Anti-il-17f antibodies and methods of use thereof.

In accordance with article 23 of the Law of the Propk from the date of submission of the application and be.

lustriaí, this patent is valid for twenty years, non-extendable, counted at the rate of the fee to keep the rights in force. '

Whoever signs this title has done so based on louks ^ uestt by | ps aSicultte 6 ° fractions III and 7 ° bis 2de4a4toy of Industrial Property (Official Gazette of the Federation (D..OF) 06/27/1991, amended 02708 / 1994, ^ / 10/1996, 26/12/1997, 17/05/1999, 26/01/2004, 16/06/2005, 25/01/2006, 06/05 / 2009,06 / 01/2010 , 49/0 ^ 2010, 06/28/2010, M / 27/2012 and 04/08/2012> articles 1^or, 3^or fraction V part a), 4^or and 12th sections I and III of the Regulations of the Mexican Institute of Industrial Property (Ó OF 1 * 12/1999, amended on 0W7 / 2Q02, 07/15/2004 07/28/2004 and 09/07/2007); items 1^or, 3’, 4^or, 5^or fraction V subsection a), 16 fractions I and lll and 30 <M Organic Statute of the Mexican Institute of Industrial Property (DOF

12/27/1999, amended 10/10/2002, 07/29/2004, 04-708 / 2Cl04 - ,. and 09/13/20Q7); .1, 3^or and 5^or Subsection a) of the Agreement that delegates powers to the Deputy General Directors, Coordinator, Dtvmonales Directors, Times <| e tas OOdnac Itegeeales, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Mexican Institute of Industrial Property: · (DOF 12/19 / 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 lll, 2 fraction V, 26 BIS and 26 TER of the Agreement establishing the guidelines for the use of the Payment and Electronic Services Portal (PASE) of the Mexican Institute of Industrial Property, in the procedures indicated.

THE DIVISIONAL DIRECTOR OF PATENTS

NAHANNY CANAL REYES

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NAHANNY MARISOL CANAL REYES | 00001000000403252793 | Administration Service

Tax | 1695 || MX / 2017/41441 MX / a / 2014/012530 | Normal patent title with divisional PCT | 1220 | RRGO | Page (s) 1 | iunSIAxdd4qE4R2oW1 NvHzlj + Go =

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Arenal No 550 Floor 1, Pueblo Santa María Tepepan. Xochimilco, 16020. Or age of Mexico (55) 53340700 www.gob.mx/impi

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ANTIBODIES ΑΝΤΙ IL-17F AND METHODS OF USE · DE ”

Field of the invention

This invention relates generally to the 5th generation of monoclonal antibodies, eg. , fully human inonoclonal antibodies, which recognize IL-17F but do not recognize IL-17A, to monoclonal antibodies, eg. , fully human monoclonal antibodies, which. recognize the heterodimeric IL-17A / IL-17F complex, and methods for using monoclonal antibodies as therapeutic agents.

Background of the invention

IL-17F (also known as ML-1) is a member of the IL-17 family of cytokines, which also includes the proteins IL-17A (also known as CTL-8, IL-17), IL-17B, IL -17C, IL-17D, IL-17E (also called IL-25). Both IL-17A and IL-17F are secreted as disulfide-linked homodimers that signal through receptors IL-17R, IL-17RC, or a multimeric receptor complex composed of IL-17R and IL-17RC. Also, both are co-expressed in the same subsets of T cells (mainly by Thl7 CD4 T cells ⁺ ). IL17A and IL-17F also interact and form a heterodimeric IL17A / IL-17F complex.

Elevated levels of IL-17F and IL2 complexes

<img file="MX348013B_D0005.tif" />

IMPI

INSTITUTO MÍXJCANO nt la n * ori £ iMD fNDVSTWIAL

17A / IL-17F have been associated with inflammatory diseases and diseases to a variety of autoimmune disorders. Accordingly, there is a need for therapies that neutralize the biological activities of IL-17F.

Summary of the invention

The present invention provides monoclonal antibodies such as fully human monoclonal antibodies that specifically bind to the IL-17F and / or IL-17A / IL-17F heterodimeric complex, but do not specifically bind IL-17A. IL-17F is typically expressed and biologically active as a heterodimeric protein. Therefore, the use of the term IL-17F and equivalents thereof refers to the heterodimeric protein IL-17F, except where otherwise indicated. The antibodies of the invention are capable of modulating, eg. , block, inhibit, reduce, antagonize, neutralize, or interfere with IL-17F-mediated pro-inflammatory cytokines and / or chemokine production.

Examples of monoclonal antibodies of the invention include, for example, the 5E12 antibody, the 41B10 antibody, the 11C5 antibody, the 21B10 antibody, the 1F1 antibody, and the 2E12 antibody. Alternatively, the monoclonal antibody is an antibody that binds to the same epitope as the 41B10 antibody, the 11C5 antibody, the 21B10 antibody, the 1F1 antibody, the 2E12 antibody, the 5D3 antibody, the 22F8 antibody, the

IMPI

ΓΝΗΤΤΙ / ΤΟ MEXICAN * LA PHOmnAB fNDUmiAl

<img file="MX348013B_D0006.tif" />

28B11 antibody, 41A4 antibody and 43G6 antibody. These antibodies are referred to herein as huIL antibodies respectively.

17F. The huIL-17F antibodies include fully human monoclonal antibodies, as well as humanized monoclonal antibodies and chimeric antibodies.

Preferably, the fully human monoclonal antibody is selected from antibody 11C5, antibody 21B10, antibody 1F1, antibody 2E12, antibody 5D3, antibody 22F8, antibody 28B11, antibody 41A4, and antibody 43G6. These antibodies exhibit greater affinity for the heterodimeric complex IL-17F and / or IL-17A / IL17F than other antibodies that bind to the heterodimeric complex IL-17F and / or IL-17A / IL-17F, such as, for example, 5E12 antibody and 41B10 antibody. These antibodies are better inhibitors of at least one biological activity or function of IL-17F than other antibodies that bind to the heterodimeric IL-17F and / or IL-17A / IL-17F complex, such as, for example, the 5E12 antibody and the 41B10 antibody. For example, antibody 11C5, antibody 21B10, antibody 1F1, antibody 2E12, antibody 5D3, antibody 22F8, antibody 28B11, antibody 41A4, and antibody 43G6 inhibit a biological activity and / or function of IL-17F. to a greater degree than the 5E12 antibody and / or the

<img file="MX348013B_D0007.tif" />

IMPI κττττντο wmcxNx M LA nrWOAD IMDUmtA * antibody 41B10. In some embodiments, the 11C5 antibody, the 21B10 antibody, the 1F1 antibody, the 2E12 antibody, the 5D3 antibody, the 22F8 antibody, the 28B11 antibody, the 41A4 antibody, and the 43G6 antibody decrease the production of a pro-inflammatory cytokine in the presence of these. antibodies to a greater degree than decreased pro-inflammatory cytokine production in the presence of other antibodies that bind to the heterodimeric complex IL-17F and / or

IL-17A / IL-17F, such as, for example, the 5E12 antibody and / or the 41B10 antibody. For example, the level of pro-inflammatory cytokine production (eg. , IL-6) in the presence of antibody 11C5, antibody 21B10, antibody 1F1, antibody 2E12, antibody 5D3, antibody 22F8, antibody 28B11, antibody 41A4 and antibody 43G6 is greater than or equal to 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100% less than the production level of pro-inflammatory cytokine of other antibodies that bind to the heterodimeric complex IL-17F and / or IL-17A / IL17F, such as, for example, the 5E12 antibody and / or the 41B10 antibody.

These antibodies show specificity for human IL-17F and / or the heterodimeric complex IL-17A / human IL-17F, and have been shown to inhibit IL-17F mediated cytokine production. These antibodies have defined specificities. In some embodiments, the hu! L-17F antibodies of the invention bind in a

IMPI Mexican institute DE LA&gt; l »Specific OFFICE '^NEITHERX '^s11I * l -

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or do not specifically bind to tt.-T '/ h r'i m jnimf nranri a ._ The huIL-17 antibodies of the invention also specifically bind to the IL-17F homodimer, but do not bind specific to IL-17A homodimer. In some embodiments, the huIL-17F antibodies of the invention specifically bind to the heterodimeric IL-17A / IL17F complex, but do not specifically bind to IL-17A or the IL-17A homodimer. In some embodiments, the huIL-17F antibodies of the invention specifically bind IL17F and the IL-17A / IL-17F heterodimeric complex, but do not specifically bind IL-17A or IL-17A homodimer. For example, antibodies 11C5, 21B10, 1F1, 2E12, 41B10, 5D3, 22F8, 28B11, 41A4, and 43G6 bind specifically to IL-17F, and these antibodies do not bind to IL-17A or the IL-17A homodimer. . Preferably, huIL-17F antibodies bind IL-17F and do not cross-react with IL-17A or the IL-17A homodimer. For example, 5E12, 11C5, 21B10, 1F1, 2E12, 41B10, 5D3, 22F8, 28B11, 41A4, and 43G6 bind IL-17F but do not cross-react with IL-17A.

Fully human antibodies of the invention contain a heavy chain variable region having the amino acid sequence of SEQ ID NO. : 10, 14, 18, 22, 26, 30, 34, 38 and 42. The human antibodies of the invention contain a light chain variable region having the

IMPI [ΝΓΓΓΤνΤΌ MUtICAJtO OtUMOriBDAD amino acid sequence of SEQ ID NO: 12,, '*^h2 ^ t⁵*2 8,

32, 36, 40 and 44. The heavy chain CDRs imulupeil UTld leqlÓff * CDR1 of V_H comprising an amino acid sequence of at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to the sequence selected from the group consisting of SEQ ID NO: 45, 48, 51, 56, 59 , 64, 67 and 70; a CDR2 region of V_H comprising an amino acid sequence of at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NO: 46, 49, 52, 54, 57 , 60, 62, 65, 68, 71 and 73; and a VH CDR3 region comprising an amino acid sequence of at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NO: 47, 50 , 53, 55, 58, 61, 63, 66, 69 and 72. The three light chain CDRs include a VL CDR1 region comprising an amino acid sequence of at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NO: 74, 77, 80, 85, 88, 91 and 94; a CDR2 region of VL comprising an amino acid sequence of at least 90%, 92%, 95%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NO: 75, 78, 81, 83, 86, 89, 92 and 96; and a VL CDR3 region comprising an amino acid sequence of at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NO: 76, 79 , 82, 84, 87, 90, 93, 95, 97, 98 and 99.

<img file="MX348013B_D0009.tif" />

IMPI

INSTITUTO MIXICAMO DI U ncPIEDAD IFQHJSTUAA

Preferably, the peaa ^ or ^ Relnyen- nner chain CDRs CDR1 region of VH comprising an amino acid sequence of at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NO: 45, 48, 51, 64, 67 and 70; a VH CDR2 region comprising an amino acid sequence of at least 90%, 92%, 95%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NO: 46, 49, 52, 54, 62, 65, 68, 71 and 73; and a CD3R region of VH comprising an amino acid sequence of at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NO: 47, 50 , 53, 55, 63, 66, 69 and 72. The three light chain CDRs include a VL CDR1 region comprising an amino acid sequence of at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NO: 74, 77, 80, 85, 91 and 94; a CDR2 region of VL comprising an amino acid sequence of at least 90%, 92%, 95%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NO: 75, 78, 81, 83, 86, 92 and 96; and a VL CDR3 region comprising an amino acid sequence of at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NO: 76, 79 , 82, 84, 93, 95, 97, 98 and 99, with the proviso that when the CDR1 of VL comprises an amino acid sequence that is

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IMPI

INSTITUTE ΜΙΧΙβΛΗΟ DE LA FIOntDAD INDUSTRIA!

at least 90%, 92%, 95%, 97% 98%, 99% o-mee — idiotic-TrIG— 'sequence of SEQ ID NO. : 85, the VL CDR2 comprises an amino acid sequence that is at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to the sequence of SEQ ID NO. : 86 and the CDR3 of VL comprises an amino acid sequence that is at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to the sequence of SEQ ID NO .: 98, and with the condition that when the VL CDR2 comprises an amino acid sequence that is at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to the sequence of SEQ ID NO. : 86, the VL CDR1 comprises an amino acid sequence that is at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to the sequence of SEQ ID NO. : 85 and the VL CDR3 comprises an amino acid sequence that is at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to the sequence of SEQ ID NO .: 98.

Antibodies of the invention immunospecifically bind IL-17F where the antibody binds to an epitope that includes one or more amino acid residues on human IL-17F. In some embodiments, the antibodies of the invention also specifically bind to the heterodimeric IL-17A / IL-17F complex but not to IL-17A, where the antibody binds to an epitope that includes one or more amino acid residues in IL- 17F human.

Antibodies of the invention also include fully human antibodies that bind in a

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INDUSTRIAL PROPERTY MEXICAN IMPI specific to IL-17F and / or the heterodimeric IL-17A / IL-17F complex where the antibody exhibits greater than 50% inhibition of IL-17F-mediated pro-inflammatory cytokine production in vitro. For example, the antibodies of the invention exhibit an inhibition greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% of IL-6 secretion by IL-17 stimulated cells. As used herein, the term "pro-inflammatory cytokine" refers to those immunoregulatory cytokines that promote inflammation and / or are associated with inflammation. Proinflammatory cytokines and chemokines include, for example, IL-6, IL-8, G-CSF, and GM-CSF. Proinflammatory chemokines include, for example, GRO-α, GRO-b, LIX, GCP-2, MIG, IP10, I-TAC, and MCP-1, RANTES, Eotaxin, SDF-1, and MIP3a.

The present invention also provides methods for treating or preventing pathologies associated with aberrant IL-17F activity (eg, aberrant pro-inflammatory cytokine production, such as for example aberrant IL-6 production), or for alleviating a symptom associated with such pathologies, by administering a monoclonal antibody of the invention (eg, a fully human monoclonal antibody) to a subject in whom such treatment or prevention is desired. The subject to be treated is, for example, a human being. The monoclonal antibody is administered in an amount

ΙΜΡΪ

ΠΦ5ΤΓΠΤΟ MBUCAN ·&gt;

enough to treat, prevent or alleviate a symptom associated with the pathology. The amount of monoclonal antibody sufficient to treat or prevent the pathology in the subject is, for example, an amount that is sufficient to reduce IL-17F signaling {eg. , IL-17F-induced production of one or more pro-inflammatory cytokines such as eg, IL-6). As used herein, the term "reduced" refers to decreased production of a pro-inflammatory cytokine in the presence of a monoclonal antibody of the invention, where the production is, for example, local pro-inflammatory cytokine production (eg. , at the site of inflamed tissue) or systemic pro-inflammatory cytokine production. IL-17F signaling (eg. , IL-17F-induced pro-inflammatory cytokine such as IL-6) decreases when the level of pro-inflammatory cytokine production (eg, IL-6) in the presence of a monoclonal antibody of the invention is greater than or equal to 5%, 10 %, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100% less than a production control level of pro-inflammatory cytokine (ie, the level of pro-inflammatory cytokine production in the absence of the monoclonal antibody). The level of pro-inflammatory cytokine production (eg IL-6) is measured eg. , by the use of IL-17F stimulated Mouse Embryonic Fibroblast (MEF) cell assays described herein.

IMPI iill ·, U — I

Those of skill in the art will appreciate that the level of pro-inflammatory cytokine production can be measured by the use of a variety of assays, including, for example, commercially available ELISA kits.

The pathologies treated and / or prevented by the use of the monoclonal antibodies of the invention {eg. , a fully human monoclonal antibody) include, for example, acute inflammation, chronic inflammation {eg. , a chronic inflammation associated with allergic conditions and asthma, a chronic inflammation associated with arthritic conditions), autoimmune diseases (eg. , Crohn's disease, multiple sclerosis, rheumatoid arthritis and other autoimmune arthritic conditions), inflammatory bowel disease, and transplant rejection.

Pharmaceutical compositions according to the invention may include an antibody of the invention and a carrier. These pharmaceutical compositions can be included in kits, such as, for example, diagnostic kits.

The present invention also provides soluble IL-17F proteins, methods for expressing IL-17F proteins, and methods for purifying such proteins in soluble form.

In some modalities, the pathology to be treated is one or more autoimmune diseases, inflammatory disorders, or

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cancers. For example, without limitation, the disease is rheumatoid arthritis and other arthritic conditions, Crohn's disease, psoriasis, multiple sclerosis, chronic obstructive pulmonary disease, and / or asthma, cancer, and angiogenesis.

Pharmaceutical compositions according to the invention may include an antibody of the invention and a carrier. These pharmaceutical compositions can be included in kits, such as, for example, diagnostic kits.

Those of skill in the art will appreciate that the antibodies of the invention have a wide variety of uses. For example, the proteins of the invention are used as therapeutic agents to prevent IL-17F receptor activation in disorders such as, for example, rheumatoid arthritis, Crohn's disease, psoriasis, multiple sclerosis, chronic obstructive pulmonary disease, angiogenesis, asthma and cancer. The antibodies of the invention are also used as reagents in diagnostic kits or as diagnostic tools, or these antibodies can be used in competition assays to generate therapeutic reagents.

Brief description of the figures

Figure 1 is a graph depicting the progression of collagen-induced arthritis clinical score

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IMPI rRSTmrro Mexicano FROM THE fROntUAO! NO''STR1A4 in mice by the use of standard ^ artri-ti ^ s ^ scoring methods.

Figures 2A to 2F are a series of graphs depicting various serum cytokine levels in mice immunized with bovine type II collagen as determined at termination (day 22). Figure 2A represents the detected serum level of tumor necrosis factor alpha (TNF-α); Figure 2B represents the detected serum level of interleukin 6 (IL-6); Figure 2C represents the detected serum level of interferon gamma (IFN-γ); Figure 2D represents the detected serum level of interleukin 1 alpha (IL-la); Figure 2E represents the detected serum level of monocytic chemotactic protein (MCP-1); and Figure 2F represents the detected serum level of the interleukin 12 / interleukin 23 heterodimeric complex (IL-12 / IL-23).

Detailed description of the invention

The present invention provides monoclonal antibodies that specifically bind IL-17F. The invention further provides monoclonal antibodies that specifically bind IL-17F and the heterodimeric IL-17A / IL17F complex (also referred to herein as IL-17A / IL-17F heterodimer). The antibody is eg. , a fully human monoclonal antibody.

IMPI iwsrmm.i Mexican Di LA ntOFIEOAD C®aOT¡p- \ rfC&gt; · _T . _ _ „, -, ___ - INDUSTRIAL- --- Ή

The antibodies of the invention bind specifically to IL-17F but not to IL-17A, whereby the antibody binds to an epitope that includes one or more amino acid residues of human IL-17F. In some embodiments, the antibodies of the invention specifically bind to IL-17F and the heterodimeric IL-17A / IL-17F complex but not to IL-17A or the IL-17A homodimer, where the antibody binds to an epitope. which includes one or more amino acid residues of human IL-17F.

The antibodies of the present invention bind to an IL-17F epitope with an equilibrium binding constant (Ká) of &lt;1 μΜ, eg, &lt;100 nM, preferably &lt;10 nM, and most preferably &lt;1 nM. For example, the huIL-17F antibodies provided herein exhibit a Ka in the range of from about &lt;1 nM to about 1 pM.

The crystal structure of IL-17F reveals that the protein adopts a cysteine ​​knot fold, suggesting a relationship to the cysteine ​​knot protein superfamily. However, the IL-17F cysteine ​​knot motif uses only four cysteines instead of the classical six cysteines to form the knot. Like other members of the cysteine ​​knot family, IL-17F also exists as a heterodimer with IL-17A. The IL-17A / IL-17F heterodimer is believed to signal via IL-17R and / or

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¹⁵ IMPI rxrrmmo m tucano THE INDUSTRIAL PROMWAD the multimeric IL-17R / IL-17RC complex. Recent evidence has shown that the cysteine ​​residues that are used for the formation of the IL-17A / IL-17F heterodimer are the same cysteines that are used for the formation of the IL-17F homodimer. These data suggest that the receptor for the IL-17F homodimer or the IL-17A / IL-17F heterodimer can bind to conserved cysteine ​​residues at the dimer interface, as can other proteins in the cysteine ​​knot family.

Numerous immunoregulatory functions have been reported for the IL-17 family of cytokines, presumably due to their induction of various immuno-signaling molecules. IL17A, expressed as the IL-17A homodimer, and IL-17F, expressed as the IL-17F homodimer, share very similar biological functions in some cases. Both promote the secretion of pro-inflammatory cytokines (eg, IL-6, IL-8, G-CSF, and GMCSF), chemokines (eg. , GRO-α, GRO-b, LIX, GCP-2, MIG, IP10, I-TAC, and MCP-1, RANTES, Eotaxin, SDF-1, and MIP3a) and prostaglandins (eg, PGE₂) from a wide variety of cells including fibroblasts, keratinocytes, macrophages, epithelial cells, and endothelial cells. Also, both have been shown to regulate the renewal of the cartilaginous matrix. The IL-17F homodimer also has biological functions distinct from those of the IL-17A homodimer such as the ability to stimulate proliferation and

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IMPI • WTTTVTO MOUCANO DE LA ERUTIEDAD industrial activation of T cells and peripheral blood mononuclear cells (PBMC), and the ability to inhibit angiogenesis.

The huIL17F antibodies of the invention serve to modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with the biological activity of IL

17F. Biological activities of IL-17F include, for example, binding to IL-17R, IL-17RC, and / or the multimeric IL-17R / IL-17RC receptor complex, and inducing cytokine and / or chemokine expression {by ex. , IL-6, IL-8, G-CSF, GM-CSF, GRO-α, GRO-b, LIX, GCP-2, MIG, IP10, I-TAC, and MCP-1, RANTES, Eotaxin, SDF- 1, and MIP3a) in target cells. For example, huIL-17F antibodies completely or partially inhibit the biological activity of IL-17F through modulation, blocking, inhibition, reduction, antagonization, neutralization, or otherwise partial or partial interference. complete with the binding of IL-17F to its receptor, or otherwise modulating, blocking, inhibiting, reducing, antagonizing, or partially or completely neutralizing IL-17F signaling activity.

HuIL-17F antibodies are considered to modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise completely interfere with the biological activity of IL-17F when the level of IL-17F activity in the presence of the huIL-17F antibody is decreased. by at least 95%, eg. , by 96%, 97%, 98%, 99% or 100% compared to the

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IMPI

MEMCANC INSTITUTE

OF THE NUNTITY

INDUSTRIAL level of IL-17F activity in the absence of a binding with a huIL-17F antibody described herein. HuIL-17F antibodies are considered to modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise partially interfere with IL-17F activity when the level of IL-17F activity in the presence of the huIL-17F antibody is decreased by at least 95%, eg. , 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90% compared to the level of IL-17F activity in the absence of binding to a huIL-17F antibody described herein.

Definitions

Unless defined otherwise, scientific and technical terms used in connection with the present invention will have meanings commonly understood by those of skill in the art. Also, unless the context requires otherwise, singular terms will include plurals and plural terms will include singular. Generally, the nomenclatures that are used in connection with, and the techniques of, cell and tissue culture, molecular biology, and protein and oligo or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the technique. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (for

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g., electroporation, lipofection).

IMPI

ΙΝΠΤΠΓΓΟ MEXICAN

OF EU INDUJTXIAL

Enzymatic reactions and purification techniques are carried out in accordance with the manufacturer's specifications or as commonly achieved in the art or as described herein. The foregoing techniques and procedures are generally carried out in accordance with conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. . See eg, Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Coid Spring Harbor Laboratory Press, Coid Spring Harbor, NY (1989)). The nomenclatures used in connection with, and laboratory techniques and procedures of, analytical chemistry, synthetic organic chemistry, and pharmaceutical medical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and administration, and patient treatment.

As used with this disclosure, the following terms, unless otherwise indicated, will be understood to have the following meanings:

As used herein, the terms Interleukin-17A, IL-17A, IL17A, IL-17, IL17, CTLA8, CTLA-8,

IMPI

MEXICAN INSTITUTE

Of LA FROT1EIIAD INDUSTRIAL cytotoxic T lymphocyte-associated antigen 8 and Interleukin-17A precursor are synonymous and can be used interchangeably. Each of these terms refers to the heterodimeric protein, except where otherwise indicated.

As used herein, the terms Interleukin-17F, IL-17F, IL17F, ML-1, ML1, Interleukin-24, IL-24, IL24, and Precursor Interleukin-17F are synonymous and can be used interchangeably. . Each of these terms refers to the heterodimeric protein IL-17F, except where otherwise indicated.

As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, that is, molecules that contain an antigen-binding site that specifically binds to ( immunoreacts with) an antigen. Binds specifically to or immunoreacts with or is directed against means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds with much lower affinity (K¿&gt; 10 '⁶). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibodies), single chain, F fragments_tob, F_ab- and F (_ab.)₂, scFvs, and an expression library F_ab.

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²⁰ IMPI

The basic structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair has a light chain (approximately 25 kDa) and a heavy chain (approximately 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that are primarily responsible for antigen recognition. The carboxy terminal portion of each chain defines a constant region primarily responsible for effector function. In general, antibody molecules obtained from humans refer to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from each other by the nature of the heavy chain present in the molecule. Certain classes also have subclasses, such as IgG₁₍ IgG₂, and others. Furthermore, in humans, the light chain can be a kappa chain or a lambda chain.

The term monoclonal antibody (MAb) or monoclonal antibody composition, as used herein, refers to a population of antibody molecules that only contain one molecular species of antibody molecule consisting of a single chain gene product. light and a single heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are Mexican νετγπγγο M LA tu JPUÍMP Ο · ηκ £ β IWD'OTWAL ^ * - i — TL.

identical in all molecules of the population. MAbs I IR I * contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity.

In general, antibody molecules obtained from humans refer to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from each other by the nature of the heavy chain present in the molecule. Certain classes also have subclasses, such as IgGi, IgG₂, and others. Furthermore, in humans, the light chain can be a kappa chain or a lambda chain.

The term "antigen-binding site" or "binding moiety" refers to the part of the immunoglobulin molecule that participates in antigen-binding. The antigen-binding site is formed by amino acid residues from the variable regions (V) of the N-terminus of the heavy (H) and light (L) chains. Three highly divergent stretches within the V regions of heavy and light chains, termed hypervariable regions, stand between more conserved flanking stretches known as framework regions, or FRs. Therefore, the term "FR" refers to amino acid sequences that occur naturally between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a chain

<img file="MX348013B_D0019.tif" />

Light and the three hypervariable regions of a heavy chain are arranged relative to each other in three-dimensional spaces to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are called complementarity determining regions, or CDRs. The amino acid assignment to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia &amp; Lesk J. Mol. Biol. 196: 901-917 (1987), Chothia et al. Nature 342: 878-883 (1989).

As used herein, the term epitope includes any protein determinant capable of specifically binding to an immunoglobulin or fragment thereof, or a T cell receptor. The term epitope includes any protein determinant capable of specifically bind to an immunoglobulin or T cell receptor. Epitopic determinants normally consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and normally have specific three-dimensional structural characteristics, as well as specific charge characteristics. I know²³ IMPI ^ mBucano institute I heard the «&lt;INDUSTRIAL piety says that an antibody binds specifically to an antigen when the dissociation constant is &lt;1 μΜ; eg 100 nM, preferably 10 nM and most preferably &lt;1 nM.

As used herein, the terms "immune binding" and "immune binding properties" refer to non-covalent interactions of the type that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Ka) of the interaction, where a lower Ka represents a higher affinity. The immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method involves measuring the rates of antigen / antigen-binding site complex formation and dissociation, where those rates depend on the concentrations of the complex partners, the affinity of the interaction, and the geometric parameters that equally influence speed in both directions. Therefore, both the constant in speed (Kon) and the constant out of speed (Koff) can be determined by calculating the concentrations and the actual rates of association and dissociation. {See Nature 361: 186-87 (1993)). The relationship

<img file="MX348013B_D0020.tif" />

de Koff / Kon allows cancellation of all parameters, .HQ ^. related to affinity, and is equal to the dissociation constant Ka. (See generally Davies et al. (1990) Annual Rev Biochem 59: 439-473). An antibody of the present invention is said to bind specifically to IL-17F and / or to the IL-17A / IL-17F heterodimer, when the equilibrium binding constant (K¿) is &lt;1 μΜ, preferably &lt; 100 nM, most preferably &lt;10 nM, and most preferably &lt;100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those with experience in The technique.

The term isolated polynucleotide as used herein will mean a polynucleotide of genomic DNA, cDNA, or of synthetic origin or some combination thereof, which by virtue of its origin the isolated polynucleotide (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is operably linked to a polynucleotide that is not found in nature, or (3) does not occur in nature as part of a longer sequence. Polynucleotides according to the invention include the nucleic acid molecules that encode the molecules of

IMPI

ΠΕ THE industrial PROPERTY heavy chain immunoglobulin represented in SEQ ID NO:

1, 5, 9, 13, 17, 21, 25, 29, 33, 37 and 41, and the nucleic acid molecules that encode the light chain immunoglobulin molecules represented in SEQ ID NO: 3, 7, 11, 15 , 19, 23, 27, 31, 35, 39 and 43.

The term "isolated protein" referred to herein means a protein of cDNA, recombinant RNA, or of synthetic origin or some combination thereof, that by virtue of its origin, or its source of derivation, the isolated protein (1) is not found. associated with naturally occurring proteins, (2) is free from other proteins from the same source, e.g. free from marine proteins, (3) is expressed by a cell of a different species, or (4) does not occur in nature.

The term "polypeptide" is used herein as a generic term to refer to a native protein, fragments, or analogs of a polypeptide sequence. Therefore, native protein fragments, and analogs are species of the genus of polypeptides. The polypeptides according to the invention comprise the heavy chain immunoglobulin molecules represented in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38 and 42, and the chain immunoglobulin molecules light represented in SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40 and 44, as well as the antibody molecules formed by means of

IMPI ^

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Μ THE PROHEOAD

INDUSTRIAL combinations comprising heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof.

The term naturally occurring as used herein when applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory or otherwise produces naturally.

The term "operatively linked" as used herein refers to positions of components so described that are in a relationship that enables them to function in their intended form. A control sequence operably linked to a coding sequence is linked such that expression of the coding sequence is achieved under conditions compatible with the control sequences.

The term "control sequence" as used herein refers to polynucleotide sequences that are necessary to effect expression.

<img file="MX348013B_D0021.tif" />

and coding sequence processing. a ή o- q ^ g ——— are linked. The nature of such control sequences differs depending on the host organism in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence in eukaryotes, generally such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, eg, leader sequences and fusion component sequences. The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, both ribonucleotides and deoxynucleotides, or a modified form of any type of nucleotide. The term includes single and double stranded forms of DNA.

The term "oligonucleotide" referred to herein includes naturally occurring nucleotides, and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a subset of polynucleotides that generally

<img file="MX348013B_D0022.tif" />

IMPI INrrrrUTO MWCAJ *&gt; Pt LA MOmpAD INDUSTRIA ·.

it comprises a length of 200 bases or less. Preferably, the oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are normally single stranded, eg, for probes, although oligonucleotides can be double stranded, eg, for use in the construction of a mutant gene. The oligonucleotides of the invention are both sense and antisense oligonucleotides.

The term "naturally occurring nucleotides" mentioned herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" mentioned herein includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" mentioned herein includes linkages of oligonucleotides such as phosphorothioate, phosphorodithioate, phosphoroselenate, phosphorodiselenate, phosphoranilothioate, phosphoranylate, phosphoramidate, and the like. See eg. , LaPlanche et al. Nucí. Acids Res. 14: 9081 (1986); Stec et al. J. Am. Chem. Soc. 106: 6077 (1984), Stein et al. Nucí. Acids Res. 16: 3209 (1988), Zon et al. Anti Cancer Drug Design 6.-539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, p. 87-108 (F. Eckstein, Ed., Oxford University Press,

Oxford England (1991)); Stec et ai. States Patent

IMPI Mexican institute of IA INDI ISTRIAL PROPERTY

States Nos. 5,151,510; Uhlmann and Peyman Chemical Reviews 90: 543 (1990). If desired, an oligonucleotide can include a tag for detection.

The term "selectively hybridize" as mentioned herein means to bind detectably and specifically. Polynucleotides, oligonucleotides and fragments thereof according to the invention selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to non-specific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions such as are known in the art and discussed herein. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments of the invention and a nucleic acid sequence of interest will be at least 80%, and more typically preferably with increasing homologies of at least 85%. %, 90%, 95%, 99%, and 100%. Two amino acid sequences are homologous if there is partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences to be paired) are allowed in maximizing the pairing; lengths of

Say LA r »OR1SDAr WDUSTUIAl gaps of 5 or less; even more than 2 or less being preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have a score of alignment greater than 5 (in units of standard deviation) the ALIGN program with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, MO, in Atlas of Protein Sequence and Structure, p. 101-110 (Volume 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, p. 1-10. The two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 50% identity when optimally aligned by use of the ALIGN program. The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (i.e., it is identical, not evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a sequence of polypeptides is identical to a reference polypeptide sequence. In contrast, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or part of a reference polynucleotide sequence. With purposes

IMPI

INSTITUTE MtUCANC

Ot THE nOMITY wnuynuAi of illustration, the TATAC nucleotide sequence corresponds to a TATAC reference sequence and is complementary to a GTATA reference sequence.

The following terms are used to describe sequence relationships between two or more polynucleotide or amino acid sequences: reference sequence, comparison window, sequence identity, percent sequence identity, and substantial identity. A reference sequence is a defined sequence used as the basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, or it may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 18 nucleotides or 6 amino acids in length, often at least 24 nucleotides or 8 amino acids in length, and more often at least 48 nucleotides or 16 amino acids in length. Since two polynucleotide or amino acid sequences can each (1) comprise a sequence (i.e., a part of the entire polynucleotide or amino acid sequence) that is similar between the two molecules, and (2) further comprise a sequence that is divergent between the two polynucleotide or amino acid sequences, the

<img file="MX348013B_D0023.tif" />

IMPI

MEJUCAMO INSTITUTE

OF INDUSTRIAL RICHNESS Sequence comparisons between two (or more) molecules are typically carried out by comparing the sequences of the two molecules with respect to a comparison window to identify and compare local regions of sequence similarity. A comparison window, as used herein, refers to a conceptual segment of at least 18 contiguous nucleotide positions, or 6 amino acids, where a polynucleotide sequence or amino acid sequence can be compared to a reference sequence. of at least 18 contiguous nucleotides or 6 amino acid sequences and where the part of the polynucleotide sequence in the comparison window may comprise additions, deletions, substitutions, and the like (ie, gaps) of 20 percent or less compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal sequence alignment to align a comparison window can be accomplished by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by means of the homology alignments algorithm of Needleman and Wunsch J. Mol. Biol. 48: 443 (1970), by searching for the -similarity method of Pearson and Lipman Proc. Nati. Acad. Sci. (USA) 85: 2444 (1988), by means of computerized implementations of these algorithms (GAP, BESTFIT, FASTA and

TFASTA in the Wisconsin Genetics Software Package Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison, Wis.), Geneworks software packages, or MacVector), or by inspection, and the best alignment is selected (ie that is, that results in the highest percentage of homology with respect to the comparison window) generated by the various methods.

The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (ie, on one nucleotide base per nucleotide or residue per residue) with respect to the comparison window. The term percent sequence identity is calculated by comparing two optimally aligned sequences with respect to the comparison window, by determining the number of positions where the identical nucleic acid base or residue (for example, A, T, C, G, U, or I) occurs in both sequences to give the number of paired positions, by dividing the number of paired positions by the total number of positions in the comparison window (ie the window size) and by multiplying the result by 100 to give the percent sequence identity. The terms substantial identity as used herein denote a characteristic of a polynucleotide or amino acid sequence, where the³⁴

INSTm / Tt MEXICAN

OF THE PROPERTY \ 7-, 7751 //

INDUSTRIAL polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more typically at least 99 percent sequence identity compared with a reference sequence with respect to a comparison window of at least 18 nucleotide positions (6 amino acids), frequently with respect to a window of at least 24 to 48 nucleotide positions (8 to 16 amino acids), where the percent sequence identity is calculated by comparing the reference sequence with the sequence that may include deletions or additions that amount to 20 percent or less of the reference sequence relative to the comparison window. The reference sequence can be a subset of a larger sequence.

As used herein, the twenty standard amino acids and their abbreviations follow standard usage. See Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland7 Mass. (1991)). Stereoisomers (eg, Damino acids) of the twenty conventional amino acids, non-natural amino acids such as α-amino acids, disubstituted amino acids, N-alkylamino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides herein.

<img file="MX348013B_D0024.tif" />

IMPL 'WTtTUTO MEXICANO nt PROMuAr · ΙΜΓ ^ ΤΤΙΙΛΙ invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-Ν, Ν, Ν-trimethylisine, ε-Ν-acetyl-lysine, O-phosphoserine, Nacetylserine, N-formylmethionine , 3-methylhistidine, 5-hydroxylysine,- Ν-methylarginine and other similar amino acids and imino acids (eg, 4-hydroxyproline). In the polypeptide notation used herein, the left hand direction is the amino terminal direction and the right hand direction is the carboxy terminal direction, in accordance with standard usage and convention.

Similarly, unless otherwise specified, the left hand end of single stranded polynucleotide sequences is the 5 'end. The left-hand direction of double-stranded polynucleotide sequences is referred to as the 5-direction¹ . Adding direction of 5¹ to 3 ¹ of nascent RNA transcripts is referred to as the direction of transcription; the sequence regions in the DNA strand that have the same sequence as the RNA and that are 5 'to the 5' end of the RNA transcript are called upstream sequences, the sequence regions in the DNA strand that have the same sequence as RNA and they are 3 'to the 3 end¹ of the RNA transcript are called downstream sequences.

ΙΜΡΪ imstttuTd m £ xican &lt;¡DE Μ ΛΟΠΕΟΑΓ! NDVST »IAl

As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as through the GAP or BESTFIT programs by the use of default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, most preferably at least 95 percent sequence identity, and with the highest preference at least 99 percent sequence identity.

Preferably, residue positions that are not identical are differentiated by conservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability of residues that have similar side chains. For example, a group of amino acids that have aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids nt the industrial raontUAO having sulfur-containing side chains is cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, valine alanine, glutamic-aspartic, and asparagine-glutamine.

As discussed herein, it is contemplated that minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are encompassed by the present invention, provided that the variations in the amino acid sequence maintain at least 75%, with highest preference at least 80%, 90%, 95%, and with the highest preference 99%. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are broadly divided into families: (1) acidic amino acids are aspartate, glutamate; (2) the basic amino acids are lysine, arginine, histidine; (3) the nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) the uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine.

IMPI MEXICAN FNSTmiTO Ot LA PfiXX'iEDAD WtJUSTRlAL

Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic hydroxy family; (ii) asparagine and glutamine, which are the amide-containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated substitution of a leucine for an isoleucine or valine, an aspartate for a glutamate, a threonine for a serine, or a similar substitution of an amino acid for a structurally related amino acid will not have a significant effect on the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can be readily determined by testing the specific activity of the polypeptide derivative. The assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of skill in the art. Preferred amino and carboxy termini of fragments or analogs occur near functional domain boundaries. The structural domains and

<img file="MX348013B_D0025.tif" />

IMPI iwrmrro mixíCano * pe LA ΒυΜΜΡ

- INDUSTRIAL functionalities can be identified by comparing nucleotide and / or amino acid sequence data with public or registered sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into a three-dimensional structure are known, Bowie et al. Science 253: 164 (1991). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the invention.

Preferred amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity to form protein complexes, (4) alter affinities for binding and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (with

IMPI

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• mnVTO MKX1CANO

DI LA ntOPItTMD INDUmui prefers conservative amino acid substitutions) in the naturally occurring sequence (preferably in the portion of the polypeptide outside the domain (s) that form intermolecular contacts). A conservative amino acid substitution should not substantially change the structural features of the original sequence (e.g., a substitution amino acid should not tend to break a helix that occurs in the original sequence, or interrupt other types of secondary structure that characterize the original sequence). Examples of art-recognized secondary and tertiary structures of polypeptides are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)), · and Thornton et at. Nature 354: 105 (1991).

The term "polypeptide fragment" as used herein refers to a polypeptide that has a deletion of the amino terminus and / or carboxy terminus, but in which the remaining amino acid sequence is identical to the corresponding positions in the sequence. inferred to occur naturally, eg, from a full-length cDNA sequence. Fragments typically have at least 5, 6, 8, or 10 amino acids of

<img file="MX348013B_D0027.tif" />

⁴¹ IMPI nWTTTUTO CX LA AVERAGE INDUSTRY *.

length, preferably at least 14 amino acids in length, more preferably at least 20 amino acids in length, usually at least 50 amino acids in length, and even more preferably at least 70 amino acids in length. The term "analog as used herein" refers to polypeptides that are composed of a segment of at least 25 amino acids that has substantial identity to a portion of a deduced amino acid sequence and that has specific binding only to IL17F or the heterodimer. IL-17A / IL-17F (ie complex), under suitable binding conditions. Typically, polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally occurring sequence. Analogs are typically at least 20 amino acids long, preferably at least 50 amino acids long or longer, and can often be as long as a full-length naturally occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to peptide template. These types of non-peptide compounds are called peptide mimetics or peptidomimetics. Fauchere, J. Adv. Drug Res. 15:29 (1986), Veber and Freidinger TINS ​​p.392 (1985); and Evans

<img file="MX348013B_D0028.tif" />

IMPI iwmvro MOLD OF THE FWOMUMB Wl'STTIfAE et al. J. Med. Chem. 30: 1229 (1987). Such —- «MRp-Uá £ utu3 £ _, ^ e ._ ^ are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide having a biochemical property or pharmacological activity) such as a human antibody, but have one or more peptide bonds optionally substituted by a bond selected from the group consisting of: - CH₂NH--, --CH₂S-, --CH₂-CH₂-, --CH = CH - (cis and trans), --COCH₂-, CH (OH) CH₂-, and -CH₂SO--, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (eg, Dlysine instead of L-lysine) can be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a variation of substantially identical consensus sequences can be generated by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61: 387 (1992)), - for example, by means of the addition of internal cysteine ​​residues capable of forming intramolecular disulfide bridges that cyclize the peptide.

The term agent is used herein to mean

IMPI 'βτπντη M RUCANO u ranncDA INDUSTRIAL denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from

<img file="MX348013B_D0029.tif" />

biological materials.

As used herein, the terms "brand" or "labeling" refer to the incorporation of a detectable marker, eg , by incorporation of a radiolabeled amino acid or binding to a polypeptide of biotinyl residues that can be detected by means of labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by means of procedures optical or colorimetric). In certain situations, the brand or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known and can be used in the art. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (eg,³H, ¹⁴C, ¹⁵N, ³⁵Yes, ⁹⁰Y, ⁹⁹Tc, ^{li: L}In, ¹²⁵I, ¹³¹I), fluorescent labels (eg, FITC, rhodamine, lanthanide phosphors), enzyme labels (eg, horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (eg, leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, labels

<img file="MX348013B_D0030.tif" />

IMPI Mexican institute DE LA FXOMSDA »industrial de epitopes). In some embodiments, .1st are joined by spacer arms of various lengths to reduce potential spherical obstruction. The term "pharmaceutical agent or drug" as used herein refers to a chemical compound or composition that can induce a desired therapeutic effect when properly administered to a patient.

Other chemistry terms herein are used in accordance with conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco ( 1985)).

The term "antineoplastic agent" is used herein to denote agents that have the functional property of inhibiting a development or progression of a neoplasm in a human, in particular a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition of metastasis is often a property of antineoplastic agents.

As used herein, "substantially pure" means that a target species is the predominant species present (i.e. on a molar basis it is more abundant than any other species of individuals in the composition), and preferably a substantially purified fraction. is a composition in which the object species

IMPI ΠΠΤΓΠΓΓΟ MEXICAN DE LA DIED EDA * INDURVUlA!

it comprises at least about 50 percent (on a molar basis) of all macromolecular species present.

Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. With the highest preference, the target species is purified to essential homogeneity (contaminating species cannot be detected in the composition by conventional detection methods), where the composition consists essentially of a single macromolecular species.

Autoimmune diseases include, for example, Acquired Immune Deficiency Syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, Addison's autoimmune disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune disease of the inner ear (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac sprue - hepetiform dermatitis; chronic fatigue syndrome and immune dysfunction (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, ridge syndrome, Crohn's disease,

Degos, juvenile dermatomyositis1, discoid lupus, essential mixed cryoglobulinemia

IMPI

IKSTTniTO MEJBCaHO. OR * THE PROPERTY

INDUSTRIAL fibromyalgia

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fibromyositis, Graves disease, GuillainBarré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Méniére's disease , mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (PSS) SS)), Sjógren's syndrome, Stiff person syndrome, Systemic lupus erythematosus, Takayasu arteritis, Temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

Inflammatory disorders include, for example, acute and chronic inflammatory disorders. Examples of inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft versus host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, diabetic retinopathy transplantation, and mechanical ventilation.

tissue injury

INDUSTRIAL and organs, vasculitis,. __— ~ JilJi • l · '* -—_ ι u ·¹·»¹· ^ Πν *. .. - r— pulmonary induced by

HuIL-17F antibodies

The monoclonal antibodies of the invention (eg, fully human monoclonal antibodies) bind IL17F, and in some embodiments, the IL17A / IL-17F heterodimeric complex, but do not bind IL-17A or IL-17A homodimer. These monoclonal antibodies have the ability to inhibit IL17F-induced pro-inflammatory cytokine production (eg, IL-6). Inhibition is determined, for example, in the IL-17F stimulated mouse embryonic fibroblast (MEF) cell assays described herein.

Examples of antibodies of the invention include, for example, the 5E12 antibody, the 41B10 antibody, the 11C5 antibody, the 21B10 antibody, the 1F1 antibody, the 2E12 antibody, the 5D3 antibody, the 22F8 antibody, the 28B11 antibody, the 41A4 and the 43G6 antibody described herein. These antibodies show specificity for human IL-17F and / or the heterodimeric IL17A / IL-17F complex, and have been shown to inhibit human IL-17F induction of the pro-inflammatory cytokine IL6 in vitro.

Each of the monoclonal huIL-17F antibodies

INSTITUTE ΜβΙΚΛΝΟ&gt;

ΟΕ PROREDA D

INDUSTRIAL ~&gt; Αdescribed herein includes a regiWr ^ 'varrtnafcire ^' Tde ^ heavy chain (VH) and a variable region of light chain (VL), as shown in the corresponding nucleic acid and amino acid sequences listed then.

The 5E12 antibody includes a heavy chain variable region (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1, and a light chain variable region (SEQ ID NO: 1). -.4) encoded by the nucleic acid sequence shown in SEQ ID NO .: 3.

&gt; 5E12 VH nucleic acid sequence (SEQ ID NO: 1)

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCT

CCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCC

AGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTACCATAGGCTATGCG

GACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGC

AAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGAACTGTATAT

CAGTGACTGGGACTCCTACTCCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACC

GTCTCCTCA:&gt; Amino acid sequence 5E12 VH (SEQ ID NO: 2)

QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGTIGYA

DSVKGRFTISRADNKNSLYLQMNSLRAEDTALYYCAKELYISDWDSYSYGMDVWGQGTTVT

VSS

<img file="MX348013B_D0032.tif" />

IMPI

IWfflVTO MBUCaNL

DE LA WOMDaO industrial&gt; Nucleic acid sequence 5E12 VL (SEQ ID NO: 3)

GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCC

TCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACC

TGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGAC

AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTG

AAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTTTCGGCGGAGGGACCAA

GGTGGAGATCAAA&gt; Amino acid sequence 5E12 VL (SEQ ID NO ..- 4) ΕΐνΕΤ03ΡσΤΕ3ΙιΞΡΟΕΕΑΤΕ30ΕΑ303ν333ΥΕΑΝΥ00ΚΡσ0ΑΡΕΕΕΙΥ6Α33ΚΑΤΟΙΡΟ RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFGGGTKVEIK

Antibody 41B10 includes a heavy chain variable region (SEQ ID NO: 6) encoded by the nucleic acid sequence shown in SEQ ID NO: 5, and a light chain variable region (SEQ ID NO: 5). : 8) encoded by the nucleic acid sequence shown in SEQ ID NO: 7.

&gt; Nucleic acid sequence 41B10 VH (SEQ ID NO .: 5) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCT CCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCC AGGGAAGGGGCTGGAATGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGGACAACAGAC TACGTTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACCCTGT

ATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTATATTACTGTACCACATCGTA

<img file="MX348013B_D0033.tif" />

IMPI

ΙΜΤητυτο MeiCANo

UF THE 'NbUSTlUAL PROPERTY

TAGCAGTTACTGGTTCCCCTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTC ·· J * ..- 11 · ΜΒ · Τ4ΛΗ »ς ττ _L TCCTCA&gt; Amino acid sequence 41B10 VH (SEQ ID NO: 6)

EVQLVE SGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTD YVAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTSYSSYWFPYYFDYWGQGTLVTV

SS&gt; Nucleic acid sequence 41B10 VL (SEQ ID NO: 7)

GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCA TCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGA GAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGG TTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAG ATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGATCACCTTCGGCCJXAGGGAC ACGACTGGAGATTAAA&gt; 41B10 VL amino acid sequence (SEQ ID NO: 8.) DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPITFGQGTRLEIK

The 11C5 antibody includes a heavy chain variable region (SEQ ID NO: 10) encoded by the nucleic acid sequence shown in SEQ ID NO. : 9, and a light chain variable region (SEQ ID NO: 12) encoded by the nucleic acid sequence shown

IMPI

INSTITUTO MeuCAJ'O DE LA FROMEDAt in SEQ ID NÚM. :eleven. The amino acid sequence of the light chain variable region for antibody 11C5 includes mutations at the 5 'end to convert residues to residues found in the corresponding human germ sequence. The non-mutated version of the light chain variable region amino acid sequence for the 11C5 antibody is shown in SEQ ID NO: 102, and the non-mutated version of the light chain variable region nucleic acid sequence for the 11C5 antibody shown in SEQ ID NO .: 103.

Nucleic acid sequence 11C5 VH (SEQ ID NO: 9) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGLAGAAGCCTGGGGCCTCAGTGAAGGT TTCCTGCAAGGCATCTGGATACACCTTCACCATCTATTATTTGCACTGGGTGCGACAGG CCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGGACAAAC TACGCACAGAAGTTCCAGGGCAGGGTCACCATGACCAGGGACCCGTCCACGAACACAGT CTACATGGAACTGAGCAGCCTGACATCTGAGGACGCGGCCGTGTATTACTGTGCGAGAG GGGAATTTAGCAGTGGCTGGCTTGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCC TOA&gt; 11C5 VH amino acid sequence (SEQ ID NO: 10.) QVQLVQSGAEVKKPGASVKVSCKASGYTFTIYYLHWVRQAPGQGLEWMGIINPSGGRTN YAQKFQGRVTMTRDPSTNTVYMELSSLTSEDAAVYYCARGEFSSGWLDYWGQGTTVTVS S&gt; Nucleic acid sequence 1105 VL (SEQ ID No.rll)

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCAC

ΙΜΡΙ ^ _

INSTITUTO MEXICANO de la noraoAL Ύ ιΐ ·

INDUSTRIAI Vn ** V

CATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCATAAAC

CAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCA

TCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA

GCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAATAGTTTCCCGCTCACTTTCG

GCGGAGGGACCAAGGTGGAGATCAAA&gt; Amino acid sequence 11C5 Vl · (SEQ ID NO: 12)

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQHKPGKAPKLLIYAASSLQSGVP

SRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK&gt; Nucleic acid sequence 11C5 VL not mutated (SEQ ID

NO. : 102)

GACATCGTGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCAC

CATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCATAAAC

CAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCA

TCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA

GCCTGI \ AGATTTTGCAACTTACTATTGTCAACAGGCTAATAGTTTCCCGCTCACTTTCG

GCGGAGGGACCAAGGTGGAGATCAAA&gt; Amino acid sequence 11C5 Vl · not mutated (SEQ ID NO: 103)

ΟΐνΜΤ03Ρ55ν3Α3ναθΡνΤΙΤσν \ 2Ο3Ι53ΝΕΆΝΥ0ΗΚΡσΚΑΡΚΕΕΙΥΆΆ23Ε03σνΡ

3ΕΡ3Ο3Ο3ΟΤΏΡΤΕΤΙ33Ε0ΡΕΟΕΑΤΥΥϋ00ΑΝ3ΡΡΕΤΡσσαΤΚνΕΙΚ

Antibody 21B10 includes a heavy chain variable region (SEQ ID NO: 14) encoded by the ⁵³ IMPI ^ Mexican Institute OF INDUSTRIAL PROPERTY - nucleic acid sequence shown in SRQ τη _ NÚM. : 13, and a light chain variable region (SEQ ID NO: 16) encoded by the nucleic acid sequence shown in SEQ ID NO: 15.

&gt; Nucleic acid sequence 21B10 VH (SEQ ID NO:

13)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT

CTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCAIGAACTGGGTCCGCCAGG

CTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTGGTGGTAGTAGTACCATATAC

TACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACT

GTATCTGCAAATGAACAGCCTGAGAGACGAGGACACGGCTGTGTATTACTGIGCGAGAG

AGGGCTATG1TTCGGGGACCTATTACAACTACTACTACGGTATGGACGTCTGGGGCCAA

GGGACCACGGTCACCGTCTCCTCA&gt; Amino acid sequence 21B10 VH (SEQ ID NO: 14)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISGGSSTIY

YADSVKGRFTISRADNKNSLYLQMNSLRDEDTAVYYCYKREGYVSGTYYlSrYYYG ^^

GTTVTVSS&gt; Nucleic acid sequence 21B10 VL (SEQ ID NO: 15)

GAAATTGTGTTGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC

CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGTTACTTAGCCTGGTACCAACAGAAAC

CTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCACCCAACAGGGCCACTGGCATCCCA

GCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGA

GCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGTCGCTCACTTTCG

<img file="MX348013B_D0034.tif" />

IMPI

MEXICAN INSTITUTE OF THE FXOHEPAt INDUSTRY!

GCGGAGGGACCAAGGTGGAGATCAAA&gt; Amino acid sequence 21B10 VL (SEQ ID NO: 16)

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAPNRATGIP

ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWSLTFGGGTKVEIK

The 1F1 antibody includes a heavy chain variable region (SEQ ID NO: 18) encoded by the nucleic acid sequence shown in SEQ ID NO: 17, and a light chain variable region (SEQ ID NO 20 ) encoded by the nucleic acid sequence shown in SEQ ID NO .: 19. The amino acid sequence of the light chain variable region for the 1F1 antibody includes mutations at the 5 'end to convert residues to residues found in the corresponding human germline sequence. The non-mutated version of the light chain variable region amino acid sequence for the 1F1 antibody is shown in SEQ ID NO: 104, and the non-mutated version of the light chain variable region nucleic acid sequence for the 1F1 antibody shown in SEQ ID NO .: 105.

&gt; 1F1 VH nucleic acid sequence (SEQ ID NO: 17)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT

CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGTCATGAGCTGGGTCCGCCAGG ⁵⁵

INSTITUT · MBtICAJO DE LA ri »« i »A» INDUSTRIAL ttccagggaaggggctggagtgggtctcagctattagtggtcgtggtggtaAcacattcu ------ TACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCT

GTATCTGCAAATGGACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAG

ATGATCGGCGTATAGCAGCAGGTAGTTTTGACTATTGGGGCCAAGGGACCACGGTCACC

GTCTCCTCA&gt; 1F1 VH amino acid sequence (SEQ ID NO: 18)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYVMSWVRQVPGKGLEWVSAISGRGGNTF

YADSVKGRFTISRDNSKNTLYLQMDSLRAEDTAVYYCAKDDRRIAAGSFDYWGQGTTVT

VSS&gt; 1F1 VL nucleic acid sequence (SEQ ID NO: 19)

GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC

CATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAAC

CAGGGAAAGCTCCTAAGCTCCTGATCTATGATGTCTCCAGTTTGGAAAGTGGGGTCCCA

TCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA

GCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCTCACTTTCG

GCGGAGGGACCAAGGTGGAGATCAAA&gt; Amino acid sequence 1F1 VL (SEQ ID NO: 20)

AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDVSSLESGVP

SRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK&gt; Nucleic acid sequence 1F1 VL not mutated (SEQ ID

NO .: 104)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC ⁵⁶ IMPI »^

MEXICAN INSTITUTE

OF LA TUOrWDAT Qykptiy 'INDUSTWAl

CATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAAC CAGGGAAAGCTCCTAAGCTCCTGATCTATGATGTCTCCA.GTTTGGAAAGTGGGGTCCCA TCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCTCACTTTCG GCGGAGGGACCAAGGTGGAGATCAAA&gt; Amino acid sequence of 1F1 VL unmutated (SEQ ID NO. 105) DIQMTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDVSSLESGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK

The 2E12 antibody includes a heavy chain variable region (SEQ ID NO: 22) encoded by the nucleic acid sequence shown in SEQ ID NO: 21, and a light chain variable region (SEQ ID NO 24 ) encoded by the nucleic acid sequence shown in SEQ ID NO .: 23.

&gt; Nucleic acid sequence 2E12 VH (SEQ ID NO: 21)

<img file="MX348013B_D0035.tif" />

GGGACCACGGTCA.CCGTCTCCTCA.

IMPI&gt; Amino acid sequence 2E12 VH (SEQ ID NO: 22)

EVQLVESGGGLVQRGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWISYISSSSSAIY

YADSVKGRFTISRADNKNSLYLQMNSLRDEDTAVYYCAREGYASGRYYNYYYGMDVWGQ

GTTVTVSS&gt; Nucleic acid sequence 2E12 VL (SEQ ID NO: 23)

<img file="MX348013B_D0036.tif" />

&gt; Amino acid sequence 2E12 VL (SEQ ID NO: 24)

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIP ARFSVSGSGTDFTLTISSLEPEDFAVYYCQQRSSWSLTFGGGTKVEIK

The 5D3 antibody includes a heavy chain variable region (SEQ ID NO: 26) encoded by the nucleic acid sequence shown in SEQ ID NO. : 25, and a light chain variable region (SEQ ID NO: 28) encoded by the nucleic acid sequence shown in SEQ ID NO: 27.

&gt; 5D3 VH nucleic acid sequence (SEQ ID NO: 25)

GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACT

<img file="MX348013B_D0037.tif" />

ΙΜΚ rwsTmrtFMExicA '; M LA FROMSBAD

INDUSTRIAL

CTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAG

CTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCAGAGGC

TATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCT

GTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAG

ATATGGTCTACGCTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA&gt; Amino acid sequence 5D3 VH (SEQ ID NO: 26)

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSRG

YADSVKGRFTISRADNKNSLYLQMNSLRAEDTALYYCAKDMVYAhDVWGQGTTVTVSS&gt; 5D3 VL Nucleic Acid Sequence · (SEQ ID NO: 27)

GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC

CCTCTCCTGCAGGGCCAGTCAGAGTTTTAGCGGCAGCTACTTAGCCTGGTACCAGCAGA

AACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATACATCCAGCAGGGCCACTGGCATC

CCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACT

GGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTACGTGGACGTTCGGCC

AAGGGACCAAGGTGGAAATCAAA&gt; Nucleic acid sequence 5D3 VL · (SEQ ID NO: 28)

EIVLTQSPGTLSLSPGERATLSCRASQSFSGSYLAWYQQKPGQAPRLLIYDTSSRATGI

PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGTWTFGQGTKVEIK

Antibody 22F8 includes a heavy chain variable region (SEQ ID NO: 30) encoded by the nucleic acid sequence shown in SEQ ID NO. : 29, and a

<img file="MX348013B_D0038.tif" />

IMPI IMSTTTUTO MEXICANO DE U FXOFlEDAn IWDVSTWlAi light chain variable region (SEQ ID NO: 32) encoded by the nucleic acid sequence shown in SEQ ID NO: 31.

&gt; Nucleic acid sequence 22F8 VH (SEQ ID NO: 29)

GAGGTGCAGTTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT

CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAACTGGGTCCGCCAGG

CTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAGTGGTCGTGGTGGTAGCATATAC

TACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCT

GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAG

AGGAGGCTACCTGGGACTTTGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA&gt; Amino acid sequence 22F8 VH (SEQ ID NO: 30)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTISGRGGSIYYA

DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEATWDFDYWGQGTTVTVSS&gt; Nucleic acid sequence 22F8 VL (SEQ ID NO: 31)

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC

CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTTCTTAGCCTGGTTCCAACAGAAAC

CTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCA

GCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGA

GCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCGACGTTCG

GCCAAGGGACCAAGGTGGAAATCAAA&gt; Amino acid sequence 22F8 VL (SEQ ID NO: 32)

EIVLTQS PATLSLSPGERATLSCRASQSVSSFLAWFQQKPGQAPRLLIYDASNRATGIP

<img file="MX348013B_D0039.tif" />

IMPI

MSeCANO INSTITUTE

MIA MortBJAD INDUSTRY »

ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTEmüWÍEiS ———— Antibody 28B11 includes a heavy chain variable region (SEQ ID NO: 34) encoded by the nucleic acid sequence shown in SEQ ID NO: 33, and a light chain variable region (SEQ ID NO .: 36) encoded by the nucleic acid sequence shown in SEQ ID NO .: 35.

&gt; Nucleic acid sequence 28B11 VH (SEQ ID NO .: 33)

CAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACT

CTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTACTACATGACCTGGATCCGCCAGG

CTCCAGGGAAGGGGCTGGAGTGGATTTCATACATTAGTAGTACTGGTGGTAACATCTAC

TACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCCAGAATTCACT

GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAG

ATGGGGGTGTAATAATCTCAACTGCTATGTTTGACTATTGGGGCCAAGGGACCACGGTC

ACCGTCTCCTCA&gt; Amino acid sequence 28B11 VH (SEQ ID NO: 34)

QVQLVESGGGLVKPGGSLRLSCAASGFTFSNYYMTWIRQAPGKGLEWISYISSTGGNIY YADSVKGRFTISRADNQNSLYLQMNSLRAEDTAVYYCARDGGVIISTAMFDYWGQGTTV TVSS ^ Nucleic Acid Sequence ID: 28 VMB 35 SEQB11 Nucleic Acid ID: 28

GCCATCCAGTTGACCCAGTCTCCCTCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC

<img file="MX348013B_D0040.tif" />

IMPI

ΙΜΠΤΤυΤΟ MEXICAN

OF U INDUSTRIAL PROPISbAtj

CATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGClTCG i Al

CAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCA.GTTTGGAAAGTGGGGTCCCA

TCAAGGCTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA

GCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCG

GCGGAGGGACCAAGGTGGAGATCAAA&gt; Amino acid sequence 28B11 VL (SEQ ID NO: 36)

AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVP

SRLSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK

Antibody 41A4 includes a heavy chain variable region (SEQ ID NO: 38) encoded by the nucleic acid sequence shown in SEQ ID NO: 37, and a light chain variable region (SEQ ID NO: 37). : 40) encoded by the nucleic acid sequence shown in SEQ ID NO .: 39.

&gt; Nucleic acid sequence VH 41A4 (SEQ ID NO .: 37) GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTGTGCCATGCACTGGGTCCGGCAAG CTCCAGGGAAGGGCCTGGAATGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCGTATAC TATGCGGACTCTGTGAAGGGCCGATTGACCATCTCCAGAGACAACGCCAAGAATTCCCT GTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTACAAAAG AAAAATACAACTGGAACGACGAGGGGGAATACTTCTACGGAATGGACGTCTGGGGCCAA

GGGACCACGGTCACCGTCTCCTCA

<img file="MX348013B_D0041.tif" />

IMPI ιιβτττυτο MEXICAN Pt LA HUJHMjAD

INDUSTRIAL&gt; Amino acid sequence 41A4 VH (SEQ ID NO: 38)

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDCAI4HWVRQAPGKGLEWVSGISWSGSVY YADSVKGRFTISRADNKNSLYLQMNSLRAEDTALYYCTKEKYNWNDEGEYFYGMDVWGQ GTTVTVSS&gt; Nucleic acid sequence VL 41A4 (SEQ ID NO .: 39) GAAATTGTGTTGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC

CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGA AACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATC CCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACT GGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCTTTCGGCGGAG GGACCAAGGTGGAGATCAAA&gt; 41A4 VL amino acid sequence (SEQ ID NO .: 40)

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRA

TGI

PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSFGGGTKVEIK

The 43G6 antibody includes a heavy chain variable region (SEQ ID NO: 4 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 41, and a light chain variable region (SEQ ID NO: .: 44) encoded by the nucleic acid sequence shown in SEQ ID NO .: 43.

₆₃ IMPI ^ fMSTnVTO MEXICAN ^^ μπιγΤ 'of the noMEOAD INDUSTRY!

&gt; 43G6 VH3 nucleic acid sequence (SeQ¹ 1U T-NO.41)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT ctcctgtgcagcctctggattcaccttcagtagctatagcatgaactgggtccgccagg CTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTGGTAGTAGTACCATATAC TACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACT GTATCTGCAAATGAACAGCCTGAGAGACGAGGACACGGCTGTGTATTACTGTGCGAGAG AGGGCTATGTTTCGGGGACCTATTACAACTACTACTACGGTATGGACGTCTGGGGCCAA GGGACCACGGTCACCGTCTCCTCA&gt; Amino acid sequence of 43G6 VH3 (SEQ ID NO .: 42) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSGSSTIY

YADSVKGRFTISRADNKNSLYLQMNSLRDEDTAVYYCAREGYVSGTYYNYYYGMDVWGQ

GTTVTVSS&gt; 43G6 VL3 Nucleic Acid Sequence (SEQ ID NO: 43)

GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGTTACTTAGCCTGGTACCAACAGAAAC CTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCACCCAACAGGGCCACTGGCATCCCA GCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGA GCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGTCGCTCACTTTCG GCGGAGGGACCAAGGTGGAGATCAAA&gt; Amino acid sequence of 43G6 VL3 (SEQ ID NO .: 44)

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAPNRATGIP

<img file="MX348013B_D0042.tif" />

IMPI u * rrruro Mexicana&gt; DE LA FHONEDAr INDUSTRIAL

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The huIL-17F antibodies of the invention also include, eg. , the heavy chain complementarity determining regions (VH CDRs) shown in Table 1, the light chain CDRs (VL CDRs) shown in Table 2, and combinations thereof.

Table 1. VH CDR Sequences of Antibody Clones That Bind to, and Neutralize, the Biological Activity of IL17F

<td>ID of clone</td><td>Heavy CDRl</td><td>Heavy CDR2</td><td>Heavy CDR3</td><td>Gene family</td>

<td>11C5</td><td>IYYLH (SEQ ID NO .: 45)</td><td>IINPSGGRTNYAQFQG (SEQ ID NO: 46)</td><td>GEFSSGWLDY (SEQ ID NO: 47)</td><td>IGHV1-46</td>

<td>21B10</td><td>SYSMN (SEQ ID NO: 48)</td><td>YISGGSSTIYYADSVKG (SEQ ID NO .: 49)</td><td>EGYVSGTYYNYYYGMDV (SEQ ID NO .: 50)</td><td>IGHV3-48</td>

<td>1F1</td><td>SYVMS (SEQ ID NO: 51)</td><td>AISGRGGNTFYADSVKG (SEQ ID NO: 52)</td><td>DDRRIAAGSFDY (SEQ ID NO: 53)</td><td>IGHV3-23</td>

<td>2E12</td><td>SYSMN (SEQ ID NO: 48)</td><td>YISSSSSAIYYADSVKG (SEQ ID NO .: 54)</td><td>EGYAS GRYYNYYYGMDV (SEQ ID NO: 55)</td><td>IGHV3-48</td>

<td>5E12 IgG4</td><td>DYAMH (SEQ ID NO .: 56)</td><td>GISWNSGTIGYADSVKG (SEQ ID NO: 57)</td><td>ELYISDWDSYSYGMDV (SEQ ID NO: 58)</td><td>IGHV3-9</td>

<td>41B10 IgG4</td><td>NAWMS (SEQ ID NO .: 59)</td><td>RIKSKTDGGTTDYVAPVKG (SEQ ID NO .: 60)</td><td>SYSSYWFPYYFDY (SEQ ID NO .: 61)</td><td>IGHV3-15</td>

<td>5D3</td><td>DYAMH (SEQ ID NO .: 56)</td><td>GISWNSGSRGYADSVKG (SEQ ID NO .: 62)</td><td>DMVYALDV (SEQ ID NO: 63)</td><td>IGHV3-9</td>

<td>22F8</td><td>SYAMN (SEQ ID NO .: 64)</td><td>TISGRGGSIYYADSVKG (SEQ ID NO: 65)</td><td>EEATWDFDY (SEQ ID NO: 66)</td><td>IGHV3-23</td>

<td>28B11</td><td>NYYMT (SEQ ID NO: 67)</td><td>YISSTGGNIYYADSVKG (SEQ ID NO: 68)</td><td>DGGVIISTAMFDY (SEQ TD NO .: 69)</td><td>IGHV3-11</td>

<img file="MX348013B_D0043.tif" />

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<td rowspan="2">41A4</td><td rowspan="2">DCAMH (SEQ ID NO .: 70)</td><td rowspan="2">GISWNSGSVYYADSVKG (SEQ ID NO: 71)</td><td>EKYNWNDEGEYFYGMDV</td><td>IGHV3- 9</td>

<td>(SEQ ID NO .: 72)</td><td></td>

<td>43G6</td><td>SYSMN (SEQ ID NO: 48)</td><td>YISSGS STIYYADSVKG (SEQ ID NO: 73)</td><td>EGYVSGTYYNYYYGMDV (SEQ ID NO: 50)</td><td>IGHV3-48</td>

Table 2. VL CDR Sequences of Antibody Clones That Bind to and Neutralize IL-17F

<td>Clone ID</td><td>CDR1 Light</td><td>CDR2 Light</td><td>CDR3 Light</td><td>Gene family</td>

<td>11C5</td><td>RASQGISSWLA (SEQ ID NO: 74)</td><td>AASSLQS (SEQ ID NO: 75)</td><td>ANSFPLT (SEQ ID NO .: 76)</td><td>IGKV1-12 IGKJ4</td>

<td>21B10</td><td>RASQSVSSYLA (SEQ ID NO: 77)</td><td>DAPNRAT (SEQ ID NO: 78)</td><td>RSNWSLT (SEQ ID NO .: 79)</td><td>IGKV3-11 IGKJ4</td>

<td>1F1</td><td>RASQGISSALA (SEQ ID NO: 80)</td><td>DVSSLES (SEQ ID NO .: 81)</td><td>FNSYPLT (SEQ ID NO .: 82)</td><td>IGKV1D-13 IGKJ4</td>

<td>2E12</td><td>RASQSVSSYLA (SEQ ID NO .: 77)</td><td>DASNRAT (SEQ ID NO: 83)</td><td>RSSWSLT (SEQ ID NO: 84)</td><td>IGKV3-11 IGKJ4</td>

<td>5E12</td><td>RASQSVSSSYLA (SEQ ID NO: 85)</td><td>GASSRAT (SEQ ID NO .: 86)</td><td>QQYGSSP (SEQ ID NO; 87)</td><td>IGKV3-20</td>

<td>41B10</td><td>RASQGISSWLA (SEQ ID NO: 88)</td><td>AASSLQS (SEQ ID NO: 89)</td><td>QQYNSYPIT (SEQ ID NO: 90)</td><td>IGKV1D-16</td>

<td>5D3</td><td>RASQSFSGSYLA (SEQ ID NO: 91)</td><td>DTSSRAT (SEQ ID NO: 92)</td><td>QQYGTWT (SEQ ID NO .: 93)</td><td>IGKV3-20</td>

<td>22F8</td><td>RASQSVSSFLA (SEQ ID NO: 94)</td><td>DASNRAT (SEQ ID NO: 83)</td><td>QQRSNWPPT (SEQ ID NO: 95)</td><td>IGKV3-11</td>

<td>28B11</td><td>RASQGISSALA (SEQ ID NO: 80)</td><td>DASSLES (SEQ ID NO .: 96)</td><td>QQFNSYPLT (SEQ ID NO: 97)</td><td>IGKV1D-13</td>

<td>4 1A4</td><td>RASQSVSSSYLA (SEQ ID NO: 85)</td><td>GASSRAT (SEQ ID NO .: 86)</td><td>QQYGSS (SEQ ID NO: 98)</td><td>IGKV3-20</td>

<td>43G6</td><td>RASQSVSSYLA (SEQ ID NO: 77)</td><td>DAPNRAT (SEQ ID NO: 78)</td><td>QQRSNWSLT (SEQ ID NO: 99)</td><td>IGKV3-11</td>

The amino acids that encompass the complementarity determining regions (CDR) are in accordance with that defined by EA Kabat et al. {See Kabat, EA, et al., Sequences of Protein of immunological interest, 5th Ed., US Department of Health and Human Services, US Government Printing Office

<img file="MX348013B_D0044.tif" />

IMPI la invcni-i nr_ vint i nnr.rpuz^- the antibodies described in the antibodies of the invention are (1991)) ·

They are also included in bind to the same epitope as present. For example, they specifically bind to the IL-17F and / or IL-17A / IL-17F heterodimeric complex, where the antibody binds to an epitope that includes one or more amino acid residues in human IL-17F (Accession No. AAH70124). In some embodiments, the antibodies of the invention specifically bind to IL-17F and the heterodimeric IL-17A / IL-17F complex, where the antibody binds to an epitope on human IL-17F {eg. , Acquisition No. AAH70124).

Those of skill in the art will recognize that it is possible to determine, without undue experimentation, whether a monoclonal antibody (eg, a fully human monoclonal antibody) has the same specificity as a monoclonal antibody of the invention {eg. , clones 5E12, 41B10, 11C5, 21B10, 1F1, 2E12, 5D3, 22F8, 28B11, 41A4 and 43G6) by determining whether the former prevents the latter from binding to IL-17F and / or the IL-complex. Heterodimeric 17A / IL-17F. If the monoclonal antibody tested competes with the monoclonal antibody of the invention, as shown by a decrease in the binding of the monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same epitope, or to one

<img file="MX348013B_D0045.tif" />

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closely related.

An alternative method to determine if a monoclonal antibody has the specificity of the monoclonal antibody of the invention is to pre-incubate the monoclonal antibody of the invention with soluble IL-17F and / or soluble heterodimeric IL17A / IL-17F complex proteins and then add the monoclonal antibody. tested to determine whether the monoclonal antibody tested is inhibited in its ability to bind IL-17F and / or the heterodimeric IL-17A / IL-17F complex. If the monoclonal antibody tested is inhibited then it is most likely to have the same epitopic specificity as the monoclonal antibody of the invention, or a functionally equivalent specificity.

Observation of the monoclonal antibodies of the invention can also be carried out, eg, by measuring the production of cytokines and / or chemokines induced by IL-17F (eg, IL-6, IL -8, G-CSF, GM-CSF, GRO-α, GRO-b, LIX, GCP-2, MIG, IP10, I-TAC, and MCP-1, RANTES, Eotaxin, SDF-1, and MIP3a) and by determining whether the test monoclonal antibody is capable of modulating, blocking, inhibiting, reducing, antagonizing, neutralize or otherwise interfere with IL-17F-induced cytokine and / or chemokine production.

Various procedures known within the art can be used for the production of monoclonal antibodies directed against the

<img file="MX348013B_D0047.tif" />

derivatives, fragments, analogs, homologues or themselves. (See, eg, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Coid Spring Harbor Laboratory Press, Coid Spring Harbor, NY, which is incorporated herein by reference). Fully human antibodies are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arises from human genes. Such antibodies are referred to as human antibodies, or fully human antibodies herein. Human monoclonal antibodies are prepared, for example, by using the procedures described in the Examples provided below. Human monoclonal antibodies can also be prepared by use of the trioma technique; the human B cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4:72); and the EBV hybridoma technique for producing human monoclonal antibodies (see Cole, et al., 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be used and produced by the use of human hybridomas (see Cote, et al., 1983. Proc Nati Acad Sci USA 80: 2026-2030) or by transforming human B cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In:

Monoclonal Antibodies and Cancer

Therapy, Alan R. Liss, Inc., p. 77-96).

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The antibodies are purified by means of well-known techniques, such as affinity chromatography using protein A or protein G, which mainly provides the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen that is the target of the sought immunoglobulin, or an epitope thereof, can be immobilized on a column to purify the immunospecific antibody by means of immunoaffinity chromatography. Immunoglobulin purification is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28) .

The antibodies of the invention (eg, 5E12, 41B10, 11C5, 21B10, 1F1, 2E12, 5D3, 22F8, 28B11, 41A4, and 43G6) are fully human monoclonal antibodies. Monoclonal antibodies that modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with IL17F-mediated pro-inflammatory cytokine production are generated, eg. , by immunizing an animal with IL-17F such as, for example, murine, rat or human IL-17F or an immunogenic fragment, derivative or variant thereof. Alternatively, the animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding IL-17F, such that IL-17F is expressed and associated with the

<img file="MX348013B_D0049.tif" />

cells transferred. Alternatively, V37 ~ ^ TT ^^ TiT ± ctteTpos are obtained by observing a library containing antibody binding domain sequences or 5 antigens to bind IL-17F. This library is prepared, e.g., in bacteriophages as protein or peptide fusions to a bacteriophage-coated protein that is expressed on the surface of assembled phage particles and the DNA-encoding sequences contained within the 0 phage particles (ie ie, phage display library). The hybridomas that result from the myeloma / B cell fusions are then observed for IL-17F reactivity.

Monoclonal antibodies are prepared, for example, by the use of hybridoma methods, such as those described by Kohler and Milstein, Nature, 256: 495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal is typically immunized with an immunizing agent to obtain lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, a fragment thereof, or a fusion protein thereof. In general, both lymphocytes of

IMPI rus ιηντο wtietMo ​​peripheral blood if spleen cell cells or lymph node cells are desired are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line by use of a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59 to 103). Immortalized cell lines are usually transformed mammalian cells, in particular myeloma cells of rodent, bovine and human origin. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused and immortalized cells. For example, if the parentel cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), the substances prevent the growth of cells deficient in HGPRT.

Preferred immortalized cell lines are those that fuse efficiently, support a stable high level of antibody expression by selected antibody-producing cells, and are

<img file="MX348013B_D0050.tif" />

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Di LA PROnFDAO INDUSTRIAL sensitive to a medium such as HAT medium. The most preferred immortalized cell lines are murine myeloma lines, available, for example, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of monoclonal antibodies. (See Kozbor, J. Immunol., 133: 3001 (1984);

Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51 to 63)).

The culture medium, in which the hybridoma cells are cultured, can then be tested for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by means of immunoprecipitation or by means of an in vitro binding assay, such as a radioimmune assay (RIA) or an enzyme-linked immunosorbent assay. (ELISA). Such techniques and tests are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107: 220 (1980). In addition, in therapeutic applications of monoclonal antibodies it is important

IMPI nwrmm MKBCAN · DE LA rBOFlEDAD,,, INDUSTRIAL identify antibodies that have a high degree of specificity and binding affinity for 'the' * target antigen.

After the desired hybridoma cells are identified, the clones can be subcloned by limited dilution procedures and cultured by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59 to

103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 Medium. Alternatively, the hybridoma cells can be cultured in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or from the ascites fluid by means of standard immunoglobulin purification procedures such as, for example, Protein A Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis. , or affinity chromatography.

Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of the invention can be easily isolated and sequenced by the use of standard procedures (e.g., by the use of

<img file="MX348013B_D0051.tif" />

<img file="MX348013B_D0052.tif" />

oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce otherwise. immunoglobulin protein, to obtain monoclonal antibody synthesis in recombinant host cells. DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of homologous murine sequences (see US Patent No. 4,816,567; Morrison , Nature 368, 812-13 (1994)) or by covalently linking to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or it can be substituted for the variable domains of an antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

Human antibodies and humanization of antibodies

Monoclonal antibodies of the invention include ⁷⁵ IMPIAS

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M LA WOR1EDAD ΐ ^ -ιι {5 · * INDUSTRIAL fully human antibodies or humanized antibodies. These antibodies are suitable for administration to humans without eliciting an immune response on the part of the human against the administered immunoglobulin.

A huIL-17F antibody is generated, for example, by using the procedures described in the Examples provided below.

In other alternative methods, a huIL-17F antibody is raised, for example, by the use of phage display methods by the use of antibodies containing only human sequences. Such approaches are well known in the art, eg. , in WO92 / 01047 and US Patent No. 6,521,404, which are incorporated herein by reference. In this approach, a combinatorial phage library carrying random pairs of heavy and light chains is observed by the use of a natural or recombinant source of IL-17F or fragments thereof. In another approach, a huIL-17F antibody can be produced by a process where at least one step of the process includes immunizing a non-human transgenic animal with the human IL-17F protein. In this approach, some of the endogenous heavy chain and / or kappa light sites of this xenogenic non-human animal have been disabled and are unable to achieve the rearrangement required to generate genes encoding immunoglobulins in response to an antigen. In addition, to

IMPI IMfTTTUT · MCX1CANO Di LAntOHEDAfi INDUSTRY!

least one human heavy chain site and at least one human light chain site have been stably transfected into the animal. Therefore, in response to an administered antigen, human sites rearrange to provide genes that encode human variable regions immunospecific for the antigen. Therefore, after immunization, the transgenic mouse produces B cells that secrete whole human immunoglobulins.

A variety of techniques for producing xenogeneic non-human animals are well known in the art. For example, see US Patent Nos. 6,075,181 and No. 6,150,584, which are incorporated herein by reference in their entirety. This general strategy was demonstrated in connection with the generation of the first XenoMouse ™ strains as published in 1994. See Green et al. Nature Genetics 7: 13-21 (1994), which is incorporated herein by reference in its entirety. See also, US Patent Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2 and European Patent No., EP 0 463 151 B1 and International Patent Application Nos. WO 94/02602, WO 96/34096, WO 98/24893, WO 00/76310 and related family members.

In an alternative approach, others have used a minisite approach in which an exogenous Ig site mimics

<img file="MX348013B_D0053.tif" />

<img file="MX348013B_D0054.tif" />

through the inclusion of pieces (individual genes) of the Ig site. Therefore, one or more VH genes, one or more D genes_h, one or more J genes_H, a mu constant region, and a second constant region (preferably a gamma constant region) are formed within a construct for insertion into an animal. See eg, US Patent Nos. 5,545,806; 5,545,807; 5,591,669; 5,612,205; 5,625,825; 5,625,126; 5,633,425; 5,643,763; 5,661,016; 5,721,367; 5,770,429; 5,789,215; 5,789,650; 5,814,318; 5,877,397; 5,874,299; 6,023,010; and 6,255,458; and European Patent No. 0 546 073 B1; and International Patent Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97 / 13852, and WO 98/24884 and related family members.

The generation of human antibodies from mice into which large pieces of chromosomes or whole chromosomes have been introduced, through microcellular fusion, has also been demonstrated. See European Patent Application Nos. 773 288 and 843 961.

Human anti-mouse antibody (HAMA) responses have led the industry to prepare chimeric or humanized antibodies by other means. While chimeric antibodies have a human constant region and an immune variable region, certain human anti-chimeric antibody (HACA) responses are expected to be observed, in particular⁷⁸ IMBI Mexican institute OF fNDVSTXIAl PROPERTY in chronic or multiple dose uses ..... of the antibody. Therefore, it would be desirable to provide fully human antibodies against IL-17F and / or the heterodimeric IL-17A / IL-17F complex in order to vitiate or otherwise mitigate the concerns and / or effects of the HAMA or HACA response.

The production of antibodies with reduced immunogenicity is also achieved through humanization, chimerization and visualization techniques by the use of appropriate libraries. It will be appreciated that murine antibodies or antibodies from other species can be humanized or primatized using techniques well known in the art. See eg, Winter and Harris Immunol Today 14:43 46 (1993) and Wright et al. Crit, Reviews in Immunol. 12125-168 (1992). The antibody of interest can be manipulated by means of recombinant DNA techniques to replace the hinge domains CH1, CH2, CH3, and / or the framework domain with the corresponding human sequence {See WO 92/102190 and US Pat. United States Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085). Also, the use of Ig cDNA for the construction of chimeric immunoglobulin genes is known in the art (Liu et al. PNAS 84: 3439 (1987) and J. Immunol. 139: 3521 (1987)). The mRNA is isolated from a hybridoma or other cell that produces the antibodies and is used to produce cDNA. The

<img file="MX348013B_D0055.tif" />

<img file="MX348013B_D0056.tif" />

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CDNA of interest can be amplified by means of the. —..........

polymerase chain by use of specific primers (US Patent Nos. 4,683,195 and 4,683,202). Alternatively, a library is made and viewed to isolate the sequence of interest. The DNA sequence encoding the variable region of the antibodies is then fused with the sequences of the human constant region. The human constant region gene sequences can be found in Kabat et al. (1991) Sequences of Proteins of immunological Interest, NIH publication no. 91-3242. Human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effector functions, such as complement fixation, or activity on antibody-dependent cellular cytotoxicity. The preferred isotypes are IgGl, IgG3 and IgG4. Any of the human, kappa, or lambda light chain constant regions can be used. The chimeric humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F (ab ')₂ and Fabs can be prepared by cleaving the intact protein, eg. , by means of a protease or chemical cleavage. Alternatively, a truncated gene can be engineered. For example, a chimeric gene that encodes a

<img file="MX348013B_D0057.tif" />

IMPI ΙΝΠΤΠ TO MEXJCAN · Df THE INDUSTRIAL PROPERTY portion of the fragment F (ab ')₂ would include the sequences। encoding the CH1 domain and H chain hinge region, followed by a translation stop codon to produce the truncated molecule.

Consensus sequences from the H and LJ regions can be used to design oligonucleotides for use as primers to introduce useful restriction sites in the J region for subsequent attachment of the V region segments to the human C region segments. The C region of the cDNA can be modified by site-directed mutagenesis to establish a restriction site at the analogous position in the human sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV-derived episomes, and the like. A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites designed so that any VL or VH sequence can be easily inserted and expressed. In such vectors, splicing typically occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also in the splice regions that occur within human CH exons. . Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The

<img file="MX348013B_D0058.tif" />

The resulting chimeric antibody can bind to any ^ strong promoter, including antiretroviral LTRs, eg, the SV-40 early promoter, (Okayama et al. Mol. Cell. Bio. 3: 280 (1983)), the LTR of Rous sarcoma virus (Gorman et al. PNAS 79: 6777 (1982)), and the LTR of Moloney murine leukemia virus (Grosschedl et al. Cell. 41: 885 (1985)). Native Ig promoters and the like can also be used, as will be appreciated.

In addition, human antibodies or antibodies to IO other species can be generated through expression type technologies, including, without limitation, phage display, retroviral expression, ribosomal expression, and other techniques, by the use of highly technical techniques. known in the art and the resulting molecules may be subjected to further maturation, such as affinity maturation, since such techniques are well known in the art. Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), Kanes and Plückthun PNAS USA 94: 4937-4942 (1997) (ribosomal expression), Parmley and

Smith Gene 73: 305-318 (1988) (phage display), Scott,

TIBS, vol. 17: 241-245 (1992), Cwirla et al. PNAS USA 87: 63786382 (1990), Russel et al. Nucí. Acids Research 21: 1081-1085 (1993), Hoganboom et al. Immunol. Reviews 130: 43-68 (1992),

Chiswell and McCafferty TIBTECH; 10: 80-8A (1992), and US Patent No. 5,733,743. If expression technologies are used to produce non-human antibodies,

IMPI πβπττυτο Mexican DE LA PROMEDA · INDURTRIAJ.

such antibodies can be humanized as described above.

By use of these techniques, antibodies can be generated to cells expressing IL-17F, IL-17F itself, forms of IL-17F epitopes or peptides thereof, and their expression libraries (See e.g., US Patent No. United States No. 5,703,057) that can be observed thereafter as described above for the activities described herein.

The huIL-17F antibodies of the invention can be expressed by means of a vector containing a DNA segment encoding the single chain antibody described above.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. Vectors include chemical conjugates such as those described in WO 93/64701, which have a target moiety (eg, a ligand for a cell surface receptor), and a nucleic acid-binding moiety (eg, polylysine), a viral vector (eg. a DNA or RNA viral vector), fusion proteins such as those described in PCT / US 95/02140 (WO 95/22618) which is a fusion protein containing a target residue (eg a cell specific antibody target) and a nucleic acid binding moiety (eg a protamine), plasmids, phage, etc. Vectors

<img file="MX348013B_D0059.tif" />

IMPI INSTITUTO mwicaNO DE LA PROPERTY INDUSTRIAL can be chromosomal, nonchromosomal or synthetic.

Preferred vectors include viral vectors fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. Viral DNA vectors are preferred. These vectors include smallpox vectors such as orthopox or avipox vectors, herpes virus vectors such as a herpes simplex virus I (HSV) vector (see Geller, AI et al., J. Neurochem, 64: 487 (1995 ); Lim, F., et al., In DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Nati. Acad. Sci .: USA 90: 7603 (1993); Geller, AI, et al., Proc Nati. Acad. Sci USA 87: 1149 (1990), Adenovirus Vectors (see LeGal LaSalle et al., Science, 259: 988 (1993); Davidson, et al., Nat. Genet 3: 219 (1993); Yang, et al. , J. Virol. 69: 2004 (1995) and adeno-associated virus vectors (see Kaplitt, MG. Et al., Nat. Genet. 8: 148 (1994).

Smallpox viral vectors introduce the gene into the cytoplasm of cells. Avipox virus vectors result in only short-term expression of the nucleic acid. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors are preferred for introducing nucleic acid into neural cells. The adenovirus vector results in a shorter term expression (approximately 2 months) than the⁸⁴ ΙΜΡΙζ ^

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INDUSTRIAL adeno-associated virus (approximately 4 months). which in turn is shorter than HSV vectors. The particular vector chosen will depend on the target cell and the condition to be treated. The introduction can be by means of standard techniques, eg. infection, transection, transduction, or transformation. Examples of modes of gene transfer include eg. , Naked DNA, CaP0 precipitation₄, DEAE dextran, electroporation, protoplastic fusion, lipofection, cell microinjection, and viral vectors.

The vector can be used to locate essentially any desired target cell. For example, stereotaxic injection can be used to direct vectors (eg, adenovirus, HSV) to a desired location. Additionally, the particles can be administered by intracerebroventricular infusion (ICV) by use of a minipump infusion system, such as a SynchroMed Infusion System. A mass flow-based method, called convection, has also proven effective in delivering large molecules to large areas of the brain and may be helpful in delivering the vector to the target cell. (See Bobo et al., Proc. Nati. Acad. Sci. USA 91: 2076-2080 (1994); Morrison et al., Am. J. Physiol. 266: 292-305 (1994)). Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, oral or other known routes of administration.

These vectors can be used to express large amounts of antibodies that can be used in a variety of ways. For example, to detect the presence of IL-17F in a sample. The antibody can also be used to attempt to bind to and disrupt signaling that is related to IL-17F.

Techniques can be adapted for the production of single chain antibodies specific for an antigenic protein of the invention (see eg, US Patent No. 4,946,778). In addition, methods for the construction of Fab expression libraries (see eg, Huse, et al., 1989 Science 246: 12751281) can be adapted to allow rapid and efficient identification of F fragments._ab monoclonals with the desired specificity for a protein or its derivatives, fragments, analogs or homologues thereof. Antibody fragments containing the idiotypes for a protein antigen can be produced by techniques known in the art including, but not limited to: (i) an F (_ab&gt;) 2 produced by pepsin digestion of an antibody molecule; (ii) an F fragment_ab generated by reducing the disulfide bridges of a fragment F (_{abl) 2}; (iii) an F fragment_ab generated by treating the antibody molecule with papain and a reducing agent and (iv) F fragments_v.

IMPIAS ΐΜππνΜΜίηαυο

OF I * nOHEDAD! N »UST1UAI

The invention also includes F fragments_{v /} Fa_b, F_tob- and F (ab ') 2 anti-yl-17f, single-chain anti-yl-T7t ~ antibodies, bispecific anti-yl-17f antibodies, and heteroconjugate anti-yl-17f antibodies.

Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for IL-17F. The second binding target is any other antigen, and is advantageously a cell surface protein or receptor or receptor subunit.

Methods for preparing bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two pairs of immunoglobulin heavy chains / light chains, in which the two heavy chains have different specificities (Milstein and Cuello, Nature, 305: 537-539 ( 1983)). Due to the random collection of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually accomplished through affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published May 13, 1993, and

<img file="MX348013B_D0060.tif" />

IMPI

MBSCANT INSTITUTE&gt;

OE LA PROflEDAP INDUSTRIAL in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

Variable domains of antibodies with desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. Preferably fusion with an immunoglobulin heavy chain constant domain, comprising part of the hinge, CH2 and CH3 regions. It is preferred that the first heavy chain constant region (CH1) contains the site necessary for attachment to the light chain present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. For additional details of the generation of bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be manipulated to maximize the percentage of heterodimers that are recovered from the recombinant cell culture. The preferred parting surface comprises at least a portion of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the parting surface of the first molecule of

IMPI »

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INDUSTRIAL ^ »· * *?

antibody are replaced by larger side chains {eg. tyrosine or tryptophan). Compensation cavities of identical or similar size to the large side chain (s) are created on the parting surface of the second antibody molecule by replacing the large amino acid side chains for smaller {eg. alanine or threonine). This provides a mechanism to increase the performance of the heterodimer over other unwanted end products such as homodimers.

Bispecific antibodies can be prepared as full-length antibodies or antibody fragments {eg. bispecific F (ab ') antibodies₂). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared by the use of chemical linkages. Brennan et al., Science 229: 81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to generate F (ab ') fragments₂. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize neighboring dithiols and prevent intermolecular disulfide formation. The Fab fragments¹ generated are then converted to thionitrobenzoate (TNB) derivatives. Then one of the Fab'-TNB derivatives is reconverted

IMPIwsTmrro mHücano OF INDITTRIAL PRORSOAD in Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Additionally, Fab fragments¹ they can be recovered directly from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody molecule F (ab ') 2. Each Fab 'fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. Therefore, the bispecific antibody formed was capable of binding to cells that overexpress the ErbB2 receptor and normal human T cells, as well as triggering lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques have also been described for preparing and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies have been produced by the use of leucine zippers. Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). Zippers Peptides

ΤΗΓΠΤϋΤΟ MEXJCAM0 from raoFiroA&gt; \ ^ e ^ s - »'4JSF XDUmiM leucine from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced in the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used for the production of antibody homodimers. The diabody technology described by Hollinger et al., Proc. Nati. Acad. Sci. USA 90: 6444-6448 (1993) has provided an alternative mechanism for preparing bispecific antibody fragments. The fragments comprise a heavy chain variable domain (V_H) connected to a light chain variable domain (V_L) by means of a link that is too short to allow pairing between the two domains on the same chain. Therefore, the domains V_H and V_L of a fragment are bound to pair with the V domains_H and V_Lcomplementary to another fragment, thus forming two antigen-binding sites. Another strategy to prepare bispecific antibody fragments through the use of Fv dimers (sFv) has also been reported. See, Gruber et al., J. Immunol. 152: 5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60 (1991).

Exemplary bispecific antibodies are

<img file="MX348013B_D0061.tif" />

IMPI Bfsrmrro Mexicano □ i LA MI QUEDAD induwelu can bind to two different epitopes, at least one of which originates from the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm that binds to a trigger molecule on a leukocyte such as a T cell receptor molecule (eg. CD2, CD3, CD28 or B7), or Fe receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) in such a way that the cellular defense mechanisms are focused so that the cell expresses the particular antigen. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing a particular antigen. These antibodies possess an antigen-binding arm and an arm that binds to a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and additionally binds tissue factor (TF).

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have been proposed, for example, to target cells of the immune system for unwanted cells (see US Patent No. 4,676,980), and for the treatment of infection.

<img file="MX348013B_D0062.tif" />

IMPI INSTrrrro mexkan · DE LA PROniDAD INDUSTRIAL by HIV (see WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include methyl iminothiolate and 4-mercaptobutyrimidate and those disclosed, for example, in US Patent No. 4,676,980.

It may be desired to modify the antibody of the invention with respect to effector function in such a way that it is enhanced, eg. , the efficacy of the antibody in the treatment of diseases and disorders associated with IL-17F signaling. For example, cysteine ​​residue (s) can be introduced into the Fe region, thus allowing the formation of interchain disulfide bonds in this region. Therefore, the homodimeric antibody generated may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (See Carón et al., J. Exp Med., 176 : 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, an antibody that has dual Fe regions can be manipulated and thus can have

<img file="MX348013B_D0063.tif" />

IMPI institute

Give THE INDUSTRIAL RRORlfcAD a complement lysis and enhanced ADCC capabilities.

(See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)).

The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof) or a radioactive isotope ( i.e. a radioconjugate).

Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain , Modecin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin and trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include²¹²Bi, ¹³¹I, ¹³¹ln, ⁹⁰And, and ¹⁸⁶Re.

Antibody and cytotoxic agent conjugates are prepared by the use of a variety of bifunctional protein coupling agents such as propionate.

IMPI

ΙΜΗΤΤνΤΟΜΚΙβΜ *

IMHTTVrOMBUeM * · Μ u FRonf »a · rNWsntiAi of N-succinimidyl-3- (2-pyridyldithiol) (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl subhydrate ), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (pazidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylene diamine), diisocyanates (such as tolylene2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MYDTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibody. (See WO94 / 11026).

Those of skill in the art will recognize that a wide variety of possible moieties can be coupled to the resulting antibodies of the invention. (See, for example, Conjugate Vaccines, Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the full contents of which are incorporated herein by reference).

Coupling can be carried out by any chemical reaction that will join the two molecules.

IMPI fNSTtTWTO MEXICANO as long as the antibody and the rest retain their respective activities. This bonding can include many chemical mechanisms, eg, covalent bonding, affinity bonding, intercalation, coordinate bonding, and complexation. However, the preferred bond is covalent bonding. Covalent attachment can be achieved both by direct condensation of existing side chains and by incorporation of external bridging molecules. Many bivalent or polyvalent binding agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylenediamines. This list is not intended to be exhaustive of the various classes of coupling agents known in the art, but is representative of the most common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133: 1335-2549 (1984); Jansen et al., Immunological Reviews 62: 185-216 (1982); and Vitetta et al., Science 238: 1098 (1987).

Preferred links are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44: 201-208 (1984) describing the use of MBS (Mmaleimidobenzoyl-N-hydroxysuccinimide ester). See also,

<img file="MX348013B_D0064.tif" />

IMPI INSTITUTE MMafiftPO DE LA PRQplEDA »INDCKTUM

US Patent No. 5,030,719, which describes the use of hydrogenated acetylhydrazide derivative coupled to an antibody via oligopeptide linkage. Particularly preferred linkages include: (i) EDC (l-ethyl-3- (3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha- (2-pridyl-dithio) toluene (Pierce Chem . Co., Cat. (21558G); (iii) Succinimidyl 6 [3- (2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co. , Cat. No. 21651G); (iv) Sulfo-LC-SPDP (6 [3- (2-pyridyldithio) propianamide] sulfosuccinimidyl hexanoate (Pierce Chem. Co. Cat. No. 2165-G); and (v) sulfo-NHS (N-hydroxysulfo -succinimide: Pierce Chem. Co., Cat. No. 24510) conjugated to EDC.

The above-described linkages contain components that have different attributes, which therefore lead to conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS esters that contain bonds are less soluble than sulfo-NHS esters. In addition, the SMPT bond contains a spherically hindered disulfide bond and can form conjugates with increased stability. In general, disulfide bonds are less stable than other bonds because the disulfide bond is cleaved in vitro, resulting in

<img file="MX348013B_D0065.tif" />

less conjugate available.

Sulfo-NHS

<img file="MX348013B_D0066.tif" />

in particular.

can enhance the stability of carbodiimide couplings. Carbodiimide couplings (such as

EDO when used in conjunction with sulfo-NHS form esters that are more resistant to hydrolysis than the carbodiimide coupling reaction alone.

The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as those described in Epstein et al., Proc. Nati. Acad. Sci. USA, 82: 3688 (1985);

Hwang et al., Proc. Nati Acad. Sci. USA, 77: 4030 (1980); and United States Patent Nos. 4,485,045 and

4,544,545. Liposomes with enhanced circulation time are disclosed in US Patent No.

5.013.556 .

Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through filters of defined pore size to give liposomes of the desired diameter. The Fab 'fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) by

<img file="MX348013B_D0067.tif" />

IMPI xrmrra u * ica&gt; or CE LA FROrtiPAP means of a disulfide exchange reaction.

Use of antibodies against IL-17F

It will be appreciated that the administration of therapeutic entities in accordance with the invention will be administered with suitable vehicles, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed. , Mack Publishing Company, Easton, PA (1975)), particularly chapter 87 by Blaug, Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (such as Lipofectin ™), DNA conjugates, anhydrous absorption pastes, oil emulsions in water and water in oil, Carbowax emulsions (polyethylene glycols of various molecular weights), semisolid gels and semisolid mixtures containing Carbowax. Any of the above mixtures may be appropriate in treatments and therapies according to the present invention, provided that the active principle in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. . See

<img file="MX348013B_D0068.tif" />

Also Baldrick P. Pharmaceutical excipient development: the need for preclinical guidance. Regul. Toxicol Pharmacol. 32 (2): 210-8 (2000), Wang W. Lyophilization and development of solid protein pharmaceuticals. Int. J. Pharm. 203 (1-2): 160 (2000), Charman WN Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts. J Pharm Sci. 89 (8): 96778 (2000), Powell et al. Compendium of excipient for parenteral formulations PDA J Pharm Sci Technol. 52: 238-311 (1998) citations inside for additional information regarding formulations, excipients and vehicles well known to pharmaceutical chemists.

In one embodiment, the antibodies of the invention, which include a monoclonal antibody of the invention {eg. , a fully human monoclonal antibody), can be used as therapeutic agents. Generally, such agents will be used to diagnose, predict, monitor, treat, alleviate, and / or prevent a disease or pathology associated with IL-17F signaling in a subject. A therapeutic regimen is carried out by identifying the subject, eg, a human patient suffering from (or at risk of developing) an inflammatory disease or disorder, using standard methods. An antibody preparation, preferably one having a high specificity and high affinity for its target antigen, is administered to the subject and will generally have a

- IMPI® * rwmvro mhjqwo 1 '• tu ϊμοηεπλο rnoumui effect due to its binding with white. The a dm ini i. irnc ~ i ÓJa. ^ GL &amp; l „antibody can suppress or inhibit or interfere with target signaling function (eg, IL-17F). Administration of the antibody can abrogate or inhibit or interfere with target binding {eg. , IL-17F) with an endogenous ligand (eg, IL-17R or IL-17RC) to which it binds naturally. For example, the antibody binds to the target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, or otherwise interferes with IL-17F-induced pro-inflammatory cytokine production.

Diseases or disorders related to IL-17F signaling include autoimmune diseases or inflammatory diseases or disorders, including but not limited to rheumatoid arthritis, Crohn's disease, psoriasis, multiple sclerosis, chronic obstructive pulmonary disease, and asthma. IL-17F was found to be regulated by accumulation in the sputum of cystic fibrosis patients {see McAllister et al., J. Immunol. 175: 404-412 (2005)), and in the colon of patients suffering from inflammatory bowel disease {see Seiderer et al., Inflamm. Bowel Dis. December 18, 2007, electronic publication prior to the print edition). IL-17A / IL-17F has been shown to play a role in the recruitment of neutrophilia from the airways, suggesting a role in the pathogenesis of respiratory diseases {see Liang et

<img file="MX348013B_D0069.tif" />

101

IMPI ίΝΤτττντ · MiuojAo DI LA nOWBM · WIXZTUAI al., J. Immunol. 179 (11): 7791-9 (2007)).

Autoimmune diseases include, for example, Acquired Immune Deficiency Syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, Addison's autoimmune disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune disease of the inner ear (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac sprue - hepetiform dermatitis; chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), scar pemphigoid, cold agglutinin disease, ridge syndrome, Crohn's disease, Degos disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia fibromyositis, Graves disease, GuillainBarré syndrome, Hashimoto thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, Insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Ménière's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis Y

<img file="MX348013B_D0070.tif" />

dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), syndrome Sjógren's, stiff person syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis.

Inflammatory disorders include, for example, acute and chronic inflammatory disorders. Examples of inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft-versus-host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, organ and tissue transplantation, vasculitis, diabetic retinopathy and mechanical ventilation-induced lung injury.

IL-17F has also been shown to be accumulated through IL-21 signaling, suggesting that the pro-inflammatory effects associated with L-21 signaling are mediated by IL-17F, in addition to IL-17A. and / or the IL-17F / IL17A complex (Wei et al., J Biol Chem. 282 (48): 34605-10 (2007)). As such, the antibodies of the invention are also useful for

103

<img file="MX348013B_D0071.tif" />

IMPI ίΝτητιτο μζχνΜ »DE LA ΗΙΟΗΚΜΡ INDUSTRIAL diagnose, predict, monitor and / or treat disorders or diseases mediated by IL-21 signaling, including, but not limited to, inflammatory / autoimmune disorders such as inflammatory bowel disease, rheumatoid arthritis, rejection transplants, and psoriasis.

The huIL-17F antibodies of the invention are useful for treating, ameliorating, delaying the onset and / or progression or otherwise reducing the severity of an arthritic condition or one or more symptoms thereof. Prior to the present invention, studies had determined that IL-17F played a small role, if any, in the development and / or progression of autoimmune arthritic conditions. {See Ishigame et al., Immunity, vol. 30: 108-119 (2009)). The experiments and data provided in the Examples herein demonstrate the unexpected and surprising result that modulation, eg. , inhibition, neutralization, or otherwise blocking the expression and / or activity of the IL-17F homodimer, but not the IL17A / IL-17F heterodimeric complex, significantly delayed the progression of the arthritic condition and reduced the level of inflammatory mediators such as TNF-α, IL-6, IFN-γ, IL-Ία, MCP-1 and IL-12 / IL-23 (p40) in collagen-induced arthritis, an animal model for rheumatoid arthritis.

Accordingly, the invention includes methods for

IMPI tNÍtnVTO MEXICMIO Pt LA nOMWAf INDUSTRIAL treat, ameliorate, delay the onset and / or progression, and / or alleviate a symptom of an arthritic condition by administering a huIL-17F antibody of the invention to a

<img file="MX348013B_D0072.tif" />

subject in need of treatment (eg, 5E12, 41B10, 11C5, 21B10, 1F1, 2E12, 5D3, 22F8, 28B11, 41A4, and 43G6) or an antibody that binds to the same or similar epitope such as a huIL-17F antibody of the invention. The huIL-17F antibody or the antibody that binds to the same epitope as a huIL-17F antibody of the invention is administered in an amount sufficient to treat, ameliorate, delay onset, delay progression, and / or alleviate one or more symptoms. of arthritic condition. The subject is, for example, a human being. In some modalities, the arthritic condition is an autoimmune arthritic condition. For example, in some modalities, the arthritic condition is rheumatoid arthritis.

Symptoms associated with inflammation-related disorders include, for example, inflammation, fever, malaise, fever, pain, often localized to the inflamed area, rapid pulse rate, joint pain (arthralgia), rapid breathing, or other breathing patterns. abnormal, chills, confusion, disorientation, agitation, dizziness, cough, dyspnea, lung infections, heart failure, respiratory failure, edema, weight gain, mucopurulent relapses, cachexia, wheezing, headache and abdominal symptoms such as, for

105

<img file="MX348013B_D0073.tif" />

IMPI • Mexican tiiuto BtMnORBAD INKKTBlAL example, abdominal pain, diarrhea or constipation. Symptoms associated with immune-related disorders include, for example, inflammation, fever, lack / loss of appetite, weight loss, abdominal symptoms such as, for example, abdominal pain, diarrhea or constipation, joint pain (arthralgia), fatigue , rash, anemia, extreme sensitivity to cold (Raynaud's phenomenon), muscle weakness, muscle fatigue, changes in skin or tissue tone, shortness of breath or other abnormal breathing patterns, chest pain or constriction of the chest muscles, abnormal heart rate (eg, elevated or decreased), sensitivity to light, blurred or otherwise abnormal vision, and reduced organ function.

An effective amount for therapeutic use of an antibody of the invention refers generally to the amount necessary to achieve a therapeutic goal. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target. The amount required to be administered will also depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is reduced from the free volume of the subject to which it is administered. Common ranges for effective dosage for therapeutic use of a^b IMPI62

MBJtiCAMO INSTITUTE your antibody, or antibody fragment of the invention may be, by way of non-limiting example, from about 0.1 mg / kg of body weight to about 50 mg / kg of body weight. Common dosing frequencies can range, for example, from twice a day to once a week.

The efficacy of the treatment is determined in conjunction with any known method for diagnosing or treating the particular inflammation-related disorder. Relief of one or more symptoms of the inflammation-related disorder indicates that the antibody confers a clinical benefit.

Methods for the observation of antibodies possessing the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.

In another embodiment, antibodies directed against IL17F and / or the IL-17F / IL17A complex can be used in methods known within the art related to the localization and / or quantification of IL-17F or the IL17A / IL-17F complex { eg. , for use in the measurement of levels of IL-17F and / or the IL-17A / IL-17F complex within appropriate physiological samples, for use in diagnostic procedures, for use in protein imaging

IMPI iirrnwo MAID OF THE ΝφΟΜΕ INDVTTIttAL and the like). In a given embodiment, antibodies specific for IL-17F and / or the IL-17A / IL-17F complex, or a derivative, fragment, analog or homologue thereof, containing the antibody-derived antigen-binding domain, are used, as pharmacologically active compounds (hereinafter referred to as Therapeutic Agents).

In another embodiment, an antibody specific to IL-17F and / or the heterodimeric IL-17A / IL-17F complex can be used to isolate an IL-17F polypeptide and / or a heterodimeric IL-17A / IL-17F complex polypeptide. using standard techniques, such as immunoaffinity,

<img file="MX348013B_D0074.tif" />

chromatography or immunoprecipitation. Antibodies directed against the IL-17F protein and / or the heterodimeric IL17A / IL-17F complex (or a fragment thereof) can be used diagnostically to monitor protein levels in tissue as part of a clinical trial procedure, e.g. ., to determine, for example, efficacy of a given treatment regimen. Detection can be facilitated by coupling (ie, physically binding) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase,¹⁰⁸ IMPIAS

INSTTTVTO ΜΓββΛΝ »

OF THE PROHSDA »

INDUSTRIAL alkaline phosphatase, β-galactosidase or acetylcholinesterase, · Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or icoerythrin, · an example of a luminescent material includes luminol; Examples of bioluminescent materials include luciferase, luciferin, and equorin, and Examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵Yes ³H.

In yet another embodiment, an antibody according to the invention can be used as an agent to detect the presence of IL-17F (or a protein fragment thereof) in a sample. In some embodiments, the antibody contains a detectable label. The antibodies are polyclonal, or more preferably monoclonal. An intact antibody, or a fragment thereof (e.g., F_ab, scFv, or F_(ab)₂) - The term labeled, with respect to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by means of a coupling (i.e., physically bound) of a detectable substance to the probe or the antibody, as well as indirect labeling of the probe or antibody through reactivity with another reagent that is directly labeled. The examples

<img file="MX348013B_D0075.tif" />

Indirect tagging includes Ha — na detection. Primary anb-irnprpn by use of a fluorescently labeled secondary antibody and labeled the end of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject. Therefore, within the use of the term biological sample, blood and a fraction or component of blood that includes blood serum, blood plasma, or lymph are included. That is, the detection method of the invention can be used to detect an analyte genomic mRNA, protein, or DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for the detection of an mRNA analyte include Northern hybridizations and in situ hybridizations. In vitro techniques for the detection of an analyte protein include enzyme-linked immunosorbent assays (ELISA), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for the detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example, in ELISA: Theory and Practice: Methods in Molecular Biology, Vol. 42,

JR Crowther (Ed.) Human Press, Totowa, NJ

1995;

<img file="MX348013B_D0076.tif" />

110

IMPI WhwroMtacANo * u ncniTY XDUSTRIAE

Immunoassay, E. Diamandis and T. ChristopgiwiII "; Aoadomie »-— Press, Inc., San Diego, CA, 1996; and Practice and Theory of Enzyme Immunoassays, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for the detection of an analyte protein include introducing a labeled anti-analyte protein antibody into a subject. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

Therapeutic Administration and HuIL-17F Antibody Formulations

The antibodies of the invention (also referred to herein as active compounds), and derivatives, fragments, analogs, and homologues thereof, can be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations involved in the preparation of such compositions, as well as a guide to the choice of components are provided, for example, in Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., Editors) Mack Pub. Co., Bastón, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug¹¹¹ PREVENT. IWnTVTOMKICANO · »la ntoriEDAO '» íix *? T »iai. Delivery (Advances In Parenteral Sciences, Vol, 4), 1991, M. Dekker, New York.

Such compositions typically comprise the antibody and a pharmaceutical acceptable carrier. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable region sequences of an antibody, peptide molecules can be designed such that they retain the ability to bind to the target protein sequence. Such peptides can be chemically synthesized and / or produced by means of recombinant DNA technology. (See, eg, Marasco et al., Proc. Nati. Acad. Sci. USA, 90: 7889-7893 (1993)).

As used herein, the term "pharmaceutical acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like, compatible with the pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such

<img file="MX348013B_D0077.tif" />

ΜΤΠυΤΟ MDUOANO

Munont «A ·

INDUSTRIAL vehicles or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and agents for active substances for pharmaceutical use is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.

Formulations to be used for in vivo administration must be sterile. This is easily done by filtration through sterile filter membranes.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, eg. , intravenous, intradermal, subcutaneous, oral {eg. , inhalation), transdermal (i.e. topical), transmucosal, and rectal. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as —Μ ***

113

<img file="MX348013B_D0078.tif" />

benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and tonicity adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be stored in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions (in which they are soluble in water) and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable vehicles include physiological saline, bacteriostatic water, Cremophor EL ™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that there is easy syrinsability. It must be stable under the conditions of manufacture and storage and must be preserved from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerin, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, through the use of a coating such as lecithin, through the maintenance of the required particle size in the case of dispersion, and through the use of surfactants. Prevention of the action of microorganisms can be achieved by means of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one component or combination of components listed above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other components from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and lyophilization which gives a powder of the active principle plus any additional desired component of a solution previously sterilized by filtration thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be kept in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared by use of a flowable vehicle for use as a mouthwash, where the compound in the flowable vehicle is applied orally and swirled and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjunct materials can be included as part of the composition. Tablets, pills, capsules, lozenges, and the like may contain any of the following components or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or cornstarch; a lubricant such as magnesium stearate or Sterotes; a glidant such as carbon dioxide

116

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colloidal silicon; a sweetener such saccharin; or a flavoring such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are administered in the form of an aerosol spray from a pressure container or dispenser containing a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, appropriate penetrants are used in the formulation to permeate the barrier. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and derivatives of fusidic acid. Transmucosal administration can be carried out by the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, balms, gels, or creams as are generally known in the art.

The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal administration.

In one embodiment, the active compounds are prepared with ¹¹⁷ IMPI

IM9WTVTO MEXICAN

M LA HOflEOAO Ομ ^ · Ε08Ιτ nrtXJSTEIAl vehicles that will protect the compound. rapid, elimination from the body, such as a sustained / controlled release formulation, which includes implants and microencapsulated delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and pollactic acid. Methods for the preparation of such formulations will be apparent to those of skill in the art.

For example, the active components can be entrapped in microcapsules prepared, for example, by means of coacervation techniques or by means of interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions.

Sustained-release preparations can be generated. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of molded articles, eg. , films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (for

118

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IMPI nfOUSTFl For example, poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Patent No.

3,773,919), L-glutamic acid and y-ethyl-L glutamate copolymers, non-degradable ethylene vinyl acetate copolymers, degradable lactic acid-glycolic acid such as LUPRON DEPOT ™ (injectable microspheres composed of lactic acid copolymer -glycolic acid and leuprorelin acetate) and poly-D - (-) - 3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time.

The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeting infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those of skill in the art, for example, as described in US Patent No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. The dosage unit form according to

<img file="MX348013B_D0081.tif" />

INDUSTRIAL refers to unit dosage units each unit a quantity

119 with what is used herein physically discrete suitable as for the subject to be treated; containing predetermined active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention is dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of combining such an active compound for the treatment of individuals. .

The pharmaceutical compositions may be included in a container, pack or dispenser together with instructions for their administration.

The formulation may also contain more than one active compound as needed for the particular indication under treatment, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise an agent that enhances its function such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for their intended purpose.

120

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Dt L * ΡΚΟίΊΒΙΜΒ r &lt;DUSTMAL

In one embodiment, the art-ive compounds rp admin / qt-ran. in combination therapy, that is, combined with other agents, eg. , therapeutic agents, which are useful for treating pathological conditions or disorders, such as autoimmune disorders and inflammatory diseases. The term in combination in this context means that the agents occur substantially contemporaneously, both simultaneously and sequentially. If given sequentially, at the beginning of the administration of the second compound, the first of the two compounds is preferably still detectable in effective concentrations at the site of treatment.

For example, combination therapy may include one or more antibodies of the invention co-formulated with, and / or co-administered with, one or more additional therapeutic agents, eg, one or more immunosuppressive, growth factor and cytokine inhibitors, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxic or cytostatic agents, as described in greater detail below. In addition, one or more antibodies described herein can be used in combination with two or more of the therapeutic agents described herein. Such combination therapies can advantageously use lower dosages of administered therapeutic agents, thus avoiding

1X1 'IX: «I»

121

IMPI Ββτπντυ MEXICANO M LA PROHiOAO,,,, rNpSTW * L _____ possible toxicities or complications associated with various monotherapies.

Preferred therapeutic agents that are used in combination with an antibody of the invention are those agents that interfere at different stages in an inflammatory response. In one embodiment, one or more antibodies described herein can be formulated and / or administered in conjunction with one or more additional agents such as other cytokine antagonists or growth factor antagonists (eg. , soluble receptors, peptide inhibitors, small molecules, ligand fusions); or antibodies or antigen-binding fragments thereof that bind to other targets (eg, antibodies that bind to other cytokines or growth factors, their receptors, or other cell surface molecules), and anti-inflammatory cytokines or agonists thereof. Non-limiting examples of agents that can be used in combination with the antibodies described herein include, but are not limited to, antagonists of one or more interleukins (ILs) or their receptors, eg. , IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-18, IL-21, and IL-22 antagonists ; antagonists of cytokines or growth factors or their receptors, such as tumor necrosis factor (TNF), LT, EMAP-II, GM-CSF, FGF and PDGF. The antibodies of the invention can also be combined with

<img file="MX348013B_D0083.tif" />

122

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IMPJ .Nmnno »dgc * NC Oí LA riWtlICAP INDUSTRIAL inhibitors of, eg, antibodies to, cell surface molecules such as CD2, CD3, CDA'VCDS, CD20 (for. _ ..... _.... . ®.. _ _. _ ____ _ _ e.g., the CD20 inhibitor rituximab (RITUXAN)), CD25, CD28,

CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, or their ligands, including CD154 (gp39 or CD4OL), or LFA-l / ICAM-1 and VLA-4 / VCAM -1 (Yusuf-Makagiansar et al. (2002) Med. Res. Rev. 22: 146-67). Preferred antagonists that can be used in combination with the antibodies described herein include antagonists of IL-1, IL-12, TNFa, IL-15, IL-18, and IL-22.

Examples of those agents include IL-12 antagonists, such as chimeric, humanized, human, or in vitro generated antibodies (or antigen-binding fragments thereof) that bind IL-12 (preferably human IL-12. ), eg, the antibody disclosed in WO 00/56772; IL-12 receptor inhibitors, eg. , antibodies to the human IL-12 receptor; and soluble IL-12 receptor fragments, eg. , human IL-12 receptor. Examples of IL-15 antagonists include antibodies (or antigen-binding fragments thereof) against IL-15 or its receptor, eg, chimeric, humanized, human, or in vitro generated antibodies to human IL-15 or its receptor. , soluble IL-15 receptor fragments, and IL-15 binding proteins. Examples of IL-18 antagonists include antibodies, eg. chimeric, humanized antibodies,

123

Human or in vitro generated IMPIs (or antigen-binding fragments thereof), for human IL-18, soluble IL-18 receptor fragments, and IL-18 binding proteins (IL-18BP). Examples of IL-1 antagonists include Interleukin 1 converting enzyme (ICE) inhibitors, such as Vx740, IL-1 antagonists, eg IL-1RA (anikinra, KINERET ™, Amgen), sILIRII (Immunex) , and anti-IL-1 receptor antibodies (or antigen-binding fragments thereof).

Examples of TNF antagonists include chimeric, humanized, human, or in vitro generated antibodies (or antigen-binding fragments thereof) to TNF (eg, human TNFa), such as (HUMIRA ™, D2E7, human TNFa antibody) , CDP-571 / CDP-870 / BAY-10-3356 (humanized anti-TNFoi antibody; Celltech / Pharmacia), cA2 (chimeric anti-TNFa antibody; REMICADe '⁵', Centocor); anti-TNF antibody fragments (eg, CPD870); soluble fragments of TNF receptors, eg. , human TNF receptors p55 or p75 or derivatives thereof, eg. , 75 kdTNFR-IgG (TNF-IgG receptor fusion protein 75, ENBREL ™; Immunex), p55 kdTNFR-IgG (TNF-IgG receptor fusion protein 55 kD (LENERCEPT °)); enzyme antagonists, eg. , TNFa converting enzyme (TACE) inhibitors (eg. , an alpha-sulfonyl-hydroxamic acid derivative, and a TACE inhibitor of N-hydroxyformamide GW 3333, -005, or -022);

124

<img file="MX348013B_D0085.tif" />

IMPI

OF INDUSTRIAL FWOWEDAD and TNF-bp / s-TNFR (soluble TNF-binding protein). Preferred TNF antagonists are soluble fragments of the TNF receptor, eg, human p55 or p75 TNF receptors or derivatives thereof, eg, 75 kdTNFR-IgG, and inhibitors of a TNFa converting enzyme (TACE) .

In other embodiments, the antibodies described herein can be administered in combination with one or more of the following: IL-13 antagonists, eg, soluble IL-13 receptors (sIL-13) and / or antibodies against IL -13; IL-2 antagonists, eg. , DAB 486-IL-2 and / or DAB 389-IL-2 (IL-2 fusion proteins, Seragen), and / or antibodies to IL2R, e.g., anti-Tac (humanized anti-IL-2R, Protein Design Labs). Still another combination includes antibodies of the invention, antagonistic small molecules, and / or inhibitory antibodies in combination with non-exhaustible anti-CD4 inhibitors (DEC-CE9.1 / SB 210396; inexhaustible primatized anti-CD4 antibody; IDEC / SmithKline). Still other preferred combinations include antagonists of the CD80 (B7.1) or CD86 (B7.2) costimulatory pathway, including antibodies, soluble receptors, or antagonistic ligands; as well as p-selectin glycoprotein ligand (PSGL), anti-inflammatory cytokines, eg. , IL-4 (DNAX / Schering); IL-10 (SCH 52000; Recombinant IL-10 / Schering XDNA); IL-13 and TGF-β, and agonists thereof (eg, agonist antibodies).

125

<img file="MX348013B_D0086.tif" />

IMPI wtituto vracM * nt laropieoao EMBUSTERIA L

In other modalities, one or more antibodies to the ——Ι — ΒΜ * / - ΜβΒΒι · ®¹ * -ILU · »&gt; · a · '··'^r- ·· · * - '* "&lt;* invention may be formulated and / or administered in conjunction with one or more anti-inflammatory, immunosuppressive, or metabolic or enzyme inhibitors. Non-limiting examples of drugs or inhibitors that can be used in combination with the antibodies described herein include, but are not limited to, one or more of: non-spheroid anti-inflammatory drugs (NSAIDs), eg, ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroids such as prednisolone; cytokine suppressing anti-inflammatory drugs (CSAIDs); nucleotide biosynthesis inhibitors, eg, purine biosynthesis inhibitors, folate antagonists (eg, methotrexate acid (N- [4 - [[(2,4-diamino-6pteridinyl) methi |] methylamino] benzoi |] -L-glutamic)); and pyrimidine biosynthesis inhibitors, eg, dihydroorotate dehydrogenase (DHODH) inhibitors. Preferred therapeutic agents for use in combination with the antibodies of the invention include NSAIDs, CSAIDs, (DHODH) inhibitors (eg. , leflunomide) and folate antagonists (eg, methotrexate). Examples of additional inhibitors include one or more of: corticosteroids (oral, inhaled, and local injection); immunosuppressants, eg cyclosporine, tacrolimus

126

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(FK-506); and mTOR inhibitors, eg. , sirolimus (rapamycin-RAPAMUNE ™ or rapamycin derivatives, eg, soluble rapamycin derivatives (eg, rapamycin ester derivatives, eg, CCI-779); agents that interfere with signaling via pro-inflammatory cytokines such as TNFa or IL-1 (eg IRAK, NIK, IKK, p3B or MAP kinase inhibitors); COX2 inhibitors, eg. , celecoxib and variants thereof, phosphodiesterase inhibitors, eg, R973401 (Type IV phosphodiesterase inhibitor);

phospholipase inhibitors, eg. , cytosolic phospholipase 2 (cPLA2) inhibitors {eg. , trifluoromethyl ketone analogs); vascular endothelial cell growth factor inhibitors or growth factor receptor, eg. , VEGF inhibitor and / or VEGF-R inhibitor; and angiogenesis inhibitors. Preferred therapeutic agents for use in combination with the antibodies of the invention are immunosuppressive, eg. , cyclosporine, tacrolimus (FK-506); and mTOR inhibitors, eg. , sirolimus (rapamycin) or derivatives of rapamycin, eg. , soluble rapamycin derivatives {eg. , rapamycin ester derivatives, by e j. , CCI-779); COX2 inhibitors, eg. , celecoxib, and variants thereof; and phospholipase inhibitors, eg. , cytosolic phospholipase 2 (cPLA2) inhibitors, eg, trifluoromethyl ketone analogs.

<img file="MX348013B_D0089.tif" />

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Additional examples of therapeutic agents that can be combined with an antibody of the invention include one or more of: 6-mercaptopurines (6-MP); Azathioprine Sulfasalazine; mesalazine; olsalazine chloroquinine / hydroxychloroquine (PLAQUENIL®); pencylamine; aurothiomalate (intramuscular and oral); azathioprine; cochicine; beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral); xanthines (theophylline, aminophylline); cromoglycate, · nedocromil; ketotifen; ipratropium and oxitropium; Mycophenolate Mofetil; adenosine agonists; antithrombotic agents; complement inhibitors; and antiadrenergic agents.

Non-limiting examples of agents for treating or preventing arthritic disorders (eg. , rheumatoid arthritis, inflammatory arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis and psoriatic arthritis), with which an antibody of the invention can be combined include one or more of the following: IL-12 antagonists as described in the present; NSAID; CSAID; TNF, eg, TNFa, antagonists as described herein; inexhaustible anti-CD4 antibodies as described herein; IL-2 antagonists as described herein; anti-inflammatory cytokines, eg. , IL-4, IL-10, IL-13 and TGFa, or agonists thereof; IL-1 antagonists or IL-1 receptor antagonists as described herein; inhibitors¹²⁸ IMPI ^

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Μ ΙΑ FHOPIIDAfi 'Ο-ί ·!'? '·¹, • rauwNM phosphodiesterase as described herein; Cox-2 inhibitors as described herein; iloprost: methotrexate; thalidomide and thalidomide-related drugs (eg, Celgen); leflunomide; plasminogen activation inhibitor, eg. , tranexamic acid; cytokine inhibitor, eg. , T614; prostaglandin El; azathioprine; an interleukin-1 converting enzyme (ICE) inhibitor; zap-70 inhibitor and / or Ick inhibitor (zap-70 tyrosine kinase inhibitor or lck); a vascular endothelial cell growth factor inhibitor or vascular endothelial cell growth factor receptor as described herein; an angiogenesis inhibitor as described herein; corticosteroid anti-inflammatory drugs (eg. , SB203580); TNFconvertase inhibitors; IL-11; IL-13; IL-17 inhibitors; gold;

penicillamine; chloroquine; hydroxychloroquine; chlorambucil; cyclophosphamide; cyclosporine; total lymphoid irradiation; anti-thymocyte globulin; CD5-toxins; orally administered peptides and collagen; disodium lobenzarite; cytokine regulatory agents (CRA) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (ΤΡΙΟ; T Cell Sciences, Inc.); prednisone; orgotein;

<img file="MX348013B_D0090.tif" />

IMPI Μ LA reOFiew * industrial glycosammoglycan polysulfate; nunocycline (MINOCIN jr anti-IL2R antibodies; marine and botanical lipids (fatty acids from plant and fish seeds); auranofin; phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immunoglobulin; zileuton; mycophenolic acid (RS-61443); tacrolimus (FK -506); sirolimus (rapamycin); amiprilose (terafectin); cladribine (2-chlorodeoxyadenosine); and azaribine. Preferred combinations include one or more antibodies of the invention in combination with methotrexate or leflunomide and in cases of moderate or severe rheumatoid arthritis, cyclosporine.

Preferred examples of inhibitors for use in combination with antibodies of the invention to treat arthritic disorders include TNF antagonists (eg, chimeric, humanized, human, or in vitro generated antibodies, or antigen-binding fragments thereof, which bind to TNF; soluble fragments of a TNF receptor, eg. , p55 or p75 human TNF receptor or derivatives thereof, e.g., 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBREL ™), p55 kD TNF receptor-IgG fusion protein ; TNF enzyme antagonists, eg, TNFa converting enzyme (TACE) inhibitors); IL-12, IL-15, IL-18, IL-22 antagonists; agents that reduce T cells and B cells (eg, anti-CD4 or anti-CD22 antibodies); small molecule inhibitors, eg. ,

<img file="MX348013B_D0091.tif" />

methotrexate and lef lunomidasirolimus (r aparn-ícínerh * and thereof, e.g., CCI-779; cox-2 inhibitors and

CPLA2; NSAIDs; p38, TPL-2, Mk-2 and NFkb inhibitors; RAGE or soluble RAGE; P-selectin or PSGLi-1 inhibitors (eg, small molecule inhibitors, antibodies thereto, eg, Pselectin antibodies); estrogen receptor beta (ERB) agonists or ERB-NFkb antagonists. More preferred additional therapeutic agents that can be administered and / or formulated in conjunction with one or more antibodies of the invention include one or more of: a soluble fragment of a TNF receptor, eg. , human TNF receptor p55 or p75 or derivatives thereof, eg. , 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBREL ™); methotrexate, leflunomide, or a sirolimus (rapamycin a) or an analog thereof, eg, CCI-779.

Non-limiting examples of agents for treating or preventing multiple sclerosis with which the antibodies of the invention can be combined include the following: interferons, eg, interferon-alpha la (eg, AVONEX; Biogen) and interferon-lb (BETASERON ™ Chiron / Berlex); Copolymer 1 (Cop-1; COPAXONE ™ Teva Pharmaceutical

Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; clabribine; TNF antagonists as described herein; corticosteroids; prednisolone;

131

IMPI »

MEXICAN WSTmrrO methylprednisolone; azathioprine; cyclophosphamide; cyclosporin A, methotrexate; 4-aminopyridine; and tizanidine. Additional antagonists that can be used in combination with the antibodies of the invention include antibodies to or antagonists of other human cytokines or other growth factors, eg, TNF, LT, IL-1, IL-2, IL-6, EL-7, IL-8, IL-12 IL-15, IL-16, IL-18, EMAP-11, GM-CSF, FGF, and PDGF. Antibodies as described herein can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their ligands. The antibodies of the invention can also be combined with agents, such as methotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, eg, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents. , complement inhibitors, adrenergic agents, agents that interfere with pro-inflammatory cytokine signaling as described herein, IL-Ib converting enzyme inhibitors (e.g. Vx740), anti-P7, PSGL, TACE inhibitors, T-cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine,

6-mercaptopurines, converting enzyme inhibitors

132

IMPI INSTITUTO MEXICANO n »la noetmnD industrial _____ angiotensin, soluble cytokine receptors and derivatives thereof, as described herein, and anti-inflammatory cytokines (eg IL-4, IL-10, IL-13 and

TGF).

Preferred examples of multiple sclerosis therapeutics with which the antibodies of the invention can be combined include interferon-β, eg, ΙΡΝβ-la and ΙΡΝβ-lb; copaxone, corticosteroids, IL-1 inhibitors, TNF inhibitors, antibodies to CD40 and CD80 ligands, IL-12 antagonists.

Non-limiting examples of agents for treating or preventing inflammatory bowel disease (eg, Crohn's disease, ulcerative colitis) with which an antibody of the invention can be combined include the following: budenoside; epidermal growth factor; corticosteroids; cyclosporine, sulfasalazine;

aminosalicylates; 6-mercaptopurine; azathioprine;

metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; monoclonal anti-IL-1 antibodies; monoclonal antibodies IL-6; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; TNF antagonists as described herein; IL-4, IL-10, IL-13, and / or TGFp cytokines or agonists thereof (e.g., antibodies

<img file="MX348013B_D0092.tif" />

IMPI Mfrnyrc M MOCA NO D »14 MiO ** DAD agonists); IL-11; glucuronide-conjugated or dextran-conjugated prodrugs of prednisolone, dexamethasone, or budesonide; ICAM-1 antisense phosphorothioate oligonucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (ΤΡΙΟ; T Cell Sciences, Inc.); slow release mesalazine; methotrexate; platelet activating factor (PAF) antagonists; ciprofloxacin; and lignocaine.

Non-limiting examples of agents for treating or preventing psoriasis with which an antibody of the invention can be combined include the following: corticosteroids; vitamin D₃ and analogs thereof; retinoiodes {eg. , soriatano); methotrexate; cyclosporine, 6-thioguanine; Accutane; hydrea; hydroxyurea; sulfasalazine; Mycophenolate Mofetil; azathioprine; tacrolimus; Fumaric acid esters; biological agents such as Amevive, Enbrel, Humira, Raptiva and Remicade, Ustekinmab, and XP-828L; phototherapy; and photochemotherapy (eg, phototherapy with combined ultraviolet and psoralen).

Non-limiting examples of agents for treating or preventing inflammatory airway / respiratory diseases (eg, chronic obstructive pulmonary disease, asthma) with which an antibody of the invention may be combined include the following: beta-adrenoceptor 2 agonists (eg, salbutamol

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134 (USAN albuterol), levalbuterol, beta adrenoceptor agonists

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INSTITUTO MEXICANO PC u PaenEDAD fíOUSTRIM terbutalina, bitolterol);

long-acting (eg, salmeterol, formoterol, bambuterol); adrenergic agonists (eg, inhaled epinephrine and ephedrine tablets); anticholinergic medications (eg, ipratropium bromide); combinations of inhaled spheroids and long-acting bronchodilators (eg. , fluticasone / salmeterol (Advair in the United States, and Seretide in the United Kingdom)) or budesonide / formoterol (Symbicort)); inhaled glucocorticoids (eg, ciclesonide, beclomethasone, budesonide, flunisolide, fluticasone, mometasone, triamcinolone); leukotriene modifiers (eg, montelukast, zafirlukast, pranlukast, and zileuton); mast cell stabilizers (eg. , cromoglycate (cromolyn), and nedocromil); antimuscarinics / anticholinergics (eg, ipratropium, oxitropium, tiotropium); methylxanthines (eg, theophylline, aminophylline); antihistamines; IgE blockers (eg, Omalizumab); muscarinic antagonists M₃ (anticholinergics) (eg, ipratropium, tiotropium); chromones (eg, cromoglycate, nedocromil); zantines (eg, theophylline); and TNF antagonists (eg, infliximab, adalimumab, and etanercept).

In one embodiment, an antibody of the invention can be used in combination with one or more antibodies directed at

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¹³⁵ IMPI

IN5TTTUTO ME1UCANI, t&gt; f the INDUSTRIAL property other targets involved in the regulation of immune responses, eg, transplant rejection.

Non-limiting examples of agents for treating or preventing immune responses with which an antibody of the invention can be combined include the following: antibodies against cell surface molecules, including but not limited to CD25 (interleukin-2 receptor-α), CDlla (LFA-1), CD54 (ICAM-1), CD4, CD45,

CD28 / CTLA4, CD80 (B7.1), eg CTLA4 Ig-abatacept (ORENCIA®)), ICOSL, ICOS and / or CD86 (B7.2). In yet another embodiment, an antibody of the invention is used in combination with one or more general immunosuppressive agents, such as cyclosporin A or FK506.

In other embodiments, antibodies are used as vaccine adjuvants against autoimmune disorders, inflammatory diseases, etc. The combination of adjuvants for the treatment of these types of disorders is suitable for use in combination with a wide variety of antigens from self-directed antigens, i.e., self-antigens, involved in autoimmunity, eg, myelin basic protein, Inflammatory autoantigens, eg. , amyloid peptide protein, or transplant antigens, eg. , alloantigens. The antigen can comprise peptides or polypeptides derived from proteins, as well as fragments of any of the following: saccharides,

IMPI • βτηντο mukn · DC LA nOHEtMD INBUSTUAl · proteins, polynucleotides or oligonucleotides, autoantigens, amyloid peptide protein, transplant antigens, allergens, or other macromolecular components. In some instances, more than one antigen is included in the antigen composition.

For example, desirable vaccines for moderating the response to allergens in a vertebrate host, which contain the adjuvant combinations of this invention, include those that contain an allergen or a fragment thereof. Examples of such allergens are described in US Patent No. 5,830,877 and published International Patent Application No. WO 99/51259, which are incorporated herein by reference in their entirety, and include pollen, insect venoms, animal dander, fungal spores, and drugs (such as penicillin). Vaccines interfere with the production of IgE antibodies, a known cause of allergic reactions. In another example, desirable vaccines for preventing or treating a disease characterized by amyloid deposition in a vertebrate host, containing the adjuvant combinations of this invention, include those containing portions of amyloid peptide protein (APP). This disease is referred to interchangeably as Alzheimer's disease, amyloidosis, or amyloidogenic disease. Therefore, vaccines of

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This invention includes the adjuvant combinations of this

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IMPI iNSTwrro mexican · DE LA PROPERTY INDUSTRIAL invention plus Αβ peptide, as well as Αβ peptide fragments and antibodies to Αβ peptide or fragments thereof.

Design and generation of other therapeutics

In accordance with the present invention and based on the activity of the antibodies produced and characterized herein with respect to IL-17F and / or the heterodimeric IL-17A / IL-17F complex, the design of other therapeutic modalities beyond antibody moieties. Such modalities include, without limitation, advanced antibody therapeutics, such as bispecific antibodies, immunotoxins, and radiolabeled therapeutics, generation of peptide therapeutics, gene therapies, particularly intracorporeal, antisense therapeutics, and small molecules.

For example, in connection with bispecific antibodies, bispecific antibodies can be generated comprising (i) two antibodies, one with a specificity for IL-17F and / or the heterodimeric IL-17A / IL-17F complex, and the other for a second. molecule that is conjugated to it, (ii) a single antibody that has a specific chain for IL-17F and / or the heterodimeric IL-17A / IL-17F complex, and a second chain specific for a second molecule, or (iii) a single chain antibody that

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IMPI

INSTITUTE MBJUCAN *)

DE LA PROPIBOAL INDUSTRIAL has a specificity for IL-17F and / or the IL complex

Heterodimeric 17A / IL-17F, and a second molecule. Such bispecific antibodies are generated by the use of techniques that are well known, for example in connection with (i) and (ii) see eg. , Fanger et al. Immunol Methods 4: 72-81 (1994) and Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), and in connection with (iii) see eg. , Traunecker et al. Int. J. Cancer (Suppl.) 7: 51-52 (1992).

In connection with immunotoxins, antibodies can be modified to act as immunotoxins using techniques that are well known in the art. See eg, Vitetta Immunol Today 14: 252 (1993). See also US Patent No. 5,194,594. In connection with the preparation of radiolabeled antibodies, such modified antibodies can also be readily prepared using techniques that are well known in the art. See eg. , Junghans et al. in Cancer Chemotherapy and Biotherapy 655-686 (2nd ed., Chafner and Longo, eds., Lippincott Raven (1996)). See also US Patent Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471, and 5,697,902. Each of the immunotoxins and radiolabeled molecules is likely to kill cells expressing IL-17F and / or the heterodimeric IL-17A / IL-17F complex.

In connection with the generation of therapeutic peptides, institute μ »κμ * ο F &amp; aláBc;

ΓΕ THE industrial PROPERTY through the use of structural information related to

IL-17F and / or the heterodimeric IL-17A / IL-17F complex and antibodies thereof, such as the antibodies of the invention or by observing peptide libraries, therapeutic peptides can be generated that are directed against IL -17F and / or the heterodimeric IL-17A / IL-17F complex. The design and observation of therapeutic peptides is discussed in connection with Houghten et al. Biotechniques 13: 412-421 (1992), Houghten PNAS USA 82: 51315135 (1985), Pinalla et al. Biotechniques 13: 901-905 (1992), Blake and Litzi-Davis BioConjugate Chem. 3: 510-513 (1992).

Immunotoxins and radiolabeled molecules can also be prepared, in a similar manner, in connection with peptide moieties as discussed above in connection with antibodies. Assuming that the IL-17F molecule and / or the heterodimeric IL-17A / IL-17F complex (or a form, such as a splice variant or an alternative form) is functionally active in a disease process, also it will be possible to design gene therapeutics and their antisense through conventional techniques. Such modalities can be used to modulate the function of IL-17F and / or the heterodimeric IL-17A / IL-17F complex. In connection therewith, the antibodies of the present invention facilitate the design and use of functional assays related thereto. A design is discussed

140

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IMPI

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OI or INDUSTRIAL PROPERTY and a strategy for antisense therapeutics in International Patent Application No. WO 94/29444. The design and strategy for gene therapy are well known. However, in particular, the use of gene therapy techniques involving intrabodies could prove to be particularly advantageous. See eg. , Chen et al. Human Gene Therapy 5: 595-601 (1994) and Marasco Gene

Therapy 4: 11-15 (1997). General design and considerations related to gene therapeutics are also discussed in International Patent Application No. WO 97/38137.

The knowledge gathered from the structure of the IL-17F molecule and / or the heterodimeric IL17A / IL-17F complex and its interactions with other molecules can be used according to the present invention, such as the antibodies of the invention, and others to rationally design additional therapeutic modalities. In this sense, rational drug design techniques such as X-ray crystallography, computer-aided (or assisted) molecular modeling (CAMM), quantitative or qualitative structure-activity relationship (QSAR), and similar technologies are used to focus the drug discovery efforts. Rational design allows the prediction of protein or synthetic structures that can interact with the molecule or specific forms of the molecule.

141

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IMPI .ptyrmrro Mexican PE THE IWmSTIiAL PROPERTY itself, which in turn can be used to modify or • · · '' Οβι / ίη-λτ? .— modulate the activity of IL-17F, and / or the IL-17A / complex Heterodimeric IL-17F. Such structures can be chemically synthesized or expressed in biological systems. This approach has been reviewed in Capsey et al. Genetically Engineered Human Therapeutic Drugs (Stockton Press, NY (1988)). In addition, combinatorial libraries can be designed and synthesized and used in observation programs, such as high-throughput observation efforts.

Observation methods

The invention provides methods (also referred to herein as observational assays) for identifying modulators, i.e., candidate or test compounds or agents (eg, peptides, peptidomimetics, small molecules, or other drugs) that modulate, block, inhibit , reduce, antagonize, neutralize or otherwise interfere with the binding of IL-17F and / or the heterodimeric IL-17A / IL-17F complex to its innate receptor, candidate or test modulating agents or compounds, they block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with the signaling function of IL-17F and / or the heterodimeric IL17A / IL-17F complex. Methods for identifying compounds useful for treating disorders associated with IL-17F and / or the signaling of the heterodimeric IL17A / IL-17F complex are also provided. The invention also includes

IMPI »

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OI THE NOTICE c and j »INDUSTRIAL compounds identified in the tests। do described herein.

In one embodiment, the invention provides assays to observe candidate or test compounds that modulate the signaling function of IL-17F and / or the heterodimeric IL-17A / IL17F complex. Test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; parallel spatially addressable solid phase or solution phase libraries; synthetic library methods that require deconvolution; the library method one bead, one compound; and synthetic library methods by the use of affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to libraries of peptide compounds, non-peptide oligomers, or small molecules. {See, eg. , Lam, 1997. Anticancer Drug Design 12: 145).

A small molecule as used herein is intended to refer to a composition having a molecular weight of less than about 5 kD and with the highest preference less than about 4 kD. Small molecules can be eg. , nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates,

143

IMPI

INSTITUTO MEXICANO m la rtontoiAC INDUmiAl lipids or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or algal extracts are known in the art and can be observed with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can be found in the art, for example, in: DeWitt, et al., 1993. Proc. Nati. Acad. Sci. USA 90: 6909; Erb, et al., 1994. Proc. Nati. Acad. Sci. USA 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Compound libraries can be presented in solution (see eg, Houghten, 1992. Biotechniques 13: 412-421), or on beads (see Lam, 1991. Nature 354: 82-84), on chips (see Fodor, 1993. Nature 364: 555-556), bacteria (see US Patent No. 5,223,409), spores (see US Patent No. 5,233,409), plasmids (see Culi, et al. , 1992. Proc. Nati. Acad. Sci. USA 89: 1865-1869) or on phages (see Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Nati. Acad. Sci. USA 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; and the

144

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OBLA MOR INOvrnUAL AGE

US Patent No. 5,233,409.).

In one embodiment, a candidate compound is introduced into an antibody-antigen complex and it is determined whether the candidate compound interferes with the antibody-antigen complex, where a disturbance of this complex indicates that the candidate compound modulates IL-17F signaling function and / or or the heterodimeric IL-17A / IL-17F complex. For example, the antibody is monoclonal antibody 5E12 (Mab05) and the antigen is IL-17F and / or the heterodimeric IL-17A / IL17F complex.

In another embodiment, the IL-17F homodimer is provided and exposed to at least one neutralizing monoclonal antibody. The formation of an antibody-antigen complex is detected, and one or more candidate compounds are introduced to the complex. If the antibody-antigen complex is disrupted following the introduction of the one or more candidate compounds, the candidate compounds are useful for treating disorders associated with IL-17F signaling.

In another embodiment, a soluble IL-17F protein is provided and exposed to at least one neutralizing monoclonal antibody. The formation of an antibody-antigen complex is detected, and one or more candidate compounds are introduced to the complex. If the antibody-antigen complex is disturbed after the introduction of the one or more candidate compounds, the candidate compounds are useful to treat

145

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disorders associated with IL-17F signaling.

Determination of the ability of the test compound to interfere with or disrupt the antibody-antigen complex can be carried out, for example, by coupling the test compound with a radioisotope or enzymatic marker, such that the binding of the Testing the antigen or the biologically active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be marked with ¹²⁵I, ³⁵Yes, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope can be detected by direct counting of the radio emission or by scintillation counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label can be detected by determining the conversion of an appropriate substrate to the product. .

In one embodiment, the assay comprises contacting an antibody-antigen complex with a test compound, and determining the ability of the test compound to interact with the antigen or otherwise disrupt the existing antibody-antigen complex. In this embodiment, the determination of the ability of the test compound to interact with the antigen and / or disrupt the complex

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Antigen-antibody INDUSTRIAL comprises determining the ability of the test compound to preferentially bind the antigen or a biologically active portion thereof, compared to the antibody.

In another embodiment, the assay comprises contacting an antibody-antigen complex with a test compound and determining the ability of the test compound to modulate the antibody-antigen complex. Determination of the ability of the test compound to modulate the antibody-antigen complex can be carried out, for example, by determining the ability of the antigen to bind to or interact with the antibody, in the presence of the test compound.

Those of skill in the art will recognize that, in any of the observation methods disclosed herein, the antibody may be a neutralizing antibody, such as for example a monoclonal antibody 5E12, 41B10, 11C5, 21B10, 1F1, 2E12, 5D3, 22F8, 28B11, 41A4, and 43G6, each of which modulates or otherwise interferes with the production of pro-inflammatory cytokines.

The observation methods disclosed herein can be carried out as a cell-based assay or as a cell-free assay. The cell-free assays of the invention are susceptible to the use of soluble IL-17F, soluble IL-17A / IL-17F complex and fragments of the

147

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themselves.

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In more than one embodiment, it may be desirable to mobilize either the antibody or the antigen to facilitate the separation of complex from non-complex forms of one or both after introduction of the candidate compound, as well as to adapt the automation of the test. Observation of the antibody-antigen complex in the presence and absence of a candidate compound can be carried out in any container suitable for containing the reagents. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to bind to a matrix. For example, GSTantibody fusion proteins or GST-antigen fusion proteins can be adsorbed onto glutathione-sepharose beads (Sigma Chemical, St. Louis, MO) glutathione-derived microtiter plates, which are then combined with the test compound, and the mixture is incubated under conditions conducive to complex formation (eg, under physiological conditions for salt and pH). After incubation, the beads or wells of the microtiter plate are washed to remove any unbound components, the matrix is ​​immobilized in the case of the beads, the complex is determined either directly or indirectly. Alternatively, complexes can

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IMPI INSTITUTE Dt LA ΛΟΠΙΟΑΟ | HDSTK1A1 _ dissociate from the matrix, and the level of antibody-antigen complex formation can be determined by using standard techniques.

Other techniques to immobilize proteins on arrays can also be used in the observational assays of the invention. For example, either the antibody {eg. 5E12, 41B10, 11C5, 21B10, 1F1, 2E12, 5D3, 22F8, 28B11, 41A4, and 43G6) or the antigen {eg. IL-17F protein or IL-17A / IL-17F complex) can be immobilized by the use of conjugation of biotin and streptavidin. Biotinylated antigen or antibody molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (eg, biotinylation kit, Pierce Chemicals, Rockford, 111.) , and immobilized in the wells of the streptavidin-coated 96-well plates (Pierce Chemical). Alternatively, other antibodies reactive with the antibody or antigen of interest, but that do not interfere with the formation of the antibody-antigen complex of interest, can be derived to the wells of the plate, and unbound antibody or antigen can become entrapped. in the wells by conjugation with antibodies. Methods for detecting such complexes, in addition to those described above for GST-immobilized complexes, include immunodetection of complexes by the use of such

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IMPI ιχττπrrti MEXICAN DE LA FUCirtEDAT rMDumuAL other antibodies reactive with the antibody or antigen.

The invention further relates to novel agents identified by any of the aforementioned observational tests and uses thereof for treatments as described herein.

Diagnostic and prophylactic formulations

The huIL-17F MAbs of the invention are used in diagnostic and prophylaxis formulations. In one embodiment, an IL-17F antagonist, such as a huIL17F MAb of the invention, is administered to patients who are at risk of developing one or more of the aforementioned autoimmune or inflammatory diseases, such as, for example, without limitation, rheumatoid arthritis and other autoimmune arthritic conditions, Crohn's disease, psoriasis, multiple sclerosis, chronic obstructive pulmonary disease, asthma, osteoarthritis, and cancer. The predisposition of a patient or an organ towards one or more of the aforementioned autoimmune or inflammatory diseases can be determined by the use of genotypic, serological or biochemical markers.

In another embodiment of the invention, an IL-17F antagonist, such as a huIL-17F antibody, is administered to human individuals diagnosed with a clinical indication.

150

IMPI i «Mexican wall BE THE industrial property associated with one or more of the autoimmune or inflammatory diseases mentioned above such as rheumatoid arthritis or other autoimmune arthritic conditions, Crohn's disease, psoriasis, multiple sclerosis, chronic obstructive pulmonary disease, asthma, osteoarthritis, and cancer. After diagnosis, an IL-17F antagonist, such as a huIL-17F antibody, is administered to mitigate or reverse the effects of the clinical indication associated with rheumatoid arthritis and other autoimmune arthritic conditions, Crohn's disease, psoriasis, multiple sclerosis disease. chronic obstructive pulmonary disease, asthma, osteoarthritis and cancer.

The antibodies of the invention are also useful in the detection of IL-17F and / or the heterodimeric IL-17A / IL-17F complex in patient samples and are therefore useful as diagnostics. For example, the huIL-17F antibodies of the invention are used in in vitro assays, eg, ELISA, to detect the levels of IL-17F and / or heterodimeric IL-17A / IL-17F complex in a patient sample.

In one embodiment, a huIL-17F antibody of the invention is immobilized on a solid support (eg, the wells of a microtiter plate). The immobilized antibody serves as a capture antibody to any IL-17F and / or any heterodimeric IL-17A / IL-17F complex that may be present in a test sample. Before putting on

IMPI INSTITUTO MKICANO DE LA PtOPIEDA »INDUSTMAL contact the immobilized antibody with a patient sample, the solid support is rinsed and treated with a blocking agent such as milk protein or albumin to prevent non-specific adsorption of the analyte.

The wells are then treated with a test sample suspected of containing the antigen, or with a solution containing a standard amount of the antigen. Such a sample is eg. , a serum sample from a subject suspected of having circulating antigen levels which is considered to be diagnostic of a pathology. After rinsing the test sample or standard, the solid support is treated with a second antibody that is detectably labeled. The second labeled antibody serves as a detection antibody. The level of detectable labeling is measured, and the concentration of IL-17F and / or the antigen heterodimeric IL-17A / IL-17F complex in the test sample is determined by comparison with a standard curve developed from the standard samples.

It will be appreciated that based on the results obtained by using the huIL-17F antibodies of the invention in an in vitro diagnostic assay, it is possible to classify a disease (eg, a clinical indication associated with ischemia, an autoimmune disorder or inflammatory) in a subject based on the expression levels of the IL17F antigen and / or the heterodimeric IL-17A / IL-17F complex. For one

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152

IMPI rxrrrnrro mmucam · dc untONEDA »industrial given disease, blood samples are taken from subjects diagnosed with various stages in the progression of the disease, and / or at various points in the therapeutic treatment of the disease. A range of antigen concentrations that can be considered characteristic of each stage is designated by the use of a sample population that provides statistically significant results for each stage of progression or therapy.

All publications and patent documents cited herein are incorporated herein by reference as if the inclusion of each publication or document herein by reference were specifically and individually indicated. Mention of publications and patent documents is not intended as an admission that any of them is of the relevant prior art, nor does it constitute an admission as to the contents or dates thereof. While the invention has been described by way of written description, those skilled in the art will recognize that the invention can be practiced in a variety of embodiments and that the preceding description and the following examples are for purposes of illustration and not limiting of the claims. following.

Examples

The following examples, which include the experiments conducted and the results achieved, are provided only

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153

IMPI iNsrm ^ o mexicam · OF INDUSTRIAL PROPERTY for illustrative purposes and should not be interRF ^ t-^tor like. Limits of the present invention.

EXAMPLE 1: Cloning, Expression and Purification of human IL-17F, rat IL-17F, cynomolgus monkey IL-17F.

Cloning

The cDNAs encoding mature human IL-17F (AF384857, aa 31-163), rat IL-17F (ΆΑΗ91568, aa 21-153) and IL-17F from cynomolgus monkeys (identical to the sequence XP_001106517 aa 31-163, ) were amplified by PCR and cloned into a PCR4TOPO vector (Invitrogen). After another PCR step, a His tag or a His tag followed by an AviTag (Avidity, Denver CO) was introduced at the Nterminal of the cytokine coding sequence. These constructs were then fused into a leader sequence and subcloned into the corresponding expression vectors.

Expression and Purification of Human IL-17F and Rat IL-17F from Baculovirus-Infected Cells

His-tagged huIL-17F or rat IL-17F preceded by the GP67 leader sequence (MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFA) (SEQ ID NO: 10 0) was subcloned into a baculovirus vector pFASTBAC Dual (Invitrogen). Following transfection into Sf9 cells, the recombinant virus was isolated and amplified. For the

154 • βΤΠνΤΟ MEXICAN

DE UA FXOMEDAD í, ^ · INtH'STMA * · * protein production, Hi5 or SF9 cells were infected with baculovirus and incubated at 27 ° C for 3 days. The cell culture medium was clarified by means of centrifugation, filtered and concentrated approximately 10 times in SartoFlow Slice 200 (Sartorius - Hydrosart cut 10 kD). After adjusting the pH to 7.0 and another centrifugation step, the concentrated protein was purified using standard procedures on Ni-NTA Superflow columns (Qiagen) or HiTrap Chelating HP columns (GE Healthcare) loaded with Ni ions.²⁺. The fractions containing IL-17F were pooled and desalted on PD-10 columns (GE Healthcare).

Human IL-17F and rat IL-17F from baculovirus-infected cells were essentially free of contaminants after a purification step, and had a predominantly disulfide-linked homodimers appearance, as demonstrated by SDS- Non-reducing PAGE. The biological activity of his-tagged human IL-17F expressed by baculovirus was comparable with the activity of commercial cytokines (huIL-17F expressed by E.Coli, Peprotech EC or R&amp;D Systems)

Expression and Purification of Human IL-17F and Rat IL-17F from CHOK1SV Cells

The rat huIL-17F or IL-17F coding sequences preceded by the CD33 leader sequence (MPLLLLLPLLWAGALAMD,

155

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SEQ ID NO ..- 101), plus a His tag, and an AviTag (Avidity, Denver CO) were placed under the control of the hCMV promoter in the expression vector pEE14.4. IL-17F was expressed from a bicistronic mRNA containing a viral internal ribosome entry site (IRES) and the GFP coding sequence as the second cistron. The vector pEE14.4. contains the glutamine synthetase (GS) gene, which is essential for the survival of cells transfected in a selection medium containing methionine sulfoximine (MSX). Stable transfectants were generated in the Lonza Biologics proprietary CHOK1SV cell line. After four weeks of culture in the presence of high expressing MSX clones, they were identified, expanded and used for the production of human or rat IL-17F.

CHOK1SV expressed human IL-17F and rat IL-17F were purified by Ni affinity chromatography²⁺. They were essentially free of contaminants and had the appearance of disulfide-linked homodimers or non-reducing SDS-PAGE gels. The biological activity of His + Avi tagged human IL-17F, expressed by CHO was significantly decreased compared to the activity of commercial huIL17F, probably due to the presence of a large double tag at the N-terminus.

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Expression and purification of human cnIL-17F IL-17F from PEAK cells

His-tagged huIL-17F or cnIL-17F coding sequences were fused to the leader sequence Gaussia princeps luciferase (AF015993) and placed under the control of the EF1 promoter in the episomal expression vector pEAK8. The cytokine coding sequence was followed by a viral internal ribosome entry site (THREE) and a second cistron (GFP). The pEAK8 vector contains the puromycin resistance gene, the EBV 1 nuclear antigen (EBNA1), and the oriP origin of replication. EBNA1 and oriP are required for the propagation of the pEAK8 vector as episomal DNA in human cells and the generation of stable transfectants.

Stably transfected cells were obtained after 7 to 10 days of culture in the presence of 2 ug / mL of puromycin. The puromycin resistant cell populations were expanded and used for cytokine production.

Those expressed by means of PEAK and purified by means of Ni affinity chromatography²⁺ they were&gt; 95% pure and found predominantly as disulfide-linked homodimers, as demonstrated by non-reducing SDS-PAGE. The biological activity of human His-tagged IL-17F expressed by PEAK was similar to the activity of huIL-17F from commercial sources.

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Example 2: immunizations

Fully human monoclonal antibodies were generated by the use of transgenic strains of mice in which the expression of the mouse antibody gene was suppressed and replaced with a human antibody gene expression. Three strains of transgenic mice were used:

1) HuMab® mouse (Medarex, Princeton NJ)

2) KM ™ mouse, a hybrid between HuMAb mouse and Kirin TC mouse (Kirin Pharma Company, Japan)

3) KM mouse (FCyRIIb-KO), a strain derived from the KM ™ mouse, in which the Fcgr2b encoding of the gene for the inhibitory Receptor IIB gamma Fe has been inactivated.

Mice were immunized with either human IL-17F or with both human IL-17F and rat IL-17F. Two forms of antigen were used for immunizations: unconjugated IL-17F or IL-17F conjugated to Megathura crenulata Hemocyanin (KLH). Immunization strategies followed standard literature protocols.

Sera from periodically immunized animals were observed by ELISA for the presence of human IgG directed against rat huIL-17F and IL-17F. Most of the animals developed high titer responses to human IL-17F. When both rat IL-17F and huIL-17 / IL-17hu were used for immunizations, most of the

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<img file="MX348013B_D0108.tif" />

animals developed high titer responses to both antigens. Cross-reactive antibodies to huIL-17A were sporadically generated in KM and KM mice (FCyRIIb-KO) immunized with huIL-17F as the sole antigen (ie, without rat IL-17F). In contrast to KM and KM (FCyRIIb-KO) mice, HuMAb mice did not develop cross-reactive titers to IL-17A, regardless of the immunization protocol used.

Example 3: generation of hybridomas

Fusion of Lymphatic Nodule Cells with SP2 / 0 Myeloma Cells

To obtain hybridomas, popliteal, inguinal, para-aortic, submandibular, cervical, axialar, and brachial lymph nodes were removed from the mice and dissolved with collagenase and DNAse. A single cell suspension of lymph node cells was mixed 1: 1 with SP2 / 0 myeloma cells and suspended in Low Conductivity Cytofusion Medium (CPS-LCMC, CytoPulse Sciences, Inc.). Fusions were performed with 30 to 60 million splenocytes in the CytoPulse CEEF50 Electrofusion apparatus as indicated by the manufacturer (Cyto Pulse Sciences, Inc). After electrofusion, cells were incubated for approximately 1 hour at 37 ° C to allow recovery.

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&gt; · ^ ·· * 1.1 · * · II.BU --Í - wvz. Hybridoma culture

The fused cells were resuspended in HAT selection medium and plated in 44 to 52 96-well plates at a cell concentration of 0.1-0.2x10.⁵ splenocytes per well in 200 μΐ medium. Hybridoma selection continued for 14 days. Fusion of lymph nodes from immunized mice resulted in the generation of hybridomas that produce antibodies specific for huIL-17F or cross-reactive antibodies specific for both hu! L-17F and IL-17A.

Hybridoma Observation

Fourteen days after fusion, the hybridoma-containing plaques were observed for the presence of human IgG binding to human IL-17F and / or human IL-17A by means of FLISA (fluorescence-linked immunosorbent assay). Briefly, 6 micron beads (Polybeads, cat. No. 07312, Polysciences Inc.) Were coated with huIL-17F (both from Peprotech EC) or BSA (Sigma) and distributed in 384-well FMAT® optical plates (Applied Biosystems ) at a density of 5,000 beads per well. The beads were mixed with a small volume of hybridoma culture supernatants (30 μΐ per

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OF THE INDUSTRIAL PROPERTY to the well) and incubated until the day if and after smearing, of the addition of goat anti-human IgG Fe (Jackson Immunoresearch No. 109-005-098) conjugated to a FMAT Blue® dye ( Applied Biosystems). After an incubation period of 2 to 8 hours, the fluorescence of the beads was measured on an analyzer of the 8200 Cell Detection System (Applied Biosystems). Human IgG producing hybridomas that bound huIL-17F, but not BSA, were expanded and subjected to further analysis.

Example 4 generation of recombinant antibodies

Hybridoma Cloned Antibody Sequence

To isolate antibody heavy and light chain variable sequences, the selected hybridoma RNA was first extracted and reverse transcribed. The VH and VL sequences were then amplified by PCR, cloned, and further analyzed by DNA sequencing. In summary, hybridoma template RNA was extracted using the RNeasy Plus kit (QIAGEN) and cDNAs were generated using ready-to-go you-prime first-strand beads (GE Healthcare, No 27-9264-01 ) with oligo dT primers for reverse transcription. The VH and VL sequences were then amplified by means of PCR.

<img file="MX348013B_D0111.tif" />

¹¹ IMPI

INSTITUTO MEXICANO Di LA PROPIEDAD INDUSTRIAD for the use of recognizing families of primer sets of human variable heavy and light chains.

The amplified sequences were cloned into a pCR®4 vector by using the TOPO® TA Sequencing Cloning Kit (Invitrogen). The selected clones were then subjected to DNA sequencing.

Antibody reform, germ line generation and expression

Heavy and light chain variable sequences were reformatted within mammalian expression vectors for antibody production and characterization. In summary, a sequence analysis was carried out with isolated VH and VL to determine their germline. Due to primer mismatches, mutations were introduced 5 'of VL 1F1 and 11C5 during the PCR amplification step. Therefore, site-directed mutagenesis, conducted with the Quikchange kit (Strategene), was used to reconvert these mutations to the human germline sequence. The VH and VL sequences were then sub-cloned into a mammalian expression system under the Human IgGl and IgKappa backbones. The corresponding antibody heavy and light chain vectors were then transected into

<img file="MX348013B_D0112.tif" />

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INSTITUTE M ETICAN # DF THE PROPERTY IKDUSTWIM PEAK cells by the use of TransTTS-T.Ti, v- ^ a rt - j vo dp transfection (Mirus Bio) and cultured in DMEM + serum depleted of IgG + Glutamine. The antibodies expressed by PEAK were then purified from the supernatant using a MabSelectSure suspension (GE Healthcare).

Example 5: cross-reactivity of huil17f antibodies

Binding test ·. huIL-17F antibodies were evaluated for their ability to bind to the other members of the IL-17 family of cytokines, as well as IL-17A and IL-17F from other species. The assay was carried out in the FLISA format, as described above. The following recombinant cytokines were bound to polystyrene beads and evaluated for their ability to bind huIL-17F antibodies: huIL17B (PeprotechEC, Cat. No. 200-28), huIL-17C (R&amp;D Systems, No. No.), huIL-17D (PeprotechEC, Cat. No. 200-27), huIL-17E (huIL-25, PeprotechEC, Cat. No. 200-24), muIL-17A (PeprotechEC, No. Cat. 210-17), mu! L-17F (PeprotechEC, Cat. No. 200-17F), rat IL-17F (His-tagged, produced internally in insect cells),

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<img file="MX348013B_D0113.tif" />

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Rat IL-17A (His-tagged, produced internally in PEAK cells), cyIL-17F (His-tagged, produced internally in cells

PEAK), and cyIL-17A (His-tagged, produced internally in PEAK cells). The ability of individual huIL-17F antibodies to bind these different cytokines is summarized below in Table 3:

Table 3: Cross-reactivity of huIL-17F antibodies as determined by FLISA (ne = not evaluated)

<td>species</td><td colspan="7">human</td><td colspan="3">cynomolgus monkey</td><td colspan="3">mouse</td><td colspan="2">rat</td>

<td>dimer: clone name</td><td>I IL-17F 1</td><td>IL-17A</td><td>IL-17A / F</td><td>IL-17B</td><td>IL-17C</td><td>IL-17D</td><td>W</td><td>IL-17F</td><td>IL-17A</td><td>IL-17A / F</td><td>IL-17F</td><td>IL-17A</td><td>IL-17A / F</td><td>IL-17F</td><td>IL-17A</td>

<td>5E12</td><td> +</td><td></td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>41B10</td><td> +</td><td> -</td><td> +</td><td></td><td> •</td><td></td><td></td><td> +</td><td></td><td> +</td><td></td><td></td><td> •</td><td></td><td></td>

<td>11C5</td><td> +</td><td></td><td> +</td><td> -</td><td></td><td></td><td></td><td> +</td><td></td><td> 4-</td><td></td><td></td><td></td><td></td><td></td>

<td>21B10</td><td> +</td><td></td><td> +</td><td> -</td><td> -</td><td></td><td></td><td></td><td></td><td> +</td><td> -</td><td></td><td></td><td></td><td></td>

<td>1F1</td><td> +</td><td></td><td> +</td><td> -</td><td></td><td></td><td></td><td> +</td><td></td><td> +</td><td></td><td> -</td><td></td><td></td><td> —</td>

<td>2E12</td><td> +</td><td></td><td></td><td></td><td></td><td> -</td><td></td><td></td><td></td><td> +</td><td></td><td></td><td></td><td></td><td></td>

<td>5D3</td><td></td><td></td><td> +</td><td></td><td></td><td></td><td></td><td> +</td><td> -</td><td> +</td><td> -</td><td></td><td></td><td></td><td></td>

<td>22F8</td><td> +</td><td></td><td> +</td><td></td><td></td><td></td><td></td><td></td><td></td><td> +</td><td></td><td></td><td></td><td></td><td></td>

<td>28B11</td><td> +</td><td></td><td> +</td><td> -</td><td> -</td><td></td><td></td><td> +</td><td> -</td><td> +</td><td> -</td><td></td><td></td><td></td><td> —</td>

<td>41A4</td><td> +</td><td> -</td><td> +</td><td></td><td></td><td></td><td></td><td> +</td><td></td><td> + /-</td><td></td><td></td><td></td><td></td><td></td>

<td>43G6</td><td> +</td><td></td><td> +</td><td></td><td></td><td></td><td></td><td> +</td><td></td><td> +</td><td></td><td></td><td></td><td></td><td></td>

<img file="MX348013B_D0114.tif" />

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Example 6: Neutralizing Potency of Antibodies-HuI ^ -17-f— IL-6 Secretion by IL-17 Stimulated Mouse Embryonic Fibroblasts

Human and cynomolgus monkey IL-17A and IL-17F bind to the corresponding mouse IL-17 receptor complex. As a consequence, mouse fibroblasts respond to human and cynomolgus monkey IL-17A and IL-17F through IL-6 secretion. Costimulation with mouse TNF was shown to synergize with IL-17 signaling (Ruddy et al. 2004, J. Biol. Chem 279: 2559), significantly increasing the sensitivity of mouse fibroblasts towards IL-17 cytokines. Therefore, mouse C57BL / 6 embryonic fibroblasts (MEF, ATCC No SCRC-1008) were used to perform assays for the neutralizing ability of huIL-17F antibodies to neutralize the biological activity of huIL-17F heterodimers, cyIL -17F, huIL-17A / F and cyIL-17A / F heterodimers.

Briefly, MEF cells that were seeded in 96-well plates in DMEM + Glutamine + 10% Fetal Bovine Serum (FBS) were cultured for 48 hours prior to the addition of IL-17 cytokines and mouse TNFα at 10 ng / ml (Peprotech EC, Cat. No. 315-01A). In assays for MAb neutralizing activity, IL-17 cytokines were pre-incubated with the antibody for 1 hour before addition to cells. After 24 hours of stimulation in

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DEunonsiMD .. industry i presence of heterodimers IL-17A / F hnn ^ pr c ÚP ypgnos cynomolgus (50 ng / ml) or after 40 hours of stimulation in the presence of human IL-17F or cynomolgus monkeys (5 ng / ml) , the supernatants were collected and the mouse IL-6 concentration was measured by means of Sandwich ELISA using rat anti mouse IL6 antibody (BD Cat. No. 554400) by capture and a second anti rat IL6 antibody. biotinylated mouse (BD 554402) in addition to streptavidin

HRP (Jackson Immunoresearch 016-030-084) by detection. No inhibition of the huIL-17A / F or cyIL-17A / F heterodimer was observed with any of the anti IL-17F antibodies tested. IC values₅₀ obtained with human and cynomolgus monkey IL-17F homodimers are synthesized in Table 4 below and obtained from IL-6 calibration curves using standard statistical techniques.

Table 4: Neutralization power (IC values₅₀) of huIL-17F antibodies in MEF cells stimulated with huIL-17F or cyIL-17F homodimers and mTNF-ot

<td>species</td><td>human</td><td>Cynomolgus monkey</td>

<td>IL-17 dimer:</td><td>IL-17F</td><td>IL-17F</td>

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<td>conc. by IL- 17</td><td>5 ng / ml</td><td>5 ng / ml</td>

<td>clone name</td><td colspan="2">IC50 (nM)</td>

<td>5E12</td><td> 1, 8</td><td> 22</td>

<td>41B10</td><td> 3,9</td><td> 18</td>

<td>11C5</td><td> 0,4</td><td> 2,9</td>

<td>21B10</td><td> 2,0</td><td> 8,7</td>

<td>1F1</td><td> 1,1</td><td> 11</td>

<td>2E12</td><td> 5,9</td><td> 26</td>

<td>5D3</td><td> 0,5</td><td> 12</td>

<td>22F8</td><td> 2,0</td><td> 10</td>

<td>28B11</td><td> 1,2</td><td> 6,3</td>

<td>41A4</td><td> 0,6</td><td> 7,2</td>

<td>43G6</td><td> 1,1</td><td> 8,4</td>

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<img file="MX348013B_D0116.tif" />

Example 7: experimental model of disease: collagen-induced arthritis (cia)

IL-17A plays an important role in the pathogenesis of arthritis, promoting the release of mediators of inflammation and destruction of cartilage. The neutralization of IL-17A has been shown to attenuate arthritis in several experimental models, including CIA (Collagen Induced Arthritis). Since IL-17F is closely related to

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IL-17A, and the fact that it is overexpressed in the synovium of rheumatoid arthritis (RA) patients, the effect of neutralizing this cytokine was explored in an RA model. For this purpose, an anti mIL-17F antibody (mouse IL-17F) was generated which potently neutralized mouse IL-17F homodimers - but not IL-17A / F heterodimers -, and the effects of this anti -mIL-17F in the CIA animal model for RA.

Briefly, 8-10 week old male DBA-1J mice were immunized with 100 micrograms of bovine type II collagen in Complete Freund's Adjuvant (CFA). Type II collagen in CFA was injected intradermally at the base of the tail. Three weeks later, 100 micrograms of type II collagen in Incomplete Freund's Adjuvant (IFA) was injected intradermally to induce disease. The first signs of disease usually appeared 4 to 10 days after the collagen-IFA stimulus. Animals that began to develop arthritis were recruited for study and distributed within the following treatment groups:

1) Isotype control (IgGlk mouse), twice weekly for three weeks at 300 micrograms per injection (n = 10)

2) Murine chimeric hamster anti TNF-alpha, once weekly for three weeks at 300 micrograms

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Mexican IMPI κπτηπο DE LA FROWEDAD INDUSTRIAL

<img file="MX348013B_D0118.tif" />

3) Anti-mouse IL-17F antibody (mouse IgClM-dee times weekly for three weeks at 300 micrograms per injection (n = ll)

The three treatment groups were balanced to contain an equivalent number of animals recruited at different clinical severity scores (1 to 3). Clinical disease scoring was conducted three times a week using standard arthritis scoring methods. The score ranged from 0 to 4 per leg (where 0 means no disease and 4 represents edema involving the entire leg) with a theoretical maximum cumulative score of 16 points per animal. In addition to the clinical severity score, serum levels of key pro-inflammatory cytokines were determined by means of Luminex at termination (day 22 after recruitment).

The progression of clinical scores and serum cytokine levels are as shown in Figures 1 and 2, respectively. Neutralization of IL-17F homodimers was sufficient to significantly delay disease progression and reduce levels of inflammatory mediators. These findings suggest that IL-17F is a candidate target for the therapy of autoimmune diseases such as rheumatoid arthritis.

Although the invention has been described in the form of

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INSTITUTO MEXICANO DE LA MOPIRDAD INDUSTRIAL written description and examples, those with experience in the art will recognize that the invention can be practiced in a variety of modalities and that the above description and examples are for purposes of illustration and not limiting of the following claims.

It is noted that in relation to this date, the best method known to the applicant for putting the aforementioned invention into practice is the one that is clear from the present description of the invention.