Tweak binding antibodies.

(54) Title: ANTIBODIES ATTACHED TO TWEAK. (54) Title: TWEAK BINDING ANTIBODIES.

(57) Summary

The present invention relates to anti-Tweak antibodies.

(57) Abstract

Anti-Tweak antibodies are described.

Institute

Mexican Property

Industrial

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of

PATENT TITLE NO. 339015¹ «*¿»5'·*··''' ·

SE} Kamaría ni kwmmia

Owner (s): BIOGEN MA INC. &gt; \? jY

Address: 250 Blnney Street, Cambridge, Massachusetts, * 02142, USA

Denomination: ANTIBODIES THAT ARE FIXED TO TWEAK.

Classification: lnt.CI.8: A61K39 / 395; C12P21 / 08

Inventor (s): LINDA C. BURKLY; ELLEN GARBER; ALEXEY LUGOVSKOY

MX / a / 2!

013 / 01385T '1

Country:

US

REQUEST sis ^:8fe 7igfe .Yes; .¡S; ·, · ““ ““ S ”K

International filing date:

May 2006

Divisional Patent Number: 324097

- - I

PRIORITY

Date:

May 2005

Number:

60/685,149

Validity: Twenty years

Expiration Date: May 25, 2026 and II t 'u': ·· ......

: ;··. .....

The reference patent is granted based on articles 1, 2 "section V; 6 “fraction III, and 59 of the Industrial Property Law.

In accordance with article 23 of the Industrial Property Law, this patent has a non-renewable term of twenty years, counted from the date of filing of the international application and will be subject to the payment of the fee to maintain the rights in force.

Whoever subscribes to this title does so based on the provisions of articles 6 fractions til and 7 bis 2 of the Industrial Property Law (Official Gazette of the Federation (DOF) 06/27/1991,. «Formed on 08/02/1994, 10/25/1996, 12/26/1997, 05/17/1999, 01/26/2004, 06/16/2005, 01/25/2006, 06/05/2009, 06/06 01/2010, 06/18/2010, 06/28/2010, 01/27/2012; and 04/09/2912); Articles 1, 3, section V, subsection a), sub subsection iii), 4th and 12th sections I and III of the Regulations of the Mexican Institute of Industrial Property (DOF 14712/1999, amended on 07/01/2002, 15 / 07/2004, 07/28/2004 and 09/07/2007); articles 1, 1, 4 * 5 fraction V inolso a), subsection iii), 16 fractions I and III and 30 of the Organic Statute of the Mexican Institute of Industrial Property (DOF 12/27/1999, amended on 10 / 10/2002, 07/29/2004, 08/04/2004 and 09/13/2007); 1st:_:.3rd and 5th paragraph a) and antepenultimate paragraph of the Agreement that. delegates powers to the Deputy Directors General, Coordinator, Divisional Directors, Head of the Regional Offices, 'SülxJIrSSores ^ DHHaioriafes, "Departmental Coordinators and other subordinates of the Mexican Institute of Industrial Property, (DOF 12/15/1999, amended on 04 / 2/2000, 07/29/2004, 08/04/2004 and 09/13/2007). -

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Issue Date: May 9, 2016

DIVISIONAL EXAMINATION OF PATENT FUND, ELECTRICAL AREAS AND INDUSTRIAL DESIGN REGISTRIES AND

PED

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Sand! No. 550, floor 1,

Goal. Pueblo Santa María Tepepan, Xochimílco, CP 16020,

Mexico City

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MX / 2016/36577

IMPI

ANTIBODIES ATTACHING TO TWEAlFSírSS

INDUSTRIAL

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Cross reference to related requests

This application claims priority of US application serial number 60 / 685,149, filed May 27, 2005, the contents of which are incorporated herein by reference in their entirety.

Background of the Invention

Tumors related to tumor necrosis factor (TNF) are a super family of proteins that have an array of functions, including those that are involved in the regulation of the immune system and in the regulation of apoptosis. TWEAK (TNF-like weak apoptosis inducer) is a member of this super family.

Brief description of the invention

Anti-TWEAK antibodies can be used to treat a variety of conditions and disorders, for example, an inflammatory disorder, a neuronal disorder, or another disorder described herein. When used to treat a human subject, the antibody is preferably a human antibody, humanized, or else an effectively human antibody.

In one aspect, the disclosure incorporates a protein that includes a first and a second immunoglobulin variable domain sequence that can include at least qna sufficient part of an immunoglobulin variable domain to form a site of

REF .: 245271 binding to TWEAK-binding antigen, per human. The protein can bind TWEAK, by affinity corresponding to a K_D less than 1O · '⁷·. M_t for example 10 ⁸, 10 ⁹, 10 ¹⁰, 1 0 ¹¹ M or better. The protein is also referred to herein as an anti-TWEAK antibody. The first and second immunoglobulin variable domain sequences can include at least a sufficient portion of an immunoglobulin variable domain to form an antigen binding site that binds TWEAK. Commonly, the first and second immunoglobulin variable domain sequences correspond to immunoglobulin variable domain sequences of a heavy and light chain, respectively, eg, a paired or compatible heavy and light chain.

The antibody can bind to an epitope in TWEAK which includes at least one, two, three, or four amino acid residues of a TWEAK epitope recognized by P2D10, to a TWEAK peptide that is bound by P2D10 (eg, a peptide less than 25, 20, or 15 amino acids in length) or to a TWEAK region recognized by P2D10. For example, the antibody specifically binds to an epitope, eg, a linear or conformational epitope, of TWEAK, in particular human TWEAK, eg, the soluble region of TWEAK. The antibody can compete with P2D 10 to bind TWEAK, eg, human TWEAK. The antibody can competitively inhibit the binding of P2D10 to TWEAK, eg, human TWEAK. In one embodiment, the

JROPIEPAD MEXICAN INSTITUTE eiemDWvsi® © ri

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epitope to be sub-erjL ·.,., ^ ____ ^r rf MEXICAN INSTITUTE OF PROPERTY&gt; · INDUSTRIAL _ at least one, two, three or four eoítooo P2D10. or an eioid of the antibody can bind to a P2D10, for example, it includes amino acids in common with when it binds, it spherically prevents TWEAK Interaction with P2D

10.

For example, the anti-TWEAK antibody can bind TWEAK and modulate, eg, Inhibit, an Interaction (eg, binding) between TWEAK and TWEAK receptor, eg, Fnl4 (eg, human Fnl4). The antibody can also reduce the signaling activity of the TWEAK receptor. The antibody can target TWEAK, hijack TWEAK, and / or modulate the in vivo stability of

TWEAK.

In one embodiment, the antibody specifically binds to at least a portion of! TWEAK site of interaction that contacts Fnl4 (eg, human Fnl4). The antibody can compete with Fnl4 to bind TWEAK, eg, a human TWEAK. The antibody can competitively inhibit the binding of Fnl 4 to TWEAK. The antibody can interact with an epitope in TWEAK which, when fixed, spherically prevents interaction between TWEAK and Fnl4 (eg, between human TWEAK and human Fnl4).

In one embodiment, the antibody can inhibit one or more TWEAK-associated activities with an IC₅₀ from about 50 nM to 5 pM, commonly about 100 to 250 pM or less. For example, the antibody can inhibit the ability of IMPI

TWEAK to promote proliferation or nedvas ^^ g ^^ n ^^ * i INDUSTRIAL endothelial. In one embodiment, the anti-TWEAK antibody reduces at least one activity associated with TWEAK, for example, such that the antibody can modulate an inflammatory condition when administered to a subject.

In other modalities, the antibody can be associated with TWEAK with kinetics in the range from 10³ up to 10⁸ M '¹s¹, commonly 10⁴ up to 10⁷ In yet another embodiment, the antibody has dissociation kinetics in the range from 10 '² up to 10'⁶ s'¹, commonly 10 '² up to 10'⁵ s'¹. In one embodiment, the antibody binds to TWEAK, eg, human TWEAK, with an affinity and / or kinetics similar to (eg, within a factor of five or ten) of, monoclonal antibody P2D10, or modified forms thereof, for example, chimeric forms or humanized forms thereof (eg, a humanized form described herein). The activity and binding kinetics of the anti-TWEAK antibody can be tested, for example, using biosensor technology (BIACORF ™).

In one embodiment, the antibody is an antigen-binding fragment of a full-length antibody, eg, a single-chain Fab, F (ab ') 2, Fv, or Fv fragment. Commonly, the antibody is a full length antibody. The antibody can be a monoclonal antibody or a specific mono antibody. For example, the antibody is in a composition that includes fewer than 20 different species of anti-TWEAK antibodies,

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for example, in a composition that does not include or anti-TWEAK antibody.

The antibody can be effectively human £ TLJri efectivamenteμυ effectively human is an antibody that includes a sufficient amount of humanoid amino acid positions such that the antibody does not trigger an immunogenic response in a normal human. Preferably, the protein does not evoke a neutralization response of! antibody, eg, the response to anti-human murine antibody (HAMA). HAMA can be problematic in a number of circumstances, for example, if it is desired that the antibodies be administered repeatedly, for example, in the treatment of a chronic or recurring disease condition. A response to HAMA can potentially render ineffective repeated administration of the antibody due to increased clearance of the antibody from serum (see, eg, Saleh et al., Cancer Immunol. Immunother., 32: 180-190 (1990)) and also due to potential allergic reactions (see, for example, LoBuglio et al. (1986) Hybridoma, 5: 5117-5123).

For example, the antibody may be a human, humanized, CDR-grafted, chimeric, mutated, affinity matured, de-immunized, synthetic, or in vitro generated antibody, and combinations thereof. In one embodiment, the antiTWEAK antibody is. a humanized antibody.

The heavy and light chains of the anti-TWEAK antibody can be substantially full length. The protein can and preferably two,

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include at least one, complete, and at least one, and preferably dcgL cadeffS ^ Nftjercfscomplete) or may include a fixation or ontgon fragment (for example, a single-chain Fab, F (ab ') 2, Fv, or Fv fragment) . In still other embodiments, the antibody has a heavy chain constant region chosen from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly chosen from, for example, lGG1, lGG2, lGG3, and lgG4, more particularly lgG1 (eg, human lqG1). Commonly, the heavy chain constant region is a human form or a modified form of a human constant region. In another embodiment, the antibody has a light chain constant region chosen, eg, from kappa or lambda, particularly, kappa (eg, human kappa).

In one embodiment, the protein includes at least one, two, and preferably three CDRs of the light or heavy chain variable region of an antibody described herein, eg, P2D10. In this context, CDRs refer to CDRs as defined by Chothia's hypervariable ties. For example, the protein includes, in the heavy variable chain domain sequence, at least one, two, or three of the following sequences within a CDR region: GFTFSRYAMS (CDR1) (SEQ ID NO: 1),

EISSGGSYPYYPDTVTG (CDR2) (SEQ ID NO: 2), VLYYDYDGDRIEVMDY (CDR3) (SEQ ID NO: 3), or a CDR that has an amino acid sequence that differs by no more than 4, 3, 2.5, 2, 1.5, 1 , or 0.5 alterations (for example, substitutions,

<img file="MX339015B_D0013.tif" />

insertions or deletions) for every 10 amino acids ((Jo¿VjpIfPU

I amount of differences that are proportional to the longiWV ^ '^ fe CDR) relative to a sequence listed above, by njc, 111 μ I ur at least one alteration, but not more than one, two or three of the following sequences within a CDR region. The heavy variable chain domain sequence may include these CDR sequences particularly in CDR3, or in at least two CDRs, eg, CDR1 and CDR3, CDR2 and CDR3, or in all three CDRs.

The protein may include, in the heavy variable chain domain sequence, at least one, two, or three of the following sequences within a CDR region (amino acids in parentheses represent alternatives for the particular position):

(i) G- (YF) - (NT) -F- (STDN) - (RY) -YA- (MIL) - (HS); (SEQ ID NO.4), (¡i) YY- (PV) -D- (TS) -V- (TK) -G; (SEQ ID NO: 5) and (iii) (VL) - (IL) - (YF) - (YF) -D- (YF) -D; (SEQ ID NO: 6) or (DE) - (RK) (ILVM) - (EQD) - (VAL) -M- (DE); (SEQ ID NO: 7).

The protein may include, in the light chain variable domain sequence, at least one, two, or three of the following sequences within a CDR region:

RSSQSLVSSKGNTYLH; (CDR1) (SEQ ID NO: 8),

KVSNRFS; (CDR2) (SEQ ID NO.9), and

SQSTHFPRT; (CDR3) (SEQ ID NO: 10), or a CDR that has an amino acid sequence that differs by no more than 4, 3, 2.5, 2, 1.5, 1, or 0.5 alterations (eg, substitutions, insertions, or deletions) ) for every 10 amino acids (for example,

ΓΜΤΪ differences that is proportional to the length of the CDR ^ '^ S'OJ ^ c

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<img file="MX339015B_D0014.tif" />

to the sequence listed above, for example, at least one alteration, but not more than two, three, or four per CDR. The light chain variable domain sequence may include these CDR sequences particularly in CDR3, or in at least two CDRs, eg, CDR1 and CDR3, CDR2 and CDR3, or in all three CDRs.

The protein may include, in the light chain variable domain sequence, at least one, two, or three of the following sequences within a CDR region (amino acids in parentheses represent alternatives for the particular position):

(i) (RK) -SSQS- (LI) - (KV) -SS- (KR) -GN- (TN) -YL- (EHDNQY);

(SEQ ID NO: 11), or (RK) -SSQS- (LI) -VSS- (KR) -GN- (TN) -YLH; (SEQ ID NO: 12) (¡i) (KE) - (LVI) -S- (NYS) - (RW) - (FAD) -S; (SEQ ID NO: 13), or K (LVI) -S- (NYS) - R- (FAD) -S; (SEQ ID NO: 14), and (iii) (SM) -Q- (GSA) - (ST) - (HEQ) - (FWL) -P; (SEQ ID NO: 15) or SQ- (GSA) - (SIT) - (HEQ) -FP; (SEQ ID NO: 16).

In a preferred embodiment, the protein includes all six closely related P2D10 CDRs or CDRs, for example, CDRs that are identical or have at least one amino acid alteration, but not more than two, three, or four alterations (eg, substitutions, deletions, or insertions), or other CDRs described here.

In yet another example, the protein includes at least one, two or three CDR regions having the same canonical structures and the corresponding CDR regions of IMPI P2D10.

INSTITUTO MEXICANO example, the same canonical structures as at 008883 $ / CDR2 of the light chain variable domains of P? D1 or

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[0020] The protein may include one of the following sequences;

• IVMTQTPLSLPVTPGEPASISCRSSQSLVSSKGNTYLHWYLQ

KPQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAED VGVYYCSQSTHFPRT (SEQ ID NO: 17) • DIVMTQTPLSLPVTPGEPASISCRSSQSLVSSKGNTYLHWYLQ

KPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCSQSTH FPRT (SEQ ID NO: 18) • DWMTQSPLSLPVTLGQPASISCRSSQSLVSSKGNTYLHWFQQ

RPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE

DVGVYYCSQSTH FPRT (SEQ ID NO: 19) • VVMTQPLSLPVTLGQPASISCRSSQSLVSSKGNTYLH WFQ

QRPGQSPRR! JYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA

EDVGVYYCSQSTH FPRT (SEQ ID NO: 20) • DIVMTQTPLSLSVTPGQPASISCRSSQSLVSSKGNTYLHWYL

QKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA

EDVGVYYCSQSTHFPRT (SEQ ID NO: 21) • DIVMTQTPLSLSVTPGQPASISCRSSQSLVSSKGNTYLH

WYLQKPGQPPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR

VEAEDVGVYYCSQSTH FPRT (SEQ ID NO: 22).

DIVMTQSPLSLPyTPGEPASISCRSSQSLVSSKGNTYL

HWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIS

RVEAEDVGVYYCSQSTH FPRT (SEQ ID NO: 2,3)

ΪΜΡΙ • DIVMTQSPLSLPVTPGEPASISCRSSQSLVSSKGÑTYLHWYLQ

KPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCSQSTH FPRT (SEQ ID NO: 24) • DIVMTQTPLSSPVTLGQPASISCRSSQSLVSSKGNTYLHW

LQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRV EAEDVGVYYCSQSTH FPRT (SEQ ID NO: 25) • DIQMTQSPSSLSASVGDRVTITCRSSQSLVSSKGNTYLH

WYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCSQSTH FPRT (SEQ ID NO: 26) or a sequence that has fewer than eight, seven, six, five, four, or two alterations (for example, substitutions, insertions, or substitutions, by by an amino acid residue P2D10, huP2D10-Ll, or huP2D10-L2). Examples of substitutions are in one of the following Kabat positions: 2, 4, 6, 35, 36, 38, 44, 47, 49, 62, 6469, 85, 87, 98, 99, 101, and 102. Substitutions For example, they can substitute one or more amino acids of P2D10 at corresponding positions in a framework region, for example, a human framework region, for example, in FR2 (for example, in position 46 for Phe according to consecutive numbering) and at FR3 (eg, at position 87 for Phe).

The protein may include one of the following sequences in the heavy chain variable domain ^

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

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QVQLVQSGAEVKKPGASVKVSCKASGFTFSRYAMSWVRQA

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PGQGLEWMGEISSGGSYPYYPDTVTGPVTMTRgT &amp; IftTl iNsrn

Czc LUA ΓΛ '.' Γ II.lRnj. '

LSRLRSDDTAVYYCAR VLYYDYDGDRIE (SEQ ID NO ^ • QVQLVQSGAEVKKPGASVKVSCKASGFTFSRYAMSW

VRQAPGQRLEWMGEISSGGSYPYYPDTVTGRVTITRDTSAST AYMELSSLRSEDTAVYYCAR VLYYDYDGDRIE (SEQ ID NO: 28) • QVQLVQSGAEVKKPGASVKVSCKASGFTFSRYAMSWVR

QATGQGLEWMGEISSGGSYPYYPDTVTGRVTMTRNTSIST AYMELSSLRSEDTAVYYCARVLYYDYDGDRIE (SEQ ID NO:

29) • QVQLVQSGAEVKKPGASVKVSCKASGFTFSRYAMSW

VRQAPGQGLEWMGEISSGGSYPYYPDTVTGRVTMTTDTSTST

AYMELRSLRSDDTAVYYCARVLYYDYDGDRIE (SEQ ID NO:

30) • QVQLVQSGAEVKKPGASVKVSCKVSGFTFSRYAMS

WVRQAPGKGLEWMGEISSGGSYPYYPDTVTGRVTMTEDTST

DTAYMELSSLRSEDTAVYYCATVLYYDYDGDRIE (SEQ ID

NO: 31) • QMQLVQSGAEVKKTGSSVKVSCKASGFTFSRYAMSW

VRQAPGQALEWMGEISSGGSYPYYPDTVTGRVTITRDRSMST

AYMELSSLRSEDTAMYYCARVLYYDYDGDRIE (SEQ ID NO: 32) • QVQLVQSGAEVKKPGASVKVSCKASGFTFSRYAMSW

VRQAPGQGLEWMGEISSGGSYPYYPDTVTGRVTMTRDTSTS

TVYMELSSLRSEDTAVYYCARVLYYDYDGDRIE (SEQ ID NO:

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³³¹ IMPI • QMQLVQSGPEVKKPGTSVKVSCKASGFTFStf? WO¡ # m ^

INDUSTRIAL

VRQARGQRLE WIG EIS SGGSYPYYPDTVTG RVTITRDM STSTA YMELSSLRSEDTAVYYCAAVLYYDYDGDRIE (SEQ ID NO: 34) • EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYAMSWV

RQAPGKGLEWVAEISSGGSYPYYPDTVTGRFTISRDNAKNS

LYLQMNSLRAEDTAVYYCARVLYYDYDGDRIE (SEQ ID NO:

35) • EVQLVESGGGLVQPGRSLRLSCAASGFTFSRYAMSW

VRQAPGKGLEWVSE1SSGGSYPYYPDTVTGRFTISRDNAKNS

LYLQMNSLPAEDTALYYCAKDVLYYDYDGDRIE (SEQ ID NO:

36) • QVQLVESGGGLVKPGGSLRLSCAASGFTFSRYAMSWI

RQAPGKGLEWVSEISSGGSYPYYPDTVTGRFTISRDNAKNSL YLQMNSLRAEDTAVYYCARVLYYDYDGDRIE (SEQ ID NO:

37) • EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYAMSW

VRQAPGKGLEWVGEISSGGSYPYYPDTVTGRFTISRDDSKNT

LYLQMNSLKTEDTAVYYCTTVLYYDYDGDRIE (SEQ ID NO:

38) • EVQLVESGGGWRPGGSLRLSCAASGFTFSRYAMSW

VRQAPGKGLEWVSEISSGGSYPYYPDTVTGRFTISRDNAKNS

LYLQMNSLRAEDTALYHCARVLYYDYDGDRIE (SEQ ID NO:

39)

EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYAMSWVRQAP

GKGLEWVSEISSGGSYPYYPDTVTGRFTISRD iUMMRJq 'MEXICAN INSTITUTE' OF PROPERTY

NSLRAEDTAVYYCAR VLYYDYDGDRIE (SBQ ID NCCTüT • EVQLLESGGGLVQPGGSLRLSCAASGFTFSRY7WSWVRTT ·

APGKGLEWVSEISSGGSYPYYPDTVTGRFTISRDNSKNTLYL QMNSLRAEDTAVYYCAK VLYYDYDGDRIE (SEQ ID NO: 41) • QVQLVESGGGWQPGRSLRLSCAASGFTFSRYAMSW

VRQAPGKGLEWVAEISSGGSYPYYPDTVTGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCAKVLYYDYDGDRIE (SEQ ID NO: 42) • QVQLVESGGGWQPGRSLRLSCAASGFTFSRYAMSWV

RQAPGKGLEWVAEISSGGSYPYYPDTVTGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCARVLYYDYDGDRIE (SEQ ID NO: 43) • QVQLVESGGGWQPGRSLRLSCAASGFTFSRYAMSW

VRQAPGKGLEWVAEÍSSGGSYPYYPDTVTGRFTISRDNSKNTL

YLQMNSLRAEDTAVYYCAKVLYYDYDGDRIE (SEQ ID NO:

44) • QVQLVESGGGWQPGRSLRLSCAASGFTFSRYAMSW

VRQAPGKGLEWVAEISSGGSYPYYPDTVTGRFTISRDNSKNTL

YLQMNSLRAEDTAVYYCARVLYYDYDGDRIE (SEQ ID NO:

45) • EVQLVESGGWVQPGGSLRLSCAASGFTFSRYAMSW

VRQAPGKGLEWVSEISSGGSYPYYPDTVTGRFTISRDNSKNS

LYLQMNSLRTEDTALYYCAKDVLYYDYDGDRIE (SEQ ID NO:

46) • EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYAMSWVR

<img file="MX339015B_D0019.tif" />

* ..

r

QAPGKGLEWVSEISSGGSYPYYPDTVTGRFtÍ £

MEXICAN INSTITUTE

QMNSLRDEDTAVYYCARVLYYDYDGDRIE • EVQI VFSGGGI VQPGRSI Rl Mr. TASfíFTFSRYAMSWF

<img file="MX339015B_D0020.tif" />

RQAPGKGLEWVGEISSGGSYPYYPDTVTGRFTISRDGSKSIAY LQMNSLKTEDTAVYYCTRVLYYDYDGDRIE (SEQ ID NO: 48) EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYAMSW

VRQAPGKGLEYVSEISSGGSYPYYPDTVTGRFTISRDNSKN

TLYLQMGSLRAEDMAVYYCARVLYYDYDGDRIE (SEQ ID NO: 49) or a sequence that has fewer than eight, seven, six, five, four, three, or two alterations (for example, substitutions, insertions, or selections, for example, conservative substitutions or a substitution for a amino acid residue at a corresponding position in P2D10). Examples of substitutions are in one of the following Kabat positions: 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104_; 106, and 107. Substitutions, for example, may replace one or more amino acids of P2D10 at positions corresponding to a framework region, eg, a human framework region.

In one embodiment, the heavy chain framework (eg, FR1, FR2, FR3, individually, or a sequence comprising FR1, FR2, and FR3, but excluding CDR) includes an amino acid sequence, which is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identical to the heavy chain framework of one of the segment V sequences of the following germ line: DP-25, DP-1, DP-12 ,

5

DP-9, DP-7, DP-31, DP-32, DP-33, DP-58, DP-54, other

FROM THE PROPHEPAP \ INDUSTRIAL germline of subgroup VH I, another germline sequence of subgroup VH III, or another V gene that is compatible with the canonical structure of class 1-3 (see, for example, Chothla et al. (1992 ) J. Mol. Biol. 227: 799-817; Tomlinson et al. (1992) J. Mol. Biol. 227: 776798). Other frameworks compatible with the canonical class structure

1-3 includes the one or more of the following residues according to Kabat numbering: Ala, Gly, Thr, or Val at position 26; Gly at position 26; Tyr, Phe, or Gly at position 27; Phe, Val, He, or Leu at position 29; Met, He, Leu, Val, Thr, Trp, or He at position 34; Arg, Thr, Ala, Lys at position 94; Gly, Ser, Asn, or Asp at position 54; and Arg at position 71.

In one embodiment, the light chain framework (eg, FR1, FR2, FR3, individually, or a sequence comprising FR1, FR2, and FR3, but excluding CDR) Includes an amino acid sequence, which is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identical to the light chain framework of a germline sequence of subgroup VK II or one of the following sequences of the terminal V segment: Al 7 . Al, Al 8, A2, A19 / A3, A23, a germline sequence of the VK I subgroup (eg, a DPK9 sequence), or another V gene that is compatible with class 4-1 canonical structure (see, for example, Tomlinson et al. (1995) EMBO J. 14: 4628). Other frames compatible with the canonical structure of class 4-1 include frames with the one or more of the following residues according to the Kabat numbering: Val or Leu or

6

IMPI

I have in position 2; Ser or Pro in position 25; (. H

MEXICAN INSTITUTE OF PROPERTY

<img file="MX339015B_D0021.tif" />

position 27b; Gly at position 29; Phe or Leu in the posicl'ffhP ^ S; and Phe at position 71. Furthermore, according to nuTTOl dLiún Kabul ·, position 48 can be He or Val.

In another embodiment, the light chain framework (eg, FR1, FR2, FR3, individually, or a sequence comprising FR1, FR2, and FR3, but excluding CDR) includes an amino acid sequence, which is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identical to! light chain frame of a terminal sequence of subgroup VK 1, for example, a sequence DPK9.

In one embodiment, the light or heavy chain variable framework (eg, the region comprising at least FR1, FR2, FR3, and optionally FR4) can be chosen from: (a) a light or heavy chain variable framework that includes at least 80%, 90%, 95%, or preferably 100% of the amino acid residues of a human light or heavy chain variable framework, for example, a residue of variable light or heavy chain framework of a mature human antibody, a human germline sequence, a human consensus sequence, or a human antibody described herein; (b) a light or heavy chain variable frame that includes from 20% to 80%, 40% to 60%, 60% to 90%, or 70% to

95% of the amino acid residues of a human light or heavy chain variable frame, for example, a light or heavy chain variable frame residue of a mature human antibody, a human germline sequence, a consensus sequence

7

Λ. '

<img file="MX339015B_D0022.tif" />

or (d) a non-human framework that has been modified, eg, to remove antigenic or clototoxic determinants, eg, deimmunized or partially humanized. In one embodiment, |, a heavy chain variable domain sequence Includes human residues or human consensus sequence residues at one or more of the following positions (preferably at least five, ten, twelve or all): (in the FR of the light chain variable domain) 4L, 35L, 36L, 38L, 43L, 44L, 58L, 46L, 62L, 63L, 64L, 65L,

66L, 67L, 68L, 69L., 7OL, 71L, 73L, 85L, 87L, 98L, and / or (in the FR of the heavy chain variable domain) 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 6OH, 67H, 68H, 69H, 7OH, 73H, 74H, 75H, 78H, 91H, 92H, 93H, and / or 103H (according to Kabat numbering).

In one embodiment, the protein includes at least one non-human CDR, eg, a murine CDR, eg, a P2D10 CDR, or a mutant thereof, and at least one framework that differs from a P2D10 framework in at least an amino acid, for example, at least 5, 8, 10, 12, 1o, or 18 amino acids. For example, proteins Include one, two, three, four, five or six of these non-human CDRs and Includes at least one amino acid difference in at least three of HC FR1, HC FR2, HC FR3, LC FR1, LC FR2, and LC

FR3.

₍In one embodiment, the protein's heavy or light chain variable domain sequence includes a sequence of

8

<img file="MX339015B_D0023.tif" />

amino acids, which is at least 80%, 85%, 90%, 95% J9) V1

INSTI

L___ or more identical to a variable domain sequence of a '^ YHWuerp described here, eg, P2D10, huP2D10-l, or · huP2D10 ^ 2, and which differs by at least 1 or 5 residues, but less than 40, 30, 20, or 10 residues, of a variable domain sequence of an antibody described herein, eg, P2D10, huP2D10-1, or huP2D10-2.

In one embodiment, one or both of the variable domains include amino acid positions in the framework region that come variably from both a murine antibody (eg, P2D10) and a humanized antibody (eg, 56-84m and Kl 07) or germline sequence. For example, the variable domain will include a number of positions at which the amino acid is identical to both the murine antibody and the human antibody (or germline sequence) because the two are identical at that position. Of the remaining framework positions where murine and human differ, at least 50, 60, 70, 80, or 90% of the variable domain positions are preferably identical to the human antibody (or germline sequence) rather than the murine. None, or at least one, two, three, or four of these remaining merco positions can be identical to the murine antibody rather than the human antibody. For example, in HC FR1, one or two of these positions can be murine; in HC FR2, one or two of these positions can be murine; in FR3, one, two, three, or four of these positions can be murine; in LC FR1, one, two, three, or four of these positions can be murine; in LC FR2, one died; in uj ¡? vl&gt; jPⁿi

MEXICAN INSTITUTE OF INDUSTRIAL RROEIEDAD

<img file="MX339015B_D0024.tif" />

domain variable oadone one amino acid sequence or two of these positions can be from these positions can be murine.

In one embodiment, the heavy or light sequence of the protein includes encoded by a nucleic acid sequence described here or a nucleic acid that hybridizes to a nucleic acid sequence described here (eg, a specific nucleic acid sequence or sequence nucleic acid encoding an amino acid sequence described herein) or its complement, for example, under low restrictive, medium restrictive, high restrictive conditions, or very high restrictive conditions.

The anti-TWEAK antibody can be derived or linked with another functional molecule, for example, another peptide, protein or compound. For example, the antibody may be functionally linked (eg, by chemical coupling, genetic fusion, non-covalent association, or other) to one or more other molecular entities, such as another antibody (eg, a bispecific antibody or a multi-antibody specific), toxins, radioisotopes, polymers, cytotoxic or cytostatic agents, among others.

In another aspect, the disclosure provides compositions, eg, pharmaceutical compositions, including a pharmaceutically acceptable carrier and an anti-TWEAK antibody, eg, an anti-TWEAK antibody described herein.

In yet another embodiment, the anti-TWEAK antibody (eg, a pharmaceutical composition thereof) was We

IMPI

<img file="MX339015B_D0025.tif" />

subject who needs anti-TWEAK antibody therapy or whose

M lili · condition could be improved by the antibody.

For example, the anti-TWEAK antibody can be administered to a subject who has or is at risk for an inflammatory disorder, immune disorder, autoimmune disorder, neuronal disorder, neoplastic disorder, or other disorder described herein. In one embodiment, an anti-TWEAK antibody described herein is used for the preparation of a medicament for the treatment of an inflammatory disorder, immune disorder, autoimmune disorder, neuronal disorder, neoplastic disorder, or other disorder described herein.

In another aspect, the disclosure incorporates a method of treating a disorder associated with TWEAK, in a subject. The method includes: administering to the subject an anti-TWEAK antibody, in an amount sufficient to treat (eg, improve or prevent) the disorder associated with TWEAK. The anti-TWEAK antibody can be administered to the subject, alone or in combination with other therapeutic modalities described herein. In one embodiment, the subject is a mammal, eg, a human, eg, a human having a disorder associated with TWEAK, eg, a disorder described herein. The antibody can be used to improve one or more symptoms of these disorders. The term "treating" refers to administering a therapy in an effective amount, manner, and / or mode to improve or prevent an associated condition, symptom, or parameter

IMPIfs with a disorder (for example, a described disorder^INS ^ {J3'Ji ^ ¥ ÍEÍ, ^ a

INDUSTRIAL prevent the onset, progression, or exacerbation of the disorder, to a statistically significant degree or to a detectable degree for a person skilled in the art. Accordingly, the treatment can achieve therapeutic and / or prophylactic benefits. An effective amount, manner, or mode can vary depending on the subject and can be adjusted to the subject. In one embodiment, an anti-TWEAK antibody described herein is used for the preparation of a medicament for the treatment of a disorder associated with

TWEAK.

In another aspect, the disclosure incorporates a method of modulating the interaction between TWEAK and the TWEAK receptor protein. For example, an anti-TWEAK antibody can be used to reduce or inhibit binding, between TWEAK and the TWEAK receptor, such as Fnl 4. The method comprises contacting TWEAK or a complex containing TWEAK with the antibody. The method can be used on cells in vitro eg in culture, eg in vitro or ex vivo. For example, cells expressing the TWEAK receptor can be cultured in vitro in culture medium and the contacting step can be performed by adding an anti-TWEAK antibody to the culture medium. Alternatively, the method can be performed on cells present in a subject, eg, as part of an in vivo procedure (eg, therapeutic or prophylactic). For example, the ^ nti-TWEAK antibody can be supplied locally or systemically. In one embodiment, an anti-TWEAK antibody described herein

DELA INDUSTRIAL PROPERTY

<img file="MX339015B_D0026.tif" />

A drug preparation was used to modulate the interaction between TWEAK and a TWEAK receptor protein.

The method may include contacting TWEAK with the TWEAK receptor complex or with a sub unit of it, under conditions that allow an interaction between TWEAK and the TWEAK receptor complex or sub unit of it, so that a TWEAK / TWEAK receiver mix. Generally, the anti-TWEAK antibody is provided in an effective amount, for example, such that contacting the TWEAK / TWEAK receptor mixture with the anti-TWEAK antibody modulates, eg, interferes with (eg, inhibits, it blocks or, if not, reduces) the interaction between TWEAK and the receptor protein or at least one function of TWEAK, for example, TWEAK-mediated signaling.

The invention also incorporates nucleic acids containing nucleotide sequences, which encode heavy and light chain variable regions of anti-TWEAK antibodies, for example, as described herein. For example, the disclosure incorporates a first and a second nucleic acid encoding P2D10 heavy and light chain variable regions, respectively. In another aspect, the disclosure incorporates host cells and vectors containing the nucleic acids described herein.

The invention also incorporates the TWEAK epitope, eg, human TWEAK, recognized by P2D10, and proteins capable of interacting with the epitope. For example, proteins and peptides that include the epitope can be used to generate.

rarlMaBi

MEXICAN INSTITUTE OF PROPERTY

<img file="MX339015B_D0027.tif" />

other binding compounds that interact with the epibid^D^^s5ⁱ,'^{TO THE}for example, proteins such as antibodies or pykutinic molecules. For example, a peptide that includes the epitope can be used as an immunogen or as a target to explore an expression library. It is also possible to evaluate compounds to determine their ability to interact with the peptide, or, or by mapping or structure determination, to evaluate compounds to determine their ability to interact with the epitope, for example, in the context of a mature TWEAK. An example of evaluation includes determining whether the compound can interact with TWEAK in the presence of a competing P2D10 antibody.

Also disclosed are methods of delivering or targeting an agent, eg, a therapeutic agent (including a genetic agent) or a cytotoxic agent, with an anti-TWEAK antibody (eg, P2D10 or another antibody described herein) to a cell, or structure that expresses TWEAK in vivo.

As used herein, the term "antibody" refers to a protein that includes at least one immunoglobulin variable region, eg, an amino acid sequence that provides an immunoglobulin variable domain or an immunoglobulin variable domain sequence. For example, an antibody may include a heavy chain variable region (H) (abbreviated here as VH), and a light chain variable region (L) (abbreviated here as VL). In another example, an antibody included

OE INDUSTRIAL PROPERTY heavy chain variables (H) and two variable chain regions. , · / Light (L). The term antibody encompasses the antigen-binding fragment of antibodies (eg, single-chain antibodies, Fab fragments, F (ab 'fragments)₂, Fd fragments, Fv fragments, and dAb fragments) as well as full-length antibodies, eg, full length, Immunoglobulins of the types IgA, IgG (eg, lGG1, lgG2, lgG3, lgG4), IgE, IgD,

IgM (as well as sub types of them). The term full-length antibody refers to an antibody that is at least 96% the length of a natural antibody that is processed to remove any signal sequences. A full length antibody may include the full length of the native antibody, eg, residues of the amino terminal residue of a native antibody (eg, to IgG1, IgG2, IgG3, IgG4) to its carboxyl terminal residue.

An immunoglobulin variable domain sequence refers to an amino acid sequence that can form the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two, or more amino acids at the N- or C-terminus, or it may include other alterations that are consistent with protein structure formation.

An isolated composition refers to a composition that is f IMPAL derived from at least 90% of at least one componentfe

INDUSTRIAL natural from which the isolated composition can be obtained. Artificially or naturally produced compositions can be compositions of at least some degree of purity if the species or species population of interest is at least 5, 10, 25, 50, 75, 80, 90, 95, 98, or 99% pure on a weight-to-weight basis.

An epitope refers to the site on a target compound that is bound by an antibody, in the case where the target compound is a protein, for example, an epitope can refer to amino acids (particularly amino acid side chains) that are linked by the antibody. Overlapping epitopes include at least one common amino acid residue, eg, at least 2, 3, 4, or 5 common amino acid residues.

As used herein, the term "hybridize under stringent, medium stringent, high stringency, or very high stringency" describes conditions for hybridization and washing. A guide to performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley &amp; Sons, NY (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either can be used. The specific hybridization conditions referenced here are as follows: 1) Low stringency sodium chloride / sodium citrate (SSC) 6X hybridization conditions at approximately 45 ° C, followed by two 0.2X SSC washes 0.1% SDS at least 50 ° C (wash temperature can be increased to 55 ° C to

<img file="MX339015B_D0028.tif" />

IMPI low restrictive conditions); 2) average industrial conditions in 6X SSC at approximately 45 ° C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60 ° C; 3) highly stringent hybridization conditions in 6X SSC at approximately 45 ° C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65 ° C; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65 ° C, followed by one or more washes with 0.2X SSC, 1% SDS at 65 ° C. High restrictive conditions (3) are the preferred and only conditions to be used unless otherwise specified.

A disorder associated with TWEAK is any disorder in which TWEAK contributes to the etiology or a disorder whose condition, symptoms, or risk of onset is altered by the provision of a TWEAK blocking agent.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by a person of ordinary skill in the art to which this invention belongs. While methods and materials similar or equivalent to those described herein may be used in practice or in the test of the invention described herein, the appropriate methods and materials are described below. Furthermore, the embodiments of the invention described with respect to Choth ^ a CDRs can also be implemented using CDR Kabat.

All publications, patent applications, patents and

<img file="MX339015B_D0029.tif" />

<img file="MX339015B_D0030.tif" />

Other references mentioned here are incorpo ^ fe ^ Mffi © cold 'reference in its entirety. In case of conflict, this specification, including definitions, prevails. Furthermore, the materials, methods and examples are illustrative only and are not intended to be limiting.

Detailed description of the invention

P2D10 is an example of a murine antibody that specifically binds to a human TWEAK and inhibits TWEAK function. Variants of the P2D10 antibody are also described, including examples of humanized variants. These antibodies, other anti-TWEAK antibodies, and other TWEAK blocking agents can be used to treat or prevent TWEA-mediated disorders, eg, inflammatory disorders and other disorders described herein.

Anti-TWEAK antibodies

This invention includes the sequences of specific examples of anti-TWEAK antibodies, such as P2D10, huP2D10-1, and huP2D10-2. Particular antibodies, such as these, can be made, for example, by preparing and expressing synthetic genes that encode the listed amino acid sequences, or by mutating human germline genes to provide a gene that encodes the listed amino acid sequences. Furthermore, these antibodies and other anti-TWEAK antibodies can be produced, for example, using one or more of the following methods.

Numerous methods are available to obtain: IMPÍ ^ _n INSTjWTO 'protein, by visualizing antibodies, particularly human antibodies, method includes scanning example expression libraries, phage or ribosome libraries. Phages are described, for example, in US 5,223,409; Smith (1985) Science 228: 1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809. Phab display of Fab is described, for example, in US Patent Nos. 5,658,727; 5,667,988; and 5,885,793.

In addition to the use of library visualization, other methods can be used to obtain an antibody that binds to TWEAK. For example, TWEAK proiein or a peptide therefrom can be used as an antigen in a non-human animal, eg, a rodent, eg, a mouse, hamster, or rat.

In one embodiment, the non-human animal includes at least a portion of a human immunoglo'oulin gene. For example, it is possible to design mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using hybridoma technology, antigen-specific monoclonal antibodies derived from genes with the desired specificity can be produced and screened. See, for example, XENOMOUSE ™, Green et al. (1994) Nature Genetics 7: 13-21, US 2003-0070185, WO 96/34096, and WO 96/33735.

In another embodiment, a monoclonal antibody is obtained from the non-human animal, and modified, eg, humanized or des

<img file="MX339015B_D0031.tif" />

» . » · ⁴⁴ · 'IMPI immunized. Winter describes an example of métd ^ oi ^^ 'jjijgii ^ g ^ • “wnorrwAt that can be used to prepare humanized antibodies described here (US 5,225,539). All or some of the CDRs of a particular human antibody can be replaced with at least a part of a non-human antibody. It may only be necessary to replace the CDRs required for binding or binding determinants of these CDRs to arrive at a useful humanized antibody that binds to TWEAK.

Humanized antibodies can be generated by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences of human Fv variable regions. General methods for producing humanized antibodies are provided in Morrison, SL (1985) Science 229: 1202-1207, by O et al. (1986) BioTechniques 4: 214, and in US 5,585,089; US 5,693,761; US 5,693,762; US 5,859,205; and US 6,407,213. These methods include isolating, manipulating, and expressing nucleic acid sequences encoding all or part of the immunoglobulin Fv variable regions of at least one of a heavy or light chain. The sources of this nucleic acid are well known to those skilled in the art, for example, they can be obtained from a hybridoma that produces an antibody against a predetermined target, as described above, of immunoglobulin germline genes, as Rewritten formerly here, from Immunoglobulin germline genes, or from

<img file="MX339015B_D0032.tif" />

synthetic constructions. Recombinant DNA from humanized antibody can then be cloned into an appropriate expression vector.

Germline sequences, for example, are described in

Tomlinson, LA. et al. (1992) J. Mol. Biol. 227: 776-798; Cook, GP et al. (1995) Immunol. Today 16: 237-242; Chothia, D. et al. (1992) J. Mol. Bio. 227: 799-817; and Tomlinson et al. (1995) EMBO J 14: 46284638. The BASE V directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, LA. Et al. MRC Center for Protein Engineering, Cambridge, UK). These sequences can be used as a human sequence source, eg, for framework and CDR regions. Human framework consensus regions can be used for example, such as those described in US Patent No. 6,300,064.

A non-human antibody that urges TWEAK can also be modified by specific deletion of cell epitopes

Human T or deimmunization by the methods described in

WO 98/52976 and WO 00/34317. Briefly, the heavy and light chain variable regions of an antibody can be analyzed by peptides that bind to MHC Class II; these peptides represent potential T cell epitopes (as defined in WO 98/52976 and WO 00/34317). For the detection of potential T-cell epitopes, a computer modeling approach called peptide threading can be applied and in addition,

<img file="MX339015B_D0033.tif" />

to explore a database of MtS-CÍVá binding days.

MEXICAN INSTITUTE OF HUMAN PROPERTY to identify motives present in the seque^ors^{r, &lt;}V^LHv_L, as described in WO 98/52976 and WO 0Ό / 343Ι7. ΕΰΙϋΰ motifs bind to any of the 18 major DR allotypes of MHC class II, and thus constitute potential T-cell epitopes. Detected potential T-cell epitopes can be removed by substituting small amounts of amino acid residues in variable regions. , or preferably, by simple amino acid substitutions. As much as possible, conservative substitutions are made. Frequently, but not exclusively, a common amino acid can be used at one position in human germline antibody sequences. After deimmunization changes are identified, V-encoding nucleic acids can be constructed_H and V_L by mutagenesis or other synthetic methods (eg de novo synthesis, cassette replacement and so on). A mutagenized variable sequence can optionally be fused to a human constant region, eg, human lgG1 or kappa constant regions.

In some cases, a potential T-cell epitope will include residues that are known or predicted to be important to the function of the antibody. For example, potential T cell epitopes are usually diverted to CDRs. Furthermore, potential T cell epitopes can appear in framework residues that are important for antibody structure and binding. Changes to remove these potential epitopes in will require more scrutiny, for example, elaborating and¹»Cvcadenas with and without the change. When possible, potential T cell epitopes that overlap on CDRs can be removed by substitutions outside of CDRs. In some cases, an alteration within a CDR is the only option, and therefore variants with and without this substitution can be tested. In other cases, the substitution required to remove a potential T cell epitope is at a residue position within the framework that could be critical for antibody binding. In these cases, variants with and without this substitution are tested. Thus, in some cases, various heavy and light chain variable regions are designed, and various heavy / light chain combinations are tested to identify the optimal deimmunized antibody. The choice of the final de-immunized antibody can then be made considering the binding affinity of the different variants in conjunction with the extent of the deimmunization, particularly the amount of potential T-cell epitopes remaining in the variable region. Deimmunization can be used to modify any antibody, for example, an antibody that includes a non-human sequence, for example, a synthetic antibody, a murine antibody other than a non-human monoclonal antibody, or an antibody isolated from a visualization library.

Other antibody methods can also be used.

<img file="MX339015B_D0034.tif" />

humanizing. For example, other methods may coniOTTupaeacAfa · PROPERTY

INDUSTRIAL 3-D structure of the antibody, frame positions that are in 3-D proximity to binding determinants, and immunogenic peptide sequences. See, eg, WO 90/07861; US Patent Numbers 5,693,762; 5,693,761; 5,585,089; 5,530,101; and 6,407,213; Tempest et al. (1991) Biotechnology 9: 266-271. Still another method is called humanization and is described, for example, in US 2005-008625.

The antibody can include a human Fe region, eg, a wild-type Fe region or a Fe region that includes one or more alterations. In one embodiment, the constant region is altered, eg, mutated, to modify the properties of the antibody (eg, to increase or decrease one or more of: Fe receptor binding, glycosylation of the antibody, the amount of cysteine ​​residues, effector cell function, or complement function). For example, the IgGI constant region may be mutated at one or more residues, for example, one or more residues 234 and 237. Antibodies may have mutations in the CH2 region of the heavy chain that reduce or alter effector function. , for example, Fe receptor binding and complement activation. For example, antibodies can have mutations such as those described in US Patent Nos. 5,624,821 and 5,648,260. Antibodies can also have mutations that stabilize the disulfide bond between the two heavy chains of an immunoglobulin, such as mutations in the hinge region of

<img file="MX339015B_D0035.tif" />

IgG4, as described in the technique (1993) Mol. Immunol. 30: 105-08). See

2005-0037000.

(for example, 4n «

INSTITUTO MEXICANO also, for example Vf&gt; fiávi'3 ^ í) S

<img file="MX339015B_D0036.tif" />

Affinity maturation

In one embodiment, an anti-TWEAK antibody is modified, for example, by mutagenesis, to provide a pool of modified antibodies. Modified antibodies are then evaluated to identify one or more antibodies that have altered functional properties (eg, improved binding, improved stability, reduced antigenicity, or increased in vivo stability). In one embodiment, deployment library technology is used to select or scan the pool of modified antibodies. Antibodies with higher affinity are then identified from the second library, for example, using more restrictive or more competitive binding and washing conditions. Other scanning techniques can also be used.

In some modalities, mutagenesis targets either known or likely to be at the binding interface. If, for example, the identified binding proteins are antibodies, then mutagenesis can be targeted to the CDR regions of the heavy or light chains as described herein. Furthermore, mutagenesis can be directed to framework regions close to or adjacent to CDRs, eg, framework regions, particularly within 10, 5, or 3 amino acids of a CDR junction. In the case of antibodies, mutagenesis can also be

V, · · ϊ limited to one or a few of the CDRs, for example

Or „-§ \ step improvements.

In one embodiment, mutagenesis is used to make a

ΓΟ MEXICAN OF INDUSTRIAL PROPERTY

<img file="MX339015B_D0037.tif" />

antibody more similar to one or more germline sequences. An example of a germline method may include: identifying one or more germline sequences that are similar (eg, most similar in a particular database) to the sequence of the isolated antibody. Mutations can then be made (at the amino acid level) in the isolated antibody, either incrementally. in combination or both. For example, a nucleic acid library is made that includes sequences that encode some or all possible germline mutations. Antibodies are then evaluated, for example, to identify an antibody that has one or more additional germline residues relative to the isolated antibody and that is still useful (eg, has functional activity). In one embodiment, as many germline residues as possible are introduced into an isolated antibody.

In one embodiment, mutagenesis is used to replace or insert one or more germline residues into a CDR line. For example, the germline residue in CDR may be from a germline sequence that is similar (eg, most similar) to the variable region being modified. After mutagenesis, the activity (eg, binding or other functional activity) of the antibody can be evaluated to determine whether the jl'd Ϊ rl residue or germline residues are tolerated. I pufe'd I'MFÍ

;. Mexican institute similar mutagenesis in the framework regions. “^ NwStÍiÍal

The selection of a germline sequence can be done in different ways. For example, a germline sequence can be selected if it meets previously determined criteria for selectivity or similarity, for example, at least some percentage identity, for example, at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity, relative to the donor non-human antibody. Selection can be performed using at least 2, 3, 5, or 10 germline sequences. In the case of CDR1 and CDR2, identification of a germline sequence may include selecting one of these sequences. In the case of CDR3, identification of a similar germline sequence may include selecting one of these sequences, but may include using two germline sequences that contribute separately to the amino terminal portion and the carboxy terminal portion. In other embodiments, more than one or two germline sequences are used, for example, to form a consensus sequence.

In other embodiments, the antibody can be modified to have an altered glycosylation pattern (ie, altered from the original natural glycosylation pattern). As used in this context, "altered" means that it has one or more deleted carbohydrate moieties, and / or that it has one or more glycosylation sites added to the original antibody. The addition of glycosylation sites

<img file="MX339015B_D0038.tif" />

Σί ..- S Λ1. ^,. ^ 5

<img file="MX339015B_D0039.tif" />

to the antibodies currently described can alter the amino acid sequence for consensus sequences at the glycosylation site; These techniques are well known in the art. Another means of increasing the amount of carbohydrate moieties in the antibodies is by chemical or enzymatic coupling of glycosides to the amino acid residues of the antibody. These methods are described, for example, in WO 87/05330, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem. 22: 259-306. The removal of any portions of carbohydrates present in the antibodies can be accomplished chemically or enzymatically as described in the art. (Hakimuddin et al. (1987) Arch. Biochem. Biophys. 259: 52; Edge et al. (1981) Anal. Biochem. 118: 131; and Thotakura et al. (1987) Meth. Enzymol. 138: 350). See, for example, US Patent No. 5,869,046 for a modification that increases half-life in vivo by providing a natural receptor binding epitope.

In one embodiment, an antibody has CDR sequences that differ only substantially from those of P2D10. Substantial differences include minor amino acid changes, such as 1 or 2 substitutions of any of commonly 5-7 amino acids in the sequence of a CDR, eg, a Chothia or Kabat CDR. Commonly an amino acid is replaced by a related amino acid that has similar charge, hydrophobic or stereochemical characteristics. These substitutions could be within the ordinary skills of a technician. Unlike in "SBwannra.xa: s *". Trt¿x.Tt. ·: -. · -----.-- »ϋ ·

<img file="MX339015B_D0040.tif" />

For CDRs, further framework region structure (FR) changes can be made without affecting adverg'SWfente · the binding properties of an antibody. Changes to TR · s include, but are not limited to, humanizing a non-human derived framework or stabilizing the binding site, for example, by changing the class or subclass of the constant region, changing specific amino acid residues that might alter a function effector such as Fe receptor binding (Lund et al. (1991) J. Immun. 147: 2657-62; Morgan et al (1995) immunology 86: 319-24), or by changing the species from which the constant region comes.

The anti-TWEAK antibody can be in the form of full length antibodies, or in the form of antibody fragments, eg, Fab, F (ab ') 2, Fd, dAb, and scFv fragments. Additional forms include a protein that includes a single variable domain, for example, a camel or camel domain. See, eg, US 2005-0079574 and Oavies et al. (1996) Protein Eng.

9(6):531-7.

Antibody production

Some antibodies, for example, Fab, can be produced in bacterial cells, for example, in E. coli cells. Antibodies can also be produced in eukaryotic cells. In one embodiment, the antibodies (eg, scFv) are expressed in a yeast cell, such as Pichia (see, eg, Powers et al. (2001) J Immunol Methods. 251: 123-35), Hanseula, or Saccharomyces.

In a preferred embodiment, the antibodies ^ JphÁiIeJ F; mexican institute

OF THE ΓΑΟΡΙΕΠΑΠ Qmammal cells. Examples of rfíWflffero host cells to express an antibody include Ováf'lü dé HálllStei Chino cells (CHO cells) (including CHO dhfr 'cells, described in Urlaub and Chasin (1980) Proc. Nati. Acad. Sci USA 77: 4216 -4220, DHFR selectable marker, for example, as described in Kaufman and Sharp (1982) Mol. Biol. 159: 601-621), lymphocytic cell lines, eg, NSO myeloma cells and SP2 cells, COS cells, and a cell from a transgenic animal, eg, a transgenic mammal. For example, the cell is a mammary epithelial cell.

In addition to the nucleic acid sequence encoding the diversified immunoglobulin domain, recombinant expression vectors may carry additional sequences, such as sequences that regulate vector replication in host cells (eg, origins of replication) and marker marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, eg, US Patent Nos. 4,399,216, 4,634,665, and 5,179,017). For example, commonly the selectable marker gene confers resistance to drugs, such as G418, hlgromicline, or methotrexate, in an amphtrione cell into which the vector has been introduced.

<img file="MX339015B_D0041.tif" />

In an example of a system for antibody expression, a recombinant expression vector that encodes both the strand

<img file="MX339015B_D0042.tif" />

antibody weighing as a light chain of a introduced into CHO dhfr 'cells by transfection «

calcium phosphate. Within the recombinant expression vector, the antibody's heavy and light chain genes are operably linked to enhancer / promoter regulatory elements (eg, derivatives of SV40, CMV, adenovirus, and the like, such as a CMV enhancer regulatory element. / AdMLP promoter or an SV40 enhancing regulatory / AdMLP regulatory element) to produce high levels of gene transfection. The recombinant expression vector also carries a DHFR gene, which allows selection of CHO cells that have been transfected with the vector using methotrexate selection / amplification. Selected transformant host cells are cultured to allow expression of the antibody heavy and light chains and the antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect host cells, select transformants, culture host cells, and recover antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a coupled protein A or protein G matrix.

For antibodies that include a Fe domain, the antibody production system preferably synthesizes antibodies in which the Fe region is glycosylated. For example, the FCj domain of IgG molecules is glycosylated at asparagine 297 in the 'iHmnii im domain CH2. This asparagine is the site for moeifi

<img file="MX339015B_D0043.tif" />

double-stranded oligosaccharides. It has been demonstrated that industrial glycosylation is required for effector functions mediated by Fcy receptors and Clq complement (Burton and Woof (1992) Adv. Immunol. 51: 1-84; Jefferis et al . (1998) Immunol. Rev. 163: 59-76). In one embodiment, the Fe domain is produced in a mammalian expression system that appropriately glycosylates the residue corresponding to asparagine 297. The Fe domain or other region of the antibody may also include other post-translational eukaryotic modifications.

Antibodies can also be produced by a transgenic animal. For example, US Patent No. 5,849,992 describes a method of expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a promoter specific for milk and nucleic acids that encode the antibody of interest and a signal sequence for secretion. The milk produced by the females of these transgenic mammals includes, secreted therein, the antibody of interest. The antibody can be purified from milk, or for some applications, it can be used directly.

Characterization

The binding properties of an antibody can be measured by any standard method, for example, one of the following methods: BIACORE ™ Enzyme Linked Immunosorbent Analysis (ELISA), Resonance Energy Transfer

<img file="MX339015B_D0044.tif" />

of Fluorescence (FRET), x-ray crystallography-ÁnAlííi Mexican institute OF PROPERTY sequence and scanning mutagenesis. The ability of a protein to inhibit one or more TWEaK activities can be evaluated in vitro or in an animal model of a trastowo, eg, a disorder described herein. Preferably, the antibody has a statistically significant effect indicating that the antibody inhibits one or more TWEAK activities.

In one embodiment, inhibition of TWEAK's ability to stimulate the production of lL-8, MMP-I, PGE2, IL-6, IP10, and RANTES in dermal fibroblasts is evaluated. See Chicheportiche et al. (2002) Arthritis Res. 4 (2): 126-133 to see the appropriate analysis conditions.

In another embodiment, an antibody is evaluated for its ability to inhibit TWEAK for stimulation of endothelial cell proliferation. See, for example, US 2003-0211993 which describes a proliferation assay (as well as other useful assays) as follows: HVECs are plated for 96-receptacle mlt clotting in subconfluence (4,000 cells per receptacle) and grown overnight in CS-medium. C without addition of supplier growth supplements. Media is replaced with full media, or with basal media. Cells are grown in basal media with or without

TWEAK (100 ng / ml), bFGF using a 1/500 to 1/1000 dilution of i growth supplement for bFGF (Clonetics) or 1 ng / ml (R&amp;D Systems), VEGF (10 ng / ml) or combinations of these factors.

í, pg / ml d-el AnltKajafcp &lt;

MEXICAN INSTITUTE OF PROPERTY .. t, INDUSTRIAL.

conTrof antibody. The 5% lasts fTS ΙΓ55 0Γ3ΰ V fe'é

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When indicated, it is also added is being tested or a cells are incubated at 37 ° C with CO₂ measured proliferation by pulsing with H-Thymidine during the last 10 hours of culture. Binding radioactivity in cells can be measured with a BETAPLATE ™ (EG&amp;G Wallac, Gaithersburg, Md.). A decrease in TWEAK-mediated proliferation or the combination of TWEAK and bFGF may indicate that the antibody is effective in blocking TWEAK activity.

Surface plasma resonance (SPR).

The binding interaction of a protein of interest and a target (eg TWEAK) can be analyzed using SPR. SPR or Blomolecular Interaction Analysis (BIA) detect biospecific interactions in real time, without marking any of the interacting agents. Changes in the mass on the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasma resonance (SPR)). Changes in refractivity generate a detectable signal, which is measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in US Patent No. 5,641,640; Raether (1988) Surface Plasmons Springer Verlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63: 2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5: 699-705 and resources on

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line provided by BIAcore International AB * (UJ

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The SPR information can be used to provide accurate and quantitative ÉNWWed of the dissociation constant on Oquilibrit »·· (Kd), and of the kinetic parameters, including K_encEndido and Kapagado, for attaching a biomolecule to a target.

Epitopes can also be directly mapped by evaluating the ability of different antibodies to compete with each other to bind TWEAK (eg, human TWEAK, particularly soluble human TWEAK) using BIAcore chromatographic techniques (Pharmacia BIAtechnoiogy Handbook, Epitope Mapping, Section 6.3.2 , (May 1994); see also Johne et al. (1993) J. Immunol. Meihods, 160: 191-198). Additional general guidance for evaluating antibodies can be found, for example, in Western detections and in immunoprecipitation analysis in Antlbodies: A Laboratory Manual, ed. by Harlow and Lañe, Coid Spring Harbor press (1988)).

Disorders Associated with TWEAK

An anti-TWEAK antibody (such as an antibody described herein) can be used to treat a variety of disorders, such as a disorder associated with TWEAK. For example, the antibody can be used to treat inflammatory, immune, or autoimmune disorders in patients, as well as neoplastic disorders. Examples of disorders associated with TWEAK include rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (including ulcerative colitis, and

Crohn's), psoriasis, or inflammatory myositis. Still

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Treatable inflammatory disorders include his'Fociíosiscte · Langerhans cells, TTS-CHST syndrome, Respiratory / broncholysis, obliterans in adults, Wegener's granulomatosis, vasculitis, cachexia, stomatitis, diopatic pulmonary fibrosls, dermatomyositis or polymyositis, scleritis non-infectious, chronic sarcoidosis with pulmonary involvement, myelodysplastic syndromes / refractory anemia with excess blasts, ulcerative colitis, severe chronic obstructive pulmonary disease, and giant cell arteritis.

A subject who is at risk, or who has been diagnosed with, or has one of these disorders, may be administered an anti-TWEAK antibody in an amount and for a time to provide a full therapeutic effect. The anti-TWEAK antibody can be administered alone or in combination with other agents. For example, USSN 60 / 679,518 describes methods of administering a TWEAK blocking agent in combination with a TNF-α blocking agent. In the case of a combination therapy, the amounts and times of administration may be those that provide, for example, a synergistic therapeutic effect. Additionally, administration of TWEAK blocking agent (with or without the second agent) can be used as a primary treatment, for example, first-line treatment, or as a secondary treatment, for example, for subjects who have an inadequate response to a previously administered therapy (i.e., a therapy other than one with a TWEAK).

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Rheumatoid arthritis (RA)

An anti-TWEAK antibody (such as an antibody described ,, here) can be used to treat rheumatoid arthritis and related disorders. Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes pain, swelling, stiffness, and loss of function, primarily in the joints. RA frequently begins in the synovium, the membrane that surrounds a joint creating a protective sac. In many individuals with RA, leukocytes leak from the circulation into the synovium, causing continuous abnormal inflammation (eg, synovitis). Consequently, the synovium becomes inflamed, causing heating, redness, swelling, and pain. Collagen in cartilage is gradually destroyed, narrowing the joint space and eventually damaging the bone. The inflammation causes erosive damage to the bone in the affected area. During this process, the synovium cells grow and divide abnormally, making the synovium normally thin, thick, and producing a swollen, spongy joint to the touch.

As RA progresses, abnormal synovial cells can invade and destroy cartilage and bone within the junction. The muscles, ligaments, and circulating tendons that support or stabilize the joint can become weak and unable to work normally. RA can also produce more generalized bone loss that can lead to osteoporosia.

MEXICAN INSTITUTE fragile bones and more prone to fracture. All é ^ Tás? SS&gt; ®ctoS ^ ES ^ produce pain, disability, and rififnrmiriarifis asnciarias nnn RA. The regions that may be affected include the wrists, knuckles, knees, and the foot joint. Often, many joints can be involved, and even the spine can be affected. In about 25% of people with RA cnn, inflammation of small blood vessels can cause rheumatoid nodules, or lumps under the skin, that often form near the joints. As the disease progresses, fluid can also accumulate, particularly in the ankles. Many RA patients also develop anemia, or a decrease in the normal number of red blood cells.

RA comprises a number of disease subtypes, such as Felty's Syndrome, seronegative RA, "classic" RA, progressive and / or relapse RA, and RA with vasculitis. Some experts classify the disease as type 1 or type 2. Type 1, the least common form, lasts for a few months, mostly, and leaves no permanent disability. Type 2 is chronic, and lasts for years, sometimes a lifetime. RA can also manifest as subcutaneous rheumatoid nodules, visceral nodules, vasculitis that produces multiple leg ulcers or mononeuritis, pleural or pericardial effusions, lymphadenopathy, Felty's syndrome, Sjogi ^ en syndrome, and episcleritls. These disease subtypes and also subjects displaying one or more of the above symptoms can be treated using the antibodies described here.

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RA can be evaluated by a vafl'ÉM'á¹# clinical measurements. Some examples of indicative error include the Total Sharp Score (TSS), Sharp Erosion Score, and the HAQ Capacity Index. The methods in this document can be used to achieve improvement for at least one of these indicators. The therapeutic properties of an antiTWEAK antibody to treat RA can be evaluated in an animal model, for example, using the mouse collagen Induced Arthritis (mCIA) piodel (see for example, Stuart et al., J. Clin, invest. 69: 673-683 (1982).

Multiple sclerosis

An anti-TWEAK antibody (such as an antibody described here) can be used to treat multiple sclerosis (MS) and related disorders. MS is a disease of the central nervous system characterized by inflammation and loss of myelin sheaths.

Patients who have MS can be identified by criteria that establish a clinically determined diagnosis of MS in the workshop on the diagnosis of MS (Poser et al., Ann. Neurol. (1983) 13: 227). In summary, an individual with clinically determined MS who has had two seizures and clinical evidence of either injury or clinical evidence of one injury and paraclinical evidence of another separate injury. Determined MS can also be diagnosed by evidence of two attacks and oligoclonal bands of IgG in the cerebrospinal fluid or by combination of a clinical attack of two lesions and oligoclonal band of

<img file="MX339015B_D0050.tif" />

cerebrospinal. The effective treatment of multiple sclerosis can be examined in several different ways. The following parameters can be used to measure the effectiveness of the treatment. Three main criteria are used; EDSS (Extended Disability Status Scale), exacerbation, or MRI (magnetic resonance imaging). EDSS is a means of graduating clinical disability due to MS (Kurtzke (1983) Neurology 33: 1444). Eight functional systems are evaluated for the type and severity of nsurological disability. Briefly, prior to treatment, patients are evaluated for disability in the following systems: pyramidal, cerebellum, brainstem, sensory, bowel and bladder, visual, cerebral, and others. Follow-ups are performed at defined intervals. The scale ranges from 0 (normal) to 10 (death due to MS). A decrease in EDSS indicates effective treatment (Kurtzke (1994) Ann. Neurol. 36: 573-79).

An example of an animal model for multiple sclerosis is the mouse autoimmune encephalitis (EAE) model, for example, that described in Tuohy et al. (J. Immunol. (1988) 141: 1126-1130), Sobel et al. (J. Immunol. (1984) 132: 2393-2401), and Traugott (Cell Immunol. (1989) 119: 114-129). Mice may be given an antibody described here prior to EAE induction. Mice are evaluated for

ΪΜΡΤ. ,. j .. -, MEXICAN INSTITUTE characteristics to determine the efficacy of the antibody »* ^ ™dap '* ™'^Γ IKT

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Cerebrovascular accident

An anti-TWEAK antibody (such as an antibody described here) can be used to treat a subject who has experienced a stroke, eg, a thromboembolic or hemorrhagic stroke (eg, within the first 48, 24 , 12, 8, or 2 hours), or to prevent a stroke, for example, in a subject who is at risk of having a stroke. Examples of methods are described in USSN 60 / 653,811. Stroke is a general term for acute brain damage that results from disease of the blood vessels. Stroke can be classified into at least two main categories: hemorrhagic stroke (resulting from blood leaking out of normal blood vessels) and ischemic stroke (cerebral ischemia due to lack of blood supply). Some events that can cause ischemic stroke include thrombosis, embolism, and systemic hypoperfusion (with ischemia and resulting hypoxia).

Stroke causes neuronal death and brain damage from oxygen deprivation and side events. The area of ​​the brain that dies as a result of a lack of blood supply or other damage is called a heart attack. In some cases, the treatments described here can be used to reduce

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or minimize the size of a heart attack, for example, re'dujfe | l ^,. ,,, _ MDFTCANO INSTITUTE secondary that cause death or neuronal damage. I5ff LA -PRÓPiEDAO * rNPUSTWAL

Blockage of a cerebral artery that results from a thrombus that has formed in the wall of a cerebral artery is generally called cerebral thrombosis. In cerebral embolism, occlusive material that blocks the cerebral artery appears downstream in the circulation (for example, an embolus that is brought into the cerebral artery from the heart). Because it is difficult to discern whether a stroke from thrombosis or embolism, the term thrombus embolism is used to cover both of these types of stroke. Systemic hypo perfusion may appear as a consequence of decreased blood levels, decreased hematocrits, low blood pressure, or the heart's inability to pump blood properly.

Additionally, an anti-TWEAK antibody can be administered as therapy.

Neuronal disorders

An anti-TWEAK antibody (such as an antibody described here) can be used to treat or prevent neuronal disorders, such as neuronal mechanical trauma and neurodegenerative disorders. Examples of mechanical trauma include spinal cord damage (SCI) and traumatic brain damage (TBI). Examples of neurodegenerative disorders include amlotropic lateral sclerosis (ALS), progressive bulbar palsy (PBP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), · '' Parkinson's disease,

MEXICAN INSTITUTE Alzheimer's disease. See, for example, US.SN.

One modality, neuronal disorder is mnrtnrbnrin— primarily by destruction or death of nerve cells, eg, motor neurons (eg, ALS), basal ganglia striatal neurons, and / or cortical neurons (eg, Huntington's disease), from substantia neurons (eg, Parkinson's disease).

Cancer

TWEAK and its receptors may be involved in the development of at least some types of cancer, for example pancreatic cancer. An anti-TWEAK antibody (such as an antibody described here) can be used to treat or prevent cancers (eg, adenocarcinomas) and other neoplastic disorders. See, for example, TREATMENT OF CANCER, USSN 60 / 685,465, filed on May 27, 2005.

Pharmaceutical compositions

An anti-TWEAK antibody (such as an antibody described herein) can be formulated as a pharmaceutical composition for administration to a subject, for example, to treat a disorder described herein. Commonly, a pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, pharmaceutically acceptable carrier includes any and all post solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like, which are physiologically co

<img file="MX339015B_D0053.tif" />

Composition may include a pharmaceutically acceptable salt, eg, an acid addition salt or a base addition salt (see eg, Berge, SM, et al. (1977) J. Pharm. Sci. 66: 119).

Pharmaceutical formulation is a well-established technique, and is further described in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20^to ed., Lippincott, Williams &amp; Wilkins (2000) (ISBN · 0683306472), Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^to Ed., Lippincott Williams &amp; Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3^to ed. (2000) (ISBN: 091733096X).

Pharmaceutical compositions can take a variety of forms. These include, for example, semi-solid and solid liquid dosage forms, such as liquid solutions (eg, injectable and infusible solutions), dispersions or suspensions, tablets, pills, iposome powders, and suppositories. The preferred form may depend on the form of administration and therapeutic application to which it is directed. Commonly, the compositions for the agents described herein are in the form of injectable or infusible solutions.

In one embodiment, the anti-TWEAK antibody is formulated with excipient materials, such as sodium chloride, dibasic sodium phosphate heptahydrate, monobasic sodium phosphate, and a stabilizer. This is buffered solution in stored at 2-8 ° C.

can provide for Τ'ΜΦϊ

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These compositions can be administered parenterally (eg, intravenous, subcutaneous, intraperitoneal, or intramuscular injection). The terms parenteral administration and parenterally administered as used herein mean different forms of administration of enteric and topical administration, usually by injection, and include, without limitation, Intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal. subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intra-sternal injection and infusion.

The composition can be formulated as a solution, microemission, dispersion, liposome, or other ordered structure suitable for stable storage at a high concentration. Sterile injectable solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating an agent described herein into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of

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sterile powders for the preparation of serum ^ o ^^

INSTITUTO MEXICANO injectables, preferred preparation methods

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vacuum and freeze-drying producing a pnlvn Hp an ogantat described here plus any desired additional ingredients from a previously filtered sterile solution of it. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of t &amp; nsoactive agents. Prolonged absorption of injectable compositions can be increased by including in the composition an agent that delays absorption by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

In certain embodiments, the anti-TWEAK antibody can be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems. It can be used: biocompatible, biodegradable polymers, such as vinyl acetate and ethylene, poly anhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Many methods of preparing these formulations are proprietary or generally known. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed.,

Marcel Dekker, Inc., New York (1978). )

An anti-TWEAK antibody can be modified, for example,

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5, 10, or 50 times. The antibody to determine if it can reach ..

With a portion that improves your circulation, for example in the example, by at least 1.5, 2, modified inflammation sites can be evaluated, for example, joints.

For example, the anti-TWEAK antibody can be associated (eg, conjugated) with a polymer, eg, a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyjetylene oxide. Appropriate polymers will vary substantially by weight. Polymers with number average molecular weights ranging from about 200 to about 35,000 Daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used.

For example, the anti-TWEAK antibody can be conjugated to a water soluble polymer, eg, a hydrophilic polyvinyl polymer, eg, polyvinyl alcohol or polyvinylpyrrolidone. Examples of these polymers include polyalkylene oxide homopolymers, such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylene polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene; polymethacrylates; branched or unbranched carbomeres and polysaccharides. IMPI

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In some embodiments, the anti-TWEAi ^ antibody^Dufamb¡erT may be coupled to or if not associated with an efficient ΌΤδΠ otrtr agent, eg, another therapeutic agent such as a cytotoxic or cytostatic agent, however, in many embodiments, this configuration is unnecessary. Examples of cytotoxic and chemotherapeutic agents include taxol, cytochalasin B, gramicidin D, vinblastine, doxorubicin, daunorubicin, a maytansinoid (for example maytansinol or the maytansinoid DMI, a sulfhydryl containing maytaginine, mitoxantrinine, dehydrotestosterone, glucocorticoids, procaine, taxane, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologs.

When the anti-TWEAK antibody is used in combination with a second agent (eg, an anti-TNF-α antibody or other agent), the two agents can be formulated separately or together. Agents can be formulated or alternatively used in a synergistically effective amount. It is also possible to use one or both of the agents in smaller amounts than could be used for monotherapy. For example, the respective pharmaceutical compositions may be mixed, eg, just prior to administration, and may be administered together or separately, eg, at the same or different times.

It is also possible to use another TWiEAK blocking agent, for example, the agents described in USSN 60 / 679,518. The agent

<img file="MX339015B_D0057.tif" />

can administer to a subject. In one embodiment, the blocking agent is a biological protein, for example, a protein having a molecular weight of between 5-300 kDa. For example, a TWEAK blocking agent can inhibit the binding of TWEAK to the TWEAK receptor. Examples of TWEAK blocking agent, other than antibodies that bind to TWEAK, include antibodies that bind to TWEAK-R and soluble forms of TWEAK-R (eg, Fn 14) that compete with TWEAK-R on the cell surface. to join TWEAK. Other therapeutic agents described herein may also be provided as a pharmaceutical composition, for example, by standard methods or methods described herein.

Administration

The anti-TWEAK antibody can be administered to a subject, eg, a human subject, by a variety of methods. For many applications, the route of administration is one of: Intravenous (IV) injection or infusion, subcutaneous (SC) injection, intraperitoneally (IP), or intramuscular injection. It is also possible to use an intra-articular supply. Other modes of parenteral administration can also be used. Examples of these modes include: intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and epidural and intrasternal injection. In some cases, administration may be directly to a site of inflammation, for example,

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and another inflamed site.

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The route and / or mode of administration of the antibody can also be adjusted for the individual case, for example, by monitoring the subject, for example, using tomographic imaging, neurological examination, and standard parameters associated with the particular disorder, for example. , criteria for evaluating rheumatoid arthritis.

The antibody can be administered as a fixed dose, or in a mg / kg dose. The dose can also be chosen to reduce or to avoid the production of antibodies against the anti-TWEAK antibody. Dose regimens are adjusted to provide the desired response, eg, a therapeutic response or a combinatorial therapeutic effect. Generally, doses of the anti-TWEAK antibody (and optionally a second agent) can be used in order to provide a subject with an agent in bioavailable amounts. For example, doses of 0.1-100 mg / kg, 0.5-100 mg / kg, 1 mg / kg -100 mg / kg, 0.5-20 mg / kg, 0.1-10 mg / kg, or 1-10 can be administered. mg / kg. Other doses can also be used.

The dosage unit form, or "fixed dose" as used herein, refers to physically appropriate discrete units as unit doses for the subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the

<img file="MX339015B_D0059.tif" />

pharmaceutical carrier required and optionally in the other agent. Can single or multiple doses be administered? Alternatively, or in addition, the antibody may be administered by continuous infusion.

A dose of anti-TWEAK antibody can be administered, for example, at a Periodic Interval for a period of time (a course of treatment) sufficient to comprise at least 2 doses, 3 doses, 5 doses, 10 doses, or more, per For example, once or twice a day, or approximately one to four times a week, or preferably weekly, biweekly, monthly, for example, for between approximately 1 and 12 weeks, preferably between 2 and 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. Factors that may influence the dose and schedule required to effectively treat a subject include, for example, the severity of the disease or disorder, formulation, route of administration, previous treatments, general greeting, and / or age. ciei subject, and other diseases present. Furthermore, treatment of a subject with a therapeutically effective amount of a compound may include a single treatment, or preferably may include a series of treatments. Animal models can also be used to determine a useful dose, for example, a starting dose of a regimen.

If a subject is at risk of developing a disorder

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inflammatory or other disorder described here, the anticuJrJVÍsí ^ í¹

INSTITUTO MEXICANO DE Ι.Λ PROPERTY administered before the total onset of the disorder, for example^,| 5t € f, '^or'eo a preventive measure. The duration of this provontive treatment · may be a single dose of the antibody or the treatment may be continued (eg multiple doses). For example, a subject who is at risk for the disorder or who is predisposed to the disorder may be treated with the antibody for days, weeks, months, or even years in order to prevent the disorder from occurring or fulminant.

A pharmaceutical composition can include a therapeutically effective amount of an agent described herein. These effective amounts can be determined based on the effect of the agent administered, or the combinatorial effect of agents if more than one agent is used. A therapeutically effective amount of an agent may also vary according to factors such as the individual's disease status, age, sex, and weight, and the ability of the compound to elicit a desired response in the individual, eg, improvement of at least an improvement parameter of at least one symptom of the disorder. A therapeutically effective amount is also one in which any toxic effects of the composition are outweighed by the therapeutically beneficial effects.

Therapy devices and equipment

Pharmaceutical compositions that include the anti-TWEAK antibody can be administered with a medical device. The device can be designed with characteristic

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OF INDUSTRIAL PROPERTY

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Ability to be portable, storage at room temperature, and ease of use, such that it can be used in emergency situations, for example, by a non-subject. trained or by emergency personnel in the field, taken from medical facilities and other medical equipment. The device can include, for example, one or more housings for storing pharmaceutical preparations that include anti-TWEAK antibody, and can be configured to deliver one or more dosage units of the antibody. The device can be further configured to administer a second agent, for example, an anti-TNF-α antibody either as a single pharmaceutical composition that also includes the anti-TWEAK antibody or as two separate pharmaceutical compositions.

For example, the pharmaceutical composition can be administered with a needleless hypodermic injection parrt device, such as the devices described in US 5,399,163; 5,383,851; 5,312,335;

5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples and well known implant modules include: US 4,487,603, which describes an implantable micro fusion pump for dispensing medication at a controlled rate; US 4,486,194, which describes a therapeutic device for administering medications through the skin; US 4,447,233, which describes a drug infusion pump for delivering drug at a precise infusion rate; US 4,447,224, which describes an apparatus for

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variable flow implantable infusion; US 4,439,196, qu £ iVLcrjO ^.¹-¹

MEXICAN INSTITUTE OF PROPERTY osmotic drug delivery system with compartment wl ^ or multiple chambers; and US 4,475,196, which describe my osiotoma of osmotic drug delivery. Many other devices, implants, delivery systems, and modules are also known.

An anti-TWEAK antibody can be provided in a kit. In one embodiment, the kit includes (a) a package containing a composition that includes the anti-TWEAK antibody, and optionally (b) information material. The information material may be descriptive, instructional, marketing, or other material that relates to the methods described here and / or the use of the agents for therapeutic benefit.

In one embodiment, the kit also includes a second agent for treating an inflammatory disorder, for example, an anti-TNF-α antibody. For example, the kit includes a first container containing a composition that includes the anti-TWEAK antibody, and a second container that includes the second agent.

The information material of the cases is not limited in form. In one modality, matter! Information may include information on the production of the compound, molecular weight of the compound, concentration, expiration date, batch or production site information, and so on. In one embodiment, the information material refers to methods of administering the anti-TWEAK antibody, eg, in an appropriate dose, dosage form, or mode of administration (eg, one dose, pari form, MaPI

MEXICAN INSTITUTE OF PROPERTY of a 'fPgratOrn

<img file="MX339015B_D0064.tif" />

Information possible.

dose, or mode of administration described here) subject who has had or is at risk for inflammatory, or other disorder described here. Provided in a variety of formats, include printed text, computer-readable material, video recording, or audio recording, or information that provides a link or address for substantive material, for example, on the internet.

In addition to the antibody, the composition in the kit can include other ingredients, such as a solvent or buffer, a stabilizer or a preservative. The antibody can be provided in any form, for example, in liquid, dry or lyophilized form, preferably substantially pure or sterile. When the agents are provided in a liquid solution, the liquid solution is preferably an aqueous solution. When the agents are provided as a dry form, reconstitution is generally by the addition of an appropriate solvent. The solvent, for example, sterile water or buffer, can optionally be provided in a kit.

The kit may include one or more containers, for the composition or compositions containing the agents. In some embodiments, the kit contains separate containers, dividers, or compartments for composition and information material. For example, the composition may be contained in a bottle, vial, or syringe, and the information material may be contained in a plastic sleeve or package. In others

<img file="MX339015B_D0065.tif" />

modalities, the separate elements of the estaniaanoenj i team within a single undivided container. For example, the cord ^ ó ^ T ^ lórT is contained in a bottle, vial, or syringe, and - σΙ material tfs ”information in the form of a label. In some embodiments, the kit includes a plurality (eg, a package) of individual containers, each with one or more dosage unit forms (eg, a dosage form described herein) of the agents. The containers may include a combination dose unit, for example, a unit that includes both the anti-TWEAK antibody and the second agent, for example, in a desired ratio. For example, the kit includes a plurality of syringes, ampoules, foil packages, ampoule packages, or medical devices, for example, each with a single combination dose unit. The containers of the cases can be hermetic, waterproof (for example, impervious to changes in humidity or evaporation), and / or opaque.

The kit optionally includes an appropriate device for administration of the composition, eg, a syringe or other appropriate delivery device. The device may be provided pre-loaded with one or both of the agents, or it may be empty, but be suitable for loading.

Aiming of cells expressing TWEAK

The anti-TWEAK antibodies described herein can be used to target a charge to a TWEAK-expressing cell or to a tissue or other structure associated with TWEAK. For example, the antibodies can be linked to a virus or to a virus that can supply an exogenous gene (ípoi ^^ gSj ^ 'SnAifc

INDUSTRIAL gene therapy) or to a liposome, for example, a liposome that

<img file="MX339015B_D0066.tif" />

encapsulates a therapeutic agent or an exogenous gene. An example method for using an antibody to target a virus is described in Roux et al. (1989) Proc Nati Acad Sci USA (1989) 86: 9079-9083. See also, for example, Curr Gene Ther. (2005) 5: 63-70 and Hum Gene Ther. (2004) 15: 1034-1044.

The anti-TWEAK antibodies of this invention can also be linked to liposomes that contain a therapeutic agent, such as chemotherapeutic agents. Antibody binding to liposomes can be accomplished by any known crosslinking agent, such as heterobifunctional crosslinking agents that have been widely used to couple toxins or chemotherapeutic agents to antibodies for targeted delivery. For example, conjugation to liposomes can be performed using the 4 (4-maleimidophenyl) butyric acid hydrazide carbohydrate crosslinking reagent (MPBH) (Duzgunes et al. (1992) J. Cell. Biochem. Abst. Suppl. 16E 77) . Liposomes containing antibodies can also be prepared by well-known methods (see for example DE 3,218,121; Epstein et al. (1985) Proc. Nati. Acad. Sci. USA, 82: 3688-92; Hwang et al. (1980 ) Proc. Nati. Acad. Sci. USA, 77: 4030-34; US 4,485,045 and 4,544,545).

Uses for diagnosis

Anti-TWEAK antibodies can be used in a method of

<img file="MX339015B_D0067.tif" />

diagnosis to detect the presence of an example TWEA, a biological sample, such as tissue, biopsy) (eg, in vivo imaging in 'Oii sujelcr). For example, one can administer human or indeed human anti-TWEAK antibodies to a subject to detect TWEAK within the subject. For example, the antibody may be labeled, for example, with a detectable MRI tag with a radio tag. The subject can be evaluated using a means to detect the detectable tag. For example, the subject can be screened to assess the location of the antibody within the subject. For example, the subject is subjected to imaging, for example, by

NMR or other means.

Examples of useful diagnostic imaging tags include radio tags such as ¹³¹1, ¹¹¹ln, ¹²³1, ^99rnTc, ³²P, ³³P, ¹²⁵1, ³H ¹⁴C, and ¹⁸⁸Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron-emitting isotopes detectable by a positron emission tomography (PET) scanner, chemiluminescents such as luciferin, and enzyme labels such as peroxidase or phosphatase. Short range radiation emitters, such as isotopes detectable by short range detector probes, can also be used. The protein ligand can be labeled with these reagents using known techniques. For example, see Wepsel and Meares (1983) Radioimmunoimaging and Radioimmunotherapy, Elsevler, New York ________ItJvo

MEXICAN INSTITUTE OF THE fROF £ DAn

<img file="MX339015B_D0068.tif" />

to review techniques related to antibody labeling, and Colcher et al. (1986) Meth. Enzymol. 121: δΟ ^ - '^ ΡΓ #¹

The subject can be subjected to VIVU imaging using known techniques, such as radionucle-a + scanning using for example a gamma camera or emission tomography. See, for example, AR Bradwell et al, Developments in Antibody Imaging, ”Monoclonal Antibodies for Cancer Detection and Therapy, RW Baldwin et al., (Eds.), Pp 65-85 (Academic Press 1985). Alternatively, a scanner with positron emission transaxial tomography, such as the so-called Pet VI located at Brookhaven National Laboratory, can be used when the radioettle emits positrons (eg, ¹¹C, ^1SF, ¹⁵O, and ¹³N).

MRI contrast agents

Magnetic resonance imaging (MRI) uses NMR to visualize internal characteristics of the living subject, and is useful for prognosis, diagnosis, treatment, and surgery. MRI can be used with radioactive tracer compounds for obvious benefit. Some MRI techniques are summarized in EPO 502 814 A.

Generally, differences related to the relaxation time constants T1 and T2 of water protons in different environments are used to generate an image.

Differences in these relaxation time constants can be enhanced by contrast agents. Examples of these contrast agents include a number of magnetic agents, paramagnetic agents (which primarily alter IT), and

<img file="MX339015B_D0069.tif" />

Ferro magnetic or super paramagnetic agents primarily alter the T2 response). _____ (for example, EDTA, DTPA, and NTA chelates) can be bound (and roduoir · the toxicity of) of some paramagnetic substances (for example, Fe³⁺, Mn²⁺, Gd³⁺). Other agents may be in the form of particles, for example, from less than 10 pm to about 10 nm in diameter).

The particles can have ferromagnetic, anti ferromagnetic, or super paramagnetic properties. Particles can include, for example, magnetite (Fe₃OR₄), and-Fe₂OR₃, ferrites, and other magnetic mineral compounds of transition elements. The magnetic particles can include one or more magnetic crystals with and without non-magnetic material. The non-magnetic material can include synthetic or natural polymers (such as sepharose, dextran, dextrin, starch, and the like).

Anti-TWEAK antibodies can also be labeled with an indicator group containing the atom ¹⁹NMR-active F or a plurality of these atoms as long as (i) substantially all naturally occurring fluorine atoms are the isotope ¹⁹F and therefore, substantially all fluorine-containing compounds are NMR active; (ii) many polyfluorinated active compounds such as trifluoroacetic anhydride are commercially available at relatively low cost, and (iii) many fluorinated compounds have been found medically acceptable for use in humans, such as perfluorinated polyethers used to transport oxygen as replacements. hemoglobin.

After allowing this incubation time, seJl¡ | ^ J? Ja ^ 2

MEXICAN INSTITUTE _Λ _, OF THE PROPERTY.

a full-body MRI using an apparatus such as urw ^ rteM-los * described by Pykett (1982) Scientific American, 346; .78- &amp; S — para. locate and form TWEAK images.

In another aspect, the invention provides a method of detecting the presence of TWEAK in an in vitro sample (eg, a biological sample, such as serum, plasma, tissue, biopsy). The method can be used to diagnose a disorder, for example, a disorder associated with immune cells. The method includes: (i) contacting the sample or a control sample with the anti-TWEAK antibody; and (i) evaluating the sample to determine the presence of TWEAK, for example, detecting the formation of a complex between the anti-TWEAK antibody and TWEAK, q detecting the presence of the antibody or TWEAK. For example, the antibody can be immobilized, for example, on a support, and retention of the antigen on the support is detected, and / or vice versa. A control sample can be included. A statistically significant change in complex formation relative to the control sample may be indicative of the presence of TWEAK in the sample. Generally, an anti-TWEAK antibody can be used in applications including fluorescence polarization, microscopy, ELISA, centrifugation, chromatography, and cell sorting (eg, fluorescence-activated cell sorting).

Example 1

The sequence of the variable domain of cadenarp \ W

<img file="MX339015B_D0070.tif" />

OF murine INDUSTRIAL PROPERTY, with the indicated CDR, is:

EVQLVESGGG LVRPGGSLKL FCAASGFTFS RYAMSWVRQS

PEKRLEWVAE

ISSGGSYPYY PDTVTGRFTI SRDNAKNTLY LEMSSLKSED

TAMYYCARVL

101 YYDYDGDRIE VMDYWGQGTA VIVSS (SEQ ID NO: 50)

This is a murine subgroup of the 3D heavy chain variable domain.

The sequence of! P2D10 light chain variable domain with underlined CDRs is:

DWMTQSPLS LSVSLGDQAS ISCRSSQSLV SSKGNTYLHW

YLQKPGQSPK

FLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVAAEDLGV

YFCSQSTHFP

101 RTFGGGTTLE IK (SEQ ID NO: 51)

This is a murine kappa 2 light chain subgroup.

The following human acceptor frameworks were chosen for the human heavy chain variable domain of subgroup 3 huP2D10: 56-84m (NCBI database access number GL33318898, Scamurra et al., Direct presentation). The sequence with underlined CDRs is as follows:

EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYWMSWVRQA PGKGLEWVAN ₍

IKQDGSEKYY VDSVKGRFTI SRDNAKNSLY LQMNSLRAED

TAVYYCARDP

101 MTTWKPSLA TNDYWGQGTL VTVSS (SEQ ID The variable domain sequence of caden-a slight fia

MEXICAN INSTITUTE

<img file="MX339015B_D0071.tif" />

subgroup 2 K107 (NCBI Gl database access number: 21669075, Akahori et al., direct submission), with underlined CDRs is:

DWMTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW

YLQKPGQSPQ

LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAFDVGV

YYCMQALQTP

101 LTFGGGTKVE IK (SEQ ID NO: 53)

In the human acceptor sequences shown, the CDRs (which are underlined) are of the same length and canonical classes as those in the muP2D10 variable domains.

The alignment of the murine PD210 heavy chain variable domains (above) and human acceptor 56-84m (below) (68.8% identical) is shown below:

EVQLVESGGGLVRPGGSLKLFCAASGFTFSEYAMSWVRQSPEKRLEWVAE 50

11111111111 i · I Η 11: | I !! l III || i 11111) -1 I 11) 11 1 EVQLVESGGGLVQPGGSLRLSCAASGFTrSSYWMSWVRQAPGKGLEWVAN 50 51 ISSGGSY PYYPDTVTGRFTIS RDNRKNTLYLEMSSLKS EDTAMYYCARVL 100

I IH ll 111111! I Η i I · H l · I · I! = · 1111 · I Η I 1 “

IKQDGSERYYVDSVKGRFTISRDNAKNSLYLQMRSLRAEPTAVYYCARDP 10 0 101 YYDYDGDRIEVMDYWGQGTAVIVSS 125 (SEQ ID NO: 54) 'ITIIÍH Mil

101 MTTWKPSLATNDYWGQGTLVTVSS 125 (SEQ ID NO: 55)

Shown below is the alignment of the murine acceptor P2D10 light chain variable domains (human arri K107 (bottom) (75.9% identical):

<img file="MX339015B_D0072.tif" />

DWMTQSPLSLSVSLGDQ715I5CRSSQSLVSSKGNTYJ..HWYLQKPGQSPK 50

DWMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLpWYLQKPGQSPQ 50

FLIYKVSMRPSGVPDRFSGSGSGTDPTLKISRVAABDLG'ZYFCSOSTHFP 100

one 1 lfl 1 I! IIII t 1 IIII) I i) II 1 1 I r ι | &gt; 111 1 'I »'

<img file="MX339015B_D0073.tif" />

101 RTFGGGTTLEIK 112 (SEQ ID NO: 56) lililí Ί II

101 LTFGGGTKVEIK 112 (SEQ ID NO: 57)

There are two versions of the huP2D10 light chain (L1 and L2). The antibody huP2D10-1 refers to an antibody that includes huP2D10 H1 and huP2D10 L1. The huP2D10-2 antibody refers to an antibody that includes huP2D10 H1 and huP2D10 L2.

The alignment of the human acceptor 56-84m (above) and the heavy chain variable domain huP2D10 H1 (below) is shown below:

III! 11111 Η 111! 11N 11 ¡U 11 lili I 111111111111111 Γ

EVQñVESGGGLVQPGGSLRLSCAASGFTFSRYaMSWROAPGKGLEWAE 50

IKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYY CARDE 100 i ii ii i-ι iiiiiiiimimiiiiiiiiiiiiimii

ISSGGSYPYYKDTVTGRFTISRDNAKRSLYLQMNSLRAEDTAVYYCARyL 100 101 MTTVVKPSLATNDYWGQGTLVTVSS 125 (SEQ ID NO: 58) = lllllllllllll

101 YYDYDGDRIEVMDYWGQGTLVTVSS 125 (SEQ ID NO: 59)

The CDRs are underlined. The huP2D 10 heavy chain is a straight CDR graft (i.e., there are no reverse mutations in the

IMPIfé

INSTITUTO MEXICANO V4 * del ac ^ WW) p% de slight.

DWMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDVIYLQKPGQSPQ 50 mm Ni-m mi mi m ii ii ii- ι i π iiiiiiiini

DWMTQSPLSLPVTPGEPASISCRSSQSLVSSKGNTYLHWYLQKPGQSPQ 50 51 LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP 10 0

NI III 11IIIIIIIIIII! 11IIIIIIIIIIIII11 N I- I fLIYKVSRRPSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYfCSQSTHFP 100

NO: 60)

NO: 61). The huP2D10 L1 light chain has two reverse mutations that are indicated in lowercase in the frame shown above · L46F in FR2 and Y87F in FR3.

The alignment of the human K107 acceptor (above) and the huP2D10 L2 light chain variable domain is shown below:

i

101

101

The CDRs are underlined. The huP2D10 L2 light chain is a straight CDR graft (without in-frame reverse mutations).

framework).

Below is the alignment of human K107 (above) and the variable domain huP2D10 L1 (below):

101 LTFGGGTKVEIK 132 (SEQ ID

I ¡I 11 N IIII

101 RTFGGGTKVEIK 112 (SEQ ID

The CDRs are underlined.

DWMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPO 5 0

IIIIIIIIIIIIII N II111N 1111 i I · I Ι ~ ΤΓΙ 11111 UNI

DW17QSPLSLPVTPGEPASISCRSSQSLVSSKGNTYLHWYL0KPG0SP0 50

LXilYLGSNRASGVPDRFSGSGSGTDFTIiKISRVEAEDVGVYYCMOALOTP 100

LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVBAEDVGVYYCSQSTHFP 100

LTFGGGTKVEIK 112 (SEQ ID NO: 62)

NINNNN

RTFGGGTKVEIK 112 (SEQ ID NO: 63)

This is an example of the heavy d amino acid sequence of mature lgG1 huP2D10 H1:

<img file="MX339015B_D0074.tif" />

EVQLVSSGGG LVQPGGSLRL SCAASGFTFS RYAMSWVRQA PGKGLEWVAE

ISSGGSYPYY PDTVTGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVL

101 YYDYDGDRIE VMDYWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL

151 GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSWTVPSSS

201 LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THTCPPCPAP ELLGGPSVFL ·

251 FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR

301 EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ 351 PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YP5DIAVEWE SNGQPNNKKHWVG DFFSSWLGDTV SFFLYSKLG

451 LSPG {SEQ ID NO: 64)

The Kabat numbering for the Vh segment of the heavy chain variable domain (SED ID NO: 65) is shown below:

Kabat No hP2D10

12345678^0

EVQLVESGGG

1234567830

LVQPGGSLRL

1234567830

SCAASGFTFS

1234567890

234567890

RYAMSWVRQA

PGKGLEWVAE

Kabat No. hP2D10

This is

12a3456789 0123456783 0123456789 012abc3456 78901234

ISSGGSYPYY PDTVTGRFTI SRDNAKNSLY. LQMNSLRAED KAVYYCAR an example of mature huP2D10 L1 light chain ae amino acid sequence:

DWMTQSPLS LPVTPGEPAS ISCRSSQSLV SSKGNTYLHW YLQKPGQSPQ

FLIYKVSNRF SGVPDRFSGS G3GTDFTLKI SRVSAEDVGV YFCSQSTHFP

101 RTFGGGTKVE IKRTVAAPSV FIPPPSDEQL KSGTASWCL LNNFYPREAK

151 VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YSKHKVYACS 201 VTHQGLSSPV TKSFNRGEC (SEQ ID NO: 66)

Kabat numbering for this segment V_L shown below (SEQ ID NO: 67):

IMPI

Kabat No hP2D10

1234567890

DWMTQSPLS

1234567890

LPVTPGEPAS

1234567abc

ISCRSSQSLV

MfXiCANO WLASOflEDAD INSTITUTE

INnuSTUAl de89012345 6789012345 SSKGNTYLHW YLQKPGQSPQ

<img file="MX339015B_D0075.tif" />

Kabat No hP2D10

6789012345

FLIYKVSNRF

6789012345

SGVPDRFSGS

6789012345

GSGTDFTLKI

6789012345 6789012345 SRVEAEDVGV YFCSQSTHFP

This is an example of the mature huP2D10 L2 light chain amino acid sequence:

<td> 1</td><td>DWMTQSPLS</td><td>LPVTPGEPAS ISCRSSQSLV SSKGNTYLHW</td><td>YLQKPGQSPQ</td>

<td> 51</td><td>LLIYKVSNRF</td><td>SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV</td><td>YYCSQSTHFP</td>

<td> 101</td><td>RTFGGGTKVS</td><td>IKRTVAAPSV FIFPPSDEQL KSGTASVVCL</td><td>LNNFYPREAK</td>

<td> 151</td><td>VQWKVDNALQ</td><td>SGNSQESVTE QDSKDSTYSL SSTLTLSKAD</td><td>YEKHKVYACE</td>

<td> 201</td><td>VTHQGLSSPV</td><td>TKSPNRGEC ISE-Q ID NO: 68)</td><td></td>

Kabat numbering for this segment V_L shown below (SEQ ID NO: 69):

Kabat No. hP2D10

1234557890

DWMTQSPLS

1234567890

LPVTPGEPAS

1234567abc

ISCRSSQSLV de89012345 € 789012345 SSKGNTYLHW YLQKPGQSPQ

Kabat No. hP2D10

6789012345

LLIYKVSNRF

6789012345

SGVPDRFSGS

6789012345

GSGTDFTLKI

6789012345

SRVEAEDVGV

6789012345

YFCSQSTHFP

Example 2

The monoclonal antibody that blocks TWEAK, mP2D10, significantly reduced clinical severity in models of multiple sclerosis, stroke, and rheumatic arthritis. The pharmacokinetics (PK) of the anti-TWEAK monoclonal antibody mP2D10, after intravenous (IV) administration was modeled.

IMPI

INSTITUT · MEXICANO I heard LA ItOPIEDAÍ »

<img file="MX339015B_D0076.tif" />

MP2D10 was administered to mice by IV injection cfeT ^ IO θ '100 mg / kg. Serum concentrations of 'mP2Di0 SU determined using ELISA. The PK time / concentration profile was analyzed using a two compartment model with first order deletion or Michaelis-Menten deletion from the central compartment with a volume of V1. The constant proportions between the two compartments were K12 (leaving compartment 1 towards 2) and K21 (leaving compartment 2 towards 1). For the first order elimination model, the constant elimination rate was K10. For the elimination model

Michaelis-Menten, the drug was eliminated at the rate of

Vm * C1 / (Km + C1), in which C1 was mP2D10 in the concentration in the central compartment, Vm and Km were constant. Data were adjusted with ADAPT II software (D'Argenio, DZ and A. Schumitzky. ADAPTII User's Guide: Pharmacokinetic / Pharmacodynamic Systems Analysis Software. Biomedical Simulations Resource, Los Angeles, 1997.) using the procedure for estimation of Maxima

Probability.

For the two-compartment linear elimination model, VI was 23.2 mL / kg, K10 was 0.0096 h-1, K12 was 2,501, and K21 was 1,053. The value of the area under the curve (AIC) was 298 and the Schwarz value was 304.2. For the two-compartment nonlinear elimination model, the V1 was 0.0235. The Vm was 9.22 mg / kg / h, the Km was 484.2 pg / mL. K12 was 2,348 h-1, and K21 was 0.966 h-1. The

<img file="MX339015B_D0077.tif" />

AlC value was 269 and Schwarz value

IMPI

MEXICAN INSTITUTE OF LA FROFIEDAI1

<img file="MX339015B_D0078.tif" />

mP2D10 was best predicted by a nonlinear model ifG '^ W'edianTé a linear model. ————- ~

The concentration-time profiles of mP2D10 were better predicted by a two-compartment model with Michaelis-Menten deletion than with first order deletion.

Those skilled in the art will recognize, or be able to determine, using nothing more than routine experimentation, many equivalents to the specific modalities described herein.

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