MX358249B

Human il-23 antigen binding proteins.

Abstract

Antigen binding proteins that bind to human IL-23 protein are provided. Nucleic acids encoding the antigen binding protein, vectors, and cells encoding the same as well as use of IL-23 antigen binding proteins for diagnostic and therapeutic purposes are also provided.

MX358249B, drawing sheet 1 — Sheet 1 of 254

Term

4.1 yearsleft in the term

Expires 26 October 2030.

23 claims: 6 independent, 17 dependent

1
CLAIMS REIVINDICACIONES 1. Una proteina de enlace al antigeno aislada que enlaza IL-23 humana nativa caracterizada porque comprende:one. An isolated antigen binding protein that binds native human IL-23 characterized in that it comprises: a heavy chain variable region comprising amino acid residues 31-35, 50-65 and 99-113 of SEQ ID NO: 31;una región variable de cadena pesada que comprende residuos de aminoácido 31-35, 50-65 y 99-113 de 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;and in y una región variable de cadena ligera que comprende residuos de aminoácido 23-36, 52-58 y 91-101 de SEQ ID NO:1;y en binds native human IL-23 characterized in that it comprises a heavy chain variable region of SEQ ID NO: 31, and a light chain variable region of SEQ ID NO: 1, and wherein said antigen binding protein binds to IL- Recombinant human 23 with a Kd that is less than or equal to 5xlO ~12M. enlaza IL-23 humana nativa caracterizada porque comprende una región variable de cadena pesada de SEQ ID NO:31, y una región variable de cadena ligera de SEQ ID NO:1, y en donde dicha proteina de enlace al antigeno se enlaza a IL-23 humana recombinante con un Kd que es menor o igual que 5xlO~12M.
2
3. The isolated antigen binding protein according to either of claims 1 or 2, characterized in that the antigen binding protein is a 3. La proteina de enlace al antigeno aislada de conformidad con cualquiera de las reivindicaciones 1 o 2, caracterizada porque la proteina de enlace al antígeno es un 169 169 ΜΡ1 ΜΡ1 INSTITUTO MEXICANO DE LA PROPIEDAD INDUSTRIAL anticuerpo. MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY antibody.
9
10. An isolated nucleic acid molecule characterized in that it encodes an antigen binding protein according to any of claims 1-2. 10. Una molécula de ácido nucleico aislada caracterizada porque codifica una proteína de enlace al antígeno de conformidad con cualquiera de las reivindicaciones 1-2.
15
16. An isolated antigen binding protein according to any of claims 1-2, 16. Una proteína de enlace al antígeno aislada de conformidad con cualquiera de las reivindicaciones 1-2, 171 characterized in that the binding protein at least one property selected from the group consisting of:171 caracterizada porque la proteína de enlace al menos una propiedad seleccionada del grupo que consiste de: a) reduce the activity of human IL-23;a) reducir la actividad de IL-23 humana;b) reduce the production of a proinflammatory cytokine;b) reducir la producción de una citoquina proinflamatoria;c) have a speed KORff of <5xl0-6 1 / s;and c) tener una velocidad KOff de < 5xl0-6 1/s;y d) have an IC50 of <400 pM. d) tener un IC50 de < 400 pM.
16
17. A pharmaceutical composition characterized in that it comprises at least one antigen binding protein according to any of claims 1-2, and a pharmaceutically acceptable excipient. 17. Una composición farmacéutica caracterizada porque comprende al menos una proteína de enlace al antígeno de conformidad con cualquiera de las reivindicaciones 1-2, y excipiente farmacéuticamente aceptable.
21
22. The antigen binding protein according to any of claims 1-2, for use in the treatment or prevention of an inflammatory disorder, a rheumatic disease, an autoimmune disorder, an oncological disorder or a gastrointestinal disorder. 22. La proteina de enlace al antigeno de conformidad con cualquiera de las reivindicaciones 1-2, para usarse en el tratamiento o prevención de un trastorno inflamatorio, una enfermedad reumática, un trastorno autoinmunitario, un trastorno oncológico o un trastorno gastrointestinal.

Independent claims6

1,764 paragraphs in 191 sections, as filed

(54) Title: HUMAN IL-23 ANTIGEN BINDING PROTEINS. (54) Title: HUMAN IL-23 ANTIGEN BINDING PROTEINS.

(57) Summary

The present invention relates to antigen binding proteins that bind to human IL-23 protein. Nucleic acids encoding the antigen binding protein, vectors, and cells encoding them are also provided as well as use of IL-23 antigen binding proteins for diagnostic and therapeutic purposes.

(57) Abstract

PATENT TITLE No. 358249

Owner (s): AMGEN INC.

Address: One Amgen Center Drive, Thousand Oaks, California, 91320, USA

Name: HUMAN IL-23 ANTIGEN BINDING PROTEINS.

Classification:

Inventor (s):

CIP: C07K16 / 24: A61K39 / 39§

CPC: C07K16 / 244; A6tK3p / S95¡ A61K2039 / 505; C07K2317 / 24; C07K2317 / 55;

C07K2317 / 76; GO7K ± 317/92; C07K2317 / 565; C07K2317 / 626

JENNIFER E. TQ.WNE; JANET D. CHENG; JASON C, O'NEILL; YU ZHANG; YU SUN 'HEATHER CERNE; DEREK E. JPÍPER; RAftJDAL R KETCHEM

REQUEST

Number:

MX / a / 2012/004868

International Presentation Date:

October 2010

<td>Country:</td><td>PRIORITY Fjkjfeiíi</td><td>Number:</td>

<td>US</td><td>October 26, 2009</td><td> 61/254,982</td>

<td>US</td><td>September 9, 2010</td><td> 61/381/287</td>

Validity: Twenty years

Expiration Date: October 26, 2030

Issue Date: From £ 201 agesto

The reference patent seigtqraa based on the anieblas 1 °, ^ frajt ^ Á V, & frái

Pursuant to article 28 of the Property Law, the PfflMie from the date of presenl

Hll, and rodela Industrial Property Law.

Article 28 of the Property Lawdjndrt ^ tja BfflW ^ e [* teMt twlfeq / Qe Agency of twenty international television stations, counted at the beginning of the international application and is $ 1 payment cf & Ufirita ^ ra rr ^ n «ner current tat rights.

Who subscribes the present title lo-tejd »: c» n foundation in the 'dls ^ ueste per tes ^ tidblaS 6 “fraceianee III and 7 ° tjis 2 of the Industrial Property Law (Official Gazette of the Federation (0.0 F. ) 08/27/1991 rearmed on 02.08.1994 »10/1996 12/26/1997, 05/17/1999 01/26/2004, 06/16/2005, 01/25/2006. 06/05 / 2009,06 / 01 / 2010,18 / 06 / 2810,28 / 06/2010, 27 / <A / 2012 9 09/04/2012 / articpkSs 1 °, 3 'fraction V subsection a), 4 ° and 12 ° sections I and III of the Regulations of the Mexican Institute dd I · PfOfMdíbd lodMtríat (3,0) = 14f1 »1999 ./(ormed on ΟΐΙ07 / 2ββ2, 07/15/2004, 07/28/2004 and 7 / 09/2007); articles 1, 3, 4. 5 'fraction V subsection a), t # factions I «.III and 30 ae + estetetefOrgánigp-deUnstitute' Mexteeno de la Propiedad Industrial (DOF 12/27/1999, amended on 10/10/2002, 29/0 / 12004,44 ^ 20 ^ 2 ^^ 8/2007): 1 ° ^ 'and St-lttCIsrart ^ t Actterdo that delegates powers to the Diiectores Generales Deputies, Coordinator, Directors Olvi ^ qnajes. Tofofares de las, Oftafas Regionales Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Instituto Mdxnteió di la-Proptedad lndU6triaí. (0 ^ 03 ^ 45/12/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

This document is signed with an advanced electronic signature (FIEL), based on articles 7 BIS 2 of the Industrial Property Law; 3 of its Regulations, and 1 fraction III, 2 fraction V, 26 BIS and 26 TER of the Agreement establishing the guidelines for the use of the Electronic Payment and Services Portal (PASE) of the Mexican Institute of Industrial Property, in the procedures indicated.

DIVISIONAL DIRECTOR OF PATENTS NAHANNY CANAL REYES

Original string.

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Digital stamp:

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Arenal No. '.iijO, Ι'Ί., Ο I, PuL-bio Santa María Tepeparn Xocbimícc ». 16020, Ciüii.ici tte Méxir r tete :) tete teO wvvvv gbb.h'ixúnipi

MX / 2018/67780

8X49

IL-2Í't ANTIGEN BINDING PROTEINS

WSTTTUTO MEXICAN PROPERTY

INDUSTRIAL

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Interleukin 23 (IL-23), a heterodimeric cytokine, is a potent inducer of proinflammatory cytokines. IL-23 is related to heterodimeric cytokine Interleukin 12 (IL-12) both to share a common p4 0 subunit. In IL-23, a single pl9 subunit is covalently linked to the p40 subunit. In IL-12, the single subunit is p35 (Oppmann et al., Immunity, 2000, 13:

713-715). IL-23 heterodimeric protein is secreted. Like IL-12, IL-23 is expressed by antigen presenting cells (such as dendritic cells and macrophages) in response to activation stimuli such as CD40 binding, Toll-like receptor agonists, and pathogens. IL-23 binds to the heterodimeric receptor comprising an IL-12RP1 subunit

ΙΜΡΪ «J1T1TOTO MEXICANO 'frt LA RRORIEDAD

INDUSTRIAL

(which is shared with the IL-12 receptor) and— «lid Single receptor lump, IL-23R. The IL-12 receptor consists of IL12Rpi and IL-12RP2. IL-23 binds its heterodimeric receptor and signals through JAK2 and Tyk2 to activate STAT1, 3, 4, and 5 (Parham et al., J. Immunol. 2002, 168: 5699-708). The receptor subunits are co-expressed predominantly in memory or activated T cells and natural eliminator cells and also at lower levels in dendritic cells, monocytes, macrophages, microglia, keratinocytes and synovial fibroblasts. IL-23 and IL-12 act on different T cell subsets and play substantially different roles in vivo.

IL-23 acts on memory and activated T cells and promotes the survival and expansion of the T cell subset, Thl7. Thl7 cells produce proinflammatory cytokines that include IL-6, IL-17, TNFa, IL-22, and GMCSF. IL-23 also acts on natural killing cells, dendritic cells, and macrophages to induce pro-inflammatory cytokine expression. Contrary to IL23, IL-12 induces differentiation of CD4 + T cells without experimenting in effector cells that produce IFNy, and induces NK and cytotoxic T cell function by stimulating IFNy production. Thl cells driven by IL-2 were previously thought to be the T cell subset

IMPI

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL pathogenic in many autoi enfermedadesτΉΤίΗ S diseases; -Slrr however, more recent animal studies in models of inflammatory bowel disease, psoriasis, inflammatory arthritis and multiple sclerosis, in which the individual contributions of IL-12 against IL-23 were evaluated, have firmly established that IL-23, not IL-12, is the key driver in autoimmune / inflammatory disease (Ahern et al., Immun. Rev. 2008 226: 147-159; Cua et al.,

Nature 2003 421: 744-748; Yago et al., Arthritis Res and Ther.

2007 9 (5): R96). IL-12 is considered to play a critical role in the development of innate and adaptive protective immune responses for many pathogens and intracellular viruses and in tumor immune surveillance. See

Kastelein, et al., Annual Review of Immunology, 2007, 25:

221-42; Liu, et al., Rheumatology, 2007, 46 (8): 1266-73;

Bowman et al., Current Opinion in Infectious Diseases, 2006

19: 245-52; Fieschi and Casanova, Eur. J. Immunol. 2003

33: 1461-4; Meeran et al., Mol. Ther cancer. 2006 5: 825-32;

Langowski et al., Nature 2006 442: 461-5. As such, a specific inhibition of IL-23 (which doses IL-12 or shares the p40 subunit) would have a potentially superior safety profile compared to double inhibition of

IL-12 and IL-23.

Therefore, the use of specific ILIMPI antagonists

MEXICAN INSTITUTE DS THE PROPERTY. INDUSTRIAL V

S 'that inhibit human IL-23 (such as antibodies that bind at least the single pl9 subunit or bind both of the pl9 and p40 subunits of IL-23) that dose IL-12 should provide efficacy equal to or greater than antagonists IL-12 or p40 antagonists without the potential risks associated with IL-12 inhibition. Humanized murine phage exhibiting antibodies selected for inhibition of recombinant IL-23 have been described; see for example US Patents

7,491,391, WIPO Publications WO1999 / 05280,

W02007 / 0244846, W02007 / 027714, WO 2007/076524, W02007 / 147019,

W02008 / 103473, WO2008 / 103432, W02009 / 043933 and W02009 / 082624.

However, there is a need for fully human therapeutic agents that are capable of inhibiting native human IL-23. Such therapeutics are highly target specific, particularly in vivo. Complete inhibition of the target in vivo can result in lower dose formulations, less frequent and / or more effective dosing which again results in reduced costs and increased efficiency. The present invention provides such IL-23 antagonists.

SUMMARY OF THE INVENTION

Antigen binding proteins are provided that bind IL-23, particularly native human IL-23. Proteins

IMPI Mexican institute

ΟΠ INDUSTRIAL PROPERTY

Binding to human IL-23 antigen can reduce, inhibit, 'interfere with, and / or modulate at least one of the biological responses related to IL-23, and as such are useful in lessening the effects of IL-related diseases or disorders -2. 3. Antigen binding proteins

IL-23 can be used, for example, to reduce, inhibit, interfere with and / or modulate IL-23 signaling, IL23 activation of Thl7 cells, IL-23 activation of NK cells, or induce the production of proinflammatory cytokines.

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

Human IL-23.

Some of the provided IL-23 binding antigen binding proteins comprise at least one heavy chain variable region comprising a CDRH1, a CDRH2 and a CDRH3 selected from the group consisting of: a CDRH1 that differs by no more of an amino acid substitution, insertion or deletion of a CDRHl as shown in

TABLE 3; a CDRH2 that differs by no more than three, two, or one amino acid substitutions, insertions, and / or deletions from a CDRH2 as shown in TABLE 3; a CDRH3 that differs by no more than three, two, or one substitutions,

ΙΜΡΙ

MEXICAN INSTITUTE BE LA ΜΓΙΙΊΕΟΛΟ

INOUSTAIAL

insertions and / or deletions of amino acids from a CDRH3 as shown in TABLE 3; and comprising at least one light chain variable region comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of: a CDRL1 that differs by no more than three, two, or one amino acid substitutions, insertions, and / or deletions of a CDRL1 as shown in TABLE 3 ;. a CDRL2 that differs by no more than one amino acid substitution, insertion, or deletion of a

CDRL2 as shown in TABLE 3; a CDRL3 that differs by no more than one amino acid substitution, insertion, or deletion of a CDRL3 as shown in TABLE 3. In one embodiment, isolated antigen binding proteins are provided comprising: a CDRH1 selected from the group consisting of SEQ ID NO: 91, 94, 97, 100, and 103; a CDRH2 selected from the group consisting of SEQ ID NO: 92, 95, 98,

101, 104, 107, and 110; a CDRH3 selected from the group consisting of SEQ ID NO: 93, 96, 99, 102, and 105; a CDRL1 selected from the group consisting of SEQ ID NO: 62, 65, 68, 71, and 74; a CDRL2 selected from the group consisting of SEQ ID NO: 63, 66, 69, 72, 75, and 78; and a CDRL3 selected from the group consisting of SEQ ID NO: 64, 67, 70 and 73. In another embodiment, isolated antigen binding protein is provided comprising: a CDRH1 selected from the group consisting of SEQ ID NO: 91, 106, 109, 112, and 115; a CDRH2 * ™r.’

IMPI

Mexican INSTITUTE

ΒΪ THE PROPERTY

INDUSTRIAL selected from the group consisting of SEQ ID NO: 113, 116, 118, 120, 121, and 122; a CDRH3 selected from the group consisting of SEQ ID NO: 108, 111, 114, 117, and 119; a CDRL1 selected from the group consisting of SEQ ID NO: 77, 80, 83,

85, 86, 87, 88, 89 and 90; a CDRL2 is SEQ ID NO: 81; and a

CDRL3 selected from the group consisting of SEQ ID NO: 76, 79, 82, and 84. In another embodiment, an isolated antigen binding protein is provided comprising at least one heavy chain variable region and at least one light chain variable region. . In yet another embodiment, an isolated antigen binding protein as described above is provided 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 tagged group.

Also provided are isolated antigen binding proteins binding IL-23 selected from the group consisting of a) an antigen binding protein having CDRH1 of

SEQ ID NO: 129, CDRH2 of SEQ ID NO: 132, CDRH3 of SEQ ID

NO: 136, and CDRL1 of SEQ ID NO: 123, CDRL2 of SEQ ID NO: 81, and

CDRL3 of SEQ ID NO: 76; b) an antigen binding protein having CDRH1 of SEQ ID NO: 131, CDRH2 of SEQ ID NO: 134, CDRH3 of SEQ ID NO: 137 and CDRL1 of SEQ ID NO: 124, CDRL2 of SEQ

ID Ν0126 and CDRL3 of SEQ ID NO: 128;

binding to the antigen having CDRH1 of SEQ ID NO: 130, CDRH2 of SEQ ID NO: 133, CDRH3 of SEQ ID NO: 99 and CDRL1 of SEQ ID NO: 68,

CDRL2. SEQ ID NO: 69, and CDRL3 of SEQ ID NO: 67; and d) an antigen binding protein having CDRH1 SEQ ID NO: 91,

CDRH2 SEQ ID NO: 135, CDRH3 SEQ ID NO: 138 and CDRL1 SEQ ID

NO: 125, CDRL2 SEQ ID NO: 127, and CDRL3 SEQ ID NO: 64.

Also provided are isolated antigen binding proteins binding 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 residues amino acids 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; a heavy chain variable region comprising amino acid residues 31-35, 50-65, and 99-1 10 of SEQ ID NO: 34 and heavy chain variable region comprising amino acid residues 31-35, 50-66, and 99- 110 of SEQ ID NO: 36; and a light chain variable region comprising amino acid residues 23-36, 52-62 and 97-105 of SEQ ID NO: 4; a heavy chain variable region comprising amino acid residues 31-35, 50-66 and 99-114 of SEQ ID NO: 38; and a light chain variable region comprising residues of

ΪΜΡΙ msTrrvro mexicana, '· de LA property t, INDUSTRIAL

amino acids 23-34, 50-61 and 94-106 of SEQ ID »0.7, Ulld lütjlW heavy chain variable comprising amino acid residues 31-35, 50-66 and 99-114 of SEQ ID NO: 40; and a light chain variable region comprising amino acid residues 24-34, 50-56 and 94-106 of SEQ ID NO: 9; a heavy chain variable region comprising amino acid residues 31-35, 50-66 and 99-114 of SEQ ID NO: 42; and a light chain variable region comprising amino acid residues 23-34, 50-61 and 94-106 of SEQ ID NO: 11; a heavy chain variable region comprising amino acid residues 31-35, 50-65 and 98-107 of SEQ ID NO: 44; and a light chain variable region comprising amino acid residues 24-34, 50-56 and 89-97 of SEQ ID NO: 13; a heavy chain variable region comprising amino acid residues 31-37, 52-67 and 100-109 of SEQ ID NO: 46 or SEQ ID

NO: 153; and a light chain variable region comprising amino acid residues 24-34., 50-56 and 89-97 of SEQ ID NO: 15;

a heavy chain variable region comprising amino acid residues 31-37, 52-67 and 100-109 of SEQ ID NO: 48; and a light chain variable region comprising amino acid residues 24-34, 50-56 and 89-97 of SEQ ID NO: 17; a heavy chain variable region comprising amino acid residues 31-37, 52-67 and 101-109 of SEQ ID NO: 50; and a light chain variable region comprising residues of • -'ί

IMP. ÍNsrrrirro Mexican 0Ε THE PROPERTY INDGSTklAI.

amino acids 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; a heavy chain variable region comprising amino acid residues 31-37, 52-67 and 100-109 of SEQ ID NO: 54; and a light chain variable region comprising amino acid residues 24-34, 50-56 and 89-97 of SEQ ID NO: 23; a heavy chain variable region comprising amino acid residues 31-37, 52-67 and 100-109 of SEQ ID NO: 56; and one

<td>variable region of</td><td>chain</td><td>light</td><td>than</td><td>understands</td><td>waste</td><td>of</td>

<td>amino acids 24-34,</td><td> 50-56</td><td>and 89-97</td><td>of</td><td colspan="2">SEQ ID NO: 25; and</td><td>a</td>

<td>variable region of</td><td>chain</td><td>heavy</td><td>than</td><td>understands</td><td>waste</td><td>of</td>

<td>amino acids 31-37,</td><td> 52-57</td><td colspan="2">and 100-109 of</td><td colspan="2">SEQ ID NO: 58; and</td><td>a</td>

<td>variable region of</td><td>chain</td><td>light</td><td>than</td><td>understands</td><td>waste</td><td>of</td>

<td>amino acids 24-34,</td><td colspan="4">500-56 and 89-97 of SEQ ID NO: 27</td><td> •</td><td></td>

<td>It is provided</td><td>in the</td><td>Present</td><td>a</td><td>protein</td><td>link</td><td>to the</td>

isolated IL-23 binding antigen comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence differs 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 from a variable region sequence of

IMPI

MEXICAN INSTITUTE OF PROPERTY. industrial

heavy chain as shown in TABLE 2; and wherein Ta light chain variable region sequence differs 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 of a light chain variable region sequence as shown in TABLE 1.

Also provided is an isolated antigen binding protein that binds to IL-23 selected from the group consisting of a) a heavy chain variable region of

SEQ ID NO: 140 and a light chain variable region of SEQ

ID NO: 30; b) a heavy chain variable region of SEQ ID

NO: 141 and a light chain variable region of SEQ ID

NO: 61; c) a heavy chain variable region of SEQ ID

NO: 142 and a light chain variable region of SEQ ID NO: 4;

and d) a heavy chain variable region of SEQ ID NO: 143 and a light chain variable region of SEQ ID NO: 139.

Also provided is an isolated antigen binding protein comprising a heavy chain variable region comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31, 34, 36, 38, -40, 42, 44, 46, 48, 50, 52, 54, 56, and 58; and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, 4, 7, 9, 11, 13, 15,

ΠΜ

MEXICAN PROPERTY WITCH

INDUSTRIAL

17, 19, 21, 23, 25 and 27. In another embodiment it is an isolated antigen binding protein comprising a heavy chain variable region selected from the group consisting of SEQ ID NO: 44, 46, 48, 50, 52, 54, 56, 58, and 153, and a light chain variable region selected from the group consisting of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, and 27. In yet another embodiment it is an isolated antigen binding protein comprising a heavy chain variable region selected from the group consisting of SEQ ID NO: 31, 34, 36, 38, 40 and 42, and a selected light chain variable region from the group consisting of SEQ ID NO: 1, 4, 7, 9 and 11.

Also provided is an isolated IL-23 binding antigen binding protein comprising a heavy chain variable region and a light chain variable region selected from the group consisting of: a) a region

<td>variable</td><td>of</td><td>chain</td><td>heavy</td><td>of</td><td>I KNOW THAT</td><td>ID</td><td>NO: 31</td><td>and</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>light</td><td>of</td><td>I KNOW THAT</td><td>ID</td><td>NO: 1;</td><td>b)</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td colspan="3">SEQ ID weighing</td><td>NOT:</td><td colspan="2">34 or 36 and</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>light</td><td>of</td><td>I KNOW THAT</td><td>ID</td><td>NO: 4;</td><td>c)</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>heavy</td><td>of</td><td>I KNOW THAT</td><td>ID</td><td>NO: 38</td><td>and</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>light</td><td>of</td><td colspan="3">SEQ ID NO: 7;</td><td>d)</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>heavy</td><td>of</td><td>I KNOW THAT</td><td>ID</td><td>NO: 40</td><td>and</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>light</td><td>of</td><td>I KNOW THAT</td><td>ID</td><td>NO: 9;</td><td>and)</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>heavy</td><td>of</td><td>I KNOW THAT</td><td>ID</td><td>NO: 42</td><td>and</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>light</td><td>of</td><td>I KNOW THAT :</td><td>[D NO: 11;</td><td>F)</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>heavy</td><td>of</td><td>I KNOW THAT</td><td>ID NO: 44</td><td>and</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>light</td><td>of</td><td>I KNOW THAT</td><td>ID NO: 13;</td><td>g)</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td colspan="5">SEQ ID NO: 46 or SEQ weighing</td><td colspan="2">ID NO: 153 and</td>

<td colspan="3">a variable region</td><td colspan="4">SEQ ID light chain</td><td>NOT</td><td> : 15;</td><td>h) one</td>

<td colspan="6">heavy chain variable region of</td><td colspan="2">SEQ ID NO: 48 and</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>light</td><td>of</td><td>I KNOW THAT</td><td>ID NO: 17;</td><td>i)</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>heavy</td><td>of</td><td>I KNOW THAT</td><td>ID NO: 50</td><td>and</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>light</td><td>of</td><td colspan="2">SEQ ID NO: 19;</td><td>j)</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>heavy</td><td>of</td><td>I KNOW THAT</td><td>ID NO: 52</td><td>and</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>light</td><td>of</td><td>I KNOW THAT</td><td>ID NO: 21;</td><td>k)</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>heavy</td><td>of</td><td>I KNOW THAT</td><td>ID NO: 54</td><td>and</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>light</td><td>of</td><td>I KNOW THAT</td><td>ID NO: 23;</td><td>i)</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>heavy</td><td>of</td><td>I KNOW THAT</td><td>ID NO: 56</td><td>and</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td colspan="4">light of SEQ ID NO: 25; and</td><td>m)</td><td>a</td><td>region</td>

<td>variable</td><td>of</td><td>chain</td><td>heavy</td><td>of</td><td>I KNOW THAT</td><td>ID NO: 58</td><td>and</td><td>a</td><td>region</td>

SEQ ID NO: 27 light chain variable.

A binding protein is also provided to the

<td>antigen</td><td>isolated bonding</td><td>to human IL-23,</td><td>in</td><td>where</td><td>the</td>

<td colspan="3">covered patch formed when protein</td><td>of </td><td>link</td><td>to the</td>

<td>antigen</td><td>binds to IL-23</td><td>human understands</td><td colspan="2">contacts</td><td>of</td>

<td>residue</td><td> 30, 31, 32, 49, 50,</td><td>52, 53, 56, 92 and</td><td> 94</td><td>from SEQ</td><td>ID</td>

NO: 15, where the residual contacts have a difference value greater than or equal to 10 A2 as determined by the

IMPI

surface area exposed to. solvent

Modality residue contacts comprise residues 31-35, 54, 58-60, 66, and 101-105 of SEQ ID NO: 46.

Also provided is an isolated antigen binding protein that binds to human IL-23, wherein the covered patch formed when the antigen binding protein binds to human IL-23 comprises residue contacts 31-34, 51, 52, 55, 68, 93 and 98 of SEQ ID NO: 1, where the residual contacts have a difference value greater than or equal to 10 A2 as determined by the surface area exposed to the solvent. Within one embodiment the residue contacts comprise residues 1, 26, 28, 31,

32, 52, 53, 59, 76, 101, 102 and 104-108 of SEQ ID NO: 31.

An isolated antigen binding protein that binds to human IL-23 is also provided, wherein when the antigen binding protein binds to human IL-23, the antigen binding protein is 5A or less from residues 32-35, 54, 58-60, 66 and 101-105 of SEQ ID

NO: 46, as determined by X-ray crystallography. In one embodiment the antigen binding protein is 5A or less from residues 31-35, 54, 56, 58-60, 66 and

101-105 of SEQ ID NO: 46.

An isolated antigen binding protein that binds to human IL-23 is also provided, where when

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OF INDUSTRIAL PROPERTY the antigen binding protein is human pnl ^ aa tt.-73, the antigen binding protein is 5A or less from residues 30-32, 49, 52,. 53, 91-94 and 96 of SEQ ID NO: 15, as determined by X-ray crystallography. In one embodiment the antigen binding protein is 5A or less from residues 30-32, 49, 50 , 52, 53, 56, 9194 and 96 of SEQ ID NO: 15.

NO: 31, as determined by X-ray crystallography. In one embodiment the antigen binding protein is 5A or less from residues 1, 26-28, 30-32, 52, 53, 59,

100, and 102-108 of SEQ ID NO: 31.

An isolated antigen binding protein that binds to human IL-23 is also provided, wherein when said antigen binding protein binds to human IL-23, said antigen binding protein is 5A or less from residues 31-34, 51, 52, 55, 68 and 93 of SEQ ID N0: 1 as determined by X-ray crystallography. In one embodiment the antigen binding protein is 5A or less from the residues 29, 31-34, 51, 52, 55, 68, and 100 of SEQ ID NO: 1.

% i ·.

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An isolated antigen binding protein is also provided 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, an F (ab') 2 fragment, an Fv fragment, a diabody, or an antibody molecule. single string. 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 IgGl, IgG2, IgG3 or IgG4 type. In yet another embodiment an isolated antigen binding protein is provided wherein the antigen binding protein is of the IgGl or IgG2 type.

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A nucleic acid molecule isolated qilfe encodes uircr antigen binding protein as described above, 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 SEQ ID NOs: 32, 35, 37, 39, 41, . 43, 45, 47, 49, 51, 53, 55,

57, 59, and 152 and at least one light chain variable region is encoded by an isolated nucleic acid molecule selected from the group consisting of SEQ ID N0s: 2, 5, 6, 8,

<td> 10, 12, 14, 16, 18,</td><td> 20, 22,</td><td> 24,</td><td>26, and</td><td>28. In another</td><td>modality</td>

<td>a</td><td>molecule</td><td>of</td><td>acid</td><td>nucleic in</td><td>where the</td>

<td>acid molecule</td><td>nucleic</td><td>I know</td><td>league</td><td colspan="2">operably to a</td>

control sequence. In another embodiment, a vector is provided comprising a nucleic acid molecule as described above. In yet another embodiment, a host cell is provided comprising the nucleic acid molecule as described above. In another embodiment, a host cell comprising the vector described above is provided. In yet another embodiment, there is provided an isolated polynucleotide sufficient to use as a hybridization probe, PCR primer, or sequence-forming primer that is a fragment of the nucleic acid molecule as described above or its complement.

Antigen binding is also provided as the step of preparing said from a host cell that secretes said antigen binding protein.

Also provided is an isolated antigen binding protein that binds to human IL-23, wherein the covered patch formed when the antigen binding protein binds to human IL-23 comprises a residue contact with residues 46-58, a residue contact with residues 112-120, and a residue contact with residues 155-163 of the human IL-23pl9 subunit as described in SEQ ID NO: 145, where the residual contact has a difference value greater than or equal to 10A2 as determined by the surface area exposed to the solvent. In one embodiment it is provided wherein the covered patch formed when the antigen binding protein binds to

Human IL-23 comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, · twelve or thirteen residue contacts within residues 46-58, one, two, three, four, five, six, seven, eight, nine, or ten residue contacts within residues 112-120, and one, two, three, four, five, six, seven, eight, or nine residue contacts within residues 155-163 of the human IL-23pl9 subunit as «Μ | Μ.

It is described in SEQ ID NO: 145. In oWd niuüülid'ael 1 or provided where the covered patch formed when the antigen-binding protein binds to human IL-23 comprises a residue contact with residues 121-125 of the human IL-23p40 subunit as described in SEQ ID

NO: 147. In a related embodiment where the covered patch formed when the antigen binding protein binds to human IL-23 comprises one, two, three, four, or five residue contacts within residues 121-125 of the IL-23p40 subunit human as described in SEQ ID NO: 147.

Within another embodiment is provided wherein the covered patch formed when the antigen binding protein binds to human IL-23 comprises residue contacts 46, 47,

49, 50, 53, 112-116, 118, 120, 155, 156, 159, 160, and 163 of

SEQ ID NO: 145. In another embodiment it is provided wherein the covered patch formed when the binding protein to

<td>antigen is</td><td>binds</td><td>to human IL-23</td><td>understands</td><td>contacts of</td>

<td>residue 46,</td><td> 47, 49,</td><td> 50, 53, 112-118,</td><td> 120, 155,</td><td> 156, 159, 160,</td>

<td>and 163 of</td><td>SEQ ID</td><td>NO: 145. Inside</td><td>of other</td><td>modality is</td>

<td>provides</td><td colspan="4">where the covered patch formed when the</td>

Antigen binding protein binds 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 it is provided where the patch

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FROM THE i-INDUSTRIAL PROPERTY shed formed when the eTTiaffe protein to the antigen binds to human IL-23 comprises contact of residue 122 of the human IL-23p40 subunit as described in SEQ ID NO: 147.

In another related embodiment it is provided wherein the covered patch formed when the antigen binding protein binds to human IL-23 comprises residue contacts 122 and 124 of the human IL-23p40 subunit as described in SEQ ID NO: 147. Still another related embodiment is provided wherein the covered patch formed when the antigen binding protein binds to human IL-23 comprises residue contact 121-123 and 125 of the subunit

Human IL-23p40 as described in SEQ ID NO: 147. In a further related embodiment is provided wherein the covered patch formed. When the antigen binding protein binds to human IL-23 comprises residue contact 121-123, 125 and 283 of the human IL-23p40 subunit as described in SEQ ID NO: 147.

An isolated antigen binding protein that binds to human IL-23 is also provided, wherein when said antigen binding protein binds to human IL-23 said antigen binding protein is 5A or less from a residue within residues 46-58, from a residue within residues 112-123, and from a residue within residues 155-163 of the IL21 subunit

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Human 23pl9 as described in SEQ IT) NO: 145 '; - C0111U sv determined by X-ray crystallography. In one embodiment, when the antigen binding protein binds to human IL-23, the antigen binding protein is 5 to or less from one, two, three, four, 'five, six, seven, eight, nine, ten, eleven, twelve or thirteen residues within residues 46-58, from one, two, three, four, five, six, seven, eight, nine or ten, waste within waste

112-123, and from one, two, three, four, five, six, seven, eight, or nine residues. Within residues 155-163 of the human IL-23pl9 subunit as described in SEQ ID NO: 145.

Within another embodiment when the antigen binding protein binds to human IL-23 the antigen binding protein is 5A or less from residues 46-50,

113-116, 120, 156, 159, 160 and 163 'of SEQ ID NO: 145. Within another embodiment when the antigen binding protein binds to human IL-23, the antigen binding protein is 5A or less from residues 46-50, 112-120, 156,

159, 160 and 163 of SEQ ID NO: 145. Within a related modality when the. antigen binding protein binds to human IL-23, antigen binding protein is 5A or less from residues 46-50, 53, 112-120, 156, 159, 160 and 163 of SEQ ID NO: 145. Within another embodiment when the antigen binding protein binds

to human IL-23, the antigen binding protein is 5A or less from residues 46-50, 53-55, 58, 113-116,

120, 121, 156, 159, 160, 162 and 163 of SEQ ID NO: 145. Within a related embodiment when the antigen binding protein binds to human IL-23, the antigen binding protein is 5A or less from residues 46-51, 53-55, 57, 58, 112 -116, 118-121, 123, 155, 156, 159, 160, 162 and 163 of SEQ ID NO: 145. Within a further embodiment when the antigen binding protein binds to human IL-23 the antigen binding protein is 5A or less from a residue within residues 121-125, of the IL-23p40 subunit human as described in SEQ ID NO: 147, as determined by X-ray crystallography. With a related embodiment when the antigen binding protein binds to human IL-23, said antigen binding protein is 5A or less from residues 122 and

124 of SEQ ID NO: 147. Within another embodiment when the antigen binding protein binds to human IL-23, the antigen binding protein is 5A or less from residues 121-123 and 125 of SEQ ID NO: 147.

An isolated antigen binding protein is also provided as described above, wherein the antigen binding protein has at least one property selected from the group consisting of: a) reducing the

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human IL-23 activity; b) reduce the production of a proinflammatory cytokine; c) bind human IL-23 with a KD of <5x10-8 M; d) that it has a Koff ratio of <5x10-6 1 / s; and d) that it has an IC50 of <400 pM.

A pharmaceutical composition comprising at least one antigen binding protein as described above and a pharmaceutically acceptable excipient is provided. In one embodiment, a pharmaceutical composition is provided which further comprises a labeled group or an effector group. In yet another embodiment, a pharmaceutical composition is provided wherein the labeled group is selected from the group consisting of isotopic labels, magnetic labels, active oxidation reduction portions, optical pigments, biotinylated groups, and predetermined polypeptide epitopes recognized by a secondary reporter. In yet another embodiment, a pharmaceutical composition is provided wherein the effector group is selected from the group consisting of a radioisotope, radionuclide, a toxin, a therapeutic group, and a chemotherapeutic group.

Also provided is a method of treating or preventing a condition associated with IL-23 in a patient, comprising administering to a patient, who requires an effective amount of at least one isolated antigen binding protein therefrom as described above. In one modality,

provides a method wherein the condition is selected from the group consisting of an inflammatory disorder, a rheumatic disorder, an immune disorder, an oncological disorder, and a gastrointestinal disorder. In yet another embodiment, a method is 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, myocarditis autoimmune; type 1 diabetes and ankylosing spondylitis. In yet another embodiment a method is provided wherein the isolated antigen binding protein is administered alone or as a combination therapy.

A method of reducing activity is also provided.

IL-23 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 said IL23 activity induces the production of a proinflammatory cytokine.

SHORT DESCRIPTION PEIAS FIGURES

FIGURE IA: Results of the luciferase STAT-reporter assay using recombinant human IL-23. All antibodies completely inhibit recombinant human IL-23

FIGURE IB: Results of the luciferase assay using native human IL-23. Only half of those antibodies that completely inhibit recombinant human IL-23 were able to completely inhibit native human IL-23.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions, kits, and methods related to antigen binding proteins.

IL-23, including IL-23 antagonizing molecules, such as anti-IL-23 antibodies, antibody fragments, and antibody derivatives, eg, 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 a portion of an IL-23 binding polypeptide, eg, a polynucleotide encoding all or part of an anti-antibody. -IL-23, antibody fragment, or derivative of antibody, plasmids and vectors comprising such nucleic acids, and cells or cell lines comprising such polynucleotides and / or vectors and plasmids. The methods provided include, for example, methods to make, INst”^ M« icano industsito the

identify or isolate IL23 antigen binding proteins, such as anti-IL-23 antibodies, methods of determining whether a molecule binds to IL-23, methods of determining whether a molecule antagonizes IL-23, methods of making compositions, such as pharmaceutical compositions, comprising an IL-23 antigen binding protein, and methods of administering an antigen binding protein

IL-23 to a subject, for example, methods for treating an IL-23 mediated condition, and for antagonizing a biological activity of IL-23, in vivo or in vitro.

Unless otherwise defined, the scientific and technical terms used in connection with the present invention will have the meanings commonly understood by those of ordinary skill in the art. Also, unless otherwise required by context, singular terms will include pluralities and plural terms will include the singular. Generally, the nomenclatures used in connection with, and techniques for, cell and tissue culture, molecular biology, immunobiology, microbiology, genetics, and nucleic acid chemistry and protein chemistry and hybridization described herein are those well known and commonly used in the art. . The methods and techniques of the present invention are generally performed in accordance with conventional methods either

IΜ Ρ ί

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OF THE PROPERTY *' ' ·"

INDUSTRIAL known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3to ed. , Coid Spring Harbor Laboratory Press, Coid Spring Harbor, NY (2001) and Ausubel et al., Current Protocols in Molecular Biology,

Greene Publishing Associates (1992), and Harlow and Lañe

Antibodies: A Laboratory Manual Coid Spring Harbor Laboratory

Press, Coid Spring Harbor, NY (1990). Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, as is commonly performed in the art, or as described herein.

The terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

All patents and other identified publications are expressly incorporated herein by reference in their entirety for the purposes of description and disclosure,

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for example, the methodologies desori tai feale »publications that can be used in connection with the information described herein.

The polynucleotide and protein sequences of the human IL-23 pl9 subunit (SEQ ID NOs: 144 and 145), the shared p40 subunit (SEQ ID NOs: 146 and 147), the heterodimeric subunits of the human IL12RP1 receptor ( SEQ ID NOs: 150 and 151) and IL-23R (SEQ ID NOs: 148 and 149), are known in the art, see for example, GenBank Accession Nos. AB030000; M65272, NM_005535, NM_144701, as are those of other mammalian species. Recombinant IL-23 and IL-23 receptor proteins including Ec and single chain proteins as well as cells expressing the IL-23 receptor have been described or are available from commercial sources, (see for example, Oppmann et al., Immunity, 2000, 13: 713-715; R&D Systems,

Minneapolis. Minnesota; United States Biological, Swampscott,

Massachusetts; WIPO Publication No. WO 2007/076524). Native human IL-23 can be obtained from human cells such as dendritic cells using methods known in the art including those described herein.

IL-23 is a heterodimeric cytokine that is comprised of a single pl9 subunit that covalently binds to a shared p40 subunit. The subunit

pl9 comprises four helices a, “A, B, and D an · one up-up-down-down portion joined by three intra helix curls between helices A and B, between helices B and C, and between helices C and D, see Oppmann et al., Immunity, 2000, 13: 713-715 and Beyer, et al., J Mol Biol, 2008. 382 (4):

942-55. The 4-helical bundle 4-cytokine A and D helices are considered to be involved with receptor binding. The p40 subunit comprises three interspersed beta-sheet domains Di, D2, and D3 (Lupardus and García,

J. Mol. Biol., 2008, 382: 931-941.

The term "polynucleotide includes both single-stranded and double-stranded nucleic acids and includes genomic DNA, RNA, mRNA, cDNA, or synthetic origin or some combination thereof that is not associated with sequences normally found in nature. Isolated polynucleotides comprising specific sequences may include, in addition to the specific sequences, coding sequences for up to ten or even up to twenty of other proteins or portions thereof, or may include operably linked regulatory sequences that control the expression of the region of encoding the recited nucleic acid sequences, and / or may include vector sequences. The nucleotides that comprise the polynucleotide can be ribonucleotides or deoxyribonucleotides or a

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OF THE PROPERTY _ , . _. ,,, INDUSTRIAL modified form of any type of nucleotide. Modifications include basetal modifications such as bromouridine and inosine derivatives, ribose modifications such as 2 ', 3'-dideoxyiribose, and internucleotide ligation modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphorousanylioate, phosphoranylate and phosphorous amidate.

The term oligonucleotide means a polynucleotide that comprises 100 or more nucleotides. In some embodiments, the oligonucleotides are 10 to 60 bases in length. In other embodiments, the oligonucleotides are 12, 13, 14,

15, 16, 17, 18, 19, or 20 to 40 nucleotides in length.

The oligonucleotides can be single-stranded or double-stranded, for example, for use in constructing a mutant gene. The oligonucleotides can be sense or antisense oligonucleotides. An oligonucleotide can include a detectable tag, such as a radio tag, a fluorescent tag, a hapten, or an antigen tag, for detection assays. Oligonucleotides can be used, for example, as PCR primers, cloning primers, or hybridization probes.

The terms polypeptide or protein mean a macromolecule that has the amino acid sequence of a native protein, that is, a protein produced by a

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naturally-occurring, nonraigit-like cell1· Or ggr produced by a genetically engineered or recombinant cell, and comprises molecules having the amino acid sequence of the native protein, or molecules having one or more deletions of, insertions to, and / or substitutions of amino acid residues of the native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogues of amino acids and. corresponding naturally occurring polymers. The terms polypeptide and protein encompass IL-23 antigen binding proteins (such as antibodies) and sequences that have one or more deletions of, additions to, and / or substitutions of the amino acid residues of the binding protein sequence to the antigen. The term "polypeptide fragment" refers to a polypeptide that has an amino terminal deletion, a carboxyl terminal deletion, and / or an internal deletion compared to the full length native protein. Such fragments can also contain modified amino acids compared to the native protein. In certain embodiments, the fragments are about five to 500 amino acids in length. For example, the fragments can be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 70, 100, 110,

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150, 200, 250, 300, 350, 400, or 450 amino acids of inngit-nH. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of an IL-23 antigen binding protein, such as. an antibody, useful fragments include but are not limited to one or more CDR regions, a variable domain of a heavy or light chain, a portion of an antibody chain, a portion of a variable region that includes less than three CDRs, and Similar.

Amino acid includes its normal meaning in the art. The twenty naturally occurring amino acids and their abbreviations follow conventional use. See,

Immunology-A Synthesis, 2to Edition, (ES Golub and DR

Gren, eds.), Sinauer Associates: Sunderland, Mass. (1991).

Stereoisomers (eg, D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as [alpha] - amino acids, disubstituted with [alpha] -, N-alkyl amino acids, and other unconventional amino acids may also be appropriate components for polypeptides . Examples of unconventional amino acids include: 4hydroxyproline, [gamma] -carboxyglutamate, [epsilon] -N, N, Ntrimethillisine, [epsilon] -N-acetyllisine, O-phosphoserine, Nacetyl Serine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, [sigma] -N-methylarginine, and other similar amino acids and imino acids (eg 4-hydroxyproline). In the

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annotation of the polypeptide 'used herein, the direction on the left side is the amino terminal address and the direction on the right side is the carboxyl terminal direction, in accordance with standard and conventional use.

The term "isolated protein" refers to a protein, such as an antigen binding protein (an example of which could be an antibody), that is purified from proteins or polypeptides or other contaminants that could interfere with its therapeutic, diagnostic use , research or other. As used herein, "substantially pure" means that the described species of molecule is the predominant species present, that is, on a molar basis it is more abundant than any other individual species in the same mixture. In certain embodiments, a substantially pure molecule is a composition where the target species comprises at least 50% (on a molar basis) of all the macromolar species present. In other embodiments, a substantially pure composition will comprise at least 80%,

85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In certain modalities, a homogeneous substance has been purified to such a degree that the contaminating species is not detected in the composition by conventional detection methods and thus the

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Composition consists of a simple clétectable macromolecular species.

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

The terms naturally occurring or native as used throughout the specification in connection with biological materials such as polypeptides, nucleic acids, host cells, and the like, refers to materials found in nature, such as native human IL23. . In certain aspects, the recombinant antigen binding proteins that bind native IL-23 are provided. In this context, a recombinant protein is a protein made using recombinant techniques, that is, through the expression of a recombinant nucleic acid as described herein. The 'methods and techniques for the production of recombinant proteins are well known in

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INDUSTRIAL technique.

The term "antibody" refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antibody, and includes, for example, fully humanized, humanized, chimeric and bispecific antibodies. An antibody as such is a kind of an antigen binding protein. 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 described below. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains such as naturally occurring antibodies in camelids that may comprise only heavy chains. Antibodies can be derived from a single source only, or can be chimeric, that is, different portions of the antibody can be derived from two different antibodies as further described below. Antigen binding proteins, antibodies, or binding fragments can be produced in hybridomas, by DNA techniques.

recombinant, or by enzymatic or chemical cleavage of intact antibodies.

The term functional fragment (or simply fragment) of an antibody or immunoglobulin chain (heavy or light chain), as used herein, is an antigen binding protein that comprises a portion (regardless of which portion is obtained or synthesizes) of an antibody lacking al. less than some of the amino acids present in a full-length chain but capable of specifically binding an antigen. Such fragments are biologically active in that they specifically bind to a target antigen and can compete with other antigen-binding proteins, including intact antibodies, for specific binding to a given epitope. In one aspect, such a fragment will maintain at least one CDR present in the full-length light or heavy chain, and in some embodiments will comprise a single heavy chain 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, which include intact antibodies. Fragments include, but are not limited to, immunologically functional fragments such as Fab, Fab ', F (ab') 2, Fv, domain antibodies, and

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single chain, and may be derived from mammalian line ctwlqui'Ul 1, including but not limited to human, mouse, rat, camelid or rabbit. It is further contemplated that a functional portion of the antigen binding proteins described herein, eg, one or more CDRs, could be covalently linked to a second protein or a small molecule to create a therapeutic agent targeting a particular target in the body, which possesses bifunctional therapeutic properties, or which has a long serum half-life.

The term "compete" when used in the context of antigen binding proteins (eg, neutralize antigen binding proteins or neutralize antibodies) means competition between antigen binding proteins as determined by an assay in which the protein binding to the antigen (for example, antibody or immunologically functional fragment thereof) under test prevents or inhibits the specific binding of a binding protein to the reference antigen '(eg, a ligand, or a reference antibody) to a common antigen (eg, an IL protein -23 or a fragment thereof). Numerous types of competitive binding assays can be used, for example: direct or indirect solid phase radioimmunoassay (RIA), direct or indirect enzyme immunoassay

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solid phase (EIA), interleaved competition assay (see, eg, Stahli et al., 1983, Methods in Enzymology 92: 242-253); Direct solid phase biotin-avidin EIA (see, eg, Kirkland et al., 1986, J. Immunol. 5 137: 3614-3619) direct solid phase labeling assay, direct phase labeling intercalation assay solid (see, for example, Harlow and Lañe, 1988, Antibodies, A

Laboratory Manual, Coid Spring Harbor Press); Solid phase direct tag RIA using tag 1-125 (see, eg, Morel et al., 1988, Molec. Immunol. 25: 7-15); Direct solid phase 'biotin-avidin EIA (see, for example,

Cheung, et al., 1990, Virology 176: 546-552); and direct tagged RIA (Moldenhauer et al., 1990, Scand. J.

Immunol. 32: 77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an undetectable test antigen binding protein, and a labeled reference antigen binding protein.

Competitive inhibition is measured by determining the amount of tag bound to the solid surface in the presence of the test antigen binding protein. Usually the test antigen binding protein is over-presented. The antigen binding proteins identified by the competition assay ('INDUSTRIAL proteins

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antigen binding competition) include antigen binding proteins that bind the same epitope as the reference antigen binding proteins and antigen binding proteins that bind an adjacent epitope sufficiently close to the epitope bound by the antigen binding protein of reference to present the spherical obstruction. Usually when the binding protein to the competition antigen is over-presented. This will inhibit the specific binding of a binding protein to the reference antigen to a common antigen by at least 40%,

45%, 50%, 55%, 60%, 65%, 70% or 75%. In some case, 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 from both contiguous and non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically maintained on exposure to denatured solvent, while epitopes formed by tertiary folding are typically lost in treatment with denatured solvents. Epitope determinants can include chemically active surface groupings of molecules

IMPI

INSTITUTO MEXICANO oe LA TOOPIF.OAO industrial

such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have three-dimensional structural characteristics, and / or specific charge characteristics. An epitope typically includes 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 using methods known in the art.

IL-23 -antigen binding proteins

An antigen binding protein as used herein means a protein that specifically binds a specified target antigen; the antigen as provided herein is IL-23, particularly human IL-23, including native human IL-23. Antigen binding proteins as provided herein interact with at least a portion of the unique pl9 subunit of IL-23, detectably linked IL-23; but not attached with any importance to IL-12 (eg IL-12 p40 and / or p35 subunits), thereby economical IL-12. As a consequence, the antigen binding proteins provided herein are capable of impacting IL-23 activity without the potential risks that inhibition of IL-12 or the shared p40 subunit may

3:¾

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incurring. Antigen binding proteins can impact the ability of IL-23 to interact with its receptor, for example by impacting receptor binding, such as by interfering with receptor association. In particular, such antigen binding proteins reduce, inhibit, interfere with, or totally or partially modulate one or more biological activities of IL-23. Such inhibition or neutralization disrupts the biological response in the presence of an antigen binding protein compared to the response in the absence of the antigen binding protein and can be determined using assays known in the art and described herein. The antigen-binding proteins provided herein inhibit IL-23-induced proinflammatory cytokine production, for example IL-23-induced IL-22 production in whole blood cells and IL-23-induced IFNy expression in NK cells and whole blood cells. The reduction in biological activity can be around 20%, 30%, 40%, 50%, 60%,

70i

80‘

90‘

92%, 93%, 94%, 95%, 96%, 97Í

98!

9 % or more.

An antigen binding protein may comprise an antigen-binding portion and, optionally, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the

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antigen binding protein to antigen. Examples of antigen binding proteins include antibodies, antibody fragments, (eg, an antigen binding portion of an antibody), 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 introduced mutations to, for example, stabilize the three-dimensional structure of the antigen-binding protein as well as fully synthetic scaffolds, for example, a biocompatible polymer. See, for example, Korndorfer et al., Proteins:

Structure, Function, and Bioinformatics, (2003) Volume 53,

Publication 1: 121-129; Roque et al., Biotechnol. Prog., 2004, 20: 639-654. In addition, peptide antibody mimics (PAMs) can be used, as well as scaffolds based on antibody mimics that utilize fiber link components as a scaffold.

Certain antigen binding proteins described herein are antibodies or are derived from antibodies. Such antigen binding proteins include, but are not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies,

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OF INDUSTRIAL TFTOHcTY

antibody mimics, 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 (eg, a Fab, a Fab ', an F (ab') 2, or a scFv). The various structures are further described and defined herein.

<td>Certain proteins of</td><td>. link</td><td>to the</td><td>antigen</td><td>than</td><td>I know</td>

<td colspan="2">provide can understand one</td><td>or</td><td>more CDRs</td><td>how</td><td>I know</td>

<td>described herein (for</td><td>example,</td><td> 1,</td><td> 2, 3, 4, 5,</td><td>6th</td><td>plus</td>

<td>CDRs). In some cases, the</td><td>protein</td><td>of</td><td>link to</td><td colspan="2">antigen</td>

it comprises (a) a polypeptide structure and (b) one or more

CDRs that are inserted into and / or attached to the polypeptide structure. The polypeptide structure can take a variety of different forms. For example, it may be, or comprise, the structure of the naturally occurring antibody, or fragment or variant thereof, or it may be fully synthetic in nature. Examples of various polypeptide structures are further described below.

An antigen binding protein of the invention is said to specifically bind its target antibody when the dissociation equilibrium constant (KD) is á 10-8 M. The antigen binding protein specifically binds, f <· »ί ·· ·· - ~ ;: '-'-. Τΐ> -i antigens with high affinity, when the KD is i 5 x 10-9 M, and iMm

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY with very high affinity when the KD is <5 x 10-10 M. In one embodiment, the antigen-binding protein would bind to human IL-23 with a KD of 5 x 10-12 M, and in yet another embodiment it will bind with a KD of <5 x 10-13 M. In another embodiment of the invention, the antigen binding protein has a KD of x 10-12 M and a Koff of about <5x10- 6 1 / s. In another embodiment, the Koff is <5xl0-71 / s.

Another aspect provides an antigen binding protein that has a half-life of at least one day in vitro or in vivo (eg, 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 four days or longer. In another embodiment, the antibody or portion thereof has a half-life of eight days or longer. In another embodiment, the antibody or antigen-binding portion thereof is derived or modified such that it has a longer half-life compared to the non-derived or unmodified antibody. In another embodiment, the antigen-binding protein contains point mutations to increase serum half-life, as described in WIPO Publication No. WO 00/09560.

In modalities where the antigen binding protein is

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used for therapeutic applications, a protein with antigen binding can reduce, inhibit, interfere with, or modulate one or more biological activities of IL-23, such as inducing the production of proinflammatory cytokines. IL-23 has many different biological effects, which can be measured in many different assays on different cell types; Examples of such tests are known and provided herein.

Some of the provided antigen binding proteins have the structure typically associated with naturally occurring antibodies. The structural units of these antibodies typically comprise one or more tetramers, each consisting of two identical polypeptide chain coupled, although some mammalian species also produce antibodies that have only a single heavy chain. In a typical antibody, each pair of couplings includes a full-length light chain (in certain embodiments, about 25 kDa) and a full-length heavy chain (in certain embodiments, around 50-70 kDa). Each individual immunoglobulin chain is composed of several immunoglobulin domains, each consisting of almost 90 to 110 amino acids and expressing a characteristic folding pattern. These domains are the basic units of which the antibody polypeptides are composed. The amino terminal portion of each rt chain

MEXICAN INSTITUTE

OWNERSHIP O »J« 2sL, g INDUSTRIAL typically includes a variable region that is responsible for antigen recognition. The carboxy terminal portion is evolutionarily more conservative than the other end of the chain and is referred to as the constant region or C region.

Human light chains are generally classified as kappa and lambda light chains, each containing a variable region and a constant domain (CLl) .z Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon chains. , and these define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subtypes, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM subtypes include IgM, and IgM2. IgA subtypes include IgAl and IgA2. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and 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 function. The number of heavy chain constant region domains will depend on the isotype. IgG heavy chains, for example, each contain three CH region domains known as CH1, CH2, and CH3. The antibodies provided can have any of these isotypes and subtypes, for example the

47.

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Mexican Institute of Industrial Property

IL-23 antigen binding protein is from the süTlLipU lijOl, IgCSy. or IgG4. If an IgG4 is desired, it may also be desired to introduce a point mutation (CPSCP-> CPPCP) in the hinge region as described in Bloom et al., 1997, Protein Science 6: 407) to alleviate a tendency to form junctions. intra H chain disulfide that can lead to heterogeneity in IgG4 antibodies. Antibodies provided herein that are of one type can be changed to a different type using subclass switching methods. See, for example, Lantto et al., 2002, Methods Mol. Biol. 178: 303-316.

In full-length light and heavy chains, the variable and constant regions are linked by a J region of about twelve or more amino acids, with the heavy chain also including a D region of about ten more amino acids. See, for example, Fundamental Immunology, 2to ed., Cap. 7 (Paul, W., ed.) 1989, New York: Raven Press. The variable regions of each light / heavy chain pair typically form the antigen binding site.

Variable Regions

Various heavy chain and light chain variable regions (or domains) provided herein are described in TABLES 1 and 2. Each of these variable regions can be linked, for example, to heavy chain constant regions.

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and light described before. In addition, each heavy and light chain dg * nm .οηοη ^ · '^ 5 thus generated can combine to form a complete antigen binding protein structure.

Antigen binding proteins are provided that contain at least one heavy chain variable region (VH) selected from the group consisting of VH1, VH2, VH3, VH4, VH5,

VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14, VH15, and VH16 and / or at least one light chain variable region (VL) selected from the group consisting of VL1, VL2, VL3, VL4, VL5 ,

VL6, VL7, VL8, VL9, VL10, VL11, VL12, VL13, VL14, VL15, and VL16 as shown in TABLES 1 and 2 below.

Each of the heavy chain variable regions listed in TABLE 2 can combine with any of the light chain variable regions shown 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 different light chain variable regions from those listed in TABLES 1 and 2. The various combinations of heavy chain variable regions can be combined with any of tirj-rnrr · various combinations of light chain variable regions,

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In other cases, the antigen binding protein contains two light chain variable regions and / or two identical heavy chain variable regions. As an example, the antigen binding protein may be an immunologically functional antibody or fragment comprising two light chain variable regions and two heavy chain variable regions in combinations of pairs of light chain variable regions and pairs of variable chain regions. heavy as listed in TABLES 1 and 2. Examples of such antigen binding proteins comprising two identical heavy chains and light chain variable regions include: Antibody A VH14 / VL14; B antibody VH9 / VL9;

VH10 / VL10 C antibody; D antibody VH15 / VL15; Antibody E VH1 / VL1, Antibody F VHll / VL11; G VH12 / VL12 antibody; H antibody VH13 / VL13; Antibody I VH8 / VL8; J VH3 / VL3 antibody; K VH7 / VL7 antibody; L VH4 / VL4 antibody; M VH5 / VL5 Antibody and N VH6 / VL6 Antibody.

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 the sequence of a 'heavy chain variable region and / or a variable region light chain selected from TABLES 1 and 2 in only 1, 2, 3, Mexican institute OF INDUSTRIAL PROPERTY

4/5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, where each such sequence difference is independently either an amino acid deletion, insertion, or substitution. The heavy and light chain variable regions, in some antigen binding proteins, 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.

Still other antigen binding proteins, eg, antibodies or immunologically functional fragments, also include variant heavy chain region forms and / or variant light chain region forms as described herein.

The term identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more polynucleotides, as, is determined by aligning and comparing the sequences. Identity Percentage means the percentage of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules to be compared.

Light Chain Region Sequences Exemplary Variant

TABLE 1

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OF THE PROPERTY

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FR1 CDRL1 FR2 CDRL2 FR3 CDRL3 FR4

QSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLlYGSGNflPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSyDSSÍ.SGW'VFGGGTRLTVL SEQ ID NO: 1

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TABLE 2

FR1 CDRH1 FR2 CDRH2 FR3 CDRH3 FR4

QVQLVESGGGWQPGRSLRLSCAASGFTFSSyGAfWWVRQAPGKGLEWVAVYWZOGSWeyyADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGyTSSWTroAFOWVGQGTMVTVSS SEQ ÍD

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For these calculations, the spaces in the alignments (if any) can be addressed by a particular computer program or mathematical model (i.e., an algorithm). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press;

Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of

Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 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 Carillo et al., 1988, SIAM J. Applied Math. 48: 1073.

When calculating percent identity, the sequences being compared are aligned in a way that gives the greatest match between the sequences. The computer program used to determine identity percentage is the GCG program package, which includes GAP (Devereux et al.,

1984, Nucí. Acid Res. 12: 387; Genetics Computer Group,

University of Wisconsin, Madison, WI). The GAP computer algorithm is used to align the two polypeptides or polynucleotides for which the percent identity of

IMPI

sequence is to be determined. The sequences are aligned for optimal pairing of their respective amino acid or nucleotide (the paired span, as determined by the algorithm). A gap opening penalty (calculated as 3x the average diagonal, where the average diagonal is the average of the diagonal of the comparison matrix used; diagonal is the record or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap gap penalty), as well as a matrix for comparison such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain modalities, a standard comparison matrix (see, Dayhoff ef al., 1978, Atlas of Protein Sequence and Structure 5: 345-352 for the PAM 250 comparison matrix; Henikoff ef al., 1992, Proc. Nati. Acad. Sci. USA 89: 10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

The recommended parameters for determining percent identity for polypeptide or nucleotide sequences using the GAP program are as follows: Algorithm: Needleman et al., 1970, J. Mol. Biol. 48: 443-453; Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra; Space penalty: 12 (but no penalty

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY β β for the final spaces), Penalty ^ o ¥ ^ on'gí'fuT ^w™and Space: 4, Similarity Threshold: 0. Certain alignment schemes to align two amino acid sequences can result in the pairing of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no relationship. important between the two full-length sequences. Accordingly, the selected alignment method (GAP program) can be adjusted if desired to result in an alignment that spans at least 50 contiguous amino acids of the target polypeptide.

The light and heavy chain variable regions described herein include consensus sequences derived from groups of related antigen-linked proteins. The amino acid sequences of the light and heavy chain variable regions were analyzed for similarities. Four emerging groups, one group having kappa light chain variable regions, (VH9 / VL9, VH10 / VL10, VH11 / VL11, VH13 / VL13, VH14/

Vl14 and Vh15 / Vl15) and three groups having lambda light chain variable regions: group 1 lambda (VH5 / VL5, VH6 / VL6 and VH7/

Vl7), group 2 lambda (VH3 VL3 and VH4 / VL4), and group 3 lambda (VH1/

VLone and VH2 / Vl2). The represented light chain germ lines include VK1 / A30 and VK1 / L19. The represented light chain lambda germ lines include VLl / le, VL3 / 3p, í IMPI <nsmuTO Mexicano

L “di '· / * ® *?

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VL5 / 5c and VL9 / 9a. The heavy chain germ lines depicted include VH3 / 3-30, VH3 / 3-30.3, VH3 / 3-33, VH3 / 3-48, VH4 / 4-31, and VH4 / 4-59. As used herein, a “consensus sequence refers to amino acid sequences that have common conserved amino acids among a number of sequences and variable amino acids that vary within the given amino acid sequences. Consensus sequences can be determined using standard phylogenic analyzes of the heavy and light chain variable regions corresponding to the IL-23 antigen-linked proteins described herein.

The light chain variable region consensus sequence for the kappa group is

DX1QX2TQSPSSVSASVGDRVTITCRASQGX3X4SX5WX6AWYQQKPGX7APX8LLIY7 \ ASSLQSGV

PSR FS GSX9SGTX10FTLTISSLQPX11DFATYX12CQQANSFPFTFGPGTKVDX13K (SEQ ID

NO: 30) where Xx is selected from I or S; X2 is selected from M or

L; X3 is selected from G or V and X4 is selected from S, F, or I; X5 is selected from S or G; X6 is selected from F or L; X7 is selected from K or Q; X8 is selected from K, No S; Xg is selected from G or

V; X10 is selected from D or E, X is selected from E or A; Xi2 is selected from Y or F; and Χχ3 is selected from I, V or F.

The light chain variable region consensus sequence for group 1 lambda is

QPXT1LTQPPSASASLGASVTLTCTLX2SGYSDYKVDWYQX3RPGKGPRFVMRVGTGGX4VGSKG X5GIPDRFSVLGSGLNRX6LTIKNIQEEDESDYHCGADHGSGX7TG

Hmpi 0 'MEXICAN INSTITUTE

OF PROPERTY '· INDUSTRIAL

ID NO: 61) where Χχ is selected from V or E; X2 is selected from N or S; X3 is selected from Q or L and X4 I know. select from I or T; X5 is selected from D or E; X6 is selected from Y or S; and X7 is selected from S or N.

The light chain variable region consensus sequence for group 3 lambda is

QSVLTQPPSVSGAPGQRVTISCTGSSSNXxGAGYDVHWYQQX2PGTAPKLLIYGSX3NRPSGVPD

RF SG SKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTX4RLTVL (SEQ ID

<td>NO: 139)</td><td>where Χχ is</td><td>select from</td><td>T</td><td>0 I; X2 is selected</td><td>from V 0</td>

<td>10 L; X3 I know</td><td>Choose</td><td>of G 0 N and X4</td><td>I know</td><td>select from R 0 K.</td><td></td>

<td>The</td><td>sequence</td><td>consensus</td><td>of</td><td>variable region of</td><td>chain</td>

<td>heavy</td><td>for</td><td>the</td><td></td><td>kappa group</td><td>is</td>

QVQLQESGPGLVKPSQTLSLTCTVSGGSIXxSGGYYWXzWIRQHPGKGLEWIGXsIX ^ iYSGXsXgY

YNP SLK

SRX7TXeSVDTSX9NQFSLXioLSSVTAADTAVYYCAXorXi2RGXi3YYGMDVWGQGTTVTVSS (SEQ ID NO: 140) where Χχ is selected from N or S; X2 is selected from S or T; X3 is selected from Y or H and X4 is selected from Y or H; X5 is selected from S or N; Χβ is selected from S or T; X7 is selected from V or I; X8 is selected from I or M;

Xg is selected from K or Q; X10 is selected from K or S, Xn is selected from R or K; Xi2 is selected from D or N; and Χχ3 it is selected from H, F or Y.

The consensus sequence of the heavy chain variable region for group 1 lambda is

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INDUSTRIAL

7AD SV KGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARRIAAAGXsXgXioYYYAXn DVWGQGTTVTVSS (SEQ ID NO: 141) where Xi is selected from A or V; X2 is selected from A or V; X3 is selected from T or S and X4 is selected from Y or F; X5 is selected from S or R; X6 is selected from R or I; X7 is selected from H, I, I; X8 is selected from P or

G; Xg is selected from W or F; X10 is selected from G or H and Xu is selected from M or L.

The consensus sequence of the heavy chain variable region for group 2 lambda is

QVQLVESGGGWQPGRSLRLSCAASGFTFSSYX1MHWVRQAPGKGLEWX2X3VISX4DGSX5KYYA

D SV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERTTLSGSYFDYWGQGTLVTVSS (SEQ ID NO: 142) where Χχ is selected from G or A; X2 is selected from V or L; X3 is selected from A or S and X4 is selected from F or H and X5 is selected from L or I.

The consensus sequence of the heavy chain variable region for group 3 lambda is

QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNXxYYADSV

KG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYX2SSWYPDAFDIWGQGTMVTVSS (SEQ ID NO: 143) where Χχ is selected from E or K and X2 is selected from T or S.

Regions that determine complementarity

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The regions that determine the σστπρ í'gfrre π tarre CDRs are embedded within a structure in the light and heavy chain variable regions where they constitute the regions responsible for binding and antigen recognition. The immunoglobulin chain variable domains of the same species, for example, generally show a similar general structure; comprising relatively conserved framework regions (FR) linked by hypervariable CDR regions. A protein bound to the antigen can have 1, 2, 3, 4, 5, 6 or more CDRs.

The variable regions discussed above, for example, typically comprise three CDRs. CDRs of heavy chain variable regions and light chain variable regions are typically aligned by the framework regions to form a structure that specifically binds in a target antibody (eg IL-23). From naturally occurring heavy and light chain variable regions, from N-terminus to C-terminus, both typically conform to the following order of these elements:

FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The CDR and FR regions of exemplary light chain variable domains and heavy chain variable domains are discarded in TABLES 1 and 2. It is recognized that the boundaries of the CDR and FR regions may vary from those highlighted. The numbering systems are

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They have created to assign numbers to amino acids that occupy positions in each of these domains. The regions that determine the complementarity and regions of structure of a given antigen-bound protein can be identified using these systems. Enumeration systems are defined in Kabat et al., Sequences of Proteins of Immunological

Interest, 5th Ed., US Dept. of Health and Human Services, PHS,

NIH, NIH Publication No. 91-3242, 1991, or Chothia & Lesk,

1987, J. Mol. Biol. 196: 901-917; Chothia et al., 1989, Nature

342: 878-883. Other enumeration systems for amino acids in immunoglobulin chains include IMGT® (ImMunoGeneTics International Information System; Lefranc et al, Dev. Comp. Immunol. 2005, 29: 185-203); and AHo (Honegger and Pluckthun, J. Mol. Biol. 2001, 309 (3): 657-670). The CDRs provided herein cannot only be used to define the antigen-bound domain of a traditional antibody structure, but can be embedded in a variety of other polypeptide structures, as described herein.

The antigen-linked proteins described herein are polypeptides in which one or more CDRs can be grafted, inserted, embedded and / or linked. An antigen-bound protein can have, for example, a heavy chain CDRl (CDRHl), and / or a heavy chain CDR2

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(CDRH2), and / or a CDR3 heavy chain CDNRHP).T—And / or un-gr

Light chain CDRl (CDRL1), and / or a light chain CDR2 (CDRL2), and / or a light chain CDR3 (CDRL3). Some antigen binding proteins include both a CDRH3 and a CDRL3. Specific modalities generally use combinations of CDRs that are not repetitive, for example, antigen binding proteins are generally not made with two CDRH.2 regions in one variable heavy chain region, etc. Antigen binding proteins can comprise one or more amino acid sequences that are

<td>identical</td><td>year</td><td>than</td><td>differ</td><td>of</td><td>the sequences</td><td>of</td><td colspan="2">amino acids</td>

<td>one or</td><td>plus</td><td>of</td><td>the CDRs</td><td colspan="2">presented in</td><td>the</td><td>TABLE 3</td><td>in</td>

<td>waste</td><td>of</td><td colspan="2">amino acids,</td><td>in</td><td>where each</td><td colspan="2">difference</td><td>of</td>

Sequence is independently either an deletion, insertion, or substitution of an amino acid. The CDRs in some antigen binding proteins comprise amino acid sequences that are at least 8.0%, 85%, 90%, 91%, 92, 93%,

94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the CDRs sequence listed in TABLE 3. In some antigen binding proteins, CDRs are embedded in a framework region , which orients the CDR (s) so that the appropriate antigen-bound properties of the

CDR (s) are reached.

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TABLE 3

CDRH and CDRL Sequences

<td colspan="3">Exemplary CDRL sequences</td>

<td>TGSSSNTGAGYDVH SEQ ID NO: 62</td><td>GSGNRPS SEQ ID NO: 63</td><td>QSYDSSLSGWV SEQ ID NO: 64</td>

<td>TGSSSNIGAGYDVH SEQ IDNO: 65</td><td>GSNNRPS SEQ ID NO: 66</td><td>MIWHSSASV SEQ ID NO: 67</td>

<td>TLRSGINVGTYRIY SEQ IDNO: 68</td><td>YKSDSDKQQGS SEQ ID NO: 69</td><td>GADHGSGSNFVYV SEQ ID NO: 70</td>

<td>TLNSGYSDYKV SEQ ID NO: 71</td><td>VGTGGIVGSKGD SEQ ID NO: 72</td><td>GADHGSGNNFVYV SEQ ID NO: 73</td>

<td>TLSSGYSDYKV SEQIDNO: 74</td><td>VGTGGIVGSKGE SEQ ID NO: 75</td><td>QQANSFPFT SEQ ID NO: 76</td>

<td>RASQGFSGWLA SEQ ID NO: 77</td><td>VGTGGTVGSKGE SEQ ID NO: 78</td><td>QQATSFPLT SEQ ID NO: 79</td>

<td>RASQVISSWLA SEQ ID NO: 80</td><td>AASSLQS SEQ ID NO: 81</td><td>QQADSFPPT SEQ ID NO: 82</td>

<td>RASQVISSWFA SEQ ID NO: 83</td><td></td><td>LQHNSYPPT SEQ ID NO: 84</td>

<td>RASQGSSSWFA SEQ ID NO: 85</td><td></td><td></td>

<td>RASQGISSWFA SEQ ID NO: 86</td><td></td><td></td>

<td>RAGQVISSWLA SEQ ID NO: 87</td><td></td><td></td>

<td>RASQGIAGWLA SEQ ID NO: 88</td><td></td><td></td>

<td>RASQGIRNDLG SEQ ID NO: 89</td><td></td><td></td>

<td colspan="3">Exemplary CDRH sequences</td>

<td>SYGMH SEQ IDNO: 91</td><td>VIWY DGS NEYYADS VKG SEQ ID NO: 92</td><td>DRGYTSSWYPDAFDI SEQ ID NO: 93</td>

<td>SYAMH SEQ ID NO: 94</td><td>VIWY DGS NKYYADS VKG SEQ ID NO: 95</td><td>DRGYSSSWYPDAFDI SEQ ID NO: 96</td>

<td>TYSMN SEQ ID NO: 97</td><td>VISFDGSLKYYADSVKG SEQ ID NO: 98</td><td>ERTTLSGSYFDY SEQ ID NO: 99</td>

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<td rowspan="2">SYSMN SEQ ID NO: 100</td><td rowspan="2">VISHDGSIKYYADSVKG SEQ ID NO: 101</td><td>RIAAAGGFHYYYALDV</td>

<td>SFSMN SEQ ID NO: 103</td><td>YISSRSSTIYIADSVKG SEQ ID NO: 104</td><td>RIAAAGPWGYYYAMDV SEQ ID NO: 105</td>

<td>SGGYYWT SEQ ID NO: 106</td><td>YISSSSSTRYHADSVKG SEQ ID NO: 107</td><td>NRGYYYGMDV SEQ ID NO: 108</td>

<td>SGGYYWS SEQ IDNO: 109</td><td>YISSRSSTIYYADSVKG SEQIDNO: 110</td><td>NRGFYYGMDV SEQ ID NO: 111</td>

<td>SYFWS SEQIDNO: 112</td><td>YIYYSGNTYYNPSLKS SEQ ID NO: 113</td><td>DRGHYYGMDV SEQ ID NO: 114</td>

<td>TYYWS SEQ ID NO: 115</td><td>HIHYSGNTYYNPSLKS SEQIDNO: 116</td><td>DRGSYYGSDY SEQ ID NO: 117</td>

<td></td><td>YIYYSGSTYYNPSLKS SEQIDNO: 118</td><td>DRGYYYGVDV SEQ ID NO: 119</td>

<td></td><td>YIYYSGSSYYNPSLKS SEQ ID NO: 120</td><td>ENTVTIYYNYGMDV SEQ ID NO: 6</td>

<td></td><td>YIYYSGSTNYNPSLKS SEQ ID NO: 121</td><td></td>

<td></td><td>LIYTSGSTNYNPSLKS SEQ ID NO: 122</td><td></td>

<td></td><td>LIWYDGSNKYYADSVKG SEQ ID NO: 90</td><td></td>

CDRI regions are provided herein comprising amino acid residues 23-34 of SEQ ID NOs: 7 and

eleven; 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 N0s: 31, 33, 34, 38, 40, 44, 52 and 60 and amino acid residues 31-37 or SEQ ID NOs: 46 ,. 48, 50, 54, 56 and 58.

CDR2 regions are provided comprising 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 NOs: 7 and

eleven; 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;

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amino acid residues 52-58 of SEQ ID NOs: 1 v 3 and r & oirin ^ of amino acids 52-67 of SEQ ID NOs: 46, 48, 50, 54, 56 and 58.

The CDR3 regions comprising 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; fifty; are also provided.

The CDRs described herein include consensus sequences derived from groups of related sequences. As previously described ,. Four groups of variable region sequences, one kappa group and three lambda groups were identified. The CDRL1 consensus sequence of the kappa group consists of RASQXiX2SX3WX4A (SEQ ID NO: 123) where Xi is selected from G or V; X2 is selected from I, F, or S; X3 is selected from S or G and X4 is selected from F or L. The group 1 lambda CDRL1 consensus sequence consists of

TLXiSGYSDYKVD (SEQ ID NO: 124) where Xx is selected from N or

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S. The group 3 lambda CDRL1 consensus sequences consist of TGSSSNXxGAGYDVH (SEQ ID NO: 125) where X is selected from I or T.

The group 1 lambda CDRL2 consensus sequence consists of VGTGGX1VGSKGX2 (SEQ ID NO: 126) where Χχ is selected from I or T and X2 is selected from D or E. The group 3 lambda CDRL2 consensus sequence consists of GSXxNRPS (SEQ ID NO: 127) where Χχ is selected from N or G.

CDRL3 consensus sequences include GADHGSGXiNFVYV (SEQ ID NO: 128) where Χχ is S or N.

The CDRH1 consensus sequence of the kappa group consists of SGGYYWXx (SEQ ID NO: 129) where Χχ is selected from S or

T. The group 1 lambda CDRH1 consensus sequence consists of XiX2SMN (SEQ ID NO: 131) where Χχ is selected from S or T and X2 is selected from Y or F. The group 2 lambda CDRH1 consensus sequence consists of SYXxMH (SEQ ID NO: 130), where Χχ is selected from G or A.

The kappa group CDRH2 consensus sequence consists of X1IX2YSGX3X4YYNPSLKS (SEQ ID NO: 132) where en is selected from Y or H; X2 is selected from Y or Η; X3 is selected from S or N and X4 is selected from T or S. The group 1 lambda consensus sequence consists of

YISSX1SSTX2YX3ADSVKG (SEQ ID NO: 134) where Χχ is selected from R or S, X2 is selected from I or R, X3 is selected from I, H

,; β; »-.- -. H

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- «so Y. The consensus sequence of group 2 lambda consists of VISX1DGSX2KYYADSVKG (SEQ ID NO: 133) where Χχ is F or H and X2 is L or T. The group 3 lambda CDRH2 consensus sequence 'consists of VIWYDGSNXxYYADSVKG (SEQ ID NO: 135) where X is selected from K or E.

The CDRH3 consensus sequence of the kappa group consists of XiRGX2YYGMDV (SEQ ID NO: 136) where Χχ is selected from N or D and X2 is selected from H, I F. The consensus sequence

Group 1 lambda CDRH3 consists of RIAAAGXxX2X3YYYAX4DV (SEQ

ID NO: 137) where Χχ is selected from G or P; X2 is selected from F or W; X3 is selected from H or G and X4 is selected from L and M. The group 3 lambda CDRH3 consensus sequence consists of 'DRGYXxSSWYPDAFDI (SEQ ID NO: 138) where Χχ is selected from S or T.

Monoclonal antibodies

Antigen binding proteins provided include monoclonal antibodies that bind to IL-23. Monoclonal antibodies can be produced using any technique known in the art, for example, by immortalizing spleen cells harvested from the transgenic animal after completion of the immunization program. Spleen cells can be immortalized using any technique known in the art, for example by fusing them with

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myleoma cells to produce hybridomas. Myeloma cells for use in fusion procedures that produce hybridoma preferably do not produce antibody, have high fusion efficiency, and enzyme deficiencies that render them unable to grow in certain selective media that support the growth of only the desired fused cells (hybridomas ). Examples of cell lines suitable for use in mouse fusions include Sp-20, P3X63 / Ag8, P3-X63-Ag8.653, NSl / l. Ag 4 1, Sp210-Agl4, FO, NSO / U,

MPC-11, MPC11-X45-GTG 1.7 and S194 / 5XXO Bui; examples of cell lines used in rat fusions include

R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210. Other useful cell lines for cell fusions are U-266, _ GM1500GRG2, LICR-LON-HMy2, and UC729-6.

In some cases, a hybridoma cell line is produced by immunization of an animal (e.g. eg, a transgenic animal having human immunoglobulin sequences) with an IL-23 immunogen; collecting the spleen cells from the immunized animal; by fusing the collected spleen cells to a myeloma cell line, therefore hybridoma cells are generated; hybridoma cell lines are established from hybridoma cells, and identifying a hybridoma cell line that produces an antibody that binds an IL-23 polypeptide without affecting IL. 68

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Dt,! A ÍKJnfDAD ¡nw / stwal

2. 3. Such hybridoma cell lines, * '^ rniyg "' STTtTcirsTpO's ~w···· monoclonal anti-IL-23 produced by them are aspects of the present application.

Monoclonal antibodies secreted by a hybridoma cell line can be purified by any method known in the art. Hybridomas or mAbs can be further filtered 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 above sequences are also provided. Monoclonal antibodies for use as therapeutic agents can be modified in various ways before use. An example is a chimeric antibody, which is an antibody composed of protein segments from different antibodies that are covalently linked to produce light or heavy chains of functional immunoglobulin or immunologically functional portions thereof. Generally, a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence of antibodies derived from a particular species or belonging to a particular class or subclass of antibody, while the rest of the chain (s)

(s) is / are identical (s) or corresponding homolog (s) of antibodies derived from other species or belonging to another class or subclass of antibodies. For methods related to chimeric antibodies, see, for example, US Patent No.0 4,816,567; and Morrison et al., 1985, Proc. Nati. Acad. Sci. USA. 81: 6851-6855. The CDR graft is described, for example, in US Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and

5,530,101.

A useful type of chimeric antibody is a humanized antibody. Generally, a humanized antibody is produced from a monoclonal antibody initially produced in a non-human animal. Certain amino acid residues in this monoclonal antibody, usually from portions that do not recognize antigens of the antibody, are modified to be homologous to the corresponding residues in a corresponding isotype human antibody. Humanization can be accomplished, for example, using various methods by substituting at least a portion of a rodent variable region for the corresponding regions of a human antibody (see, eg, US Patent Nos. 5,585,089, and N

5,693,762; Jones et al., 1986, Nature 321: 522-525; Riechmann

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et al., 1988, Nature 332: 323-27; Verhoeyen et al., 1988, Science 239: 1534-1536).

In certain embodiments, the constant regions of non-human species can be used in conjunction with the variable human region (s) to produce hybrid antibodies.

Fully human antibodies

Fully human antibodies are also provided.

Methods are available for the manufacture of fully human specific antibodies to a given antigen without exposing humans to the antigen (fully human antibodies). One of the specific means envisaged for the application of fully human antibody production is the humanization of the mouse humoral immune system. The introduction of immunoglobulin sites. Human (Ig) in mice in which the endogenous' Ig genes have been inactivated is a means of producing fully human monoclonal antibodies (mAbs) in the mouse, an animal that can be immunized with any desirable antigen. Immunogenic and allergic responses that can sometimes be caused by administration of mouse-derived mAbs to humans as therapeutic agents can be minimized using fully human antibodies.

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Fully human antibodies can be produced by immunizing transgenic animals (usually mice) that are capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production. Antigens for this purpose generally have six or more contiguous amino acids and, optionally, are conjugated to a carrier, for example a hapten. See, for example, Jakobovits EF. / 1993, Proc.

Nati. Acad. Sci. USA 90: 2551-2555; Jakobovits et al., 1993,

Nature 362: 255-258; And EF Bruggermann et al., 1993, Year in

Immunol. 07:33. In one example of such a method, transgenic animals are produced by disabling endogenous mouse immunoglobulin sites encoding mouse heavy and light immunoglobulin chains therein, and inserted into large mouse genome fragments of genome DNA. human · containing the sites encoding human heavy and light chain proteins. Partially modified animals, having less than the full complement of human immunoglobulin sites, are bred to obtain an animal with all desired immune system modifications. When administered an immunogen, these transgenic animals produce antibodies that are immunospecific to the immunogen but have more human than murine amino acid sequences, including variable regions. For details of these methods, see, for example, WIPO Patent Publications W096 / 33735 and W094 / 02602. Additional methods related to transgenic mice to produce human antibodies are described in US patent. No. «ΐΧ. A, -.χ

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5,545, 807, 6, 713,610, 6, 673,986, 6, 162,963, 5,545,807,

6,300,129, 6,255,458, 5,877,397, 5,874,299 and 5,545,806, in WIPO publications, patent WO91 / 10741, W090 / 04036, and in EP 546073B1 and EP 546073A1 '.

The transgenic mice described above contain a human immunoglobulin gene mini-site that encodes the rearranged human heavy and light chain immunoglobulin sequences ([MU] and [gamma]) and [kappa], along with specific mutations that inactivate sites of the endogenous chain [MU] and [kappa] (Lonberg et al.,.

1994, Nature 368: 856-859). Accordingly, mice exhibit reduced expression of mouse IgM or [kappa], and in response to immunization, and introduced human heavy and light chain transgenes undergo a kind of switching and somatic mutation to generate high antibody affinity human IgG monoclonal [kappa] (Lonberg et al., supra; Lonberg and Huszar, 1995, Rev. Intern Immunol 13: 65-93; Harding and Lonberg, 1995, Ann N. & Acad Sci 764: 536546). The preparation of such mice is described in detail.

in Taylor et al., 1992, Nucleic Acids .Research 20: 628Τ = Έ295; Chen et al / 1993, International Immunology 5:

Tuaillon et al., 1994., J. Immunol. 152: 2912-2920; Lonberg et al., 1994, Nature 368: 856-859; Lonberg, 1994, Handbook of Exo. Pharmacology 113: 49-101; Taylor et al., 1994, International Immunology 6: 579-591; Lonberg and Huszar 1995, Int. Rev. Immunol. 13: 65-93; Harding and Lonberg, 1995, Ann.

NY Acad. Sci. 764: 536-546; Fishwild et al., 1996, Nature

Biotechnology 14: 845-85 .. See, US Patents

<td>No.</td><td>5,874,299 and</td><td>No.</td><td> 5,770,429,</td><td>So</td><td>like the patent</td><td>of</td><td>state</td>

United No. 5,545,807; WIPO numbers WO 93/1227, WO 92/22646 and WO 92/03918. The technologies used for the production of human antibodies in these transgenic mice are also disclosed in WIPO publication No. WO 98/24893, and Méndez et al., 1997, Nature Genetics 15: 146-156. For example, the transgenic mouse strains HCo7 and HCol2 can be used to generate anti-IL-23 antibodies.

Using hybridoma technology, antigens specific to human mAbs with the desired specificity can be produced and selected from transgenic mice, such as those described above. These antibodies can be cloned and expressed by

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a suitable vector and the hnsppdpr ^ n Iqb antibody cell can be harvested from cultured hybridoma cells.

Fully human antibodies can also be derived from phage display libraries (as described in Hoogenboom et al., 1991, J. Mol Biol. 227: 381;

Marks et al., 1991, J. Mol Biol. 222: 581; publication of the

WIPO No. WO 99/10494). Phage display techniques mimic immune selection by sampling the antibody repertoires on the surface of the filamentous bacteriophage, and subsequent selection of phages for binding to an antigen of choice.

Proteins bound to bifunctional or biospecific antigen

A "biospecific," double-specific, or "bifunctional antigen binding protein or antibody is an antigen binding protein or antibody, respectively, having two different binding antigen sites, such as one or more CDRs or one or more variable regions. as described above. In some cases they are a hybrid artificial antibody that has two different pairs of the heavy / light chain and two different binding sites. The binding protein of the multispecific antigen or multispecific antibody is one that targets more than one antigen or

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epitope. Proteins bound to the antgayo — bieopoolfieog — and antibodies are a kind of multispecific antigen-binding protein antibody and can be produced by a variety of methods that include, but are not limited to, hybridoma fusion or Fab fragments. See, eg, Songsivilai and Lachmann, 1990, Clin. Exp.

Immunol. 79: 315-321; Kostelny et al., 1992, J. Immunol.

148:1547-1553.

Immunological fragments

Bound proteins. to the antigen also include immunological fragments of an antibody (eg, a

Fab, a Fab ', an F (ab')2<or a scFv). A Fab fragment is composed of a light chain (the variable region of the light chain (VL) and its corresponding constant domain (CL)) and a heavy chain (the heavy chain variable region (VH) and the first constant domain (CH one)) . The heavy chain of one Fab molecule cannot form a disulfide bond with another heavy chain molecule. A Fab 'fragment contains a light chain and a portion of a heavy chain that also contains the region between C domainsHone and CH2, such that an interchain disulfide bond can be formed between the two heavy chains of the Fab fragments to form an F (ab ') molecule2. A fragment F (ab ')2 therefore it is made up of two Fab 'fragments that are held together by one.

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disulfide bond between the two chains passed to an Fv fragment consists of the light chain variable region and the single arm heavy chain variable region of an antibody. ScFv single-chain antibodies are Fv molecules in which the variable heavy and light chain regions have been connected by a flexible ligation to form a single polypeptide chain, which forms an antigen binding region. Single chain antibodies are discussed in detail in the WIPO publication

No. WO 88/01649, US Patents. numbers 4,946,778 and

5,260,203; Bird, 1988, Sci. 242: 423; Huston et al., 1988,

Proc. Nati. Acad. Sci. USA 85: 5879, Ward et al., 1989, Nature

334: 544, de Graaf et al., 2002, Methods Mol. Biol. 178: 379387; Kortt et al., 1997, Prot. Eng. 10: 423; Kortt et al,

2001, Biomol. Eng. 18: 95-108 and Kriangkum et al., 2001, Biomol. Eng. 18:31 -40. A Fe region contains two heavy chain fragments that comprise the C domainsHone and CH2 of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C domains.H3.

Also included are domain antibodies, which are immunologically functional fragments of immunoglobulin that contain only the variable region of a heavy chain or the variable region of a light chain. In some cases, i ivi PI

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two or more V regionsH are covalently linked *1'with' ^ mf '' linker peptide to create a 'bivalent' domain antibody. The two regions VH of a bivalent domain antibody can target the same or different antigens. Diabodies are bivalent antibodies that contain two polypeptide chains, where each polypeptide chain comprises the V domains.H and VL linked by a linker that is too short to allow pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, eg, Holliger et al., Proc. Nati. Acad. USA 90: 6444-48,

1993 and Poljak et ali, Structure 02:01 121-23, 1994). Similarly, tribodies and tetrabodies are antibodies that comprise three and four polypeptide chains, respectively, and form three and four antigen binding sites, respectively, that may be the same or different. The mabodies comprise bivalent scFvs covalently related to the IgGi Fe region (see, for example, Fredericks et al., 2004, Protein Engineering Design & Section, 17: 95-106. Powers et al., 2001, Journal of Immunological Methods , 251: 123-135; Shu et al., 1993, Proc Nati Acad Sci USA 90: 7995-7999; Hayden et al., 1994, Therapeutic Immunology 1: 3-15).

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_ || «Γ ---------- Other various forms

Variant forms of the antigen-bound proteins described above are also provided, some of the antigen-bound proteins have, 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.

The amino acids that. occur naturally can be divided into classes based on the common properties of hydrophobic side chains: (norleucine, Met, Ala, Val,

Leu, He); neutral hydrophilic (Cys, Ser, Thr, Asn, Gln); acids (Asp, Glu); basic (His, Lys, Arg); residues influencing chain orientation (Gly, Pro); and aromatics (Trp,

Tyr, Phe).

Conservative amino acid substitutions may involve exchanging a member of one of these classes with another member of the same class. Conservative amino acid substitutions can encompass unnaturally occurring amino acid residues, which are generally incorporated by chemical synthesis of peptides rather than by synthesis in biological systems. These include peptidomimetics and other inverted or reserved forms of amino acid portions.

Such substantial changes in characteristics

and / or biochemical functions of the protein "SynZa3as aT antigen" described in this document can be achieved by creating substitutions in the amino acid sequence of the heavy and light chains that differ significantly in their effect maintaining (a) the structure of the molecular skeleton in the substitution zone, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain.

Non-conservative substitutions may involve exchanging a member of one of the previous classes for a member of another class. Such substituted residues can be introduced into regions of the antibody that are homologous with human antibodies, or into non-homologous regions of the molecule.

In making such changes, according to certain modalities, the hydropathic amino acid index can be considered. The hydropathic profile of a protein is calculated by assigning a numerical value to each amino acid (hydropathy index), and then an average of these values is repeatedly performed along the peptide chain. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and loading characteristics. These are: isoleucine (+4.5);

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valine (+4.2); leucine (+3.8); phenylalanine 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); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5);

asparagine (-3.5); Usin (-3.9) and arginine (-4.5).

The importance of the hydropathic profile in conferring interactive biological function on a protein is understood in the art (see, eg, in Kyte et al., 1982, J. Mol.

Biol. 157: 105-131). It is known that certain amino acids can be replaced by other amino acids that have a similar hydropathic index or score and that still retain similar biological activity. When making changes based on the hydropathic index, in certain modalities, the substitution of amino acids whose hydropathic indexes are within ± 2 is included. In some respects, those within ± are included, and in other respects, those within ±

0.5 are included.

It is also understood in the art that similar amino acid substitution can be done effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thus created is intended for use in immunological modalities, as in the present case. In certain modalities, the highest local hydrophilicity

Average of a protein, which is governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and the binding to the antigen or immunogenicity, that is, with a biological property of the protein.

The following hydrophilicity values have been assigned to these amino acid residues: arginine (+ 3.0); Usina (+3.0); aspartate (+3.0 ± 1); gluta mato (+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); cistern (-1.0); methionline (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);

phenylalanine (-2.5) and tryptophan (-3.4). When making changes based on similar hydrophilicity values, in certain modalities, the substitution of amino acids whose hydrophilicity values are within ± 2 is included, in other modalities, which are within ± 1 are included, and in other modalities, those within ± 0.5 are included. In some cases, epitopes of primary amino acid sequences can also be identified on the basis of hydrophilicity. These regions are also known as epitopic central regions.

Examples of conservative amino acid substitutions are listed in TABLE 4.

TABLE 4

Conservative substitutions of aminoaciWDiS

<td>Residue</td><td>Sub</td><td rowspan="4"></td><td>Residue</td><td>Sub</td><td rowspan="4"></td><td>Residue</td><td>Sub</td><td rowspan="4"></td><td>Residue</td><td>Sub</td>

Waste: Original Waste

Sub = Exemplary Substitution

One of skill in the art will be able to determine suitable polypeptide variants as set forth herein using well known techniques. One of skill in the art can identify suitable areas of the molecule that can be changed without destroying activity by targeting the regions it creates are not important to activity. The person skilled in the art will also be20 Able to identify residues and portions of molecules that are conserved among similar polypeptides. In future modalities, even in areas that may be important for biological activity or structure, they may be subject to amino acid substitutions 'S2L ·'.

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FROM THE iV®INOUSTRIAL PROPERTY conservative, without destroying the biological activity or without negatively affecting the polypeptide structure.

In addition, the person skilled in the art can review structure-function studies by identifying residues in similar polypeptides that are important for activity or structure.

In view of such a comparison, the importance of amino acid residues in a protein corresponding to amino acid residues important for the activity or structure of similar proteins can be predicted. The person skilled in the art may choose chemically similar amino acid substitutions for such important predicted amino acid residues.

The person skilled in the art can also analyze the three-dimensional structure and the amino acid sequence in relation to said structure in similar polypeptides. In view of such information, one skilled in the art can predict the possible alignment of the amino acid residues of an antibody with respect to its three-dimensional structure. The person skilled in the art may choose not to make radical changes in the amino acid residues that are expected to be on the surface of the protein, since said residues may be involved in important interactions with other molecules. Additionally, one skilled in the art can generate assay variants that contain a single

amino acid substitution at each desired amino acid residue?

These variants can then be examined by assays for IL-23 activity, (see examples below), thus performance information as to which amino acids can and cannot be changed. In other words, based on information obtained from routine experiments, those skilled in the art can easily determine amino acid positions where future substitutions should be avoided either alone or in combination with other mutations.

A number of scientific publications have been devoted to predicting secondary structure. See, Moult, 1996,

Curr. Op. In Biotech. 7: 422-427; Chou et al., 1974, Biochem.

13: 222-245; Chou et al., 1974, Biochemistry 113: 21 1-222;

Chou et al., 1978, Adv. Enzymol. Relat. Areas Mol. Biol.

47: 45-148; Chou et al., 1979, Ann. Rev. Biochem. 47: 251-276, and EF Chou et al., 1979, Biophys. J. 26: 367-384. Furthermore, computer programs are currently available to assist in predicting secondary structure. A secondary structure prediction method is based on homology modeling. For example,

<td>two polypeptides</td><td>or</td><td>protein</td><td colspan="2">who have an identity</td><td>of</td>

<td>sequence over</td><td>of</td><td colspan="2">30%, or greater similarity</td><td>40%, for</td><td>the</td>

<td>general possess</td><td colspan="2">topologies</td><td>structural</td><td>Similar.</td><td>The</td>

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Recent growth of the structural data base — pi; uLuíiiaL '(AP) has provided greater predictability of the secondary structure, including the potential number of folds within a polypeptide or protein structure. See, Holm et al., 1999, Nucí. Acid. Res. 27: 244247. It has been suggested (Brenner et al. 1997, Curr. Operative.

Struct. Biol. 7: 369-376) that there are a limited number of folds in a given protein or polypeptide and that once a critical number of structures has been resolved, the structural prediction will be dramatically more accurate.

Other methods of. secondary structure prediction include strand formation (Jones, 1997, Curr Opin

Struct Biol. 7: 377-387; Sippl et al., 1996, Structure 4: 1519), Profile Analysis (Bowie et al., 1991, Science

253: 164-170 ;. Gribskov et al., 1990, Meth Enzym 183: 146-159;

Gribskov et al., 1987, Proc Nat Acad Sci 84: 4355-4358), and the linkage of evolution (See, Holm, 1999, supra, and

Brenner, 1997, supra).

In some embodiments, amino acid substitutions are made so that: (1) susceptibility to proteolysis is reduced, (2). the susceptibility to oxidation is reduced, (3) the binding affinity is altered to form protein complexes, (4) the affinities of · «are altered

ligand or antigen binding, and / or (4) other physicochemical or functional properties are conferred or modified on said polypeptides, such as maintaining the structure of the molecular skeleton in the substitution area, for example, a sheet or helical conformation; maintaining or altering the charge or hydrophobicity of the molecule at the target site, or maintaining or altering the voluminosity of a side chain.

For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be performed in the naturally occurring sequence. Substitutions can be made in the portion of the antibody that is outside the domain (s) that form the intermolecular contacts). In such modalities, conservative amino acid substitutions can be used so that they do not substantially change the structural characteristics of the matrix sequence (for example, one or more substitution amino acids that do not disturb the secondary structure that characterizes the matrix or the protein bound to the native antigen). Examples of the secondary and tertiary structures of polypeptides recognized in the art are described in

Proteins, Structures and Molecular Principles (Creighton, ed.), 1984, WH New York: Freeman and Company, Introduction · / ·

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to Protein Structure (Branden and Tooze, eds? ')', 1991; —tftaore

York: Garland Publishing, and Thornton et al., 1991, Nature

354:105.

Additional variants include cysteine variants in which one or more cysteine residues in the matrix or the native amino acid sequence are removed or replaced by another amino acid (eg, serine). Cysteine variants are useful, inter alia when the antibodies (for example) must be withdrawn into a biologically active conformation. .Cysteine variants may have fewer cysteine residues than the native protein, and generally have an even number to minimize the interactions resulting from unpaired cysteines.

The described heavy and light chain variable region and CDR can be used to prepare antigen-linked proteins that contain an antigen-binding region that can specifically bind to an IL-23 polypeptide. The region that binds to the antigen means a protein, or a portion of a protein, that specifically binds to a specific antigen, such as the area that contains the amino acid residues that interact with an antigen and gives the antigen-bound protein its specificity. and affinity for the target antigen. An antigen binding region can include one or more CDRs and certain

¡ΤΓγ ^ ΗΤ1Antigen binding areas also include one or more framework regions. For example, one or more of the CDRs listed in Table 3 can be incorporated into a molecule (eg, a polypeptide) covalently or non-covalently to make an immunoadhesion. An immunoadhesion can incorporate the CDR (s) as part of a larger polypeptide chain, or can covalently link the CDR (s) to another polypeptide chain, or can incorporate the CDR (s) non-covalently. The CDR (s) allows immunoadhesion to specifically bind to a particular antigen of interest (eg, an IL-23 polypeptide).

Other antigen-bound proteins include mimetics (eg, mimetic peptides or Peptidomimetics) based on variable regions and CDRs that are

15, described in this document. These analogs can be peptides, non-peptides, or combinations of the peptide and non-peptide regions. Fauchere, 1986, Adv. Drug Res.

15:29; Veber and Freidinger, 1985, TINS. p. 392; and Evans et al., 1987, J. Med. Chem. 30: 1229. Peptide mimetics 20 that are structurally similar to therapeutically useful peptides can be used to produce a similar therapeutic or prophylactic effect. Such compounds have been developed with the help of computerized molecular modeling. Generally, peptidomimetics are

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proteins structurally similar to a J> rotein bound to the antigen showing a desired biological activity, such as the · ability to bind to IL-23, but peptidomimetics have one or more peptide bonds optionally substituted by a bond selected from, for example : - CH2NH-, -CH2S-, -CH2-CH2-, -CH-CH- (cis and trans), -COCH2

-, - CH (OH) CH2-, and -CH2SO-, by means of methods well known in the art. Systematic replacement of one or more amino acids in a consensus sequence with a D-amino acid of the same type (eg, D-lysine instead of L-lysine) can be used in certain embodiments to generate more stable proteins. Furthermore, limited peptides comprising a consensus sequence or a substantially identical consensus sequence variation can be generated by methods known in the art (Rizo and

Gierasch, 1992, Ann Rev. Biochem 61: 387), for example, by the addition of internal cysteine residues capable of forming intramolecular disulfide bridges which cycle the peptide.

Derivatives of the antigen-bound proteins described in this document are also provided. Derived proteins bound to the antigen can comprise any molecule or substance that imparts a desired property to the protein bound to the antigen or fragment, such

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As an increased half-life in a pari-i ,,, 11¾ .-- ρίίΐΐΐϋιΒ -? - · bound to the derived antigen it may comprise, for example, a detectable (or labeled) portion (eg, a radioactive, colorimetric antigenic , or enzyme molecule, a detectable bead (eg, a magnetic or electrodense bead (eg, gold), or a molecule that binds to another molecule (eg, biotin or streptavidin)), a therapeutic or diagnostic fraction ( for example, a radioactive residue, cytotoxic, or pharmaceutically active), or a molecule that enhances the suitability of the antigen-bound protein for a particular use (eg, administration to a subject, such as a human subject, or other uses in vivo or in vitro). Examples of molecules that can be used to derive an antigen-bound protein include albumin (eg, human serum albumin) and polyethylene glycol (PEG). The linked and PEGylated albumin derivatives of the antigen-bound proteins can be prepared using methods well known in the art. In one embodiment, the antigen-bound protein is conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly (N-vinyl pyrrolidone), polyethylene glycols,

polyoxyethylated polyols and homopolymers of propylene glycol, polypropylene / ethylene oxide, polyvinyl alcohols.

Other derivatives include covalent conjugates or aggregates of proteins bound to the IL-23 antigen with other proteins or polypeptides, such as the expression of recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-terminus of an IL-23 protein. binding to the antigen. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide, eg, the yeast alpha factor leader, or a peptide such as an epitope tag. An IL-23 antigen-bound protein containing fusion proteins may comprise added peptides to facilitate purification or identification of the IL-23 antigen-linked protein (eg, poly-His). A protein bound to the IL-23 antigen may also be related to the FLAG peptide as described in Hopp et al., 1988, Bio / Technology 6: 1204; and US Patent No. 5,011,912. The FLAG peptide is highly antigenic and provides a reversibly linked epitope via a specific monoclonal antibody (MAB), allowing for rapid assay and easy purification of the expressed recombinant protein. Useful reagents for the preparation of fusion proteins in which the

FLAG peptide with a polypeptide gives ^^ se1 GTló'UG'flLl'iTTT commercially available (Sigma, St. Louis, MO).

Oligomers containing one or more proteins bound to the IL-23 antigen can be used as IL-23 antagonists. The oligomers can be in the form of covalently linked or non-covalently linked dimers, trimers, oligomers, or higher. Oligomers comprising two or more proteins bound to the binding IL-23 antigen are contemplated for use, with one example being a homodimer. Others include heterodimeric oligomers, heterotrimers and homotrimers, homotetramers, heterotetramers, etc. Also included are oligomers comprising multiple IL-23 binding proteins linked covalently or non-covalently between peptide moieties fused to IL-23 antigen bound proteins. Such peptides can be peptide linkers (spacers), or peptides that have the property of promoting oligomerization. Suitable peptide linkages include those described in US Patent No. 4,751,180 and 4935233. Leucine zippers and certain antibody-derived polypeptides are among the peptides that can promote oligomerization of IL-23 antigen-bound proteins it. Examples of leucine zipper domains suitable for protein production

----1

IMPí / g "INDUSTRIAL soluble oligomers are described in WIPO publication N 0 WO 94/10308; Hoppe et al., EBSLetters

344: 191; and Fanslow et al., 1994, Semin. Immunol. 6: 267-278. In one method, the recombinant fusion proteins comprising a fragment of the antigen binding protein

IL-23 or derivative fused to a leucine zipper peptide are expressed in suitable host cells, and the fragments or derivatives of protein bound to the soluble oligomeric IL-23 antigen that are formed are recovered from the culture supernatant.

Such oligomers can comprise from two to four proteins bound to the IL-23 antigen. The antigen-bound protein portions of the oligomer can be in any of the forms described above, eg, variants or fragments. Preferably, the oligomers comprise proteins bound to the IL-23 antigen that have the IL-23 binding activity. Oligomers can be prepared using immunoglobulin derived polypeptides. The preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of polypeptide antibody derivatives (including Fe area) has been described, for example, by Ashkenazi et al., 1991, Proc. Nati. Acad. Sci. USA. 88: 10535; Byrn et al., 1990, Nature 344: 677; and ι ινι r ι

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Hollenbaugh et al., 1992 Construction of Immunoglobulin Fusion Proteins, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.1 1.

Also included are dimers that comprise two fusion proteins created by the fusion of a protein bound to the Fe region antigen of an antibody. The dimer can be made, for example, by inserting a fusion gene encoding the fusion protein into an appropriate expression vector, expressing the fusion gene in host cells transformed with the recombinant expression vector, and allowing the protein to Expressed fusion is assembled as antibody molecules, whereby inter-chain disulfide bonds are formed between the Fe portions to produce the dimer. Such Fe polypeptides include native and mutein forms of polypeptides derived from the Fe region of an antibody. Truncated forms of such polypeptides containing the hinge region that promote dimerization are also included. Fusion proteins comprising portions (Fe and oligomers formed thereof) offer the advantage of easy purification by affinity chromatography on protein A or protein G columns. A suitable Fe polypeptide, which is described in WIPO Publication No. WO

93/10151 and US Patent Nos. 5,426,048 and 5,262,522, is twerm / το Mexican Oí.Cá μοριεοαγ »INDUSTRIAL

a single chain polypeptide that extends from the hinge N-terminal region to the native C-terminus of the Fe region of a human IgGl antibody. Another useful Fe polypeptide is the Fe mutein described in US Patent No. 5,457,035, and in Baum et al., 1994, EMBO J. 13: 3992-4001. The amino acid sequence of this mutein is identical to that of the native Fe sequence presented in the Publication of the

WIPO No. WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, 'amino acid 20 has been changed from

Leu to Glu, and amino acid 22 has been changed from Gly to Ala.

The mutein shows reduced affinity for Fe receptors.

The antigen-bound protein may have a glycosylation pattern that is different or modified from that found in native species. As is known in the art, glycosylation patterns can depend both on the sequence of the protein (eg, the presence or absence of particular glycosylation amino acid residues, discussed below), or on the host cell or organism in the that protein is produced. Particular systems of expression are discussed below.

Generally, glycosylation of polypeptides is N-linked or O-linked. N-linked refers to the binding of the carbohydrate moiety to the side chain of a residue of

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asparagine. The tri-peptide sequences aspara ^ PW ^ -ks and asparagine-X-threonine, where X is any ammo except proline, are the recognition sequences for the enzymatic binding of the carbohydrate moiety to the asparagine side chain. In this way, the presence of any of these tri-peptide sequences in a polypeptide creates a potential glycosylation site. 0-linked glycosylation refers to the binding of one of the N-acetylgalactosamine, galactose, or xylose sugars to a hydroxyamino acid, more commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

Addition of glycosylation sites to the antigen-bound protein is conveniently done by altering the amino acid sequence so that it contains one or more of the above-described tri-peptide sequences (for glycosylation sites linked to

N). Alteration can also be done by adding, or replacing, one or more serine or threonine residues to the starter sequence (for O-linked glycosylation sites). For convenience, the amino acid sequence of the antigen-bound protein can be altered by changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases of

7Γ '' 'Pur.

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FROM THE FROF1EUAII C »m INDUSTRIAL way we generate the codons that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate servings in the protein bound to the antigen is by chemical or enzymatic coupling of glycosides to the protein. These procedures are advantageous because they do not require production of the protein in a host cell that has capabilities for N-linked glycosylation and O-linked glycosylation. Depending on the coupling mode used, the sugar (s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as cistern compounds, (d) free hydroxyl groups , such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, tryptophan, or (f) the amide group of glutamine. These methods are described in the publication

PCT number WO 87/05330, and in Aplin and Wriston, 1981, CRC Crit.

Rev, Biochem., Pp 259-306.

The elimination of portions of carbohydrates present in the protein bound to the starting antigen can be carried out chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in

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cleavage of most or all of the sugars, except the binding sugar (N-acetylglucosamine or Nacetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259: 52 and by

Edge et al., 1981, Anal. Biochem. 118: 131. Enzymatic cleavage of carbohydrate portions into polypeptides can be accomplished through the use of a variety of endo and exoglycosidases as described by Thotakura et al., 1987, Meth.

Enzymol. 138: 350. Glycosylation at potential glycosylation sites can be prevented by using the compound tunicamycin described by Duskin et al., 1982, J.

Biol. Chem. 257: 3105. Tunicamycin blocks the formation of protein-N-glucoside bonds.

Therefore, aspects include glycosylation variants of antigen-bound proteins in which the number and / or type of glycosylation site (s) have been altered compared to the amino acid sequences of the polypeptide matrix. In certain embodiments, variants of the antigen-bound protein comprise more or fewer N-linked glycosylation sites than the parent polypeptide. Substitutions that remove or alter this sequence will prevent the addition of an N-linked carbohydrate chain present

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in the precursor polypeptide. For example, ..... Ía ...... ^ - Llaas'ilation. ' it can be reduced by deleting an Asn or replacing the Asn with a different amino acid. Antibodies generally have an N-linked glycosylation site in the Fe region.

Labels and effector groups

Proteins bound to the antigen can comprise one or more tags. The term tag or tag group refers to any detectable tag. In general, the labels are subdivided into a variety of classes, depending on the test in which they have detected: a) isotopic labels, which can be radioactive or heavy isotopes, b) magnetic labels (for example, magnetic particles), c) redox active portions; d) optical dyes, enzyme groups (eg, horseradish root peroxidase, β-galactosidase, luciferase, alkaline phosphatase), e) biotin groups, and f) predetermined polypeptide epitopes - recognized by a secondary reporter (eg , leucine zipper pair sequences, the binding of sites for secondary antibodies, metal binding areas, epitope tags, etc.). In some embodiments, the tagged group is coupled to the antigen-bound protein through spacer arms of various lengths to reduce steric hindrance

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potential. Various methods for prnt-p rrnr * -dq tag4are known in the. technique. Examples of suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (eg.3H

N, S, Y, Te, In, I, I), fluorescent groups (eg, with FITC, rhodamine, lanthanide phosphors), enzyme groups (eg, horseradish root peroxidase, β-galactosidase, luciferase, alkaline phosphatase ), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (eg, leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags). In some embodiments, the tagged pool is coupled to the antigen-bound protein through spacer arms of various lengths to reduce the potential spherical hindrance. Various methods of labeling proteins are known in the art and can be used depending on how appropriate they are.

The term effector group means that any group coupled to an antigen-bound protein acts as a cytotoxic agent. Examples of suitable effector groups are radioisotopes or radionuclides (eg.3H 14C, 15N, 35Yes, 90AND,

Tea, llxIn, X25T, 131I)

Other suitable groups

101

include toxins, therapeutic groups ... r upo u chemotherapeutic. Examples of suitable groups include calicheamycin, auristatins, geldanamicin, and maytansine. In some embodiments, the effector group is coupled to the antigen-bound protein - via spacer arms of various lengths to reduce the potential spherical hindrance.

Polynucleotides that encode proteins bound to the IL-23 antigen

Also provided are polynucleotides encoding the antigen-bound proteins described herein, or portions thereof, including polynucleotides encoding one or both chains of an antibody or a fragment, derivative, mutein, or variant thereof, polynucleotides that encode heavy chain variable regions or CDRs only, enough polynucleotides for use as. hybridization, PCR primers or sequencing primers to identify, analyze, mutate, or amplify a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibition of expression of a polynucleotide, and sequences complementary to the foregoing. The polynucleotides can be of any length. They can be, for example, 5, 10,

102

4-5«

175, 200, 250, 300, 350, 400 , 450, 500, 750, 1000, 1500

3,000, 5,000 or more nucleic acids in length, including all intermediate values, and / or may comprise one or more additional sequences, eg, regulatory sequences, and / or be part of a larger polynucleotide, eg, a vector. The polynucleotides can be single-stranded or double-stranded and can comprise RNA and / or DNA nucleic acids and artificial variants thereof (eg, peptide nucleic acids).

Polynucleotides' that encode certain proteins bound to the antigen, or portions thereof (eg, full-length antibody, heavy or light chain, variable domain, or a CDRH1, CDRH2, CDRH3, CDRL1,

CDRL2, or CDRL3) can be isolated from B cells of mice that have been immunized with IL-23 or an immunogenic fragment thereof. The polynucleotide can be isolated by means of conventional procedures such as polymerase chain reaction (PCR). Phage display is another example of a known technique by which antibody derivatives and other antigen-bound proteins can be prepared. In one approach, polypeptides that are components of a protein bound to the antigen of interest are expressed in any

103

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Adequate recombinant expression, and the expressed polypeptides are allowed to assemble to form antigen-bound protein molecules. Phage display is also used to derive antigen-bound proteins that have different properties (eg, varying affinities to the antigen to which they bind) through chain mixing, see Marks et al., 1992, Biotechnology

10:779.

Due to the degeneracy of the genetic code, each of the polypeptide sequences described herein are also encoded by a large number of other polynucleotide sequences in addition to those provided. For example, the heavy chain variable domains contained in this document may be encoded by polynucleotide sequences SEQ ID NO: 32, 35, 37, 39, 41, 43, 45, 47,

49, 51, 53, 55, 57, or 59. Light chain variable domains can be encoded by means of polynucleotide sequences SEQ ID NO: 2, 5, 6, 8, 10, 12, 14, 16, 18, 20 , 22, 24, 26, or 28. One skilled in the art will appreciate that the present application provides a suitable written description and is possible for each nucleotide sequence encoding each protein bound to the antigen.

A further aspect provides polynucleotides that hybridize to other polynucleotide molecules under others.

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104

Particular hybridization conditions wiJ UTO 11 'very well in this technique the methods for nucleic acid hybridization, the basic parameters that affect the choice of hybridization conditions and the orientation for the elaboration of the appropriate conditions. See, for example, Sambrook, Fritsch 'and Maniatis (2001, Molecular Cloning: A Laboratory Manual, Coid Spring Harbor Laboratory

Press, Coid Spring Harbor, NY and Current Protocols in

Molecular Biology, 1995, Ausubel et al., John Wiley & Sons,

Inc. As defined here, a moderately rigorous hybridization condition uses a solution containing 5x prewash sodium chloride / sodium citrate (SSC), SDS at

0.5%, a 0.0 mM EDTA (pH 8.0), a hybridization buffer of approximately 50% formamide, 6x SSC, and a hybridization temperature of 55 ° C (or other similar hybridization solutions, such as one containing approximately 50% formamide, with a hybridization temperature of 42 ° C), and washing conditions of 60 ° C, in 0.5 x SSC, 0.1% SDS. A severe hybridization condition in 6x SSC at 45 ° C, followed by one or more washes in 0.1 x SSC, 0.2% SDS at 68 ° C. Furthermore, one skilled in the art can manipulate hybridization and / or wash conditions to increase or decrease the severity of hybridization such that the polynucleotides comprising the sequences

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5 DS LA PHOflEDAO

INDUSTRIAL nucleic acids that are at least 65%, 70%, 75%, 80%, 85%. . 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to each other, including all intermediate values, generally remain hybridized to each other.

Changes can be introduced by mutation in a polynucleotide, leading to changes in the amino acid sequence of a polypeptide (eg, an antigen-linked protein or antigen-linked protein derivative) that it encodes. Mutations can be introduced using any method known in the art, such as site-directed mutagenesis and random mutagenesis. The mimic polypeptides can be expressed and selected for a desired property. Mutations can be introduced into a polynucleotide without significantly altering the biological activity of a coding polypeptide. For example, substitutions on non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a polynucleotide that selectively changes the biological activity of a polypeptide that it encodes. For example, the mutation can quantitatively or qualitatively change biological activity, such as increase, decrease, or eliminate activity, and change the antigenic specificity of an antigen-bound protein.

106

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Another aspect provides polynucleotides that are suitable for use as primers or hybridization probes for detection of nucleic acid sequences. A polynucleotide may comprise, only a portion of a nucleic acid sequence encoding a full length polypeptide, eg, a fragment that can be used as a probe or primer, or a fragment encoding an active portion (eg, IL -23 a binding portion) of a polypeptide. Probes based on the sequence of a nucleic acid can be used to detect nucleic acid or similar nucleic acids, for example, transcripts encoding a polypeptide. The probe may comprise a tag group, for example, a radioisotope, a fluorescent compound, an enzyme, or a cofactor enzyme. Such probes can be used to identify a cell that expresses the polypeptide.

Expression methods of antigen-bound proteins

The antigen-bound proteins provided herein can be prepared by means of a number of conventional techniques. For example, IL-23 antigen binding proteins can be produced by means of recombinant expression systems, using any method known in the art. See for example, Monoclonal

Antibodies, Hybridomas: A New- Dimension in Biological

107

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Analyzes, Kennet et al. (eds.) Plenum Pressy — Wcw ΥιίΐΊί »(lQflQ)». and Antibodies: A Laboratory Manual, Harlow and Lañe (eds.), Coid Spring Harbor Laboratory Press, Coid Spring Harbor, NY

(1988).

Constructs and expression systems in the form of plasmids, expression vectors, expression cassettes or transcription that comprise at least one polynucleotide as previously described are also provided herein, as well as host cells comprising such constructs or expression systems.

As used herein, "vector" means any molecule or entity (eg, nucleic acid, plasmid, bacteriophage 'or virus) suitable for use to transfer protein-encoding information into a host cell. The effects of vectors include, but are not limited to, plasmids, viral vectors, non-cpisomal mammalian vectors, and expression vectors, eg, recombinant expression vectors. Expression vectors, such as recombinant expression vectors, are useful for the transformation of a host cell and contain nucleic acid sequences that direct and / or control (along with the host cell) the expression of one or more coding areas he 'theologians operatively connected to that place. An expression construct can include, but

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it is not limited to sequences that λ i'i.t.'Li-ln A transcription, translation, and, if introns are present, affects the RNA by coupling a coding region operably connected to that site. Operably connected means that the components to which the term is applied have a relationship that allows them to carry their inherent functions.

For example, a control sequence, eg, a promoter, in a vector that is operably linked to a protein coding sequence is organized such that normal activity of the control sequence leads to transcription of the protein coding sequence. resulting in the expression of the encoded protein.

Another aspect provides host cells into which an expression vector, such as a recombinant expression vector, has been introduced. A host cell can be any prokaryotic cell (eg, E.coli) or eukaryotic cell (eg, mammalian, insect, or fungal cells (eg, CHO cells)). The DNA vector can be introduced into prokaryotic or eukaryotic cells by means of conventional transformation or transection techniques. For the stable transfection of mammalian cells, it is known that, depending on the expression vector and the transfection technique used, only a small fraction of cells can integrate the external DNA into their genome. To identify

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and to select these integrants, a gene encoding a selected trwn «i · - - marker (eg, for antibiotic resistance) is generally introduced into the host cells alongside the gene of interest. Preferred selectable markers include those that confer drug resistance, such as G418, hygromycin, and methotrexate. Stably transferred cells with the introduced polynucleotide can be identified by drug selection (eg, cells that have the selected marker gene incorporated will survive, while the other cells will die), among other methods.

Proteins bound to the antigen can be expressed in *

hybridoma cell lines (eg, in particular antibodies it may be expressed in hybridomas) or in cell lines other than hybridomas. The expression constructs encode the antigen-bound proteins can be used to transform a mammal, insect, or microbe host cell. Transformation can be performed using any known method to introduce polynucleotides into a host cell, including, for example, packaging the polynucleotide into a virus or bacteriophage and transducing a host cell with the construct by means of transfection processes known in the art, as exemplified by US Patent Nos.

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4,399,216; 4,912,040; 4,740,461; 4,959,455. The optimal transformation process used will depend on which type of host cell is transformed. Methods for the introduction of mammalian cells into polynucleotides 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 the polynucleotide (s) in liposomes, nucleic acid mixture with positively charged lipids, and direct microinjection of DNA into the nucleus.

Recombinant expression constructs generally comprise a polynucleotide encoding a polypeptide. The polypeptide may comprise one or more of the following: one or more CDRs as provided herein: a light chain variable region: a heavy chain variable region, a light chain constant region, a heavy chain constant region, (for example, CH1 Ch2 and / or Ch3); and / or another scaffolding portion of a protein bound to the IL-23 antigen. These nucleic acid sequences are inserted into an appropriate expression vector using standard ligation techniques. In one embodiment, the heavy or light chain constant region is attached to the C-terminus of the heavy chain variable region

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<td colspan="3">or light provided in</td><td>the present</td><td>document and</td><td>binds</td><td>in</td>

<td>a vector</td><td colspan="2">expression,</td><td>. For the</td><td>general the</td><td>vector</td><td>I know</td>

<td>Choose</td><td>for</td><td colspan="2">be functional in</td><td>the cell</td><td colspan="2">host</td>

<td>particular</td><td>(by</td><td>example</td><td>, the vector</td><td colspan="2">it's compatible with</td><td>the</td>

<td>machinery</td><td>of</td><td>cell</td><td>host,</td><td colspan="2">can happen</td><td>the</td>

gene amplification and / or expression). In some embodiments, vectors are used in fragment-protein complementation assays using protein reporters, such as dihydrofolate reductase (see, for example,

USA No. 6,270,964). Suitable expression vectors can be purchased, for example, from Invitrogen Life Technologies (Carlsbad, CA) or BD Biosciences (San Jose, CA). Other useful vectors for the cloning and expression of antibodies and fragments include those described in Bianchi and McGrew,

2003, Biotech. Biotechnol. Bioeng. 84: 439-44. Additional suitable expression vectors are discussed, for example, in Methods Enzymol., Vol. ' 185 (DV Goeddel, ed.), 1990, New

York: Academic Press.

Generally, the expression vectors used in any of the host cells contain sequences for plasmid maintenance and for the cloning and expression of exogenous nucleotide sequences. Such sequences, correlatively referred to as flanking sequences in certain modalities will generally include a

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or more of the following i'iuult'ú'LidUL sequences. 'HIT promoter, one or more enhancer sequences, an origin or replication, a transcriptional termination sequence, a complete intron sequence containing an acceptor and donor center site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polyconnection area for inserting the polynucleotide encoding the polypeptide to be expressed, and a selectable marker element.

The provided expression vectors can be constructed from a starter vector as a commercially available vector. Such vectors may or may not contain all of the various desired sequences. Where one or more of the desired sequences described in this document are no longer present in the vector, they can be individually obtained and connected to the vector. The methods used to obtain each diverse sequence are well known to a person skilled in the art.

Optionally, the vector may contain a tag coding sequence, eg, an oligonucleotide molecule located at the end of positions 5 'or 3' of the coding sequence for the protein bound to the IL-23 antigen; the oligonucleotide sequence encodes polyHis (such as hexaHis), or other

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labeled as FLAG®, HA (influenza virus hemagglutinin), or myc, for which there is an antibody commercially. This tag is generally fused to the polypeptide on it. expression of the polypeptide, and can serve as a means for the detection or affinity purification of the protein bound to the IL-23 antigen of the host cell. Affinity purification can be performed, for example, by means of column chromatography using antibodies against the label as a purification matrix. Optionally, the tag can be subsequently removed from the purified IL-23 antigen binding protein in many ways such as using certain peptidases for cell culture.

Flanking sequences may be homologous (eg, from the same species and / or strain as the host cell), heterologous (eg, from a species or strain other than that of the host cells), hybrid (eg, a combination of flanking sequences from more than one source), synthetic or native. As such, the source of a diverse sequence may be 'any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence functions in the organism, and that it can be activated by, the host cell machinery.

1.552^

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The flanking sequences that are useful in the "vectors" can be obtained by any of the many methods well known in the art. Generally, the flanking sequences useful herein will have been previously identified by restriction endonuclease allocation and / or digestion and can then be isolated from the tissue source itself using the appropriate restriction endonucleases. In some cases, the total nucleotide sequence of a flanking sequence may be known. Here, the flanking sequence can be synthesized using the methods described herein for nucleic acid synthesis or cloning.

Whether all or only a portion of the flanking sequence is known, it can be obtained using a polymerase chain reaction (PCR) and / or by screening a genomic library with a suitable probe as an oligonucleotide and / or flanking sequence fragments from the same or other species. Where the flanking sequence is not known, a DNA fragment containing a flanking sequence can be isolated from a longer piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation can be achieved by restriction endonuclease digestion to produce the fragment itself

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DNA followed by isolation using gelr -ije, pg, pi,tf, l-cation of agarose, Qiagen® column chromatography (Qiagen, Chatsworth, CA), or other methods known to the skilled technician. The selection of suitable enzymes to accomplish this purpose will be apparent to one skilled in the art.

An origin of replication is generally a part of those commercially acquired prokaryotic expression vectors, and the origin aids in amplification in a host cell. If the vector of choice does not contain an origin of replication site, one can be chemically synthesized based on a known sequence, and linked to the vector. For example, the origin of replication of plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most gram-negative bacteria, and various viral origins (eg, SV40, polyoma, adenovirus, vesicular stoma virus ( VSV) or papillomavirus such as HPV or BVP) are useful for cloning vectors into mammalian cells. In general, the origin of the replication component is not needed for mammalian expression vectors (for example, the SV40 origin is frequently used only because it also contains the early promoter virus).

A transcription termination sequence is generally placed 3 'at the end of an area

* ΙΜΡΙ

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<td>encoder</td><td>of</td><td>polypeptides and serves</td><td>to end</td><td>the</td>

<td>transcription</td><td>. By</td><td colspan="2">usually the termination sequence</td><td>of</td>

<td>transcription</td><td colspan="2">in prokaryotic cells it is a</td><td>rich shard</td><td>in .</td>

<td>GC followed</td><td>by</td><td>a poly-T sequence.</td><td>While</td><td>the</td>

<td>sequence it</td><td>clone</td><td colspan="2">easily from a library or even</td><td>I know</td>

Commercially acquired as part of a vector, it can also be synthesized using methods for nucleic acid synthesis such as those described in this document.

A selection marker gene encodes a protein necessary for the survival and growth of a growing host cell in a selective culture medium.

Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, eg, ampicillin, tetracycline, or kanamycin for prokaryotic host cells; (b) complement autotrophic cell deficiencies; or (c) supply critical nutrients not available from defined or complex media.

Specific selected markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, a neomycin resistance gene can also be used for selection in both host cells, prokaryotes and eukaryotes.

Other selected genes can be used to

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amplify the gene that will be expressed. The process in which genes required for the production of a protein critical for cell growth or survival are repeated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase genes without promoters. Transformed mammalian cells are placed under selection pressure where only transformed ones are particularly adapted to survive by virtue of the selectable gene present in the vector. The selection pressure is imposed by culturing the transformed cells under conditions in which the selection concentration agent in the medium is increased satisfactorily, thus leading to amplification of both the selectable gene and the DNA encoding another gene. , as an antigenic binding protein that binds to IL-23. As a result, increased amounts of a polypeptide as an antigen-bound protein are synthesized from the amplified DNA.

A ribosome binding site is generally necessary for the initiation of messenger RNA translation and is characterized by a Shine-Dalgarno (prokaryotic) or · «·· sequence.

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a Kozak (eukaryote) sequence. The elelTTéhto 'is generally located at a position 3' to the promoter and at a position 5 'to the coding sequence of the polypeptide to be expressed.

In some cases, such as when glycosylation is desired in a host cell expression system, one can manipulate the many pre- or pro-sequences to enhance glycosylation or culture. For example, one can alter the peptidase cleavage site of a particular signal peptide, or add prosequences, which can also affect glycosylation. The final protein product may have, at position -1 (relative to the first amino acid of the mature protein), one or more additional incident amino acids for expression, which may not have been fully removed. For example, the final protein product may have one or two amino acid residues found at the peptidase cleavage site, attached to the amino terminus. Alternatively, the use of some peptidase cleavage sites may result in a slightly truncated form of the polypeptide, if the enzyme cleaves in a certain area within the mature polypeptide.

Expression and cloning will generally contain a promoter that is recognized by the host organism and is operatively connected to the molecule encoding a protein bound to the IL-23 antigen. The promoters are

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Localized untranscribed sequences I commented áilllJU (poof example, a position 5 ') · at the beginning of the codon of a structural gene. Promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters begin by increasing their levels of DNA transcription under their control in response to some changes in culture conditions, such as the presence or absence of a nutrient or a change in temperature. On the other hand, the constitutive promoters uniformly transcribe a gene to which they are operatively connected, this with little or no control over gene expression. A large number of promoters, recognized by a variety of potential host cells, are well known. A suitable promoter is operably linked to DNA by encoding a heavy chain variable region or a light chain variable region of a protein bound to the IL-23 antigen by removing the promoter from the DNA source by restriction enzyme digestion and inserting the desired promoter sequence in the vector.

Promoters suitable for use with yeast hosts are also well known. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for the use of mammalian host cells are well known and included, but not

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they are limited to those obtained from the genomes of viruses such as polyoma virus, poultry smallpox virus, adenoviruses (such as adenovirus 2). Bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and Simian Virus (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, eg, heat shock promoters and the actin promoter.

Additional promoters which may be of interest include, but are not limited to: the early promoter

SV40 (Thornsen et al., 1984, Proc. Nati. Acad. USA 81: 659663); the CMV promoter (Benoist and Chambón, 1981, Nature

290: 304-310); the promoter contained in the 3 'long terminal repeat of the Sarcoma Rous virus (Yamamoto et al., 1980, Cell 22: 787-797); the herpes thymidine kinase promoter (Wagner et al., 1981, Proc.

Nati. Acad. Sci. USA 78: 1444-1445); the promoter and regulatory sequences of the metallothionein gene (Prinster et al., 1982, Nature 296: 39-42); and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Nati. Acad. Sci. USA 75: 3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Nati. Acad. Sci. USA 80: 2125). Also of interest are the following regions of animal transcriptional control, which exhibit

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Tissue specificity and the elastase I gene control region that is active in pancreatic acinar cells have been used in transgenic animals (Swift et al., 1984, Cell 38: 639-646; Ornitz et al., 1986, Coid Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald, 1987, Hepatology 7: 425-515); the insulin gene control region that is active in beta cells (Hanahan, 1985, Nature

315: 1 15-122); the immunoglobulin gene control region that is active in lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature 318: 533-538; Alexander et al., 1987,

Mol. Cell. Biol. 7: 1436-1444); the mouse mammary tumor virus control region that is active in mast, lymphoid, testicular, and breast cells (Leder et al.,

1986, Cell 45: 485-495); the albumin gene control region that is active in the liver (Pinkert et al., 1987,

Genes and Devel. 1: 268-276); the protein-fetus-alpha gene control region that is active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol ·. 5: 1639-1648; Hammer et al., 1987,

Science 253: 53-58); the control region of the 1-antitrypsin alfa gene that is active in the liver (Kelsey et al.,

1987, Genes and Devel. 1: 161-171); the beta-globin gene control region that is active in myeloid cells (Mogram et al., 1985, Nature 315: 338-340; Kollias et al.,

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1986, Cell 46: 89-94); the basic pioLéiliar control gene region that is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48: 703-712); the control region of the myosin light chain-2 gene that is active in skeletal muscle (Sani, 1985, Nature 314: 283-286); and the control region of the gonadotropin-releasing hormone gene that is active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378).

An enhancer sequence can be inserted into the vector to increase transcription by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually around 10-300bp in length, that act on the promoter to increase transcription. Enhancers are relatively independent in position and orientation having been found in both the 5 'and 3' positions to the transcription unit.

Available enhancer sequences of mammalian genes are known (eg, globin, elastase, albumin, protein-fetus-alpha, and insulin). However, a virus enhancer is generally used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, the adenovirus enhancers known in the art are examples of enhancer elements for the activation of promoters of

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eukaryotes. While an enhancer may be positioned in the vector at either a 5 'or 3' position for a coding sequence, it is generally located at the 5 'site of the promoter. A sequence encoding an appropriate native signal sequence or a heterologous (leader sequence or signal peptide) can be incorporated into an expression vector, to promote extracellular secretion of the antibody. The choice of signal peptide or leader depends on the type of host cells in which the antibody is produced, and a heterologous signal sequence can replace the native signal sequence. Examples of signal peptides that are functional in mammalian host cells include the following: the signal sequence for interleukin 7 described in US Patent No. 4,965,195;

the signal sequence for interleukin 2 described in Cosman et al., 1984, Nature 312: 768; the interleukin-4 receptor signal peptide described in EP Patent No. 0367 566; the interleukin-1 type 1 receptor signal peptide in US Patent No. 4,968,607; the interleukin-1 type II receptor signal peptide described in EP Patent No. 0 460

846.

After the vector has been constructed, the completed vector can be inserted into. a host cell suitable for polypeptide expression and / or amplification. The

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124 Transformation of an expression vector for an antigen-bound protein into a selected host cell can be accomplished by means of well-known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-mediated transfection. dextran, or other known methods. The selected method will be in part a function of the type of host cell to be used. These methods and other suitable techniques are well known to a person skilled in the art, and are established, for example, in

Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Coid Spring Harbor Laboratory Press, Coid Spring Harbor,

NY (2001).

When a host cell is cultured under the appropriate conditions, it synthesizes the protein that can be subsequently collected from the culture medium (if the host cell secretes in the medium) or directly from the host cell producing it (if it is not secreted). Selection of an appropriate host cell will depend on various factors, such as desired expression levels, modifications of the polypeptide that are desirable or necessary for activity (such as glycosylation or phosphorylation) and easy to incorporate into a biologically active molecule. .

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Mammalian cell lines available as hosts for expression 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 hamster ovarian cells. Chinese (CHO), HeLa cells, baby hamster kidney cells (BHK), monkey kidney cells (COS), human hepatocellular carcinoma cells (for example, Hep G2) and a number of other cell lines. In certain embodiments, cell lines can be screened to determine which cell lines possess expression levels and constitutively produce antigen-bound proteins. IL-23. In other embodiments, a cell line may also be selected from the B cell lineage that does not possess its own antibody but has the ability to make and secrete a heterologous antibody.

Use of IL-23 Antigen Bound Proteins for Therapeutic and Diagnostic Purposes

Antigen-bound proteins are useful for detecting IL-23 in biological samples and for identifying cells or tissues that produce IL-23. Antigen-bound proteins that specifically bind to IL-23 can be used in diagnosis and / or treatment of IL-23 related diseases in a

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patient who needs it. For some, the IL-23 antigen-linked proteins can be used in diagnostic tests, for example, binding tests to detect and / or qualify IL-23 expressed in blood, serum, cells or tissue. Furthermore, the antigen-bound 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, the antigen-bound proteins that bind to

IL-23 may have therapeutic use in illnesses related to IL-23.

Indications

The present invention also relates to the use of antigen-bound proteins for use in the prevention or therapeutic treatment of medical disorders, such as those disclosed herein. Proteins bound to the IL-23 antigen are useful in treating a variety of conditions in which 11-23 is associated with or plays a role in contributing to or contributing to the underlying disease or disorder. another way to a negative symptom.

Conditions effectively treated by proteins bound to the IL-23 antigen play a role in the inflammatory response. Such inflammatory disorders include periodontal disease, lung disorders like asthma, skin disorders like psosiaris, dermatitis

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atopic, contact dermatitis, rheumatic disorders like rheumatoid arthritis, progressive systematic sclerosis (scleroderma); systemic lupus erythematosus;

spondyloarthritis including ankylosing spondylitis, psoriatic arthritis, · enteropathic arthritis and reactive arthritis. Uveitis including Vogt-Koyanagi-Harada disease, idiopathic anterior and posterior uveitis, and uveitis associated with spondyloarthritis are also contemplated. The use of the 11-23 antigen-bound proteins is also contemplated for the treatment of autoimmune disorders including multiple sclerosis;

autoimmune myocarditis: type 1 diabetes and autoimmune thyroiditis.

Degenerative conditions of the gastrointestinal system are treatable or preventable with proteins bound to the IL-23 antigen. Such gastrointestinal disorders include inflammatory bowel disease: Crohn's disease, ulcerative colitis, and Celiac disease.

Also included is the use of proteins bound to the antigen in treatments for graft-host disease, and complications such as implant rejection, resulting from organ transplants, such as heart, liver,

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skin, kidney, lung or others before - 1 ^ '- l uy cndu 1'1' · ° · * · bone marrow transplants.

Methods for the use of proteins bound to the IL-23 antigen are also provided in this document to treat various oncological disorders including various forms of cancer including colon, stomach, prostate, renal, cervical and ovarian cancer (SCLC and NSCLC ). Also included are solid tumors including sarcoma, osteosarcoma, and carcinoma, such as adenocarcinoma and squamous cell carcinoma, esophageal cancer, gastric cancer, bladder carcinoma, leukemia, including acute myelogenous leukemia, chronic myelogenous leukemia, myeloid leukemia, leukemia acute lymphoblastic and hairy cell leukemia, and multiple myeloma.

Diagnostic methods

The described antigen-bound proteins can be used for diagnostic purposes to detect, diagnose, or monitor IL-23 associated diseases and / or conditions. Examples of methods useful in detecting the presence of IL-23 include immunoassays, such as the enzyme linked to the immunosorbent test (ELISA) and the radioimmunoassay (RIA).

For diagnostic applications, the protein bound to the antigen will usually be labeled with a

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detectable labeling group. Suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (for example,3H 14C, 15N, 35S, 9 ° Y, ηιΐη, 125I 131I), fluorescent groups (for example,

FITC, rhodamine, lanthanide phosphors), enzyme groups (eg horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminous groups, biotinylated groups, or predetermined polypeptide epitopes recognized by a secondary reporter (eg sequences leucine zipper, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the tagged pool is coupled to the antigen-bound protein through spacer arms of various lengths to reduce the potential spherical hindrance. Various methods for labeling proteins are known in the art.

Other diagnostic methods are provided to identify a cell or cells that express IL-23. In a specific embodiment, the antigen-bound protein is labeled with a labeling set and the IL-23 antigen-bound protein is detected. In a future embodiment, binding of the antigen-bound protein to IL-23 is detected in vivo. . In a future embodiment, the IL-23 antigen-bound protein is isolated and measured using

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methods known in the art. See, for example, Harlow and Lane, 1988, Antibodies: A Laboratory Manual, New York: Coid Spring Harbor (ed. 1991 and periodic supplements); John E. Coligan, ed., 1993, Current Protocols In Immunology New York: John Wiley & Sons.

Other methods are provided to detect the presence of a test molecule that competes for binding to IL-23 with the provided antigen-bound protein. An example of one such test would involve detecting the free amount of antigen-bound protein in a solution containing an amount of IL-23 in the presence or absence of the test molecule. An increase in the free amount of antigen-bound protein (eg, non-IL-23-bound antigen-bound protein) would indicate that the test molecule is capable of competing for an IL-23 binding with the antigen-bound protein. In another embodiment, the antigen-bound protein is tagged with a tag group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of a protein bound to the antigen.

Treatment methods: Pharmaceutical formulas, Administration routes

Pharmaceutical compositions are provided which

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OWNERSHIP comprise a therapeutically therapeutic amount of one or a plurality of antigen-bound proteins and a pharmaceutically acceptable excipient, diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. In addition, methods of treating a patient by administering said pharmaceutical composition are included.

The term patient includes human patients. The terms treat and treatment encompass relief or prevention of at least one symptom or other aspect of a disorder, or reduction of a serious disease, and the like. The term "therapeutically effective amount" or "effective amount" refers to the determined amount of protein bound to the IL-23 antigen to produce any therapeutic response in a mammal. Such therapeutically effective amounts are readily determined by one skilled in the art.

A protein bound to the antigen needs not to influence a complete cure, or eradicate every symptom or manifestation of a disease, to 'constitute a viable therapeutic agent. As recognized in the relevant field, drugs used as therapeutic agents can reduce the severity of the given disease state, but need not suppress every manifestation of the disease in order to be considered as useful therapeutic agents. Similarly, a prophylactically administered treatment

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it does not need to be completely effective in preventing the onset of a condition to be a viable prophylactic agent. Simply reducing the impact of a disease (for example, reducing the number or severity of its symptoms, or increasing the effectiveness of another treatment, or producing another benefit), or reducing the probability that the disease in a subject will occur or worsen, It's enough. Certain methods provided herein comprise administration of an antagonist

11-23 to the patient (such as the antigen-bound proteins disclosed in this document) in an amount and for long enough to induce sustained improvement over the baseline of an indicator that reflects the severity of the particular disorder.

As understood in the relevant field, pharmaceutical compositions comprising the molecules of the invention are administered to the patient in a manner appropriate to the indication. The pharmaceutical compositions can be administered by any appropriate technique, including but not limited to, parental, topical, or inhalation. If injected, the pharmaceutical composition may be administered, for example, intra-articularly, intravenously, intramuscularly, intralesionally, intraperitoneally, or subcutaneously, by bolus injection, or infusion • r —¡Λ <· *

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keep going. Administration., Loca-crippled, for example, at a site of disease or injury, is contemplated, as long as they are transdermal delivery and sustained release from implants. Delivery by inhalation, eg, oral or nasal inhalation, use of a nebulizer, inhalation of the antagonist in the form of an aerosol, and the like. Other alternatives include eye drops, oral preparations including pills, syrups, dragees, or chewing gum; and topical preparations such as lotions, gels, sprays, and ointments. .

The use of antigen-bound proteins in ex vivo procedures is also contemplated. For example, a patient's blood or other body fluid can be connected to an antigen-binding protein 11-23 ex vivo. The antigen-bound protein may be intended for an insoluble matrix or solid support material.

Advantageously, the antigen-bound proteins are administered in the form of a composition composed of one or more additional components as a physiologically acceptable carrier, excipient, or diluent. Optionally, the composition additionally comprises one or more physiologically active agents for combination therapy. A pharmaceutical composition may comprise a protein bound to the IL-23 antigen together with one or more substances.

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selected from the group consisting of a neutralizer. An antioxidant such as ascorbic acid, a low molecular weight polypeptide (such as those with less than 10 amino acids), a protein, an amino acid, a carbohydrate such as glucose, sucrose, or dextrins, a chelating agent such as EDTA, glutathione, a stabilizer , and an excipient. Neutral saline or saline mixed with specific serum albumin are examples of suitable solvents.

According to appropriate industry standards, preservatives like benzyl alcohol can also be added. The composition can be formulated as a lyophilisate using appropriate excipient solutions (eg, sucrose) as solvents. The suitable components are not toxic to the containers in the dosages and concentrations used. Future examples of the components that can be used in pharmaceutical formulas are presented in any of the Remington Pharmaceutical Sciences, including 21 st Ed. (2005), Mack Publishing Company, Easton, PA.

Kits used by professional physicians include a protein bound to the IL-23 antigen and a label or other instruction for use in treating any of the conditions discussed herein. In one embodiment, the kit includes a sterile preparation of one or

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more proteins bound to the IL-23 antigen, which may be in composition form as disclosed above, and may be in one or more vials.

Dosages and frequency of administration may vary according to such factors as the route of administration, the particular antigen-bound proteins employed, 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. Appropriate dosages can be determined by procedures known in the relevant art, for example, in clinical experiments that may involve dose intensification studies.

A typical dosage can be in the range of about 0.1 g / kg to about 30 mg / kg or more, depending on the factors mentioned above. In specific embodiments, the dosage range is from about 0.1 g / kg to about 30 mg / kg, optionally from 1 g / kg to about 30 mg / kg, optionally from 10 g / kg to about 10 mg / kg, optionally 0.1 mg / kg to 5 mg / kg, or optionally 0.3 mg / kg to 3 mg / kg.

The frequency of dosing will depend on the pharmacokinetic parameters of the protein bound to the

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particular human 11-23 antigen in the formula used. Typically, a clinician administers the composition until a dosage reaches the desired effect. Therefore, the composition can be administered as a single dosage, or as two or more dosages (which may or may not contain the same amount of desired molecule) over time, or as a continuous infusion by means of a catheter implantation or device. Appropriate dosages can be determined through the use of appropriate dosage data. A protein bound to the antigen of the invention can be administered, for example, once or more times than one, for example, at regular intervals over a period of time. In particular embodiments, an IL-23 binding protein is administered over a period of at least four months or more, for example, for one, two, or three months or even indefinitely. For the treatment of chronic conditions, long-term treatment is generally mostly effective. However, for the treatment of acute conditions, administration for short periods, for example, one to six weeks, may suffice. In general, the antigen-bound protein is administered until the patient exhibits a medically relevant grade or improvement over baseline for the selected indicator (s).

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If it is contemplated that a protein bound to the IL-23 antigen is administered to the patient in an amount and a time sufficient to induce an improvement, preferably a prolonged improvement, in at least one indicator that reflects the severity of the disorder or condition being treated . Various indicators that reflect the extent of the disease, illness or condition can be evaluated determining if the amount and time of treatment is sufficient. Such indicators include, for example, clinically recognized indicators of disease severity, symptoms, or manifestations of the disorder in question. In one modality, improvement is considered to have been prolonged if the subject shows improvement on at least two occasions two to four weeks apart. The degree of improvement is usually determined by a physicist, who can also use questionnaires that are administered to the subject, such as quality of life questionnaires developed for a given disease.

Particular embodiments of methods and compositions of the invention involve the use of a protein bound to the IL-23 antigen and one or more additional IL-23 antagonists, eg two or more proteins bound to the antigen of the invention, or a protein bound to the antigen of the invention and one or more IL-23 antagonists. In the same way

138 Institutomexica. ^, ' w Industrial provide proteins bound to the IL-23 antigen administered alone or in combination with other agents useful for the treatment of the afflicting condition of the patient. Examples of such agents include both protein and non-protein drugs. Such agents include therapeutic portions that possess anti-inflammatory properties (eg, non-steroidal anti-inflammatory agents, spheroids, immunomodulators, and / or other cytokine inhibitors such as those that antagonize, eg, IFN-γ, GM-CSF, IL-6, IL-8, IL-17, IL-22, and TNFs), or from an antigen-bound protein and one or more other treatments (eg, surgery, ultrasound, or effective treatment to reduce inflammation). When multiple therapists are co-administered, the dosages can be adjusted accordingly, as recognized or known in the art. Useful agents that can be combined with proteins bound to the IL-23 antigen include those used for treatment, for example, Crohn's disease or ulcerative colitis, such as aminosalicylate (for example, mesalazine), corticosteroids (including prednisone), antibiotics such as metronidazole or ciprofloxacin (or other antibiotics useful for the treatment, for example, patients with fistulas), and immunosuppressants such as azathioprine, 6-mercaptopurine, methotrexate, tacrolimus and

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cyclosforin. Said agents can be administered orally or by other means, for example via suppository or enema. Agents that can combine with IL-23 antigenic binding proteins in the treatment of psoriasis include corticosteroids, calcipotriene and other vitamin D derivatives, acetretin and other rethionic acid derivatives, methotrexane, tacrolimus, and cyclosporine used topically or systematic. Said agents can be administered simultaneously, consecutively, alternatively or according to any other regimen that allows the full course of therapy for its effectiveness.

In addition to human patients, 11-23 antigen-bound proteins are useful in the treatment of non-human animals, such as domestic pets (dogs, cats, birds, primates, etc.), farm domestic animals (cattle horses, sheep, pigs, birds, etc.). In such cases, an appropriate dosage can be determined according to the weight of the animal. For example, a dosage of 0.2-1 mg / kg can be used. Alternatively, the dosage is determined according to the surface area of the animal, an example dosage ranges from 0.1-20 mg / m2, or more preferably 5-12 mg / m2. For small animals, such as dogs or cats, an ideal dosage is 0.4 mg / kg. The protein bound to the IL140 antigen

ΐΜ «ΤΓΤυΤθ MEXICANO“ (V LA ΕΧΟΜΕ »*?

INDUSTRIAL (preferably constructed from genes derived from · receptor species) is administered by injection or other suitable route one or more times per week until the condition of the animal has improved, or may be administered indefinitely.

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

EXAMPLES

Example 1

Generation of Human IL-23 Antibody

XenoMouse ™ technology (Amgen, Thousand Oaks, CA) was used to develop human monoclonal antibodies that recognize and inhibit native human IL-23 activity while sparing human IL-12. The antibodies also recognize and inhibit recombinant cynomologous IL-23 but do not recognize murine or rat IL-23.

Antibodies were selected for recognition and complete inhibition of native human IL-23 obtained from human monocyte derived dendritic cells (MoDCs), using the STAT-luciferase reporter assay described below. Human monocytes were isolated from cells

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mononuclear cells from healthy donors using negative selection '(Monocyte II Isolation Kit, Miltenyi Biotec, Auburn, CA). MoDCs were generated by culturing monocytes with human GM-CSF (50 ng / ml) and human IL-4 (100

<td>ng / ml)</td><td>during</td><td>Ί days</td><td>in</td><td>RPMI</td><td>1640 with medium</td><td>full</td><td>of</td>

<td>serum</td><td colspan="2">fetal bovine</td><td>to the</td><td> 10%.</td><td>MoDCs are then</td><td>washed</td><td>two</td>

<td>times</td><td>with PBS</td><td>Following</td><td>by</td><td colspan="3">stimulation with human CD40L</td><td> (1</td>

<td>pg / ml)</td><td>during</td><td colspan="3">about 48 hours</td><td>additional. The</td><td colspan="2">supernatant</td>

CD40L-stimulated MoDC contains IL-23, IL-12, and IL12 / 23p40. ELISAs are used to determine the amount of

IL-12p70 (R&D System, Minneapolis, MN), IL-23 (eBiosciences,

San Diego, CA) and IL-12 / 23p40 (R&D Systems). The STATluciferase assay responds to IL-23 and not IL-12 or free IL-12 / 23p40, therefore the assay can be used with crude supernatants to assess IL-23 activity. For use in the NK cell assay, described below, the crude supernatant

Native human IL-23 was purified using an IL-23 affinity column followed by size exclusion chromatography. Concentration was determined using an IL-23 specific ELISA (eBiosciences).

The purified antibody supernatants were also tested against recombinant human IL-23 (rhu) and recombinant cynomolgous IL-23 (cyno) in the STAT-luciferase assay. Of the proven antibodies that completely inhibit IL-

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Recombinant human, only half of those antibodies recognize and completely inhibit native human IL-23. Recognition and complete inhibition of recombinant human IL-23 was not predicted of, nor correlated for, recognition and complete inhibition of native human IL-23.

As shown in FIGURES IA and IB, of the antibody supernatants that completely inhibit recombinant human IL-23, only half of those antibodies fully inhibit native human IL-23. Those antibodies that fully recognize and inhibit native human IL-23 were selected for further characterization.

EXAMPLE 2

Functional tests

a) STAT-luciferase assay

IL-23 is known to bind its signals and heterodimeric receptor through JAK2 and Tyk2 to activate STAT 1,

3, 4 and 5. In this assay, cells transfected with a STAT / luciferase reporter gene are used to assess the ability of IL-23 antibodies to inhibit IL-23-induced bioactivity.

Chinese hamster ovary cells expressing the human IL-23 receptor are transiently transfected with STAT-luciferase reporter overnight. Antibodies

IL-23 are serially diluted (12 points of 1: 4 serial dilutions starting at 37.5 pg / ml) in 96-well plates. Native human IL-23 (the method of preparation described in Example 1) is added to each well at a concentration of 2 ng / ml and incubated at room temperature for 15-20 minutes. Transiently transfected cells aggregate (8x103 cells) at a final volume of 100 μΐ / ροζο and incubate for 5 hours at 37 ° C, 10% CO2. After incubation, cells are used using 100 pL / well of Glo lysis buffer (1x) (Promega, Madison, Wisconsin) at room temperature for 5 minutes. Fifty microliters of cell lysate is added to a 96-well plate along with 50 pL of Bright-Glo luciferase substrate (Promega) and read on a luminometer.

Statistical analysis can be performed using software

GraphPad PRISM (GraphPad Software, La Jolla, CA). Results can be expressed as the mean ± standard deviation (SD).

As shown in TABLE 5, all IL23 antibodies potently and completely inhibit the reporter

STAT / luciferase induced by native human IL-23 in a dose dependent manner. The antibodies also potently and completely inhibit recombinant human IL-23 (rhu) and recombinant IL-23 cyno (cyno). Antibodies all

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144 _ have IC values50 in the picomolar interval.

TABLE 5. Table of mean IC50 values (pM) for antibodies

IL-23 in the STAT-luciferase assay.

<td></td><td colspan="2">huIL-23 native</td><td colspan="2">rhuIL-23</td><td colspan="2">IL-23 Cyno</td>

<td>Antibody</td><td>IC50 +/- SD</td><td>Repeat- tions</td><td>IC50 +/- SD</td><td>Repeat- tions</td><td>IC50 +/- SD</td><td>Repeat- tions</td>

b) NK cell assay

IL-23 is known to act on natural killer cells to induce proinflammatory cytokine expression, such as inferred γ (IFN γ). In this assay, human primary natural killer (NK) cells are used to assess the ability of IL-23 antibodies to inhibit IFNy activity induced by

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IL-23 in cells expressing the native receptor for human tt.-9 7.

NK cells are isolated from multiple human donors by negative selection (NK Cell Isolation Kit, Miltenyi Biotec, Auburn, CA). Purified NK cells are added (1 x 106 cells / ml) to 6-well plates in RPMI 1640 plus complete medium of 10% fetal bovine serum supplemented with recombinant human IL-2 (lOng / ml, R&D

Systems, Minneapolis, MN), at a final volume of 10ml / well.

Cells are cultured for 1 day at 37 ° C, CO2 at 5%. IL-2 activated NK cells are then stimulated with rhulL-23 or IL-23 cyno (10 ng / ml) and recombinant human IL-18 (20ng / ml, R&D Systems, Minneapolis, MN) in the presence of serial dilutions (11 points of 1: 3 dilutions in series starting at

3pg / ml) of IL-23 antibodies for 24 hours. IFNy levels in the supernatant are measured by ELISA IFNy (R&D Systems,

Minneapolis, MN) according to the manufacturer's instructions.

Statistical analysis can be performed using software

GraphPad PRISM. Results can be expressed as the mean ± standard deviation (SD).

As seen in TABLE 6, all antibodies potently inhibit IL-23 cyno and rhulL-23 induced IFNy expression in NK cells in a dose dependent manner.

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Antibodies all have IC values50 in the picomolar interval. The assay was performed on a subset of antibodies using native human IL-23 (30pg / ml, the preparation method is described in Example 1) and rhulL-18 (40ng / ml, R&D Systems) and provided the results shown in TABLE 6. Consistent with selection for specific IL-23 antibodies, these anti-IL-23 antibodies have no effect on IL-12-stimulated IFN production in cells.

NK using the assay described above, while a specific neutralizing antibody IL-12p35, mAb219 (R&D

Systems, Minneapolis, MN) potently inhibits recombinant human IL-12.

TABLE 6. Table of mean IC50 values (pM) for antibodies

IL-23 in the NK cell assay.

<td></td><td colspan="2">huIL-23 native</td><td colspan="2">rhuIL-23</td><td colspan="2">IL-23 Cyno</td>

<td>Antibody</td><td>ic50+/- sd</td><td>Repeat- tions</td><td>ic50+/- sd</td><td>Repeat- tions</td><td>IC50 +/- SD</td><td>Repeat- tions</td>

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c) Human Whole Blood Test

Human whole blood is collected from multiple healthy donors using Refludan® (Bayer Pittsburgh,

PA) as an anti-coagulant. The final concentration of

Refludan® in whole blood is 10 pg / ml. A stimulation mixture of rhulL-23 or cyno.IL-23 (final concentration ng / ml) + rhulL-18 (final concentration 20 ng / ml) + rhulL-2 (final concentration 5 ng / ml) in RPMI 1640 + FBS 10% added to a 96-well plate, final volume 20 μΐ / ροζο. Serially diluted IL-23 antibodies (11 points of 1: 3 serial dilutions starting from 3pg / ml) are added in 20pl / well and incubated with the stimulation mixture for 30 minutes at room temperature. Then whole blood (120 µΐ / ροζο) is added and the final volume is adjusted to 200 µΐ / ροζο with RPMI 1640 + 10% FBS. The final concentration of whole blood is 60%. Plates are incubated for 24 hours at 37 ° C, CO2 at 5%. Cell-free supernatants are harvested and IFNy levels of the supernatants are measured by IFNy ELISA (R&D Systems) according to the manufacturer's instructions.

Statistical analysis can be performed using /

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GraphPad PRISM software. The results pü'gggn ..... • ggpTg'ga'T5g as the mean ± standard deviation (SD).

As seen in TABLE 7, all antibodies potently inhibit rhulL-23-induced and cyno-IL-23-induced IFNy expression in whole blood cells in a dose-dependent manner. Antibodies all have IC values50 in the picomolar interval.

TABLE 7. Table of IC values50 antibody media (pM)

IL-23 in the IFNy human whole blood assay

<td></td><td colspan="2">rhuIL-23</td><td colspan="2">IL-23 Cyno</td>

<td>Antibody</td><td>IC50+/- SD</td><td>Reps</td><td>IC50 +/- SD</td><td>Reps</td>

d) IL-22 assay

IL-23 is known to be a potent inducer of proinflammatory cytokines. IL-23 acts on activated and memory T cells and promotes the survival and expansion of Thl7 cells that produce proinflammatory cytokines including IL22. In this assay, human whole blood is used to assess the ability of IL-23 antibodies to inhibit

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IL-23 induced IL-22 production.

A whole blood assay is conducted in the same manner as described above with the modification of using rhulL-23 or cynolL-23 at 1 ng / ml and rhulL-18 at lOng / ml to induce IL-22 production. IL-22 concentration is determined by ELISA IL-22 (R&D Systems, Minneapolis, MN).

As seen in TABLE 8, the antibodies potentially inhibit rhulL-23-induced and cyno IL-23-induced IL-22 production in whole blood cells in a dose-dependent manner. The antibodies all have IC50 values in the picomolar range.

TABLE 8. Table of IC values50 antibody media (pM)

IL-23 in the IL-22 human whole blood assay

<td></td><td colspan="2">rhulL-23</td><td colspan="2">IL-23 Cyno</td>

<td>Antibody</td><td>IC50 +/- SD</td><td>Reps</td><td>ic50+/- sd</td><td>Reps</td>

Example 3

Determine the Equilibrium Dissociation Constant (KD) for Anti-IL-23 Antibodies Using KinExA Technology

The binding affinity of rhulL-23 for IL-23 antibodies is evaluated using a kinetic exclusion assay

150 (KinExA trial, Sapidyne Instruments, T nr, B oit C7 — ID), Sepharose 4 rapid flow beads activated with normal human serum (NHS) (Amersham Biosciences, part of GE Healthcare, Uppsala, Sweden), are precoated with rhulL-23 and blocked with 1 m Tris buffer with lOmg / mL of

BSA. 50pM of IL-23 antibody is incubated with rhulL-23 (12 points of 1: 2 dilutions starting from 800 pM) at room temperature for 72 hours before it is run through the rhulL-23 coated Sepharose beads. The amount of the bead-bound antibody is quantified by fluorescently labeled goat anti-human-Fc antibody (Cy5) (Jackson Immuno Research, West Grove, Pa.). The binding signal is proportional to the amount of free antibody in equilibrium.

The dissociation equilibrium constant (KD) and the association speed (Kon) are obtained from a curve fit using KinExA Pro software. The dissociation rate (Koff) is derived from: KD = Koff/ Kon

As seen in TABLE 9, the antibodies have high affinity for binding to human IL-23. They all have K valuesD in the low range for sub pM.

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TABLE 9. Table of speeds KD (pM), Kon (1 / MS) and Koff (1 / s)

<td>Antibody</td><td>KD (pM)</td><td>Kon (1 / MS)</td><td>Koff (1 / s)</td>

Example 4 1θ Structure determination using X-ray Crystallography

One way to determine the structure of an antibody antigen complex is by using ray crystallography.

X, see for example, Harlow and Lañe Antibodies: A Laboratory

Coid Spring Harbor Laboratory Press Manual, Coid Spring - * - 5 Harbor, NY (1990), p.23. The crystal structure of IL-23 has been determined, (see Lupardus and García, J Mol Biol, 2008,

382: 931-941) and the crystal structure of an IL23 / Fab complex has been described, (see Beyer et al. J Mol Biol, 2008.

382 (4): 942-55). The structural determination of

IL-23 with Fab fragments of antibodies claimed herein using X-ray crystallography.

Protein for crystallization

A human IL-23 heterodimer was used recombinantly »·

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derived for crystallization studies (see Beyer et al., supra). The sequence of the human pl9 subunit comprises 20-189 residues of SEQ ID NO: 145, the signal sequence of SEQ ID NO: 154 and a C-terminal 6-His tag SEQ ID NO: 155. The sequence of the subunit Human p40 was mutated from asparagine to glutamine at position 222 of SEQ ID NO: 147 in order to prevent glycosylation at this site (Beyer, et al., supra).

Fabs derived from Antibody B and Antibody E were expressed on an IgGl scaffold incorporating a caspase cleavage site. Fabs were processed by protease cleavage.

Complex formation and crystallization

The IL-23-Antibody B Fab complex was made by mixing a 2X molar excess of the 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 -12 mg / ml for crystallization. The IL-23-Antibody B Fab complex crystallizes from 0.1 M Hepes pH 7, 8% PEG 8000.

The IL-23-Antibody E Fab complex was made by mixing a 2X molar excess of the Antibody E Fab with the human heterodimeric IL-23 described above. The complex is methyl

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using a JBS Methylation Kit according to Ins manufacturer instructions (Jena Bioscience, Jena, Germany). The complex was then treated with PNGase to de-glycosylate the protein. After these treatments, the complex was purified by size exclusion chromatography to remove excess Fab from Antibody E and concentrated to 13.5 mg / ml for crystallization. The Fab complex of IL-23-Antibody E crystallized in 0.1 M Tris pH 8.5, 0.2 M magnesium chloride, 15% PEG 4000.

Data collection and structure determination

The Fab crystals of IL-23-Antibody B grow in the group of space P2i with the unit cell dimensions a = 70.93, b = 71.27, c = 107.37 Ά, β = 104.98 ° and diffract at 2.0 Á resolution. The Fab structure of IL-23-Antibody B was resolved by molecular replacement with the MOLREP program (CCP4, The CCP4 suite: programs for protein crystallography.

Acta Crystallogr D Biol Crystallogr, 1994. 50 (Pt 5): p. 7603) using the IL-23 structure (Beyer et al. Supra) as the starting search model. By keeping the IL-23 solution fixed, an antibody variable domain was used as a search template. By keeping the IL-23-antibody variable domain solution fixed, an antibody constant domain was used as a search template. The complete structure

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improved with multiple rounds of model building with

Quanta and refinement with cnx (Brunger, et al., Acta Crystallogr D Biol Crystallogr, 1998, 54 (Pt 5): p. 905-21).

The distances between the protein atoms were calculated using the PyMOL program (DeLano, WL The PyMOL Graphics System. Palo Alto, 2002) (Schrodinger, LLC; New York, NY)). Amino acids were chosen if at least one atom was within the threshold distance required for the companion protein.

The boundaries of the A, B, C and D helices of the pl9 subunit of IL-23 when binding the Antibody B Fab include

28-47 helix A residues, 86-105 helix B residues, 119 134 of helix C residues and 154-187 of helix D residues of SEQ ID NO: 145.

Interaction regions in the IL-23pl9 subunit when linked to the Antibody B Fab include residues within Ser46-Glu58, Glull2-Glul23 and Prol55-Phel63 of SEQ ID.

NO: 145.

IL-23pl9 subunit amino acid residues with 4A atoms or less from the Antibody B Fab include Ser4 6, Ala47, Hís48, Prc49, Leu50, His53, Met54, Asp55, Glu58, Prol

13, Serll4, Leull5, Leull6, Prol20, Vall21, Trpl56, Leul59,

Leul60, Argl62 and Phel63 of SEQ ID NO: 145. The IL-23pl9 amino acid residues with atoms between 4 Á and 5 Á of the Fab of

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Antibody B include Val51, Arg57, Glull2, Aspll8, Serll9,

Glnl23, Prol55 of SEQ ID NO: 145.

IL-23p40 subunit amino acid residues with 4A atoms or less from the Antibody B Fab include Glul22 and

Lysl24 of SEQ ID NO: 147.

Antibody B Fab heavy chain amino acid residues with 4A atoms or less of the IL-23 heterodimer include Gly32, Gly33, Tyr34, Tyr35, His54, Asn58, Thr59,

Tyr60, Lys66, ArglOl, Glyl02, Phel03, Tyrl04 and Tyrl05 from SEQ

ID NO: 46. Antibody B Fab heavy chain amino acid residues with <5A atoms of the IL-23 heterodimer include Ser31, Gly32, Gly33, Tyr34, Tyr35, His54, Ser56,

Asn58, Thr59, Tyr60, Lys66, ArglOl, Glyl02, Phel03, Tyrl04 and

Tyrl05 from SEQ ID NO: 46.

Fab light chain amino acid residues of

Antibody B with 4A atoms or less of the IL-23 heterodimer include Ser30, Ser31, Trp32, Tyr49, Ser52, Ser53, Ala91,

Asn92, Ser93, Phe94, and Phe96 of SEQ ID NO: 15. Fab light chain amino acid residues of Antibody B with <5A atoms of the IL-23 heterodimer include Ser30, Ser31, Trp32, Tyr49, Ala50, Ser52, Ser53, Ser56, Ala91, Asn92,

Ser93, Phe94, and Phe96 of SEQ ID NO: 15.

Crystals of the Fab complex of IL-23-Antibody E grow in the group of space P222i with cell dimensions

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unit a = 61.60, b = 97.59, c = 223.95 Á and are diffracted for resolution 3.5 Á. The Fab complex structure of IL-23Antibody E was resolved by molecular replacement with the Phaser program (CCP4, supra) using the IL-23 structure, an antibody variable domain, and an antibody constant domain as the three search models for start, as described above. The entire structure was enhanced with multiple rounds of model construction with Quanta and refinement with cnx (Brunger, et al., Supra). The Fab constant domain of Antibody E was left out of the final refined structure due to each poor electron density for such a portion of the protein.

Interaction regions in the IL-23pl9 subunit identified when binding to the E Antibody Fab include residues within Ser46-His53, Glul12-Vall20 and Trpl56-Phel63 of SEQ ID NO: 145.

IL-23pl9 amino acid residues with 4A atoms or less from the E Antibody Fab include Ser46, Ala47, His48, Pro49, Leu50, Glull2, Proll3, Serll4, Leull5, Leull6, Proll7, Aspll8, Serll9, Prol20, Trpl56, Leul59 , Leul60 and Phel63 of SEQ ID NO: 145. The IL-23pl9 amino acid residues with atoms between 4 Á and 5 Á of the Antibody E Fab include His53 of SEQ ID NO: 145.

IL-23p40 amino acid residues with 4A atoms or

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minus Fab of Antibody E include Lysl21J__. Qlul22-r-AND!-roi23 · and Asnl25 of SEQ ID NO: 147.

Antibody E Fab heavy chain amino acid residues with 4A atoms or less of the IL-23 heterodimer include Gly26, Phe27, Thr28, Ser31, Tyr53, Tyr59, Tyrl02, Serl04, Serl05, Trpl06, Tyrl07, and Prol08 from SEQ ID N0: 31. The Fab heavy chain amino acid residues of Antibody E with <5A atoms of the IL-23 heterodimer include Glnl, Gly26, Phe27, Thr28, Ser30, Ser31, Tyr32, Trp52, Tyr53, Tyr59,

ArglOO, Tyrl02, Thrl03, Serl04, Serl05, Trpl06, Tyrl07, and

Prol08 of SEQ ID NO: 31.

Fab light chain amino acid residues of Antibody E with 4A atoms or less of the IL-23 heterodimer include Ala31, Gly32, Tyr33, Asp34, Tyr51, Gly52, Asn55,

Lys68, and Tyr93 of SEQ ID NO: 1. Fab light chain amino acid residues of Antibody B with <5A atoms of the IL-23 heterodimer include Thr29, Ala31, Gly32, Tyr33, Asp34,

Tyr51, Gly52, Asn55, Lys68, Tyr93, and TrplOO of SEQ ID NO: 1.

Example 5

Determination of Contact Residues of IL-23Antibody Complex through Surface Area Differences

Solvent Accessible

The residue is contacted in the paratope (the portion

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of the antibody recognizing the antigen) and the portion of the antigen that binds the binding through the paratope in a Fab complex of human IL-23-Antibody B and in a Fab complex of human IL-23 Antibody E were determined using accessible surface area differences solvent. Solvent accessible surface area calculations were performed using Molecular Operating Environment (Chemical Computing Group, Montreal,

Quebec).

Differences in solvent accessible surface area of the paratope residues in the Fab complex of IL-23 Antibody B were calculated by adjusting the Fab residues of

Antibody B as the desired setting. The structural information obtained in Example 4 for the Fab complex of IL-23Antibody B was used and the solvent accessible surface area of the residue from the amino acid residues of the Antibody Fab.

B in the presence of the IL-23 heterodimer were calculated and plotted the binding areas for fit.

The solvent accessible surface area of the residue from each of the Antibody B Fab residues in the absence of IL-23 antigen were calculated and plotted free areas.

The bonding areas were then subtracted from the free areas resulting in the difference in surface area exposed to the solvent for each residue in the fit. The

IMPI ιηπτπ.τό Mexican OF THE INDUSTRIAL FRCtWAO Antibody B Fab residues that have not changed in surface area, or a zero difference, do not have contact with the residues of the IL-23 antigen when complexed. The Fab residues of Antibody B that have a difference value> 10 A2 were considered to be in significant contact with residues in the IL-23 antigen such that these Antibody B Fab residues were at least partially to completely occlude when the 'Antibody B Fab bound to human IL-23. This adjustment of Antibody B Fab residues makes the patch coat, the residues involved in the interface structure when the Antibody B Fab binds to human IL-23, see Tables 10 and 11. Fab residues of Antibody B in this coat patch cannot be involved in bound interactions with residues of the IL-23 antigen, but mutation of any single residue within the coat patch can introduce energetic differences that must impact binding

Fab from Antibody B to human IL-23. With the exception of Tyr49, all of the residues' are located in the CDR regions of the Fab light and heavy chains of Antibody B. These residues were also within 5A or less of the 11-23 antigen when binding to the Fab of Antibody B, as described in

Example 4.

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Table 10. Differences in Surface Area -of ... Solvent Accessibility for Fab Light Chain of Antibody B

<td>AHO number of waste</td><td>Waste Position SEQ ID NO: 15</td><td>Difference of the surface area exposed to the solvent (Á2)</td>

Table 11. Differences in Solvent Accessibility Surface Area

<td>AHO number of waste</td><td>Waste Position SEQ ID NO: 46</td><td>Difference of the surface area exposed to the solvent (Á2)</td>

Differences in accessible surface area of

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Solvent residues in the Fab complex of IL-23Antibody E were calculated as described above. The Fab residues of Antibody E having a difference value £ 10 Á2 se. were considered to be in major contact with residues in the IL-23 antigen and those Antibody E Fab residues were at least partially to completely occlude when the Antibody E Fab bound to human IL-23. This Fab residual adjustment of

Antibody E makes the cover patch, the residues involved in the interface structure when the Fab's

Antibody E binds to human IL-23, see Tables 12 and 13.

The Fab residues of Antibody E in this coat patch cannot be involved in bound interactions with residues of the .IL-23 antigen, but mutation of any single residue within the coat patch can introduce energetic differences that must impact the Fab binding of E antibody to human IL-23. For the most part, these cover patch residues were located within the CDR regions of light and heavy chains.

Antibody E Fab. These residues were also within 5A or less of the IL-23 antigen when bound to the E Antibody Fab, as described in Example 4.

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Table 12. Differences in Solvent Accessibility Surface Area for Fab Light Chain of Antibody E

<td>AHO number of waste</td><td>Waste Position SEQ ID NO: 1</td><td>Difference of the surface area exposed to the solvent (Á2)</td>

Table 13. Differences in Solvent Accessibility Surface Area for Heavy Chain Fab of Antibody E

<td>AHO number of waste</td><td>Waste Position SEQ ID NO: 31</td><td>Difference of the surface area exposed to the solvent (Á2)</td>

163

Differences in solvent surface area — trceesi'ble · ο · ί of the IL-23 heterodimer binding portion by the antibody B Fab paratope were calculated by adjusting the IL-23 heterodimer residues as the desired fit. The structural information obtained in Example 4 for the Antibody B Fab-IL-23 complex was used and the solvent accessible surface area of the residue of the amino acid residues of the IL-23 heterodimer in the presence of the Antibody B Fab were calculated and represent the binding areas for adjustment.

The solvent accessible surface area of the residue from each of the IL-23 heterodimer residues in the absence of the Antibody B Fab were calculated and plotted the free areas of the fit.

As described above, the binding areas were subtracted from the free areas resulting in the difference in surface area exposed to solvent for each IL23 residue. IL-23 heterodimer residues that have no change in surface area, or a zero difference, have no contact with residues of the Antibody B Fab when complexed. The IL-23 heterodimer residues having a difference value ^ 10 Á2 were considered to be in significant contact with residues of the Antibody B Fab and these residues of the 11-23 heterodimer were

164 at least partially to occlude human IL-23 heterodimer bound

ΙΜΡΪ

MEXICAN INSTITUTE

PIP THE PPOP1ΕΓΑ1 '

INDUSTRIAL fully when the Fab of Antibody B.

This adjustment of residues of the IL-23 heterodimer makes the envelope patch, the residues involved in the interface structure when the human IL-23 heterodimer binds to the E Antibody Fab, see Table 14. The heterodimer residues

11-23 in this cover patch not everyone can engage in residue-bound interactions on the Fab of

Antibody B, but mutation of any single residue within the envelope patch may introduce energetic differences that must impact the Antibody Fab binding

B to human IL-23. These residues are also within 4A or less of the Antibody B Fab, as described in the Example.

IMPI

MEXICAN INSTITUTE Say LA «OÍIEOAIΊ

INDUSTRIAL

165

Table 14. Solvent Accessibility Surface Area Differences for IL-23 heterodimer residues

<td>Waste p19 (SEQ ID NO: 145)</td><td>Difference of surface area exposed to solvent (Á2)</td>

<td>waste p40 (SEQ ID NO: 147)</td><td></td>

Differences in the solvent accessible surface area of the IL-23 heterodimer binding portion by the Antibody E Fab paratope were calculated as described above. Residues of the IL-23 heterodimer that have a difference value ^ 10 Á2 were considered to be in significant contact with residues of the Fab of

Antibody E and these residues of heterodimer 11-23 were at least partially to completely occlude when the

166 τΡΙ. . «- VU * A Κ} ΓΊ

Human IL-23 heterodimer bound to Falg_ Ha. Mtiouiipu ET this residue fit of the IL-23 heterodimer made the patch coat, the residues involved in the interface structure when the human IL-23 heterodimer binds to the E-Antibody Fab, see Table 15. The heterodimer residues

11-23 in this cover patch not everyone can engage in residue-bound interactions on the Fab of

Antibody E, but mutation of any single residue within the · patch coat can introduce energetic differences that must impact the Fab binding of Antibody E to human IL-23. These residues are also within 5A or less of the E Antibody Fab, as described in the Example.

167 iN '+ tT'.m miwcaí *;

JWWJWRiAi

Table 15. Solvent Accessibility Surface Area Differences for IL-23 heterodimer residues.

<td>residues p19 (SEQ ID NO: 145)</td><td>Difference of surface area exposed to solvent (Á2)</td>

<td>waste p40 (SEQ ID NO: 147)</td><td></td>

IMPI

-ir OR MEXICAN INSTITUTE

168 OF THE «OHEDAP

INDUSTKIAL

NOVELTY OF THE INVENTION _________

Having described the present invention, it is considered as a novelty, and therefore the content of the following is claimed as property:

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94 members in 37 offices

This recordMember, by publication datePriority claim27 more offices are in the list below

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Priority claims
Document	Office	Kind	Date
25498209	United States of America	P	2009-10-26
25498209	United States of America	P	2009-10-26
61254982	United States of America	–	2009-10-26
38128710	United States of America	P	2010-09-09
38128710	United States of America	P	2010-09-09
61381287	United States of America	–	2010-09-09
2010054148	United States of America	W	2010-10-26
2010054148	United States of America	W	2010-10-26
61254982	–	–	–
61381287	–	–	–
PCTUS2010054148	–	–	–
US20090254982P	–	–	–
US20100381287P	–	–	–
WO2010US54148	–	–	–

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