: 46), or SEQ ID NO: 62 amino acid residues 47-56, and

(c) a CDRH3, the CDRH3 comprising: sequence ARFGQRYA (SEQ ID NO: 47), or SEQ ID NO: 62 amino acid residues 96-103, and

(d) CDRL1, the CDRL1 comprising the sequence SISSYLA (SEQ ID NO: 25),

(e) CDRL2, the CDRL2 comprising the sequence GASSRAS (SEQ ID NO: 27), and

(f) CDRL3, the CDRL3 comprising the sequence YYSSPLT (amino acid residues 91-97 of SEQ ID NO: 20) or SYSSPLT (amino acid residues 91-97 of SEQ ID NO: 61).

2. The antibody or antigen-binding fragment of claim 1, comprising:

(a) CDRH1, said CDRH1 comprising the sequence TSYEIS (SEQ ID NO: 45),

(b) CDRH2, the CDRH2 comprising the sequence WVGSIYLWGG (SEQ ID NO: 46), and

(c) CDRH3, the CDRH3 comprising the sequence ARFGQRYA (SEQ ID NO: 47), and

(d) CDRL1, the CDRL1 comprising the sequence SISSYLA (SEQ ID NO: 25), and

(e) CDRL2, the CDRL2 comprising the sequence GASSRAS (SEQ ID NO: 27), and

(f) CDRL3, the CDRL3 comprising the sequence YYSSPLT (amino acid residues 91-97 of SEQ ID NO: 20).

3. The antibody or antigen-binding fragment of claim 1, comprising:

(a) CDRH1, said CDRH1 comprising the sequence SEQ ID NO: 62 of the amino acid residues 30 to 35,

(b) CDRH2, said CDRH2 comprising SEQ ID NO: 62 amino acid residues 47-56, and

(c) CDRH3, said CDRH3 comprising SEQ ID NO: 62 amino acid residues 96-103, and

(d) CDRL1, the CDRL1 comprising the sequence SISSYLA (SEQ ID NO: 25),

(e) CDRL2, the CDRL2 comprising the sequence GASSRAS (SEQ ID NO: 27), and

(f) CDRL3, the CDRL3 comprising SYSSPLT (amino acid residues 91-97 of SEQ ID NO: 61).

4. An antibody or antigen-binding fragment thereof that binds IL-17A, comprising a heavy chain variable region that differs from the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 62 and the light chain variable region has at least 95% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 61 have at least 95% sequence identity.

5. The antibody or antigen-binding fragment of claim 4, comprising a heavy chain variable region that is substantially identical to the amino acid sequence of SEQ ID NO: 22 and the light chain variable region has at least 95% sequence identity to SEQ ID NO: 20 have at least 95% sequence identity.

6. The antibody or antigen-binding fragment of claim 5, comprising a heavy chain variable region that is substantially identical to the amino acid sequence of SEQ ID NO: 22 and the light chain variable region has at least 98% sequence identity to SEQ ID NO: 20 have at least 98% sequence identity.

7. The antibody or antigen-binding fragment of claim 6, comprising a heavy chain variable region that is substantially identical to the amino acid sequence of SEQ ID NO: 22 and the light chain variable region has at least 99% sequence identity to SEQ ID NO: 20 have at least 99% sequence identity.

8. The antibody or antigen-binding fragment of claim 4, comprising the amino acid sequence of SEQ ID NO: 22 and SEQ ID NO: 20 light chain variable region.

9. The antibody or antigen-binding fragment of claim 4, comprising a heavy chain variable region that is substantially identical to the amino acid sequence of SEQ ID NO: 62 and the light chain variable region has at least 95% sequence identity to SEQ ID NO: 61 have at least 95% sequence identity.

10. The antibody or antigen-binding fragment of claim 9, comprising a heavy chain variable region that is substantially identical to the amino acid sequence of SEQ ID NO: 62 and the light chain variable region has at least 98% sequence identity to SEQ ID NO: 61 have at least 98% sequence identity.

11. The antibody or antigen-binding fragment of claim 10, comprising a heavy chain variable region that is substantially identical to the amino acid sequence of SEQ ID NO: 62 and the light chain variable region has at least 99% sequence identity to SEQ ID NO: 61 have at least 99% sequence identity.

12. The antibody or antigen-binding fragment of claim 4, comprising the amino acid sequence of SEQ ID NO: 62 and SEQ ID NO: 61, light chain variable region.

13. The antibody or antigen-binding fragment of any one of claims 1-12, which is monoclonal.

14. The antibody or antigen binding fragment of claim 13, which is chimeric, humanized or human.

15. The antibody or antigen-binding fragment of any one of claims 1-12, which is bispecific.

16. The antibody or antigen binding fragment of claim 15, which is chimeric, humanized or human.

17. The antibody or antigen binding fragment of claim 14, wherein the fragment is selected from the group consisting of: fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

18. The antibody or antigen binding fragment of claim 16, wherein the fragment is selected from the group consisting of: fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

19. The antibody or antigen-binding fragment of claim 13, which inhibits a biological function of IL-17A.

20. The antibody or antigen-binding fragment of claim 15, which inhibits a biological function of IL-17A.

21. An isolated nucleic acid molecule encoding the heavy chain of the antibody of claim 13, or an antigen-binding fragment thereof.

22. An isolated nucleic acid molecule encoding the light chain of the antibody of claim 13, or an antigen-binding fragment thereof.

23. A recombinant host cell comprising the nucleic acid of claim 21.

24. A recombinant host cell comprising the nucleic acid of claim 22.

25. A recombinant host cell comprising the nucleic acid of claim 21 and claim 22.

26. The recombinant host cell according to any one of claims 23-25, which is a eukaryotic host cell.

27. The recombinant host cell of claim 26, wherein the eukaryotic host cell is a Chinese Hamster Ovary (CHO) cell.

28. The recombinant host cell according to any one of claims 23-25, which is a prokaryotic host cell.

29. The recombinant host cell according to claim 28, wherein said prokaryotic host cell is an e.

30. A pharmaceutical composition comprising the antibody or antibody fragment of claim 14 in admixture with a pharmaceutically acceptable excipient.

31. A pharmaceutical composition comprising the antibody or antibody fragment of claim 15 in admixture with a pharmaceutically acceptable excipient.

32. The bispecific antibody or antigen-binding fragment of claim 15 for use in the preparation of a medicament for the treatment of an inflammatory or immune-related disease.

33. The bispecific antibody or antigen-binding fragment of claim 32, wherein the inflammatory disease or immune-related disease is selected from the group consisting of: systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathy (dermatomyositis, polymyositis), sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Graves ' disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes, immune-mediated nephropathy (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous system including multiple sclerosis, idiopathic demyelinating polyneuropathy or acute idiopathic polyneuritis and chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases, including infectious hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viral hepatitis), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, Inflammatory Bowel Disease (IBD), including ulcerative colitis, Crohn's disease, gluten-sensitive bowel disease, and Whitler's disease, autoimmune or immune-mediated skin diseases, including bullous skin disease, erythema multiforme and contact dermatitis, psoriasis, allergic diseases, including asthma, allergic rhinitis, atopic dermatitis, food allergies and urticaria, immunological diseases of the lung, including eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation-related diseases, including graft rejection and graft-versus-viral host disease, infectious diseases, including graft diseases, including AIDS (HIV infection) and hepatitis A, hepatitis B, hepatitis C, hepatitis D and E, herpes, bacterial infections, fungal infections, protozoal infections and parasitic infections.

34. The bispecific antibody or antigen-binding fragment of claim 33, wherein the inflammatory disease or immune-related disease is selected from the group consisting of Rheumatoid Arthritis (RA), Inflammatory Bowel Disease (IBD), and asthma.

35. An article of manufacture, comprising: (a) a container; (b) a label on the container; and (c) a composition of matter comprising the antibody or antigen-binding fragment of claim 15 contained with the container, wherein the label on the container indicates that the composition of matter is useful for treating an inflammatory disease or an immune-related disease.

36. Use of an antibody or antigen-binding fragment according to claim 15 in the manufacture of a medicament for the treatment of an inflammatory or immune-related disease.

37. A pharmaceutical composition for treating an inflammatory disease or an immune-related disease comprising the antibody or antigen-binding fragment of claim 15.