HK40016846A - Human il-23 antigen binding proteins - Google Patents

Description

Human IL-23 antigen binding proteins

The application is a divisional application, the application date of the original application is 26/10/2010, the application number is 201510065612.3, and the invention name is 'human IL-23 antigen binding protein'.

Cross Reference to Related Applications

The present application claims the benefit of U.S. patent application No. 61/254,982 filed on 26/10/2009 and U.S. patent application No. 61/381,287 filed on 9/2010, which are incorporated herein by reference, in accordance with 35 u.s.c.119 (e).

Background

Interleukin 23 (IL-23), a heterodimeric cytokine, is a potent inducer of proinflammatory cytokines. IL-23 is associated with the heterodimeric cytokine interleukin 12 (IL-12), both of which share a common subunit of p 40. In IL-23, a unique p19 subunit is covalently bound to a p40 subunit. In IL-12, the unique subunit is p35 (Oppmann et al, Immunity, 2000,13: 713-715). The IL-23 heterodimeric protein is secreted. Like IL-12, IL-23 is expressed by antigen presenting cells (e.g., dendritic cells and macrophages) in response to activating stimuli such as CD40 ligation, Toll-like receptor agonists, and pathogens. IL-23 binds to a heterodimeric receptor comprising an IL-12R β 1 subunit (which is shared with the IL-12 receptor) and a distinct receptor subunit IL-23R. The IL-12 receptor is composed of IL-12R β 1 and IL-12R β 2. IL-23 binds to its heterodimeric receptor and signals through JAK2 and Tyk2 to activate STATs 1,3, 4, and 5 (Parham et al, J. Immunol. 2002, 168: 5699-708). The receptor subunits are mainly co-expressed on activated or memory T cells and natural killer cells, and also expressed at lower levels on dendritic cells, monocytes, macrophages, microglia, keratinocytes and synovial fibroblasts. IL-23 and IL-12 on different T cell sub-groups play a basically different role in vivo.

IL-23 acts on activated and memory T cells and promotes the survival and expansion of the T cell subset Th 17. Th17 cells produce proinflammatory cytokines including IL-6, IL-17, TNF α, IL-22, and GM-CSF. IL-23 also acts on natural killer cells, dendritic cells and macrophages to induce expression of proinflammatory cytokines. Unlike IL-23, IL-12 induces the differentiation of naive CD4+ T cells into mature IFN γ -producing effector cells Th1 and induces the function of NK and cytotoxic T cells by stimulating IFN γ production. Th1 cells previously thought to be driven by IL-12 in many autoimmune diseases are a subset of pathogenic T cells, however, recent animal studies in models of inflammatory bowel disease, psoriasis, inflammatory Arthritis and multiple sclerosis, in which the individual contribution of IL-12 compared to IL-23 was assessed, firmly established that IL-23, but not IL-12, is a key driver in autoimmune/inflammatory diseases (Ahern et al, Immun. Rev. 2008226: 147-159; Cua et al, Nature 2003421: 744-748; Yago et al, Arthritis Res and Ther. 20079 (5): R96). IL-12 is thought to play a key role in the development of protective innate and adaptive immune responses against many intracellular pathogens and viruses and in tumor immune surveillance. See Kastellein et al, Annual Review of Immunology, 2007, 25: 221-42; liu, et al, Rheumatology, 2007, 46(8): 1266-73; bowman et al, Current Opinion Infectious diseases, 200619: 245-52; fieschi and Casanova, Eur. J. Immunol. 200333: 1461-4; meeran et al, mol. Cancer ther. 20065: 825-32; langowski et al, Nature 2006442: 461-5. Thus, specific inhibition of IL-23 (either broadly avoiding IL-12 or the shared p40 subunit) should have potentially superior safety profiles compared to dual inhibition of IL-12 and IL-23.

Thus, the use of an IL-23-specific antagonist that inhibits human IL-23 while sparing IL-12 (e.g., an antibody that binds at least the unique p19 subunit of IL-23 or both the p19 and p40 subunits of IL-23) should provide equal or greater potency than an IL-12 antagonist or a p40 antagonist without the potential risk associated with inhibiting IL-12. The selection of murine, humanized and phage display antibodies for inhibition of recombinant IL-23 has been described; see, e.g., U.S. patent 7,491,391, WIPO publication Nos. WO1999/05280, WO2007/0244846, WO2007/027714, WO2007/076524, WO2007/147019, WO2008/103473, WO 2008/103432, WO2009/043933, and WO 2009/082624. However, there is a need for fully human therapeutics capable of inhibiting native human IL-23. The therapeutic agents are highly specific for the target, especially in vivo. Complete inhibition of the target in vivo may result in lower doses of the formulation, less frequent and/or more effective dosing, which in turn results in reduced cost and improved efficacy. The present invention provides such IL-23 antagonists.

Brief description of the drawings

Antigen binding proteins that bind IL-23, particularly native human IL-23, are provided. The human IL-23 antigen binding proteins can reduce, inhibit, interfere with and/or modulate at least one biological response associated with IL-23, and as such, are useful for ameliorating the effects of an IL-23 associated disease or disorder. IL-23 antigen binding proteins may be used, for example, to reduce, inhibit, interfere with and/or modulate IL-23 signaling, IL-23 activation of Th17 cells, IL-23 activation of NK cells or to induce pro-inflammatory cytokine production.

Expression systems, including cell lines, are also provided for the production of IL-23 antigen binding proteins, and methods for diagnosing and treating diseases associated with human IL-23 are provided.

Some antigen binding proteins that bind IL-23 are provided that comprise at least one heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of: a CDRH1 that differs by no more than 1 amino acid substitution, insertion or deletion from the CDRH1 shown in table 3; a CDRH2 that differs by no more than 3, 2 or 1 amino acid substitutions, insertions and/or deletions from the CDRH2 shown in table 3; a CDRH3 that differs by no more than 3, 2 or 1 amino acid substitutions, insertions and/or deletions from the CDRH3 shown in table 3; the at least one light chain variable region comprises a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of: a CDRL1 that differs by no more than 3, 2, or 1 amino acid substitutions, insertions, and/or deletions from the CDRL1 shown in table 3; a CDRL2 that differs by no more than 1 amino acid substitution, insertion or deletion from the CDRL2 shown in table 3; a CDRL3 that differs from the CDRL3 shown in table 3 by no more than 1 amino acid substitution, insertion or deletion. In one embodiment, an isolated antigen binding protein is provided comprising: CDRH1 selected from the group consisting of SEQ ID NOs 91, 94, 97, 100 and 103; CDRH2 selected from the group consisting of SEQ ID NOs 92, 95, 98, 101, 104, 107 and 110; 93, 96, 99, 102 and 105 selected from the group consisting of CDRH 3; CDRL1 selected from SEQ ID NOs 62, 65, 68, 71 and 74; 63, 66, 69, 72, 75 and 78 selected from the group consisting of CDRL2 of SEQ ID NOs; and a CDRL3 selected from SEQ ID NOs 64, 67, 70 and 73. In another embodiment, an isolated antigen binding protein is provided comprising: CDRH1 selected from the group consisting of SEQ ID NOs 91, 106, 109, 112 and 115; CDRH2 selected from the group consisting of SEQ ID NOs 113, 116, 118, 120, 121 and 122; CDRH3 selected from the group consisting of SEQ ID NOs 108, 111, 114, 117 and 119; CDRL1 selected from SEQ ID NOs 77, 80, 83, 85, 86, 87, 88, 89 and 90; CDRL2 of SEQ ID NO: 81; and a CDRL3 selected from SEQ ID NOs 76, 79, 82 and 84. In another embodiment, an isolated antigen binding protein comprising at least one heavy chain variable region and at least one light chain variable region is provided. In yet another embodiment, there is provided an isolated antigen binding protein as described above, comprising at least two heavy chain variable regions and at least two light chain variable regions. In yet another embodiment, an isolated antigen binding protein is provided, wherein the antigen binding protein is coupled to a labeling group.

Also provided is an isolated antigen binding protein that binds IL-23, selected from the group consisting of: a) an antigen binding protein having a CDRH1 of SEQ ID NO 129, a CDRH2 of SEQ ID NO 132, a CDRH3 of SEQ ID NO 136, a CDRL1 of SEQ ID NO 123, a CDRL2 of SEQ ID NO 81, and a CDRL3 of SEQ ID NO 76; b) an antigen binding protein having a CDRH1 of SEQ ID NO:131, a CDRH2 of SEQ ID NO: 134, a CDRH3 of SEQ ID NO: 137, a CDRL1 of SEQ ID NO: 124, a CDRL2 of SEQ ID NO: 126, and a CDRL3 of SEQ ID NO: 128; c) a) an antigen binding protein having a CDRH1 of SEQ ID NO 130, a CDRH2 of SEQ ID NO 133, a CDRH3 of SEQ ID NO 99, a CDRL1 of SEQ ID NO 68, a CDRL2 of SEQ ID NO 69, and a CDRL3 of SEQ ID NO 67; and d) an antigen binding protein having CDRH1 of SEQ ID NO 91, CDRH2 of SEQ ID NO 135, CDRH3 of SEQ ID NO 138, CDRL1 of SEQ ID NO 125, CDRL2 of SEQ ID NO 127 and CDRL3 of SEQ ID NO 64.

Also provided are isolated antigen binding proteins that bind IL-23, comprising at least one heavy chain variable region and at least one light chain variable region selected from the group consisting of: a heavy chain variable region comprising amino acid residues 31-35, 50-65, and 99-113 of SEQ ID NO. 31, and a light chain variable region comprising amino acid residues 23-36, 52-58, and 91-101 of SEQ ID NO. 1; the heavy chain variable region comprising amino acid residues 31-35, 50-65, and 99-110 of SEQ ID NO 34, the heavy chain variable region comprising amino acid residues 31-35, 50-66, and 99-110 of SEQ ID NO 36, and the light chain variable region comprising amino acid residues 23-36, 52-62, and 97-105 of SEQ ID NO 4; the heavy chain variable region comprising amino acid residues 31-35, 50-66 and 99-114 of SEQ ID NO. 38 and the light chain variable region comprising amino acid residues 23-34, 50-61 and 94-106 of SEQ ID NO. 7; the heavy chain variable region comprising amino acid residues 31-35, 50-66 and 99-114 of SEQ ID NO. 40 and the light chain variable region comprising amino acid residues 24-34, 50-56 and 94-106 of SEQ ID NO. 9; the heavy chain variable region comprising amino acid residues 31-35, 50-66 and 99-114 of SEQ ID NO. 42 and the light chain variable region comprising amino acid residues 23-34, 50-61 and 94-106 of SEQ ID NO. 11; the heavy chain variable region comprising amino acid residues 31-35, 50-65, and 98-107 of SEQ ID NO. 44, and the light chain variable region comprising amino acid residues 24-34, 50-56, and 89-97 of SEQ ID NO. 13; 46 or 153 and light chain variable region comprising amino acid residues 24-34, 50-56 and 89-97 of SEQ ID NO 15; the heavy chain variable region comprising amino acid residues 31-37, 52-67 and 100-109 of SEQ ID NO. 48 and the light chain variable region comprising amino acid residues 24-34, 50-56 and 89-97 of SEQ ID NO. 17; the heavy chain variable region comprising amino acid residues 31-37, 52-67 and 101-109 of SEQ ID NO:50 and the light chain variable region comprising amino acid residues 24-34, 50-56 and 89-97 of SEQ ID NO: 19; a heavy chain variable region comprising amino acid residues 31-35, 50-65, and 98-107 of SEQ ID NO 52, and a light chain variable region comprising amino acid residues 24-34, 50-56, and 98-107 of SEQ ID NO 21; the heavy chain variable region comprising amino acid residues 31-37, 52-67 and 100-109 of SEQ ID NO: 54 and the light chain variable region comprising amino acid residues 24-34, 50-56 and 89-97 of SEQ ID NO: 23; the heavy chain variable region comprising amino acid residues 31-37, 52-67 and 100-109 of SEQ ID NO: 56 and the light chain variable region comprising amino acid residues 24-34, 50-56 and 89-97 of SEQ ID NO: 25; and the heavy chain variable region comprising amino acid residues 31-37, 52-57 and 100-109 of SEQ ID NO: 58 and the light chain variable region comprising amino acid residues 24-34, 500-56 and 89-97 of SEQ ID NO: 27.

Provided herein are isolated antigen binding proteins that bind IL-23, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence differs from the heavy chain variable region sequence shown in table 2 by no more than 13, 12, 11, 10, 9,8, 7, 6,5, 4,3, 2, or 1 amino acid substitutions, additions, and/or deletions; and wherein the light chain variable region sequence differs from the light chain variable region sequence set forth in Table 1 by no more than 13, 12, 11, 10, 9,8, 7, 6,5, 4,3, 2, or 1 amino acid substitutions, additions, and/or deletions.

Also provided is an isolated antigen binding protein that binds IL-23, selected from the group consisting of: a) 140 heavy chain variable region and 30 light chain variable region of SEQ ID NO; b) the heavy chain variable region of SEQ ID NO 141 and the light chain variable region of SEQ ID NO 61; c) the heavy chain variable region of SEQ ID NO: 142 and the light chain variable region of SEQ ID NO: 4; and d) the heavy chain variable region of SEQ ID NO 143 and the light chain variable region of SEQ ID NO 139.

Also provided are isolated antigen binding proteins comprising a heavy chain variable region consisting of an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs 31, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, and 58; the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NOs 1, 4,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27. In another embodiment, the isolated antigen binding protein comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs 44, 46, 48, 50, 52, 54, 56, 58, and 153, and a light chain variable region selected from the group consisting of SEQ ID NOs 13, 15, 17, 19, 21, 23, 25, and 27. In yet another embodiment, the isolated antigen binding protein comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs 31, 34, 36, 38, 40, and 42 and a light chain variable region selected from the group consisting of SEQ ID NOs 1, 4,7, 9, and 11.

Also provided is an isolated antigen binding protein that binds IL-23 comprising a heavy chain variable region and a light chain variable region selected from the group consisting of: a) the heavy chain variable region of SEQ ID NO. 31 and the light chain variable region of SEQ ID NO. 1; b) the heavy chain variable region of SEQ ID NO 34 or 36 and the light chain variable region of SEQ ID NO 4; c) the heavy chain variable region of SEQ ID NO 38 and the light chain variable region of SEQ ID NO 7; d) the heavy chain variable region of SEQ ID NO 40 and the light chain variable region of SEQ ID NO 9; e) the heavy chain variable region of SEQ ID NO 42 and the light chain variable region of SEQ ID NO 11; f) the heavy chain variable region of SEQ ID NO 44 and the light chain variable region of SEQ ID NO 13; g) 46 or 153 heavy chain variable region and 15 light chain variable region of SEQ ID NO; h) the heavy chain variable region of SEQ ID NO 48 and the light chain variable region of SEQ ID NO 17; i) the heavy chain variable region of SEQ ID NO 50 and the light chain variable region of SEQ ID NO 19; j) the heavy chain variable region of SEQ ID NO 52 and the light chain variable region of SEQ ID NO 21; k) the heavy chain variable region of SEQ ID NO 54 and the light chain variable region of SEQ ID NO 23; l) the heavy chain variable region of SEQ ID NO 56 and the light chain variable region of SEQ ID NO 25; and m) the heavy chain variable region of SEQ ID NO 58 and the light chain variable region of SEQ ID NO 27.

Also provided are isolated antigen binding proteins that bind human IL-23, wherein a covered patch (covered patch) formed when the antigen binding protein binds human IL-23 comprises residues at positions 30, 31, 32, 49, 50, 52, 53, 56, 92, and 94 of SEQ ID NO:15, wherein the residue contact has greater than or equal to 10 ŲAs determined by the solvent exposed surface area. In one embodiment, the residue contacts residues 31-35, 54, 58-60, 66 and 101-105 of SEQ ID NO 46.

Also provided are isolated antigen binding proteins that bind human IL-23, wherein a patch overlay formed when the antigen binding protein binds human IL-23 comprises a contact of residues 31-34, 51, 52, 55, 68, 93, and 98 of SEQ ID NO:1, wherein the contact of residues has greater than or equal to 10 ŲAs determined by the solvent exposed surface area. In one embodiment, the residue contacts residues 1, 26, 28, 31, 32, 52, 53, 59, 76, 101, 102 and 104-108 comprising SEQ ID NO 31.

Also provided are isolated antigen binding proteins that bind human IL-23, wherein the antigen binding protein is located at or closer to residues 5 Å of residues 32-35, 54, 58-60, 66 and 101-105 of SEQ ID NO 46 when the antigen binding protein binds human IL-23 as determined by X-ray crystallography.

Also provided are isolated antigen binding proteins that bind human IL-23, wherein the antigen binding proteins are located at or closer to residues 5 Å at positions 30-32, 49, 52, 53, 91-94 and 96 of SEQ ID NO. 15 when the antigen binding protein binds human IL-23, as determined by X-ray crystallography in one embodiment, the antigen binding proteins are located at or closer to residues 5 Å at positions 30-32, 49, 50, 52, 53, 56, 91-94 and 96 of SEQ ID NO. 15.

Also provided are isolated antigen binding proteins that bind human IL-23, wherein the antigen binding protein is located at or closer to residues 5 Å at positions 26-28, 31, 53, 59, 102, and 104 of SEQ ID NO. 31 when the antigen binding protein binds human IL-23 as determined by X-ray crystallography, hi one embodiment, the antigen binding protein is located at or closer to residues 5 Å at positions 1, 26-28, 30-32, 52, 53, 59, 100, and 102 of SEQ ID NO. 31.

Also provided are isolated antigen binding proteins that bind human IL-23, wherein the antigen binding proteins are at or closer to residues 5 Å at positions 31-34, 51, 52, 55, 68, and 93 of SEQ ID NO. 1 when the antigen binding protein binds human IL-23, as determined by X-ray crystallography in one embodiment, the antigen binding proteins are at or closer to residues 5 Å at positions 29, 31-34, 51, 52, 55, 68, 93, and 100 of SEQ ID NO. 1.

Also provided is an isolated antigen binding protein as described above, wherein the antigen binding protein is an antibody. In one embodiment, an isolated antigen binding protein is provided, wherein the antibody is a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment thereof. In another embodiment, an isolated antigen binding protein is provided, wherein the antibody fragment is a Fab fragment, a Fab 'fragment, a F (ab')2 fragment, a Fv fragment, a diabody (diabody), or a single chain antibody molecule. In yet another embodiment, an isolated antigen binding protein is provided, wherein the antigen binding protein is a human antibody. In yet another embodiment, an isolated antigen binding protein is provided, wherein the antigen binding protein is a monoclonal antibody. In another embodiment, an isolated antigen binding protein is provided, wherein the antigen binding protein is of the IgG1 type, IgG2 type, IgG3 type, or IgG4 type. In yet another embodiment, an isolated antigen binding protein is provided, wherein the antigen binding protein is of the IgG1 class or IgG2 class.

Isolated nucleic acid molecules encoding the antigen binding proteins described above are also provided. In one embodiment, an isolated nucleic acid molecule is provided wherein at least one heavy chain variable region is encoded by an isolated nucleic acid molecule selected from the group consisting of: 32, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 152, at least one light chain variable region encoded by an isolated nucleic acid molecule selected from the group consisting of: 2,5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28. In another embodiment, a nucleic acid molecule is provided wherein the nucleic acid molecule is operably linked to a regulatory sequence. In another embodiment, a vector comprising a nucleic acid molecule as described above is provided. In yet another embodiment, a host cell comprising a nucleic acid molecule as described above is provided. In another embodiment, a host cell comprising a vector as described above is provided. In yet another embodiment, an isolated polynucleotide is provided that is a fragment of a nucleic acid molecule as described above or the complement thereof sufficient for use as a hybridization probe, a PCR primer, or a sequencing primer.

Also provided is a method of making an antigen binding protein as described above, the method comprising the step of preparing the antigen binding protein from a host cell that secretes the antigen binding protein.

Also provided are isolated antigen binding proteins that bind human IL-23, wherein a covering patch formed when the antigen binding protein binds human IL-23 comprises a contact of residues within residues 46-58, 112-120 and 163 of the human IL-23p19 subunit as set forth in SEQ ID NO 145, wherein the contact of residues has a length greater than or equal to 10 ŲAs determined by the solvent exposed surface area. In one embodiment, there is provided wherein the overlay patch formed when the antigen binding protein binds to human IL-23 comprises 1,2, 3,4, 5,6, 7, 8, 9, 10, 11, 12 or 13 residues within residues 46-58 of the human IL-23p19 subunit as set forth in SEQ ID NO 145, 1,2, 3,4, 5,6, 7, 8, 9 or 10 residues within residues 112 and 120, and 1,2, 3,4, 5,6, 7, 8, 9 or 9 residues within residues 155 and 163. In another embodiment, provides wherein the antigen binding protein and human IL-23 binding formed when the coveringThe patch comprises a contact at residues within residues 121-125 of the human IL-23p40 subunit as set forth in SEQ ID NO: 147. In related embodiments, the patch covering that is formed when the antigen binding protein binds to human IL-23 comprises contact at1, 2, 3,4, or 5 residues within residues 121-125 of the human IL-23p40 subunit as set forth in SEQ ID NO: 147. In another embodiment, there is provided wherein the overlay patch formed upon binding of the antigen binding protein to human IL-23 comprises residues at positions 46, 47, 49, 50, 53, 112, 116, 118, 120, 155, 156, 159, 160 and 163 of SEQ ID NO. 145. In another embodiment, there is provided wherein the overlay patch formed upon binding of the antigen binding protein to human IL-23 comprises the contact of residues 46, 47, 49, 50, 53, 112 and 118, 120, 155, 156, 159, 160 and 163 of SEQ ID NO. 145. In another embodiment, there is provided wherein the overlay patch formed upon binding of the antigen binding protein to human IL-23 comprises residues 46, 47, 49, 50, 53-55, 57, 58, 112, 116, 118, 120, 155, 156, 159, 160, 162 and 163 of SEQ ID NO 145. In a related embodiment, there is provided wherein the overlay patch formed upon binding of the antigen binding protein to human IL-23 comprises a contact of residue 122 of the human IL-23p40 subunit as set forth in SEQ ID NO: 147. In another related embodiment, there is provided wherein the overlay patch formed upon binding of the antigen binding protein to human IL-23 comprises a contact of residues at subunits 122 and 124 of human IL-23p40 as set forth in SEQ ID NO: 147. In yet another related embodiment, there is provided wherein the overlay patch formed upon binding of the antigen binding protein to human IL-23 comprises the contact of residues 121-123 and 125 of the human IL-23p40 subunit as set forth in SEQ ID NO: 147. In yet another related embodiment, there is provided wherein the overlay patch formed upon binding of the antigen binding protein to human IL-23 comprises contact at residues 121-123, 125 and 283 of the human IL-23p40 subunit as set forth in SEQ ID NO: 147.

Also provided are isolated antigen binding proteins which bind to human IL-23, wherein when said antigen binding protein binds to human IL-23, the antigen binding protein is located at or closer to residues 46-58, 112-123 and 5 Å -163 of the human IL-23p19 subunit as set forth in SEQ ID NO 145. in one embodiment, when the antigen binding protein binds to human IL-23, the antigen binding protein is located at or closer to residues 1,2, 3,4, 5,6, 7, 8, 9, 10, 11, 12 or 13 of the human IL-23p19 subunit as set forth in SEQ ID NO 145. 1,2, 3,4, 5,6, 7, 8, 9 or 10 residues 163, 160, 156, 120, 112-123, and when the antigen binding protein binds to human IL-23, 120, 160-156, 120, 160-55, 160, 120, 55, 160, 55, 160, 55, 160, 55, 160, or 55, 160, 55, 160, 55, or 55, 160, 55, or 55, 160, 55, or 55, 160, 55, 160, or 55, 160, 55, 160, or 55, 160, 55, or 55, 160, 55, 160, or 55, 160, or 55, 160, 55, 160, 7, 160, or more residues of the relevant residues of the amino acid binding protein in one of the amino acid binding protein associated.

Also provided is an isolated antigen binding protein as described above, wherein the antigen binding protein has a sequence selected from the group consisting ofAt least one characteristic: a) reducing human IL-23 activity; b) reducing the production of proinflammatory cytokines; c) binding human IL-23 with KD less than or equal to 5 × 10-8M; d) k of ≤ 5 × 10-61/s_offA rate; and d) IC50 with ≦ 400 pM.

Pharmaceutical compositions are provided comprising at least one antigen binding protein as described above and a pharmaceutically acceptable excipient. In one embodiment, provided pharmaceutical compositions further comprise a labeling group or an effector group. In yet another embodiment, pharmaceutical compositions are provided wherein the labeling group is selected from the group consisting of: isotopic labels, magnetic labels, redox-active moieties, optical dyes, biotinylated groups, and predetermined polypeptide epitopes recognized by a second reporter. In yet another embodiment, pharmaceutical compositions are provided wherein the effector group is selected from the group consisting of: radioisotopes, radionuclides, toxins, therapeutic groups, and chemotherapeutic groups.

Also provided are methods for treating or preventing a condition associated with IL-23 in a patient, comprising administering to a patient in need thereof an effective amount of at least one isolated antigen binding protein as described above. In one embodiment, methods are provided wherein the condition is selected from the group consisting of: inflammatory disorders, rheumatic disorders, autoimmune diseases, neoplastic disorders and gastrointestinal disorders. In yet another embodiment, methods are provided wherein the condition is selected from the group consisting of: multiple sclerosis, rheumatoid arthritis, cancer, psoriasis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, psoriatic arthritis, autoimmune myocarditis; type 1 diabetes and ankylosing spondylitis. In yet another embodiment, methods are provided wherein the isolated antigen binding proteins are administered alone or as a combination therapy.

Also provided are methods of reducing IL-23 activity in a patient comprising administering an effective amount of at least one antigen binding protein as described above. In one embodiment, a method of reducing IL-23 activity is provided, wherein the IL-23 activity induces the production of pro-inflammatory cytokines.

Brief Description of Drawings

FIG. 1A: results of STAT-luciferase reporter assay with recombinant human IL-23. All antibodies completely inhibited recombinant human IL-23.

FIG. 1B: results of STAT-luciferase reporter assays with native human IL-23. Only half of the antibodies that completely inhibit recombinant human IL-23 are capable of completely inhibiting native human IL-23.

Detailed description of the invention

The present invention provides compositions, kits and methods relating to IL-23 antigen binding proteins, including molecules that antagonize IL-23, such as anti-IL-23 antibodies, antibody fragments and antibody derivatives, such as antagonistic anti-IL-23 antibodies, antibody fragments or antibody derivatives. Also provided are: polynucleotides and derivatives and fragments thereof comprising a nucleic acid sequence encoding all or part of a polypeptide that binds IL-23, e.g., polynucleotides encoding all or part of an anti-IL-23 antibody, antibody fragment, or antibody derivative; plasmids and vectors comprising said nucleic acids; and cells or cell lines comprising the polynucleotides and/or vectors and plasmids. The provided methods include, for example, methods of making, identifying, or isolating an IL-23 antigen binding protein (e.g., an anti-IL-23 antibody), methods of determining whether a molecule binds to IL-23, methods of determining whether a molecule antagonizes IL-23, methods of making compositions, e.g., pharmaceutical compositions, comprising an IL-23 antigen binding protein, and methods for administering an IL-23 antigen binding protein to a subject, e.g., methods for treating a condition mediated by IL-23 and for antagonizing the biological activity of IL-23 in vivo or in vitro.

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. The terminology used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are generally well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods as described in various general and more specific references that are well known in the art and that are cited and discussed throughout the present specification. See, e.g., Sambrook et al, molecular cloning: A Laboratory Manual, 3 rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and Ausubel et al, Current Protocols in molecular biology, Greene Publishing Associates (1992); and Antibodies by Harlow and Lane, oral Manual Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990). Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well known and commonly employed in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

All patents and other publications identified are expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the information described herein.

The polynucleotide and protein sequences of human IL-23p19 subunit (SEQ ID NOS: 144 and 145), consensus p40 subunit (SEQ ID NOS: 146 and 147), human IL-23 receptor heterodimer subunits IL-12R β 1 (SEQ ID NOS: 150 and 151), and IL-23R (SEQ ID NOS: 148 and 149) are known in the art, see, e.g., GenBank accession numbers AB030000, M65272, NM-005535, NM-144701, as are those from other mammalian species. Recombinant IL-23 and IL-23 receptor proteins, including single chain and Fc proteins, as well as cells expressing the IL-23 receptor have been described or are available from commercial sources. (see, e.g., Oppmann et al, Immunity, 2000,13: 713-. Native human IL-23 can be obtained from human cells, such as dendritic cells, using methods known in the art, including the methods described herein.

IL-23 is a heterodimeric cytokine consisting of a unique p19 subunit covalently bound to a common p40 subunit. The p19 subunit contains four α -helices "A", "B", "C" and "D" in an up-down motif connected by three internal helical loops between the A and B helices, between the B and C helices and between the C and D helices, see Oppmann et al, Immunity, 2000,13: 713-715 and Beyer et al, J Mol Biol, 2008.382 (4): 942-55. The a and D helices of the 4 helix bundle cytokines are thought to be involved in receptor binding. The p40 subunit contains 3 β -sheet sandwich domains D1, D2 and D3 (Lupardus and Garcia, J. MOL. biol., 2008, 382: 931-.

The term "polynucleotide" includes both single-and double-stranded nucleic acids, and includes genomic DNA, RNA, mRNA, cDNA, or synthetic sources or some combination thereof not associated with sequences normally found in nature. An isolated polynucleotide comprising a particular sequence may comprise, in addition to the particular sequence, a coding sequence for up to 10 or even up to 20 other proteins or portions thereof, or may comprise operably linked regulatory sequences that control expression of the coding region of the nucleic acid sequence, and/or may comprise vector sequences. The nucleotides that make up the polynucleotide may be ribonucleotides or deoxyribonucleotides or any type of modified form of a nucleotide. Modifications include base modifications, such as bromouridine and inosine derivatives; ribose modifications, such as 2',3' -dideoxyribose; and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate (phosphoroselenoate), phosphorodiselenoate (phosphorodiselenoate), phosphoroanilothioate (phosphoroanilothioate), phosphoroanilate (phosphoroaniladate), and phosphoramidate.

The term "oligonucleotide" means a polynucleotide comprising 100 nucleotides or less. In some embodiments, the oligonucleotide is 10-60 bases long. In other embodiments, the oligonucleotide is 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 nucleotides in length. The oligonucleotides may be single-stranded or double-stranded, for example for use in the construction of mutant genes. The oligonucleotide may be a sense or antisense oligonucleotide. The oligonucleotide may include a detectable label for use in a detection assay, such as a radioactive label, a fluorescent label, a hapten or an antigenic label. The oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization probes.

The term "polypeptide" or "protein" means a macromolecule having the amino acid sequence of a native protein, that is, a protein produced by a naturally occurring and non-recombinant cell; or a molecule produced by a genetically engineered or recombinant cell and comprising an amino acid sequence of a native protein; or a molecule that has one or more amino acid residues deleted from the native sequence, inserted into the native sequence, and/or substituted for one or more amino acid residues of the native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of corresponding naturally occurring amino acids and polymers. The terms "polypeptide" and "protein" include IL-23 antigen binding proteins (e.g., antibodies), and sequences in which one or more amino acid residues have been deleted from, added to, and/or substituted for one or more amino acid residues of the antigen binding protein sequence. The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxy-terminal deletion, and/or an internal deletion as compared to the full-length native protein. The fragments may also contain modified amino acids as compared to the native protein. In certain embodiments, fragments are about 5-500 amino acids in length. For example, a fragment can be at least 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of IL-23 antigen binding proteins, such as antibodies, useful fragments include, but are not limited to, one or more CDR regions, variable domains of heavy or light chains, portions of antibody chains, portions of variable regions comprising less than 3 CDRs, and the like.

"amino acid" includes its ordinary meaning in the art. The 20 naturally occurring amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis, 2 nd edition, (edited by E.S. Golub and D.R. Gren), Sinauer Associates: Sunderland, Mass. (1991). Stereoisomers of 20 conventional amino acids, unnatural amino acids such as [ alpha ] -, [ alpha ] -disubstituted amino acids, N-alkyl amino acids and other non-conventional amino acids (e.g., D-amino acids) may also be suitable polypeptide components. Examples of unconventional amino acids include: 4-hydroxyproline, [ gamma ] -carboxyglutamic acid, [ epsilon ] -N, N, N-trimethyllysine, [ epsilon ] -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, [ sigma ] -N-methylarginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide labeling approach used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction, consistent with standard usage and convention.

The term "isolated protein" refers to a protein (e.g., an antigen binding protein, an example of which may be an antibody) that is purified from the protein or polypeptide or other contaminant that would interfere with its therapeutic, diagnostic, prophylactic, research or other use. As used herein, "substantially pure" means that the molecular species is the predominant species present, that is, it is more abundant on a molar basis than any other individual species in the same mixture. In certain embodiments, a substantially pure molecule is a composition in which the species of interest makes up at least 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition comprises at least 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In certain embodiments, the substantially homogeneous mass is purified to the extent that contaminating species cannot be detected in the composition by conventional detection methods, and thus the composition consists of a single detectable macromolecular species.

A "variant" of a polypeptide (e.g., an antigen binding protein, e.g., an antibody) comprises the amino acid sequence: wherein one or more amino acid residues are inserted into, deleted from and/or substituted for one or more amino acid residues in the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins. A polypeptide "derivative" is a polypeptide that has been chemically modified in some manner other than an insertion, deletion or substitution variant, for example, via conjugation to another chemical moiety.

The term "naturally-occurring" or "native" as used throughout this specification in connection with a biological substance, e.g., a polypeptide, nucleic acid, host cell, etc., refers to a substance found in nature, e.g., native human IL-23. In certain aspects, recombinant antigen binding proteins that bind native IL-23 are provided. In this context, a "recombinant protein" is a protein prepared using recombinant techniques, i.e., by expression of a recombinant nucleic acid as described herein. Methods and techniques for producing recombinant proteins are well known in the art.

The term "antibody" refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with an intact antibody for specific binding to a target antigen, including, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. Thus, antibodies are a class of antigen binding proteins. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments and muteins thereof, examples of which are set forth below. A whole antibody typically comprises at least two full length heavy chains and two full length light chains, but in some cases may comprise fewer chains, for example an antibody naturally occurring in a camelid (camelid), which may comprise only heavy chains. Antibodies may be obtained from only a single source, or may be "chimeric," i.e., different portions of an antibody may be obtained from two different antibodies, as further described below. Antigen binding proteins, antibodies or binding fragments can be produced in hybridomas by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.

As used herein, the term "functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain) is an antigen-binding protein that comprises a portion of the antibody (regardless of how the portion is obtained or synthesized) that lacks at least some of the amino acids present in the full-length chain, but is capable of specifically binding to an antigen. The fragments are biologically active in that they specifically bind to the target antigen and can compete with other antigen binding proteins (including whole antibodies) for specific binding to a given epitope. In one aspect, the fragments retain at least one CDR present in a full-length light or heavy chain, and in some embodiments, comprise a single heavy and/or light chain or portion thereof. These biologically active fragments can be produced by recombinant DNA techniques or can be produced by enzymatic or chemical cleavage of antigen binding proteins, including whole antibodies. Fragments include, but are not limited to, immunologically functional fragments, such as Fab, Fab ', F (ab')2, Fv, domain antibodies, and single chain antibodies, and can be obtained from any mammalian source, including, but not limited to, human, mouse, rat, camel, or rabbit. It is further contemplated that functional portions of the antigen binding proteins disclosed herein, such as one or more CDRs, can be covalently bound to a second protein or to a small molecule to create a therapeutic agent that is directed against a specific target in vivo and has bifunctional therapeutic properties or has an extended serum half-life.

The term "competition," when used in the context of an antigen binding protein (e.g., a neutralizing antigen binding protein or neutralizing antibody), means competition between antigen binding proteins as determined by an assay in which an antigen binding protein (e.g., an antibody or immunologically functional fragment thereof) in an assay prevents or inhibits specific binding of a reference antigen binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., IL-23 protein or fragment thereof). Numerous types of competitive binding assays can be used, for example: solid phase direct or indirect Radioimmunoassays (RIA), solid phase direct or indirect Enzyme Immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al, 1983, Methods in Enzymology 92: 242-; solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al, 1986, J. Immunol. 137:3614-3619), solid phase direct labeling assays, solid phase direct labeling sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, Laboratory Manual, Cold Spring Harbor Press); direct labeling of RIA with an I-125 labeled solid phase (see, e.g., Morel et al, 1988, mol. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung et al, 1990, Virology 176: 546-552); and direct labeling of RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32: 77-82). Typically the assay involves the use of purified antigen bound to a solid surface or cells bearing any of the following antigen binding proteins: unlabeled test antigen binding protein and labeled reference antigen binding protein.

Competitive inhibition is measured by determining the amount of label bound to a solid surface or cells in the presence of the test antigen binding protein. Typically, the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assays (competing antigen binding proteins) include antigen binding proteins that bind the same epitope as a reference antigen binding protein, and antigen binding proteins that bind to a proximal epitope sufficiently close to the epitope bound by the reference antigen binding protein to be sterically hindered. Typically, when a competing antigen binding protein is present in excess, it inhibits specific binding of a reference antigen binding protein to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an antigen binding protein binds. Epitopes can be formed by adjacent amino acids or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. Epitope determinants may include chemically active surface clusters (groupings) of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and may have specific three-dimensional structural characteristics and/or specific charge characteristics. Epitopes typically comprise at least 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 amino acids in a unique spatial conformation. Epitopes can be determined by methods known in the art.

IL-23 antigen binding proteins

As used herein, "antigen binding protein" means a protein that specifically binds to a particular target antigen; the antigen provided herein is IL-23, particularly human IL-23, including native human IL-23. The antigen binding proteins provided herein interact with at least a portion of the unique p19 subunit of IL-23, detectably binding IL-23; but does not bind to IL-12 (e.g., the p40 and/or p35 subunits of IL-12) in any significant way, thus "sparing IL-12". Thus, the antigen binding proteins provided herein are capable of affecting IL-23 activity without the potential risk of inhibiting IL-12 or the consensus p40 subunit. Antigen binding proteins may affect the ability of IL-23 to interact with its receptor, for example by affecting binding to the receptor, for example by interfering with receptor association. In particular, the antigen binding proteins reduce, inhibit, interfere with, or modulate, in whole or in part, one or more biological activities of IL-23. Such inhibition or neutralization in the presence of an antigen binding protein disrupts a biological response, as compared to a response in the absence of the antigen binding protein, which can be determined using assays known in the art and described herein. The antigen binding proteins provided herein inhibit IL-23-induced pro-inflammatory cytokine production, such as IL-23-induced IL-22 production in whole blood cells and IL-23-induced IFN γ expression in NK and whole blood cells. The reduction in biological activity can be about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

The antigen binding protein may comprise a moiety that binds to an antigen, optionally comprising a scaffold or framework moiety that allows the antigen binding moiety to adopt a conformation that facilitates binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include antibodies, antibody fragments (e.g., antigen binding portions of antibodies), antibody derivatives, and antibody analogs. The antigen binding protein may comprise an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to: antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of an antigen binding protein; and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, e.g., Korndorfer et al, Proteins: Structure, Function, and AndBioinformatics, (2003), Vol.53, No. 1: 121-; roque et al, Biotechnol. prog., 2004,20: 639-654. In addition, peptide antibody mimetics ("PAM") as well as scaffolds based on antibody mimetics, which use a fibronectin component as a scaffold, can be used.

Certain antigen binding proteins described herein are antibodies or derived from antibodies. Such antigen binding proteins include, but are not limited to: monoclonal antibodies, bispecific antibodies, minibodies (minibodies), domain antibodies, synthetic antibodies, antibody mimetics, chimeric antibodies, humanized antibodies, human antibodies, antibody fusions, antibody conjugates, single chain antibodies, and fragments thereof, respectively. In some cases, the antigen binding protein is an immunological fragment of an antibody (e.g., Fab ', F (ab')2, or scFv). Various structures are further described and defined herein.

Certain antigen binding proteins provided can comprise one or more CDRs (e.g., 1,2, 3,4, 5,6, or more CDRs) described herein. In some cases, the antigen binding protein comprises: (a) a polypeptide structure; and (b) one or more CDRs inserted into and/or attached to the polypeptide structure. The polypeptide structure may take a number of different forms. For example, it may be or comprise the framework of a naturally occurring antibody or fragment or variant thereof, or may be virtually completely synthetic. Examples of various polypeptide structures are further described below.

When the dissociation equilibrium constant (KD) is less than or equal to 10^-8M, the antigen binding protein of the invention is considered to "specifically bind" to its target antigen. When KD is less than or equal to 5 multiplied by 10^-9When M is higher than the above range, the antigen binding protein specifically binds to antigen with "high affinity", and KD is less than or equal to 5X 10^-10M, the antigen binding protein specifically binds to the antigen with "very high affinity". In one embodiment, the antigen binding protein will be at ≤ 5 × 10^-12M KD binds to human IL-23, which in yet another embodiment will have a KD ≦ 5 × 10^-13And M is combined. In another embodiment of the invention, the antigen binding protein has ≤ 5 × 10^-12KD of M and about ≦ 5 × 10^-61/s K_off. In another embodiment, K_offIs less than or equal to 5 multiplied by 10^-71/s。

Another aspect provides antigen binding proteins having a half-life of at least one day in vitro or in vivo (e.g., when administered to a human subject). In one embodiment, the antigen binding protein has a half-life of at least three days. In another embodiment, the antibody or portion thereof has a half-life of 4 days or more. In another embodiment, the antibody or portion thereof has a half-life of 8 days or more. In another embodiment, the derivatized or modified antibody or antigen-binding portion thereof has a longer half-life as compared to the underivatized or unmodified antibody. In another embodiment, the antigen binding protein contains a point mutation to increase serum half-life, for example as described in WIPO publication No. WO 00/09560.

In embodiments in which the antigen binding protein is used for therapeutic applications, the antigen binding protein may reduce, inhibit, interfere with, or modulate one or more biological activities of IL-23, such as inducing pro-inflammatory cytokine production. IL-23 has many different biological effects, which can be measured in many different assays in different cell types; examples of such assays are known and are provided herein.

Some antigen binding proteins are provided that have a structure that is normally associated with naturally occurring antibodies. The structural units of these antibodies typically comprise one or more tetramers, each consisting of two identical pairs of polypeptide chains, but some species of mammals also produce antibodies with only a single heavy chain. In a typical antibody, each pair or pair includes one full length "light" chain (in certain embodiments, about 25 kDa) and one full length "heavy" chain (in certain embodiments, about 50-70 kDa). Each individual immunoglobulin chain is composed of several "immunoglobulin domains," each consisting of approximately 90-110 amino acids and exhibiting a characteristic folding pattern. These domains are the basic units that make up antibody polypeptides. The amino-terminal portion of each chain typically includes a variable region responsible for antigen recognition. The carboxy-terminal portion is evolutionarily more conserved than the other ends of the strand, and is referred to as the "constant region" or "C-region". Human light chains are generally divided into kappa and lambda light chains, each of which contains a variable region and a constant domain (CL 1). The z heavy chain is typically divided into mu, delta, gamma, alpha or epsilon chains, and these define the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subtypes, including but not limited to IgG1, IgG2, IgG3, and IgG 4. The IgM subtypes include IgM and IgM 2. IgA subtypes include IgA1 and IgA 2. In humans, IgA and IgD isotypes contain four heavy chains and four light chains; IgG and IgE isotypes contain two heavy chains and two light chains; the IgM isotype contains five heavy chains and five light chains. The heavy chain constant region (CH) typically comprises one or more domains that may be responsible for effector functions. The number of heavy chain constant region domains depends on the isotype. For example, IgG heavy chains each contain 3 CH region domains, designated CH1, CH2, and CH 3. The antibodies provided may be of any of these isotypes and subtypes, for example, the IL-23 antigen binding protein is of the IgG1, IgG2 or IgG4 subtype. If IgG4 is desired, it may also be desirable to introduce a point mutation (CPSCP- > CPPCP) into the hinge region (as described in Bloom et al, 1997, Protein Science 6: 407) to reduce the tendency to form intra-H chain disulfide bonds that can lead to heterogeneity of IgG4 antibodies. Subclass switching methods can be used to change an antibody provided herein that belongs to one class to a different class. See, e.g., Lantto et al, 2002, Methods mol. biol. 178: 303-.

In full-length light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, wherein the heavy chain also includes a "D" region of about 10 more amino acids. See, e.g., Fundamental Immunology, 2 nd edition, chapter 7 (Paul, W. ed.) 1989, New York: Raven Press. The variable region of each light/heavy chain pair typically forms an antigen binding site.

Variable region

The various heavy and light chain variable regions (or domains) provided herein are described in tables 1 and 2. Each of these variable regions may be linked, for example, to the heavy and light chain constant regions described above. In addition, each of the heavy and light chain sequences so produced can combine to form a complete antigen binding protein structure.

Providing an antigen binding protein comprising at least one heavy chain variable region (VH) and/or at least one light chain variable region (VL), said heavy chain variable region being selected from: VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14, VH15, and VH 16; the light chain variable region is selected from: VL1, VL2, VL3, VL4, VL5, VL6, VL7, VL8, VL9, VL10, VL11, VL12, VL13, VL14, VL15 and VL16, which are shown in tables 1 and 2 below.

Each of the heavy chain variable regions listed in table 2 can be combined with any of the light chain variable regions listed in table 1 to form an antigen binding protein. In some cases, the antigen binding protein includes at least one heavy chain variable region and/or one light chain variable region from those listed in tables 1 and 2. In some cases, the antigen binding protein includes at least two different heavy chain variable regions and/or light chain variable regions from those listed in tables 1 and 2. Various combinations of heavy chain variable regions may be combined with various combinations of any light chain variable region.

In other cases, the antigen binding protein contains two identical light chain variable regions and/or two identical heavy chain variable regions. For example, the antigen binding protein may be an antibody or immunologically functional fragment comprising two light chain variable regions and two heavy chain variable regions in a combination of light chain variable region pairs and heavy chain variable region pairs as listed in tables 1 and 2. Examples of such antigen binding proteins comprising two identical heavy and light chain variable regions include: antibody A VH 14/VL 14; antibody B VH 9/VL 9; antibody C VH 10/VL 10; antibody D VH 15/VL 15; antibody E VH 1/VL 1; antibody F VH 11/VL 11; antibody G VH12/VL 12; antibody H VH 13/VL 13; antibody I VH 8/VL 8; antibody J VH 3/VL 3; antibody K VH 7/VL 7; antibody L VH4/VL 4; antibody M VH 5/VL 5 and antibody N VH 6/VL 6.

Some antigen binding proteins provided comprise a heavy chain variable region and/or a light chain variable region comprising an amino acid sequence that differs from a sequence selected from the heavy chain variable regions and/or the light chain variable regions of tables 1 and 2 only by 1,2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, wherein each of the sequence differences is independently a deletion, insertion, or substitution of one amino acid. In some antigen binding proteins, the light and heavy chain variable regions comprise amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences provided in tables 1 and 2. Other antigen binding proteins, e.g., antibodies or immunologically functional fragments, also include variant heavy chain region forms and/or variant light chain region forms described herein.

The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more polynucleotides, as determined by aligning and comparing the sequences. "percent identity" means the percentage of residues that are identical between amino acids or nucleotides of the molecules being compared, calculated based on the size of the smallest molecule being compared

。

For these calculations, the nulls (if any) in the alignment should be accounted for by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in:Computational Molecular Biology(Lesk, A. M. eds.), 1988, New York: Oxford University Press; biocomputing information and Genome Projects, (Smith, D. W. eds.), 1993, New York: Academic Press; computer Analysis of Sequence Data, Part I, (Griffin, A. M. and Griffin, edited by H. G.), 1994, New Jersey: human Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; sequence Analysis Primer, (Gribskov, M. and Devereux, J. eds.), 1991, New York: M. Stockton Press; and also Carillo et al, 1988,SIAM J. Applied Math. 48:1073。

in calculating percent identity, the sequences being compared are aligned in a manner that provides the greatest match between the sequences. The computer program used to calculate percent identity is the GCG package, which includes GAP (Devereux et al, 1984,Nucl. Acid Res. 12387; genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align two polypeptides or polynucleotides whose percentage sequence identity is to be determined. The sequences are aligned for the best match ("matched span," which is determined by an algorithm) of their respective amino acids or nucleotides. Gap opening penalty (calculated as 3 times the average diagonal), where "average diagonal" is the ratio adoptedComparing the average of the diagonal of the matrix; "diagonal" is the score or value assigned to each perfect amino acid match by a particular comparison matrix) and a gap extension penalty (typically 1/10 for the gap opening penalty) and a comparison matrix such as PAM 250 or BLOSUM 62 are used with the algorithm. In certain embodiments, the algorithm also uses a standard comparison matrix (for PAM 250 comparison matrices, see Dayhoff et al, 1978,Atlas of Protein Sequence and Structure 5345 and 352; for BLOSUM 62 comparison matrices, see Henikoff et al, 1992,Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919)。

the recommended parameters for determining percent identity of a polypeptide or nucleotide sequence using the GAP program are as follows: the algorithm is as follows: the results of Needleman et al, 1970,J. MOL. Biol. 48443-; comparing the matrixes: BLOSUM 62 from Henikoff et al, 1992, supra; gap penalties: 12 (but no end gap penalty); gap length penalty: 4; similarity threshold: 0. certain alignment schemes for aligning two amino acid sequences can result in only short region matches of the two sequences, such small alignment regions can result in very high sequence identity even without significant relationship between the two full-length sequences. Thus, if desired, the selected alignment method (GAP program) can be adjusted to result in an alignment of at least 50 contiguous amino acids across the target polypeptide.

The heavy and light chain variable regions disclosed herein include consensus sequences derived from the group of related antigen binding proteins. The amino acid sequences of the heavy and light chain variable regions were analyzed for similarity. Four groups are presented, one with the kappa light chain variable region (V)_H9/ V_L9、V_H10/ V_L10、V_H11/ V_L11、V_H13/ V_L13、V_H14/ V_L14 and V_H15/ V_L15) Three groups have λ light chain variable regions: lambda set 1 (V)_H5/ V_L5、V_H6/ V_L6 and V_H7/ V_L7) Lambda group 2 (V)_H3/ V_L3 and V_H4/ V_L4) And lambda set 3 (V)_H1/ V_L1 and V_H2/ V_L2). Representative light chain germline include VK1/A30 and VK 1/L19. RepresentsThe light chain lambda germline of (a) includes VL1/1e, VL3/3p, VL5/5c and VL9/9 a. Representative heavy chain germline includes VH3/3-30, VH3/3-30.3, VH3/3-33, VH3/3-48, VH4/4-31 and VH 4/4-59. As used herein, "consensus sequence" refers to an amino acid sequence having conserved amino acids that are common among multiple sequences and variable amino acids that vary within a given amino acid sequence. The consensus sequences were determined using standard phylogenetic analyses for the light and heavy chain variable regions corresponding to the IL-23 antigen binding proteins disclosed herein.

The light chain variable region consensus sequence of the kappa group is DX₁QX₂TQSPSSVSASVGDRVTITCRASQGX₃X₄SX₅WX₆AWYQQKPGX₇APX₈LLIYAASSLQSGVPSRFSGSX₉SGTX₁₀FTLTISSLQPX₁₁DFATYX₁₂CQQANSFPFTFGPGTKVDX₁₃K (SEQ ID NO: 30), wherein X₁Is selected from I or S; x₂Is selected from M or L; x₃ Selected from G or V; and X₄Selected from S, F or I; x₅ Selected from S or G; x₆Is selected from F or L; x₇ Is selected from K or Q; x₈Selected from K, N or S; x₉Selected from G or V; x₁₀ Selected from D or E; x₁₁Selected from E or A; x₁₂ Selected from Y or F; and X₁₃ Selected from I, V or F.

The light chain variable region consensus sequence of lambda group 1 is QPX₁LTQPPSASASLGASVTLTCTLX₂SGYSDYKVDWYQX₃RPGKGPRFVMRVGTGGX₄VGSKGX₅GIPDRFSVLGSGLNRX₆LTIKNIQEEDESDYHCGADHGSGX₇NFVYVFGTGTKVTVL (SEQ ID NO: 61), wherein X₁Selected from V or E; x₂Is selected from N or S; x₃ Is selected from Q or L; x₄Is selected from I or T; x₅ Selected from D or E; x₆Selected from Y or S; and X₇ Selected from S or N.

The light chain variable region consensus sequence of lambda group 3 is QSVLTQPPVPSVSGAPGQRVTISCTGTSSSNX₁GAGYDVHWYQQX₂PGTAPKLLIYGSX₃NRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTX₄RLTVL (SEQ ID NO: 139), wherein X₁Selected from T or I; x₂Is selected from V or L; x₃ Selected from G or N; and X₄Is selected from ROr K.

The heavy chain variable region of kappa group has the consensus sequence QVQLQEGSGPGLVKPSQTLTCTVSGGSIX₁SGGYYWX₂WIRQHPGKGLEWIGX₃IX₄YSGX₅X₆YYNPSLKSRX₇TX₈SVDTSX₉NQFSLX₁₀LSSVTAADTAVYYCAX₁₁X₁₂RGX₁₃YYGMDVWGQGTTVTVSS (SEQ ID NO: 140), wherein X₁Is selected from N or S; x₂Selected from S or T; x₃ Selected from Y or H; x₄Selected from Y or H; x₅ Selected from S or N; x₆Selected from S or T; x₇ Selected from V or I; x₈Is selected from I or M; x₉Is selected from K or Q; x₁₀ Is selected from K or S; x₁₁Is selected from R or K; x₁₂ Selected from D or N; and X₁₃ Selected from H, F or Y.

The common sequence of heavy chain variable regions of lambda set 1 is EVQLVESGGGLVQPGGSLRLSCX₁X₂SGFTFSX₃X₄SMNWVRQAPGKGLEWVSYISSX₅SSTX₆YX₇ADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARRIAAAGX₈X₉X₁₀YYYAX₁₁DVWGQGTTVTVSS (SEQ ID NO: 141), wherein X₁Is selected from A or V; x₂Is selected from A or V; x₃ Selected from T or S; and X₄Selected from Y or F; x₅ Selected from S or R; x₆Is selected from R or I; x₇ Selected from H, Y or I; x₈Is selected from P or G; x₉Selected from W or F; x₁₀ Selected from G or H; and X₁₁Is selected from M or L.

The heavy chain variable region consensus sequence of lambda set 2 is QVQLVESGGGVQPGRSLRLSCAASGFTFSSYX₁MHWVRQAPGKGLEWX₂X₃VISX₄DGSX₅KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERTTLSGSYFDYWGQGTLVTVSS (SEQ ID NO: 142), wherein X₁Selected from G or A; x₂Is selected from V or L; x₃ Selected from A or S; and X₄Selected from F or H; and X₅ Is selected from L or I.

The common sequence of heavy chain variable regions of lambda group 3 is QVQLVESGGGVQPGRSLRLSCAASGFTFSSYGMHWVRQACPGKGLEWVAVIYDGSNX₁YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYX₂SSWYPDAFDIWGQGTMVTVSS (SEQ ID NO: 143), wherein X₁Is selected from E or K; and X₂Selected from T or S.

Complementary assay region

The complementary assay regions or "CDRs" are embedded in the framework of the heavy and light chain variable regions, where they constitute the regions responsible for antigen binding and recognition. Variable domains of immunoglobulin chains of the same species, for example, typically exhibit similar overall structures; comprising relatively conserved Framework Regions (FRs) linked by hypervariable CDR regions. The antigen binding protein may have 1,2, 3,4, 5,6 or more CDRs. For example, the variable regions discussed above typically comprise three CDRs. The CDRs from the heavy and light chain variable regions are typically positioned by framework regions to form a structure that specifically binds on the target antigen (e.g., IL-23). Both the naturally occurring light and heavy chain variable regions generally follow the following sequence of elements from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. The CDR and FR regions of exemplary light and heavy chain variable domains are highlighted in tables 1 and 2. It is recognized that the boundaries of the CDR and FR regions may vary from those highlighted. The numbering system is designed to assign numbers to the amino acids that occupy positions in each of these domains. These systems can be used to identify the complementarity determining regions and framework regions of a given antigen binding protein. The numbering system is defined in the following documents: kabat et al, Sequences of Proteins of immunological Interest, 5 th edition, United states department of Health and public Services (US Dept. of Health and human Services), PHS, NIH, NIH publication No. 91-3242, 1991; or Chothia& Lesk, 1987, J. MOL. Biol.196: 901-; the results of Chothia et al, 1989,Nature342:878-883. Other numbering systems for amino acids in immunoglobulin chains include IMGT (the International immunogenetics information System), Lefranc et al,Dev. Comp. Immunol2005, 29:185- > 203); and AHo (honeygger and Pluckthun,J. MOL. Biol. 2001, 309(3):657-670). The CDRs provided herein can be used not only to define the antigen binding domains of traditional antibody structures, but can also be embedded in a variety of other polypeptide structures described herein.

An antigen binding protein disclosed herein is a polypeptide to which one or more CDRs may be grafted, inserted, embedded, and/or linked. The antigen binding protein may have, for example: a heavy chain CDR1 ("CDRH 1"), and/or a heavy chain CDR2 ("CDRH 2"), and/or a heavy chain CDR3 ("CDRH 3"), and/or a light chain CDR1 ("CDRL 1"), and/or a light chain CDR2 ("CDRL 2"), and/or a light chain CDR3 ("CDRL 3"). Some antigen binding proteins comprise both CDRH3 and CDRL 3. Particular embodiments typically utilize combinations of CDRs that are not repetitive, e.g., antigen binding proteins are typically formed without two CDRH2 regions in one variable heavy chain region, and so forth. An antigen binding protein may comprise one or more amino acid sequences that are identical to the amino acid sequence of one or more CDRs presented in table 3, or that differ from the amino acid sequence of one or more CDRs presented in table 3 by only 1,2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, wherein each of said sequence differences is independently a deletion, insertion or substitution of one amino acid. In some antigen binding proteins, a CDR comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a CDR sequence set forth in table 3. In some antigen binding proteins, the CDRs are embedded in a "framework" region that orients one or more CDRs so as to achieve the appropriate antigen binding properties of the CDRs.

TABLE 3

Exemplary CDRH and CDRL sequences

Provided herein are CDR1 regions comprising the following amino acid residues: amino acid residues 23-34 of SEQ ID NO 7 and 11; amino acid residues 24-34 of SEQ ID NOs 9, 13, 15, 17, 19, 21, 23, 25, 27 and 29; amino acid residues 23-36 of SEQ ID NOS 1,3 and 4; amino acid residues 31-35 of SEQ ID NOs 31, 33, 34, 38, 40, 44, 52 and 60; and amino acid residues 31-37 of SEQ ID NOS 46, 48, 50, 54, 56 and 58.

Providing a CDR2 region comprising the following amino acid residues: amino acid residues 50-56 of SEQ ID NOs 9, 13, 15, 17, 19, 21, 23, 25, 27 and 29; amino acid residues 50-61 of SEQ ID NO 7 and 11; amino acid residues 52-62 of SEQ ID NO 4; amino acid residues 50-65 of SEQ ID NOS 31, 33, 44 and 52; amino acid residues 50-66 of SEQ ID NOS 36, 38, 40, 42 and 60; amino acid residues 52-58 of SEQ ID NOS: 1 and 3; and amino acid residues 52-67 of SEQ ID NOs 46, 48, 50, 54, 56 and 58.

Also provided are CDR3 regions comprising the following amino acid residues: amino acid residues 89-97 of SEQ ID NOs 13, 15, 17, 19, 21, 23, 25, 27 and 29; amino acid residues 91-101 of SEQ ID NOS 1 and 3; amino acid residues 94-106 of SEQ ID NOS 7, 9 and 11; amino acid residues 98-107 of SEQ ID NOS 44 and 52; amino acid residues 97-105 of SEQ ID NO 4; amino acid residues 99-110 of SEQ ID NOS 34 and 36; amino acid residues 99-112 of SEQ ID NO. 112; amino acid residues 99-113 of SEQ ID NOS 31 and 33; amino acid residues 99-114 of SEQ ID NOS 38, 40 and 42; amino acid residues 100-109 of SEQ ID NOS 46, 48, 54, 56 and 58; and amino acid residues 101-019 of SEQ ID NO: 50.

CDRs disclosed herein include consensus sequences derived from a related sequence set. Four groups of variable region sequences were identified, as described previously, namely the kappa group and the three lambda groups. CDRL1 consensus sequence from the kappa group consisting of RASQX₁X₂SX₃WX₄A (SEQ ID NO: 123) wherein X₁Selected from G or V; x₂Selected from I, F or S; x₃ Selected from S or G; and X₄Selected from F or L. CDRL1 consensus sequence from Lambda group 1 consists of TLX₁SGYSDYKVD (SEQ ID NO: 124), wherein X₁Is selected from N or S. The CDRL1 consensus sequence from lambda group 3 is represented by TGSSSNX₁GAGYDVH (SEQ ID NO: 125), wherein X₁Is selected from I or T.

CDRL2 consensus sequence from Lambda group 1 is represented by VGTGGX₁VGSKGX₂ (SEQ ID NO126) in which X₁Selected from I or T, X₂Is selected from D or E. CDRL2 consensus sequence from Lambda group 3 consisting of GSX₁NRPS (SEQ ID NO: 127) wherein X₁Is selected from N or G.

CDRL3 consensus sequences include GADHGSGX₁NFVYV (SEQ ID NO:128), where X₁Is S or N.

CDRH1 consensus sequence from the kappa group is represented by SGGYYWX₁(SEQ ID NO: 129) wherein X₁Selected from S or T. CDRH1 consensus sequence from lambda set 1 is represented by X₁X₂SMN (SEQ ID NO: 131) wherein X₁Selected from S or T, X₂Selected from Y or F. CDRH1 consensus sequence from Lambda group 2 was SYX₁MH (SEQ ID NO: 130), wherein X₁Is selected from G or A.

CDRH2 consensus sequence from the kappa group consisting of X₁IX₂YSGX₃X₄YNPSLKS (SEQ ID NO: 132) composition, wherein X₁Selected from Y or H; x₂Selected from Y or H; x₃ Selected from S or N, X₄Selected from T or S. The consensus sequence from lambda set 1 is represented by YISSX₁SSTX₂YX₃ADSVKG (SEQ ID NO: 134) with X₁Selected from R or S, X₂Selected from I or R, X₃Selected from I, H or Y. From lambda group 2 the consensus sequence is represented by VISX₁DGSX₂KYYADSVKG (SEQ ID NO: 133), wherein X₁Is F or H, X₂Is L or T. CDRH2 consensus sequence from lambda set 3 was VIWYDGSNX₁YYADSVKG (SEQ ID NO: 135), wherein X₁Is selected from K or E.

CDRH3 consensus sequence from the kappa group consisting of X₁RGX₂YYGMDV (SEQ ID NO: 136) wherein X₁Selected from N or D, X₂Selected from H, Y or F. CDRH3 consensus sequence from Lambda group 1 is represented by RIAAAAGX₁X₂X₃YYYAX₄DV (SEQ ID NO: 137) in which X₁Is selected from G or P; x₂Selected from F or W; x₃ Selected from H or G; and X₄Selected from L and M. CDRH3 consensus sequence from lambda set 3 was represented by DRGYX₁SSWYPDAFDI (SEQ ID NO: 138), wherein X₁Selected from S or T.

Monoclonal antibodies

Antigen binding proteins provided include monoclonal antibodies that bind to IL-23. Monoclonal antibodies can be produced by any technique known in the art, for example, by immortalizing spleen cells harvested from a transgenic animal following completion of an immunization protocol. The spleen cells may be immortalized by any technique known in the art, for example by fusing them with myeloma cells to produce hybridomas. Myeloma cells used in hybridoma-producing fusion procedures preferably do not produce antibodies, have high fusion efficiency and lack enzymes, which makes them incapable of growing in certain selective media that support the growth of only the desired fused cells (hybridomas). Examples of suitable cell lines for mouse fusion include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 41, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7, and S194/5XXO Bul; examples of cell lines for rat fusion include R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B 210. Other cell lines that can be used for cell fusion are U-266, GM1500-GRG2, LICR-LON-HMy2, and UC 729-6.

In some cases, hybridoma cell lines are generated as follows: by immunizing an animal (e.g., a transgenic animal having human immunoglobulin sequences) with an IL-23 immunogen; harvesting splenocytes from the immunized animal; fusing the harvested splenocytes with a myeloma cell line, thereby producing hybridoma cells; hybridoma cell lines were established from the hybridoma cells and hybridoma cell lines producing antibodies that bind IL-23 polypeptide while sparing IL-12 were identified. The hybridoma cell lines and anti-IL-23 monoclonal antibodies produced therefrom are aspects of the present application.

Monoclonal antibodies secreted by hybridoma cell lines can be purified by any technique known in the art. Hybridoma cells or mabs can be further screened to identify mabs with particular properties, such as the ability to inhibit IL-23-induced activity.

Chimeric and humanized antibodies

Chimeric and humanized antibodies based on the foregoing sequences are also provided. Monoclonal antibodies for use as therapeutic agents can be modified in a variety of ways prior to use. One example is a chimeric antibody, which is a chimeric antibody derived from a diabodyAn antibody consisting of protein segments of the same antibody covalently linked to produce a functional immunoglobulin light or heavy chain or immunologically functional portion thereof. Typically, portions of the heavy and/or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass. For methods related to chimeric antibodies, see, e.g., U.S. Pat. nos. 4,816,567; and Morrison et al, 1985,Proc. Natl. Acad. Sci. USA81:6851-6855. CDR grafting is described, for example, in U.S. Pat. nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101.

One useful type of chimeric antibody is a "humanized" antibody. Typically, humanized antibodies are generated from monoclonal antibodies originally produced in non-human animals. Certain amino acid residues in the monoclonal antibody, typically those from the non-antigen-recognizing portion of the antibody, are modified to be homologous to corresponding residues of a human antibody of the corresponding isotype. Humanization can be carried out, for example, by various methods by substituting at least a portion of the rodent variable regions for corresponding regions of human antibodies (see, e.g., U.S. Pat. Nos. 5,585,089 and 5,693,762; Jones et al, 1986,Nature321: 522-525; riechmann et al, 1988,Nature332: 323-27; verhoeyen et al, 1988,Science 239:1534-1536)。

in certain embodiments, the hybrid antibody can be produced using constant regions from a species other than human, together with human variable regions.

Fully human antibodies

Fully human antibodies are also provided. Methods are available for making fully human antibodies specific for a given antigen ("fully human antibodies") without exposing the human to the antigen. One specific means for implementing the production of fully human antibodies is to "humanize" the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated is one means of generating fully human monoclonal antibodies (mabs) in mice that can be immunized with any desired antigen. The use of fully human antibodies minimizes immunogenic and allergic reactions that can sometimes result from administration of a mouse mAb or mouse-derived mAb to a human as a therapeutic agent.

Fully human antibodies can be produced by immunizing a transgenic animal (typically a mouse) capable of producing a human antibody repertoire in the absence of endogenous immunoglobulin production. Antigens used for this purpose typically have 6 or more adjacent amino acids and are optionally conjugated to a carrier, such as a hapten. See, e.g., Jakobovits et al, 1993,Proc. Natl. Acad. Sci. USA2551, 2555; jakobovits et al, 1993,Nature362: 255-258; and Bruggermann et al, 1993,Year in Immunol.7:33. In one example of the method, a transgenic animal is produced by incapacitating an endogenous mouse immunoglobulin locus encoding a mouse heavy and light chain immunoglobulin chain therein and inserting human genomic DNA containing the locus encoding a human heavy and light chain protein into a large fragment of the mouse genome. The partially modified animals having less than the complete complement of the human immunoglobulin locus are then crossed to obtain animals having all of the desired immune system modifications. When administered to an immunogen, these transgenic animals produce antibodies immunospecific for the immunogen but having amino acid sequences (including variable regions) that are human rather than murine. For further details on the process see, for example, WIPO patent publication Nos. WO96/33735 and WO 94/02602. Additional methods related to transgenic mice for making human antibodies are described in the following references: U.S. patent nos. 5,545,807; 6,713,610, respectively; 6,673,986, respectively; 6,162,963, respectively; 5,545,807, respectively; 6,300,129, respectively; 6,255,458, respectively; 5,877,397, respectively; 5,874,299 and 5,545,806; WIPO patent publication Nos. WO91/10741, WO90/04036 and EP 546073B1 and EP 546073A 1.

The transgenic mice contain a gene encoding unrearranged human heavy chain ([ mu ])]And [ gamma ]]) And [ kappa ]]Human immunoglobulin Gene minilocus (minimus) of light chain immunoglobulin sequences, and the endogenous [ mu ] s]And [ kappa ]]Targeted mutations of inactivation of the chain locus (Lonberg et al, 1994,Nature368:856-859). Thus, the mice exhibited a decreaseLow mouse IgM or [ kappa ]]Expression and reactivity to immunity, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high affinity human IgG [ kappa ]]Monoclonal antibodies (Lonberg et al, supra; Lonberg and Huszar, 1995,Intern. Rev. Immunol.13: 65-93; harding and Lonberg, 1995,Ann. N.Y Acad. Sci. 764:536-546). The preparation of such mice is elaborated in the following literature: the total weight of the polymer in Taylor et al, 1992,Nucleic Acids Research6287-6295; chen et al, 1993,International Immunology647-656; the general knowledge of Tuaillon et al, 1994,J. Immunol.152: 2912-2920; the number of drops in the water of Lonberg et al, 1994,Nature 368:856-859；Lonberg, 1994, Handbook of Exp. Pharmacology113: 49-101; taylor et al, 1994,International Immunology579-; lonberg and huskzar, 1995,Intern. Rev. Immunol.13: 65-93; harding and Lonberg, 1995,Ann. N.Y Acad. Sci.764, 536-546; fishwild et al, 1996,Nature Biotechnology14:845-85. See also the following documents: U.S. Pat. nos. 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; 5,789,650, respectively; 5,877,397, respectively; 5,661,016, respectively; 5,814, 318; 5,874,299, respectively; and 5,770,429; and U.S. patent No. 5,545,807; WIPO publication No. WO 93/1227; WO 92/22646; and WO 92/03918. Techniques for producing human antibodies in these transgenic mice are also disclosed in WIPO publication Nos. WO 98/24893 and Mendez et al, 1997,Nature Genetics15: 146-. For example, HCo7 and HCo12 transgenic mouse strains can be used to generate anti-IL-23 antibodies.

Using hybridoma technology, antigen-specific human mAbs of the desired specificity can be generated and selected from transgenic mice, such as those described above. The antibody can be cloned and expressed with suitable vectors and host cells, or can be harvested from cultured hybridoma cells.

Fully human antibodies can also be derived from phage display libraries (e.g., as disclosed in Hoogenboom et al, 1991,J. MOL. Biol.227: 381; marks et al, 1991,J. MOL. Biol.222: 581; WIPO publication No. WO 99/10494). The phage display technique is carried out by displaying antibody repertoires on the surface of filamentous phage and then by using the antibody repertoiresBinding of the selected antigen selected phages were selected to mimic immunoselection.

Bispecific or bifunctional antigen binding proteins

A "bispecific", "dual specificity" or "bifunctional" antigen binding protein or antibody, respectively, is a hybrid antigen binding protein or antibody, having two different antigen binding sites, e.g., one or more CDRs or one or more variable regions as described above. In some cases, they are artificial hybrid antibodies with two different heavy/light chain pairs and two different binding sites. A multispecific antigen-binding protein or "multispecific antibody" is an antigen-binding protein or antibody that targets more than one antigen or epitope. Bispecific antigen binding proteins and antibodies are a class of multispecific antigen binding protein antibodies that can be produced by a variety of methods including, but not limited to, hybridoma fusion or ligation of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990,Clin. Exp. Immunol.79: 315-; the general formula of Kostelny et al, 1992,J. Immunol.148:1547-1553。

immunological fragments

Antigen binding proteins also include immunological fragments of antibodies (e.g., Fab ', F (ab')₂Or scFv). The "Fab fragment" consists of a light chain (variable region of the light chain (V))_L) And its corresponding constant domain (C)_L) And a heavy chain (heavy chain variable region (V))_H) And a first constant domain (C)_H1) ) is prepared. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab' fragment" comprises a light chain and a portion of a heavy chain, which also comprises C_H1 and C_H2 such that an interchain disulfide bond can be formed between the two heavy chains of the two Fab 'fragments to form F (ab')₂A molecule. Thus, "F (ab')₂Fragment "consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The "Fv fragment" consists of the variable light and heavy chain regions of a single arm of an antibody. A single-chain antibody "scFv" is an Fv molecule in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chainThe chains form the antigen binding region. Single chain antibodies are discussed in detail in the following references: WIPO publication No. WO 88/01649; U.S. Pat. nos. 4,946,778 and 5,260,203; the number of copies of Bird, 1988,Science242: 423; the results of the Huston et al, 1988,Proc. Natl. Acad. Sci. U.S.A5879, 85: 5879; the results of Ward et al, 1989,Nature334:544, de Graaf et al, 2002, Methods Mol Biol.178: 379-; the amount of the acid required for Kortt et al, 1997,Prot. Eng.10: 423; the amount of oxygen present in the mixture of Kortt et al, 2001,Biomol. Eng.18:95-108 and Kriangkum et al, 2001,Biomol. Eng.18:31-40. The "Fc" region contains a C comprising an antibody_H1 and C_H2 domain. Two heavy chain fragments through two or more disulfide bonds and through C_HThe hydrophobic interactions of the 3 domains remain together.

Also included are domain antibodies, i.e., immunologically functional immunoglobulin fragments that contain only the heavy chain variable region or the light chain variable region. In some cases, two or more V_HThe regions are covalently linked to a peptide linker to form a bivalent domain antibody. Two V of bivalent Domain antibody_HThe regions may target the same or different antigens. Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises a V connected by a linker_HAnd V_L(ii) domains which are too short to allow pairing between two domains of the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al,Proc. Natl. Acad. Sci. USA90:6444-48, 1993 and Poljak et al,Structure2:1121-23, 1994). Similarly, three-chain antibodies (tribodies) and four-chain antibodies (tetrabodies) are antibodies comprising three and four polypeptide chains, respectively, forming three and four antigen binding sites, respectively, which may be the same or different, and maxibodies comprising an IgG and IgG, respectively₁Covalently linked bivalent scFv of (a) (see, e.g., Fredericks et al, 2004,Protein Engineering, Design & Selection17: 95-106; powers et al, 2001,Journal of Immunological Methods,251: 123-; shu et al, 1993,Proc. Natl. Acad. Sci. USA90: 7995-; hayden et al, 1994,Therapeutic Immunology 1:3-15)。

various other forms

Also provided are variant forms of the above-disclosed antigen binding proteins, some having, for example, one or more conservative amino acid substitutions in one or more of the heavy or light chains, variable regions or CDRs listed in tables 1 and 2.

Naturally occurring amino acids can be classified based on common side chain properties as: hydrophobic (norleucine, Met, Ala, Val, Leu, Ile); neutral hydrophilic (Cys, Ser, Thr, Asn, Gln); acidic (Asp, Glu); basic (His, Lys, Arg); residues affecting chain orientation (Gly, Pro); and aromatic (Trp, Tyr, Phe).

Conservative amino acid substitutions may involve the exchange of a member of one of these classes for another member of the same class. Conservative amino acid substitutions may include non-naturally occurring amino acid residues, which are typically introduced by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics (peptidomimetics) and other inverted or inverted forms of the amino acid moiety. Such substantial modification of the functional and/or biochemical properties of the antigen binding proteins described herein can be achieved by making substitutions in the heavy and light chain amino acid sequences that differ significantly in their effect on maintaining: (a) the structure of the molecular framework in the substituted region, e.g., as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the size of the side chain (bulkiness).

Non-conservative substitutions may involve the exchange of a member of one of the above classes for a member of another class. The substituted residues may be introduced into a region of an antibody homologous to a human antibody, or into a non-homologous region of the molecule.

In making such changes, according to certain embodiments, the hydropathic index of amino acids may be considered. The hydrophilic character of a protein is calculated by assigning a numerical value ("hydrophilicity index") to each amino acid and then repeatedly averaging these values along the peptide chain. Each amino acid is assigned a hydropathic index based on its hydrophobic and charge characteristics. It comprises the following steps: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine/cystine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of hydrophilic character in conferring interactive biological function on proteins is understood in the art (see, e.g., Kyte et al, 1982,J. MOL. Biol.157:105-131). It is known that certain amino acids may be substituted with other amino acids having similar hydropathic indices or scores and still retain similar biological activity. Where changes are made based on hydropathic index, substitutions of amino acids whose hydropathic index is within ± 2 are included in certain embodiments. In some aspects includes substitutions of amino acids within ± 1, in other aspects includes substitutions of amino acids within ± 0.5.

It is also understood in the art that substitution of like amino acids can be made efficiently based on hydrophilicity, particularly where the biologically functional protein or peptide thereby produced is intended for use in immunological embodiments, as in the context of the present invention. In certain embodiments, the maximum local average hydrophilicity of a protein, governed by the hydrophilicity of its adjacent amino acids, is correlated with its immunogenicity and antigen binding or immunogenicity, that is, correlated with the biological properties of the protein.

The following hydrophilicity values assigned to these amino acid residues: arginine (+ 3.0); lysine (+ 3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). Where changes are made based on similar hydrophilicity values, substitutions of amino acids having hydrophilicity values within + -2 are included in certain embodiments, substitutions of amino acids within + -1 are included in other embodiments, and substitutions of amino acids within + -0.5 are included in yet other embodiments. In some cases, epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions".

Exemplary conservative amino acid substitutions are shown in table 4.

TABLE 4

Conservative amino acid substitutions

The skilled person will be able to determine suitable variants of the polypeptides shown herein using well known techniques, such as those described above. One skilled in the art can identify suitable regions of the molecule that can be altered by targeting regions not believed to be important for activity, but without disrupting activity. The skilled artisan is also able to identify residues and portions of the molecule that are conserved among similar polypeptides. In other embodiments, even regions important to biological activity or structure may be subjected to conservative amino acid substitutions without disrupting biological activity or adversely affecting polypeptide structure.

In addition, one skilled in the art can review structure-function studies that identify residues in similar polypeptides that are important for activity or structure. In view of such comparisons, the importance of amino acid residues in proteins corresponding to amino acid residues important for activity or structure in similar proteins can be predicted. One skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze 3-dimensional structures and amino acid sequences related to 3-dimensional structures in similar polypeptides. Given this information, one skilled in the art can predict the arrangement of amino acid residues of an antibody based on its 3-dimensional structure. One skilled in the art may choose not to make significant changes to amino acid residues predicted to be located on the surface of a protein, as such residues may participate in important interactions with other molecules. In addition, one skilled in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using assays directed to IL-23 activity (see examples below), thereby obtaining information on which amino acids can be altered and which cannot be altered. In other words, based on the information gathered from such routine tests, the skilled person can easily determine amino acid positions where further substitutions alone or in combination with other mutations should be avoided.

Many scientific publications are dedicated to predicting secondary structure. See, Moult, 1996,Curr. Op. in Biotech. 7: 422-; chou et al，1974，Biochemistry13: 222-245; the results of Chou et al, 1974,Biochemistry113: 211-222; chou et al, 1978,Adv. Enzymol. Relat. Areas Mol. Biol. 47: 45-148; chou et al, 1979,Ann. Rev. Biochem. 47: 251-276; and the list of applications by Chou et al, 1979,Biophys. J. 26:367-384. Furthermore, computer programs are currently available that assist in predicting secondary structure. One method of predicting secondary structure is based on homology modeling. For example, 2 polypeptides or proteins with sequence identity greater than 30% or similarity greater than 40% typically have similar topologies. Recent growth in protein structure databases (PDBs) has increased the predictability of secondary structure, including the likely number of folds within a polypeptide or protein structure. See, Holm et al, 1999,Nucl. Acid. Res. 27:244-247. Studies have shown (Brenner et al)，1997，Curr. Op. Struct. Biol. Biol. 7:369-376), there are a limited number of folds in a given polypeptide or protein, and once the cutoff value of the structure is resolved, the structure prediction becomes significantly more accurate.

Other methods of predicting secondary structure include the "threading method" (Jones, 1997,Curr. Opin. Struct. Biol. 7: 377-87; sippl et al, 1996,Structure4: 15-19); "Profile analysis" (Bowie et al)，1991，Science253: 164-; gribskov et al, 1990,Meth. Enzym. 183: 146-; gribskov et al, 1987,Proc. Nat. Acad. Sci. 4355-; and Brenner，1997, supra).

In some embodiments, amino acid substitutions are made that: (1) reducing susceptibility to proteolysis; (2) reducing susceptibility to oxidation; (3) altering the binding affinity of the protein complex formed; (4) altering ligand or antigen binding affinity; and/or (4) confer or alter other physicochemical or functional properties of the polypeptide, for example, maintain the structure of the molecular scaffold in the region of substitution, for example as a sheet or helical conformation; maintaining or altering the charge or hydrophobicity of the molecule at the target site; or to maintain or change the size of the side chain.

For example, single or multiple amino acid substitutions (in certain embodiments conservative amino acid substitutions) may be made in the naturally occurring sequence. Substitutions may be made in portions of the antibody that are outside of one or more domains that form intermolecular contacts. In such embodiments, conservative amino acid substitutions may be used that do not substantially alter the structural characteristics of the parent sequence (e.g., do not disrupt the substituted amino acid(s) that characterize the secondary structure of the parent or native antigen binding protein). Examples of art-recognized secondary and tertiary structures of polypeptides are described in the following references: proteins, Structures and molecular Principles (Creighton, ed.), 1984, W.H. New York: Freeman and company; introduction to Protein Structure (edited by Branden and Tooze), 1991, New York: Garland Publishing; and Thornton et al, 1991,Nature 354:105。

additional variants include cysteine variants in which one or more cysteine residues are deleted from the parent or native amino acid sequence or substituted with another amino acid (e.g., serine). Cysteine variants are particularly useful when the antibody, for example, must be refolded into a biologically active conformation. Cysteine variants may have fewer cysteine residues than the native protein, usually an even number to minimize interactions due to unpaired cysteines.

The disclosed heavy and light chain variable regions and CDRs can be used to prepare antigen binding proteins comprising an antigen binding region that specifically binds to an IL-23 polypeptide. By "antigen binding region" is meant a protein or portion of a protein that specifically binds a particular antigen, e.g., a region containing amino acid residues that interact with an antigen and confer its specificity and affinity for a target antigen on the antigen binding protein. Antigen binding regions may include one or more CDRs, and certain antigen binding regions also include one or more "framework" regions. For example, one or more of the CDRs listed in table 3 can be incorporated, covalently or non-covalently, into a molecule (e.g., a polypeptide) for immunoadhesion. Immunoadhesions can incorporate CDRs as part of a larger polypeptide chain, can have CDRs covalently linked to another polypeptide chain, or can incorporate CDRs non-covalently. The CDRs enable the immunoadhesion to specifically bind to a particular antigen of interest (e.g., an IL-23 polypeptide).

Other antigen binding proteins include mimetics (e.g., "peptide mimetics" or "peptidomimetics") based on the variable regions and CDRs described herein. These analogs can be peptidic, non-peptidic, or a combination of peptidic and non-peptidic regions. The results of the Fauchere, 1986,Adv. Drug Res.15: 29; veber and Freidinger, 1985,TINSpage 392; and the results of Evans et al, 1987,J. Med. Chem.30:1229. Peptidomimetics that are structurally similar to therapeutically useful peptides can be used to produce similar therapeutic or prophylactic effects. The compounds are typically developed by means of computerized molecular modeling. A peptidomimetic is typically a protein that is structurally similar to an antigen binding protein that exhibits a desired biological activity (e.g., the ability to bind IL-23), but has one or more peptide linkages, optionally replaced by a linkage selected from, for example: -CH₂NH-、-CH₂S-、-CH₂-CH₂-, -CH-CH- (cis and trans) -, -COCH₂-、-CH(OH)CH₂-and-CH₂SO-. Systematic substitution of one or more amino acids of the consensus sequence with the same type of D-amino acid (e.g., D-lysine in place of L-lysine) can be utilized in certain embodiments to produce more stable proteins. In addition, constrained peptides comprising a consensus sequence or substantially identical consensus sequence variations can be generated by methods known in the art (Rizo and giarasch, 1992,Ann. Rev. Biochem61:387), for example by adding internal cysteine residues capable of forming intramolecular disulfide bonds which cyclize the peptide.

Derivatives of the antigen binding proteins described herein are also provided. The derivatized antigen binding protein may comprise any molecule or substance that confers a desired property (e.g., increased half-life in a particular use) to the antigen binding protein or fragment. The derivatized antigen binding protein can comprise, for example, a detectable (or labeled) moiety (e.g., a radioactive molecule, a colorimetric molecule, an antigenic molecule, or an enzymatic molecule, a detectable bead (e.g., a magnetic or electron dense (e.g., gold) bead), or a molecule that binds to another molecule (e.g., biotin or streptavidin)), a therapeutic or diagnostic moiety (e.g., a radioactive moiety, a cytotoxic moiety, or a pharmaceutically active moiety), or a molecule that increases the suitability of the antigen binding protein for a particular use (e.g., administration to a subject, such as a human subject, or other in vivo or in vitro use). Examples of molecules that can be used to derivatize antigen binding proteins include albumin (e.g., human serum albumin) and polyethylene glycol (PEG). Albumin linked and pegylated derivatives of antigen binding proteins can be prepared using techniques well known in the art. In one embodiment, the antigen binding protein is conjugated or otherwise linked To Transthyretin (TTR) or a TTR variant. TTR or TTR variants may be chemically modified with a chemical substance selected, for example, from: dextran, poly (n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylenated polyol, and polyvinyl alcohol.

Other derivatives include covalent or aggregative conjugates of IL-23 antigen binding proteins with other proteins or polypeptides, for example by expression of a recombinant fusion protein comprising a heterologous polypeptide fused to the N-terminus or C-terminus of the IL-23 antigen binding protein. For example, the conjugate peptide can be a heterologous signal (or leader) polypeptide, such as a yeast alpha-factor leader peptide, or a peptide such as an epitope tag or the like. Fusion proteins containing IL-23 antigen binding proteins may include peptides (e.g., poly-His) added to facilitate purification or identification of IL-23 antigen binding proteins. IL-23 antigen binding proteins may also be linked to FLAG peptides as described in: the number of copies of Hopp et al, 1988,Bio/Technology6: 1204; and U.S. patent No. 5,011,912. The FLAG peptide is highly antigenic, providing an epitope that is reversibly bound by a specific monoclonal antibody (mAb), which enables rapid assay and easy purification of expressed recombinant proteins. Commercially available for preparing a peptide in which the FLAG peptide is fused to a given polypeptideReagents for the fusion proteins in question (Sigma, St. Louis, MO).

Oligomers containing one or more IL-23 antigen binding proteins can be used as IL-23 antagonists. The oligomer may be in the form of a covalently linked or non-covalently linked bimer, trimer or higher oligomer. Oligomers comprising two or more IL-23 antigen binding proteins are contemplated for use, one example being homodimers. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, and the like. Also included are oligomers comprising a plurality of IL-23 binding proteins linked via covalent or non-covalent interactions between peptide moieties fused to the IL-23 antigen binding protein. The peptide may be a peptide linker (spacer), or a peptide with properties that promote oligomerization. Among suitable peptide linkers are those described in U.S. patent nos. 4,751,180 and 4,935,233. Leucine zippers and certain polypeptides derived from antibodies belong to peptides that can promote oligomerization of the IL-23 antigen binding protein to which they are linked. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in the following documents: WIPO publication No. WO 94/10308; the results of the reactions of Hoppe et al, 1994,FEBS Letters344: 191; and Fanslow et al, 1994,Semin. Immunol.6:267-278. In one approach, a recombinant fusion protein comprising an IL-23 antigen-binding protein fragment or derivative fused to a leucine zipper peptide is expressed in a suitable host cell, and the resulting soluble oligomeric IL-23 antigen-binding protein fragment or derivative is recovered from the culture supernatant.

The oligomer may comprise two to four IL-23 antigen binding proteins. The IL-23 antigen binding protein portion of the oligomer can be in any of the forms described above, e.g., a variant or a fragment. Preferably, the oligomer comprises an IL-23 antigen binding protein having IL-23 binding activity. Oligomers can be prepared from polypeptides derived from immunoglobulins. The preparation of fusion proteins comprising certain heterologous polypeptides fused to portions of antibody-derived polypeptides, including Fc domains, is described, for example, in the following references: ashkenazi et al, 1991,Proc. Natl. Acad. Sci. USA88: 10535; byrn et al, 1990,Nature344: 677; and Hollenbaugh et al, 1992, "ConstrThe construction of Immunoglobulin Fusion Proteins is described in Current Protocols in Immunology, supplement 4, pp 10.19.1-10.19.11.

Also included are dimers comprising two fusion proteins produced by fusing an IL-23 antigen binding protein to the Fc region of an antibody. The dimer can be prepared as follows: the dimer is obtained by, for example, inserting a gene fusion encoding the fusion protein into a suitable expression vector, expressing the gene fusion in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble very like an antibody molecule, thereby forming interchain disulfide bonds between the Fc portions. The Fc polypeptides include native and mutein forms of polypeptides derived from the Fc region of an antibody. Also included are truncated forms of the polypeptides that include a hinge region that promotes dimerization. Fusion proteins comprising an Fc portion (and oligomers formed therefrom) offer the advantage of being easily purified by affinity chromatography on a protein a or protein G column. One suitable Fc polypeptide, described in WIPO publication No. WO 93/10151 and U.S. patent nos. 5,426,048 and 5,262,522, is a single chain polypeptide that extends from the N-terminal hinge region to the natural C-terminus of the Fc region of human IgG1 antibody. Another useful Fc polypeptide is that described in U.S. patent No. 5,457,035 and Baum et al, 1994,EMBO J.3992 and 4001. The amino acid sequence of this mutein is identical to the amino acid sequence of the native Fc sequence presented in WIPO publication No. WO 93/10151, but amino acid 19 was changed from Leu to Ala, amino acid 20 was changed from Leu to Glu, and amino acid 22 was changed from Gly to Ala. The muteins exhibit reduced Fc receptor affinity.

Glycosylation

Antigen binding proteins may have a glycosylation pattern that is different from or altered from that found in the native species. As is known in the art, the glycosylation pattern can depend on both the protein sequence (e.g., the presence or absence of specific glycosylated amino acid residues described below) or the host cell or organism in which the protein is produced. Specific expression systems are described below.

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

The addition of glycosylation sites to the antigen binding protein is suitably achieved by altering the amino acid sequence to contain one or more of the above-described tri-peptide sequences (for N-linked glycosylation sites). Changes (for O-linked glycosylation sites) can also be made by adding one or more serine or threonine residues to the starting sequence or substituting the starting sequence with such residues. For simplicity, the antigen binding protein amino acid sequence can be altered by changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases so as to produce codons that translate to the desired amino acids.

Chemical or enzymatic coupling of glycosides to proteins is another means of increasing the number of carbohydrate moieties on antigen binding proteins. These methods are advantageous because they do not require the production of proteins in host cells with glycosylation capability for N-linked and O-linked glycosylation. Depending on the coupling mode used, the sugar may be linked to: (a) arginine and histidine; (b) a free carboxyl group; (c) free sulfhydryl groups, such as the sulfhydryl group in cysteine; (d) free hydroxyl groups, such as the hydroxyl groups in serine, threonine, or hydroxyproline; (e) aromatic residues, such as residues in phenylalanine, tyrosine or tryptophan; or (f) an amide group of glutamine. Such methods are described in PCT publication nos. WO 87/05330 and Aplin and Wriston,1981,CRC Crit. Rev, Biochem.page 259-306.

Removal of the carbohydrate moiety present on the starting antigen binding protein may be achieved chemically or enzymatically. Chemical deglycosylation requires contacting the protein with the compound trifluoromethanesulfonic acid orEquivalent compounds. This treatment results in the cleavage of most or all of the sugars except the linked sugar (N-acetylglucosamine or N-acetylgalactosamine) while preserving the integrity of the polypeptide. The results obtained by Hakimuddin et al, 1987,Arch. Biochem. Biophys.259:52 and Edge et al, 1981,Anal. Biochem.118:131 illustrates chemical deglycosylation. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by using a variety of endo-and exoglycosidases, such as Thotakura et al, 1987,Meth. Enzymol.138: 350. Glycosylation at potential glycosylation sites can be prevented by the use of the compound tunicamycin, e.g., Duskin et al, 1982,J. Biol. Chem.257: 3105. Tunicamycin blocks the formation of protein-N-glycosidic bonds.

Thus, aspects include glycosylated variants of the antigen binding protein in which the number and/or type of glycosylation sites is altered compared to the amino acid sequence of the parent polypeptide. In certain embodiments, the antigen binding protein variant comprises a greater or lesser number of N-linked glycosylation sites than the parent polypeptide. Substitutions that eliminate or alter this sequence will prevent the addition of an N-linked carbohydrate chain present in the parent polypeptide. For example, glycosylation can be reduced by deleting Asn or by substituting Asn with a different amino acid. Antibodies typically have N-linked glycosylation sites in the Fc region.

Labelling and effector groups

The antigen binding protein may comprise one or more labels. The term "label" or "labeling group" refers to any detectable label. Generally, labels are classified into a variety of categories depending on the assay in which they are detected: a) an isotopic label, which can be radioactive or a heavy isotope; b) magnetic labels (e.g., magnetic particles); c) a redox active moiety; d) an optical dye; enzyme groups (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase); e) a biotinylated group; and f) a predetermined polypeptide epitope recognized by a second reporter molecule (e.g., a leucine zipper pair sequence, a second antibody binding site, a metal binding domain, an epitope tag, etc.). In some embodiments, the labeling group is coupled to the antigen binding protein via spacer arms of various lengthsTo reduce possible steric hindrance. Various methods for labeling proteins are known in the art. Examples of suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g. of the type³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I) A fluorescent group (e.g., FITC, rhodamine, lanthanide phosphors), an enzyme group (e.g., horseradish peroxidase, β -galactosidase, luciferase, alkaline phosphatase), a chemiluminescent group, a biotin group, or a predetermined polypeptide epitope recognized by a second reporter (e.g., leucine zipper pair sequence, a second antibody binding site, a metal binding domain, an epitope tag). In some embodiments, the labeling group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used as deemed appropriate.

The term "effector group" means any group coupled to an antigen binding protein that functions as a cytotoxic agent. Examples of suitable effector groups are radioisotopes or radionuclides (e.g. as³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I) In that respect Other suitable groups include toxins, therapeutic groups, or chemotherapeutic groups. Examples of suitable groups include calicheamicins, auristatins, geldanamycin (geldanamycin) and maytansine. In some embodiments, the effector group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance.

Polynucleotides encoding IL-23 antigen binding proteins

Also provided are polynucleotides encoding the antigen binding proteins described herein, or portions thereof, comprising: a polynucleotide encoding one or both chains of an antibody or a fragment, derivative, mutein or variant thereof; a polynucleotide encoding a heavy chain variable region or only a CDR; a polynucleotide sufficient for use as a hybridization probe, PCR primer, or sequencing primer for identifying, analyzing, mutating, or amplifying a polynucleotide encoding a polypeptide; an antisense nucleic acid that inhibits expression of the polynucleotide; and the complement of the aforementioned polynucleotides. The polynucleotide may be of any length. They may be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleic acids in length (including all values in between), and/or may comprise one or more additional sequences, such as regulatory sequences and/or be part of a larger polynucleotide, such as a vector. Polynucleotides may be single-stranded or double-stranded, and may comprise RNA and/or DNA nucleic acids and artificial variants thereof (e.g., peptide nucleic acids).

Polynucleotides encoding certain antigen binding proteins or portions thereof (e.g., full length antibodies, heavy or light chains, variable domains or CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3) can be isolated from B-cells of mice immunized with IL-23 or an immunogenic fragment thereof. Polynucleotides can be isolated by conventional procedures, such as Polymerase Chain Reaction (PCR). Phage display is another example of a known technique by which antibody derivatives and other antigen binding proteins can be prepared. In known methods, polypeptides that are components of an antigen binding protein of interest are expressed in any suitable recombinant expression system and the expressed polypeptides are assembled to form an antigen binding protein molecule. Antigen binding proteins with different properties (i.e., altered affinity for the antigen to which they bind) are also obtained via chain shuffling using phage display, see Marks et al，1992, BioTechnology 10:779。

Due to the degeneracy of the genetic code, each of the polypeptide sequences depicted herein is also encoded by a large number of other polynucleotide sequences in addition to the polynucleotide sequences provided. For example, the heavy chain variable domains provided herein can be encoded by polynucleotide sequences SEQ ID NOs 32, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 59. The light chain variable domain may be encoded by the polynucleotide sequence of SEQ ID NO 2,5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28. Thus, one of ordinary skill in the art will appreciate that the present application provides a written description of sufficient and allows each degenerate nucleotide sequence to encode each antigen binding protein.

One aspect further provides polynucleotides that hybridize to other polynucleotide molecules under specific hybridization conditions. Methods for hybridizing nucleic acids, basic parameters that influence the choice of hybridization conditions, and guidelines for designing suitable conditions are well known in the art. See, e.g., Sambrook, FRitsch, and Maniatis (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Current Protocols in Molecular Biology, 1995, Ausubel et al, eds., John Wiley & Sons, Inc., as defined herein, medium stringency hybridization conditions use a prewashing solution containing 5x sodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer of about 50% formamide, 6x SSC, and a hybridization temperature of 55 ℃ (or other similar hybridization solutions such as those containing about 50% formamide and a hybridization temperature of 42 ℃), and washing conditions of 60 ℃ in 0.5x SSC, 0.1% SDS, a washing condition of 60 ℃ followed by one more washes in 6x SSC, 0.68 ℃ in 0.68% SSC, one of skill in the art can manipulate hybridization and/or wash conditions to increase or decrease hybridization stringency such that polynucleotides comprising nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (including all values therebetween) identical to each other are typically maintained hybridized to each other.

Changes can be introduced into a polynucleotide by mutation, thereby resulting in a change in the amino acid sequence of the polypeptide (e.g., antigen binding protein or antigen binding protein derivative) that it encodes. Mutations can be introduced by any technique known in the art, such as site-directed mutagenesis and random mutagenesis. The mutant polypeptide may be expressed and selected for a desired property. Mutations can be introduced into a polynucleotide without significantly altering the biological activity of the polypeptide it encodes. For example, substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a polynucleotide that selectively alter the biological activity of the polypeptide encoded thereby. For example, mutations can quantitatively or qualitatively alter biological activity, e.g., increase, decrease or eliminate activity and alter the antigen specificity of an antigen binding protein.

Another aspect provides polynucleotides suitable for use as primers or hybridization probes to detect nucleic acid sequences. A polynucleotide may comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide, e.g., a fragment that can be used as a probe or primer or a fragment encoding an active portion of a polypeptide (e.g., an IL-23 binding portion). Nucleic acid sequence-based probes can be used to detect the nucleic acid or similar nucleic acids, e.g., to detect transcripts encoding polypeptides. The probe may comprise a labelling group, such as a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor. Such probes can be used to identify cells expressing the polypeptide.

Methods of expressing antigen binding proteins

The antigen binding proteins provided herein can be prepared by any of a variety of conventional techniques. For example, the IL-23 antigen binding protein may be produced by recombinant expression systems using any technique known in the art. See, e.g., monoclonal antibodies, hybrids: A New Dimension in Biological analytes, Kennet et al (eds.) Plenum Press, New York (1980); and Antibodies A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988).

Also provided herein are expression systems and constructs in the form of plasmids, expression vectors, transcription cassettes, or expression cassettes comprising at least one polynucleotide as described above, and host cells comprising the expression systems or constructs. "vector" as used herein means any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) suitable for transferring protein-encoding information into a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors. Expression vectors, such as recombinant expression vectors, may be used to transform a host cell and contain (with the host cell) nucleic acid sequences that direct and/or control the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, if introns are present, RNA splicing of coding regions operably linked thereto. "operably linked" means that the components to which the term applies are in a relationship permitting them to perform their inherent function. For example, a regulatory sequence such as a promoter that is "operably linked" to a protein coding sequence is arranged in a vector such that normal activity of the regulatory sequence results in transcription of the protein coding sequence, which results in recombinant expression of the encoded protein.

Another aspect provides host cells into which expression vectors, such as recombinant expression vectors, have been introduced. The host cell may be any prokaryotic cell (e.g., E.coli: (E.coli) (E.coli))E. coli) Or eukaryotic cells (e.g., yeast, insect, or mammalian cells (e.g., CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques。For stable transfection of mammalian cells, it is known that, depending on the expression vector and transfection technique used, only a small fraction of cells can integrate the foreign DNA into their genome. To identify and select these integrants, a gene encoding a selectable marker (e.g., for resistance to antibiotics) is typically introduced into the host cell along with the gene of interest. Preferred selectable markers include markers that confer resistance to drugs such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced polynucleotide can be identified by methods such as drug selection (e.g., cells into which the selectable marker gene is introduced will survive while the remaining cells die).

The antigen binding protein may be expressed in a hybridoma cell line (e.g., in particular, antibodies may be expressed in a hybridoma) or a cell line other than a hybridoma. Expression constructs encoding antigen binding proteins can be used to transform mammalian, insect or microbial host cells. Transformation can be carried out by any known method for introducing a polynucleotide into a host cell, including, for example, packaging the polynucleotide into a virus or phage, and transfecting the host cell with the construct by transfection procedures known in the art (such as those exemplified in U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461; 4,959,455). The optimal transformation procedure used will depend on the type of host cell being transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides within liposomes, mixing of nucleic acids with positively charged lipids, and microinjection of DNA directly into the nucleus of a cell.

Recombinant expression constructs typically comprise a polynucleotide encoding a polypeptide. The polypeptide may comprise one or more of: one or more CDRs, e.g., as provided herein; a light chain variable region; a heavy chain variable region; a light chain constant region; heavy chain constant region (e.g., C)_H1、C_H2 and/or C_H3) (ii) a And/or another scaffold moiety of an IL-23 antigen binding protein. These nucleic acid sequences are inserted into a suitable expression vector using standard ligation techniques. In one embodiment, the heavy or light chain constant region is appended to the C-terminus of the heavy or light chain variable region provided herein and ligated into an expression vector. Vectors are typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery, allowing gene amplification and/or expression to occur). In some embodiments, vectors are used that employ protein-fragment complementation assays with protein reporters such as dihydrofolate reductase (see, e.g., U.S. Pat. No. 6,270,964). Suitable expression vectors can be purchased, for example, from Invitrogen Life technologies (Carlsbad, Calif.) or BD Biosciences (San Jose, Calif.). Other useful vectors for cloning and expressing antibodies and fragments include those described in Bianchi and McGrew, 2003,Biotech. Biotechnol. Bioenga support of 84: 439-44. Further suitable expression vectors are described, for example, inMethods EnzymolVol.185 (edited by D.V. Goeddel), 1990, New York: Academic Press.

Generally, any expression vector used in a host cell contains sequences for the maintenance of the plasmid and for cloning and expression of the exogenous nucleotide sequence. In certain embodiments, such sequences, collectively referred to as "flanking sequences," typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a polynucleotide encoding the polypeptide to be expressed, and a selectable marker element. The provided expression vectors can be constructed from starting vectors, such as commercially available vectors. Such vectors may or may not contain all of the desired flanking sequences. When one or more of the flanking sequences described herein are not already present in the vector, they may be obtained separately and ligated into the vector. Methods for obtaining each flanking sequence are well known to those skilled in the art.

Optionally, the vector may contain a sequence encoding a "tag", i.e., an oligonucleotide molecule located 5 'or 3' to the coding sequence for the IL-23 antigen binding protein; the oligonucleotide sequence encodes a poly-His (e.g., a hexameric His) or another "tag" such as FLAG^®HA (hemagglutinin influenza virus) or myc, for which commercially available antibodies are present. The tag is typically fused to the polypeptide upon expression of the polypeptide and can be used as a means for affinity purification or detection of the IL-23 antigen binding protein from the host cell. Affinity purification can be performed, for example, by column chromatography with an antibody against the tag as an affinity matrix. Optionally, the tag can be subsequently removed from the purified IL-23 antigen binding protein by various means, such as cleavage with certain peptidases.

The flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), heterozygous (i.e., a combination of flanking sequences from more than one source), synthetic, or natural. Thus, the source of the flanking sequences may be any prokaryote or eukaryote, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences are functional in and activatable by the host cell machinery.

The flanking sequences useful in the vector may be obtained by any of several methods well known in the art. Typically, the flanking sequences useful herein have been previously identified by mapping and/or by restriction endonuclease digestion, and thus may be isolated from a suitable tissue source using a suitable restriction endonuclease. In some cases, the complete nucleotide sequence of the flanking sequences may be known. Here, the flanking sequences may be synthesized using the methods described herein for nucleic acid synthesis or cloning.

Whether all or only a portion of the flanking sequences are known, they may be obtained using Polymerase Chain Reaction (PCR) and/or by screening genomic libraries with appropriate probes (e.g., oligonucleotides and/or flanking sequence fragments from the same species or another species). If the flanking sequences are not known, a DNA fragment containing the flanking sequences can be isolated from a larger piece of DNA that may contain, for example, the coding sequence or even another gene or genes. Can be digested by restriction endonucleases to generate suitable DNA fragments, followed by agarose gel purification, Qiagen^®Separation is achieved by column chromatography (Qiagen, Chatsworth, CA) or other methods known to the skilled person for separation. The selection of suitable enzymes to achieve this is readily apparent to those of ordinary skill in the art.

The origin of replication is usually part of those prokaryotic expression vectors which are commercially available and which facilitate the amplification of the vector in the host cell. If the selected vector does not contain an origin of replication site, one can be chemically synthesized based on the known sequence and ligated to the vector. For example, the origin of replication of the plasmid pBR322 (New England Biolabs, Beverly, Mass.) is suitable for most gram-negative bacteria, while various viral origins (e.g., SV40, polyoma, adenovirus, Vesicular Stomatitis Virus (VSV) or papilloma viruses such as HPV or BPV) can be used to clone vectors in mammalian cells. For mammalian expression vectors, an origin of replication component is generally not required (e.g., the SV40 origin is often used simply because it also contains a viral early promoter).

Transcription termination sequences are typically located 3' to the polypeptide coding region and are used to terminate transcription. Transcription termination sequences in prokaryotic cells are usually G-C rich fragments followed by poly-T sequences. Although this sequence is readily purchased from library clones or even commercially as part of a vector, it can also be readily synthesized using nucleic acid synthesis methods such as those described herein.

Selectable marker genes encode proteins necessary for the survival and growth of host cells grown in selective media. Typical selectable marker genes encode the following proteins: (a) conferring resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin) to prokaryotic host cells; (b) complement the auxotrophy of the cell; or (c) supplying key nutrients not available from complex or defined media. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, the neomycin resistance gene can also be used for selection in both prokaryotic and eukaryotic host cells.

Other selection genes may be used to amplify the gene to be expressed. Amplification is a process in which genes necessary for the production of proteins essential for growth or cell survival are repeated back and forth within the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for use in mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase genes. Mammalian cell transformants are placed under selective pressure, where only the transformants are uniquely suited for survival due to the selection gene present in the vector. Selection pressure is applied by culturing the transformed cells under conditions in which the concentration of the selection agent in the culture medium is gradually increased, resulting in amplification of both the selection gene and the DNA encoding another gene, e.g., an antigen binding protein that binds IL-23. As a result, an increased amount of polypeptide (e.g., antigen binding protein) is synthesized from the amplified DNA.

The ribosome binding site is usually necessary for the initiation of rnRNA translation and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is generally located 3 'to the promoter and 5' to the coding sequence for the polypeptide to be expressed.

In some cases, e.g., where glycosylation is desired in a eukaryotic host cell expression system, various pre-sequences (sequences) or pro-sequences (sequences) may be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide can be altered, or a prosequence that also affects glycosylation can be added. The final protein product may have one or more additional amino acids at the-1 position (corresponding to the first amino acid of the mature protein) that are susceptible to expression, which may not be completely removed. For example, the final protein product may have one or two amino acid residues present at the peptidase cleavage site attached to the amino terminus. Alternatively, if the enzyme cuts such regions within the mature polypeptide, the use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide.

Expression and cloning may typically comprise a promoter that is recognized by the host organism and operably linked to the molecule encoding the IL-23 antigen binding protein. The promoter is a non-transcribed sequence, located upstream (i.e.5') of the start codon of the structural gene (typically in the range of about 100-1000 bp), which controls transcription of the structural gene. Promoters are conventionally classified into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions (e.g., the presence or absence of nutrients or a change in temperature). Constitutive promoters, on the other hand, transcribe genes operably linked to them consistently, that is, have little or no control over gene expression. Many promoters recognized by a variety of potential host cells are well known. The appropriate promoter is operably linked to the DNA encoding the heavy chain variable region or the light chain variable region of the IL-23 antigen binding protein by removing the promoter from the source DNA by digestion with a restriction enzyme and inserting the desired promoter sequence into the vector.

Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus and Simian Virus 40(SV 40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

Other promoters that may be of interest includeBut are not limited to: the SV40 early promoter (Benoist and Chambon, 1981,Nature290: 304-; the CMV promoter (Thornsen et al, 1984,Proc. Natl. Acad. U.S.A.659-; the promoter contained in the 3' long terminal repeat of rous sarcoma virus (Yamamoto et al, 1980,Cell22: 787-; the herpes thymidine kinase promoter (Wagner et al, 1981,Proc. Natl. Acad. Sci. U.S.A.78: 1444-; promoter and regulatory sequences from the metallothionein (metallothionein) gene (Prinster et al, 1982,Nature296: 39-42); and prokaryotic promoters, such as the beta-lactamase promoter (Villa-Kamaroff et al, 1978,Proc. Natl. Acad. Sci. U.S.A.75: 3727-3731); or the tac promoter (DeBoer et al, 1983,Proc. Natl. Acad. Sci. U.S.A.80:21-25). Also of interest are the following animal transcriptional regulatory regions that are tissue specific and have been used in transgenic animals: the elastase I gene regulatory region, which is active in pancreatic acinar cells (Swift et al, 1984, Cell639-646; ornitz et al, 1986,Cold Spring Harbor Symp. Quant. Biol. 50:399-409；MacDonald, 1987, Hepatology7: 425-; the insulin gene regulatory region active in pancreatic beta cells (Hanahan, 1985,Nature315: 115-122); immunoglobulin gene regulatory regions active in lymphoid cells (Grosschedl et al, 1984, Cell38: 647-; the general procedure of Adames et al, 1985,Nature318: 533-; alexander et al, 1987, Mol. Cell. Biol.7: 1436-; mouse mammary tumor virus regulatory regions active in testicular, mammary, lymphoid and mast cells (Leder et al, 1986, Cell45: 485-; the albumin gene regulatory region active in the liver (Pinkert et al, 1987,Genes and Devel.1: 268-276); the alpha-fetoprotein gene regulatory region that is active in the liver (Krumlauf et al, 1985,Mol. Cell. Biol.5: 1639-1648; the average particle size of the particles obtained by Hammer et al, 1987,Science253: 53-58); the alpha 1-antitrypsin gene regulatory region which is active in the liver (Kelsey et al, 1987,Genes and Devel.1: 161-171); the beta-globin gene regulatory region active in myeloid cells (Mogram et al, 1985,Nature315: 338-; the molecular weight distribution of Kollias et al, 1986,Cell 46:89-94); the myelin basic protein gene regulatory region, which is active in oligodendrocytes in the brain (readhaed et al, 1987, Cell48: 703-); myosin light chain-2 gene regulatory region active in myeloid muscle (sai, 1985,Nature314: 283-; and the gonadotropin-releasing gene regulatory region active in the hypothalamus (Mason et al, 1986,Science 234:1372-1378)。

enhancer sequences can be inserted into the vector to increase transcription by higher eukaryotes. Enhancers are cis-acting elements of DNA that act on a promoter to increase transcription, and are generally about 10-300 bp in length. Enhancers are relatively orientation and position independent, and have been found both at the 5 'and 3' positions of a transcriptional unit. Several enhancer sequences are known that can be obtained from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically an enhancer from a virus is used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancers known in the art are exemplary enhancing elements for activating eukaryotic promoters. While enhancers can be located 5 ' or 3' to the coding sequence in a vector, they are usually located 5 ' to the promoter site. Sequences encoding suitable native or heterologous signal sequences (leader sequences or signal peptides) may be incorporated into the expression vector to facilitate extracellular secretion of the antibody. The choice of signal peptide or leader sequence depends on the type of host cell in which the antibody is to be produced, and the heterologous signal sequence may replace the native signal sequence. Examples of signal peptides that are functional in mammalian host cells include the following: the signal sequence of interleukin-7 described in U.S. patent No. 4,965,195; as described in Cosman et al, 1984,Natureinterleukin-2 receptor signal sequence of 312: 768; interleukin-4 receptor signal peptide described in european patent No. 0367566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; the interleukin-1 receptor type II signal peptide described in European patent No. 0460846.

After the vector is constructed, the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression. The expression of antigen binding proteins can be carried out by well-known methodsThe vector is transformed into the selected host cell by methods including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection or other known techniques. The method selected may vary depending on the type of host cell to be used. These and other suitable methods are well known to the skilled person and are proposed, for example, in the following documents: sambrook et al，Molecular Cloning A Laboratory Manual, 3 rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).

When cultured under suitable conditions, the host cell synthesizes the protein, which can then be collected from the culture medium (if the host cell secretes it into the culture medium) or directly from the host cell from which it is produced (if not secreted). The selection of an appropriate host cell will depend on a variety of factors such as the desired level of expression, the modification (e.g., glycosylation or phosphorylation) of the polypeptide required or necessary for activity, and the ease of folding into a biologically active molecule.

Mammalian cell lines available as expression hosts are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese Hamster Ovary (CHO) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a variety of other cell lines. In certain embodiments, cell lines can be selected by determining which cell lines have high expression levels and constitutively produce an antigen binding protein with IL-23 binding properties. In another embodiment, cell lines from a B cell lineage that do not produce their autoantibodies, but have the ability to produce and secrete heterologous antibodies, may also be selected.

Use of human IL-23 antigen binding proteins for diagnostic and therapeutic purposes

Antigen binding proteins can be used to detect IL-23 in biological samples and to identify cells or tissues that produce IL-23. Antigen binding proteins that specifically bind to IL-23 can be used to diagnose and/or treat IL-23-associated diseases in a patient in need thereof. For example, IL-23 antigen binding proteins can be used in diagnostic assays, such as binding assays, to detect and/or quantify IL-23 expressed in blood, serum, cells, or tissues. In addition, IL-23 antigen binding proteins can be used to reduce, inhibit, interfere with, or modulate one or more biological activities of IL-23 in a cell or tissue. Thus, an antigen binding protein that binds to IL-23 may have therapeutic utility in alleviating IL-23-associated diseases.

Indications of

The invention also relates to the use of an IL-23 antigen binding protein for the prophylactic or therapeutic treatment of a medical disorder, such as a medical disorder disclosed herein. IL-23 antigen binding proteins can be used to treat a variety of conditions in which IL-23 is associated with or functions to promote an underlying disease or disorder or otherwise promotes negative symptoms.

Conditions that are effectively treated by IL-23 antigen binding proteins play a role in inflammatory responses. Such inflammatory disorders include: periodontal disease; pulmonary disorders, such as asthma; skin disorders such as psoriasis, atopic dermatitis, contact dermatitis; rheumatism such as rheumatoid arthritis, progressive systemic sclerosis (scleroderma); systemic lupus erythematosus; spondyloarthritis, including ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis and reactive arthritis. Uveitis is also contemplated, including Vogt-Koyanagi-Harada disease, idiopathic anterior and posterior uveitis, and uveitis associated with spondyloarthritis. Also contemplated is the use of an IL-23 antigen binding protein for the treatment of autoimmune diseases, including multiple sclerosis; autoimmune myocarditis; type 1 diabetes and autoimmune thyroiditis.

IL-23 antigen binding proteins may be used to treat or prevent degenerative conditions of the gastrointestinal system. The gastrointestinal disorders include inflammatory bowel diseases: crohn's disease, ulcerative colitis, and celiac disease.

Also included is the use of IL-23 antigen binding proteins in the treatment of graft versus host disease and complications such as transplant rejection resulting from solid organ transplantation (e.g., heart, liver, skin, kidney, lung or other transplantation, including bone marrow transplantation).

Also provided herein are methods of using IL-23 antigen binding proteins for the treatment of various neoplastic disorders, including various forms of cancer, including colon, gastric, prostate, renal cell, cervical and ovarian cancer and lung cancer (SCLC and NSCLC). Also included are solid tumors including sarcomas, osteosarcomas, and carcinomas such as adenocarcinoma and squamous cell carcinoma, esophageal carcinoma, gastric carcinoma, gallbladder carcinoma, leukemias including acute myelogenous leukemia, chronic myelogenous leukemia, chronic or acute lymphoblastic leukemia, and hairy cell leukemia, and multiple myeloma.

Diagnostic method

The antigen binding proteins can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or conditions associated with IL-23. Examples of methods that can be used to detect the presence of IL-23 include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and Radioimmunoassays (RIAs).

For diagnostic applications, antigen binding proteins are typically labeled with a detectable labeling group. Suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g. of the type³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I) A fluorescent group (e.g., FITC, rhodamine, lanthanide phosphors), an enzyme group (e.g., horseradish peroxidase, β -galactosidase, luciferase, alkaline phosphatase), a chemiluminescent group, a biotin group, or a predetermined polypeptide epitope recognized by a second reporter (e.g., leucine zipper pair sequence, a second antibody binding site, a metal binding domain, an epitope tag). In some embodiments, the labeling group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known and can be used in the art.

Other diagnostic methods for identifying a cell or cells that express IL-23 are provided. In particular embodiments, the antigen binding protein is labeled with a labeling group and the binding of the labeled antigen binding protein to IL-23 is detected. At another placeIn particular embodiments, the binding of an antigen binding protein to IL-23 is detected in vivo. In another specific embodiment, IL-23 antigen binding proteins are isolated and measured using techniques known in the art. See, e.g.Harlow and Lane, 1988,Antibodies: A Laboratory Manual,New York, Cold Spring Harbor (compiled in 1991 and periodically with additional costs);john E. Coligan editing, 1993, Current Protocols In Immunology New York: John Wiley& Sons。

Other methods are provided for detecting the presence of a test molecule that competes with a provided antigen binding protein for binding to IL-23. One example of such an assay would involve detecting the amount of free antigen binding protein in a solution containing an amount of IL-23 in the presence or absence of a test molecule. An increased amount of free antigen binding protein (i.e., antigen binding protein that does not bind to IL-23) would indicate that the test molecule is capable of competing with the antigen binding protein for binding to IL-23. In one embodiment, the antigen binding protein is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence and absence of antigen binding protein.

The treatment method comprises the following steps: pharmaceutical formulation, route of administration

Pharmaceutical compositions are provided comprising a therapeutically effective amount of one or more antigen binding proteins and pharmaceutically acceptable excipients, diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants. Additionally, methods of treating a patient by administering the pharmaceutical compositions are included. The term "patient" includes human patients. The term "treating" includes alleviating or preventing at least one symptom or other aspect of the condition or lessening the severity of the disease or the like. The term "therapeutically effective amount" or "effective amount" refers to the amount of IL-23 antigen binding protein determined to produce any therapeutic response in a mammal. Such therapeutically effective amounts are readily determined by one of ordinary skill in the art.

Antigen binding proteins need not play a complete curative role or eradicate every symptom or manifestation of the disease, to constitute a viable therapeutic. As is recognized in the relevant art, drugs used as therapeutic agents may reduce the severity of a given disease state, but need not eradicate every manifestation of the disease and are considered useful therapeutic agents. Likewise, to constitute a viable prophylactic, a prophylactically administered treatment need not be completely effective in preventing the onset of the condition. It is sufficient to reduce the impact of the disease alone (e.g., by reducing the number or severity of its symptoms or by increasing the effectiveness of another treatment or by producing another beneficial effect) or to reduce the likelihood that the disease will occur or worsen in the subject. Certain methods provided herein comprise administering an IL-23 antagonist (e.g., an antigen binding protein disclosed herein) to a patient in an amount and for a time sufficient to induce a sustained improvement over baseline that reflects an indicator of the severity of a particular condition.

As is understood in the relevant art, a pharmaceutical composition comprising a molecule of the invention is administered to a patient in a manner appropriate for the indication. The pharmaceutical composition may be administered by any suitable technique including, but not limited to, parenterally, topically, or by inhalation. If injected, the pharmaceutical composition may be administered by bolus injection or continuous infusion, e.g., via intra-articular, intravenous, intramuscular, intralesional, intraperitoneal, or subcutaneous routes. Topical administration, e.g., at the site of disease or injury, is contemplated, as are transdermal delivery and sustained release from the implant. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation of the antagonist in aerosol form, and the like. Other alternatives include eye drops; oral preparations including pills, syrups, lozenges or chewing gum; and topical formulations such as lotions, gels, sprays and ointments.

The use of antigen binding proteins in ex vivo procedures is also contemplated. For example, the patient's blood or other bodily fluid may be contacted ex vivo with an antigen binding protein that binds IL-23. The antigen binding protein may be bound to a suitable insoluble matrix or solid support material.

Advantageously, the antigen binding protein is administered in the form of a composition comprising one or more additional components, such as physiologically acceptable carriers, excipients or diluents. Optionally the composition additionally comprises one or more physiologically active agents for use in combination therapy. The pharmaceutical composition may comprise an IL-23 antigen binding protein together with one or more substances selected from the group consisting of: a buffering agent; antioxidants, such as ascorbic acid; low molecular weight polypeptides (e.g., peptides having less than 10 amino acids), proteins, amino acids; carbohydrates, such as glucose, sucrose or dextrins; chelating agents such as EDTA; glutathione, a stabilizer and an excipient. Neutral buffered saline or saline mixed with the same serum albumin are examples of suitable diluents. Preservatives such as benzyl alcohol may also be added in accordance with appropriate industry standards. The composition may be formulated as a lyophilized product with a suitable excipient solution (e.g., sucrose) as a diluent. Suitable components are nontoxic to recipients at the dosages and concentrations employed. Further examples of components that can be used in Pharmaceutical formulations are presented in any of the versions Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., including 21 st edition (2005).

Kits for use by a medical practitioner include an IL-23 antigen binding protein and a label or other instructions for use in treating any of the conditions discussed herein. In one embodiment, the kit comprises a sterile formulation of one or more IL-23 binding antigen binding proteins, which may be in the form of a composition as disclosed above, and may be in one or more vials.

The dosage and frequency of administration may vary depending on, for example, the following factors: the route of administration, the particular antigen binding protein used, the nature and severity of the disease to be treated, whether the condition is acute or chronic and the size and general condition of the subject. Suitable dosages can be determined, for example, by procedures known in the relevant art in clinical trials, which may involve dose escalation studies.

Typical dosages may range from about 0.1. mu.g/kg up to about 30 mg/kg or more, depending on the factors discussed above. In particular embodiments, the dose may be between 0.1 μ g/kg and up to about 30 mg/kg, optionally 1 μ g/kg and up to about 30 mg/kg, optionally 10 μ g/kg and up to about 10 mg/kg, optionally about 0.1 mg/kg to 5 mg/kg, or optionally about 0.3 mg/kg to 3 mg/kg.

The frequency of administration depends on the pharmacokinetic parameters of the particular human IL-23 antigen binding protein in the formulation used. The clinician administers the composition until a dosage is reached that achieves the desired effect. Thus, the composition may be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion through an implanted device or catheter. Appropriate dosages can be determined by using appropriate dose response data. The IL-23 antigen binding proteins of the invention can be administered, e.g., once or more than once, e.g., at regular intervals over a period of time. In particular embodiments, the IL-23 antigen binding protein is administered over a period of at least 1 month or more, e.g., 1,2, or 3 months, or even indefinitely. For the treatment of chronic conditions, long-term treatment is generally most effective. However, for the treatment of acute conditions, administration over a shorter period, e.g. 1 to 6 weeks, may be sufficient. In general, the antigen binding protein is administered until the patient exhibits a medically relevant degree of improvement relative to baseline of the selected indicator or indicators.

It is contemplated that the IL-23 antigen binding protein is administered to the patient in an amount and for a time sufficient to induce an improvement, preferably a sustained improvement, in at least one indicator reflecting the severity of the condition to be treated. Different indicators reflecting the extent of the disease, disorder or condition in the patient can be evaluated for determining whether the amount and time of treatment is sufficient. Such indicators include, for example, clinically recognized indicators of disease severity, symptoms, or manifestations of the condition in question. In one embodiment, an improvement is considered to be sustained if the subject exhibits the improvement over at least two periods separated by 2-4 weeks. The degree of improvement is typically determined by a physician who can make this determination based on signs, symptoms, biopsies, or other test results, or who can also use a questionnaire given to the subject, such as a quality of life questionnaire developed for a given disease.

Particular embodiments of the methods and compositions of the invention involve the use of an IL-23 antigen binding protein and one or more additional IL-23 antagonists, e.g., two or more antigen binding proteins of the invention, or one antigen binding protein of the invention and one or more other IL-23 antagonists. Also provided are IL-23 antigen binding proteins administered alone or in combination with other agents useful in treating a condition suffered by a patient. Examples of such agents include both proteinaceous and non-proteinaceous drugs. The agents include therapeutic moieties having anti-inflammatory properties (e.g., non-steroidal anti-inflammatory agents, steroids, immunomodulators and/or other cytokine inhibitors, such as inhibitors that antagonize, e.g., IFN-. gamma., GM-CSF, IL-6, IL-8, IL-17, IL-22 and TNF) or IL-23 antigen binding proteins and one or more other therapies (e.g., surgery, ultrasound or therapies effective in reducing inflammation). When multiple treatments are co-administered, the dosage can be adjusted accordingly in accordance with methods recognized or known in the relevant art. Useful agents that can be combined with an IL-23 antigen binding protein include: agents useful in the treatment of, for example, crohn's disease or ulcerative colitis, such as aminosalicylates (e.g., mesalamine), corticosteroids (including prednisone), antibiotics such as metronidazole or ciprofloxacin (or other antibiotics that may be useful in treating, for example, patients suffering from fistulas), and immunosuppressants such as azathioprine, 6-mercaptopurine, methotrexate, tacrolimus, and cyclosporine. The agent may be administered orally or by another route, e.g., via suppository or enema. Agents that may be used in combination with IL-23 binding proteins for the treatment of psoriasis include corticosteroids, calcipotriene and other vitamin D derivatives, isotretinoin (acetoretin) and other retinoic acid derivatives, methotrexate, tacrolimus, and cyclosporine, for local or systemic use. The agents may be administered simultaneously, sequentially, alternately, or according to any other regimen that results in an effective total course of treatment.

In addition to human patients, IL-23 antigen binding proteins may also be used to treat non-human animals, such as domestic pets (dogs, cats, birds, primates, etc.), domestic farm animals (horses, cattle, sheep, pigs, birds, etc.). In such cases, the appropriate dosage can be determined according to the animal's weight. For example, dosages of 0.2-1 mg/kg may be used. Alternatively, the dosage is determined according to the surface area of the animal, with an exemplary dosage between 0.1-20 mg/m²Or more preferably 5 to 12 mg/m². For small animals, such as dogs or cats, a suitable dose is 0.4 mg/kg. The IL-23 antigen binding protein (preferably constructed from a gene derived from the recipient species) is administered by injection or other suitable route one or more times per week until the condition of the animal is improved, or it may be administered indefinitely.

The following examples, including experiments performed and results obtained, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims.

Examples

Example 1

Production of human IL-23 antibodies

By XenoMouse^TMTechniques (Amgen, Thousand Oaks, Calif.) were developed to identify and inhibit native human IL-23 activity while sparing human IL-12. The antibody also recognizes and inhibits recombinant cynomolgus (cynomologous) IL-23, but does not recognize murine or rat IL-23.

The following STAT-luciferase reporter assay was used to select antibodies that recognize and completely inhibit native human IL-23 obtained from human monocyte-derived dendritic cells (MoDC). Human monocytes were isolated from peripheral blood mononuclear cells of healthy donors by negative selection (monocyte isolation kit II, Miltenyi Biotec, Auburn, CA). The MoDC was generated by culturing monocytes with human GM-CSF (50 ng/ml) and human IL-4 (100 ng/ml) in RPMI 1640 complete medium containing 10% fetal bovine serum for 7 days. The MoDC was then washed twice with PBS followed by restimulation with human CD40L (1. mu.g/ml) for 48 hours. CD 40L-stimulated MoDC supernatant contained IL-23, IL-12 and IL-12/23p 40. The amounts of IL-12p70(R & D SYSTEMs, Minneapolis, MN), IL-23 (eBiosciences, San Diego, Calif.) and IL-12/23p40 (R & D SYSTEMs) were determined by ELISA. The STAT-luciferase assay is responsive to IL-23 and non-responsive to IL-12 or free IL-12/23p40, and therefore, the assay can be used on crude supernatants to assess IL-23 activity. For use in the NK cell assay described below, crude supernatants of native human IL-23 were purified using IL-23 affinity column followed by size exclusion chromatography. Concentrations were determined by IL-23 specific ELISA (ebiosciences).

Purified antibody supernatants were also tested in STAT-luciferase assays against recombinant human (rhu) IL-23 and recombinant cynomolgus (cyno) IL-23. Of the antibodies tested that completely inhibited recombinant human IL-23, only half of the antibodies recognized and completely inhibited native human IL-23. Recognition and complete inhibition of recombinant human IL-23 was not predictive of, nor was it relevant to, recognition and complete inhibition of native human IL-23. As shown in FIGS. 1A and 1B, only half of the antibody in the supernatant of the antibody completely inhibiting recombinant human IL-23 completely inhibited native human IL-23. Antibodies that recognize and completely inhibit native human IL-23 were selected for further characterization.

Example 2

Functional assay

a) STAT-luciferase assay

IL-23 is known to bind its heterodimeric receptor and signal through JAK2 and Tyk2 to activate STATs 1,3, 4 and 5. In this assay, cells transfected with STAT/luciferase reporter genes were used to assess the ability of IL-23 antibodies to inhibit IL-23-induced biological activity.

Chinese hamster ovary cells expressing the human IL-23 receptor were transiently transfected with STAT-luciferase reporter overnight. IL-23 antibody was serially diluted (12 points starting with a 1:4 serial dilution from 37.5. mu.g/ml) into 96-well plates. Native human IL-23 (preparation described in example 1) was added to each well at a concentration of 2 ng/ml and incubated for 15-20 minutes at room temperature. Transient transfected cells were added (8X 10)³Cells) to a final volume of 100. mu.l/well and 10% CO at 37 ℃₂Incubate for 5 hours. After incubation, cells were lysed with 100 μ L/well Glo lysis buffer (1 ×) (Promega, Madison, Wisconsin) for 5 min at room temperature. 50 microliters of cell lysate was added to a 96-well plate along with 50. mu.L of Bright-Glo luciferase substrate (Promega) and read on a luminometer.

Statistical analysis can be performed using GraphPad PRISM Software (GraphPad Software, La Jolla, Calif.). The results can be expressed as mean ± Standard Deviation (SD).

As seen in Table 5, all IL-23 antibodies effectively and completely inhibited the native human IL-23 induced STAT/luciferase reporter in a dose-dependent manner. The antibodies also effectively and completely inhibit recombinant human (rhu) IL-23 and recombinant cynomolgus (cyno) IL-23. All of the antibodies have IC in picomolar range₅₀The value is obtained.

TABLE 5 average IC of IL-23 antibodies in STAT-luciferase assay₅₀Tables of (pM) values

b) NK cell assay

IL-23 is known to act on natural killer cells to induce the expression of proinflammatory cytokines such as interferon gamma (IFN γ). In this assay, human primary Natural Killer (NK) cells were used to evaluate the ability of IL-23 antibodies to inhibit IL-23-induced IFN γ activity in cells expressing the human IL-23 natural receptor.

NK cells were isolated from various human donors via negative selection (NK cell isolation kit, Miltenyi Biotec, Auburn, CA). Purified NK cells (1X 10)⁶Individual cells/ml) were added to a 6-well plate of RPMI 1640 plus 10% fetal bovine serum in complete medium supplemented with recombinant human IL-2 (10ng/ml, R) to a final volume of 10 ml/well&D SYSTEMs, Minneapolis, MN). Cells were allowed to incubate at 37 ℃ with 5% CO₂Cultured for 7 days. Then, rhuIL-23 or cyno IL-23 (10 ng/ml) and recombinant human IL-18 (20ng/ml, R)&D SYSTEMs, Minneapolis, MN) stimulated IL-2 activated NK cells for 24 hours in the presence of serial dilutions of IL-23 antibody (11 points at 1:3 serial dilutions starting from 3. mu.g/ml). By IFN γ ELISA (R)&D Sysetms, Minneapolis, MN) IFN γ levels in the supernatants were measured according to the manufacturer's instructions.

Statistical analysis can be performed using GraphPad PRISM software. The results can be expressed as mean ± Standard Deviation (SD).

As seen in Table 6, all antibodies were effective in a dose-dependent manner in NK cells to inhibit rhuIL-23 and cynoIL-23 induced IFN γ expression. All antibodies had IC in the picomolar range₅₀The value is obtained. With native human IL-23 (30. mu.g/ml, preparation described in example 1) and rhuIL-18 (40 ng/ml, R)&D Systems) were performed to determine the antibody subpopulation, and the results shown in table 6 were obtained. Consistent with the selection of antibodies specific for IL-23, using the above assay, these anti-IL-23 antibodies had no effect on IL-12-stimulated IFN γ production in NK cells, while IL-12p 35-specific neutralizing antibody mAb219 (R)&D Systems, Minneapolis, MN) was effective in inhibiting recombinant human IL-12.

TABLE 6 mean IC of IL-23 antibodies in NK cell assay₅₀Of (pM) valueTable form

c) Human whole blood assay

Using Refludan^®(Bayer Pittsburgh, Pa.) human whole blood was collected from multiple healthy donors as an anticoagulant. Refludan in whole blood^®The final concentration of (2) was 10. mu.g/ml. Stimulation mixtures of rhuIL-23 or cynoIL-23 (final concentration 1 ng/ml) + rhuIL-18 (final concentration 20 ng/ml) + rhuIL-2 (final concentration 5 ng/ml) in RPMI 1640 + 10% FBS were added to 96-well plates in a final volume of 20. mu.l/well. Serial dilutions of IL-23 antibody (11 points in a 1:3 dilution series starting at 3. mu.g/ml) were added at 20. mu.l/well and incubated with the stimulation mix for 30 minutes at room temperature. Whole blood (120. mu.l/well) was then added and the final volume was adjusted to 200. mu.l/well with RPMI 1640 + 10% FBS. The final concentration of whole blood was 60%. The plates were allowed to stand at 37 ℃ with 5% CO₂Incubate for 24 hours. Cell-free supernatants were harvested by IFN γ ELISA (R)&D Systems) IFN γ levels were measured from the supernatant according to the manufacturer's instructions.

Statistical analysis can be performed using GraphPad PRISM software. The results can be expressed as mean ± Standard Deviation (SD).

As seen in Table 7, all antibodies were effective in inhibiting rhuIL-23-induced and cyno-IL-23-induced IFN γ expression in whole blood cells in a dose-dependent manner. Antibodies all have IC in picomolar range₅₀The value is obtained.

TABLE 7 average IC of IL-23 antibodies in IFN γ human Whole blood assay₅₀Tables of (pM) values

d) IL-22 assay

IL-23 is known to be a potent inducer of pro-inflammatory cytokines. IL-23 acts on activated T cells and memory T cells and promotes the survival and expansion of Th17 cells that produce pro-inflammatory cytokines including IL-22. In this assay, human whole blood was used to assess the ability of IL-23 antibodies to inhibit IL-23-induced IL-22 production.

Whole blood assays were performed in the same manner as described above, with the modification that 1ng/ml of rhuIL-23 or cynoIL-23 and 10ng/ml of rhuIL-18 were used to induce IL-22 production. IL-22 concentration was determined by IL-22 ELISA (R & D Systems, Minneapolis, MN).

As seen in Table 8, the antibodies were effective in inhibiting rhuIL-23-induced and cynoIL-23-induced IL-22 production in whole blood cells in a dose-dependent manner. Antibodies all have IC in picomolar range₅₀The value is obtained.

TABLE 8 mean IC of IL-23 antibody in IL-22 human Whole blood assay₅₀Tables of (pM) values

Example 3

Determination of the equilibrium dissociation constant (K) of anti-IL-23 antibodies by the KinExA technique_D)

The binding affinity of rhuIL-23 to IL-23 antibodies was assessed using a dynamic exclusion assay (kinetic exclusion assay, KinExA assay, Sapidyne Instruments, Inc., Boise, ID). Normal Human Serum (NHS) activated Sepharose 4 fast beads (Amersham Biosciences, part of GE Healthcare, Uppsala, Sweden) were pre-coated with rhuIL-23 and blocked with 10mg/mL BSA in 1m Tris buffer. 50pM of IL-23 antibody and rhuIL-23 (from 800 pM starting 1:2 dilution of 12 points) at room temperature were incubated for 72 hours, then let through the rhuIL-23 coated agarose beads. The amount of antibody bound to the beads was quantified by a fluorescently (Cy5) labeled goat anti-human-Fc antibody (Jackson ImmunoResearch, West Grove, Pa.). The binding signal is proportional to the amount of free antibody at equilibrium.

Dissociation equilibrium constant (K) was obtained from curve fitting using KinExA Pro software_D) And rate of binding (K)_on). Off rate (K)_off) Derived from: k_D=K_off/K_on

As can be seen in Table 9, the antibody has high affinity binding to human IL-23. All K_DValues range from low pM to subpm.

TABLE 9K_D (pM)、K_on(1/MS) and K_off (1/s) Rate

Antibodies	KD (pM)	Kon (1/MS)	Koff (1/s)
				E	0.131	9.12E+05	1.4E-07
D	0.126	1.72E+06	2.2E-07
				B	3.99	1.17E+06	4.7E-06
C	2.56	1.36E+06	4.1E-06
				F	2.62	5.69E+05	1.5E-06
L	1.08	3.34E+06	3.7E-06
				G	2.00	4.00E+05	8.1E-07

Example 4

Determination of structure by X-ray crystallography

One method of determining the structure of an antibody-antigen complex is by X-ray crystallography, see, e.g., Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), page 23. The crystal structure of IL-23 has been determined (see Lupardus and Garcia, J Mol Biol, 2008, 382:931-941) and the crystal structure of the IL-23/Fab complex has been revealed (see Beyer et al, J Mol Biol, 2008.382 (4): 942-55). Structural determination of IL-23 containing Fab fragments of the antibodies claimed herein was obtained using X-ray crystallography.

Proteins for crystallization

Recombinant sources of human IL-23 heterodimers were used for crystallization studies (see Beyer et al, supra). The human p19 subunit sequence consisted of residues 20-189 of SEQ ID NO:145, the signal sequence of SEQ ID NO: 154 and the C-terminal 6-His tag SEQ ID NO: 155. The human p40 subunit sequence was mutated from asparagine to glutamine at position 222 of SEQ ID NO:147 to prevent glycosylation at this site (Beyer et al, supra).

Fab derived from antibody B and antibody E were expressed on IgG1 scaffolds incorporating caspase cleavage sites. Fab was treated by protease cleavage.

Complex formation and crystallization

The IL-23-antibody B Fab complex was prepared by mixing a 2X molar excess of antibody B Fab with the human heterodimeric IL-23 described above. The complex was purified by size exclusion chromatography to remove excess antibody B Fab and concentrated to about 12 mg/ml for crystallization. The IL-23-antibody B Fab complex was crystallized in 0.1M Hepes pH 7, 8% PEG 8000.

The IL-23-antibody E Fab complex was prepared by mixing a 2X molar excess of antibody E Fab with the human heterodimeric IL-23 described above. The complex was methylated using the JBS methylation kit according to the manufacturer's instructions (Jena Bioscience, Jena, Germany). The complex is then treated with PNG enzyme to deglycosylate the protein. After these treatments, the complex was purified by size exclusion chromatography to remove excess antibody E Fab and concentrated to 13.5 mg/ml for crystallization. The IL-23-antibody E Fab complex was crystallized in 0.1M TrispH 8.5, 0.2M magnesium chloride, 15% PEG 4000.

Data acquisition and structural determination

P2 having a unit cell size of a =70.93, b =71.27, c =107.37 Å, β =104.98 °₁The IL-23-antibody B Fab crystals were grown in space groups and diffracted at a resolution of 2.0 Å by using the procedure molrrep (CCP4,The CCP4 suite: programs for protein crystallography.analysis of the structure of the Fab of the IL-23-antibody B by molecular replacement carried out by Acta Crystallogr D Biol Crystallogr, 1994.50 (Pt 5): pages 760-3) using the IL-23 structure (Beyer et al, supra) as the initial search model. The IL-23 solution was kept stationary, using antibody variable domains as a search model. IL-23-antibody variable domain solutions were kept fixed, using antibody constant domains as a search model. The complete structure was improved by multiple rounds of model construction using Quanta and refinement with cnx (Brunger, et al, Actacrystallogr D Biol Crystallogr, 1998, 54(Pt 5): pages 905-21).

The program PyMOL (Delano, W.L. The PyMOL Graphics System. Palo Alto, 2002) (Schrodinger, LLC; New York, NY)) was used to calculate The distances between protein atoms. The amino acid is selected if at least one atom is within a desired distance threshold of the partner protein.

When bound to the antibody B Fab, the A, B, C and D helix boundaries of the p19 subunit of IL-23 include residues 28-47 of the A helix, residues 86-105 of the B helix, residues 119-134 of the C helix, and residues 154-187 of the D helix of SEQ ID NO 145.

The interaction region on the subunit of IL-23p19 when bound to the antibody B Fab includes residues within Ser46-Glu58, Glu112-Glu123 and Pro155-Phe163 of SEQ ID NO: 145.

The IL-23p19 subunit amino acid residues containing atoms 4 Å or closer to the antibody B Fab include Ser46, Ala47, His48, Pro49, Leu50, His53, Met54, Asp55, Glu58, Pro113, Ser114, Leu115, Leu116, Pro120, Val121, Trp156, Leu159, Leu160, Arg162 and Phe163 of SEQ ID NO 145 the IL-23p19 subunit amino acid residues containing atoms between 4 Å and 5 Å away from the antibody B Fab include Val51, Arg57, Glu112, Asp118, Ser119, Gln123, Pro155 of SEQ ID NO 145.

The amino acid residues of the IL-23p40 subunit containing atoms 4 Å or closer to the antibody B Fab include Glu 122 and Lys 124 of SEQ ID NO: 147.

Antibody B Fab heavy chain amino acid residues containing an atom at or closer to IL-23 heterodimer 4 Å include Gly32, Gly33, Tyr34, Tyr35, His54, Asn58, Thr59, Tyr60, Lys66, Arg101, Gly102, Phe103, Tyr104, and Tyr105 of SEQ ID NO 46 antibody B Fab heavy chain amino acid residues containing an atom at or less than 5 Å from IL-23 heterodimer include Ser31, Gly32, Gly33, Tyr34, Tyr35, His54, Ser56, Asn58, Thr59, Tyr60, Lys66, Arg101, Gly102, Phe103, Tyr104, and Tyr105 of SEQ ID NO 46.

Antibody B Fab light chain amino acid residues containing atoms at or closer to 4 Å from the IL-23 heterodimer include Ser30, Ser31, Trp32, Tyr49, Ser52, Ser53, Ala91, Asn92, Ser93, Phe94, and Phe96 from SEQ ID NO15 antibody B Fab light chain amino acid residues containing atoms at or less than 5 Å from the IL-23 heterodimer include Ser30, Ser31, Trp32, Tyr49, Ala50, Ser52, Ser53, Ser56, Ala91, Asn92, Ser93, Phe94, and Phe96 from SEQ ID NO 15.

Large unit cell with a =61.60, b =97.59, c =223.95 ÅSmall P222₁The IL-23-antibody E Fab complex structure was resolved by molecular replacement performed with the program Phaser (CCP4, supra) using the IL-23 structure, antibody variable domains and antibody constant domains as three initial search models as described above.

The interaction region on the subunit of IL-23p19 identified when bound to the antibody E Fab includes residues within Ser46-His53, Glu112-Val120 and Trp156-Phe163 of SEQ ID NO. 145.

The amino acid residues of IL-23p19 containing atoms 4 Å or closer to the antibody E Fab include Ser46, Ala47, His48, Pro49, Leu50, Glu112, Pro113, Ser114, Leu115, Leu116, Pro117, Asp118, Ser119, Pro120, Trp156, Leu159, Leu160 and Phe163 of SEQ ID NO 145 the amino acid residues of IL-23p19 containing atoms between 4 Å and 5 Å from the antibody E Fab include His53 of SEQ ID NO 145.

The amino acid residues of IL-23p40, which contain an atom 4 Å or closer to the antibody E Fab, include Lys121, Glu 122, Pro123 and Asn 125 of SEQ ID NO: 147.

Antibody E Fab heavy chain amino acid residues containing atoms 4 Å or closer to the IL-23 heterodimer include Gly26, Phe27, Thr28, Ser31, Tyr53, Tyr59, Tyr102, Ser104, Ser105, Trp106, Tyr107, and Pro108 of SEQ ID NO. 31 antibody E Fab heavy chain amino acid residues containing atoms 5 Å from the IL-23 heterodimer include Gln1, Gly26, Phe27, Thr28, Ser30, Ser31, Tyr32, Trp52, Tyr53, Tyr59, Arg100, Tyr102, Thr103, Ser104, Ser105, Trp106, Tyr107, and Pro108 of SEQ ID NO. 31.

Antibody E Fab light chain amino acid residues containing an atom at or closer to IL-23 heterodimer 4 Å include Ala31, Gly32, Tyr33, Asp34, Tyr51, Gly52, Asn55, Lys68 and Tyr93 of SEQ ID NO1 antibody B Fab light chain amino acid residues containing an atom at or less than 5 Å from IL-23 heterodimer include Thr29, Ala31, Gly32, Tyr33, Asp34, Tyr51, Gly52, Asn55, Lys68, Tyr93 and Trp100 of SEQ ID NO 1.

Example 5

Determination of IL-23-antibody complex contact residues by solvent accessible surface area differences

The difference in solvent accessible surface area is used to determine the paratope (the portion of the antibody that recognizes the antigen) that is in contact with the residues of the portion of the antigen that is bound by the paratope in the human IL-23-antibody B Fab complex and the human IL-23-antibody E Fab complex. Solvent accessible surface area calculations were performed with a Molecular Operating Environment (Chemical Computing Group, Montreal, Quebec).

The solvent accessible surface area difference of the paratope in the IL-23-antibody B Fab complex was calculated by setting the antibody B Fab residues according to the desired set (set). Using the structural information of the IL-23-antibody B Fab complex obtained in example 4, the residue solvent accessible surface area of the amino acid residues of the antibody B Fab in the presence of IL-23 heterodimers was calculated, which represents the "binding area" of the pool.

The solvent accessible surface area of residues for each antibody B Fab residue in the absence of IL-23 antigen was calculated and represents the "free area" of the pool.

The "bound area" is then subtracted from the "free area" to obtain the "solvent exposed surface area difference" for each residue in the pool, antibody B Fab residues with no change or zero difference in surface area, which did not contact IL-23 antigen residues when complexed, are considered to have a value of 10 or more 10 ŲThe difference antibody B Fab residues are in operative contact with residues in the IL-23 antigen such that when the antibody B Fab binds to human IL-23, these antibody B Fab residues are at least partially to fully occluded. This collection of antibody B Fab residues constitutes the "overlay patch" which participates in the interface structure when the antibody B Fab is bound to human IL-23, see tables 10 and 11. Antibody B Fab residues in the overlay patch may not participate in binding interactions with IL-23 antigen residues, but any single residue mutation within the overlay patch may introduce energy that will affect binding of the antibody B Fab to human IL-23These residues are also within 5 Šor less from the Il-23 antigen when bound to the antibody B Fab, as described in example 4.

TABLE 10 solvent accessible surface area difference of antibody B Fab light chains

TABLE 11 solvent accessible surface area difference of antibody B Fab heavy chains

The difference in solvent accessible surface area of residues in the IL-23-antibody E Fab complex was calculated as described above, and is believed to be 10 Å or more²The difference antibody E Fab residues make significant contact with residues in the IL-23 antigen that are at least partially to completely occluded when the antibody E Fab binds to human IL-23 this collection of antibody E Fab residues constitutes a cover patch that is involved in the interface structure when the antibody E Fab binds to human IL-23, see tables 12 and 13.

TABLE 12 solvent accessible surface area difference of antibody E Fab light chains

TABLE 13 solvent accessible surface area difference of antibody E Fab heavy chains

The difference in solvent accessible surface area of the IL-23 heterodimer moiety bound by the paratope of the antibody B Fab was calculated by setting the IL-23 heterodimer residues to the desired set. Using the structural information of the antibody B Fab-IL-23 complex obtained in example 4, the residue solvent accessible surface area of the amino acid residues of the IL-23 heterodimer in the presence of the antibody B Fab was calculated, which represents the "binding area" of the pool.

The solvent accessible surface area of the residues for each IL-23 heterodimer residue in the absence of the antibody B Fab was calculated and represents the "free area" of the pool.

As described above, subtracting the "binding area" from the "free area" yields the "solvent exposed surface area difference" for each IL-23 residue, IL-23 heterodimer residues with no change or zero difference in surface area, were not contacted with antibody B Fab residues upon complexation, were considered to have ≧ 10 ŲThe different IL-23 heterodimer residues are in significant contact with residues of the antibody B Fab and are at least partially to completely occluded when human IL-23 heterodimer binds to the antibody B Fab this collection of IL-23 heterodimer residues constitutes a "cover patch" which is involved in the interface structure when human IL-23 heterodimer binds to the antibody E Fab, see Table 14. IL-23 heterodimer residues in this cover patch may not all be involved in binding interactions with antibody B Fab residues, but any single residue mutation within the cover patch may introduce an energetic difference that will affect binding of antibody BFab to human IL-23. these residues are also within a distance of or less from antibody B Fab 4 Å, as described in example 4.

TABLE 14 solvent accessible surface area differences of IL-23 heterodimer residues

Residue p19 (SEQ ID NO: 145)	Difference in solvent exposed surface area (Å)2)
		Ser46	26.5
Ala47	12.7
		Pro49	59.6
Leu50	122.2
		His53	47.8
Met54	13.9
		Asp55	20.5
Arg57	14.6
		Glu58	96.5
Glu112	29.7
		Pro113	64.8
Ser114	30.0
		Leu115	31.4
Leu116	60.0
		Asp118	14.4
Ser119	19.7
		Pro120	64.7
Pro155	19.4
		Typ156	61.9
Leu159	72.8
		Leu160	27.0
Arg162	14.4
		Phe163	67.5
Residue p40 (SEQ ID NO: 147)
		Glu122	29.1
Lys124	60.9

The difference in solvent accessible surface area of the IL-23 heterodimer moiety bound by the paratope of the antibody E Fab was calculated as described above, and is believed to be 10 Å or more²The different IL-23 heterodimer residues are in significant contact with antibody E Fab residues and when human IL-23 heterodimer binds to antibody E Fab, these IL-23 heterodimer residues are at least partially to completely occluded the collection of IL-23 heterodimer residues constitutes a covering patch that is involved in the interface structure when human IL-23 heterodimer binds to antibody E Fab, see Table 15. the IL-23 heterodimer residues in the covering patch may not all be involved in binding interactions with antibody E Fab residues, but any single residue mutation within the covering patch may introduce an energetic difference that will affect binding of antibody E Fab to human IL-23. these residues are also within 5 Å or less distance from antibody E Fab, as described in example 4.

TABLE 15 solvent accessible surface area differences of IL-23 heterodimer residues

。

<110> Amm's Co Ltd

<120> human IL-23 antigen binding proteins

<130> A-1529-WO-PCT

<140> pending assignment

<141> 2010-10-26

<150> 61/381,287

<151> 2010-09-09

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Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asp Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 14

<211> 321

<212> DNA

<213> human

<400> 14

gacatccagt tgaccccgtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggtattgcc ggctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcaacag gctgacagtt tccctcccac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 15

<211> 107

<212> PRT

<213> human

<400> 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Ser Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Val Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Phe Lys

100 105

<210> 16

<211> 321

<212> DNA

<213> human

<400> 16

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca ggttattagc agctggttag cctggtatca gcagaaacca 120

gggaaagccc ctagcctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtgtatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcaacag gctaacagtt tcccattcac tttcggccct 300

gggaccaaag tggatttcaa a 321

<210> 17

<211> 107

<212> PRT

<213> human

<400> 17

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ser Ser Ser Trp

20 25 30

Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 18

<211> 321

<212> DNA

<213> human

<400> 18

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggaagtagc agctggtttg cctggtatca gcagaaacca 120

gggaaagccc caaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagac ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcaacag gctaacagtt tcccattcac tttcggccct 300

gggaccaaag tggatatcaa a 321

<210> 19

<211> 107

<212> PRT

<213> human

<400> 19

Asp Ser Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Phe Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 20

<211> 321

<212> DNA

<213> human

<400> 20

gacagccaga tgacccagtc tccatcttcc gtgtctgcct ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggtattagc agctggtttg cctggtatca gcagaaacca 120

gggcaagccc ctaacctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcaacag gctaacagtt tcccattcac tttcggccct 300

gggaccaaag tggatatcaa a 321

<210> 21

<211> 107

<212> PRT

<213> human

<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Gly Gln Val Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Thr Ser Phe Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 22

<211> 321

<212> DNA

<213> human

<400> 22

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgggtca ggttattagc agctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatcg 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gacgattttg caacttacta ttgtcaacag gctaccagtt ttcccctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 23

<211> 107

<212> PRT

<213> human

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Phe Ser Gly Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 24

<211> 321

<212> DNA

<213> human

<400> 24

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggttttagc ggttggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ctgtcaacag gctaacagtt tcccattcac tttcggccct 300

gggaccaaag tggatatcaa a 321

<210> 25

<211> 107

<212> PRT

<213> human

<400> 25

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val Ile Ser Ser Trp

20 25 30

Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Ala Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ala Asn Ser Phe Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Val Lys

100 105

<210> 26

<211> 321

<212> DNA

<213> human

<400> 26

gacatccagt tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca ggttattagc agctggtttg cctggtatca gcagaaacca 120

gggaaagccc ctaacctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gcagattttg caacttactt ttgtcaacag gctaacagtt tcccattcac tttcggccct 300

gggaccaaag tggatgtcaa a 321

<210> 27

<211> 107

<212> PRT

<213> human

<400> 27

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ser Ser Ser Trp

20 25 30

Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 28

<211> 321

<212> DNA

<213> human

<400> 28

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggtagtagc agctggtttg cctggtatca acagaaacca 120

gggaaagccc caaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcaacag gctaacagtt tcccattcac tttcggccct 300

gggaccaaag tggatatcaa a 321

<210> 29

<211> 107

<212> PRT

<213> human

<400> 29

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu

100 105

<210> 30

<211> 108

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Xaa can be Ile or Ser

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Xaa can be Met or Leu

<220>

<221> MISC_FEATURE

<222> (29)..(29)

<223> Xaa can be Gly or Val

<220>

<221> MISC_FEATURE

<222> (30)..(30)

<223> Xaa can be Ser, Phe or Ile

<220>

<221> MISC_FEATURE

<222> (32)..(32)

<223> Xaa can be Ser or Gly

<220>

<221> MISC_FEATURE

<222> (34)..(34)

<223> Xaa can be Phe or Leu

<220>

<221> MISC_FEATURE

<222> (43)..(43)

<223> Xaa can be Lys or Gln

<220>

<221> MISC_FEATURE

<222> (46)..(46)

<223> Xaa can be Lys, Asn or Ser

<220>

<221> MISC_FEATURE

<222> (67)..(67)

<223> Xaa can be Gly or Val

<220>

<221> MISC_FEATURE

<222> (71)..(71)

<223> Xaa can be Asp or Glu

<220>

<221> MISC_FEATURE

<222> (82)..(82)

<223> Xaa can be Glu or Ala

<220>

<221> MISC_FEATURE

<222> (88)..(88)

<223> Xaa can be Tyr or Phe

<220>

<221> MISC_FEATURE

<222> (107)..(107)

<223> Xaa can be Ile, Val or Phe

<400> 30

Asp Xaa Gln Xaa Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Xaa Xaa Ser Xaa

20 25 30

Trp Xaa Ala Trp Tyr Gln Gln Lys Pro Gly Xaa Ala Pro Xaa Leu Leu

35 40 45

Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Xaa Ser Gly Thr Xaa Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Xaa Asp Phe Ala Thr Tyr Xaa Cys Gln Gln Ala Asn Ser Phe Pro

85 90 95

Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Xaa Lys

100 105

<210> 31

<211> 124

<212> PRT

<213> human

<400> 31

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Glu Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Tyr Thr Ser Ser Trp Tyr Pro Asp Ala Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 32

<211> 372

<212> DNA

<213> human

<400> 32

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa tgaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagatcgg 300

gggtatacca gtagctggta ccctgatgct tttgatatct ggggccaagg gacaatggtc 360

accgtctctt ca 372

<210> 33

<211> 124

<212> PRT

<213> human

<400> 33

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Tyr Ser Ser Ser Trp Tyr Pro Asp Ala Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 34

<211> 121

<212> PRT

<213> human

<400> 34

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Phe Asp Gly Ser Leu Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Arg Thr Thr Leu Ser Gly Ser Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 35

<211> 363

<212> DNA

<213> human

<400> 35

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatcatttg atggaagtct taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa caccctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagaacgg 300

actactttaa gtgggagcta ctttgactac tggggccagg gaaccctggt caccgtctcc 360

tca 363

<210> 36

<211> 121

<212> PRT

<213> human

<400> 36

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Val Ile Ser His Asp Gly Ser Ile Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Arg Thr Thr Leu Ser Gly Ser Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 37

<211> 363

<212> DNA

<213> human

<400> 37

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt agctatgcca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg gttgtcagtt atatcacatg atggaagtat taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagaacgg 300

actactctaa gtgggagcta ctttgactac tggggccagg gaaccctggt caccgtctcc 360

tca 363

<210> 38

<211> 125

<212> PRT

<213> human

<400> 38

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Arg Ser Ser Thr Ile Tyr Ile Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ile Ala Ala Ala Gly Gly Phe His Tyr Tyr Tyr Ala Leu

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 39

<211> 375

<212> DNA

<213> human

<400> 39

gaggtgcagc tggtggagtc tgggggaggc ctggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt agctatagta tgaactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtttcgtac attagtagta ggagtagtac catatacatc 180

gcagactctg tgaagggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240

ctgcaaatga acagcctgag agacgaagac acggctgtgt attactgtgc gagacggata 300

gcagcagctg gtgggttcca ctactactac gctttggacg tctggggcca agggaccacg 360

gtcaccgtct cctca 375

<210> 40

<211> 125

<212> PRT

<213> human

<400> 40

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Ser Thr Arg Tyr His Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ile Ala Ala Ala Gly Pro Trp Gly Tyr Tyr Tyr Ala Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 41

<211> 375

<212> DNA

<213> human

<400> 41

gaggtgcagc tggtggagtc tgggggaggc ttggtacaac ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt acctatagca tgaactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtttcatac attagtagca gtagtagtac cagataccac 180

gcagactctg tgaagggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240

ctgcaaatga acagcctgag agacgaggac acggctgtgt attactgtgc gagacgtata 300

gcagcagctg gtccgtgggg ctactactac gctatggacg tctggggcca agggaccacg 360

gtcaccgtct cctca 375

<210> 42

<211> 125

<212> PRT

<213> human

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Val Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Arg Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ile Ala Ala Ala Gly Pro Trp Gly Tyr Tyr Tyr Ala Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 43

<211> 375

<212> DNA

<213> human

<400> 43

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgtag tctctggatt caccttcagt agttttagca tgaactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtttcatac attagtagtc gtagtagtac catatactac 180

gcagactctg tgaagggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240

ctgcaaatga acagcctgag agacgaggac acggctgtgt attattgtgc gagacgtata 300

gcagcagctg gtccgtgggg ctactactac gctatggacg tctggggcca agggaccacg 360

gtcaccgtct cctca 375

<210> 44

<211> 118

<212> PRT

<213> human

<400> 44

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Thr Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Ala Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Tyr Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Met Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Arg Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Arg Gly Tyr Tyr Tyr Gly Val Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 45

<211> 354

<212> DNA

<213> human

<400> 45

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt acttactact ggagctggat ccggcagccc 120

gccgggaagg gactggagtg gattgggctt atctatacca gtgggagcac caactacaac 180

ccctccctca agagtcgagt caccatgtca ttagacacgt ccaagaacca gttctccctg 240

aggctgacct ctgtgaccgc cgcggacacg gccgtttatt actgtgcgag agatcgtggg 300

tactactacg gtgtggacgt ctggggccag gggaccacgg tcaccgtctc ctca 354

<210> 46

<211> 120

<212> PRT

<213> human

<400> 46

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly His Ile His Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Lys Asn Arg Gly Phe Tyr Tyr Gly Met Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 47

<211> 360

<212> DNA

<213> human

<400> 47

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtggtt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggcacatcc attacagtgg gaacacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaatcagttc 240

tccctgaaac tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgaaaaat 300

cgcgggttct actacggtat ggacgtctgg ggccaaggga ccacggtcac cgtctcctca 360

<210> 48

<211> 120

<212> PRT

<213> human

<400> 48

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Asn Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Ser Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Gln Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Arg Gly His Tyr Tyr Gly Met Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 49

<211> 360

<212> DNA

<213> human

<400> 49

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcaac agtggtggtt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagctcctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctca gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagat 300

cgggggcact actacggtat ggacgtctgg ggccaaggga ccacggtcac cgtctcctca 360

<210> 50

<211> 120

<212> PRT

<213> human

<400> 50

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Arg Gly His Tyr Tyr Gly Met Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 51

<211> 360

<212> DNA

<213> human

<400> 51

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt agtggtggtt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacattt attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagat 300

cggggccact actatggaat ggacgtctgg ggccaaggga ccacggtcac cgtctcctca 360

<210> 52

<211> 118

<212> PRT

<213> human

<400> 52

Gln Val Gln Leu Gln Glu Ser Gly Pro Arg Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Ser Ser Tyr

20 25 30

Phe Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Ile Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr

85 90 95

Arg Asp Arg Gly Ser Tyr Tyr Gly Ser Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 53

<211> 354

<212> DNA

<213> human

<400> 53

caggtgcagc tgcaggagtc gggcccaaga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtga ctccatcagt agttacttct ggagctggat ccggcagccc 120

ccagggaagg gactggagtg gcttgggtat atctattaca gtgggagcac caactacaac 180

ccctccctca agagtcgagt caccatatca atagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc tgcggacacg gccgtgtatt actgtacgag agatcggggg 300

agctactacg gatctgacta ctggggccag ggaaccctgg tcaccgtctc ctca 354

<210> 54

<211> 120

<212> PRT

<213> human

<400> 54

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Thr Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Ile Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Ser Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asn Arg Gly Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 55

<211> 360

<212> DNA

<213> human

<400> 55

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtggtt actactggac ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gaacacctac 180

tacaacccgt ccctcaagag tcgaattacc atatcagtgg acacgtctaa gaaccagttc 240

tccctgagcc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagaaat 300

cgcgggtact actacggtat ggacgtctgg ggccaaggga ccacggtcac cgtctcctca 360

<210> 56

<211> 120

<212> PRT

<213> human

<400> 56

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Lys Asn Arg Gly Phe Tyr Tyr Gly Met Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 57

<211> 360

<212> DNA

<213> human

<400> 57

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtggtt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atgtcagtag acacgtctaa gaaccagttc 240

tccctgaaac tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgaaaaat 300

cgcgggttct actacggtat ggacgtctgg ggccaaggga ccacggtcac cgtctcctca 360

<210> 58

<211> 120

<212> PRT

<213> human

<400> 58

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Asn Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Ser Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Arg Gly His Tyr Tyr Gly Met Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 59

<211> 360

<212> DNA

<213> human

<400> 59

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcaat agtggtggtt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcagctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagttg acacgtctaa gaaccagttc 240

tccctgaagc tgagttctgt gactgccgcg gacacggccg tgtattactg tgcgagagat 300

cgggggcact actacggtat ggacgtctgg ggccaaggga ccacggtcac cgtctcctca 360

<210> 60

<211> 123

<212> PRT

<213> human

<400> 60

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asn Thr Val Thr Ile Tyr Tyr Asn Tyr Gly Met Asp Val

100 105 110

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 61

<211> 116

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Xaa can be Val or Glu

<220>

<221> MISC_FEATURE

<222> (25)..(25)

<223> Xaa can be Asn or Ser

<220>

<221> MISC_FEATURE

<222> (38)..(38)

<223> Xaa can be Gln or Leu

<220>

<221> MISC_FEATURE

<222> (55)..(55)

<223> Xaa can be Ile or Thr

<220>

<221> MISC_FEATURE

<222> (61)..(61)

<223> Xaa can be Asp or Glu

<220>

<221> MISC_FEATURE

<222> (77)..(77)

<223> Xaa can be Tyr or Ser

<220>

<221> MISC_FEATURE

<222> (101)..(101)

<223> Xaa can be Ser or Asn

<400> 61

Gln Pro Xaa Leu Thr Gln Pro Pro Ser Ala Ser Ala Ser Leu Gly Ala

1 5 10 15

Ser Val Thr Leu Thr Cys Thr Leu Xaa Ser Gly Tyr Ser Asp Tyr Lys

20 25 30

Val Asp Trp Tyr Gln Xaa Arg Pro Gly Lys Gly Pro Arg Phe Val Met

35 40 45

Arg Val Gly Thr Gly Gly Xaa Val Gly Ser Lys Gly Xaa Gly Ile Pro

50 55 60

Asp Arg Phe Ser Val Leu Gly Ser Gly Leu Asn Arg Xaa Leu Thr Ile

65 70 75 80

Lys Asn Ile Gln Glu Glu Asp Glu Ser Asp Tyr His Cys Gly Ala Asp

85 90 95

His Gly Ser Gly Xaa Asn Phe Val Tyr Val Phe Gly Thr Gly Thr Lys

100 105 110

Val Thr Val Leu

115

<210> 62

<211> 14

<212> PRT

<213> human

<400> 62

Thr Gly Ser Ser Ser Asn Thr Gly Ala Gly Tyr Asp Val His

1 5 10

<210> 63

<211> 7

<212> PRT

<213> human

<400> 63

Gly Ser Gly Asn Arg Pro Ser

1 5

<210> 64

<211> 11

<212> PRT

<213> human

<400> 64

Gln Ser Tyr Asp Ser Ser Leu Ser Gly Trp Val

1 5 10

<210> 65

<211> 14

<212> PRT

<213> human

<400> 65

Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His

1 5 10

<210> 66

<211> 7

<212> PRT

<213> human

<400> 66

Gly Ser Asn Asn Arg Pro Ser

1 5

<210> 67

<211> 9

<212> PRT

<213> human

<400> 67

Met Ile Trp His Ser Ser Ala Ser Val

1 5

<210> 68

<211> 14

<212> PRT

<213> human

<400> 68

Thr Leu Arg Ser Gly Ile Asn Val Gly Thr Tyr Arg Ile Tyr

1 5 10

<210> 69

<211> 11

<212> PRT

<213> human

<400> 69

Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser

1 5 10

<210> 70

<211> 13

<212> PRT

<213> human

<400> 70

Gly Ala Asp His Gly Ser Gly Ser Asn Phe Val Tyr Val

1 5 10

<210> 71

<211> 11

<212> PRT

<213> human

<400> 71

Thr Leu Asn Ser Gly Tyr Ser Asp Tyr Lys Val

1 5 10

<210> 72

<211> 12

<212> PRT

<213> human

<400> 72

Val Gly Thr Gly Gly Ile Val Gly Ser Lys Gly Asp

1 5 10

<210> 73

<211> 13

<212> PRT

<213> human

<400> 73

Gly Ala Asp His Gly Ser Gly Asn Asn Phe Val Tyr Val

1 5 10

<210> 74

<211> 11

<212> PRT

<213> human

<400> 74

Thr Leu Ser Ser Gly Tyr Ser Asp Tyr Lys Val

1 5 10

<210> 75

<211> 12

<212> PRT

<213> human

<400> 75

Val Gly Thr Gly Gly Ile Val Gly Ser Lys Gly Glu

1 5 10

<210> 76

<211> 9

<212> PRT

<213> human

<400> 76

Gln Gln Ala Asn Ser Phe Pro Phe Thr

1 5

<210> 77

<211> 11

<212> PRT

<213> human

<400> 77

Arg Ala Ser Gln Gly Phe Ser Gly Trp Leu Ala

1 5 10

<210> 78

<211> 12

<212> PRT

<213> human

<400> 78

Val Gly Thr Gly Gly Thr Val Gly Ser Lys Gly Glu

1 5 10

<210> 79

<211> 9

<212> PRT

<213> human

<400> 79

Gln Gln Ala Thr Ser Phe Pro Leu Thr

1 5

<210> 80

<211> 11

<212> PRT

<213> human

<400> 80

Arg Ala Ser Gln Val Ile Ser Ser Trp Leu Ala

1 5 10

<210> 81

<211> 7

<212> PRT

<213> human

<400> 81

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 82

<211> 9

<212> PRT

<213> human

<400> 82

Gln Gln Ala Asp Ser Phe Pro Pro Thr

1 5

<210> 83

<211> 11

<212> PRT

<213> human

<400> 83

Arg Ala Ser Gln Val Ile Ser Ser Trp Phe Ala

1 5 10

<210> 84

<211> 9

<212> PRT

<213> human

<400> 84

Leu Gln His Asn Ser Tyr Pro Pro Thr

1 5

<210> 85

<211> 11

<212> PRT

<213> human

<400> 85

Arg Ala Ser Gln Gly Ser Ser Ser Trp Phe Ala

1 5 10

<210> 86

<211> 11

<212> PRT

<213> human

<400> 86

Arg Ala Ser Gln Gly Ile Ser Ser Trp Phe Ala

1 5 10

<210> 87

<211> 11

<212> PRT

<213> human

<400> 87

Arg Ala Gly Gln Val Ile Ser Ser Trp Leu Ala

1 5 10

<210> 88

<211> 11

<212> PRT

<213> human

<400> 88

Arg Ala Ser Gln Gly Ile Ala Gly Trp Leu Ala

1 5 10

<210> 89

<211> 11

<212> PRT

<213> human

<400> 89

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 90

<211> 17

<212> PRT

<213> human

<400> 90

Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 91

<211> 5

<212> PRT

<213> human

<400> 91

Ser Tyr Gly Met His

1 5

<210> 92

<211> 17

<212> PRT

<213> human

<400> 92

Val Ile Trp Tyr Asp Gly Ser Asn Glu Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 93

<211> 15

<212> PRT

<213> human

<400> 93

Asp Arg Gly Tyr Thr Ser Ser Trp Tyr Pro Asp Ala Phe Asp Ile

1 5 10 15

<210> 94

<211> 5

<212> PRT

<213> human

<400> 94

Ser Tyr Ala Met His

1 5

<210> 95

<211> 17

<212> PRT

<213> human

<400> 95

Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 96

<211> 15

<212> PRT

<213> human

<400> 96

Asp Arg Gly Tyr Ser Ser Ser Trp Tyr Pro Asp Ala Phe Asp Ile

1 5 10 15

<210> 97

<211> 5

<212> PRT

<213> human

<400> 97

Thr Tyr Ser Met Asn

1 5

<210> 98

<211> 17

<212> PRT

<213> human

<400> 98

Val Ile Ser Phe Asp Gly Ser Leu Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 99

<211> 12

<212> PRT

<213> human

<400> 99

Glu Arg Thr Thr Leu Ser Gly Ser Tyr Phe Asp Tyr

1 5 10

<210> 100

<211> 5

<212> PRT

<213> human

<400> 100

Ser Tyr Ser Met Asn

1 5

<210> 101

<211> 17

<212> PRT

<213> human

<400> 101

Val Ile Ser His Asp Gly Ser Ile Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 102

<211> 16

<212> PRT

<213> human

<400> 102

Arg Ile Ala Ala Ala Gly Gly Phe His Tyr Tyr Tyr Ala Leu Asp Val

1 5 10 15

<210> 103

<211> 5

<212> PRT

<213> human

<400> 103

Ser Phe Ser Met Asn

1 5

<210> 104

<211> 17

<212> PRT

<213> human

<400> 104

Tyr Ile Ser Ser Arg Ser Ser Thr Ile Tyr Ile Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 105

<211> 16

<212> PRT

<213> human

<400> 105

Arg Ile Ala Ala Ala Gly Pro Trp Gly Tyr Tyr Tyr Ala Met Asp Val

1 5 10 15

<210> 106

<211> 7

<212> PRT

<213> human

<400> 106

Ser Gly Gly Tyr Tyr Trp Thr

1 5

<210> 107

<211> 17

<212> PRT

<213> human

<400> 107

Tyr Ile Ser Ser Ser Ser Ser Thr Arg Tyr His Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 108

<211> 10

<212> PRT

<213> human

<400> 108

Asn Arg Gly Tyr Tyr Tyr Gly Met Asp Val

1 5 10

<210> 109

<211> 7

<212> PRT

<213> human

<400> 109

Ser Gly Gly Tyr Tyr Trp Ser

1 5

<210> 110

<211> 17

<212> PRT

<213> human

<400> 110

Tyr Ile Ser Ser Arg Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 111

<211> 10

<212> PRT

<213> human

<400> 111

Asn Arg Gly Phe Tyr Tyr Gly Met Asp Val

1 5 10

<210> 112

<211> 5

<212> PRT

<213> human

<400> 112

Ser Tyr Phe Trp Ser

1 5

<210> 113

<211> 16

<212> PRT

<213> human

<400> 113

Tyr Ile Tyr Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 114

<211> 10

<212> PRT

<213> human

<400> 114

Asp Arg Gly His Tyr Tyr Gly Met Asp Val

1 5 10

<210> 115

<211> 5

<212> PRT

<213> human

<400> 115

Thr Tyr Tyr Trp Ser

1 5

<210> 116

<211> 16

<212> PRT

<213> human

<400> 116

His Ile His Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 117

<211> 10

<212> PRT

<213> human

<400> 117

Asp Arg Gly Ser Tyr Tyr Gly Ser Asp Tyr

1 5 10

<210> 118

<211> 16

<212> PRT

<213> human

<400> 118

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 119

<211> 10

<212> PRT

<213> human

<400> 119

Asp Arg Gly Tyr Tyr Tyr Gly Val Asp Val

1 5 10

<210> 120

<211> 16

<212> PRT

<213> human

<400> 120

Tyr Ile Tyr Tyr Ser Gly Ser Ser Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 121

<211> 16

<212> PRT

<213> human

<400> 121

Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 122

<211> 16

<212> PRT

<213> human

<400> 122

Leu Ile Tyr Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 123

<211> 11

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Xaa can be Gly or Val

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> Xaa can be Ile, Phe or Ser

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> Xaa can be Ser or Gly

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> Xaa can be Phe or Leu.

<400> 123

Arg Ala Ser Gln Xaa Xaa Ser Xaa Trp Xaa Ala

1 5 10

<210> 124

<211> 12

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Xaa can be Asn or Ser

<400> 124

Thr Leu Xaa Ser Gly Tyr Ser Asp Tyr Lys Val Asp

1 5 10

<210> 125

<211> 14

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> Xaa can be Ile or Thr

<400> 125

Thr Gly Ser Ser Ser Asn Xaa Gly Ala Gly Tyr Asp Val His

1 5 10

<210> 126

<211> 12

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> Xaa can be Ile or Thr

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> Xaa can be Asp or Glu

<400> 126

Val Gly Thr Gly Gly Xaa Val Gly Ser Lys Gly Xaa

1 5 10

<210> 127

<211> 7

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Xaa can be Asn or Gly

<400> 127

Gly Ser Xaa Asn Arg Pro Ser

1 5

<210> 128

<211> 13

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> Xaa can be Ser or Asn

<400> 128

Gly Ala Asp His Gly Ser Gly Xaa Asn Phe Val Tyr Val

1 5 10

<210> 129

<211> 7

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> Xaa can be Ser or Thr

<400> 129

Ser Gly Gly Tyr Tyr Trp Xaa

1 5

<210> 130

<211> 5

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Xaa can be Gly or Ala

<400> 130

Ser Tyr Xaa Met His

1 5

<210> 131

<211> 5

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Xaa can be Ser or Thr

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Xaa can be Ser or Thr

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Xaa can be Tyr or Phe

<400> 131

Xaa Xaa Ser Met Asn

1 5

<210> 132

<211> 16

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Xaa can be Tyr or His

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Xaa can be Thr or His

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> Xaa can be Ser or Asn

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> Xaa can be Thr or Ser

<400> 132

Xaa Ile Xaa Tyr Ser Gly Xaa Xaa Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 133

<211> 17

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Xaa can be Phe or His

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> Xaa can be Leu or Thr

<400> 133

Val Ile Ser Xaa Asp Gly Ser Xaa Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 134

<211> 17

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Xaa can be Arg or Ser

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> Xaa can be Ile or Arg

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> Xaa can be Ile, His or Try

<400> 134

Tyr Ile Ser Ser Xaa Ser Ser Thr Xaa Tyr Xaa Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 135

<211> 17

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> Xaa can be Lys or Glu

<400> 135

Val Ile Trp Tyr Asp Gly Ser Asn Xaa Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 136

<211> 10

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Xaa can be Asn or Asp

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Xaa can be His, Tyr or Phe

<400> 136

Xaa Arg Gly Xaa Tyr Tyr Gly Met Asp Val

1 5 10

<210> 137

<211> 16

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> Xaa can be Gly or Phe

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> Xaa can be Phe or Trp

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> Xaa can be His or Gly

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> Xaa can be Leu and Met

<400> 137

Arg Ile Ala Ala Ala Gly Xaa Xaa Xaa Tyr Tyr Tyr Ala Xaa Asp Val

1 5 10 15

<210> 138

<211> 15

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Xaa can be Ser or Thr

<400> 138

Asp Arg Gly Tyr Xaa Ser Ser Trp Tyr Pro Asp Ala Phe Asp Ile

1 5 10 15

<210> 139

<211> 112

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (29)..(29)

<223> Xaa can be Ile or Thr

<220>

<221> MISC_FEATURE

<222> (41)..(41)

<223> Xaa can be Val or Leu

<220>

<221> MISC_FEATURE

<222> (54)..(54)

<223> Xaa can be Gly or Asn

<220>

<221> MISC_FEATURE

<222> (107)..(107)

<223> Xaa can be Arg or Lys

<400> 139

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Xaa Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Xaa Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Ser Xaa Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser

85 90 95

Leu Ser Gly Trp Val Phe Gly Gly Gly Thr Xaa Arg Leu Thr Val Leu

100 105 110

<210> 140

<211> 120

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (30)..(30)

<223> Xaa can be an nd or Ser

<220>

<221> MISC_FEATURE

<222> (37)..(37)

<223> Xaa can be Ser or Thr

<220>

<221> MISC_FEATURE

<222> (52)..(52)

<223> Xaa can be Tyr or His

<220>

<221> MISC_FEATURE

<222> (54)..(54)

<223> Xaa can be Tyr or His

<220>

<221> MISC_FEATURE

<222> (58)..(58)

<223> Xaa can be Ser or Asn

<220>

<221> MISC_FEATURE

<222> (59)..(59)

<223> Xaa can be Ser or Asn

<220>

<221> MISC_FEATURE

<222> (69)..(69)

<223> Xaa can be Ser or Thr

<220>

<221> MISC_FEATURE

<222> (71)..(71)

<223> Xaa can be Val or Ile

<220>

<221> MISC_FEATURE

<222> (77)..(77)

<223> Xaa can be Ile or Met

<220>

<221> MISC_FEATURE

<222> (83)..(83)

<223> Xaa can be Lys or Gln

<220>

<221> MISC_FEATURE

<222> (99)..(99)

<223> Xaa can be Arg or Lys

<220>

<221> MISC_FEATURE

<222> (100)..(100)

<223> Xaa can be Asp or Asn

<220>

<221> MISC_FEATURE

<222> (103)..(103)

<223> Xaa can be His, Phe or Try

<400> 140

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Xaa Ser Gly

20 25 30

Gly Tyr Tyr Trp Xaa Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Xaa Ile Xaa Tyr Ser Gly Xaa Xaa Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Xaa Thr Xaa Ser Val Asp Thr Ser Xaa Asn Gln Phe

65 70 75 80

Ser Leu Xaa Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Xaa Xaa Arg Gly Xaa Tyr Tyr Gly Met Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 141

<211> 125

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (23)..(23)

<223> Xaa can be Ala or Val

<220>

<221> MISC_FEATURE

<222> (24)..(24)

<223> Xaa can be Ala or Val

<220>

<221> MISC_FEATURE

<222> (31)..(31)

<223> Xaa can be Thr or Ser

<220>

<221> MISC_FEATURE

<222> (32)..(32)

<223> Xaa can be Tyr or Phe

<220>

<221> MISC_FEATURE

<222> (54)..(54)

<223> Xaa can be Ser or Arg

<220>

<221> MISC_FEATURE

<222> (58)..(58)

<223> Xaa can be Arg or Ile

<220>

<221> MISC_FEATURE

<222> (60)..(60)

<223> Xaa can be His, Try or Ile

<220>

<221> MISC_FEATURE

<222> (105)..(105)

<223> Xaa can be Pro or Gly

<220>

<221> MISC_FEATURE

<222> (106)..(106)

<223> Xaa can be Trp or Phe

<220>

<221> MISC_FEATURE

<222> (107)..(107)

<223> Xaa can be Gly or His

<220>

<221> MISC_FEATURE

<222> (112)..(112)

<223> Xaa can be Met or Leu

<400> 141

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Xaa Xaa Ser Gly Phe Thr Phe Ser Xaa Xaa

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Xaa Ser Ser Thr Xaa Tyr Xaa Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ile Ala Ala Ala Gly Xaa Xaa Xaa Tyr Tyr Tyr Ala Xaa

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 142

<211> 121

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (33)..(33)

<223> Xaa can be Gly or Ala

<220>

<221> MISC_FEATURE

<222> (48)..(48)

<223> Xaa can be Val or Leu

<220>

<221> MISC_FEATURE

<222> (49)..(49)

<223> Xaa can be Ala or Ser

<220>

<221> MISC_FEATURE

<222> (53)..(53)

<223> Xaa can be Phe or His

<220>

<221> MISC_FEATURE

<222> (57)..(57)

<223> Xaa can be Leu or Ile

<400> 142

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Xaa Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Xaa

35 40 45

Xaa Val Ile Ser Xaa Asp Gly Ser Xaa Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Arg Thr Thr Leu Ser Gly Ser Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 143

<211> 124

<212> PRT

<213> Artificial

<220>

<223> consensus sequence

<220>

<221> MISC_FEATURE

<222> (58)..(58)

<223> Xaa can be Glu or Lys

<220>

<221> MISC_FEATURE

<222> (103)..(103)

<223> Xaa can be Thr or Ser

<400> 143

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Xaa Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Tyr Xaa Ser Ser Trp Tyr Pro Asp Ala Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 144

<211> 1026

<212> DNA

<213> human

<400> 144

aactcggtga acaactgagg gaaccaaacc agagacgcgc tgaacagaga gaatcaggct 60

caaagcaagt ggaagtgggc agagattcca ccaggactgg tgcaaggcgc agagccagcc 120

agatttgaga agaaggcaaa aagatgctgg ggagcagagc tgtaatgctg ctgttgctgc 180

tgccctggac agctcagggc agagctgtgc ctgggggcag cagccctgcc tggactcagt 240

gccagcagct ttcacagaag ctctgcacac tggcctggag tgcacatcca ctagtgggac 300

acatggatct aagagaagag ggagatgaag agactacaaa tgatgttccc catatccagt 360

gtggagatgg ctgtgacccc caaggactca gggacaacag tcagttctgc ttgcaaagga 420

tccaccaggg tctgattttt tatgagaagc tgctaggatc ggatattttc acaggggagc 480

cttctctgct ccctgatagc cctgtgggcc agcttcatgc ctccctactg ggcctcagcc 540

aactcctgca gcctgagggt caccactggg agactcagca gattccaagc ctcagtccca 600

gccagccatg gcagcgtctc cttctccgct tcaaaatcct tcgcagcctc caggcctttg 660

tggctgtagc cgcccgggtc tttgcccatg gagcagcaac cctgagtccc taaaggcagc 720

agctcaagga tggcactcag atctccatgg cccagcaagg ccaagataaa tctaccaccc 780

caggcacctg tgagccaaca ggttaattag tccattaatt ttagtgggac ctgcatatgt 840

tgaaaattac caatactgac tgacatgtga tgctgaccta tgataaggtt gagtatttat 900

tagatgggaa gggaaatttg gggattattt atcctcctgg ggacagtttg gggaggatta 960

tttattgtat ttatattgaa ttatgtactt ttttcaataa agtcttattt ttgtggctaa 1020

aaaaaa 1026

<210> 145

<211> 189

<212> PRT

<213> human

<400> 145

Met Leu Gly Ser Arg Ala Val Met Leu Leu Leu Leu Leu Pro Trp Thr

1 5 10 15

Ala Gln Gly Arg Ala Val Pro Gly Gly Ser Ser Pro Ala Trp Thr Gln

20 25 30

Cys Gln Gln Leu Ser Gln Lys Leu Cys Thr Leu Ala Trp Ser Ala His

35 40 45

Pro Leu Val Gly His Met Asp Leu Arg Glu Glu Gly Asp Glu Glu Thr

50 55 60

Thr Asn Asp Val Pro His Ile Gln Cys Gly Asp Gly Cys Asp Pro Gln

65 70 75 80

Gly Leu Arg Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile His Gln Gly

85 90 95

Leu Ile Phe Tyr Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu

100 105 110

Pro Ser Leu Leu Pro Asp Ser Pro Val Gly Gln Leu His Ala Ser Leu

115 120 125

Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Gly His His Trp Glu Thr

130 135 140

Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp Gln Arg Leu Leu

145 150 155 160

Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln Ala Phe Val Ala Val Ala

165 170 175

Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Ser Pro

180 185

<210> 146

<211> 1399

<212> DNA

<213> human

<400> 146

ctgtttcagg gccattggac tctccgtcct gcccagagca agatgtgtca ccagcagttg 60

gtcatctctt ggttttccct ggtttttctg gcatctcccc tcgtggccat atgggaactg 120

aagaaagatg tttatgtcgt agaattggat tggtatccgg atgcccctgg agaaatggtg 180

gtcctcacct gtgacacccc tgaagaagat ggtatcacct ggaccttgga ccagagcagt 240

gaggtcttag gctctggcaa aaccctgacc atccaagtca aagagtttgg agatgctggc 300

cagtacacct gtcacaaagg aggcgaggtt ctaagccatt cgctcctgct gcttcacaaa 360

aaggaagatg gaatttggtc cactgatatt ttaaaggacc agaaagaacc caaaaataag 420

acctttctaa gatgcgaggc caagaattat tctggacgtt tcacctgctg gtggctgacg 480

acaatcagta ctgatttgac attcagtgtc aaaagcagca gaggctcttc tgacccccaa 540

ggggtgacgt gcggagctgc tacactctct gcagagagag tcagagggga caacaaggag 600

tatgagtact cagtggagtg ccaggaggac agtgcctgcc cagctgctga ggagagtctg 660

cccattgagg tcatggtgga tgccgttcac aagctcaagt atgaaaacta caccagcagc 720

ttcttcatca gggacatcat caaacctgac ccacccaaga acttgcagct gaagccatta 780

aagaattctc ggcaggtgga ggtcagctgg gagtaccctg acacctggag tactccacat 840

tcctacttct ccctgacatt ctgcgttcag gtccagggca agagcaagag agaaaagaaa 900

gatagagtct tcacggacaa gacctcagcc acggtcatct gccgcaaaaa tgccagcatt 960

agcgtgcggg cccaggaccg ctactatagc tcatcttgga gcgaatgggc atctgtgccc 1020

tgcagttagg ttctgatcca ggatgaaaat ttggaggaaa agtggaagat attaagcaaa 1080

atgtttaaag acacaacgga atagacccaa aaagataatt tctatctgat ttgctttaaa 1140

acgttttttt aggatcacaa tgatatcttt gctgtatttg tatagttaga tgctaaatgc 1200

tcattgaaac aatcagctaa tttatgtata gattttccag ctctcaagtt gccatgggcc 1260

ttcatgctat ttaaatattt aagtaattta tgtatttatt agtatattac tgttatttaa 1320

cgtttgtctg ccaggatgta tggaatgttt catactctta tgacctgatc catcaggatc 1380

agtccctatt atgcaaaat 1399

<210> 147

<211> 328

<212> PRT

<213> human

<400> 147

Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu

1 5 10 15

Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val

20 25 30

Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu

35 40 45

Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln

50 55 60

Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys

65 70 75 80

Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val

85 90 95

Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp

100 105 110

Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe

115 120 125

Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp

130 135 140

Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg

145 150 155 160

Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser

165 170 175

Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu

180 185 190

Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile

195 200 205

Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr

210 215 220

Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn

225 230 235 240

Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp

245 250 255

Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr

260 265 270

Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg

275 280 285

Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala

290 295 300

Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser

305 310 315 320

Glu Trp Ala Ser Val Pro Cys Ser

325

<210> 148

<211> 2826

<212> DNA

<213> human

<400> 148

acaagggtgg cagcctggct ctgaagtgga attatgtgct tcaaacaggt tgaaagaggg 60

aaacagtctt ttcctgcttc cagacatgaa tcaggtcact attcaatggg atgcagtaat 120

agccctttac atactcttca gctggtgtca tggaggaatt acaaatataa actgctctgg 180

ccacatctgg gtagaaccag ccacaatttt taagatgggt atgaatatct ctatatattg 240

ccaagcagca attaagaact gccaaccaag gaaacttcat ttttataaaa atggcatcaa 300

agaaagattt caaatcacaa ggattaataa aacaacagct cggctttggt ataaaaactt 360

tctggaacca catgcttcta tgtactgcac tgctgaatgt cccaaacatt ttcaagagac 420

actgatatgt ggaaaagaca tttcttctgg atatccgcca gatattcctg atgaagtaac 480

ctgtgtcatt tatgaatatt caggcaacat gacttgcacc tggaatgctg ggaagctcac 540

ctacatagac acaaaatacg tggtacatgt gaagagttta gagacagaag aagagcaaca 600

gtatctcacc tcaagctata ttaacatctc cactgattca ttacaaggtg gcaagaagta 660

cttggtttgg gtccaagcag caaacgcact aggcatggaa gagtcaaaac aactgcaaat 720

tcacctggat gatatagtga taccttctgc agccgtcatt tccagggctg agactataaa 780

tgctacagtg cccaagacca taatttattg ggatagtcaa acaacaattg aaaaggtttc 840

ctgtgaaatg agatacaagg ctacaacaaa ccaaacttgg aatgttaaag aatttgacac 900

caattttaca tatgtgcaac agtcagaatt ctacttggag ccaaacatta agtacgtatt 960

tcaagtgaga tgtcaagaaa caggcaaaag gtactggcag ccttggagtt cactgttttt 1020

tcataaaaca cctgaaacag ttccccaggt cacatcaaaa gcattccaac atgacacatg 1080

gaattctggg ctaacagttg cttccatctc tacagggcac cttacttctg acaacagagg 1140

agacattgga cttttattgg gaatgatcgt ctttgctgtt atgttgtcaa ttctttcttt 1200

gattgggata tttaacagat cattccgaac tgggattaaa agaaggatct tattgttaat 1260

accaaagtgg ctttatgaag atattcctaa tatgaaaaac agcaatgttg tgaaaatgct 1320

acaggaaaat agtgaactta tgaataataa ttccagtgag caggtcctat atgttgatcc 1380

catgattaca gagataaaag aaatcttcat cccagaacac aagcctacag actacaagaa 1440

ggagaataca ggacccctgg agacaagaga ctacccgcaa aactcgctat tcgacaatac 1500

tacagttgta tatattcctg atctcaacac tggatataaa ccccaaattt caaattttct 1560

gcctgaggga agccatctca gcaataataa tgaaattact tccttaacac ttaaaccacc 1620

agttgattcc ttagactcag gaaataatcc caggttacaa aagcatccta attttgcttt 1680

ttctgtttca agtgtgaatt cactaagcaa cacaatattt cttggagaat taagcctcat 1740

attaaatcaa ggagaatgca gttctcctga catacaaaac tcagtagagg aggaaaccac 1800

catgcttttg gaaaatgatt cacccagtga aactattcca gaacagaccc tgcttcctga 1860

tgaatttgtc tcctgtttgg ggatcgtgaa tgaggagttg ccatctatta atacttattt 1920

tccacaaaat attttggaaa gccacttcaa taggatttca ctcttggaaa agtagagctg 1980

tgtggtcaaa atcaatatga gaaagctgcc ttgcaatctg aacttgggtt ttccctgcaa 2040

tagaaattga attctgcctc tttttgaaaa aaatgtattc acatacaaat cttcacatgg 2100

acacatgttt tcatttccct tggataaata cctaggtagg ggattgctgg gccatatgat 2160

aagcatatgt ttcagttcta ccaatcttgt ttccagagta gtgacatttc tgtgctccta 2220

ccatcaccat gtaagaattc ccgggagctc catgcctttt taattttagc cattcttctg 2280

cctcatttct taaaattaga gaattaaggt cccgaaggtg gaacatgctt catggtcaca 2340

catacaggca caaaaacagc attatgtgga cgcctcatgt attttttata gagtcaacta 2400

tttcctcttt attttccctc attgaaagat gcaaaacagc tctctattgt gtacagaaag 2460

ggtaaataat gcaaaatacc tggtagtaaa ataaatgctg aaaattttcc tttaaaatag 2520

aatcattagg ccaggcgtgg tggctcatgc ttgtaatccc agcactttgg taggctgagg 2580

taggtggatc acctgaggtc aggagttcga gtccagcctg gccaatatgc tgaaaccctg 2640

tctctactaa aattacaaaa attagccggc catggtggca ggtgcttgta atcccagcta 2700

cttgggaggc tgaggcagga gaatcacttg aaccaggaag gcagaggttg cactgagctg 2760

agattgtgcc actgcactcc agcctgggca acaagagcaa aactctgtct ggaaaaaaaa 2820

aaaaaa 2826

<210> 149

<211> 629

<212> PRT

<213> human

<400> 149

Met Asn Gln Val Thr Ile Gln Trp Asp Ala Val Ile Ala Leu Tyr Ile

1 5 10 15

Leu Phe Ser Trp Cys His Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly

20 25 30

His Ile Trp Val Glu Pro Ala Thr Ile Phe Lys Met Gly Met Asn Ile

35 40 45

Ser Ile Tyr Cys Gln Ala Ala Ile Lys Asn Cys Gln Pro Arg Lys Leu

50 55 60

His Phe Tyr Lys Asn Gly Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile

65 70 75 80

Asn Lys Thr Thr Ala Arg Leu Trp Tyr Lys Asn Phe Leu Glu Pro His

85 90 95

Ala Ser Met Tyr Cys Thr Ala Glu Cys Pro Lys His Phe Gln Glu Thr

100 105 110

Leu Ile Cys Gly Lys Asp Ile Ser Ser Gly Tyr Pro Pro Asp Ile Pro

115 120 125

Asp Glu Val Thr Cys Val Ile Tyr Glu Tyr Ser Gly Asn Met Thr Cys

130 135 140

Thr Trp Asn Ala Gly Lys Leu Thr Tyr Ile Asp Thr Lys Tyr Val Val

145 150 155 160

His Val Lys Ser Leu Glu Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser

165 170 175

Ser Tyr Ile Asn Ile Ser Thr Asp Ser Leu Gln Gly Gly Lys Lys Tyr

180 185 190

Leu Val Trp Val Gln Ala Ala Asn Ala Leu Gly Met Glu Glu Ser Lys

195 200 205

Gln Leu Gln Ile His Leu Asp Asp Ile Val Ile Pro Ser Ala Ala Val

210 215 220

Ile Ser Arg Ala Glu Thr Ile Asn Ala Thr Val Pro Lys Thr Ile Ile

225 230 235 240

Tyr Trp Asp Ser Gln Thr Thr Ile Glu Lys Val Ser Cys Glu Met Arg

245 250 255

Tyr Lys Ala Thr Thr Asn Gln Thr Trp Asn Val Lys Glu Phe Asp Thr

260 265 270

Asn Phe Thr Tyr Val Gln Gln Ser Glu Phe Tyr Leu Glu Pro Asn Ile

275 280 285

Lys Tyr Val Phe Gln Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp

290 295 300

Gln Pro Trp Ser Ser Leu Phe Phe His Lys Thr Pro Glu Thr Val Pro

305 310 315 320

Gln Val Thr Ser Lys Ala Phe Gln His Asp Thr Trp Asn Ser Gly Leu

325 330 335

Thr Val Ala Ser Ile Ser Thr Gly His Leu Thr Ser Asp Asn Arg Gly

340 345 350

Asp Ile Gly Leu Leu Leu Gly Met Ile Val Phe Ala Val Met Leu Ser

355 360 365

Ile Leu Ser Leu Ile Gly Ile Phe Asn Arg Ser Phe Arg Thr Gly Ile

370 375 380

Lys Arg Arg Ile Leu Leu Leu Ile Pro Lys Trp Leu Tyr Glu Asp Ile

385 390 395 400

Pro Asn Met Lys Asn Ser Asn Val Val Lys Met Leu Gln Glu Asn Ser

405 410 415

Glu Leu Met Asn Asn Asn Ser Ser Glu Gln Val Leu Tyr Val Asp Pro

420 425 430

Met Ile Thr Glu Ile Lys Glu Ile Phe Ile Pro Glu His Lys Pro Thr

435 440 445

Asp Tyr Lys Lys Glu Asn Thr Gly Pro Leu Glu Thr Arg Asp Tyr Pro

450 455 460

Gln Asn Ser Leu Phe Asp Asn Thr Thr Val Val Tyr Ile Pro Asp Leu

465 470 475 480

Asn Thr Gly Tyr Lys Pro Gln Ile Ser Asn Phe Leu Pro Glu Gly Ser

485 490 495

His Leu Ser Asn Asn Asn Glu Ile Thr Ser Leu Thr Leu Lys Pro Pro

500 505 510

Val Asp Ser Leu Asp Ser Gly Asn Asn Pro Arg Leu Gln Lys His Pro

515 520 525

Asn Phe Ala Phe Ser Val Ser Ser Val Asn Ser Leu Ser Asn Thr Ile

530 535 540

Phe Leu Gly Glu Leu Ser Leu Ile Leu Asn Gln Gly Glu Cys Ser Ser

545 550 555 560

Pro Asp Ile Gln Asn Ser Val Glu Glu Glu Thr Thr Met Leu Leu Glu

565 570 575

Asn Asp Ser Pro Ser Glu Thr Ile Pro Glu Gln Thr Leu Leu Pro Asp

580 585 590

Glu Phe Val Ser Cys Leu Gly Ile Val Asn Glu Glu Leu Pro Ser Ile

595 600 605

Asn Thr Tyr Phe Pro Gln Asn Ile Leu Glu Ser His Phe Asn Arg Ile

610 615 620

Ser Leu Leu Glu Lys

625

<210> 150

<211> 2100

<212> DNA

<213> human

<400> 150

ggtggctgaa cctcgcaggt ggcagagagg ctcccctggg gctgtggggc tctacgtgga 60

tccgatggag ccgctggtga cctgggtggt ccccctcctc ttcctcttcc tgctgtccag 120

gcagggcgct gcctgcagaa ccagtgagtg ctgttttcag gacccgccat atccggatgc 180

agactcaggc tcggcctcgg gccctaggga cctgagatgc tatcggatat ccagtgatcg 240

ttacgagtgc tcctggcagt atgagggtcc cacagctggg gtcagccact tcctgcggtg 300

ttgccttagc tccgggcgct gctgctactt cgccgccggc tcagccacca ggctgcagtt 360

ctccgaccag gctggggtgt ctgtgctgta cactgtcaca ctctgggtgg aatcctgggc 420

caggaaccag acagagaagt ctcctgaggt gaccctgcag ctctacaact cagttaaata 480

tgagcctcct ctgggagaca tcaaggtgtc caagttggcc gggcagctgc gtatggagtg 540

ggagaccccg gataaccagg ttggtgctga ggtgcagttc cggcaccgga cacccagcag 600

cccatggaag ttgggcgact gcggacctca ggatgatgat actgagtcct gcctctgccc 660

cctggagatg aatgtggccc aggaattcca gctccgacga cggcagctgg ggagccaagg 720

aagttcctgg agcaagtgga gcagccccgt gtgcgttccc cctgaaaacc ccccacagcc 780

tcaggtgaga ttctcggtgg agcagctggg ccaggatggg aggaggcggc tgaccctgaa 840

agagcagcca acccagctgg agcttccaga aggctgtcaa gggctggcgc ctggcacgga 900

ggtcacttac cgactacagc tccacatgct gtcctgcccg tgtaaggcca aggccaccag 960

gaccctgcac ctggggaaga tgccctatct ctcgggtgct gcctacaacg tggctgtcat 1020

ctcctcgaac caatttggtc ctggcctgaa ccagacgtgg cacattcctg ccgacaccca 1080

cacagaacca gtggctctga atatcagcgt cggaaccaac gggaccacca tgtattggcc 1140

agcccgggct cagagcatga cgtattgcat tgaatggcag cctgtgggcc aggacggggg 1200

ccttgccacc tgcagcctga ctgcgccgca agacccggat ccggctggaa tggcaaccta 1260

cagctggagt cgagagtctg gggcaatggg gcaggaaaag tgttactaca ttaccatctt 1320

tgcctctgcg caccccgaga agctcacctt gtggtctacg gtcctgtcca cctaccactt 1380

tgggggcaat gcctcagcag ctgggacacc gcaccacgtc tcggtgaaga atcatagctt 1440

ggactctgtg tctgtggact gggcaccatc cctgctgagc acctgtcccg gcgtcctaaa 1500

ggagtatgtt gtccgctgcc gagatgaaga cagcaaacag gtgtcagagc atcccgtgca 1560

gcccacagag acccaagtta ccctcagtgg cctgcgggct ggtgtagcct acacggtgca 1620

ggtgcgagca gacacagcgt ggctgagggg tgtctggagc cagccccagc gcttcagcat 1680

cgaagtgcag gtttctgatt ggctcatctt cttcgcctcc ctggggagct tcctgagcat 1740

ccttctcgtg ggcgtccttg gctaccttgg cctgaacagg gccgcacggc acctgtgccc 1800

gccgctgccc acaccctgtg ccagctccgc cattgagttc cctggaggga aggagacttg 1860

gcagtggatc aacccagtgg acttccagga agaggcatcc ctgcaggagg ccctggtggt 1920

agagatgtcc tgggacaaag gcgagaggac tgagcctctc gagaagacag agctacctga 1980

gggtgcccct gagctggccc tggatacaga gttgtccttg gaggatggag acaggtgcaa 2040

ggccaagatg tgatcgttga ggctcagaga gggtgagtga ctcgcccgag gctacgtagc 2100

<210> 151

<211> 662

<212> PRT

<213> human

<400> 151

Met Glu Pro Leu Val Thr Trp Val Val Pro Leu Leu Phe Leu Phe Leu

1 5 10 15

Leu Ser Arg Gln Gly Ala Ala Cys Arg Thr Ser Glu Cys Cys Phe Gln

20 25 30

Asp Pro Pro Tyr Pro Asp Ala Asp Ser Gly Ser Ala Ser Gly Pro Arg

35 40 45

Asp Leu Arg Cys Tyr Arg Ile Ser Ser Asp Arg Tyr Glu Cys Ser Trp

50 55 60

Gln Tyr Glu Gly Pro Thr Ala Gly Val Ser His Phe Leu Arg Cys Cys

65 70 75 80

Leu Ser Ser Gly Arg Cys Cys Tyr Phe Ala Ala Gly Ser Ala Thr Arg

85 90 95

Leu Gln Phe Ser Asp Gln Ala Gly Val Ser Val Leu Tyr Thr Val Thr

100 105 110

Leu Trp Val Glu Ser Trp Ala Arg Asn Gln Thr Glu Lys Ser Pro Glu

115 120 125

Val Thr Leu Gln Leu Tyr Asn Ser Val Lys Tyr Glu Pro Pro Leu Gly

130 135 140

Asp Ile Lys Val Ser Lys Leu Ala Gly Gln Leu Arg Met Glu Trp Glu

145 150 155 160

Thr Pro Asp Asn Gln Val Gly Ala Glu Val Gln Phe Arg His Arg Thr

165 170 175

Pro Ser Ser Pro Trp Lys Leu Gly Asp Cys Gly Pro Gln Asp Asp Asp

180 185 190

Thr Glu Ser Cys Leu Cys Pro Leu Glu Met Asn Val Ala Gln Glu Phe

195 200 205

Gln Leu Arg Arg Arg Gln Leu Gly Ser Gln Gly Ser Ser Trp Ser Lys

210 215 220

Trp Ser Ser Pro Val Cys Val Pro Pro Glu Asn Pro Pro Gln Pro Gln

225 230 235 240

Val Arg Phe Ser Val Glu Gln Leu Gly Gln Asp Gly Arg Arg Arg Leu

245 250 255

Thr Leu Lys Glu Gln Pro Thr Gln Leu Glu Leu Pro Glu Gly Cys Gln

260 265 270

Gly Leu Ala Pro Gly Thr Glu Val Thr Tyr Arg Leu Gln Leu His Met

275 280 285

Leu Ser Cys Pro Cys Lys Ala Lys Ala Thr Arg Thr Leu His Leu Gly

290 295 300

Lys Met Pro Tyr Leu Ser Gly Ala Ala Tyr Asn Val Ala Val Ile Ser

305 310 315 320

Ser Asn Gln Phe Gly Pro Gly Leu Asn Gln Thr Trp His Ile Pro Ala

325 330 335

Asp Thr His Thr Glu Pro Val Ala Leu Asn Ile Ser Val Gly Thr Asn

340 345 350

Gly Thr Thr Met Tyr Trp Pro Ala Arg Ala Gln Ser Met Thr Tyr Cys

355 360 365

Ile Glu Trp Gln Pro Val Gly Gln Asp Gly Gly Leu Ala Thr Cys Ser

370 375 380

Leu Thr Ala Pro Gln Asp Pro Asp Pro Ala Gly Met Ala Thr Tyr Ser

385 390 395 400

Trp Ser Arg Glu Ser Gly Ala Met Gly Gln Glu Lys Cys Tyr Tyr Ile

405 410 415

Thr Ile Phe Ala Ser Ala His Pro Glu Lys Leu Thr Leu Trp Ser Thr

420 425 430

Val Leu Ser Thr Tyr His Phe Gly Gly Asn Ala Ser Ala Ala Gly Thr

435 440 445

Pro His His Val Ser Val Lys Asn His Ser Leu Asp Ser Val Ser Val

450 455 460

Asp Trp Ala Pro Ser Leu Leu Ser Thr Cys Pro Gly Val Leu Lys Glu

465 470 475 480

Tyr Val Val Arg Cys Arg Asp Glu Asp Ser Lys Gln Val Ser Glu His

485 490 495

Pro Val Gln Pro Thr Glu Thr Gln Val Thr Leu Ser Gly Leu Arg Ala

500 505 510

Gly Val Ala Tyr Thr Val Gln Val Arg Ala Asp Thr Ala Trp Leu Arg

515 520 525

Gly Val Trp Ser Gln Pro Gln Arg Phe Ser Ile Glu Val Gln Val Ser

530 535 540

Asp Trp Leu Ile Phe Phe Ala Ser Leu Gly Ser Phe Leu Ser Ile Leu

545 550 555 560

Leu Val Gly Val Leu Gly Tyr Leu Gly Leu Asn Arg Ala Ala Arg His

565 570 575

Leu Cys Pro Pro Leu Pro Thr Pro Cys Ala Ser Ser Ala Ile Glu Phe

580 585 590

Pro Gly Gly Lys Glu Thr Trp Gln Trp Ile Asn Pro Val Asp Phe Gln

595 600 605

Glu Glu Ala Ser Leu Gln Glu Ala Leu Val Val Glu Met Ser Trp Asp

610 615 620

Lys Gly Glu Arg Thr Glu Pro Leu Glu Lys Thr Glu Leu Pro Glu Gly

625 630 635 640

Ala Pro Glu Leu Ala Leu Asp Thr Glu Leu Ser Leu Glu Asp Gly Asp

645 650 655

Arg Cys Lys Ala Lys Met

660

<210> 152

<211> 360

<212> DNA

<213> human

<400> 152

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtggtt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggcacatcc attacagtgg gaacacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaatcagttc 240

tccctgaaac tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgcgaaat 300

cgcgggttct actacggtat ggacgtctgg ggccaaggga ccacggtcac cgtctcctca 360

<210> 153

<211> 120

<212> PRT

<213> human

<400> 153

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly His Ile His Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asn Arg Gly Phe Tyr Tyr Gly Met Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 154

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> bee melittin signal

<400> 154

Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile

1 5 10 15

Ser Tyr Ile Tyr Ala Ala Ala

20

<210> 155

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> His tag

<400> 155

His His His His His His

1 5

Claims (24)

1. An isolated antigen binding protein that binds IL-23, comprising:

a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID No. 31 and a light chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID No. 1.

2. The isolated antigen binding protein of claim 1, wherein said antigen binding protein is an antibody.

3. The isolated antigen binding protein of claim 2, wherein the antibody is a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment thereof.

4. The isolated antigen binding protein of claim 3, wherein said antibody fragment is a Fab fragment, a Fab 'fragment, a F (ab')₂Fragments, Fv fragments, diabodies or single chain antibody molecules.

5. The isolated antigen binding protein of claim 3, wherein said antigen binding protein is a human antibody.

6. The isolated antigen binding protein of claim 3, wherein said antigen binding protein is a monoclonal antibody.

7. The isolated antigen binding protein of claim 2, wherein the antigen binding protein is of the IgG1 type, IgG2 type, IgG3 type, or IgG4 type.

8. The isolated antigen binding protein of claim 7, wherein said antigen binding protein is of the IgG1 type or IgG2 type.

9. An isolated nucleic acid molecule encoding the antigen binding protein of claim 1.

10. The nucleic acid molecule of claim 9, wherein the nucleic acid molecule is operably linked to a regulatory sequence.

11. A vector comprising the nucleic acid molecule of claim 9.

12. A host cell comprising the nucleic acid molecule of claim 9.

13. A host cell comprising the vector of claim 11.

14. A method of making the antigen binding protein of claim 1, comprising the step of preparing the antigen binding protein from a host cell that secretes the antigen binding protein.

15. The isolated antigen binding protein of claim 1, wherein said antigen binding protein has at least one property selected from the group consisting of:

a) reducing human IL-23 activity;

b) reducing the production of proinflammatory cytokines;

c) at most 5X 10^-8K of M_DBinds to human IL-23; and

d) has a value of ≤ 5 × 10^-61/s ofK_offThe rate.

16. A pharmaceutical composition comprising the antigen binding protein of claim 1 and a pharmaceutically acceptable excipient.

17. The pharmaceutical composition of claim 16, further comprising a labeling group or an effector group.

18. The pharmaceutical composition of claim 17, wherein the labeling group is selected from the group consisting of: isotopic labels, magnetic labels, redox-active moieties, optical dyes, biotinylated groups, and predetermined polypeptide epitopes recognized by a second reporter.

19. The pharmaceutical composition of claim 17, wherein the effector group is selected from the group consisting of: radioisotopes, radionuclides, toxins, therapeutic groups, and chemotherapeutic groups.

20. The isolated antigen binding protein of claim 1, wherein said antigen binding protein is coupled to a labeling group.

21. Use of the isolated antigen binding protein of claim 1 in the manufacture of a medicament for treating or preventing a condition associated with IL-23 in a patient.

22. The use of claim 21, wherein the isolated antigen binding protein is administered alone or as a combination therapy.

23. Use of the isolated antigen binding protein of claim 1 in the manufacture of a medicament for reducing IL-23 activity in a patient.

24. The use of claim 23, wherein the IL-23 activity induces the production of a pro-inflammatory cytokine.