CA2890586A1

CA2890586A1 - Treatment of tnf.alpha. related disorders

Info

Publication number: CA2890586A1
Application number: CA2890586A
Authority: CA
Inventors: Subhashis Banerjee; Lori K. Taylor; Clive E. Spiegler; Daniel Edward Tracey; Elliot Keith Chartash; Rebecca S. Hoffman; William T. Barchuk; Philip Yan; Anwar Murtaza; Jochen G. Salfeld; Steven Fischkoff
Original assignee: AbbVie Biotechnology Ltd
Current assignee: AbbVie Biotechnology Ltd
Priority date: 2002-07-19
Filing date: 2003-07-18
Publication date: 2004-01-29

Abstract

Methods of treating TNF.alpha.-related disorders comprising administering TNF.alpha.
inhibitors, including TNF.alpha. antibodies are described.

Description

DENIA.NDES OU BREVETS VOLUNIINEUX
LA PRtSENTE PARTIE DE CETTE DENIA.NDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
CECI EST LE TOME DE a NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
THIS IS VOLUME _ OF ci NOTE: For additional volumes please contact the Canadian Patent Office.

TREATMENT OF TNITa RELATED DISORDERS
RELATED APPLICATIONS
10 In addition, this application is related to U.S. Patent Nos.
6,090,382, 6,258,562, and 6,509,015. This application is also related to -U.S.
Publication No.
2003-0092059 filed March 7, 2001; U.S. Publication No. 2003-0219438 filed November 22, 2002; U.S. Publication No. 2003-0235585 filed June 2, 2002; and U.S.
Publication No. 2003-0206898 filed April 26, 2002.
This application is related to U.S. utility applications (2004-0126372A1) entitled "Treatment of TNFa-Related Disorders Using TNFa Inhibitors," (2004-0131614A I) entitled "Treatment of Pulmonary Disorders Using TNFa Inhibitors,"

(2004-0126373A1) entitled "Treatment of Coronary Disorders Using TNFoi Inhibitors," (2004-0151722A1) entitled "Treatment of Metabolic Disorders Using TN-For Inhibitors," (2004-0136991A1) entitled "Treatment of Anemia Using TNFa Inhibitors," (2004-0136990A1) entitled "Treatment of Pain Using TNFa Inhibitors,"
(2004-0219142A1) entitled "Treatment of Skin and Nail Disorders Using TNFa Inhibitors," (2004-0136989A1) entitled "Treatment of Vasculitides Using TNFa Inhibitors," and (2004-0126372A1) entitled "Treatment of TNFa Related Disorders Using TNFce Inhibitors," all of which are filed on even date herewith.

BACKGROUND OF THE INVENTION
Cytoki nes, such as intcrleukin-1 (1L-1)and turner necrosis factor (TNF) are molecules produced by a variety of cells, such as monocytes and macrophages, which have been identified as mediators of infhumnatory processes. Cytokines, including TNP, regulate the intensity and duration of the inflammatory response which occurs as the result of an injury or infection. TNFa (also referred to as INF) has been implicated in the pathophysiology of a variety of human diseases and disorders, including sepsis, infections, autoimmune diseases, transplant rejection and graft-versus-host disease (see e.g., Moeller et al. (1990) Cytolcine 2:162; U.S. Patent No, 5,231,024 to IVIoeller et al.;
European Patent Publication No. 260 610 B1 by Moeller, A. et al.; Vasilli (1992)Annu.
Rev. intniunot. 10:411; Tracey and Ccrarni (1994) Aim. Rev. Med. 45:491).
SUMMARY OF THE INVENTION
There is a need to treat TNFa-related disorders, where TNI7cc activity is detrimental, in a safe and effective manner. The present invention includes methods for safe and effective treatment of TNFa-related disorders where TNFa activity is detrimental.
One aspect of the invention describes a method of treating a TNFa-related disorder in a subject comprising administering to the subject a therapeutically effective amount of a neutralizing, high affinity TNFa antibody, such that said disorder is treated.
hi one embodiment the TNI7a-related disorder is a spondyloarthropathy, a pulmonary disorder, a coronary disorder, a metabolic disorder, anemia, pain, a hepatic disorder, a skin disorder, a nail disorder, or vasculitis. In another embodiment, the TN-Fa-related disorder is age-related cachexia, Alzheimer's disease, brain edema, inflammatory brain injury, chronic fatigue syndrome, dermatomyositis, drug reactions, edema in and/or around the spinal cord, familial periodic fevers, Felty's syndrome, fibrosis,.

glomerulonephritides (e.g. post-streptococcal glomerulonephritis or IgA
nephropathy), loosening of prostheses, microscopic polyangiitis, mixed connective tissue disorder, multiple myeloma, cancer and cachexia, multiple organ disorder, myelo dysplastic syndrome, orchitism osteotysis, pancreatitis, including acute, chronic, and pancreatic abscess, periodontal disease polymyositis, progressive renal failure, psenclogout, pyoderrna gangrenostun, reiapsing polychonclritis, rheumatic heart disease, sarcoidosis,

- 2 -sclerosing cbolangitis, strolce, thoracoabdominal aortic aneurysm repair (TAAA), TNF
receptor associated periodic syndrome (TRAPS), symptoms related to Yellow Fever vaccination, inflammatory diseases associated with the ear, chronic ear inflammation, or pediatric ear inflammation. In still another embodiment of the invention, the TNFa-related disorder is a Crohn's disease-related disorder, juvenile arthritis/Still's disease (IRA), uveitis, sciatica, prostatitis, endornetriosis, choroidal neovascularization, lupus, Sjo glen's syndrome, and wet macular degeneration.
In one embodiment, the antibody of the invention is an isolated human antibody, or an antigen-binding portion thereof, that dissociates from human TNFa with a Kd of 1 x 10-8 M or less and a Koff rate constant of 1 x 10-3 s-1 or less, both determined by surface plasmon resonance, and neutralizes human TNFoc cytotoxicity in a standard in vi fro L929 assay with an IC56 of 1 x 10-7 M or less.
In another embodiment of the invention, the antibody is an isolated human antibody, or an antigen-binding portion thereof which dissociates from human TNFa with a Koffrate constant of 1 x 10-3 s-1 or less, as determined by surface plasmon resonance; has a light chain CDR3 domain comprising the amino acid sequence of SEQ
ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9; and has a heavy chain CDR3 domain coinprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 1.1 or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.
In another embodiment of the invention, the antibody is an isolated human antibody, or an antigen-binding portion thereof, with a light chain variable region (LCVR) cotnptising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region (11CVR) comprising the amino acid sequence of SEQ 1D NO: 2.
In a further embodiment of the invention, the antibody is D2E7, also referred to as HUMIRA or adalimumab.
Another aspect of the invention includes a method of treating a subject suffering froin a TNFa-related disorder comprising administering a therapeutically effective amount of a TNFa antibody, or an antigen-binding fragment thereof, to the subject, wherein the antibody dissociates from lumian TNFa with a Kd of 1 x 10-8 M or less and

- 3 -a Koff rate constant of 1 x 10-3 s-1 or less, both determined by surface plasmon resonance, and neutralizes human TNFa, cytotoxicity in a standard in vitro L929 assay with an 1050 of 1 x 10-7 M or less, such that said TNFa-related disorder is treated.
Still another aspect of the invention includes a method of treating a subject suffering from a TNFa-rclated disorder, comprising administering a therapeutically effective amount a TNFa antibody, or an antigen-binding fragment thereof, wherein the antibody dissociates from human TNFa with a Koff rate constant of 1 x 10-3 s-I
Or less, as determined by surface plasmon resonance; has a light chain CDR3 domain comprising the amino acid sequence of SEQ lD NO: 3, or modified frona SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9; and has a heavy chain CDR3 domain comprising the amino acid sequence of SEC? ID NO: 4, or mortified from SEQ ID
NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12, such that said TNFa-related disorder is treated.
A further aspect of the invention features a method of treating a subject suffering from a TNFa-related disorder, comprising administering a therapeutically effective amount a TNFa. antibody, or an antigen-binding fragnent thereof, with a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ
ID
NO: 2, such that said TNFa-related disorder is treated. In one embodiment, the TNFa antibody, or antigen binding fragment thereof, is D2E7. In another embodiment, the TNFor antibody is administered with at least one additional therapeutic agent.
Yet another aspect of the invention features a method for inhibiting human TNFa activity in a human subject suffering from a TNFa-related disorder, comprising administering a therapeutically effective amount of a TN-Fa antibody, or an antigen -binding fragment thereof, to the subject, wherein the antibody dissociates from human TNFcc with a Kd of 1 x 10-8 M or less and a Koff rate constant of 1 x 10-3 s-1 or less, both determined by surface plasmon resonance, and neutralizes htunan TNFa, cyto toxicity in a standard hi vitro L929 assay with an IC50 of 1 x 10-7 M or less. In one embodiment, the TNFa antibody, or antigen-binding fragment thereof, is D2E7.

-4-.

Yet another aspect of the invention includes a method of treating a subject suffering from a TNFet-related disorder, comprising administering a therapeutically effective amount of D2E7, or an antigen-binding fragment thereof, to the subject, such that the disease is treated.
Still another aspect of the invention includes a method of treating a subject suffering from a TNFa-rclated disorder, comprising administering a therapeutically effective amount of D2E7, or an antigen-binding fragment thereof, to the subject, such that the disease is treated in one embodiment of the invention, D2E7 (also referred to as HUMIRA. or adalirntunab) is administered with at Least one additional therapeutic agent.
Another aspect of the invention is a kit comprising a pharmaceutical coniposition comprising a TNFa antibody, or an antigen binding portion thereof, and a pharmaceutically acceptable carrier; and instructions for administering to a subject the 'INFcc antibody pharmaceutical composition for treating a subject who is suffering from a TNFa-related disorder. In one embodiment, the TNFcc antibody, or an antigen binding portion thereof, is D2E7 (I-IUMIRA ).
DETAILED DESCRIPTION OF THE INVENTION
This invention pertains to methods of treating TNFaTrelated disorders in which TNFa, activity, e.g., human TNFet activity, is detrimental. 'The methods include administering to the subject a therapeutically effective amount of a TNFa inhibitor, such that the TNFa-related disorder is treated. The invention also pertains to methods wherein the TNFa inhibitor is administered in combination with another therapeutic agent to treat a TNFa-related disorder. Various aspects of the invention relate to treatment with antibodies and antibody fragments, and pharmaceutical compositions comprising a TNFct inhibitor, and a pharmaceutically acceptable carrier for the treatrnent of TNFa-related disorders.

-5 -.Definitions ln order that the present invention may be more readily understood, certain terms are first defined.
The term "human TNFa" (abbreviated herein as laNFa., or simply liTNF), as used herein, is intended to refer to a human cytokine that exists as a 17 kD
secreted form and a 26 ItD membrane associated form, the biologically active form of which is composed of airimer of noncovalcntly bound 17 IcD molecules. The structure of hTNFo.
is described thither in, for example, Pennica, D., et al. (1984) Nature 312:724-729;
Davis, J.M., et al. (1987) 33iocheinisiry 26:1322-1326; and Jones, E.Y., et al. (1989) Nature 338:225-228. The term human TNFa is intended to include recombinant human TNFa. (rliTNFoc.), which can be prepared by standard recombinant expression methods or purchased commercially (R & D Systems, Catalog No. 210-TA, Minneapolis, MN).
TNFa is also referred to as INF.
The term "TNFa inhibitor" includes agents which inhibit TNFa. Examples of TNFa inhibitors include etanercept (Enbrel , Amgen), infliximab (Remicade , Johnson and Johnson), human anti-TNF nionoclonal antibody (D2E7/1-IUMI1iA , Abbott Laboratories), CDP 571 (Celltech), and CDP 870 (Celltech) and other compounds which inhibit TNFa .activity, such that when administered to a subject suffering nom or at risk of suffering from a disorder in which TNFa activity is denimental, tbe disorder is treated. In one embodiment, a TNFee, inhibitor is a compound, excluding etanercept and iniliximah, which inhibits TNFa activity. In another embodiment, the TNFa inhibitors of thc invention are used to treat a TNFa-related disorder, as described in more detail in section 11 In one embodiment, the TN-Fa inhibitor, excluding etanercept and infliximab, is used to treat a TN-Fa-related disorder. ìn another embodiment, the TNFa.
inhibitor, excluding etanercept and intliximab, is used to treat ankylosing spondylitis.
The term also includes each of the anti-TNFa human antibodies and antibody portions described herein as well as those described in U.S. Patent Nos. 6,090,382; 6,258,562;

6,509,015, and in U.S. Patent Application Serial Nos. 2003-009205 9 and 2003-0219438.
The term "antibody", as used herein, is intended to refer to irnmuneglobulin molecules comprised of four polypeptide chains, two heavy (IT) chains and two light (L) chains inter-connected by disulfide bonds. Bach heavy chain is comprised of a heavy chain variable region (abbreviated herein as 1-ICVR. or VII) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VII and VL regions can be further subdivided into regions of hyperv at-lability, termed complementarity determining regions (CDR), interspersed with regions that are MOM conserved, termed frameworlc regions (FR).
Each VI-I and VL is composed of three CDRs and four FRs, arranged from amino-tenninus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibodies oldie invention are described in further detail in U.S.
Patent Nos. 6,090,382; 6,258,562; and 6,509,015, and in U.S. Publication Nos.

. and 2003/0219438.
The teal' "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., hTNFa). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Exainples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;
(iii) a lid fragment consisting of the VII and CI-I1 domains; (iv) a Fv fragment consisting of the VL and VII domains of a single arm of an antibody, (v) a clAb fí-agment (Ward et al., (1989) Nature 341:544_5L16), which consists of a VII domain; and (vi) an isolated completnentarity detenitining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VII, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VII regions pair to form monovalent molecules (known as single chain Fv (scPwv); sec e.g., Bird et al. (1988) Science 242:423-'126; and Huston et al. (1988) Proc. Natl. Acad. Set, USA 85:5879-5883) . Such single chain antibodies are also intended to be encompassed within thc term "antigen--binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies are

- 7 -also encompassed. Diabodies are bivalent, bispecific antibodies in which VI-1 and VI, domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the sine chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) PrOC, Natl. Acad. Sci.
USA 90:6444-6448; Poljak, RI, et al. (1994) Structure 2:1121-1123). The antibody portions of the invention are described in fw.ther detail in U.S. Patent Nos. 6,090,382, 6,253,562, 6,509,015, and in U.S. :Publication Nos. 2003/0092059 and 2003/0219438.
Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact inummoglobnlins. Binding fragments include Fab, Fab', F(al12,Fabc, Fv, single chains, and single-chain antibodies. Other than "bispecific" Or "bifunctional" inummoglobulins or antibodies, arj immunoglobulin or antibody is understood to have each of its binding sites identical. A
"bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecifiu antibodies can be produced by a = variety of melhods including ftision of bybridomas or linking of Fab fragnents. See, e.g., Songsivilai & Lachmann, Clin. Exp. Inununol. 79:315-321 (1990); Kostelny et 01,, J. finnutnol. 148, 1547-1553 (1992).
= A "conservative amino acid substitution", as used herein, is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, senile, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, lencine, isoleucine, proline, phenylalanine, methionine, Uyptophan), beta-branched side chains (e.g., threonine, valine, isolencine) and aromatic side chains (e.g., tyrosine, phenylatanine, tryptophan, histidinc).
The term "limnan antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglohutin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by .3 -random or site-specitic mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector lransfected into a host cell (described Ihrther below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenie for human immunogiobulin genes (see = e.g., Taylor, L.D. et al. (1992)Nucl. Acids Res. 20:6287) or antibodies prepared,.
expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived fiorn human germline immunoglobulin sequences. In certain enibociiments, however, such recombinant human = antibodies are subjected to in vitro rnutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V1-1 and VL regions of the recombinant antibodies are sequences that, while derived from and related to hm-nan gennline VI-I and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds hTNFa is substantially free of antibodies that specificallybind antigens other than hTNFa). An isolated antibody that specifically binds liTNFa may, however, have cross-reactivity to other antigens, such as TNFa molecules from other species (discussed in thither detail below). Moreover, an isolated antibody may be substantially free of other-cellular material and/or chemicals.
A "neutralizing antibody", as used herein (or an "antibody that neutralized hTNFct activity"), is intended to refer to an antibody whose binding to hTNFa results in inhibition of the biological activity ofhTNFa. This inhibition of the biological activity of liTNFa can be assessed by measuring one or more indicators of hTNFot biological activity, such as I-ITN-Fa-induced cytotoxicity (either in vitro or in vivo), hTNFet-induced cellular activation and liTNPct binding to h'INFa receptors. These indicators of hTN-17,c( biological activity can be assessed by one or more of several standard in vitro or in vivo assays known in the art (see (J.S. Patent No. 6,090,382). Preferably, the ability of an antibody to neutralize hTNFcc activity is assessed by inhibition of liTNFa-induced cytotoxicity of L929 cells. As an additional or alternative parEuneter of liTNFoc activity, the ability of an antibody to inhibit hTNFec-induced expression of ELAM-1 on HUVEC, as a measure of hTNFa.-induced cellular activation, can be assessed.
The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecifie interactions by detection or alterations in protein concentrations within a biosensor matrix, for example using the BlAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For fitrther descriptions, see Example 1 ofU.S. Patent 6,258,562 and Jonsson et al.
(1993) Ann. Biol. Clin. 51:19; Jonsson et al. (1991) Biotechniques 11:620-627;
Tohnsson et al. (1995) .1. Mol. Recognit. 8:125; and Johnnson et al. (1991) Anal.
Biochem.198:268.
The term "Koff', as used herein, is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex.
The term "K.d", as sused herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction.
The term. "IC50" as used herein, is intended to refer to the concentration of the inhibitor required to inhibit the biological endpoint of interest, e.g., neutralize cytotoxicity activity.
The term "nucleic acid molecule", as used herein, is intended to include DNA
inolecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
The term "isolated nucleic acid molecule", as used herein in reference to nucleic acids encoding antibodies or antibody portions (e.g., VH, VL, CDR3) that bind liTNFa, is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than hTNEet, which other sequences may naturally Bank the nucleic acid in human genomic DNA.
Thus, for example, an isolated nucleic acid of the invention encoding a VII region of an anti-liTNI7cx antibody contains no other sequences encoding other VH regions that bind antigens other than liTNFa.
The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another micleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligatcd. Another type of vector is a viral vector, wherein additional DNA segnents may be ligated into the viral geoome, Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episornal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the gnome of a host cell upon introduction into the host cell, and thereby are replicated along with the host gcnome. Moreover, certain vectors are capable of dixecting the expression of genes to which they are operatively linked. Such vectors are referred to herein as ''recombinant expression vectors" (or simply, "expression vectors").
In =
general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmicls. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasraid is the most commonly used form of vector.
However, the invention is intended to include such other fonus of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may liot, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.
The term "closing", as used herein, refers to the administration of a substance (e.g., an anti-TN-Fa. antibody) to achieve a therapeutic objective (e.g,, the treatment of a TNFcc-associated disorder).

The terms "biweekly dosing regimen", "biweekly dosing", and "biweekly adniinistration", as used herein, refer to the time course of administering a substance (e.g., an anti-TNI-7a antibody) to a subject to achieve a therapeutic objective (e.g., the treatment of a TNFa-associated disorder). The biweekly dosing regimen is not intended to include a weekly dosing regimen. Preferably, the substance is administered every 9-19 days, more preferably, every 11-17 days, even more preferably, every 13-15 days, and most preferably, every 14 days.
The term "combination" as in the phrase "a first agent in combination with a second agent" includes co-administration of a first agent and a second agent, which for example may he dissolved or intermixed in the same pharmaceutically acceptable carrier, or administration of a first agent, followed by the second agent, or administration of the second agent, followed by the first agent. 'The present invention, therefore, includes methods of combination therapeutic treatment and combination pharmaceutical compositions.
The term "concomitant" as in the phrase "concomitant therapeutic treatment"
includes administering an agent in the presence of a second agent. A
concomitant therapeutic treatment method includes methods in which the first, second, third, or additional agents are co-administered. A concomitant therapeutic treatment method also includes methods in which the first or additional agents are administered in the presence of a second or additional agents, wherein the second or additional agents, for example, rnay have been previously administered. A concomitant therapeutic treatment method may he executed step-wise by different actors. For example, one actor may adtninister to a subject a first agent and a second actor may to administer to the subject a second agent, and the adniinistering steps may be executed at the same time, or nearly the same time, or at distant times, so long as the first agent (and additional agents) arc after administration in the presence of the second agent (and additional agents).
The actor and the subject may be the same entity (e.gõ human).
The term "combination therapy", as used herein, refers to the administration of two or more therapeutic substances, e.g., an anti-TNFa antibody and another drug, such as a DMARD or NSADD. The other drug(s) may be administered concomitant with, prior to, or following the administration of an anti-TNFo: antibody.

The term "TN-Fa-mediated condition" or "TNFa-related disorder" refers to a local and/or systemic physiological disorder where TNFc is a primary mediator leading to the manifestation of the disorder.
The term "inflammatory disorder'' or "inflammatory disease," as used interchangeably herein, refers to an inflarnmation-nicdiated malady, whether or not also immune mediated. Inflammatory disorders are disorders in which an excessive or unregulated inflammatory response leads to excessive inflammatory symptoms, host tissue damage, or loss of tissue function. Examples include rheumatoid arthritis and spondyloardiropatbies, In one embodiment, the inflammatory disorder of the invention refers to an inflammation-mediated malady excluding osteoartlaritis and rheumatoid spondylitis.
The term "pulmonary diseaSe" as used herein refers to any idiopathic interstitial lung disease arid/or chronic obstructive airway disorder. In one embodiment of the invention, the tem pulmonary disease includes any hmg disease and/or chronic obstructive airway disorder excluding shock lung, chronic pulmonary inflammatory disease, pulmonary sacroidosis, pulmonary fibrosis, and silicosis.
= The tetra " idiopathic interstitial lung disease" or "idiopathic interstitial lung disorder," as used interchangeably herein, refers to any one of several diseases of unknown etiology with similar clinical features, producing diffuse pathologic changes primarily in interalveolar interstitial tissue. Examples of idiopathic interstitial lung :diseases include, but are not limited to, interstitial pulmonary fibrosis (TPF). Iii one embodiment, idiopathic interstitial lung diseases include any one of several diseases of u_uknown etiology with similar clinical features, producing diffuse pathologic changes prima:61y in interalveolar interstitial tissue but exclude shock lung, chronic pulnaonary inflammatory disease, pulmonary sacroidosis, pulmonary fibrosis, and silicosis.
The term "chronic obstructive airway disorder" as used herein, refers to pulmonary diseases due to physiologically determined chronic airflow obstruction, regardless of etiology. Examples of chronic obstructive airway disorders include, but are not limited to, astluna and chronic obstructive pulmonary disease (COPD). In one embodiment, the term chronic obstructive airway disorder includes pulmonary diseases due to physiologically determined chronic airflow obstruction but excludes shock hang, chronic pulmonary inflanunatory disease, pulmonary sacroidosis, pulmonary fibrosis, and silicosis The term "airway obstruction" refers to an increased resistance to airflow exhibited by characteristic spirometric findings.
The terra "cardiovascular disorder" or "coronary disorder" as used.
interchangeably herein, refers to any disease, disorder, or state involving the cardiovascular systcna, e.g., the heart, the blood vessels, and/or the blood.
A coronary disorder is generally characterized by a narrowing of the blood vessels that supply blood and oxygen to the heart (coronary arteries). Coronary disease usually results from the build up of fatty material and plaque. As the coronary arteries narrow, the flow of blood to the heart can slow or stop. Coronary disorders of the invention can apply to any abnonnality of an artery, whether structural, histological, biochemical or any other abnormality. An example of coronary heart disease is restenosis. In one enabodiment, a coronary disorder refers to any disease, disorder, or state involving the cardiovascular system excluding ischemia of the heart and heart insufficiency.
The term "restenosis" as used herein refers to the recurrence of stenosis, which is the narrowing or constriction of an artery. Restenosis oflen occurs as a preocclusive lesion that develops following a reconstructive procedure in a diseased blood vessel.
The twin is not only applied to the recurrence of a pre-existing stenosis, but also to previously normal vessels that become partially occluded following vascular bypass. In another embodiment, the invention provides a method of treating restenosis comprising administering the antibody, or antigen binding portion thereof, of the invention to a subject who has or is at risk of developing restenosis.
The term "steal" as used herein refers to a structure that is inserted into the lumen of an anatomical vessel, e.g. an artery, especially to keep a formerly blocked passageway open. Stent is used to maintain the flow of fluids (e.g., blood) from one portion of a vessel to another, and an enclovascular scaffolding or stent which holds open a body passageway and/or supports the graft or wrap. A stent is often used -following balloon angioplasty, although they can also be used as direct therapy for heating stenosis.
In one embodiment of the invention, the stent is drug-eluting. The term "drug-chi ting" refers to a stent which is coated with a slow-to-moderate release drug formulation. The terms "drug-eluting" or "drug-releasing" or "drug-coatod" are used interchangeably herein. A stcnt can be coated with any drug which treats coronary heart disease, including, for example, the antibody, or antigen-binding fragment thereof, of the invention. In another embodiment, the stent delivers D2E7, In a further embodiment, the stent delivers D2E7 in combination with another drug used to treat coronary disorders, including dexamethasone, allceran, cytoxau, leukeran, cis-platinum, BiCNI.J, adriamycin, doxorubicin, cerubidine, idamycin, mithracin, muta.mycin, Iluorouracil, methotrexate, thoguanine, toxotere, etoposide, vincristine, irinotecan, hycarnptin, matulane, vumon, hexalin, hydroxyurea, gen-tzar, oncovin, etophophos, tacrolimus (1K506), and the following analogs of sirolimus: SDZ-RAID, CCI-779, 7-epi-rapainyciii, 7-thiomethyl-rapamycin, 7-epi-trimethoxyphenyl-- rapamycin, 7-epi-thiomethyl-rapamycin, 7-dernethoxy-rapamycin, 32-demethoxy, 2-desmethyl and proline.
The tcrm "metabolic disorder," as used herein, refers to diseases or disorders which affect bow thc body processes substances needed to carry out physiological functions. Examples of tnetabolic disorders include, but are not limited to, diabetes and obesity. In one embodiment of the invention, the term "metabolic disorder" is used to = refer to disorders which affect how the body processes substances needed to carry out physiological functions, excluding autoirnimme diabetes.
The term "diabetes" or "diabetic disorder" or "diabetes mellitus," as used interchangeably herein, refers to a disease which is marked by elevated levels of sugar (glucose) in the blood. Diabetes can be caused by too little insulin (a chemical produced by the pancreas to regulate blood sugar), resistance to insulin, or both.
The Phrase "disorders associated with diabetes," as used herein, refers to conditions and other diseases which are commonly associated with or related to diabetes.
Example of disorders associated with diabetes include, for example, hyperglycemia, hyperinsulinaemia, hyperlipidaemia, insulin resistance,, impaired glucose metabolism, obesity, diabetic retinopathy, macular degeneration, cataracts, diabetic ileptiropathy, glomeruloselerosis, diabetic neuropathy, erectile dysfunction, prernenstnial syndrome, vascular restenosis, ulcerative colitis, coronary heart disease, hypertension, angina pectoris, myocardial infarction, stroke, skin and connective tissue disorders, foot ulcerations, metabolic acidosis, arthritis, and osteoporosis.

The term "obesity" as used herein, refers to a condition in which the subject.
has an excess of body fat relative to lean body mass. In one embodiment, obesity refers to a condition in which an individual weighs at least about 20% or nacre over the maxiinum desirable for their height. When an adult is more than 100 pounds overweight, be or she is considered to be "morbidly obese." In another embodiment, obesity is defined as 13MI (body mass index) over 30 lcg/m2.
The tenn "anemia" as used herein, refers to an abnormally low number of circulating red cells or a decreased concentration of hemoglobin in the blood.
'1'he tenn "pain" as used herein, refers to all types of pain. The term shall refer to acute and chronic pains, such as neuropathic pain aud post-operative pain, chronic lower back pain, cluster headaches, herpes neuralgia, phantom limb pain, central pain, dental pain, opioid-resistant pain, visceral pain, surgical pain, bone injury pain, pain during labor and delivery, pain resulting fiorn burns, including sunburn, post partum pain, migraine, angina pain, an.d genitourinary tract-related pain including cystitis. The term also includes nociceptive pain or nociception.
As used herein, the term "hepatic disorder" refers to a mammalian and preferably a human liver disease or condition associated with hepatocellular injury or a biliary tract disorder. In one embodiment, hepatic disorders refers to a human liver disease or condition associated with hepatocellular injury or a biliary tract disorder excluding hepatitis, alcoholic hepatitis, and viral hepatitis.
The term "sldn disorder" or "skin disease" as used interchangeably herein, refers to abnormalities, other than injury wounds, of the skin which have induced a state of inflammation. In one embodiment, the skin disorder of the invention is an inflammatory skin disorder, wherein the skin is characterized by capillary dilatation, leukocytic infiltration, redness, heat, and/or pain. Examples of skin disorders include, but are not limited to, psoriasis, pemplaigus vulgaris, scleroderma, atopic dermatitis, sarcoidosis, erythema nodostun, hidradenitis suppurative, lichen planus, Sweet's syndrome, and vitiligo.
The term "psoriasis" as used herein, refers to skin disorders associated with epidegual hyperplasia. Example of psoriasis include, but are not limited to, chronic plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, psoriasis vulgaris, and erythrodennic psoriasis. Psoriasis can also be associated with other infiamniatory disorders, including inflammatory bowel disease (II3D) and rheumatoid arthritis (RA).
The tenn "healthy skin" or "normal skin" refers to non-lesional skin, i.e., with no visually obvious erythema, edema, hyper-, hypo-, or uneven pigmentations, scale formation, xerosis, or blister formation. Histologically, healthy or normal skin refers to skin tissue with a morphological appearance comprising well-organized basal, spinous, arid granular layers, and a coherent multi-layered stratum comeum.
The term "nail disorder" or "nail disease" as used herein, refers to conditions wherein the fingeniails or toenails to abnormal color, shape, texture, or thickness.
The term "vasculitis" or "vasculitides" as used interchangeably herein, refers to a group of disorders which ne characterized by the inflammation of blood vessels. Blood vessels of all sizes may be affected, from the largest vessel in the body (the aorta) to the smallest blood vessels in the skin (capillaries). The size of blood vessel affected varies according to the specific type of vasculitis.
= The term "kit" as used herein refers to a packaged product comprising components with which to administer the TNFa antibody of the invention for treatment of a TNFa¨related disorder. The kit preferably comprises a box or container that holds the components of the kit. The box or container is affixed with a label or a Food and Drug Administration approved protocol. The box or container holds components of the invention which are preferably contained within plastic, polyethylene, polypropylene, ethylene, or propylene vessels. The vessels can be capped-tubes or bottles.
The kit can also include instructions for administering the TNFa antibody of the invention.
Various aspects of the invention are described in further detail herein_ I. TNFa Inhibitors of the Invention This invention provides a method of treating a TNFa-related disorder in which the administration of a TN-Fa inhibitor is beneEcial. In one embodiment, these methods include administration of isolated human antibodies, or antigeu-binding portions thereof, that bind to human TNFa with high affinity and a low off rate, and have a high neutralizing capacity. Preferably, the human antibodies of the invention are recombinant, neutralizing human anti-hTNFa antibodies. The most preferred recombinant, neutralizing antibody of the invention is referred to herein as D2B7, also refeired to as HUMItsRA and alaliniumab (the amino acid sequence of the D2E7 VL
region is shown in SEQ ID NO: 1; the aini o acid sequence of the D2E7 VH
region is shown SEQ ID NO: 2). The properties of D2E7 (I-fUM1IA ) have been described in Salfeld el al., U.S. patent No. 6,090,382, In one embodiment, the treatment of the invention includes the administration of D2E7 antibodies and antibody portions, D2E7-related antibodies and antibody portions, and other liwnan antibodies and antibody portions with equivalent properties to D2E7, such as high affinity binding to hTNEa with low dissociation kinetics and high neutralizing capacity. In one embodiment, the invention provides treatment with an isolated human antibody, Or an antigen-binding portion thereof, that dissociates from human TNEa with a Kd of 1 x 10-8 M or less and a Koff rate constant of 1 x 10-3 s-1 or less, both determined by surface plasrnon resonance, and _neutralizes human TNFoc cytotoxicity in a standard in vitro L929 assay with an IC50 of 1 x 10-7 M
or.less. More preferably, the isolated human antibody, or antigen-binding portion thereof, dissociates from human TNFcc with a Koff of 5 x 10-4 s-1 or less, or even more preferably, with a Koff of 1 x 10-4 s-1 or less. More preferably, the isolated human antibody, or antigen-binding portion thereof, neutralizes human TNEa cylotoxicity in a standard in vitro L929 assay with an IC50 of 1 x 10-8 M or less, even more preferably with an IC50 of 1 x 10-9 M or less and still more preferably with an 1050 of 1 x 10-10 M or less. hi a preferred embodinient, the antibody is an isolated human recombinant antibody, or an antigen-binding portion thereof.
It is well known in the art that antibody heavy and light chain CDR3 domains play an important role in the binding specificity/affinity of an antibody for an antigen.
Accordingly, in another aspect, the invention pertains to methods of treating a TN.Fa-related disorder in which the TNTa activity is detriniental by administering btunan antibodies that have slow dissociation kinetics for association with liTNFa'ancl that have light and heavy chain CDR3 domains that structurally are identical to or related to those of D2E7. Position 9 of the D2E7 VL CDR3 can he occupied by Ala or Thr without substantially affecting thc Koff Accordingly, a consensus motif for the D2E7 VI, CDR3 comprises the amino acid sequence: Q-R-Y-N-R-A-P-Y-(T/A) (SEQ ID NO: 3).
Additionally, position 12 of the D2E7 VH CDR3 can be occupied by Tyr or Asn, without substantially affecting the Koff. Accordingly, a consensus motif for the D2E7 CDR3 comprises the amino acid sequence: V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQ
ID NO: 4). Moreover, as demonstrated in Example 2 of U.S. Patent No.
6,090,382, the CDR3 domain of the D2E7 heavy and light chains is amenable to substitution with a single alanine residue (at position 1, 4, 5, 7 or 8 within the VL CDR3 or at position 2, 3, 4, 5, 6, 8, 9,10 or 11 within the CDR3) without substantially affecting the Koff.
Still farther, the skilled artisan will appreciate that, given the amenability of the D2E7 VL and VH CDR3 domains to substitutions by alanine, substitution of other amino acids within the CDR3 domains may be possible while still retaining the low off rate constant of the antibody, in particular substitutions with conservative amino acids.
Preferably, no more than one to five conservative amino acid substitutions are made within the D2E7 VL and/or VH CDR3 domains. More preferably, no more than one to three conservative amino acid substitutions are made within the D2E7 VL and/or VH

domains, Additionally, conservative amino acid substitutions should not be made at amino acid positions critical for binding to hTNFcc. Positions 2 and 5 of the CDR3 and positions 1 and 7 of the D2E7 VH CDR3 appear to be critical for interaction with liTI\FFcc and thus, conservative amino acid substitutions preferably are not made at these positions (although an alanine substitution at position 5 of the D2E7 VL
(21JR3 is acceptable, as described above) (see U.S. Patent No. 6,090,382).
Accordingly, in another embodiment, the invention provides methods of treating a TNFoc-related disorder by the administration of an isolated human antibody, or antigen-binding portion thereof, The antibody or antigen-binding portion thereof preferably contains the following characteristics:
a) dissociates dom. human TNEct with a Koff rate constant of 1 x 10-3 CI or less, as determined by surface plasmon resonance;
b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ
ID NO: 3, or modifier) from SEQ 1D NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;
c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ
ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.

More preferably, the antibody, or antigen-binding portion thereof, dissociates from human TNFo, with a Koff of 5 x 10-4 s-1 or less. Even more preferably, the antibody, or antigen-binding portion thereof, dissociates :from human TNITa with a Koff of 1 x 10-4 s-1. or less.
In yet another embodiment, the invention provides methods of treating a TNFa.-related disorder by the administration of an isolated human antibody, or antigen-binding portion thereof. The antibody or antigen-binding portion thereof preferably contains a Light chain variable region (LCVR) having a CDR3 domain comprising the amino acid SECIllelACC of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8, and with a heavy chain variable region (IICVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. Preferably, the LCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5 (i.e., the 02B7 VL CDR2) and the HCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6 (i.e., the D2E7 VII
CDR2). Evan more preferably, the LCVR further has CDRI domain comprising the amino acid sequence of SEQ JD NO: 7 (i.e., the 02E7 VL CDRI) and the TICVR has a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8 (i.e., the D2E7 VII
CDRI). The framework regions for VL preferably are from the Vic1 human germline family, naore preferably from the A20 human gennline Vk gene and n3ost preferably from. the D2E7 VL framework sequences shown in Figures lA and 1B of U.S.
Patent No. 6,090,382. The framework regions for VII preferably are from the V1.13 human germane family, J110Te preferably from the DP-31 human gerniline VH gene and most preferably from the D2E7 VII framework sequences shown in Figures 2A and 2I3 of *U.S. Patent No. 6,090,382.
Accordingly, in another embodiment, the invention provides methods of treating a TN-Ea-related disorder by the administration of an isolated human antibody, or antigen-binding portion thereof. The antibody or antigen-binding portion thereof preferably contains a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 (i.e., the D2E7 VL) and a heavy chain variable region (HCV10 comprising the amino acid sequence of SEQ ED NO: 2 (i.e., the D2E7 VH). Iu certain embodiments, the antibody comprises a heavy chain constant region, such as an IgGl, IgG2, 1gG3, Ig04, IgA, IgE, IgM or IgD constant region. Prefe,rably, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. Furthermore, the antibody can cotnprise a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region.
Preferably, the antibody comprises a kappa light chain constant region. Alternatively, the antibody portion can be, for example, a Fab fragment or a single chain Fv fragment.
In still other embodiments, the invention provides methods of treating a TNFec-related disorder in which the administration of an anti-TNFcc antibody is beneficial . administration of an isolated human antibody, or an antigen-binding portions thereof.
The antibody or antigen-binding portion thereof preferably contains D2E7-related VL
and VII CDR3 domains, for example, antibodies, or antigen-binding portions thereof, with a light chain variable region (LCVR) having a CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO:
11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ED NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ lD NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 : or with a heavy chain variable region (1-ICVR) having a CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID
NO:
27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:
32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.
In another embodiment, the TN-Fa inhibitor of the invention is etanercept (described in WO 91/03553 and WO 09/406476), infliximab (described in U.S.
Patent No. 5,656,272), CDP571 (a Inunanized monoclonal anti-TNF-alpha IgG4 antibody), CDP 870 (a humanized monoclonal anti-TNE-alpha antibody fragment), D2E7 (a human anti-TI\T inAb), soluble TNF receptor Type I, or a pe.gylated soluble TNT
receptor Type I (PEGs TNE-R1).
The INFoc antibody of the invention can be modified. In some embodiments, the TNFa antibody or antigen binding fragments thereof, is chemically modified to provide a desired effect. For example, pegylation of antibodies and antibody fragments of the invention may be carried out by any of the pegylation reactions known in the art, as described, for example, in the following references: Focus on Growth Factors 3:4-1() (1992); EP 0 154 316; and EP 0 401 384 Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer). A preferred water-soluble polymer for pegylation of the antibodies and antibody fragments of the invention is polyethylene glycol (PEG). As used herein, "polyethylene glycol" is meant to encompass any of the forms of PEG that have been used to clerivatize other proteins, such as mono (01-010) alkoxy- or aryloxy-polyethylene glycol.
Methods for preparing pegylated antibodies and antibody fragments of the invention will generally comprise the steps of (a) reacting the antibody or antibody fragment with polyethylene glycol, such as a reactive ester or aldehyde derivative of PEG, under conditions whereby the antibody or antibody fragment becomes attached to one or more PEG groups, and (h) obhining the reaction products. lt will be apparent to one of ordinary skill in the art to select the optimal reaction conditions or the acylation reactions based on known parameters and the desired result.
Pegylated antibodies and antibody fragments may generally be used to treat TNFct-related disorders of the invention by administration of the TNFa antibodies and antibody fragments described herein. Generally the pegyInted antibodies and antibody fragments have increased half-life, as compared to the nonpegylated antibodies and antibody frafpnents. The pegylated antibodies and antibody fragments may be employed alone, together, or in combination with other pharmaceutical compositions.
In yet another embodiment of the invention, TNFa antibodies or fragments thereof can be altered wherein the constant region of the antibody is modified to reduce at least one constant region-mediated biological effector function relative to an unmodified antibody. To modify an antibody of the invention such that it exhibits reduced binding to the Fc receptor, tbe immanoglobulin constant region segment of the antibody can be mutated at particular regions necessary for Fc receptor (FcR) interactions (see e.g., Canfield, S.M. and S.L. Monison (1991) J. Exp. Med.
173:1483-1491; and Lund, J. et al. (1991)J. of Inzawnol. 147:2657-2662). Reduction in FcR
= binding ability of the antibody may also reduce other effector functions which rely on FcR interactions, such as opsonization and phagocytosis and antigen-dependent cellular cytotoxicity.
_ An antibody or antibody portion oftlte invention can be derivatized or linked to another functional molecule (e.g., another peptide or protein). Accordingly, the antibodies and antibody portions of the invention are intended to include derivatized arid otherwise modified forms of the human anti-hTNFa antibodies described herein, including immunoadhesion molecules. For example, an iittibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic tbsion, noticovalent association or otherwise) to one or more other inolecular entities, such as another antibody (e.g., a bispecifie antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate associate of the, antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies).
Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunetional (e.g., disuccinimidyl suberate).
Such linkers are available from Pierce Chemical Company, Rockford, EL.
Useful detectable agents with which an antibody or antibody portion of the invention may be derivatized include fluorescent compounds. Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, dimethylainine-l-napthalenesulfonyt chloride, phycoerythrin and the like. An antibody may also be derivatized with detectable enzymes, sucb as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxiclase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable. An antibody may also be derivatized with biotin, arid detected through indirect measurement of avidin or streptavidin binding.
An antibody, or antibody portion, of the invention can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell.
To express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglohutin light and heavy chains of the antibody such that the tight and heavy chains are expresseci in the host cell and, preferably, secreted into the medium in which the host cells Lu.e cultured, from which medium the antibodies can be recovered. Standard recombinant DNA methodologies iu-c used to obtain antibody heavy and light chain genes, incoiporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F.M. et al. (cds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Patent No. 4,816,397 by Boss et al.
To express D2E7 or a D2E7-related antibody, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by amplification and modification of gennline light and heavy chain variable sequences using the polytnerase chain reaction (PCR). Gerniline DNA sequences for human heavy and light chain variable region genes are known in the art (see e.g., the "Vbase" human germane sequence database; see also Kabat, B.A., et at. (1991) Sequences of Proteins of Immunological interest, Fifth Edition, U.S. Department o ['Health and Human Services, Publication No. 91-3242; Tomlinson, 1.M., et al. (1992) "The Repertoire of Human Gennline VH Sequences Reveals about Fifty Groups of V1-1 Segments with Different Hypervariable Loops" J. Mot, Biol. 227:776-798; and Cox, J.P.L. et al. (1994) "A
Directory of Human Genn-line V78 Segments Reveals a Strong Bias in their Usage"
Eur. Innnunol. 2A:827-836.
To obtain a DNA fragment encoding the heavy chain variable region of D2137, or a D2E7-rclated antibody, a member of the VH3 family of human gem-dine VII genes is runplified by standard PCR. Most preferably, the DP-31 VPI
germline sequence is amplified. To obtain a DNA fragment encoding the light chain variable region of D2E7, or a D2E7-related antibody, a inember of the VK1 family of human germline VL genes is amplified by standard PCR. Most preferably, the A20 VI.
germline SeqUellCE is amplified. PCR primers suitable for use in amplifying the DP-31 gerinline 'VII and A20 germline VL sequences can be designed based on the nucleotide sequences disclosed in the references cited supra, using standard methods.

Once the germline VII and VL fragments are obtained, these sequences can be mutated to encode the D2E7 or D2E7-related amino acid sequences disclosed herein.
The amino acid sequences encoded by the germline VEI and VL DNA sequences are first compared to the D2E7 or D2E7-related VH and VL amino acid sequences to identify amino acid residues in the D2E7 or D2E7-related sequence that differ from gennline.
Then, the appropriate nucleotides of the gennline DNA sequences are mutated such that the mutated gennline sequence encodes the D2E7 or D2E7-related amino acid sequence, using the genetic code to determine which nucleotide changes should be made.
Mutagencsis of the gennline sequences is carried out by standard methods, such as PCR-mediated mu tagenesis (in which the mutated nucleotides are incorporated into the PCR
primers such that the PCR product contains the inutations) or site-directed mutagenesis.
Once DNA fragments encoding D2E7 or D2E7-related VH and VL segments are obtained (by amplification and mutagenesis Egon-aline VH arid VL genes, as described above), these DNA fragments can be further manipulated by standard recombinant DNA
techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA frawnent encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked", as used in this context, is intended to mean that the two DNA
fragments are joined such that the amino acid sequences encoded by the two DNA
fragments remain in-frame.
The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA
molecule encoding heavy chain constant regions (CHI, CH2 and CI-13). The sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat, B.A., et al. (1991) Sequences ofPro(eins of Imnuazological Interest, Fifth Edition,U.S.
Department of Health and Human Services, NIfi Publication No. 91-3242) and DNA

fragments encompassing these regions can be obtained by standard PCR
amplification.
The heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgB, IgIVI or IgD constant region, but most preferably is an IgG1 or IgG4 constant region.
For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VI, region can be converted to i full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g., Kabat, E,A., et al. (1991) Sequences of Proteins of immunological Interest, Fifth Edition, U.S. Department of Health and Iltunan Services, NIH Publication No.
91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR
amplification. The light chain consttml region can be a kappa or lambda constant region, but most preferably is a kappa constant region.
To create a scFv gene, the VEI- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (G1y4.-Ser)3, such that the VII and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).
= To express the antibodies, or antibody portions oldie invention, DNAs encoding partial or full-length light and heavy chains, obtained as described above, are inserted into.expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control. sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible ,,vith the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fraginent and vector, or blunt end ligation if no restriction sites are present). Prior to insertion of the D2E7 or D2E7-related light or heavy chain sequences, the expression vector may already carry antibody constant region sequences. For example, one approach to converting the D2E7 or D2E7-related VH and VL sequences to full-length antibody genes is to insert them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the VH
segment is operatively linked to the CH scgmeut(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can he cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-irnmunoglobulin protein).
In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes iii a host cell. The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g,, polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, iu Goeddel; Gene Expression Technology:
ilifethods in Enzymology 185, Academic Press, San Diego, CA (1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CIVIV
promoter/enhancer), Simian Virus 40 (SV40) (such as the 5V40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMI.P)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Patent No.
5,168,062 by Stinslci, U.S. Patent No. 4,510,245 by Bell et al. and U.S. Patent No.
4,968,615 by Schaffner et al.
In addition to the antibody chair) genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as =
sequences that regulate replication of the vector in host cells (e.g., origins ofreplication) and selectable marker genes. The selectable marker gene facilitates selection of h.ost cells into which the vector has been introduced (see e.g., U.S. Patents Nos.
4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable rniu-ker genes include the dihydrolblate reductase (DEFR) gene (for use in dlifr host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
For expression of the light and heavy chaius, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotie host cells, expression of antibodies in eukaryotie cells, and most preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. Prokaryotic expression of antibody genes has been reported to be ineffective for production of high yields of active aritibody (Boss, M.A.
and Wood, C. R. (1985) Immunology Today 6:12-13).
= Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO
cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHPR selectable marker, e.g., as described in R.J. Kaufman and P.A.
Sharp (1982) Mal. Biol. 159:601-624 NSO myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.
Antibodies can be recovered from the culture medium using standard protein purification methods.
Host cells can also he used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. It is understood that variations on the above procedure are within the scope of the present invention. For example, it may be desirable to transfect a host cell wilh DNA encoding either the light chain or the heavy chain (but not both) of an antibody of this invention. RecombinaraDNA technology may also be used to remove some or all of the DNA encoding either Or both of the light and heavy chains that is not necessary for binding to laNra. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, bifunctional antibodies may be produced in which 011e heavy and one light chain are an antibody of the invention and the other heavy and light chain are specine for an antigen other than liTNFct by crosslinking an antibody of the invention to a second antibody by standard chemical crosslinking methods.
lir a preferred system for recombinant expression of an antibody, or antigen-1 0 binding portion thereof, of the invention, a.recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CEIO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy ancl light chain genes are each operatively linked to CMV
enhariccr/AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DIOR. gene, which allows for selection of CHO cells that have been transfectecl with the vector using rnethotroxate selection/amplification. The selected transfonnant host cells are culture to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium.
Recombinant human antibodies of the invention in addition to D2E7 or an antigen binding portion thereof, or D2E7-related antibodies disclosed herein can be isolated by screening of a recombinant combinatorial antibody library, preferably a scFv phage display library, prepared using human VL and VII cDNAs prepared from rnRNA
derived from 'human lymphocytes. Methodologies for preparing and screening such libraries are lcnown in the art. in addition to commercially available kits for generating pbage display libraries (e.g., the Phatotacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SuriZAPTM phage display kit, catalog no.
240612), examples of methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in, for example, Ladner et al. U.S.
Patent No. 5,223409; Kang et al. PCT Publication No. WO 92/18619; Dower et aL
PCT
'79 Publication No. WO 91/17271; Winter et al. PCT Publication No. WO 92/20791;
Marlchmd et al. PCT Publication No. WO 92/15679; Breitling el al. PCT
Publication No.

8; McCafferty et al. PCT Publication No. WO 92/01047; Garrard et al.
PCT
Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-.1372;
Hay et al. (1992)1-ban Antibod Hybridonzas 3:81-35; Huse et al. (1989) Science 246;1275-1281; McCafferty el al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J
12:725-734; 1-Tawkins et al. (1992)J Mol Biol 226:889-896; Clacicson et al.
(1991) Nature 352:624-623; Grarn et al. (1992) P.WAS 89:3576-3580; Garrard el a/.
(1991) Bio/Technology 9:1373-1377; Hoogenboorn et al. (1991) Nuc Acid Res 19:4133-4137;
and Barbas et all (1991) PNAS 88:7978-7932. Methods of isolating human antibodies with high affinity and a low off rate constant for hTNFa are described in U.S.
Patent Nos. 6,090,382, 6,258,562, and 6,509,015, EL Uses of TNFa. Inhibitors of the Invention In an embodiment, the invention provides a method for inhibiting TNFa activity in a subject suffering from a TNFa-related disorder in which TNFa activity is detrimental. In one embodiment, the TNFa inhibitor is 1)2F,7, also referred to as HUMIRA (adalhnurnab).
TNFa has been implicated in the pathophysiology of a wide variety of a TNFa-related disorders including sepsis, infections, autoimmune diseases, transplant rejection and graft--versus-host disease (see e.g., Moeller, A., et al. (1990) Cytolcine 2:162-169;
U.S. Patent No. 5,231,024 to Moeller et al.; European Patent Publication No.

B1 by Moeller, A., el al. Vasilli, P. (1992) Amu. Rev. Anntenol, 10:411-452;
Tracey, K.J.
and Ceraini, A. (1994) AMU!. Rel,, Med. 45:491-503), The invention provides methods for inhibiting TNFa activity in a subject suffering from a TNFa-related disorder, which method comprises administering to thc subject an antibody, antibody portion, or other TNFa inhibitor such that TN-Fa activity in tbe subject suffering from the TN-Fa-related disorder is inhibited. The invention also provides methods for inhibiting or decreasing TNFa. activity in a subject with vasculitis, comprising administering to the subject an antibody, or antibody portion, or other TNFa inhibitor of the invention such that TNFcc activity in the subject is inhibited or decreased. Preferably, the TN-Fa is human TNI7a and the subject is a human subject. Alternatively, the subject can be a mammal expressing a TNFr.c with which an antibody of the invention cross-reacts.
Still finther the subject can be a mammal into which has beerc introduced liTNFa (e.g., by =
administration of liTNEct or by expression of an IfINFrx transgene). An antibody of the invention can be administered to a human subject for therapeutic purposes (discussed further below).
Moreover, an antibody of the invention can be administered to a non-human manunal expressing a TNEa with which the antibody cross-reacts (e_g.,.a primate, pig or mouse) for veterinary purposes or as an animal model of human disease.
Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention (e.g., testing of dosages and time courses of administration), Examples of animal models used to study spondyloarthropathies include tank/ma transgenic mice, HLA-B27 tzansgenic rats (see Taurog et al. (1998) The Spo Ildylarth ritides. Oxford:Oxford University Press), Examples of animal models used for evaluating the -therapeutic efficacy of au agent for treating a hepatic disorder include the chimpanzee hepatitis C virus model (see Shimizu et al. (1990) PrOC Nall Acad Sci. USA 87:6441). Examples of animal models used to study skin and nail disorder disorders include, for example, the severe combined immunodeficient (SCID) mouse model (psoriasis) and the Smith line (SL) chicken and depigmenting mouse (vitiligo) (see Nickoloff (2000) Investig Dermatol Symp Proc.5:67;
Austin et al. (1995)Am J Palhol. 146:1529; Lerner et al. (1986).1 Invest Dermatol.
87:299), Examples of animal models for evaluating the efficacy of a TNEcc antibody for the treatment of a metabolic disorder include NOD tansgenic mice, Akita mice, NS-S.7"
transgenic mice and ob/ob mice (see Baeder et al. (1992) Clin Exp Ininntnol.
89:174; , flaseyama et al. (2002) Tohoku J Exp Med. 198:233; Makin et al. (1980):
Exp.Anim.
29:1; Kolb (1987) Diabetes/Metabolism Reviews 3:751; Hanaada et al. (2001) Metabolism. 50:1282; Coleman, (1978) Diabeto1ogia, 14:141; Bailey et al_ (1982) int.J.Obesity 6:11). Examples of animal models used to study vasculitis includes the mouse HSV model (Beheet's disease), the mouse L. casei model (Kawasaki's disease), and the mouse ANCA model (Kawasaki's disease). Other models of vaseulitis include the ..Mc1-15-/prl/pr strain (Nose, M., et al. (1996) Am. J. Path. 149:1763) and the SCG/Kj strain of mice (Kinjoh, et al. (1993) Proc. Nat(. Acad. Sci., USA 90:3413).
These mice strains spontaneously develop crescentic glomerulonephritis and necrotizing vasculitis of the small arteries and arterioles of the spleen, stomach, heart, uterus and ovaries. These a ni 11 als develop hyperganunaglobulinemia and ANCA autoantibodies that react with myeloperoxidase (1100). Additionally, immunization of rats with human MPO
results in ANCA-associated necrotizing crescentic glomerulonephritis (Brouwer, E., et al.
(1993) J. Exp. Med. 177:905).
Examples of animal models used to study idiopathic interstitial lung disease and chronic obstructive airway disorders include ovalbumin (OVA) induced allergic asthma mice and cigarette smoke induced chronic obstructive pulmonary disease mice (see Hesse], EM., et al. (1995) Eur JPharmacol. 293:401; Keast D, et al. (1981) J.
Pathol.
135:249) Commonly used animal models for studying coronary disorders, including restenosis, include the rat or mouse carotid artery ligation model and the carotid artery injury model (Ferns et al, (1991) Science 253:1.129; Clowes et al. (1983) Lab.
Invest.
49:208; Lindner el al. (1993) Circ Res. 73:792). In the carotid artery ligation model, arterial blood 'low is disrupted by ligation of the vessel near the distal bifurnation. As described in Cowes et al., the carotid artery injury model is performed such that the common carotid artery is denuded of endothelium by the intralmninal passage of a balloon catheter introduced through the external carotid artery. At 2 weeks, the carotid artery is markedly narrowed due to smooth muscle cell constriction, but between 2 and 12 weeks the inthnal doubles in thickness leading to a decrease in lumina]
size. Any of these models can be used to determine the potential therapeutic action of the Tl\l-Fa antibodies of the invention in the prevention and treatment of restenosis in humans.
Examples of animal models used to study anemia include rats inoculated with peptidolglyclui-polysaccharide polymers (see Coccia et al., (2001) Exp Hematology.
29;1201-1209). Examples of animal models used to study pain are well known in the int, and include the rat sciatic nerve ligation model, arid the rat segmental spinal nerve ligation model (see Bennett and Zie, (1988) Pain. 33:87-107; Kim and Chung, (1992) Pain 50:355-363).

As used herein, thc term " TNFa-rclated disorder in which TN17a, activity is cletritnental" is intended to include TNI7a-rclated diseases and other disorders in which the presence of TNFcc in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder, e.g., juvenile rheumatoid arthritis.
Accordingly, TNFa-related disorders in which TNFa activity is detrimental are disorders in which inhibition of Ti\TFa activity is expected to alleviate the symptoms and/or progression of the disorder. Such disorders may be evidenced, for example, by an increase in the concentration of TNra in a biological fluid of a subject suffering from the disorder (e.g., an increase in the concentration of TNFcc in serum, plasma, synovial Fluid, etc. of the subject), which can be detected, for example, using an anti-TNFa antibody as described above. The use of thc antibodies, antibody portions, and other TNIkc inhibitors of the invention in the treatment of specific TNFa-related disorder in which TNITa. activity is detrimental, is discussed further below. In certain embodiments, the antibody, antibody portion, or other TNFcc inhibitor of the invention is administered to the subject in combination with another therapeutic agent, as described below in Section III.
A. Spondyloarthroputhies ?() TNFa, has been implicated in the pathophysiology of a wide variety of disorders, including inflammatory diseases such as spondyloarthopathi es (see e.g., Moeller, A., et al. (1990) Cytolcine 2:162-169; U.S. Patent No. 5,231,024. to Moeller et al.;
European Patent Publication No. 260 610 B1 by Moeller, A). The invention provides methods for TNFa, activity in a subject suffering from such a disorder, which method comprises adininistering to the subject an antibody, antibody portion, or other TNFa inhibitor such that TNFo: activity in the subject suffering from a spondyloarthropathy is inhibited. In one embodiment, the invention provides a method of treating spondyloarthopathies.
As used herein, the term "spondyloarthropathy" or "spondyloarthropathies" is used to refer to any one of several diseases affecting the joints of the spine, wherein such diseases share common clinical, radiological, and histological features. A
number of spondyloarthropathies share genetic characteristics, i.e. -they are associated with the - 33..

IlLA-1327 allele. In onc embodiment, the term spondyloartlu-opathy is used to refer to any one of several diseases affecting the joints of the spine, excluding ardcylosing spondylitis, wherein such diseases share common clinical, radiological, and histological features. Examples of spondyloarthropathies include ankylosing spondylitis, psoriatie arthritis/spondylitis, enteropathic arthritis, _reactive arthritis or Reiter's syndrome, and undifferentiated spondyloarthropathies.
The TNEec antibody of the invention can also be used to treat subjects who are at risk of developing a spondyloaithropathy. Examples of subjects who are at risk of having spondyloarthropathies include launans suffering from arthritis.
Spondyloarthropathies can be associated with other forrns of arthritis, including rheumatoid arthritis. In one embodiment, the antibody of the invention is used to treat a subject who suffers from a spondyloanhropathy associated with rheumatoid arthritis.
Examples of spondyloarthropathies which can be treated with the TI\IFct.
antibody of the invention are described below:
1. Ankylosing Spondylitis (AS) Tumor necrosis factor has been implicated in the pathophysiology of ankylosing spondylitis (see Verjans et al, (1991) Arthritis Rheum, 34(4):486; Verjans et al. (1994) Clin Krp Immunol. 97(1):45; Kaijtzel et at. (1999) Hinz Inununol. 60(2): 140).
AnIcylosing spondylitis (AS) is an inflammatory disorder involving inflammation of one or more vertebrae. AS is a chronic inflammatory disease that affects the axial skeleton and/or peripheral joints, including joints between the vertebrae of the spine and sacroiliac joints and the joints between the spine and the pelvis. AS can eventually cause the affected vertebrae to fuse or grow together. Spondyarthropathies, including AS, can be associated with psoriatic arthritis (PsA) and/or inflammatory bowel disease (113D), including ulcerative colitis and Crohn's disease.
Early manifestations of AS can be determined by radiographic tests, including CT scans and IvIRI scans. Early manifestations of AS often include scroiliitis and changes in the sacroliac joints as evidenced by the binning of the cortical margins of the subehrondral bone, followed by erosions and sclerosis. Fatigue has also been noted as a common symptom of AS (Duffy et a/. (2002).ACR 66th Annual Scientific Mecling Abstract). Accordingly, the antibody, or antigen-binding fragment thereof, of the invention cai be used to treat AS. In onc embodiment, the TNFa. antibody, or antigen-binding fragment thereof, of the invention is used to treat spondyloarthropathy associated with IBD, including AS
AS is often treated with nonsteroidal anti-inflammatory medications (NSAIDs), such as aspirin or indernethacin- Accordingly, the TNFcc antibody of the invention may also be administered in combination with agents commonly used to reduce inflammation and pain conunonly associated with ankylosing spontlylitis.
2. PSOriatiC arthriiis l 0 TIMIOr necrosis factor has been implicated in the pathophysiology of psoriatic arthritis (Partsch et al. (1998) A/171 Rheum Ms. 57:691; Ritchlin et al.
(1998) .1 Rheionatol. 25:1544). As referred to herein, psoriatic arthritis (PsA) or psoriasis associated with the skin, refers to chronic inflammatory arthritis which is associated with psoriasis. Psoriasis is a common chronic skin condition that causes red patches on the body. About 1 in 20 individuals with psoriasis will develop arthritis along with the skin condition, and in about 75% of cases, psoriasis precedes the arthritis. PsA
exhibits itself in a variety of ways, ranging from mild to severe arthritis, wherein the arthritis usually affects the fingers and the spine. When the spine is affected, the symptoms are sirnilar to those of ankylosing spondylitis, as described above. The TNFcc. antibody, or antigen-binding fragment thereof, of the invention can be used to treat PsA.
PsA is sometimes associated with arthritis mutilans. Arthritis inutilans refers to a disorder which is characterized by excessive bone erosion resulting in a gross, erosive deformity which mutilates the joint. In one embodiment, the TNFa antibody, or antigen-binding fragment thereof, of the invention can be -used to treat arthritis mutilans.
3. Reactive arthritis / Reiter's syndrome Tumor necrosis factor has been implicated in the pathophysiology of reactive arthritis, which is also referred to as Reiter's syndrome (Braun et al.
(1999)/1i-thrills Rheum. 42(10):2039). Reactive artluitis (ReA) refers to arthritis which complicates an infection elsewhere in the body, often following enteric or urogenital infections. ReA is often characterized by certain clinical symptoms, including inflammation of the joints (arthritis), urethritis, conjunctivitis, and lesions of the skin and mucous membranes. In addition, ReA can occurs following infection with a sexually transmitted disease or dysenteric infection, including chlarnydia, campylobacter, salmonella, or yersinia.
Accordingly, the TNFo. antibody, or antigen-binding fragment thereof, of the invention can be used to treat ReA, 4. Undifferentiated spondyloarthropathies In one embodiment, the TNFa antibodies of the invention are used to treat subjects suffering from undifferentiated spondyloarthropathies (see Zeidler et al. (1992) Rheum Dis Clin North Ain. 18:187). Other terms used to describe undifferentiated spondyloarthropathies include seronegative oligoarthritis and undifferentiated oligoarthritis. Undifferentiated spondyloarthropathies, as used herein, refers to a disorder wherein the subject demonstrates only some of the symptoms associated with a spondyloarthropathy. This condition is usually observed in young adults who do not have IBD, psoriasis, or the classic symptoms of AS or Reiter's syndrome. In some instances, undifferentiated spondyloarthropathies may be an early indication of AS. In one embodiment, the TN-Fa antibody, or antigen-binding fragment thereof, of the = invention can be used to treat undifferentiated spondyloarlhropathies.
=
B. Pii/ntonary Disorders TNFa has been implicated in the pathophysiology of a wide variety of puhnonary disorders, including pulmonary disorders such as idiopathic interstitial lung disease and chronic obstructive airway disorders (see e.g., Piquet PF et al. (1989)J.Ex1, Med.
170:655-63; Whyte M, et al. (2000) AIll IRespir Crit Care Med. 162:755-8;
Anticevich SZ, et al. (1995) Eta- Pharmacol. 284:221-5). The invention provides methods for That activity in a subject suffering from such a pulmonary disorder, which method comprises administering to the subject an antibody, antibody portion, or other TNFcc inhibitor such that TNFa activity in the subject suffering from idiopathic interstitial lung disease or a chronic obstructive airway disorder is inhibited. Exqmples of idiopathic interstitial lung diseases and chronic obstructive airway disorders in which TNFo:
activity is detrimental are discussed further below.

1. Idiopathic interstitial lung disease In one embodiment, the TNFcc antibody of the invention is used to treat subjects who have an idiopathic interstitial lung disease. Idiopathic interstitial lung diseases affect the lungs in three ways: first, the lung tissue is damaged in sonic known or unknown way; second, the walls of the air sacs in the lung become inflamed;
and finally, scarring (or fibrosis) begins in the interstitium (or tissue between the air sacs), and the lung becomes stiff. Examples of idiopathic interstitial lung diseases are described below.
a. Idiopathic pulozonaiy fibrosis am Minor necrosis factor has been implicated in the pathophysiology of idiopathic pulmonary fibrosis (IPF) (see Piquet PF, et al. (1989)J Erp Med. 170:655-63;
Whyte M, et al. (2000) Ain J Respir Crit Care Med 162:755-8; Corbett EL, et al.
(2002) Ain J
Respir Crit Care Med. 165:690-3). For example, it has been found that 113F
patients have increased levels of TNF expressiojn in macrophages and in type 11 epithelial cells (Piquet et al. (1993) Ant J Pathol 143:651; Nash et al. (1993) Histopathology 22:343;
Zhang et al. (1993) .1 Inentunol 150:4188). Certain genetic polymorphisms are also associated with increased TNF expression, and are implicated in playing a role in rpF
and silicosis (Whyte et al., supra; Corbett EL, et al., supra).
The term "idiopathic pulmonary fibrosis" or "IPF" refers to a group of disorders characterized by inflammation and eventually scarring of the deep lung tissues, leading to shortness of breath. The scarring of the alveoli (air sacs) and their supporting structures (the interstitium) in IPF eventually leads to a loss of the functional alveolar units and a reduction of the transfer of oxygen from air to blood_ IPF is also referred to as diffuse parenchymal lung disease; alveolitis; cryptogenic fibrosing alveolitis (CEA);
idiopathic pulmonary pnewnonitis (IPP); and usual interstitial pneumonitis (UN). IPF is often used synonymously with UEP ('IPF/UIP") because ULP is the most common cellular pattern seen in the pathologic diagnosis of IPF.
Patients with IPF often exhibit certain symptoms, including a dry cough, chest pain, and/or shortness of breath. Commonly used drugs for the treatment of HT
are preclnisone and cytoxan, although only a fraction of patients improve vvitb continued use of these drugs (American Thoracic Society (2000) Ain. J. Respir. Crit. Care Med.

161:646). Oxygen administration and transplantation of the lung are other choices for treatment. In one embodiment, the TNFcc antibody of the invention is administered to the subject in combination with another therapeutic agent, for example oxygen, for the treatment of idiopathic pulmonary fibrosis,.
2. Chronic obstructive airway disorder In one embodiment, the TN-Fa antibody of the invention is used to treat a subject who has a chronic obstructive airflow disorder. In these diseases, airflow obstruction may be chronic and persistent or episodic and recurrent. Airflow obstruction is -usually determined by forced expiratory spirometry, which is the recording of exhaled volume against time during a maximal expiration. In a subject who does not have an obstructed airflow, a full forced expiration usually takes between 3 and 4 seconds. In a Patient with chronic obstructive airflow disorder, Wherein airflow is obstructed, it usually takes up to to 20 seconds and may be limited by breath-holding time. The normal forced 15 expiratory volume in the first second of expiration (FEVI) is easily measured and accurately predicted on the basis of age, sex, and height. The ratio of FEVI
to forced vital capacity (FEV1/FVC) normally exceeds 0.75. Recording airflow against volume during forced expiration and a subsequent forced inspiration¨the flow-vohune loop--is also useful, mainly for distinguishing upper from lower airway nan-owing.
Examples of chronic obstructive airway disorders are described below.
a. Asthma Tumor necrosis factor has been implicated in the pathophysiology of asthma, (A_nticevich SZ, et al. (1995) Eur Pharnzacol. 284:221-5; Thomas PS, et al.
1995. Am J Respir Crit Care Ailed. 152:76-80; Thomas PS, Heywood G. (2002) Thorax.
57:774-8).
For example, acute asthma attacks have been found to be associated with pulmonary neutrophilia and elevated BAL TNF levels (Ordonez CL. et al. (2000) Am .1 Respir Crit Care !vied 161:1185). It bias been found that the severity of asthma symptoms correlates with enclotoxin levels in house dust. In rats, anti-TNI? antibodies reduced endotoxin-induced airway changes (Kips et al. (1992) Am Rev 1?espir Dis 145:332).

te.1111 "asIhrmi" as used herein, refers lo a disorder in which inflamminion of the airways causes airflow into and out of the lungs to be restricted. Asthma is also referred to as bronchial asthma, exercise, induced asthma - bronchial, and reactive airways disease, (RAD). In some, instances, asthma is associated with allergies and/or is familial. Asthma includes a condition which is characterized by widespread fluctuations iri ale diameter or caliber of bronchial airways over short 'Periods of time, resulting in changes in lung function. The resulting increased -resistance to air flow produces symptoms in the affected subject, including breathlessness (dyspnea), chest constriction or "tightness," and wheezing.
1?aticnts with astluna are characterized according to NTH guidelines, are described as mild intermittent, mild persistent, moderate persistent, and severe persistent (see NAEPP Expert Panel Report Guidelines Thr the 1Diagnosis and Management of Asthma-Update on Selected Topics 2002. JAC1 2002; 110: S141-S209; Guidelines for the Diagnosis ancl Management of Asthma. NTH Publication 97-4051, 'July 1997).
Patients diagnosed with moderate persistent asthma are often 'treated with inhaled corticosteroids.
Patients diagnosed with severe persistent asthma are often treated with high dose inhaled corticosteroids and p.o, corticostcroids.
b. Chronic obstructive pain-loamy disease (COPD) 'rumor necrosis factor has been implicated in the pathophysiology of elaronic obstructive, puhnonary disease, (Keatings VAC (2000) Chest. 115:971-5; Salcao S, et al.( 2001) ./.11.n Respir Crit Care Med. 163:420-22; Sakti S, et al. (2002) Chest.
122:416-20). The term " chronic obstructive pulmontu-y disease" or ''COPD" as used interchangeably herein, refers to a group of lung diseases characterized by limited airflow with variable degrees of air sack enlargement and lung tissue destruction. The term (2,0PD includes chronic bronchitis (mucous hyperseoretion with goblet cell subanicosal gland hyperplasia), chronic obstructive bronchitis, or emphysema (destruction of airway parenchyma), or combinations of these conditions.
Empliyscina and chronic bronchitis are the most common forms of chronic obstructive pulmonary disease. COPD is defined by irreversible airflow obstruction.

ln COPD, chronic inflammation leads to fixed narrowing of small airways and lung parenchyma and alveolar wall destruction (emphysema). This is characterized by increased numbers of alveolar macrophages, neutrophils, and cytotoxic T
lymphocytes, and the release of multiple inflammatory mediators (lipids, chemolcines, cytokines, growth factors). This inflammation leads to fibrosis with a narrowing of the small airways and lung parenchymal destruction. There is also a high level of oxidative stress, which may tunplify this inflammation.
C. Coronaiy Disorders TNFcx has been implicated in the pathophysiology of a wide variety of coronary disorders, including restenosis (see e.g., Clausen et al.. (1994), supra;
Medan et cd.
(1997) Heart 78(3):273). As used herein, the term "a coronary disorder in which TNFa activity is detriinental" is intended to include coronary and cardiovascular diseases in which the presence of TNFa in a subject suffering from the disorder has been shown to be or is suspected of being either -responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder, including cardiovascular disorders, e.g., rcstcnosis. Coronary disorders in which TNFa activity is detrimental often result from a blockage in an artery. Such a blockage can be caused by a clot, which usually forms in a coronary artery that has been previously narrowed from changes usually related to atherosclerosis. For example, Utile atherosclerotic plaque inside the arterial wall cracks, it can trigger the formation of a thrombus, or clot. Such disorders may be evidenced, for example, by an increase in the concentration of TNFa. in a biological fluid of a subject suffering from the disorder (e.g., an increase in the concentration of TNIkt. in scrum, plasma, synovial fluid, etc. of the subject), which can be detected, for example, using an anti-TM:a antibody as described above. A corollary disorder can be also caused by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. Coronary disorders includes both coronary artery disease and peripheral vascular disease.
There are numerous examples of coronary disorders in which. TNFec activity is detrimental, including restenosis. The use of the antibodies, antibody portions, and other TN-Fa inhibitors of the invention lathe treatment of specific coronary disorders are discussed further below. In certain embodiments, the antibody, antibody portion, or other TNFa inhibitor of the invention is administered to the subject in combination with another therapeutic agent, as described below The invention provides a method for inhibiting TNFct activity in a subject with a coronary disorder. The invention provides methods for inhibiting or decreasing TNFa activity in a subject with a coronary disorder, comprising administering to the subject an antibody, or antibody portion, or other TNFa inhibitor of the invention such that TNIkt activity in the subject is inhibited or decreased. Preferably, the TNFa is human TNFa and the subject is a human subject. Alternatively, the subject can be a mammal expressing a TNFa with which an antibody of the invention cross-reacts. Still further the subject can be a imumnal into which has been introduced liTNFa (e.g., by administration of liTNFri or by expression of an hTNFo. transgene). .An antibody of the invention can be adntinistered to a human subject for therapeutic purposes (discussed further below). Moreover, an antibody of the invention can be administered to a non-human mammal expressing a TNFcc with which the antibody cross-reacts (e.g., a primate, pig or naouse) for veterinaty purposes or as an animal model of human disease.
Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention (e.g., testing of dosages and tinac courses of administration).
Commonly used animal models for studying coronary disorders, including restenosis, include the rat or mouse carotid artery ligation model and the carotid artery injury model (Ferns et al. (1991) Science 253:1129; Clowes et al. (1983) Lab.
Invest.
49:203; Lindner et al. (1993) Circ Res. 73:792). In the carotid artery ligation model, arterial blood flow is disrupted by ligation of the vessel near the distal bifurnation. As described in Clowes et al., the carotid artery injury model is perfornaed such that the common carotid artery is denuded of endothelium by the intralurninal passage of a balloon catheter introduced through the external carotid artery. At 2 weeks, the carotid artery is markedly narrowed due to smooth muscle cell constriction, but between 2 and 12 weeks the intirnal doubles in thickness leading to a decrease in luminal size. Any of these models can be used to determine the potential therapeutic action of the TNFa antibodies of the invention in the prevention and treatment of restenosis in humans.
-'U -The antibody of the invention can be used to treat cardiovascular disorders in which TNFa. activity is detrimental, wherein inhibition of TNEcc activity is expected to alleviate the symptoms and/or progression of the coronary disease or to prevent the coronary disease. Subjects suffering from or at risk of developing coronary disorders can be identified through clinical symptoms. Clinical symptoms in coronary disease often include chest pain, shortness of breath, weakness, fainting spells, alterations in consciousness, extremity pain, paroxysmal nocturnal clyspnea, transient ischemic attacks and other such phenomena experienced by the patient. Clinical signs of coronary disease can also include EKG abnormalities, altered peripheral pulses, arterial bruits, abnormal heart sounds, rates and wheezes, jugular venous distention, neurological alterations and other such findings discerned by the clinician. Coronary disorders may also be evidenced, for example, by an increase in the concentration of TNFcc in a biological fluid of a subject suffering from the disorder (e.g., an increase in the concentration. of TNIT7a in serum, plasma, synovial fluid, etc. of the subject).
1.5 Examples of a cardiovascular disorder include, but are not limited to, coronary artery disease, angina pectoris, myocardial infarction, cardiovascular tissue damage caused by cardiac arrest, cardiovascular tissue damage caused by cardiac bypass, cardio genic shock, and hypertension, atherosclerosis, coronary artery spasm, coronary artery disease, valvular disease, arrhythmias, and carcliomyopathies. The use of the antibodies, antibody portions, and other TNFa inhibitors of the invention in the treatment of specific cardiovascular diseases are discussed further below. In certain embodiments, the antibody, antibody portion, or other TNFcc inhibitor of the invention is administered to the subject in combination with another therapeutic agent, as described below in section DI.
1. Restenosis TNFa has been implicated in the pathophysiology of restenosis (see Zhou et al.

(2002) Atherosclerosis. 161:153; Javed et al. (2002) Exp and Mot Pathol 73:104). For example, in the murinc wire carotid model, INF -/- mice demonstrated a seven-fold reduction in lanai hyperplasia compared to wild type mice (Zimmerman et al.
(2002) Am .1 Plisiol Regul Integr Comp Physiol 283:R505). Restenosis cau occur as the result of any type of vascular reconstruction, whether in the coronary vasculature or in the periphery (Colburn and Moore (1998) Myointimal Iiyperplasia pp. 690-709 in Vascular Surgeiy: C'ompreliensive Review Philadelphia.: Saunders). For example, studies have repotted symptomatic restenosis rates of 30-50% following coronary angioplasties (see Berk and Harris (1995) Adv. Intern. Med. 40455-501). After cnotid endarterectomies, as a firther example, 20% of patients studied had a luminal narrowing greater than 50%
(Clagelt et al. (1986) J. Vase. Sing. 3:10-23). Restenosis is evidenced in different degrees of symptomatology which accompany preocclusive lesions in different anatomical locations, due to a combination of factors including the nature of the vessels involved, the extent of residual disease, and local hemodynamics.
"Stenosis," as used herein refers to a narrowing of an artery as seen in occlusive disorder or in restenosis. Stenosis can be accompanied by those syniptoms reflecting a = decrease in blood flow past the narrowed arterial segment, in which case the disorder giving rise to the stenosis is termed a disease (i.e., occlusive disease or restenosis disease). Stenosis can exist asymptoinatically in a vessel, to be detected only by a diagnostic intervention such as an angiography or a vascular lab study.
The antibody of the invention can be used to treat a subject suffering from or at risk of developing restenosis. A subject at risk of developing restenosis includes a subject who has undergone PTCA. The subject may have also had a stent inserted to prevent restenosis. The TNFec antibody of the invention can be used alone or in combination with a stent to prevent the re-occurrence of stenosis in a subject suffering from cardiovascular disease.
2. Congestive Heart Failure TNFct has been implicated in the pathophysiOlogy of congestive heart failure (see Zhou et al. (2002) Atherosclerosis =161:153). Sentra levels of TN-Fa are elevated in pati cuts with congestive heart failure in an-tanner which is directly proportional to the severity of the disease (Levine et al. (1990)N Engl J Med 323:236; Torre-Amioue el al.
(1996)JAnt Coll Cardiol 27:1201). In addition, inhibitors of TNFa have also been shown to improve congestive heart failure symptoms (Chung et al. (2003) Circulation 107:3133).

As used herein, the term "congestive heart failure" includes a condition characterized by a diminished capacity of the heart to supply the oxygen demands of the body. Symptoms and signs of congestive heart failure include diminished blood flow to the various tissues of the body, accumulation of excess blood in the various organs, e.g., when the heart is unable to pump out the blood returned to it by the great veins, exertional dyspnea, fatigue, and/or peripheral edema, e.g., peripheral edema resulting from loll ventricular dysfunction. Congestive heart failure may be acute or chronic. The manifestation of congestive heart failure usually occurs secondary to a variety of cardiac or systemic disorders that share a temporal or permanent loss of cardiac function.
Examples of such disorders include hypertension, coronary artery disease, valvular disease, and cardiomyopathies, e.g., hypertrophic, dilative, or restrictive cardioniyopathies.
A "subject who has or is suffering from congestive heart failure" is a subject who has a disorder involving a clinical syndrome of diverse etiologies linked by the common denominator of impaired heart pumping in which the heart cannot punip blood commensurate with the requirements of the metabolizing tissues, or can do so only from an elevated filling pressure. A "subject at risk of developing congestive heart failure" is a subject who has a propensity of developing congestive heart failure because of certain factors affecting the cardiovascular system of the subject. It is desirable to reduce the risk of or prevent the development of congestive heart failure in these subjects. The phrase "with congestive heart failure" includes patients who are at risk of suffering from this condition relative to the general population, even though they may not have suffered from it yet, by virtue of exhibiting risk factors. For example, a patient with untreated hypertension n3ay not have suffered from congestive heart failure, but is at risk because of his or her hypertensive condition. In one embodiment of the invention, thc antibody D2E,7 is used to -treat a subject at risk of developing congestive heart failure.
3. ilcute coronaly syndromes TNFcc has been implicated in the pathophysiology of acute coronary syndromes (sec Libby (1995) Circulation 91:2844 ). Acute coronary syndromes include those disorders wherein the subject experiences pain due to a blood flow restriction resulting in not enough oxygen reaching the heart. Studies have found that "ft\TRY.
plays a rote in acute coronary syndromes. For example, in a novel rat heterotropie cardiac transplantation-coronary ligation model capable of inducing myocardial infarction in the absence of downstream lie,modynamic effects, administration of chimeric soluble TNT' receptor (sTNFR) abolished transient LV remodeling and dysfunction (Nakamura, et al.
(2003) .1. Cartliol. 41:41). ìt was also found that direct injection of an sTNFR
expression plasmid to the myocarditun, resulted in a reduction in the infarction size in acute myocardial infarction (AMI) experimental rats (Sugano et al. (2002) 16:1421).
in one embodiment, TNFa antibody of thc invention is used to treat or prevent an acute coronary syndrome in a subject, wherein the acute coronary syndrome is a myocardial infarction or angina.
As used herein, the ten-El "myocardial infarction" or "MI" refers to a heart attack.
A myocardial infarction involves the necorsis or pennanent damage of a region of the heart due to an inadequate supply of oxygen to that area. This necrosis is typically caused by an obstruction in a coronary artery from either atherosclerosis or an embolis.
Nils which are treated by the TNFa, antibody of the invention include both Q-wave and non-Q-wave myocardial infarction. Most heart attacks are caused by a clot that blocks one of the coronary arteries (the blood vessels that bring blood and oxygen to the heart muscle). For example, a clot in the coromuy artery interrupts the flow of blood and oxygen to the heart muscle, leading to the death of heart cells in that area.
The damaged heart muscle pemvanently loses its ability to contract, and the remaining heart muscle needs to compensate for it. An MI can also be caused by overwhelming stress in the individual.
The term "angina" refers to spasmodic, choking, or suffocative pain, and especially as denoting angina pectoris which is a paroxysmal thoracic pain due, most often, to anoxia of the myocardiuna. Angina includes both variant angina and exerhonal angina. A subject having angina has ischemic heart disease which is manifested by sudden, severe, pressing substemal pain that often radiates to the left shoulder and along the left arm. TNFcc has been implicated in angina, as TN-17a levels are upregulated in patients with both MI and stable angina (Balbay et al (2001).Angiolo 52109).

.Arillerosclernsis "Atherosclerosis" as used herein refers to a condition in which fatty material is deposited along the walls of arteries. This fatty material thickens, hardens, and may eventually block the arteries. Atherosclerosis is also referred to arteriosclerosis, hardening of the arteries, and arterial plaque buildup. Polyclonal antibodies directed against TIVa have been shown to be effective at neutralizing TNFa.
activity.resulting in inflammation and restenosis in the rabbit atherosclerotic model (Zhou aril., supra).
Accordingly, the TNFcc antibody of the invention can be used to treat or prevent subjects afflicted with or at risk of having atherosclerosis.
5. Cardioinyopathy The term ''cardiomyopathy" as used herein is used to define diseases of the nvocardiurn wherein the heart muscle or myocardium is weakened, usually resulting in inadequate heart pumping. Cardiomyopathy can be caused by viral infections, heart attacks, alcoholism, long-term, severe hypertension (high blood pressure), or by autoiminune causes..
In approximately 75-80% of heart failure patients coronary artery disease is the underlying cause of the eardiomyopathy and is designated "ischeinic cardioniyopathy."
Ischemic cardiomyopathy is caused by heart attacks, which leave scars in the heart muscle or myocardium. The affected myocardium is then unable to contribute to the heart pumping function. The larger the scars or the more numerous the heart attacks, the higher the chance there is of developing ischemic cardiornyopatby.
Cardioniyopathies that are not attributed to underlying coronary artery disease, and are designated "non-isehernic ctu-dioniyopathies." Non-ischemic cardiomyopathics include, but are not limited to idiopathic cardiomyopathy, hypertrophic cardiornyopathy, alcoholic cardicnnyopathy, dilated cardiomyopathy, periparturn cardiomyopathy, and restrictive cardioniyopathy.
D. Metabolic Disorders TN-Foc has been implicated in the patliophysiology of a wide variety of disorders, including metabolic disorders, such as diabetes and obesity (Spiegelrnan and Hotamisligil (1993) Ce// 73:625; Chu et al. (2000) Int J has Relat Metab Disord.

24:1085; Ishii et al. (2000) Metabolism. 49:1616). The invention provides methods for TNFa. activity in a subject suffering from such a metabolic disorder, which method comprises administering to the subject an antibody, antibody portion, or other TiNIFcc inhibitor such that TN-Fa activity in the subject suffering from a metabolic disorder is inhibited. The TNFct antibody of the invention can also be used to treat subjects who are at risk of developing a metabolic disorder. Metabolic disorders arc often associated with arthritis, including rheumatoid arthritis. In one embodiment, the antibody o.r the invention is used to treat a subject who suffers from a metabolic disorder associated with rheumatoid arthritis. In another embodiment, the TNFct antibody of the invention is used to treat disorders associated with diabetes or obesity Metabolic disorders affect how the body processes substances needed to carry out physiological functions. A number of metabolic disorders of the invention share certain characteristics, i.e. they are associated the insulin resistance, lack of ability to regulate blood sugar, weight gain, and increase in body mass index. Examples of metabolic disorders include diabetes and obesity. Examples of diabetes include type 1 diabetes mellitus, type 2 diabetes mellitus, diabetic neuropathy, peripheral neuropathy, diabetic retinopathy, diabetic ulcerations, rctinopathy ulcerations, diabetic macrovasculopathy, and obesity. Examples of metabolic disorders which can be treated with the TNFo-antibody o f the invention are described in more detail below:
1. Diabetes Tumor necrosis factor has been implicated in the pathophysiology of diabetes.
(see e.g., Navarro J,F., Mora C., Maca, Arn J Kidney Dis. 2003 Jul;42(1):53-61; Daimon M et al., Diabetes Care. 2003 Jul;26(7):2015-20; Zhang M et al., J Toni IVIed Univ.
1999;19(3):203-5, Barbieri M et aL, Am J Hypertens. 2003 Jul;16(7):537-43.) For example, 'FNFcc is implicated in the pathophysiology for insulin resistance.
It has been found that scrum TNF levels in patients with gastrointestinal cancer correlates with . insulin resistance (see e.g., McCall, J. et aL Br. I. Stag. 1992; 79:
1361-3).
Diabetes includes the two most common typos of the disorder, namely type I
diabetes and type 11 diabetes, which both result from the body's inability to regulate insulin. Insulin is a hormone released by the pancreas in response to increased levels of blood sugar (glucose) in the blood.

11c term "type 1 diabetes," as used herein, refers to a chronic disease that occurs when the pancreas produces too little insulin to regulate blood sugar levels appropriately.
Type I diabetes is also referred to as ingulin.-dependent diabetes mellitus, IDM1v1, juvenile, onset diabetes, and diabetes - type I. Type -1 diabetes represents is the result o f a progressive autoinnuttne destruction of the pancreatic 13-ce11s with subsequent insulin deficiency.
The tenn "type 2 diabetes," refers to a chronic disease that occurs when the pancreas does not make enough insulin to keep blood glucose levels normal, often because the body does not respond well to the insulin. Type 2 diabetes is also referred to as noninsulin-dcpendent diabetes mellitus, N.DDM, and diabetes - type 11 Diabetes is can be diagnosed by the administration of a glucose tolerance test.
Clinically, diabetes is ollen divided into several basic categories. Primary examples of these categories include, autoinumme diabetes mellitus, non-insulin-dependent diabetes mellitus (type 1 NDDM), insulin-dependant diabetes mellitus (type 2 IDDM), non-autoinimune diabetes mellitus, u.on-insulin-dependant diabetes mellitus (type NIDDM), and maturity-onset diabetes of the young (MODY). A further category, often referred to as secondary, refers to diabetes brought about by some identifiable condition which causes or allows a diabetic syndrome to develop. Examples of secondary categories include, diabetes caused by pancreatic disease, hormonal abnormalities, drug-or chemical-induced diabetes, diabetes caused by insulin receptor abnormalities, diabetes associated with genetic syndromes, and diabetes of other causes. (see e.g., Harrison's (1996) 14th ed., New York, McGraw-Hill).
Diabetes manifests itself in the foregoing categories and can cause several complications that are discussed in the following sections. Accordingly, the antibody, or antigcn-bincling fragnent thereof, of the invention can be used to treat diabetes. In one embodiment, the TNEcc autibody, or antigen-binding fragment thereof, of the invention is used to treat diabetes associated with the above identified catagores.
Diabetes is aften treated with diet, insulin dosages, and various medications described herein. Accordingly, the TNFct, antibody of the invention may also be administered in combination with agents corrunonly used to treat metabolic disorders and pain commonly associated with diabetes.

In one embodiment, the TNFa antibody of the invention can also be -used to treat disorders associated with diabetes. Diabetes manifests itself in many complications and conditions associated with diabetes, including the following catagorics:
a. Diabetic Neuropathy and Peripheral Neuropathy Tumor necrosis factor has been implicated in the patbophysiolo,gy of diabetic neuropathy and peripheral neuropathy. (See Denjaheld et al. (2001) _Diabetes Care.
24:753; Qiang, X. et al. (1998) Diabe1ologia.41:1321-6; Pfeiffer el al. (1997) florin Metab Res. 29:111).
The term "neuropathy," also referred to as nerve damage-diabetic, as used herein, refers to a cominon complication of diabetes in which nerves are damaged as a result of hyperglycemia (high blood sugar levels). A variety of diabetic neuropathies are recognized, such as distal sensorimotror polyneuropathy, -focal motor neuropathy, and autonomic neuropathy.
The term "peripheral neuropathy," also lcnown as peripheral neuritis and diabetic neuropathy, as used herein, refers to the failure of the nerves to carry information to and from the brain and spinal cord. Peripheral neuropathy produces symptoms such as pain, loss of sensation, and the inability to control muscles. In some cases, the failure of nerves to control blood vessels, intestinal function, and other organs results in abnormal blood pressure, digestion, and Joss of other basic involuntary processes.
Peripheral neuropathy inay involve damage to a single nerve or nerve group (mononeuropathy) or may affect multiple nerves (polyneuropathy).
Newnpathies that affect small myelinated arid unnayelinated fibers of the syr pathetic and parasympathetic nerves are known as "peripheral neuropathies."
Furthermore, the related disorder of peripheral neuropathy, also 'mown as peripheral neuritis and diabetic neuropathy, refers to the failure of the nerves to carry information to and from the brain and spinal cord. This produces symptoms such as pain, loss of sensation, and the inability to control muscles. In some cases, failure of nerves controlling blood vessels, intestinal function, and other organs results in abnormal blood pressure, digestion, and loss of other basic involuntary processes. Peripheral neuropathy may involve damage to a single nerve or nerve group (mononeuropatliy) or may affect multiple nerves (polyneuropathy).

The term "diabetic neuropathy" refers to a common complication of diabetes in which nerves are damaged as a result of hyperglycemia (high blood sugar levels).
Diabetic neuropathy is also referred to as neuropathy and nerve damage-diabetic. A
variety of diabetic neuropathies are recognized, such as distal sensorimotror polyneuropathy, focal motor neuropathy, and autonomic neuropathy.
b. Diabetic Retinopathy Tumor necrosis factor has been iniplicated in the pathophysiology of diabetic retinop thy (Scholz et al. (2003) Trends Microbiol. 11:171). The lean "diabetic retinopathy" as used herein, refers to progressive damage to the eye's retina caused by long-term diabetes. Diabetic retinopathy, includes proliferative retinopathy.
Proliferative neuropathy iii turn includes includes neovascularization, pertinal hernmorrhave and retinal detachement In advanced retinopathy, small vessels proliferate on the surface of the retina_ These blood vessels are fragile, tend to bleed and can cause peretinal hemorrhages. The hemorrhage can obscure vision, and as the hemorrhage is resorbed fibrous tissue forms predisposing to retinal detaclunents and loss of vision. In addition, diabetic retinopathy includes prolferative retinopathy which includes neovascularization, pertinal hemmorrhave and retinal detachment. Daibetic retinopathy also includes "background retinopathy" which involves changes occuring with the layers of the retina.
c. Diabetic Ulcerations and Retinopathy'Ulcerations Tumor necrosis factor has been implicated in the pathophysiology of diabetic ulcerations, (see Lee et al. (2003) lintn Inununol, 64:614; Navarro et aL
(2003) Ain .1 Kidney Dis. 42:53; Daimon et al (2003)Diabetes Care. 26:2015; Zhang et al.
(1999)J
Tongji Med Univ. 19:203; Barbieri et al. (2003) A7/I J Hypertens. 16:537; Venn et al.
(1993) Arthritis 1?heunz. 36:819; Westacott et al. (1994) JRheutnatol.
21:1710).
The term "diabetic ulcerations," as used herein, refers to an ulcer which results as a complication of diabetes. An ulcer is a crater-like lesion on the skin or mucous membrane caused by an inflammatory, infectious, malignant condition, or metabolic disorder. Typically diabetic ulcers cart be found on limbs and extremeties, more typically the feet. These ulcers, caused by diabetic conditions, such as neuraptly and a .

vacualr insuffciency, can lead to ischernia and poor wound healing. More extensive ulcerations may progress to ostemyelitis. Once ostemyelitis develops, it may be dificulte to eradicate with antiboties alonda lid amputation mayb e necessary..
The term "retinopathy ulcerations," as used herein refers to an ulcer which causes or results in damages to the eye and the eye's retina. Retinopathy ulcerations may include conditions such has retinoathic herninorages.
d. Diabetic Placrovasculopathy Tumor necrosis factor has been implicated in the pathophysiology of diabetic inacrovasculopathy (Devaraj et al. (2000) Circulation. 102:191; Hattori Y et al. (2000) Cardiovasc Res. 46:188; Clausen N et al. (1999) Cardiovasc Pathol.8:145). The term "diabetic macrovasculopathy," also referred to as "macrovascular disease," as used herein, refers to a disease of the blood vessels that results :from diabetes.
Diabetic macrovasculopathy complication occurs when, for example, fat and blood clots build up in the large blood vessels and stick to the vessel walls. Diabetic macrovasculopathies includediseases such as coronary disease, cerebrovascular disease, and peripheral vascular disease, hyperglycaemia and cardiovascular disease, and strokes.
2. Obesity Tumor necrosis factor has been implicated in the pathophysiology of obesity (see e.g, Pililajamalci J et aL (2003) Obes Res.11:912; Barbieri et al. (2003) Am J
Hypertens.
16:537; Tsuda et al. (2003) J Nutr. 133:2125). Obesity increases a person's risk of illness and death due to diabetes, stroke, coronary artery disease, hypertension, high cholesterol, and kidney and gallbladder disorders. Obesity may also increase the risk for some types of cancer, and may be a risk factor for the development of osteoarthritis and sleep apnea. Obesity can be treated with the antibody of the invention alone or in combination with other metabolic disorders, including diabetes.
E. Anemia TN17cc has been implicated in the pathophysiology of a wide variety of anemias (see e.g., Jongen-Lavrencic M., et al. (1997)J. Rheurnato1.24(8):1504-9;
Demeter J., et al. (2002).Ami Hematal. 81(10):566-9; DiCato M., (2003) The Oncologist 8 (suppl 1):19-21). The invention provides methods for inhibiting TN-Fcc activity in a subject suffering from such a disorder, which method comprises administering to the subject an antibody, antibody portion, or other TNFcc inhibitor of the invention such that TNFcc activity in the subject suffering from anemia is inhibited. In one embodiment, the anemia is associated with rheumatoid arthritis.
The term "anemia" as used herein, refers to an abnormally low number of circulating red cells or a decreased concentration of hemoglobin in the blood.
Examples of anemia related to rheumatoid arthritis include, for example, anemia of chronic = disease, iron deficiency anemia, and autoimmune hemolytic anemia. hi one embodiment, the invention provides a method of treating anemias related to, for example, anemias related to rheumatoid arthritis, anemias of infection and chronic inflammatory diseases, iron deficiency anemia, autoinunune hemolytic anemia, myeloplithisic anemia, aplastic anemia, hypoplastic anemia, pure red cell aplasia and anemia associated with renal failure or endocrine disorders, megaloblastic anemias, defects in heme or globin synthesis, anemia caused by a structural defect in red blood "
cells, e.g., sickle-cell anemia, and anemias of unlcnown origins such as sideroblastie anemia, anemia associated with chronic infections such as malaria, trypanosomiasis, HIV, hepatitis virus or other viruses, and myclophthisic anemias caused by marrow deficiencies.
P. Pain TNFo. has been implicated in the pathophysiology of a wide variety of pain syndromes (see e.g., Sorkin, LS. et al., (1997) Neuroscience. 81(1):255-62;
Huygen FJ., et al. (2002) Mediators Inflamin. 11(1):47-5I; Parada CA., et al. (2003) Ellr Neurosei.17(9):1847-52). The invention provides inethods for inhibiting TNFcc activity in a subject suffering from such a pain disorder, which method comprises administering to the subject an antibody, antibody portion, or other TNFcc inhibitor of the invention such that TNFec activity in the subject suffering from pain is inhibited. Pain has been defined in a variety of ways, including nociceptive pain and netu-opalliic paiu. The most commonly experienced form of pain may be defined as the effect of a stimulus ou nerve endings, which results in the transmission of impulses to the cerebnun. Pain is also commonly associated with inflammatory disorders, including, for example, rheumatoid arthritis. ht one embodiment, the antibody of the invention is used to treat a subject who suffers from pain associated with rheumatoid arthritis. Examples of pain disorders in which TNITa activity is detrimental are discussed further below.
=
1, iVeuropathic Pain Tumor necrosis factor has been implicated in the pathophysiology of neuropathic pail) (see Sommer C., (1999) SC11111C17. 13(5):315-23; Empl M et al., (2001) Neurology.
56(10):1371-7; Schafers M et al., (2003)J Neurosci. 23(7):3028-38). As used herein the term "neuropathic pain" refers to pain -that results from injury to a nerve, spinal cord, or brain, and often involves neural supersensitivity. Examples of neuropathic pain include chronic lower back pain, pain associated with arthritis, cancer-associated pain, herpes neuralgia, phantom limb pain, central pain, opioid resistant neuropathic pain, bone injury pain,.and pain during labor and delivery. Other exanples of neuropathic pain include post-operative pain, cluster headaches, dental pain, surgical pain, pain resulting from severe, for example third degree, burns, postpartum pain, angina pain, genitourinary tract related pain, and including cystitis.
Neuropathic pain is distinguished Jioin nociceptive pain. Pain involving a nociceptive mechanism usually is limited in duration to the period of tissue repair and generally is alleviated by available analgesic agents or opioids (Myers, Regional Anesthesia 20:173-184 (1995)). Neuropathic pain typically is long-lasting or chronic and often develops days or months following an initial acute tissue injury.
Neuropathic pain can involve persistent, spontaneous pain as well as allodynia, which is a painful response to a stimulus that normally is not painful. Neuropathic pain also can be characterized by hyperalgesia, in which there is an accentuated response to a painful stimulus that usually 95 is trivial, such as a pin prick. Unlike nociceptive pain, neuropathic pain generally is resistant to opioid therapy (Myers, supra, 1995). Accordingly, the antibody, or antigen-binding fragment thereof, of the invention can be used to treat neuropathic pain.

9. Nociceptive pain As used herein the term "nociceptivc pain" refers to pain that is transmitted across intact neuronal pathways, i.e., pain caused by injury to the body. Nociceptive pain includes somatic sensation and normal function of pain, and informs the subject of impending tissue damage. The nociceptive pathway exists for protection of the subject, e.g., the pain experienced in response to a burn). Nociceptive pain includes bone pain, visceral pain, and pain associated with soft tissue.
Tumor necrosis factor has been implicated in the pathophysiology of visceral pain (see Coelho A., et al. (2000) Am J Physic)] Gastrointest Liver Physiol.
279:G781-G790; Coelho A, et al. (2000) Brain Res Bull. 52(3):223-8). Visceral pain is used to refer to nociceptive pain that is mediated by receptors on A-delta and C nerve fibers. A-delta and C-netn fibers are which are located in skin, bone, connective tissue, muscle and viscera. Visceral pain can be vague in distribution, spasmodic in nature and is usually described as deep, aching, squeezing and colicky in nature. Examples of visceral pain include pain associated with a heart attack, wherein the visceral pain can be felt in the arm, neck and/or back, and liver capsule pain, wherein the visceral pain can be felt in the back and/or right shoulder. Accordingly, the TNEct. antibody, or antigen-binding fragment thereof, of the invention can be used to treat visceral pain.
G. Hepatic Disorders TNFa has been implicated in the pathophysiology of a wide variety of hepatic disorders (see e.g., Colletti L1\4,, et al. (1990)J Clin Invest. 85(6):1936-43; Tiegs G.
(1997) Acta Gastroenterol Belg. 60(2):176-9; Fernandez ED., el aL (2000)J
Endotaxin Res. 6(4):321-8). The invention provides methods for TNFct activity in a subject suffering from such a hepatic disorder, which method cotnprises administering to the subject an antibody, antibody portion, or other TNFa inhibitor of the invention such that TNFet activity in the subject suffering from a hepatic disorder is inhibited.
As used herein, tbe term "a hepatic disorder in which TNFcc activity is detrimental" is intended to include diseases and other disorders of the liver in which the presence of TNFa in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a hepatic disorder in which TNFcc activity is detrimental is a disorder in which inhibition of TNFa.
activity is expected to -alleviate the symptoms and/or progression of the hepatic disorder.

Hepatic disorders include many diseases and disorders wherein the liver functions improperly or ceases to function. Hepatocellular injuries can include alcoholic cirrhosis, al antitypsin deli.eiency, autoimmune cirrhosis, cryptogenic cirrhosis, fulmittant hepatitis, hepatitis B and C, and steatohepatitis. Examples of biliary tract disorders include cystic fibrosis, primary biliary cirrhosis, sclerosing cholantis and biliary obstruction (Wiesner, R. ll, Current Indications, Gonna Indications and Timing for Liver Transplantation (1996), in Transplantation of the Liver, Saunders (publ.);
Busuttil, R. W. and Klintmalm, G. B. (cds.) Chapter 6, e.g., Tables 6-3 tind 6-5 as well as FIGS. 6-11; Klein, A. W., (199S) Partial Hypertension: The Role of Liver Transplantation, Musby (publ.) in Current Surgical Therapy 6^th Ed.
Cameron, J.
(ed).
The term "hepatitis" refers to inflammation of the liver. Hepatitis can be caused by infections with various organisms, including bacteria, viruses (Hepatitis A, B, C, etc.), or parasites. Chemical toxins such as alcohol, drugs, or poisonous mushroonas can also damage the liver and cause it to become inflamed. A rare but extremely dangerous cause of hepatitis results frora overdose of acetaminophen (Tylenol), which can be deadly. In addition, immune cells in the body may.attack the liver and cause autoimmane hepatitis. Hepatitis may resolve quickly (acute hepatitis), or cause long-terrn disease (chronic hepatitis). In some instances, progressive liver damage or liver failure may result. The incidence and severity of hepatitis vary depending on many factors, including the cause of the liver damage and any underlying illnesses in a patient.
In one enabodimeut, the invention features a method for treating a hepatic disorder in which TNFcc activity is detrimental, comprising administering to a subject an effective ainount of a TNFa inhibitor, such that said disorder is treated. In one embodiment, the hepatic disorder is selected from the group consisting of hepatitis C
virus, autoimmunc hepatitis, fatty-liver disease, hepatitis I3 virus, hepatotoxicity, and non-alcoholic hepatitis, including non-alcoholic steatohepatitis (NASH).
Examples of hepatic disorders are further described below.

1. Hepatitis C Virus (HCV) Tumor necrosis factor has been implicated in the pathophysiology of the hepatitis C virus (see Gonzalez-Amaro. (1994) JExp Med. 179:841-8; Nelson DR, et al.
(1997) Dig Dis S'ei 42:2487-94; Kallinowski B, et al. (1998) Clin EAp inznzuzzol.
111:269-77).
The term. "hepatitis C virus" or "HCV" is used to describe the hepatitis virus which is the causative agent of non-A, non-B hepatitis. Hepatitis C virus causes an inflammation of the liver. HCV infection causes hepatitis C. Hepatitis C in the acute stage is, in general, milder than hepatitis B, but a greater proportion of such infections become chronic. HCV
is a major cause of acute hepatitis and chronic liver disease, including cirrhosis and liver cancer. FICV is one of the viruses (A, B, C, D, and E), which together account for the vast majority of cases of viral hepatitis. It is an enveloped RNA virus in the flavivixidae family which appears to have a narrow host range. An important feature of the virus is the relative mutability of its genome, which in turn is probably related to the high propensity (80%) of inducing cluonic infection. HCV is clustered into several distinct genotypes which may be important in determining the severity of the disease and the response to treatment. In one embodiment, the TN-Fa antibody, or antigen-binding fragnient thereof, of the invention can be used to treat HCV.
In one embodiment, subjects who are infected with HCV are treated with the TNFa. antibody of the invention. Symptoms oI HCV infection (hepatitis C) include at least one of the following: jaundice, abdominal pain (especially in the right upper abdomen), fatigue, loss of appetite, nausea-and vomiting, low-grade fever, pale or clay-colored stools, dark urine, and generalized itching. However, it should be noted that many people -who are infected with the hepatitis C do not have symptoms, as hepatitis C
is often detected during blood tests for a routine physical or other medical procedure.
2. Atitohnnzune Hepatitis (AII-I) Tumor necrosis factor has been implicated in the pathophysiology of autoimmune hepatitis (see Cookson S. et al., (1999) Hepatology 30(4):851-6;
Jazrawi S.
et al., (2003) Liver. .Transpl. 9(4):377-82). As used herein, "autoimmune hepatitis" refers to a hepatic disorder characterized by inflammation of the liver caused by rogue immune cells that mistake the liver's normal cells for a foreign tissue or pathogen (disease-causing agent). Autoimmune hepatitis is often responsible for a progressive destruction of the hepatic parenchyma with a high mortality if !eft untreated (Johnson P.
J. et al., (1993) Hepatology, 18:998-1005). One of the characteristics of au toimmune hepatitis is the presence of circulating autoantibodies in almost 90% of patients' sera.
Such antibodies can be used to identify subjects who have autoimmune hepatitis.
Clinical and serological differences between patients have lead to the classification of AII-I into two types. Type 1 is characterized by the presence of zmti-smooth muscle (SMA) and/or anti-nuclear antibodies (ANA) in patients' sera, while sera from. 'Typc patients show anti-liver kidney mierosomal antibodies type 1 (LICIVI1) (Homberg S. C. et al., (1987) Hepatology, 7:1333-1339; 'Maggiore G. el at, (1993) J.
__ Pediatr. Gastroenterol Nutr., 17:376-381). A serological marker, anti-liver cytosol type 1 antibodies (LC1), has been identified in 30% of patients with an A111 type IL In addition, i.C1 proved to be the only serological marker in 10% of patients tested (Martini E. et al., (1988) Hepatology, 8:1662-1666). In one embodiment, the INFa antibody, or antigen-binding fragment thereof, of the invention is used to treat AIH.
= 3. Fatly-liver disease Tumor necrosis factor has been implicated in the pathophysiology of fatty-iiver disease (see Val6nti L. et al., (2002) Gastmenerology 122(2):2'74-80; Li Z. et al., (2003) Hepatology 37(2):343-50). Fatty-liver disease refers to a disease wherein fat __ (hepatocytes) is excessively accumulated in the liver. Fatty liver disease is believed to be caused by supemutrition, hyperingestion of alcohol, diabetes and side effects due to administration of pharmaceuticals. Fatty liver disease can cause severe discase,s such as chronic hepatitis and hepatic cirrhosis. In patients with fatty liver disease, lipids, particularly neutral fat, accumulate in hepatocytes to the extent that the amou-nt exceeds __ the physiologically permissible range. From a biochemical point of view, a standard for judgment of fatty liver is that the weight of neutral fat is about 10% (100 mg/g wet weight) or more of the wet weight of hepatic tissue. Jn one embodiment, the TNFa antibody, or antigen-binding fragment thereof, of the invention can be used to treat fatty liver disease.

4. Hepatitis B Yirns (HBV) Tumor necrosis factor has been implicated in the pathophysiology of hepatitis B
virus (see Kasahara S. et al., (2003) J Virol. 77(4):2469-76; Wang F.S., (2003) World J
Gastroenterol. 9(4):641-4;Bieriner M. et al., (2003)J Virol. 77(7):4033-42).
The tenn "hepatitis B virus" (1113V) is used to describe the virus (sertun hepatitis virus) which produces viral hepatitis type B in humans. This is a viral disease with a long incubation period (about 50 to 160 days) in contrast to hepatitis A virus (infectious hepatitis virus) which has a short incubation period. The hepatitis B virus is usually transmitted by injection of infected blood or blood derivatives or merely by use of contaminated needles, lancets or other instruments. Clinically and pathologically, the disease is similar to viral hepatitis type A; however, there is no cross-protective immunity.
Viral antigen (HBAg) is found in the serum alter infection.
-Hepatitis B virus infects humans at a very high rate. Most people, who become infected with Hepatitis B get rid of the virus within 6 months, wherein a short infection is ls..nown as an ''acute" case of Hepatitis B. It is es tiinated that at least about 300 million people are chronic carriers of BBV. Infection with the virus results in a range of clinical symptoms including minor flu-like symptoms to death. In one embodiment, the TNFcc antibody, or antigen-binding fragment thereof, of the invention can be used to treat I-113V
infection.
5. Hepatotoxicity Tumor necrosis factor has been implicated in the pathophysiology of hepatotoxicity (see Bruccoleri A. et al., (1997) Hepatology 25(1):133-41;
Luster M.I. et al., (2000) Ann NY Acad Sci. 919:214-20; Simeonova P. et al., (2001) Toxicol Appl Pharniaeol. 177(2):112-20). The temi hepatotoxicity refers to liver damage caused by medications and other chemicals or drugs. The best indicator for identifying liver toxicity in a subject is the elevation of certain enzyrne measurements in the blood, such as AST (aspartate arninotransferase), ALT (alanine aminotransferase), and GOT
(glutamate oxalacetate transaminase).
Bcpatotoxicity can cause permanent injury and death. Initial symptoms of hepatotoxieity Call include acute gastrointestinal symptoms, e.g., severe diarrhea. The second phase of hepatotoxicity is characterized by abatement of symptoms.
During this apparent subsidence, biochemical evidence of hepatic injury appears. Oliguria (decreased urine output) is usual during the second phase. The third phase, that of overt hepatic damage, becomes clinically apparent 3 to 5 days after ingestion of the chemical, with the appearance ofjaundice. Renal failure may also occur. The symptoms of chcruically-induced (drug-induced) hepatitis are siinilar to that of infectious hepatitis. In one embodiment, the TNFa antibody, or antigen-binding fragment thereof, of the invention can be used to treat hepatotoxicity.
6. Liver failure (e.g. chronic liver failure) Tumor necrosis factor has been implicated in the pathophysiology of liver failure (e.g. chronic liver failure) (see Takenaka K. et al., (1998) Dig Dis Sal.
43(4):887-92;
Nagaki M. et al., (1999) J Heptztol. 31(6):997-1005; Streetz K. et al., (2000) Gastroenterology. 119(2):446-60. Liver failure, including chronic liver failure, usually develops over a period of years and is caused by a repeated insult to the liver (such as alcohol abuse, or infection with hepatitis virus) which slowly damages the organ. Less commonly, liver failure is acute, and occurs over a period of days or weeks.
Causes of acute liver failure include hepatitis virus infections, drugs, pregnancy, autoirnmune disease, and sudden low blood flow to the liver. In one embodiment, the TNFcc antibody, or antigen-binding fragment thereof, of the invention can be used to liver failure.
7. Non-alcoholic hepatitis, including NASH
Tumor necrosis factor has been implicated in the pathophysiology of non-alcoholic hepatitis, including nonalcoholic steatohepatitis (see Crespo I. el al., (2001) Hepatology. 34(6):1158-63;Pessa.yre D. et al., (2002) 282(2):G193-9). The term "nonalcoholic steatohepatitis" or "NASH" refers to the development of histologic changes in the liver that arc comparable to those induced by excessive alcohot intake, but in the absence of alcohol abuse. NASH is characterized by macrovesicular and/or microvesicular steatosis, lobular and portal inflammation, and occasionally Mallory bodies with fibrosis and cirrhosis. NASH is also commonly associated with hyperlipidemia, obesity, and type 11 diabetes mellitus.
- 59..

Additional clinical conditions which characterize hepatic steatosis and inflammation include excessive fasting, j ejunoi teal bypass, total parental nutrition, chronic hepatitis C, Wilson's disease, arid adverse drug effects such as those from corticosteroids, calcium channel blockers, high dose synthetic estrogens, niethotrexate and amiodarone. Thus, the term "nonalcoholic steatoliepatitis" can be used to describe those patients who exhibit these biopsy findings, coupled with the absence of (a) significant alcohol consumption, (b) previous surgery for weight loss, (c) history of drug use associated with sleatoliepatitis, (d) evidence of genetic liver disease or (e) chronic hepatitis C infection (see, Ludwig, J. R. et al., (1980) lvfayo Clin. Proc., 55:434; Powell E. et aL , (1990) flepatot, 11:74). In one embodiment, the TNFa antibody, or antigen-binding fragment thereof, of the invention can be used to treat NASH.
H. Skin and Nail Disorders In one ambodiment, the TNFa. antibodyof the invention is used to treat sldn and = 15 nail disorders. As used herein, the term "skin and nail disorder in which TNFa. activity is detrimental" is intended to include skin and/or nail disorders and other disorders in which the presence of TNFa in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the patbophysiology of the disorder or a factor that contributes to a worsening of the disorder, e.g., psoriasis.
Accordingly, skin and nai1 disorders in which TNFa activity is detrimental are disorders in which inhibition of TN-Fa. activity is expected to alleviate the symptoms and/or progression of the disorder. The use of the antibodies, antibody portions, and other TN-Fcc inhibitors of the invention in the treatment of specific skin and nail disorders is discussed further below. In certain embodiments, the antibody, antibody portion, or other TN-Fec inhibitor or the invention is administered to the subject in con.thination -with another therapeutic agent, as described below in Section M. In one embodiment, the TNFcc antibody of the invention is administered to the subject in combination with another therapeutic agent for the treatment of psoriasis and the treatment of psoriasis associated with arthritis.

1. Psoriasis Tumor necrosis =factor has been implicated in the pathophysiology of psoriasis (Taketnatsu et al. (1989) Arch Dermatol Res. 281:398; Victor and Gottlieb (2002) J
Drugs Dermatol. 1(3):2641). Psoriasis is described as a skin inflammation (irritation and redness) characterized by frequent episodes of redness, itching, and thick, dry, silvery scales on the skin. In particular, lesions are forrned which involve primary and secondary alterations in epidermal proliferation, inflartunatory responses of the skin, and an expression of regulatory molecules such as lympholcines and inflammatory factors.
Psoriatic skin is morphologically characterized by an increased. turnover of epidermal cells, thickened epidermis, abnorrnalkeratinization, inflammatory cell hifiltrates into the epidermis and polymorphonuclear leukocyte and lymphocyte infiltration into the epiderinis layer resulting in an increase in the basal cell cycle. Psoriasis often involves the nails, which frequently exhibit pitting, separation of the nail, thickening, and discoloration. Psoriasis is often associated with other inflammatory disorders, for example arthritis, including rheumatoid arthritis, inflammatory bowel disease (IBD), and Crohn's disease.
Evidence of psoriasis is most commonly seen 011 the trunk, elbows, knees, scalp, skin folds, or Engernails, but it may affect any or all parts of the skin.
Normally, it takes about a month for new skin cells to move up from the lower layers to the surface. In psoriasis, this process takes only a few clays, resulting in a build.-up of dead sldn cells and formation of thick scales. Symptoms of psoriasis include: skin patches, that are dry or red, covered with silvery scales, raised patches of skin, accompanied by red borders, that 'nay crack and become painful, and that are usually lovated on the elbows, knees, tnink, scalp, and hands; skin lesions, including pustules, cracking of the skin, and skin redness; joint pain or aching which may be associated with of arthritis, e.g., psoriatic arthritis.
Treatment for psoriasis often includes a topical corticosteroids, vitamin D
analogs, and topical or oral retinoids, or combinations thereof. In one embodiment, the TI\TFcc inhibitor of the invention is administered in combination with or the presence of one of these common treatments. Additional therapeutic agents which can also be combined with the TNFcc inhibitor of the invention for treatment of psoriasis are described in more cictail in Section ftl.B.

The diagnosis of psoriasis is usually based on the appearance of the skin.
Additionally a skin biopsy, or scraping tnd culture of skin patches may be needed to rule out other skin disorders. An x-ray may be used to check for psoriatic arthritis if joint pain is present and persistent.
In one embodiment of the invention, a rINFa inhibitor is used to treat psoriasis, including chronic plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, pernphigus vulgaris, crythrodennic psoriasis, psoriasis associated with inflammatory bowel disease (I13D), and psoriasis associated with rheumatoid arthritis (RA).
Specific types of psoriasis included in the treatment methods of the invention are described in detail below:
a. Chronic plaque psoriasis Tumor necrosis factor has been implicated in the pathophysiology of chronic plaque psoriasis (Asadullah et al. (1999) .Br J Derma to1.141:94). Chronic plaque psoriasis (also referred to as psoriasis vulgaris) is tbc most common form of psoriasis.
Chronic plaque psoriasis is characterized by raised reddened patches of skin, ranging from coin-sized to much larger. In chronic plaque psoriasis, the plaques may be single or multiple, they may vary in size from a few Inillimeters to several centimeters. The plaques are usually red with a scaly surface, and reflect light when gently scratched, creating a "silvery" effect. Lesions (which are often synnmehical) from chronic plaque psoriasis occur all over body, but with predilection for extensor surfaces, including the knees, elbows, lumbosacral regions, scalp, and nails. Occasionally chronic plaque psoriasis can occur on the penis, vulva and flexures, but scaling is usually absent.
Diagnosis of patients with chronic plaque psoriasis is usually based on the clinical features described above. In particular, the distribution, color and typical silvery scaling of the lesion in chronic plaque psoriasis are characteristic of chronic plaque psoriasis.
b. Guttate psoriasis Guttate psoriasis refers to a form ofpsoriasis with characteristic water drop shaped scaly plaques. Flares of guttate psoriasis generally follow an infection, most notably a streptococcal throat infection. Diagnosis of guttate psoriasis is usually based on the appearance of the skin, and the fact that there is often a history of recent sore throat.
c. /7 verse psoriasi.s.
Inverse psoriasis is a form of psoriasis in which the patient has smooth, usually moist areas of slcin that arc red and inflammed, which is unlike the scaling associated with plaque psoriasis. Inverse psoriasis is also referred to as intertiginous psoriasis or flexural psoriasis. Inverse psoriasis occurs mostly in the armpits, groin, under the breasts and in other skin folds around the genitals and buttocks, and, as a result of the locations of presentation, rubbing and sweating can iniate the affected areas.
d. Piolular psoriasis Pustular psoriasis is a form of psoriasis that causes pus-filled blisters that vary in size and location, but oflen occur on the hands and feet. The blisters may be localized, or spread over large areas of the body. Pustular psoriasis can be both tender and painful, can cause fevers.
e. Other psoriasis disorders Other examples of psoriatic disorders which can be treated with the TNFu antibody of the invention include crythrodermic psoriasis, vulgaris, psoriasis associated with IDD, and psoriasis associated with arthritis, including rheumatoid arthritis.
2. Pemphigus vulgaris Pemphigus vulgaris is a serious autoinunune systemic dermatologic disease that often affects the oral mucous membrane and skin. The pathogenesis of pemphigus vulgaris is thought to be an autoirarnune process that is directed at skin and oral mucous membrane desmosomes. Consequentially, cells do not adhere to each other. The disorder manifests as large fluid-filled, rupturc-prone bullae, and has a distinctive histologic appearance. Anti-inflammatory agents are the only effective therapy for this disease which has a high mortality rate. Complications that arise in patients suffering from peniphigus vulgaris arc intractable pain, interference with nutrition and fluid loss, ;mil infections.
3. A/op ic dermatitis /eczema Atopic dermatitis (also referred to as eczema) is a chronic; skin disorder categorized by scaly and itching plaques. People with eczema often have a family history of allergic conditions like asthma, hay fever, or eczema. Atopic dermatitis is a hypersensitivity reaction (similar to an allergy) which occurs in file skin, causing chronic 'inilinumation. Tin, inflammation causes the skin to become itchy and scaly.
Chronic irritation and scratching can CallSe the skin to thicken and become leathery-textured.
Exposure to environmental irritants can worsen symptoms, as can dryness of the skin, exposure to water, temperature changes, and stress.
Subjects with atopie dermatitis can be identified by certain symptoms, which often include intense itching, blisters with oozing and crusting, skin redness or inflammation around the blisters, rash, dry, leathery skin areas, raw areas of the skin ham scratching, and car discharges/bleeding.
4. Sarcoidosis, Sarcoidosis is a disease in which granulowatous inflammation occurs in the lymph nodes, lungs, liver, eyes, skin, ind/or other tissues. Sareonlosis includes cutaneous sarcoidosis (sarcoidosis of the skin) and nodular sarcoidosis (sarcoidosis of the lymph nodes). Patients with sincoidosis can be identified by the symptoms, which often include general discomfort, uneasiness, or an ill feeling; fever; skin lesions.
5. iftytheiiia nodoszinz Erytherna nodositin refers to an inflammatory disorder that is characterized by tender, red nodules -under the skin, typically on the anterior lower legs.
Lesions associated with erythema nodosum often begin as flat, but firm, hot red painful lumps (approximately an inch across). Within a few days the lesions may become purplish, and then Over several weeks fade to a brownish flat patch.
- 6,1-In sonic instances, erythema nodosum may be associated with infectialIS
including, streptococcus, coccidioidomycosis, tuberculosis, hepatitis 113, syphilis, cat scratch disease, tudaremia, yersinia, leptospirosis psittacosis, Itistoplasmosis, mononucleosis (EBV). In other instances, erythema nodosrum may be associated with sensitivity to certain medications including, oralcontraccptives, penicillin, sulfonainide.s, sulfoiccs, barbiturates, hydantoin, plienacctin, salicylates, iodides, and progestin.
Erythema nodostun is ellen associated with other disorders including, leukemia, sarcoidosis, rheinnatic fever, and ulcerative colitis.
Symptoms of etythcma nodosum usually present themselves on the shins, but ID lesions may also occur OT1 other areas of the body, including the buttocks, calves, ankles, thif;lis and upper e,xtremitics. Other symptc-nns in subjects with erythema nodosurn can include fever and malaise.
6. = Ilidradonitis suppurative 1.5 I=Edradenitis suppurativa refers to a skin disorder in which swollen, painful, inflamed lesions or lumps develop in the groin and sometimes under the arms and under the breasts. flidradcnitis suppurativa occurs when apocrine gland outlets become blocked by perspiratio.n or arc unable to drain normally 'because of incomplete gland development. Secretions trapped in the gltuids force perspiration and bacteria. into 20 surrounding tissue, causing subcutaneous induration, inflammation, and infection, suppurativa is confined to areas of the body that contain apocrine gla.ncls.
These areas are the axillae, areola of the nipple, groin, perineurn, circurnanal, and periumbilical regions.
25 7. Lichen planus Tumor necrosis factor has been irnplicated in the pathophysiology olliehen plaints (Sklavounott et al. (2000)J Oral Pathol Med. 29:370). Lichen plarms refers to a disorder of the skin and the mucous membranes resulting in inflammation, itching, and distinctive skin lesions. Lichen pianus may be associated with hepatitis C or certain 30 medications.

8. Sweet's syndrome inflammatory cytokines, including tumor necrosis factor, have been implicated in tile pathophysiology of Sweet's syndrome (Reuss-Borst et al. (1993) Br J
Haentatol.
84:356). Sweet's syndrome, which was described by R.D. Sweet in 1964, is characterized by the sudden onset of fever, leukocytosis, and cutaneous eruption. The eruption consists of tender, erythematous, well-demarcated papules and plaques which show dense neutrophilic infiltrates microscopically. The lesions may appear anywhere, but favor the upper body including the face. The individual lesions are often described as pseudovcsicular or pseudopustular, but may be fraalcly pustular, bullous, or ulcerative, Oral and cye involvement (conjunctivitis or episcleritis) have also been frequently reported in patients with Sweet's syndrome. Leukemia has also been associated with Sweet's syndrome.
=
9. Tlitiligo Vitiligo refers to a skin condition in which there is loss of pitynent from areas of skin resulting in irregular white patches with normal skin texture. Lesions characteristic of vitiligo appear as flat depigmented areas. The edges adze lesions are sharply defined but irregular. Frequently affected areas in subjects with vitiligo include the face, elbows and lames, hands and feet, and genitalia.

10. Seleroderma Tumor necrosis factor has been implicated in the pathophysiology of scleroderma.
(Ttituneu Z et al. (2002) Clin Exp Rheinnatol. 20(6 Stipp' 28):S146-51;
Mackiewicz Z et = al. (2003) Clin Rip Rheumatol. 21(0:41-8; lvlurota 1-1 et al. (2003) Arthritis Rhezun.
48(4):1117-25). Sclerodenua refers to a a diffuse connective tissue disease characterized by changes in the skin, blood vessels, skeletal muscles, and internal organs.
Scleroderma is also referred to as CREST syndrome or Progressive systemic sclerosis, and usually affects people between the ages 30-50. Women are affected more often than men.
The cause of scleroderma is unknown. The disease may produce local or systemic symptoms. The course and severity of the disease varies widely in those affected.Excess collagen deposits in the skin and other organs produce the symptoms.

Damage to small blood vessels within the skin and affected organs also occurs.
In the skin, ulceration, calcification, and changes in pigmentation may occur.
Systemic features may include fibrosis and degeneration of the heart, Itmgs, kidneys and gastrointestinal tract.
Patients suffering from sclerodemia exhibit certain clinical features, including, blanching, blueness, or redness of fingers and toes in response to heat and cold (Raynaud's phenomenon), pain, stiffness, and swelling of fingers and joints, skin Thicloming and shiny hands arid forearm, esophageal reflux or heartburn, difficulty swallowing, and shortness of breath. Other clinical sypintonis used to diagnose sclerodenna include, an elevated erythrocyte sedirnentaion rate (BSR), an elevated rheumatoid factor (RF), a positive antinuclear antibody test, urinalysis that shows protein and microscopic blood, a chest X-ray that may show fibrosis, and pulmonary funtion studies that show restricitive lung disease.

11. Nail disorders Nail disorders include any abnormality of the nail. Specific nail disorders include, but are not limited to, pitting, koilonychia, Beau's lines, spoon nails, onycholysis, yellow nails, pterygium (seen in lichen planus), and leukonychia.
Pitting is characterised by the presence of small depressions on the nail surface. Ridges or linear elevations can develop along the nail occurring in a "lengthwise" or "crosswise"
direction. Beau's lines are linear depressions that occur "crosswise"
(transverse) in the = fingernail. Leukonychia describes white streaks or spots on the nails.
Koilonychia is an abnormal shape of the fingernail where the nail has raised ridges and is thin and concave = Koilonychia is often associated with iron deficiency.
Nail disorders which can be treated with the TNFa. antibody of the invention also include psoriatic nails. Psoriatic nails include changes in nails which are attributable to psoriasis. In some instances psoriasis may occur only in the nails and nowhere else on the body. Psoriatic changes in nails range from mild to severe, generally reflecting the extent of psoriatic involvement of the nail plate, nail matrix, i.e., tissue from which the nail grows, nail bed, i.e., tissue under the nail, and skin at the base of the nail. Damage to the nail bed by the pustular type ofpsoriasis can result in loss of the nail. Nail changes in psoriasis fall into general categories that may occur singly or all together. In one category of psoriatic nails, the nail plate is deeply pitted, probably due to defects in nail growth caused by psoriasis. 1N another categot-y, the nail has a yellow to yellow-pink discoloration, probably due to psoriatic involvement of the nail bed. A
third subtype of psoriatic nails are characterized by white areas which appear under the nail plate. The white areas are actually air bubbles marking spots where the nail plate is becoming detached from the nail bcd. There may also be reddened skin around the nail.
A fourth category is evidenced by the nail plate crumbling in yellowish patches, Le., onyehodystropliy, probably due to psoriatic involvenient in the nail matrix. A
fifth category is characterized by the loss of the nail M its entirety due to psoriatic involvement of the nail matrix and nail bed.
The TN-Fa. antibody of (he invention can also be used to treat nail disorders often associated with lichen planus. Nails in subjects with lichen planus often show thinning and surface roughness of the nail plate with longitudinal ridges or pterygiurn.
The TNI7a. antibody of the invention can be used to treat nail disorders, such as those described herein. Often nail disorders are associated with skin disorders. Ii one embodiment, the invention includes a method of treatment for nail disorders with a TN-Fec antibody. In another embodiment, the nail disorder is associated with another disorder-, including a slcin disorder such as psoriasis. In another embodiment, the disorder associated with a nail disorder is arthritis, including psoriatic arthritis.

12. Other Skin and Nail Disorders The TN-Fcc antibody of the invention can be used to treat other skin and nail disorders, such as chronic actinic dermatitis, bullous pemphigoid, and alopecia areata_ Chronic actinic dermatitis (CAD) is also referred to as photosensitivity dermatitis/actinic reticuloid syndrome (PD/AR). CAD is a condition in which the skin becomes inflamed, particularly in areas that have been exposed to sunlight or artificial light.
Commonly, CAD patients have allergies to certain substances that come into contact with their skin, pal ticularly various flowers, woods, per-Runes, sunscreens and rubber compounds.
Bullous pernphigoid refers to A skin disorder characterized by the formation of large blisters ou tlie trunk and extremities. Alopecia areata refers to bair loss characterized by round patches of complete baldness in the scalp or beard.

I. Vasculitides TNFa has been implicated in the pathophysiology of a variety of vasculitides, (see e.g., Degueln et al. (1989) Lancet. 2:745). In one embodiment, the invention provides it method for inhibiting TNFa. activity in a subject suffering from a vasculitis in which TNFa activity is detrimental.
As used herein, the term "a vasculitis in which TNFa activity is detrimental"
is intended to include vasculitis in which the presence of TNFa in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Such disorders may be evidenced, for example, by an increase in the concentration of TNFo. in a biological fluid of a subject suffering from the disorder (e.g., an increase in the concentration of TNFa in senun, plasma, synovial fluid, etc.. of the subject), which can be detected, for example, using an anti-TNFa antibody as described above, There are numerous examples of vasculitides in which TNFa activity is = dettirnental, including Delicet's disease. The use of the antibodies, antibody portions, and other TNFa inhibitors of the invention in the treatment of specific vasculitides are discussed further below. In certain embodiments, the antibody, antibody portion, or other TNFa inhibitor of the invention is administered to the subject in combination with another therapeutic agent, as described below The antibody of the invention can be used to treat vasculitis in which TNFa .
activity is detrimental, wherein inhibition of TNFa activity is expected to alleviate the symptoms and/or progression of the vasculitis or to prevent the vasculitis.
Subjects suffering from or at risk of developing vasculitis can be identified through clinical symptoms aud tests. For example, subjects with vasculitides ollen develop antibodies to certain proteins in the cytoplasm of neutrophils, antineutrophil cytoplasmic antibodies (ANCA). Thus, in some instances, vasculitides may be evidenced by tests (e.g., ELISA), which measure ANCA presence.
Vasculitis and its consequences may be the sole manifestation of disease or it may be a. secondary component of another primary disease. Vasculitis niay be confined to a single organ or it may simultaneously affect several organs. and depending on the syndrome, arteries and veins of all sizes can be affected. Vasculitis can affect auy organ in the body.
It) vasculitis, the vessel, lumen is usually compromised, which is associated with ischemia of the tissues supplied by the involved vessel. The broad range of disorders .5 that may result from this process is due to the fact that any type, size and location of vessel (e.g., artery, vein, artetiole, verrule, capillary) can be involved.
Vasculitides are generally classified according to the size of the affected vessels, as described below. It should be noted that some small and large vessel vasculitides may involve medium-sized arteries; but large and medium-sized vessel vasculitides do not involve vessels smaller than arteries. Large vessel disease includes, but is not limited to, giant cell arteritis, also known as temporal arteritis or cranial arteritis, polymyalgia rheumatics, and Takayasu's disease or arteritis, which is also known as aortic arch syndrome, young female arteritis and Pulseless disease. Medium vessel disease includes, but is not limited to, classic polyarteritis nodosa and Kawasaki's disease, also known as mucocutaneous lymph node syndrome. Non-limiting examples of small vessel disease arel3clicet's Syndrome, Wegner's granulomatosis, microscopic polyangitis, hypersensitivity vasculitis, also known as cutaneous vasculitis, stn.all vessel vasculitis, Henoch-Schonleinpurpura, allergic granulamotosis and vasculitis, also known as Churg Strauss syndrome.
Other vasculitides include, but are not limited to, isolated central nervous system vasculitis, and thromboangitis obliterans, also known as Buerger's disease. Classic Polyarteritis nodosa (PAN), microscopic PAN, and allergic granulomatosis are also alien grouped = together and are called the systemic necrotizing vasculitides. A further description of vasculitis is described below:
1. Large vessel vasculitis in one embodiment, the TNFa antibody of the invention is used to treat subjects who have large vessel vasculitis. The term "large vessel(s)" as used herein, refers to the aorta and the largest branches directed toward major body regions. Large vessels include, for example, the aorta, and its branches and corresponding veins, e.g., the subclavian artery; the brachiocephalic artery; the common carotid artery; the innonimate vein; internal and external jugular veins; the pulmonary arteries and veins;
the venae =

cavac; the renal arteries and veins; the femoral arteries and veins; and the carotid arteries. Examples of large vessel vascnlitides arc described below.
a. Giant cell arteritis (GC/I) Tumor necrosis factor has been implicated in the pathophysiology of giant cell arteritis (Sneller, M.C. (2002) Cleve. Clin. i Med. 69:ST140-3; Schett, G., et al. (2002) Ann. Rheinn. Dis. 61:463). Giant cell arteritis (GCA), refers to a vasculitis involving inflammation and damage to blood vessels, particularly the large or medium arteries that branch from the external carotid artery of the neck. GCA is also referred to as temporal arteritis or cranial arteritis, and is the most common primary vasculitis in the elderly. It almost exclusively affects individuals over 50 years of age, however, there are well-documented cases of patients 40 years and younger. GCA usually affects.extracranial arteries. GCA can affect the branches of the carotid arteries, including the temporal artery. GCA is also a systemic disease which can involve arteries in multiple locations, . Iiistopathologically, GCA is a panarteritis with inflammatory mononuclear cell infiltrates within the vessel wall with frequent Langban.s type giant cell formation. There is proliferation of the intima, granulomatous inflammation and fragmentation of the internal elastic lamina. The pathological findings in organs is the result of ischernia related to the involved. vessels.
Patients suffering from GCA exhibit certain clinical symptoms, including fever, -headache, anemia and high erythrocyte sedimentation rate (ESR). Other typical indications of GCA include jaw or tongue claudication, scalp tenderness, constitutional symptoms, pale optic disc edema (particularly 'chalky white' disc edema), and vision disturbances. The diagnosis is confirmed by temporal artery biopsy.
b. Polymyalgia rheumatic Tumor necrosis factor has been implicated in the pathophysiology of polynayalgia rheumatica (Straub, 12.11., et al. (2002)1Meuinatology (Oxford) 41:423;
Uddhammar, A., et al. (1998) Br. 1?hetanato1.37:766). Polymyalgia dieumatica refers to a rheumatic disorder that is associated with moderate to severe muscle pain and stiffness in the neck, shoulder, and hip, most noticeable in the morning. IL-6 and IL-113 expression has also been detected in a majority of the circulating monocytes in patients with the polymyalgia rhetimatica. Polyinyalgia rheumatica may occur independently, or it may coexist with or precede GCA, which is an inflammation of blood vessels.
c. Takayasies Arleritis Tumor necrosis factor has been implicated in thc pathophysiology of Takayasu's arteritis (Kobayashi, Y. and Numano, F. (2002) Inteni. Me(L 41:44; Fraga, A.
and Medina F. (2002) CWT. Rhettillai01. .Rep.4:30). Talcayasu's arteritis refers to a vascu.litis characterized by an inflammmation of the aorta and its major branches.
Takayasu's artcritis (also known as Aortic arch syndrome, young female arteritis and.
Pulseless disease) affects the thoracic and abdominal aorta and its main branches or the pulmonary arteries. Fibrotic thickening of the aortic wall and its branches (e.g., carotid, inominate, and subclavian arteries) can lead to reduction of lumen size of vessels that arise from the aortic arch. This condition also typically affects the renal arteries.
= Takayastes arteritis primarily affects young women, usually aged 20-40 years old, particularly of Asian descent, and may be manifested by malaise, arthralgias and the gradual onset of extremity claudication. Most patients have asymmetrically reduced pulses, usually along with a blood pressure differential in the arms. Coronary and/or renal artery stenosis may occur.
The clinical features of Takayasu's arteritis may be divided into the features of the early inflammatory disease and the features of the later disease. The clinical features of the early infla_mmatory stage of Takayasu's disease ate: malaise, low grade fever, weight loss, myalgia, arthrala, and erythema multiforme. Later stages of Takayasu's disease are characterised by fibrotic stenosis of arteries and thrombosis. The main resulting clinical features are ischaenuic phenomena, e.g. weak and asymmetrical arterial pulses, blood pressure discrepancy between the arms, visual disturbance, e.g.
scotomata and hemianopia, other neurological features including vertigo and syncope, hemiparesis or stroke. The clinical features result nom ischaemia due to arterial stenosis and thrombosis_ 2. Medium Vessel Disease In one embodiment, the TNFa antibody of the invention is used to treat subjects who have medium vessel vasculitis. The term "medium vessel(s)" is used to refer to - '12 -those blood vessels which are the main visceral arteries. Examples of mecii-um vessels include the mesenteric arteries and veins, the iliac arteries and veins, and the maxillary arteries and veins. Examples of medium vessel vasculitides are described below.
a. Polyarteritis Nodosa Tumor necrosis factor has been-implicated ili ihe pathophysio logy of polyarteritis nodosa (DiGirolamo, N., et al. (1997)J. Leukoc. Biol. 61:667). Polyarteritis noclosa, or periarteritis nodosa refers to vasculitis which is a serious blood vessel disease in which small and medium-sized arteries become swollen and damaged because they are attacked by rogue immune cells. Polyarteritis nodosa usually affects adults more frequently than children. It damages the tissues supplied by the affected arteries because they don't receive enough oxygen and nourishment without a proper blood supply.
Symptoms which are exhibited in patients with polyarteritis nodosa generally result from damage to affected organs, often the skin, heart, lcidneys, and nervous system. Generalized syinptorns of polyarteritis nodosa include fever, fatigue, weakness, loss o appetite, and weight loss. Muscle aches (myalgia) and joint aches(arthralgia) are common, The skin of subjects with polyarteritis nodosa may also show rashes, swelling, ulcers, and lumps (nodular lesions).
Classic PAN (polyarteritis nodosa) is a systemic arteritis of small to medium muscular arteritis in which involvement of renal and visceral arteries is COMITIOU.
Abdominal vessels have aneurysms or occlusions in 50% of PAN patients. Classic PAN
does not involve the puhnonary arteries although the bronchial vessels may be involved.
Granulomas, significant eosinophilia and an allergic diathesis are not part of the syndrome. Although any organ system may be involved, the most common manifestations include peripheral neuropathy, mononeuritis multiplex, intestinal ischemia, renal ischemia, testicular pain and livedo reticularis.
b. .Kawasaki's Disease Tumor necrosis factor has been implicated in the pathophysialogy of Kawasaki's disease (Sundel, R.P. (2002) Curr. .Rheurnatol. Rep. 4:474; Gedalia, A.(2002) Curr.
Rheionatol. Rep. 4:25). Although the cause of Kawasaki's disease is unknown, it is associated with acute inflammation oftbe coronmy arteries, suggesting that the tissue damage associated with this disease may be mediated by proinflaramatory agents such as TN-Foc. Kawasaki's disease refers lo a vasculitis that affects the mucus membranes, lymph nodes, lining of the blood vessels, and the heart. Kawasaki's disease is also often referred to as mucocutaneous lymph node syndrome, mucocutaneous lymph node disease, and infantile polyarteritis. Subjects afflicted with Kawasaki's disease develop vasculitis often involving the coronary arteries which can lead to myocarrlitis and pericarditis. Often as the acute inflamniation diminishes, the coronary arteries may develop aneurysm, thrombosis, and lead to myocardial infarction_ Kawasaki's disease is a febrile systemic vasculitis associated with edema in the palms and the soles of the feet, with enlargement of cervical lymph nodes, cracked lips and "strawberry tongue". Although the inflammatory response is found in vessels throughout the body, the most common site of end-organ damage is the coronary arteries. Kawasaki's Disease predominantly affects children under the age of 5. The highest incidence is in Japan but is becoming increasingly recognized in the West and is now the leading cause of acquired heart disease in US children. The most serious complication of Kawasaki disease is coronary arteritis and aneurysm formation that occurs in a third of untreated patients.
3. Small vessel disease In one: embodiment, the TN-Fa antibody of the invention is used to treat subjects who have small vessel vasculitis. The term "small vessel(s)" is used to refer to arterioles, venules and capillaries. Arterioles are arteries that conta.in only 1 or 2 layers of sooth muscle cells and are terminal to and continuous with the capillary network.
Venules carry blood from the capillary network to veins and capillaries connect arterioles and venules. Examples of small vessel vasculitides are described below.
a. Behcet's Disease Tumor necrosis factor bas been implicated in the pathophysiology of Beticet's disease (Sfikaids, P.P. (2002) Ann. Rheum_ Dis. 61 :ii51-3; Dogan, D. and Farah, C.
(2002) Oftalrnologia. 52:23). Behcet's disease is a chronic disorder that involves inflammation of blood vessels throughout the body. Behcet's disease may also cause various types of slcin lesions, arthritis, bowel inflammation, and meningitis (infiamimition of the membranes of the brain and spinal cord). As a result of Beheet's disease, the subject with the disonler may have inflammation in tissues and organs throughout the body, including the gastrointestinal tract, central nervous system, vascular system, lungs, and kidneys. Behcet's disease is three times more common in males than females and is more common in the cast Mediterranean and Japan.
Subjects who have Behcet's disease may show clinical symptoms including recurrent oral ulcers (resembling canker sores), rccun-ent genital ulcers, and eye inflammation. Scrum levels of TI\l"Fcc, IL-8, IL-1, IL-611\fF-y and IL-12 are elevated in Belicet's patients, and the production of these factors has been shown to be elevated in the monocytes of Beheet's patients (see, e.g., Inflammatoty Disease of Blood Vessels (2001) Marcel Dekker, Inc., eds. G.S. Hoffman and C.M. Weyand, p. 473).
b. Wegener's granulomatosis Tumor necrosis factor has been implicated in the pathophysiology of Wegener's gr, -anulomatosis (Marquez, J., et al. (2003) Curr. Rheumatol. Rep. 5:128; I-Iarman, L.E.
and Margo, C.E. (1998) Sun). Oplahahnol. 42:458). Wegener's granulomatosis refers to a vasculitis that causes inllainmation of blood vessels in the upper respiratory tract (nose, sinuses, ears), lungs, and kidneys. Wegener's granulomatosis is also referred to as niidline granulomatosis. Wegener's granulomatosis includes a granulomatous inflammation involving the respiratory tract, and necrotizing vasculitis affecting small to medium-sized vessels. Subjects who have Wegener's granulonaatosis often also have arthritis (joint i ntlanunation). Glomerulonephritis may also be present in affected subjects, but virtually any organ may be involved.
Patients affected with Wegener's granulomatosis typically show clinical symptoms comprising recurrent sinusitis or epistaxis, mucosal ulcerations, otitis media, cough, hemoptysis and dyspnea. The first symptoms of Wegener's granulomatosis frequently include upper respiratory tract symptoms, joint pains, weakness, and tiredness.
c. Churg-Strauss syndrome Tumor necrosis factor has been implicated in the patbophysiology of Churg-Strauss syndrome (Gross, W.L (2002) Curr. Opin. Rheumatol. 14:11; Churg, W.A.(2001) Mod. Pathol, 14:1284). Churg-Strauss syndrome refers to a vasculitis that is systemic and shows early manifestation signs of asthma and eosinophilia.
Churg-Strauss syndrome is also referred to as allergic granulontatosis and angiitis, and occurs in the setting of allergic rhinitis, asthma and eosinophilia. Sinusitis and pulmonary infiltrates also occur in Churg-Strauss syndrome, primarily affecting the lung and heart.
Peripheral neuropathy, coronary arteritis and gastrointestinal involvement are common.
Patients afflicted with Churg-Strauss syndrome can be diagnosed according to criteria established by the American College of Rheumatology (ACR). These criteria were intended to distinguish CSS from other tbrrns of vasculitis. Not all patients meet every criterion. Some, in fact, may have only 2 or 3 criteria, yet they are still classified as Churg-Strauss syndrome. The ACR selected 6 disease features (criteria) as being those that best distinguished Churg-Strauss syndrome frorn other vasculi tides.
These criteria include: 1) asthma; 2) eosinophilia [>10% on differential WBC count]; 3) mononeuropathy; 4) transient pulmonary infiltrates on chest X-rays; 5) paranasal .sinus abnormalities; and 6) biopsy containing a blood vessel with extravascular eosinophils.
J. Other TNEct-Related Disorders in one embodiment, the invention features a method for -heating a TNFa-related disorder in which TNFa activity is detrimental, comprising administering to a subject an effective amount of a TNFa inhibitor, such tbat said TNFot-related disorder is treated.
Examples of TNFa-related disorders in which TNFa activity is detrimental, are discusse,d further below.
1. Crohn's Disease-Related Disorders Tumor necrosis factor has been implicated in the pathophysiology of inflammatory bowel disorders (1BD), including Crohn's disease (see e.g., Tracy, KJ., et al. (1986) Science 234:470-474; S11.11, X-M., et al. (1988)J. Clin. Invest.
81:1328-1331;
MacDonald, T.T., et al. (1990) Clin. Exp. Imnzunol. 81:301-305).
In one embodiment, the TNFa inhibitor of the invention is used to treat disorders often associated with IBD and Crohn's disease. The term "inflammatory bowel disorder (I3D)-re1ated disorder" or "Crohn's disease-related disorder," as used interchangeably herein, is used to describe conditions and complications commonly associated with lBD

and Crohn's disease. Examples of Crohn's disease-related disorders include fistulas iu the bladder, vagina, and skin; bowel obstructions; abscesses; nutritional deficiencies;
complications from corticosteroid use; inflatnrnation of the joints; erythern nodosum;
pyoderma guagrenosimi; and lesions of the eye. Other disorders commonly associated with Crohn's disease include Crohn's-related arthralgias, ftstulizing Crohn's, indctenninant colitis, and pouchitis.
2. .11-0811ile Tumor necrosis factor has been implicated in the pathophysiology of juvenile arthritis, including juvenile rheumatoid arthritis (Grom et al. (1996) Arthritis Rheum.
39:1703; Mangge el al. (1995) Arthritis Rheum. 8:211). In one embodiment, the TNFoc antibody of the invention is used to treat juvenile rheumatoid arthritis.
The term "juvenile rheumatoid arthritis" or "IRA" as used herein refers to a chronic, inflammatory disease which occurs before age 16 that may cause joint or connective tissue damage. SRA is also referred to as juvenile chronic polyarthritis and Still's disease.
SRA causes joint inflammation and stiffness for more than 6 weeks in a child of 16 years of age or less. Inflammation causes redness, swelling, warmth, and soreness in the joints. Any joint can be affected and inflammation may limit the mobility of affected joints. One type of.TRA can also affect the internal organs.
jRA is often classified into three types by the number of joints involved, the symptoms, and the presence or absence of certain antibodies found by a blood test.
These classifications help the physician determine how the disease will progress and whether the internal organs or skin is affected. The classifications of SRA
include the following a. Pauciarticular JRA, wherein the patient has four or fewer joints are affected. Pauciarticular is the most common form ofJRA, and typically affects large joints, such as the knees.
b. Polyarticular HRA, wherein five or more joints are affected. The small joints, such as those in the hands and feet, are most commonly involved, but the disease may also affect large joints.

Systemic JRA is characterized by joint swelling, fever, a light skin rash, and may also affect internal organs such as the heart, liver, spleen, and lymph. nodes.
Systemic JRA is also refen-ed to as it Still's disease. A small percentage of these children develop arthritis in many joints and can have severe arthritis that continues into adulthood.
3. Entionzetriosis Tumor necrosis factor has been implicated in the pathophysiology of endometriosis, as women with endometriosis have elevated peritoneal levels of TNF
(Eiserrnann J, et al. (1988) Feral Stoll 50:573; Halrne I. (1989) A nz J
Obstet Gynecol 161:1718; Mori H, et al. (1991)/Im J Reprod I-limn/m[26:62; .Taketani Y, et al. (1992) Ant J Obstet Gyitecol 167:265; Overton C, et al. (1996) Hunt Reprod 1996;11:380). In one embodiment, the TNFa antibody of the invention is used to treat endornettiosis.
The term "endometriosis" as used herein refers to a condition in which the tissue that normally lines the uterus (endometrium) grows in other areas of the body, causing pain, irregular bleeding, and frequently infertility.
4. Prostatitis Minor necrosis factor has been implicated in the pathophysiology of prostatitis, as Men with chronic prostatitis and chronic pelvic pain have significantly higher levels of TNF and IL-1 in semen compared to controls (Alexander RB, et al. (1998) Urology 52:744; Nadler RB, et al. (2000)J Urol 164:214; Orhan et al. (2001) Int J Urol 8:495) Furthermore, in a rat model of prostatitis TNF levels were also increased in comparison to controls (Asakawa K, et al. (2001) .Flinyolcilca Kiyo 47:459; Harris et al.
(2000) Prostate 44:25). In one embodiment, the TNFcc antibody of the invention is used to treat prostatitis.
The term "prostatitis" as used herein refers to an inflatunation of the prostate.
Prostatitis is also referred to as peMc pain syndrome. Prostzttitis manifests itself in a variety of forms, including nonbacterial prostatitis, acute prostatitis, bacterial prostatitis, and acute prostatitis. Acute prostatitis refers to an intlamnaation of the prostate gland that develops suddenly. Acute prostatitis is usually caused by a bacterial infection of the prostate gland. Chronic prostatitis is an inflammation of the prostate gland that develops gradually, continues for a prolonged period, and typically has subtle symptoms. Chronic prostatitis is also usually caused by a bacterial infection.
5. Autoimm line disorders Tumor necrosis factor has been implicated in the pathophysiology of many autoimmune disorders, including lupus (Shvidel et al. (2002) 1-fematol J.
3:32;
Studnicka-Benke et al. (1996) Br J Rheumatol. 35:1067). In one embodiment, the TNFa antibody of the invention is used to treat autoinamune disorders such as lupus, multisystem au toiramtme diseases, and autoimmune hearing loss.
The term "lupus" as used herein refers to a chronic, inflammatory autoirnmune disorder called lupus erythematosus that may affect many organ systems including the skin, joints and internal organs. Lupus is a general term which includes a number of specific types of lupus, including systemic lupus, lupus nephritis, and lupus cerebritis. In.
systemic lupus (SLE), the body's natural defenses are ttu-ned against the body and rogue immune cells attack the body's tissues. Antibodies may be produced that can react against the body's blood cells, organs, and tissues. This reaction leads to immune cells attacking the affected systems, producing a chronic disease. Lupus nephritis, also referred to as lupus glomerular disease, is kidney disorder that is usually a complication .20 of SLE, and is characterized by damage to the glomendus and progressive loss of kidney function. Lupus eerebritis refers to another con3plication of ST.F, which is inflammation of the brain and/or central nervous system.
6. = Choroidal neovascularization 95 Tumor necrosis factor bas been implicated in the pathophysio logy of choroidal neovascularization. For example, in surgically excised choroidalneovascular membranes, neovasculax vessels stained positive for both TNF and M-1 (Oh FI et al.
(1999)1-avast Ophthahnol Vis Sci 40:1891). In one embodiment, the TNFa antibody of the invention is used to treat choroidal neovascularization. The term "choroidal 30 neovascularization" as used herein refers to the growth of new blood vessels that originate from the choroid through a break in the Bruch membrane into the sub¨retinal - '79 -pigment epithelium (sub-RPE) or subretinal space. Choroidal neovascularization (CNV) is a major cause of visual loss inpatients with the condition.
7. Sciatica Tumor necrosis factor has beeu implicated in the pathophysiology of sciatica (Ozak.tay ee al. (2002) Eur Spine J. 11:467; Brisby et al. (2002) Eur Spine J.
11:62). In one embodiment, the TNFoc antibody of the invention is -used to treat sciatica. The term "sciatica" as used herein refers to a condition involving impaired movement and/or sensation in the leg, caused by damage to the sciatic nerve. Sciatica is also commonly referred to as neuropathy of the sciatic nerve and sciatic nerve dysfunction.
Sciatica is a form of peripheral netu-opathy. Itoccurs when there is damage to the sciatic nerve, located in the back of the leg. The sciatic nerve controls the muscles of the back of the knee and lower leg and provides sensation to the back of the thigh, part of the lower leg and the sole of the foot. Sciatica can be indicative of another disorder, including a lumbar herniated disc, spinal stenosis, degenerative disc disease, isthmic spondyloisthesis and piniformis syndrome.
8. Sjogren's syndrome Tumor necrosis factor has been implicated in the pathophysiology of Sjogren's syndrome (Koski et al. (2001) Clin Exp Rheumatol. 19:131). In one embodiment, the TNEcc antibody of the invention is used to treat Sjogren's syndrome. The term "Sjogren's syndrome" as used herein refers to a systemic inflammatory disorder characterized by dry mouth, decreased tearing, and other dry mucous membranes, and is often associated .
with autoimmune rheumatic disorders, such as rheumatoid arthritis. Dryness of the eyes and mouth are the most common symptoms of this syndrome. The symptoms may occur alone, or with symptoms associated with rheumatoid arthritis Or other connective tissue diseases. There may be an associated enlargement of the salivary glands. Other organs may become affected. The syndrome may be associated with rheumatoid arthritis, systemic lupus erythernatosus, sclerodenna, polymyositis, and other diseases.
- SO -9. Uveitis Tumor necrosis factor has been implicated in the pathophysiology of uveitis (Wakefield and Lloyd (1992) Cytokine 4:1; Woon et al. (1998) Curr Eye Res.
17:955).
In one embodiment, the TNFcc antibody of the invention is used to treat -uveitis. The term "uveitis" as used herein refers to an inflamrnati Oil of the the uvea, which is the layer between the sclera and the retina, which includes the iris, ciliary body, and the choroid.
Uveitis is also commonly referred to as iritis, pars planitis, chroiditis, chorioretinitis, anterior uveitis, and posterior uveitis. The most conaraon form of uveitis is anterior uveitis, which involves 'inflammation in the front part of the eye, which is usually isolated to the iris. This condition is often called intis. In one embodiment, the tenn uveitis refers to an inflamniation of the the uvca which excludes inflammation associated with an antoimmune disease, i.e., excludes autoinunune uveitis.
10. V1/et macular degeneration Tumor necrosis factor has beeu implicated in the pathophysiology of wet macular degeneration. In one embodiment, the TNFa antibody of the invention is used to heat wet macular degeneration. The term "wet inacular degeneration" as used herein refers to a disorder that affects the macula (the central part of the retina of the eye) and causes decreased visual acuity and possible loss of central vision_ Patients with wet macular degeneration develop new blood vessels under the retina, which causes hemorrhage, swelling, and scar tissue.
11. Osteoporosis Turnor necrosis factor has been implicated in the pathophysiology of osteoporosis, (Tsutsumimoto et al. (1999) J Bone Miner Res. 14:1751).
Osteoporosis is used to refer to a disorder characterized by the progressive loss of bone density and thinning of bone tissue. Osteoporosis occurs when the body fails to form enough new bone, or when too much old bone is reabsorbed by the body, or both. The TNFot antibody, or antigen-binding fragment thereo of the invention can be used to treat osteoporosis.

12. Osleoarthrais Tumor necrosis factor has been implicated in the pathophysiology of ostcoarthritis, (Venn et al. (1993) Arthritis Rheum. 36:819; Westacott et al.
(1994)J
Rhetanatol. 21:1710). Osteoarthritis (OA) is also referred to as hypertrophic ostcoarthritis, osteoarthrosis, and degenerative joint disease_ OA is a chronic degenerative disease of skeletal joints, which affects specific joints, commonly knees, hips, band joints and spine, in adults of all ages. OA is characterized by a number of the following manifestations including degeneration and thinning of the articular cartilage with associated development of ''ulcers'' or craters, osteophyte formation, hypertrophy of bone at the margins, and changes in the snyovial membrane and enlargement of affected joints. Furthermore, osteoarthritis is accompanied bypain and stiffness, particularly after prolonged activity. The antibody, or antigen-binding fragment thereof, of the invention can be used to teat osteoardaritis. Characteristic radiographic features of osteoarthritis include joint space mirrowing, subchondral sclerosis, osteophytosis, subchondral cyst formation, loose osseous body (or 'joint mouse").
Medications used to treat osteoa_rthritis include a variety of nonsteroidal, anti-inflammatory drugs (NSAIDs). In addition, COX 2 inhibitors, including Celebrex, Vioxx, and Dextra, aand Etoricoxib, are also used to treat OA. Steroids, which are injected directly into the joint, may also be used to reduce inflammation and pain. In one embodiment of the invention, TN17cL antibodies of the invention are administered in combination with a NSAEDs, a COX2 inhibitor, and/or steroids.

13. Other The antibodies, and antibody portions, of the invention, also can be used to treat various other disorders in which TNFcc activity is detrimental. Examples of other diseases and disorders in which TNFa activity has been implicated in the pathophysiology, and thus which can be treated using an antibody, or antibody portion, of the invention., include age-related cachexia, Alzheimer's disease, brain edema, inflammatory brain injury, cancer, cancer and cachexia, chronic fatigue syndrome, dermatornyositis, drug reactions, such as Stevens-Johnson syndrome and Jarisch-lientheimer reaction, edema in and/or around the spinal cord, familial periodic fevers, Felty's syndrome, fibrosis, glomerulonephritides (e.g. post-streptococcal glomerulonephritis or IgA nephropathy), loosening of prostheses, microscopic polyangiitis, mixed connective tissue disorder, multiple myeloma, cancer and cachexia, multiple organ disorder, myeto d.ysplastic s-yndrome, orchilism osteolysis, pancreatitis, including acute, chronic, and pancreatic abscess, periodontal disease polymyositis, progressive renal failure, pseudogout, pyodenna gangrenosum, relapsing polychondritis, rheumatic heart disease, sarcoidosis, sclerosing cholangitis, stroke, thoracoabdominal aortic aneurysm repair (TAAA), TNF receptor associated periodic syndrome (TRAPS), symptoms related to Yellow Fever vaccination, inflammatory diseases associated with the ear, chronic ear inflammation, chronic otitis m.edia with or without cholesteatoma, pediatric ear inflammation, myotosis, ovarian cancer, colorectal cancer, disorders associated with transplantation, therapy associated with inducecl inflammatory syndrome (e.g., syndromes follow/33g IL-2 administration), and a disorder associated with a reperfussion injury.
It is understood that all of the above-mentioned TNFa-related disorders include both the adult and juvenile forms of the disease where appropriate. It is also understood that all of the above-mentioned disorders include both chronic and acute forms of the disease. In addition, the TNI7n antibody of the invention can be used to treat each of the above-mentioned 'TNFa-related disorders alone or in combination with one another, e.g., a subject who is suffering from uveitis and lupus.
111. Pharmaceutical Compositions and Pharmaceutical_ Administration 'cnnpositions and Adntinistration The antibodies, antibody-portions, and other TNFct inhibitors of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody, antibody portion, or other TNTµa inhibitor of the invention and a pharmaceutically acceptable carrier. As used herein, "phamiaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as maunitol, sorbitol, or sodium chloride in the composition..
Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody, antibody portion, or other TNFa inhibitor.
The compositions of this invention may be in a variety of forms. These include, for example, liquid, seini-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred forrn depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solUtions, such as compositions similar to those used for passive immmlization of humans with other antibodies or other TNFa inhibitors.
The prefen-ed mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). in a preferred embodiment, the antibody or other TNFct inhibitor is administered by intravenous infusion or injection. In another preferred embodiment, the antibody or other TNFa inhibitor is administered by intramuscular or subcutaneous injection_ Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be fornaulated as a solution, microernulsion, dispersion, liposoine, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody, antibody portion, or other TNFa inhibitor) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. 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 surfactants. Prolonged absorption of injectable compositions can bc brought about by including in the composition an agent that delays absorption, for example, monostettrate salts and gelatin.
Supplementary active compounds can also be incorporated into the compositions.
In certain embodiments, an antibody or antibody portion of the invention is coformulated with and/or coadministered with one or more additional therapeutic agents. For example, au anti-hTNFa antibody or antibody portion of the invention may be coformulated and/or coadministered with one or more DM.ARD or one or more or one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytolcines or that bind cell surface molecules), one or more cytolines, soluble TNFa receptor (see e.g., PCT Publication No. WO 94/06476) and/or one or more chemical agents that inhibit hTNFa production or activity (such as cyclohexane-ylidene derivatives as described in PCT Publication No. WO 93/19751) or any combination thereof. Furthermore, one or more antibodies of the invention may be used in combination with two or more of thc foregoing therapeutic agents. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible side effects, complications or low level of response by the patient associated with the various monotherapies.
in one embodiment, the invention includes pharmaceutical compositions comprising an effective amount of a Mira inhibitor and a pharmaceutically acceptable carrier, wherein the effective amount of the TNFct inhibitor may be effective to treat a TNFa-related disorder, including, for example, sciatica, endometriosis, and prostatitis.
The antibodies, antibody-portions, and other TNFcc inhibitors of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is intravenous injection or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodn' /lents, the active compound may be prepared with a carrier that will protect the compowid against rapid release, such as a controlled release formulation, including implants, transderrnal patches, and rnicroencapsulated delivery systems. Biodegradable, biocompatible polyrners can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such foimulations are patented or generally lcnow to those skilled in the art.
See, e.g., Susta.imed and C'ontrolled Release Diug Deliveiy Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
The TNPa antibodies of thc invention can also be administered in the form of protein crystal formulations which include a combination of protein crystals encapsulated within a polymeric carrier to form coated particles. The coated particles of the protein crystal formulation may have a spherical morphology and be microspheres of up to 500 -micro meters in diameter or they may have some other morphology and be microparticulates. The enhanced concentration of protein crystals allows the antibody of the inve,ntion to be delivered subcutaneously. In one embodiment, the TNFi .
antibodies of the invention are delivered via a protein deliveiy system, wherein one or more of a protein ciystal formulation or composition, is administered to a subject with a TNFa-related disorder. Compositions and methods ofpreparing stabilized formulations of whole antibody crystals or antibody fragment custals are also described in WO
02/072636.
fu one embodiment, a formulation comprising the crystallized antibody fragments described in Examples 37 and 38 are used to treat a TNFa-relatecl disorder.
In certain embodiments, an antibody, antibody portion, or other TNFa inhibitor of the invention may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the lilce. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
The pharmaceutical compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective ainount" of an antibody or antibody portion of the invention. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody, antibody portion, or other TNFoc inhibitor may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody, antibody portion, other TNFa.
inhibitor to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody, antibody portion, or other TNIa inhibitor are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount Dosage regimens may be adjusted to provide the optirnum desired response (elg., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of adminisiration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetemnried quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of tbe invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is 10-150 mg, more preferably 20-80 nag and most preferably about 40 mg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated.
It is to be further tmderstood that for any particular subject, specific dosage reghnens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Ranges intermediate to the above recited concentrations, e.g., about 6-144 mWinl, are also intended to be part of this invention_ For example, ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included The invention also pertains to packaged pharmaceutical compositions which comprise a INFn inhibitor of the invention and instructions for using the inhibitor to treat TNFa-related disorders, as described above.
Another aspect of the invention pertains to kits containing a pharmaceutical composition comprising an anti-TNFa antibody and a pharmaceutically acceptable carrier and one or more pharmaceutical compositions each comprising a drug useful for treating a TN-Fa-related disorder and a pharmaceutically acceptable carrier.
Alternatively, the kit comprises a single pharmaceutical composition comprising an anti-TNFa antibody, one or more dnzgs useful for treating a TNFa-related disorder and a pharmaceutically acceptable carrier. The kits contain instructions for dosing of the pharmaceutical compositions for the treatment of a TNFa-related disorder in which the administration of an anti-TNFa antibody is beneficial, such as lupus.
The invention also pertains to packaged pharmaceutical compositions or kits which comprise a TNFcc inhibitor of the invention and instructions for using the inhibitor to treat a particular disorder in which TNFa activity is detrimental, as described above. The package or kit alternatively can contain the TNFcc inhibitor and it can be promoted for use, either within the package or through accompanying information, for the uses or treatment of the disorders described herein. The packaged pharmaceuticals or kits further can include a second agent (as described herein) packaged with or copromoted with instructions for using the second agent with a first agent (as described herein).
B. Additional therapeutic agents The invention pertains to pharmaceutical compositions and methods of use thereof for the treatment of a TNFa-related disorder. The pharmaceutical compositions cotnprise a first agent that prevents or inhibits a TNFa-related disorder. The pharmaceutical composition also may comprise a second agent that is an active pharmaceutical ingredient; that is, the second agent is therapeutic and its function is beyond that of an inactive ingredient, such as a pharmaceutical carrier, preservative, diluent, or buffer. The second agent may be usefid in treating or preventing TNFcc-related disorders. The second agent may diminish or treat at least one synaptom(s) associated with the targeted disease. The first and second agents may exert their biological effects by similar or tunclated mechanisms of action; or either one or both of the first and second agents may exert their biological effects by a multiplicity of mechanisms of action. A pharmaceutical composition may also comprise a third compound, or even more yet, wherein the third (and fourth, etc.) compound has the same characteristics of a. second agent.
It should be understood that the pharmaceutical compositions described herein may have the ftrst and second, third, or additional agents in the same pharmaceutically acceptable carrier or in a different pharmaceutically acceptable carrier for each described embodiment. It further should be understood that the first, second, third and additional agent may be administered simultaneously or sequentially within described embodiments. Alternatively, a first and second agent may be administered simultaneously, and a third or additional agent may be administered before or after the first two agents.
The combination of agents used within the methods and pharmaceutical compositions described herein rimy have a therapeutic additive or synergistic effect on the condition(s) or disease(s) targeted for treatment. The combination of agents used within the methods or pharmaceutical compositions described herein also may reduce a detrimental effect associated with at least one of the agents when administered alone or without the other agent(s) of the particular phannaceutical composition. For example, the toxicity of side effects of one agent maybe attenuated by another agent of the composition, thus allowing a higher dosage, improving patient compliance, and improving therapeutic outcome. The additive or synergistic effects, benefits, and advantages of the compositions apply to classes of therapeutic agents, either structural or functional classes, or to individual compounds themselves.
Supplementary active compounds can also be incorporated into the compositions.
In certain embodiments, an antibody or antibody portion of the invention is cofonmulated with and/or coadministered with one or more additional therapeutic agents that are useful for treating TI\TFcc-related disorder in which TNFa activity is detrimental. For example, an anti-hTNFa antibody, antibody portion, or other TNFa inhibitor of the invention may be cofonnulated and/or coadministercd with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules), one or more cytokines, soluble TNFa receptor (see e.g., PCT
Publication No. WO 94/06476) and/or one or more chemical agents that inhibit hTNFa production or activity (such as cyclohexane-yliclene derivatives as described in PCT
Publication No. WO 93/19751). Furthermore, one or more antibodies or other TNFa inhibitors of the invention maybe used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible to-xicities or complications associated with the various monotherapies. Specific therapeutic agent(s) w.e generally selected based on the particular TNFa-related disorder being treated, as discussed below.
Nonlimiting examples of therapeutic agents with which an antibody, antibody portion, or other TINTFa inhibitor of the invention can be combined include the following:
non-steroidal anti-inflaminatory drug(s) (1'ISA1Ds); cytokine suppressive anti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanized anti-TNFoc antibody; Celltech/Bayer); cA2/infliximab (chimeric anti-TNFa antibody;
Centocor); 75 kdDIFR-IgG/etanercept (75 kD TNF receptor-IgG fusion protein; Immunex; see e.g., Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNF-1gG (55 kD TNF recepcor-IgG fusion protein; Hoffmann-LaRoche); MEC-CE9.1/SB 210396 (non-deplcting primatized anti-CD4 antibody;
IDEC/SinithRiline; see e.g., Arthritis & Rheumatism (1995) Vol. 38, S185); DAB 486-1L-2 and/or DAB

2 (11.-2 fusion proteins; Seragen; see e.g., Arthritis & Rheumatisni (1993) Vol. 36,1223);
Anti-Tac (humanized anti-IL-2Ra; Protein Design Labs/Roche); 1L-4 (anti-inflammatory cytokine; DNAX./Schering); EG-10 (SCH 52000; recombinant LL-10, anti-inflammatory cytolcine; DNAX/Schering); IL-4;11..40 and/or 1L-4 agonists (e.g., agonist antibodies);
11,1RA (11,1 receptor antagonist; Synergen/Amgen); TNF-bp/s-TNF (so)uble TNT' binding protein; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284; Allier. J Physiol. - Heart and Circulatory Physiology (1995) Vol. 268, pp. 37-42);
R973401 (phosphodiesterase Type IV inhibitor; see e.g., Arthritis cti 1?heuniatism (1996) Vol. 39, No. 9 (supplement), S282); MK-966 (COX-2 Inhibitor; see e.g., Arthritis &

= Rheumatism (1996) Vol. 39, No. 9 (supplement), S81); lloprost (see e.g., Arthritis &
Rheumatism (1996) Vol. 39, No. 9 (supplement), 882); methotrexate; thalidomide (see e.g., Arthritis & Rheumatism (1996) Vol. 39 No. 9 (supplement), S282) and thalidomide-related clings (e.g., CeIgen); leflunornide (anti-inflammatory and cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S131;
Inflammation Research (1996) Vol. 45, pp. 103-107); tranexamic acid (inhibitor of plasminogen activation; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (suppleme)it), S284); T-614 (cytokine inhibitor; sec e.g,, Arthritis &
_Rheumatism (1996) Vol. 12, No. 9 (supplement), S282); prostaglandin El (see e.g., Arthritis &
Rheuntatisnt (1996) Vol. 39, No. 9 (supplement), S282); Teniclap (non-steroidal anti-inflammatory drug; see e.g., Arthritis & Rheumatism (1996) Vol. 39 No. 9 (supplement), S280);
Naproxen (non-steroidal anti-inflammatory drug; see e.g., Netiro Report (1996) Vol. 7, pp. 1209-1213); Meloxicam (non-steroidal anti-inflaminatory drug); Ibuprofen (non-steroidal anti-inflanimatory drug); Piroxicam (non-steroidal anti-inflammatory drug);
rtvt TM
Diclofenac (non-steroidal anti-inflarrunatory drug); Indomethacin (non-steroidal anti-inflammatory drug); Sulfasalazine (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No.
9 (supplement), S281); Azathioprine (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S281); ICE inhibitor (inhibitor of the enzyme interleulcin-converting enzyme); zap-70 and/or lck inhibitor (inhibitor of the tyrosine kinase zap-70 or lck); VEGF inhibitor and/or VEGF-R inhibitor (inhibitos of vascular endothelial cell growth factor or vascular endothelial cell growth factor receptor; inhibitors of angiogenesis); corticosteroid anti-inflammatory drugs (e.g., SI3203580); TNF-convertase inhibitors; anti-11-12 antibodies; anti-IL-18 antibodies; interleukin-11 (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S296); interlenkin-13 (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), 8308); interleukin-inhibitors (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S120);
gold; penicillamine; cbloroquine; hydroxychloroquine; chlorambucil;
cyclophosphamide; cyclosporine; total lymphoid irradiation; anti-thymocyte globulin;
anti-CD4 antibodies; CDS-toxins; orally-administered peptides and collagen;
lobenzarit clisodium; Cytokine Regulating Agents (CRAs) II1P228 and 1-113466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucicotides (ISIS
2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulphate;
minocycline;
anti-IL2R antibodies; marine and botanical lipids (fish and plant seed fatty acids; see e.g., DeLuca et al. (1995) Rheum. Dis. Clin. North Am. 21:759-777); auranofin;

phenylbutazone; meclofenamic acid; flufenamic acid; intravenous inunune globulin;
zileuton; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin);
amiprilose (therafectin); cladribine (2-chlorodeoxyadenosine); azaribine;
methotrexate;
antivirals; and immune modulating agents. Any of the above-mentioned agents can be administered in coinbination with the TNFa antibody of the invention to treat an TNFa-related disorder.
In one embodiment, the TNFa antibody of the invention is administered in combination with one of the following agents for the treatment of rheumatoid arthritis:
small molecule inhibitor of KDR (ABT-123), small naolecule inhibitor of Tie-2;

methotrexate; prednison.e; c,elecoxib; folic acid; hydroxychloroquine sulfate;
rofecoxib;
etanercept; inflixiniab; leffimomide; naproxen; valdec,oxib; sulfasaIazine;
naethylprednisolone; ibuprofen; meloxicarn; methylprednisolone acetate; gold sodium thiomalate; aspirin; azathioprine; triamcinolone acetonide; propxyphene napsylate/vap;
folatc; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodiurn;
oxaprozin;
oxycodone hcl; hydrocodone bitartrate/apap; diclofenac sodium/misoprostol;
fentanyl;
anakinra, human recombinant; tramadol hcl; salsalate; sulindac;
cyanocobalarnin/fa/pyridoxine; acetaminophen; alendronate sodiurn;
prednisolone;
morphine sulfate; lidocaine hydrochloride; indomethacin; glucosamine sulfate/chondroitin; cyclosporine; amitriptyliue hcl; sulfadiazine; oxyc,odone hellacetaminophen; olopatadine licl; misoprostol; uaproxen sodium;
orneprazole;
mycophenolate naofetil; cyclophosphamide; rituximab; IL-1 TRAP; MRA; CTLA4-IG;
IL-18 BI?; ABT-874; Al3T-325 (anti-IL 18); anti-IL 15; NLRB-796; SC10-469; VX-702;
AMG-548; VX-740; Roflumilast; IC-485; CDC-801; and mesopram_ In another embodiment, the TNFa antibody of the invention is administered for the treatment of a TNFa related disorder in combination with one of the above mentioned agents for the treatment of rheurnatoid arthritis.
In one embodiment, the TNFa antibody of the invention is administered in combination with one of the following agents for the treatment of a l'NFec-related disorder in which TNFcc activity is detrimental: anti-11L12 antibody (ABT
874); anti-IL18 antibody (ABT 325); small molecule inhibitor of1_,CK; small molecule inhibitor of COT; anti-11,1 antibody; small molecule inhibitor of MK2; anti-CD19 antibody;
small molecule inhibitor of CXCR3; sniall molecule inhibitor of CCR5; small molecule inhibitor of CCR11 anti-E/L selectin antibody; small molecule inhibitor of P2X7; small molecule inhibitor of IRAIC.--el; small molecule agonist of glucocorticoid receptor; anti-C5a receptor antibody; small molecule inhibitor of C5a receptor; anti-CD32 antibody;
and CI)32 as a therapeutic protein.
In yet another embodiment, the TNFo, antibody of the invention is administered in combination with an antibiotic or antiinfective agent. Antiinfective agents include those agents known in the art to treat viral, timgal, parasitic or bacterial infections. The term, "antibiotic," as used herein, refers to a chemical substance that inhibits the growth of, or kills, microorganisms. Encompassed by this term are antibiotic produced by a microorganism, as well as synthetic antibiotics (e.g., analogs) known in the art.
Antibiotics include, but are not limited to, clarithromycin (Biaxine), ciprofloxacin (Ciproe), and nnetronidazole (Flagyle).
In another embodiment, the TNFa antibody oldie invention is administered in combination with an additional therapeutic agent to treat sciatica or pain.
Examples of agents which can be used to reduce or inhibit the symptoms of sciatica or pain include hydrocodone bitartrate/apap, rofecoxib, cyclobenzaprine bcl, methylprednisolone, naproxen, ibuprofen, oxycodone hcUacetaminophen, cele,coxib, valdecoxib, methylprednisolone acetate, prednisone, codeine phosphate/apap, tramadol hcl/acetarninophen, metaxalone, ineloxicam, methocarbanaol, lidocaine hydrochloride, diclofenac sodium, gabapentiu, dexamethasone, carisoprodol, ketorolac tromethamine, indornethacin, acetaminophen, diazepam, nabumetone, oxycodone hcl, tizaaidine hcl, diclofenac sodium/misoprostol, propoxyphene napsylate/apap, asa/oxycod/oxycodoue ter, ibuprofen/hyclrocodone bit, tramadol hcl, ctodolac, propoxyphene het, arnitriptyline carisoprodol/codeine phos/asa, morphine sulfate, multivitamins, naproxen sodium, orphenadrine citrate, and temazepam =
In yet another embodiment, the TNFa-related disorder is treated with the TNFa antibody of the invention in combination with hemodialysis.

In another embodiment, the TNFa antibody of the invention is used in combination with a drug used to treat Crohn's disease or a Crohn's-related disorder.
Examples of therapeutic agents which can be used to treat Crohn's include mesalamine, prednisone, azathioprine, mercaptopurine, infliximab, budesonide, sulfasalazine, methylprednisolone sod succ, diphenoxylate/atrop su1 loperamide hydrochloride, tnethotrexate, otneprazole, folate, ciprofloxacin/dextrose-water, hydrocodone bitartrate/apap, tetracycline hydrochloride, fluocinouide, metronidazole, thiraerosal/boric acid, eholestyramine/suerose, eiproftoxacba hydrochloride, hyoscyamine sulfate, meperidine hydrochloride, midazolam hydrochloride, oxycodone hcllacetarninophen, promethazine hydrochloride, sodium phosphate, sulfarnethoxazole,/trimethoprim, celecoxib, polycarbophil, propoxyphene napsylate, hydrocortisone, multivitamins, balsalazide disodium, codeine phosphate/apap, colesevelam hcl, cyanocobalatnin, folic acid, levofloxacin, methylprednisolone, nataliannab, and interferon-ganu-na.
= In another embodiment, the TNra antibody of the invention is administered in combination with an additional therapeutic agent to treat asthma. Examples of agents which can be used to reduce or inhibit the symptoms of asthma include the following:
albuterol; salmeterol/fluticasone; sodium; fluticasone propionate; budesonide;

prednisone; salrneterol xinafoate; levalbuterol hcl; sulfate/ipratropium;
prednisolone sodium phosphate; triamcinolone acetonide; beclomethasone dipropionate;
ipratropium bromide; .Azithromycin; pirbuterol acetate, prednisolone, theophylline anhydrous, rnethylprednisolone sod succ, clarithromycin, zafalukast, formoterol fumarate, influenza virus vaccine, methylprednisolone, trihydrate, flunisolide, allergy injection, cromolyn sodium, fexofenadine hydrochloride, flunisolide/ruenthol, amoxicillin/clavulanate, levofloxacin, inhaler assist device, guaifenesin, dexaniethasone sod phosphate; moxifloxacin hcl; hyclate; guaifencsin/d-methorphan;
pephedrinekod/chlotplienir; gatifloxacin; cetirizine hydrochloride;
tnometasoue furoate;
salmeterol xinafoate; benzonatate; cephalexitr, pe/hydrocodone/chlorphenir;
cetirizine hclipseudoephed; phenylephrine/codipromethazine; codeine/promethazine;
cefprozil;
dexamethasone; guaifenesin/psettdoephedrine, chlorpheniramine/hydrocodone, ne;docromil sodium, tcrbutaline sulfate, epinephrine and methylprednisolone, metaproterenol sulfate.

In another embodiment, the TNFQ antibody of the invention is administered in combination with an additional therapeutic agent to treat COPD. Examples of agents which can be used to reduce or inhibit the symptoms of COPD include, albuterol sulfatdipratropiuna; ipratropium bromide; salmeterol/fluticasone; albuterol;
salmeterol;
xinafoate; fluticasone propionate; prednisone; theophylline anhydrous;
methylprednisolone sod succ;.montelulcast sodium; budesonicle; forrnoterol furnarate;
triamcinolone acetonide; levofloxacin; guaifenesin; azithromycin;
beclornethasone;
clipropionate; levalbuterol hcl; flunisolide; sodium; trihydrate;
gatifloxacin; zafirlukast;
amoxicillin/clavulanate; flunisolide/menthol; chlorpheniramine/hydrocodone;
metaproterenol sulfate; methylprednisolone; furoate; -ephedrinekod/chlorphenir;
pirbutexol acetate; -ephedrine/loratadine; terbutaline sulfate; tiotropium bromide;(R,R)-formoterol; TgAAT; Cilomilast and Rofiumilast In another embodiment, the TNIkc antibody of the invention is administered in combination with an additional therapeutic agent to treat 1PF. Examples of agents which can be used to reduce or inhibit the symptoms of PP include prednisone;
azathioprine;
albuterol; c,olclaicines; sulfate; digoxim gammainterferon;
raethylprednisolone sod succ;
furosemide; lisinopril; nitroglycerin; spironolactone; cyclophosphamide;
ipratropiurn bromide; actinomycin d; alteplase; fluticasone propionate; levofloxacin;
metaproterenol sulfate; morphine sulfate; oxycodone hcl; potassium chloride; trhuncinolone acetonide;
tacrolimus anhydrous; calcium; interferon-alpha; methotrexate; mycophenolate mofetil.
In one embodiment of the invention, a TNFcc antibody is administered in combination with an agent which is commonly used to treat spondyloarthropathies.
Examples of such agents include nonsteroidal, anti-inflammatory drugs (NSAIDs), COX
2 inhibitors, including Celebrex , Vioxe, and Bextra , aand etoricoxib.
Physiotherapy is also commonly used to treat spondyloarthropathies, usually in conjunction With non-steoidal inflanunatory drugs.
in another embodiment, the TNFcc antibody of the invention is administered in.

combination with an additional therapeutic agent to treat ankylosing spondylitis.
Examples of agents which can be used to reduce or inhibit the symptoms of ankylosing spondylitis include ibuprofen, diclofenac and misoprostol, naproxen, meloxicana, indomethacin, diclofenac, celecoxib, rofecoxib, sulfasalaz-ine, prednisone, raethotrexate, azathioprine, rainocyclin, prednisone, etanercept, and infliximab.

ln another embodiment, the TNFa antibody of the invention is administered in combination with an additional therapeutic agent to treat psoriatic arthritis.
Examples of agents which can be used to reduce or inhibit the symptoms of psoriatic arthritis include inethotrexate; etanercept; rofecoxib; celecoxib; folic acid; sulfasalazine;
naproxen;
leflunotnide; methylpreduisolone acetate; iudomethacin; hydroxychloroquine satiate;
sulindac; prednisone; betamethasone diprop augmented; infliximab;
methotrexate;
folate; triamcinolone acetonide; dielofenac; dimethylsulfoxide; pirmcicam;
diclofenac sodium; lcetoprofen; meloxicam; prednisone; methylprednisolone; nabumetone;
tolmetin sodium; calcipotriene; cyclospoxine; diclofenat; sodium/misoprostol;
fluocirionide;
glucosamine sulfate; gold sodium thiomalate; hydrocodone; bitartratciapap;
ibuprofen;
risedronate sodium; sulfadiazine; thioguanine; valdecoxib; alefacept; and efalizumab.
In one embodiment the TNFcc inhibitor is administered following an initial procedure for treating coronary heart disease. Examples of such procedures include, but =
are not limited to coronary artery bypass grafting (CABG) and Percutaneous transluminal coronary balloon angioplasty (PTCA) or angioplasty. In one embodiment, the TNFcc inhibitoris administered in order to prevent stenosis from re-occurring. In another embodiment of the invention, the TNFcc inhibitor is administered in order to prevent or treat restenosis. The invention also provides a method of treatment, wherein the TNFric inhibitor is administered prior to, in conjunction with, or following the insertion of a stent in the artery of a subject receiving a procedure for treating coronary heart disease. hi one embodiment the stent is administered following CABG or PTCA.
A wide variety of stent grafts may be utilized within the context of the present invention, depending on the site and nature of treatment desired. Stant grafts may be, for example, bifiucn ted or tube grafts, cylindrical or tapered, self-expandable or balloon-expandable, unibody, or, modular. Moreover, the stent grail may be adapted to release the drug at only the distal ends, or along the entire body of the stent graft. The TNFcc inhibitor of the invention can also be administered on a stent. In one embodiment, the TNFcc antibody of the invention, including, for example, D2E7/1-1-UMURA is administered by a drug-eluting stent.

The TNFa antihOdy of the invention can be administered in combination with an additional therapeutic agent to treat restenosis. Examples of agents which can be used to treat or prevent restenosis include strolimus, paclitaxel, everolimus, tacrolimus, ABT-578, and acetaminophen.
The TNFcr antibody of the invention can be administered in combination with an additional therapeutic agent to treat myocardial infarction. Examples of agents which can be used to treat or prevent myocardial infarction include aspirin, nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparin sodium, clopidogrel bisulfate, carvedilol, atenolol, morphine sulfate, metoprolot succinate, warfarin sodium, lisinopril, isosorbide mononitrate, digoxin, furosemide, simvastatin, ramipril, tenecteplase, enalapril naaleate, torscmide, retavase, losartan potassium, quinapril hcl/rnag carb, burnetanide, alteplase, enalaprilat, annodarone hydrochloride, tirofiban hcl in-hydrate, diltiazem hydrochloride, captopril, irbesartan, valsartan, propranolol hydrochloride, fosinopdl sodium, lidocaine hydrochloride, eptifibatide, cefazolin sodium, atropine sulfate, aminocaproic acid, spironolactone, interferon, sotalol hydrochloride, potassiuna chloride, docusate sodium, dobutamine hcl, alprazolam, pravastatin sodium, atorvastatin calcitun, mirlarolam hydrochloride, meperidine hydrochloride, isosorbide dinitrate, epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin, ezetimibe/simvastatin, avasimibe, abciximab, and cariporide.
The TNFcc antibody of the invention can be administered in combination with an additional therapeutic agent to treat angina. Examples of agents which can be used to treat or prevent angina include: aspirin; nitroglycerin; isosorbide mononitrate;
metoprolol succinate; atenolol; metoprolol tartrate; amlodipine besylate, dilitiazem hydropchloride, isesorbide dinitrate; clopidogrel bisulfate; nife,dipine;
atorvastatin calcium; potassium chloride; furosemide; simvastatin; verapamil lacl; digoxin;
propranolol hcl; carvedilo; lisinopril; sprionolactone; hydrochlorothiazide;
enalapril maleate; inadolol; ramipril; enoxaparin sodium; heparin sodium; valsartan;
sotatol hydrochloride; fenofibrate; ezetimibe; bumetanide; losartan potassium;
lisinopril/hydrochlorothiazide; felodipine; captopril; and bisoprolol fumarate.
In one embodiment of the invention, a TNFcc antibody is administered in combination with an agent which is commonly used to treat hepatitis C virus.
Examples of such agents include Interferon-apllaa-2a, Interferon-alpha-2b, Interferoa-alpha conl, - 9.7 -Interfero-aopha-nl, Pegylated interferon-alpha-2a, Pegylated interferon-alplia-2b, Ribavirin, Peginterferon alfa-2b and ribavirin, Ursodeoxycholic Acid, Glycyrrhizic Acid, TM
Thymalfasin, Maxamine, and VX-497.
The TNFz antibody of the invention is administered in combination with topical corticosteroids, vitamin D analogs, and topical or oral retinoicls, or combinations thereof, for the treatment of psoriasis. In addition, the TNFrx antibody of the invention is administered in combination with one of the following agents for the 'treatment of psoriasis: small molecule inhibitor of KDR (ABT-123), small molecule inhibitor of Tie-2, calcipotriene, clobetasol propionate, triamcinolone acctonicle, halobetasolpropionate, tazarotene, inethotrexate, fluouinonicle, hetamethasone diprop augmented, fluocinolone, aeetonide, acitretirt, tar shampoo, betarnethasone valeratc, mometasone fbroate, ketoconazole, pramoxine/fluocinolone, hydrocortisone valerate, flurandrenolide, urea, . . betaniethasone, clobetasol propionate/emoll, fluticasonc propionate, azithromycin, hydrocortisone, moisturizing formula, folic acid, desonide, coal tar, diflorasone diacetate, etanercept, folate, lactic acid, methoxsalen, hc/bisinuth subgal/znox/resor, inethylprednisolone acetate, prednisone, sunscreen, salicylic acid, halcinonide, anthralin, elocortolone pivalate, coal extract, coal tar/salicylic acid, coal tar/salicylic acid/sulfur, clesoximetasone, diazepam, emollient, pirnecrolimus emollient, fluocinonide/emollient , mineral oil/castor oil/na Int, mineral oil/peanut oil, petroleturdisopropyl myristate, psoralen, salicylic acid, soap/tribromsalan, lhimerosal/boric acid, celecoxib, infliximab, ilefacept, efalizumab, tacrolimus, pimecrolimus, PUVA, UVB, and sulfasatazinc.
An antibody, antibody portion, or other TINIFa inhibitor of the invention can be used in combination with other agents to treat skin conditions. For example, an tunibody, antibody portion, or other T1\117a inhibitor of the invention is combined with PUVA therapy. PUVA is a combination of psoralen (P) and long-wave ultraviolet radiation (UVA) that is used to treat many different skin conditions. The antibodies, antibody portions, or other TNFa inhibitors of the invention can also be combined with piniecrolimus. In another embodiment, tbe antibodies of the invention are used to treat psoriasis, wherein the antibodies are administered in combination with tacrolimus. In a fin-titer embodiment, tacrolimus and TI\IPiz inhibitors arc administered in combination with methotrexate and/or cyclosporine. In still another embodiment, the T317a inhibitor of tbe invention is administered with excimer laser treatment for treating psoriasis.

Nonlimiting examples of other therapeutic agents with which a TNI7a. inhibitor can be combined to treat a slcin or nail disorder include UVA and UVB
phototherapy.
Other nonlimiting examples which can be used in combination with a TN-Fa inhibitor include anti-M-12 and anti-M-18 therapeutic agents, including antibodies.
In one embodiment, the TNFa antibody of the invention is administered in combination with ui additional therapeutic agent in the treatment of Belieet'.s disease.
Additional therapeutic agents which can be used to treat Belicers disease include, but TM
are not limited to, prednisone, cyclophosphamide (Cytoxan), Azathioprine (also called imuran, methotrexatc, timethoprim/sulfamethoxazole (also called bactrim or septrii) and folic acid.
Any one of the above-mentioned therapeutic agents, alone or in combination therewith, can be administered to a subject suffering from a TNFa-related disorder in which TNFa is detrimental, in combination with the TNFa antibody of the invention. In one embodiment, any one of the above-mentioned therapeutic agents, alone or in combination therewith, can be administered to a subject suffering from rheumatoid arthritis in addition to a TNFcc antibody to treat a TNI7a-related disorder.
This invention is thriller illustrated by the following examples which should not be construed as limiting.
. .
EXAMPLES
Example 1: TNIla Inhibitor in Rat Model for AnIcylosing Spondylitis Administration of TNF antibody to human leukocyte antigen-1327(711,21-B27) rats to test inhibition of progressive ankylosis Fisher 344 rats genetically engineered to ca.riy high-copy numbers of the human major histocompatibility complex class 1 allele B27 and the (32-microglobulin genes exhibit symptoms sirniku- to liwnan spondyloarthopathies particularly ankylosing sporidylitis (AS) (Zhang et al. Carr Rhewnatol Rep. 2002: 4:507). Male transgenic hwnan leuolcocyte antigen-B27 (I-ILA-B27) rats are obtained at 10 weeks of age and are housed in an animal facility until they are 40 weeks of age. A group of Fisher 344 rats are obtained and serve as nontransgenic controls. The control rats are purchased at 36 weeks and are housed in the animal facility under the same conditions for an additional 3 to 4 weeks.
Prior to the experimental treatment, body weights are measured for both the ITLA-B27 transgenic rats, and the control rats to make sure there is no significant difference between the two. The rats are then administered intraperitoneally (i.p.) doses of either a placebo or a monoclonal anti-TNPa antibody that is known to bind and neutralize rat TNFa., e.g., antibody TN3 (T1\13-19.12) (see Marzi et al.
(1995) Shock 3:27; Williams et al. (1992) Proc Natl Acad Sci USA. 89:9784; BD Biosciences Pharmingen). Rats are evaluated for symptoms of AS using the following tests beginning at roughly 36 weeks of age and continuing throughout the study:
weight, forepaw grasp of a wire grid, ability to cling to an inverted wire grid, gait, thorax flexibility, spinal mobility, and appearance of eyes, skin, nails, genitals, and peripheral and axial skeletal joints with respect to redness and swelling, joint deformity, and mobility. Rats are also examined for evidence of arthritis, particularly decreases in AS
symptoms in the treated rats, and are closely observed for growth characteristics and changes in slcin and nails. At 4, 6, 8, 10, 12, 16, and 20 weeks, rats are sacrificed for radiographic and microscopic analysis.
E_xampIe 2: TNF Inhibitor Effects on AS Symptoms Anlrylosing Spondylitis- Clinical Considerations Patients who eXhibit symptoms commonly associated with AS are examined and tested to determine if they suffer from AS, and thus qualify for the study.
Symptoms commonly associated with AS are low Niel( pain that is worse after inactivity, stiffness and limited motion in the low back, hip pain and stiffness, limited expansion of the chest, limited range of motion (especially involving spine and hips), joint pain arid joint swelling in the shoulders, lalees, and ankles, neck pain, heel pain, chronic stooping to relieve symptoms, fatigue, fever, low grade, loss of appetite, weight loss, and/or eye inflammation. Patients are given a physical examination to cletemiine whether or not they axhibit any of the characteristic symptoms indicative of limited spine motion or chest expansion associated with AS. Examples of tests which indicate AS
include X-rays of sacroiliac joints and vertebrae a which show characteristic findings associated with AS.
Ankylosing spondylitis is diagnosed using the modified New York criteria (Moll et al. (1973) Ann Rheum Dis 32:354; Van der Linden et al. (1984) Arthritis Rhetun 27:361). The New York criteria for anlcylosing spondylitis is a modification of the Rome criteria as proposed at the CIOMS Symposituat in New York during 1966. It combines both clinical criteria and radiographic findings of the sacroiliac joint.
Clinical criteria of New York criteria:
(a) Limitation of motion of the lumbar spine in all 3 planes (anterior flexion lateral flexion extension). Skin markings to aid in the examination are shown in Moll, supra;
(b) A history of pain or the presence of pain at the dorsolumbar junction or in the lumbar spine; and (c) Limitation of chest expansion to 1 inch (2.5 cm) or less measured at the level of the fourth intercostal space.
Scoring Index for New York Criteria Radiographic Changes in the Sacroiliac Grade Joint(s) normal 0 suspicious 1 minimal sacroihitis 2 moderate sacroiliitis = 3 ankylosis 4 The clinical course of AS is measured by using any number of instnunents to evaluate =
various AS symptoms. Some of the commonly used scales include the Assessment in AnIcylosing Spondylitis (ASAS), the Bath Ankylosing Spondylitis Disease Activity Index. (BASDA1) (Garrett et al. (1994)./Rheumato/ 21:2286), the Bath Ankylosiilg Spondylitis Metrology index (BASMI) (Jenkinson et al. (1994) J Rhewnatol 21:1694), and the Bath Anlcylosing Spondylitis Functional Index (BASFI) (Cahn et al.
(1994).1 Rhezonatol 21:2281). These indices can be used to monitor a patient over lime and to determine improvement. Each of these scales is described further below:
Criteria for Measuring the Clinical Course of AS
1. The Assessment in AnIcylosing Spondylitis (ASAS20) is the primary endpoint in the pivotal Phase 3 AS studies. A..20% improvement and absolute improvement of units (scale of 0-100) in ?_.3 of 4 domains: Subject Global Assessment, Pain, Function, and Inflammation. There must be an absence of deterioration in the potential remaining domain (deterioration is defined as a change for the worse of _20%
and a net worsening of units (scale of 0-100).
2. The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) can be used to evaluate the level of disease activity in a patient with AS. BASDAI focuses upon signs and symptoms of the inflammatory aspects of AS, nocturnal 'and total back pain, the patient's global assessment and actual physical measurements of spinal mobility such as the Schober's test, chest expansion score and occiput to wall measurement.
B.ASDA1 measures disease activity on the basis of six questions relating to fatigue, spinal pain, peripheral arthritis, enthesitis (inflammation at the points where tendons/ligaments/joint capsule enter the bone), and morning stiffness. These questions are answered on a 10-CM horizontal visual analog scale measuring severity of fatigue, spinal and peripheral joint pain, localized tenderness, and morning stiffness (both qualitative and quantitative).
The final BASDAI score has a range of 0 to 10.
3. The Bath Ankylosing Spondylitis Functional index (BASF1) measures the physical function impairment caused by AS, and is a self-assessment instrument that consists of 8 specific questions regarding function in AS, and 2 questions reflecting the patient's ability to cope with everyday life. Each question is answered on a 10-cm horizontal visual analog scale, the mean of which gives the BASFI score (0-10).
4. The Bath Ankylosing Spondylitis Metrology Index (BASMI) consists of 5 simple clinical measurements that provide a composite index and define disease status in AS.
Analysis of metrology (20 meastu-ements) identified these 5 measurements as most accurately reflecting axial status: cervical rotation, tragus to wall distance, lateral flexion, modified Schober's test, and intermalleolar distance. The BASIv11 is quick (7 minutes), reproducible, and sensitive to change across the entire spectrum of disease.
The BASMI index comprises 5 measures of hip and spinal mobility in AS. The five BASMI measures, sealed 0 (mild) to 10 (severe), include traps to wall distance, cervical rotation, lumbar flexion, lumbar side flexion, and intennolleolar distance.
Combinations of the above-mentioned criteria are used to evaluate patients. In addition, radiographic, MA and bone and cartilage degradation markers can be used to determine disease activity in AS patients.
Clinical studies examining D2E7 in hwnan subjects with active AS
Patients are administered a dose of D2E7 s.c in a placebo-controlled clinical trial over a period of weeks, and re-examined every 2-6 weeks for the next year to determine if AS symptoms are reduced or treated. A dose of 40 mg every other week, which is effective and safe in treating rheumatoid arthritis, is used in the study.
Only patients who have a confirmed diagnosis of active AS, as defined by ha-ving 2 of the following 3 criteria- BASDA1 index, a visual analog scale (VAS) for pain and the presence of naorning stiffness- are chosen for the study. The BASDAI index is described in more detail above. In order to enroll in this study, patients must have signincant pain at screening and at baselineõ a pain score of> 4 on a 10-cm VAS, and a BASDAI
score of 4.
Disease-modifying antirheuniatic drugs (DMARDS) or other immimosuppressive agents are allowed in the study. Patients are allowed to enroll if they are, on an equivalent dose of- 10 nag of prednisone per day.
Screening examinations are performed prior to the study enrollment in order to document each patient's medical history and current findings. The following information is obtained from each patient: morning stiffness (duration and severity), occurrence of anterior uveitis (number of episodes and duration), and the number of inflamed peripheral joints. For each patient, radiographs of the vertebral coltu-nn and the sacroiliac joints are obtained. Magnetic resonance imaging can also be used to document the spinal column of the patients enrolled Patients are randomly divided into experimental aid placebo groups, and are administered either D2E7 or the placebo once every two weeks in a blinded fashion until week 12 or week 24. D2E7 has been administered at doses of 20 to 80 mg that have been used effectively to treat rheumatoid arthritis; a 40 mg dose was determined to be effective. A higher dose might be necessary to treat spinal inflammation, so a higher dose (40 mg weeldy in those patients who are nonresponders and who are not on methotrexate) is used in the study. The percentage of patients who achieve an is calculated.
Example 3: TNI? Inhibitor in Clinical Study for Psoriatic Arthritis D2E7 in human subjects .with psoriatic arthrits Patients with moderate to severe psoriatic arthritis of any subtype (arthritis of the distal fiaterphalangeal joints, arthritis mutilans, symmetric polyartluitis, asymmetric oligoarthritis and/or spoyloarthropathy) are selected for the study. Patients have either failed or exhibited intolerance to non-steroidal antiinflamatory drugs (NSAIDs) or disease modifying anti-rheumatic drugs (DIVIARDs). Therapy is given alone and/or in combination with NSAEDs and DMARDs.
Dosage ranges being evaluated include 40 mg every other week, which is the D2E7 dose which has been found to be most effective at treating rheumatoid arthritis in patients. Higher dose (40 mg every week) is also being studied. Studies are a comparison to placebo for 12 to 24 weeks followed by open label therapy to determine long temi safety and effitacy.
Patients are examined clinically at screening, baseline, and frequently during treatment. The primary efficacy for signs and symptoms is measured via American College of Rheurnatology preliminary criteria for improvement (ACR20) at 12 weeks.
An additional primaty endpoint includes evaluation of raliologic changes over 6 to 12 months to assess changes in structural damage. Multiple other evaluations are performed during treatment including Psoriatie Arthritis Response Criteria (PsARC), quality of life measurements, and skin evaluations to determine efficacy on psoriasis lesions (psorasis area severity index (PAS1) and target lesion evaluations).
- 10,1 -Example 4: TN-Fa Inhibitor in Mouse Model for Asthma YNF antibody study using ovallmmin (OVA)-induced allergic asthma mice The mouse OVA model of allergic asthma (Hessel, E.M., et al. (1995) Eur.
Pharmacol. 293:401; Daphne, T., et al. (2001) .4nz.J.Rcípir. Cell Alol. Biol.
25:751, is used in the following study for treating allergic astluna.
All mice are sensitized to OVA (chicken egg albumin, crude grade V; Sigma, St.
Louis, MO). Active sensitization is performed without an adjuvant by giving seven intraperitoueal injections of 101.1.g OVA in 0.5 nal pyrogen-free saline on alternate days (one injection per day). Three weeks after the last sensitization, mice are exposed to either 16 OVA challenges (2 mg/nal in pyrogen-free saline) or 16 saline aerosol challenges =for 5 min on consecutive days (one aerosol per day). An additional group of mice first receive eight OVA aerosols, followed by eight saline aerosols (OVA/saline, spontaneous resolution group).
For the experiment in the more severe ongoing model of allergic asthma, all mice are sensitized to OVA by active sensitization with two intraperitoneal injections (7 d apart) of 0.1 ml alum-precipitated antigen, comprising 1011g OVA adsorbed onto 2.25 mg alum (Alumlinject; Pierce, Rockford, IL). Two weeks after the second sensitization, mice are exposed to either six OVA challenges (10 mg/nal in pyrogen-free saline) or six saline aerosol challenges for 20 min every third day (one aerosol every third day). An additional group of mice first receive three OVA aerosols, followed by three saline aerosols (OVA/saline, spontaneous resolution group).
The aerosol treatment is perfonned in a plexiglas exposure chamber (5 liter) coupled to a Pari LC Star nebulizer (PARI Respiratory Equipment, Richmond, VA;
particle size 2.5-3.1 nm) driven by c,ompressecl air at a flow rate of 6 liters/min. Aerosol is given in groups composed of no more than eight animals.
A monoclonal anti-TNEa antibody which is known to bind and neutralize mouse TNFa, e.g., antibody r1"1\13 (TN3-19.12) (see Marzi et al. (1995) Shock 3:27;
Williams et al. (1992) Proc Natl Acad Sci USA. 89:9784; BD Biosciences Pharmingen) is administered to the OVA sensitized mice in a range of doses after the second sensitization according to standard protocols known in the art. Appropriate placebo controls are also administered.
Airway responsiveness is measured in conscious, unrestrained mice using barometric whole-body pleth.ysmography by recording respiratory pressure curves in response to inhaled methacholine (acetyl-P-methylcholine chloride; Sigma).
Briefly, mice are placed in a whole-body chamber, and basal readings ai-e obtained and averaged for 3 min. Aerosolized saline, followed by doubling concentrations of methacholine (ranging from 1.6-50 mg/ml saline), are nebulized for 3 min, and readings are taken and averaged for 3 min after each nebulization. Dose-response curves (DRCs) to rnethacholine are statistically analyzed by a general linear model of repeated measurements followed by post-hoc comparison between groups. Data are LOG transformed before analysis to equalize variances in all groups.
. After measurenaent of in vivo airway responsiveness, mice are sacrificed by intraperitoneal injection of 1 nal 10% urethane in pyrogen-free saline (Sigma).
Subsequently, mice are bled by cardiac puncture, and OVA-specific IgE is measured by ELISA. Briefly, naicrotiter plates (Nunc A/S, Roskilde, Denmark) are coated overnight at 4 C with 2 Itg/inlrat anti-mouse IgE (clone EM95) diluted in phosphate-buffered saline (PBS). The next day, the ELISA is performed at room temperature. After blocking with ELISA buffer (PBS containing 0.5% bovine serum albumin [Sigma], 2 niM EDTA, 136.9 raM NaCl, 50mM Tris, 0.05% Tween-20 [Merck, Whitehouse Station, NJ] pH
7.2) for 1 h, serum samples and a duplicate standard curve (starting 1:10), diluted in ELISA buffer, are added for 2 h. An OVA-specific IgE reference standard is obtained by intraperitoneal immunizationwith OVA and arbitrarily assigned a value of 10,000 experimental units/nd (BU/m1). After incubation, 1 itg/m1 of OVA
coupled to digoxigenin (DIG), which is prepared from a kit containing DIG-3-o-methylcarbony1-5 -aminocaproie acid-N-hydroxy-suceinimide-ester (Roche Diagnostics, Basel, Switzerland) in ELISA buffer, is added for 1.5 h, followed by incubation with anti-DIG-Fab fragments coupled to horseradish peroxiclase (Roche Diagnostics) diluted 1:500 in ELISA buffer for 1 hour. Color development is performed with o-phenylenediamine-dichloride substrate (0.4 mg/ml, Sigma) and 4 inM H202 in PBS and stopped by adding 4 M H2SO4. The optical density is read at 492 run, using a Benchmark microplate reader (13io-Rad Laboratories, Hercules, CA). The detection limit of the ELISA is 0.5 EUhnl IgB.
' Bronchial alveolar lavage (BAL) is performed immediately after bleeding of the mice. Briefly, the ainvays are lavaged five times through a tracheal carmula with 1-ml aliquots of pyrogen-ftee saline warmed to 37 C. The recovered lavage :fluid is pooled, and cells are pelleted (32 x g, 4 C, 5 min) and resuspended in 150 p.1 cold PBS. The total number of cells in the BALF is determined using a Biirker-Tink counting-chamber (Karl -Hecht Assistent KG, SondheimfRolun, Germany). For differential BALF cell counts, cytospiu preparations are made and stained with Diff-Quick (Dade AG, Dtidingen, Switzerland). Per cytospin, 400 cells are counted and differentiated into mononuclear cells (monocytes, macrophages, and lymphocytes), eosinophils, and neutrophils by standard morphology. Statistical analysis is performed using the nonparametric Mann-Whitney Utest.
Cytokine production by antigen-restinnilated T cells in lung tissue is deternained as described previously (Hofstra, C.L., et al. (1999) litflanun. Res. 48:602).
Briefly, the lungs are lavaged as described above and perfused via the right ventlicle with 4 ml saline containing 100 U/nal heparin to remove any blood and intravascular leukocytes.

Complete lung tissue is removed and transferred to cold sterile PBS. Lungs are then minced and digested in 3 nal RPMI 1640 containing 2.4 mg/nal collagenase A and DNase I (grade LI) (both from RocheDiagnostics) for 30 min at 37 C. Collagenase activity is stopped by adding fetal calf serum (FCS). The lung tissue digest is filtered through a 70-um nylon cell strainer (Becton Dicicinson Labware, Franklin Lakes, Ni) with 10 rn1 RP1VIT 1640 to obtain a single-cell suspension. The lung-cell suspension is washed, resuspended in culture medium (RPIVE 1640 containing 10% FCS, 1% glutamax I, arid gentarnicin from Life Technologies, Gaithersburg, MD]) and 50 mIVI li-mercaptoethanol (Sigma), and the total number of lung cells is determined using a Btirker-Ttirk counting-chamber. Lung cells (8 x 105 lung cells/well) are cultured in round-bottom 96-well plates (Greiner Bio-One GmbH, Kremsamenster, Austria) in the presence of OVA (101_tg/m1) or medium only. As a positive control, cells are cultured in the presence of plate-bound rat anti-mouse CD3 (clone 17A2, 50 n.g/nil, coated overnight at 4"C). Each in vitro stimulation is performed in triplicate. Afier 5 days of eulture at 37 C, the supernatant is harvested, pooled per stimulation, and stored at ¨20 C until cytokine levels were determined by ELISA.
The 113N-y, IL-4, IL-5, IL-10, and IL-13 ELISAs are performed according to the manufacturer's instructions (PharMingen, SanDiego, CA). The detection limits of the ELISAs are 160 pg/ml for IFN-y, 16 pg/mlforIL-4, 32 pg/ml for TL-5, and 100 pg/ml for M-10 and EL-13.
In all experiments, airway responsiveness to methacholine, OVA-specific IgE
levels in sertun, cellular infiltration in theBALF, and T-cell responses in lung tissue are measured 24 hours after the last challenge in each mouse.
Improvements in asthma in the experimental mice are marked byn decrease in the number of mononuclear cells (including monocytes, macrophages, and lymphocytes), eosinophils, and neutrophils in the BALF, a decrease in the airway hyperresponsiveness, and a decrease in the cytokine production by antigen-restirnulated T cells in the lung tissue.
Example 5: TNFcc Inhibitor in Mouse Model of Chronic Ostructive Puhnonary Disease (COPD).
Study examining treatment for alveolar enlargement and inflammation The following study is perfonned using a cigarette smoke induced COPD mouse model ((east, D.et al. (1981) J. Pathol. 135:249; Hautmaki, R.D., et al.
(1997) Science 277:2002). In response to cigarette smoke, inflammatory cell recruitment into the lungs followed by pathologic chan. ges characteristic of emphysema have been observed.
Previous studies have shown that progressive inflammatory cell recruitment begins within the first month of smoking followed by air space enlargement after 3 to 4 months of cigarette exposure (Hautmaki et al. (1997) Science 277:2002).
Mice are exposed to smoke from two non-filtered cigarettes per day, 6 days per week, for 6 months, with the use of a smoking apparatus with the chamber adapted for mice. Nonsmoking, age-matched animals are used as controls. After 6 months of exposure to smolce as desciibed above, a inonoclonal anti-TNFot antibody which is lcnown to bind and neutralize mouse TNPa, e.g., antibody TN3 (TN3-19.12) (see Marzi et al. (1995) Shock 3:27; Williams el al. (1992) Proc Acad Sci U S A.
89:9784; BD
Biosciences Phaimingen) is administered in a range of doses according to standard protocols known in the. art. An appropriate placebo control is also administered. Mice are achninistered the antibody treatment for a period of 21 days. Mice are sacrificed, followed by examination of lung volume and compliance, cylokine measurement, histological mucus index measurement, alveolar duct enlargement, air space measurement, alveolar and interstitial macrophage counts and alveolar size, as described below.
Following antibody treatment, bronchiolar lavage is performed on euthanized aninaals; the trachea is isolated by blunt dissection, and small caliber tubing is inserted and secured in the airway. Two volumes of 1.0 ml of PBS with 0.1% BSA are instilled, gently aspirated, and pooled. Each BAL fluid sample is centrifuged, and the supernatants are stored in ¨700 -until used. Cytoldne measurements are as described in Example 5.
To determine lung volume aud conapliance, animals are anesthetized, the trachea is cannulated,. and the lungs are ventilated with 100% 02 via a "T" piece attachment. The = trachea is then clamped and oxygen absorbed in the face of ongoing pulmonary perfusion. At the cnd of this degassing, the lungs and heart are removed en bloc and inflated with PBS at gradually increasing pressures from 0 to 30 cm. The size of the lung at each 5-cm interval is evaluated via volume displacement An increase in the Itmg volume of treated arnmals compared to placebo treated control animals indicates an improvement in COPD.
For histological analysis, animals are sacrificed and a median stemotomyis performed, and right heart perfusion is accomplished with calcium- and magnesium-free PBS to clear the puhnonary intravascular space. The lungs are then -fixed to pressure (25 crn) with neutral buffered 10% Formant], fixed overnight in 10% formalin, embedded in paraffin, sectioned at 5 p.m and stained with FIematoxylin and eosin (H&E) and periodic acid-Schiff with diastase (D-PAS).
The histological mucus index (HMI) provides a measurement of the percentage of epithelial cells that are D-PAS per unit airway basement membrane. It is calculated from D-PAS¨stained sections (Cohn, L., et al. (1997) .1 Exp. Med. 186:1737). A

decrease in the Blvil of heated animals compared to placebo treated control animals indicates an improvement in COPD.
Lm, an indicator of air space size, is calculated for each mouse from 15 random fields at x200 by means of a 50-line counting pid (10-nun total length). The results are the average of measurements of two independent investigators. An increase in air space size of treated animals compared to placebo treated control animals indicates an improvement in COPD.
To determine alveolar duct enlargement, the proximal surface areas from the terminal bronchiole-alveolar duct transition extending 250 pin into the duct using Optinaus 5.2 image analysis software (Optimus, Bothell, WA) is measured. A
decrease in alveolar duct size of treated anin3als compared to placebo treated control animals indicates an improvement in COPD.
Alveolar and interstitial macrophages are quantitated by counting macrophages identified by murine Mac-3 (rat antibody to mouse (0.5 mg/nil), used at 1:4000 dilution;
FharMingen, San Diego, CAO immunostaining using the avidin-biotin alkaline. A
decrease in the number of alveolar and interstitial macrophages of treated animals compared to placebo treated control animals indicates an improvement in COPD.
Alveolar size is estimated from the mean cord length of the airspace (Ray, P., et al. (1997) .1 Clin. Invest. 100:2501). This measurement is similar to the mean linear intercept, a standard measure of air space size, but has the advantage that it is independent of alveolar septa' thickness. Sections are prepared as described above. To obtain images at random for analysis, each glass slide is placed on a printed rectangular .
grid and a series of clots placed on the coverslip at the intersection of the grid lines, i.e., at intervals of approximately 1 min. Fields as close as possible to each dot axe acquired by systematically scanning at 2-nun intervals. Fields containing identifiable artifacts or non-alveolated structures such as bronchovascular bundles or pleura are discarded.
A minimum of ten fields from each mouse lung are acquired into a Macintosh G3 computer (Apple Computer Inc., Cupertino, California, USA) through a franiegrabber board. Images are acquired in 8-bit gray-scale at a final magnification of 1.5 pixels per micron. The images are analyzed on a Macintosh computer using the public domain NTH
linage program written by Wayne Rasband using a custom-written macro available from the web site (http://rsbinfo.nili.govinill-image). Images are manually tluesholded and then smoothed and inverted. The image is then subject to sequential logical image match "and" operations with a horizontal and then vertical grid.
At least 300 nleasurements per field are made for each animal. The overlying air space air is averaged as the mean chord length. Chord length increases with alveolar enlargement.
An increase in alveolar size of treated animals compared to placebo treated control animals indicates an improvement in COPD.
Example 6: TNFcc Inhibitor in Idiopathic Puhnonary Fibrosis an?) Mouse Model.
Study ofilPF treatment using bleomycin induced lung fibrosis mouse model The following study is performed using the bleornycin induced lung fibrosis mouse model (reviewed in Bowden, D.H. (1984) Lab. Invest. 50:487; Tokuda, A., et al.
(2000) J. Immunol. 164:2745).
Bleomycin sulfate is administered to C57BL/6.1 female mice aged 8-10 weeks.
Briefly, C57BL/61 mice are anesthetized with 200 ul of 5 mg/m1pentobarbital injected.
i.p., followed by intratracheal instillation of 3 mg/kg bleomycin sulfate in 50 Ir.1 sterile saline. = =
A monoclonal anti-TNFa antibody which is known to bind and neutralize mouse TNFa, e.g., antibody TN3 (TN3-19.12) (see Marzi et al. (1995) Shock 3:27;
Williams et al. (1992) Proc Nail Acad Sci U S A. 89:9784; 13D Bio sciences Plimmingen) is administered to the blemnycin induced lung fibrosis mice in a range of doses, after intratracheal instillation of bleomycin as described above. An appropriate placebo control is also administered. Mice ar treated twice daily for 14 days.
Mice are sacrificed 20 and 60 clays following bleomycin treatment. Tissues are fixed in 10% buffered fonnalin and embedded in paraffin. Sections are stained with hematoxylin and eosin and examined by light microscopy. Lung-infiltrating leukocyte counts, cytokine measurements, and total lung collagen content is determined as described below.
BAL cells and lung-infiltrating leukocytes are prepared as described in Smith et al. (1994) J. Immunol. 151:4704. 1n brief, following anesthesia, 1 ml PBS is instilled and withdrawn five times from the lung via an intratracheal cannula. The BAL
fluids are collected and after RBC lysis total leukocyte counts arc determined. Cell differentials are determined after cytospin centrifuge. Specimens are stained with Diff-Quik products (Baxter, Miami, FL).
To isolate lung-infiltrating leukocytes, lungs are perfused with saline, dissected from the chest cavity, and then minced with scissors. Each sample is incubated for 30 minutes at 37 C on a rocker in 15 nil digesting buffer (10% FCS in RPM 1640 with 1%
collagenase; Wako Pure Chemical, Osaka, Japan). Next, the sample is pressed through nylon mesh and suspended in 10%FCS-RPM11640 after being rinsed. The cell suspension is heated with 1-Jistopaque-1119 (Sigma, St. Louis, MO) and centrifuged at 2000 rpm for 20 min to remove lung parenchymal cells and RBC. The pellet is resuspended in 2.5% FCS-PBS after being rinsed. After cell counts are performed, flow cytometric immunofluorescence analyses are conducted.
Immunofluorescence analyses of peripheral blood leukocytes and lung-infiltrating leukocytes are performed with the use of an Epics Elite cell sorter (Coulter Electronics, Hialeah, FL) as described previously (Yoneyama et al. (1998)J.
Clin.
Invest. 102:1933; Murai et al. (1999)J. Clin. Invest. 104:49). Peripheral blood leukocytes are prepared from normal mice with RBC lysis buffer. .After incubation with Fc Block (anti-CD16/32; PharMingen, San Diego, CA) for 10 min, cells are stained with PE-conjugated rnAb against CD3, CD4, CD8, CD11b, CD11 c, and Gr-1 (PharMingen), and also stained with 20 cg/m1 of rabbit anti-CCR1 polyclonal Ab followed by staining with FITC-conjugated goat anti-rabbit IgG (Leine Technologies, St. Louis, MO). Before analyses propidium iodide (Sigma) staining is performed to remove the dead cells. A
decrease in the number of lung-infiltrating leukocytes of treated animals compared to placebo treated control animals indicates an improvement in 113F.
Inummohistochemistry of lung samples is carded out as follows: lung specimens are prepared as described previously (Yoneyama et al. (1998)J. Clin. Invest.
102:1933;
Murai et al. (1999)J. Clin. Invest. 104:49). Briefly, lung specirnens are fixed in periodate-lysine-paraformaldehyde, washed with PBS containing sucrose, embedded in Tissue-Tek OCT compound (Miles, Elkhart, IN), frozen in liquid nitrogen, and cut into 7-uin-thick sections with a cryostat. After inhibition of endogenous peroxidase activity, the sections are incubated with the first Ab. The Abs used are rabbit anti-CCR1 Ab, rat anti-F4/80 (BMA I3ionieclicals, Geneva, Switzerland), rat anti-CD4, rat anti-CD8, rat anti-Gr-1 (Pharlvfingen), rat anti-nonlymphoid dendritic cell (NLDC)-145, and rat anti-WIC class II (BMA Biomedicals). As a negative control either a rabbit IgG or a rat IgG
is used, respectively. They are treated sequentially with either BP-conjugated goat anti-rabbit IgG (Cedarlane Laboratories, Hornby, Ontario, Canada) or a BRP-conjugated goat anti-rat IgG (BioSource International, Camarillo, CA). After staining with 3,3'-diarninobenzidine (Wako Pure Chemical) or 3-amino-9-ethylcarbazole substrate kit (Vector Laboratories, Burlingame, CA), samples are cowiterstained with Mayer' s hematoxylin. A decrease in CCR1, and decreases in the number of CD4-1- T
cellsõ
CDS+ T cells, nonlymphoiddendritic cell (NLDC), and WIC class 11 bearing cells of treated animals compared to placebo treated control animals indicates an improvement in Total lung collagen content is determined by assaying total soluble collagen using .the Sircol Collagen Assay kit (Biocolor, Northern Ireland) according to the manufacturer's instructions. Briefly, lungs are harvested at day14 after bleomycin administration andhomogenized in 10 nil 0.5 M acetic acid containing about 1 mg pepsin/10111g tissue residue. Each sample is incubated for 24 h at 4 C with stirring. After centrifugation, 200 I of each supernatant is assayed. One milliliter of Sircol dye reagent that binds to collagen is added to each sample and then mixed for 30 rrin.
After centrifugation, the pellet is suspended in 1 ml of the alkali reagent included in the kit and read at 540 nna by a spectrophotometer. Collagen standard solutions are utilized to construct a standard curve. Collagens contain about 1µ1% hydroxyproline by weight, and collagen contents obtained with this method correlate well with the hydroxyproline content according to the manufacturer's data. A decrease on lung collagen content of treated animals compared to placebo treated control animals indicates an improvement in IPF
Using the bleomycin induced lung fibrosis mouse model, mice are examined for a decrease in the BAL cell counts, a decrease in the peripheral blood leukocytes arid lung infiltrating leukocytes. Mice are also examined for a decrease in the total lung collagen content in D2E7 treated mice as compared to placebo treated mice.

Example 7: TNEct Inhibitor in Treatment of Asthma Clinical study of D2E7 in human subjects with asthma Patients 12 to 65 years of age are eligible for the study if they have had a documented diagnosis of asthma of at least 2 years duration and have also had demonstrable reversible bronchospasm with an increase in FEV1 of 15% or greater after the administration of albuterol within the previous six months. Additional inclusion criteria include, a baseline FEV1 between 50% and 80% of predicted nounal, absence of any clinically significant disease other than asthma, a history of daily use of inhaled corticosteroicls and cessation of all 132-agonist use 30 days prior to the beginning of the study.
A baseline visit occurs within 7 days after the screening visit. All patients undergo evaluation of FEV1 and have a complete physical examination. Pulmonary auscultation and oropharyngeal examinations are performed, and asthma symptoms are assesses. Patients who qualify are randomly assigned to a treatment group including a ' placebo group.
Following baseline measurements, patients begin receiving treatment. They are randomized and treated with either D2E7 or placebo in a blinded fashion. At days 15 and 29, all examinations perfomied at the baseline visit are repeated. A 12-lead ECG is also performed. Diary cards are reviewed with patients regarding the use of other medications and any adverse events.
Improvements are determined on spirometry tests measured at each visit. These include FEV1, peak expiratory flow rate (PEFR), Forced Vital Capacity (FCV), and forced expiratory flow at 25% to 75% of FVC. FEV1 at the final visit is regarded as the primary measure of efficacy. Twice-daily PEFR tests performed by the 'patient are compared and the number of inhalations of rescue medication is calculated.
Patient/physician evaluations of asthma symptoms (wheezing, tightness in the chest, shortness of breath and cough) are characterized by severity. Compliance is assessed by review of the patient's diary cards and by collecting unused study medication.

Example 8: TNFa, Inhibitor in Treatment of CON) Clinical study examining D2E7 ill human subjects with COPD
The study population is male and female subjects who are 40 to 80 years of age with a diagnosis of COPD. Subjects must have a best FEVi/FVC ratio A).70 liters, fixed airway obstniction, defined by 5% or 00 inl (or both) increase in FEV1 after the administration of albuterol and a post-albuterol FEV1 between 30 and 70%
of predicted. Subjects must also be current or previous smokers with a history of smoking 0 pack years.
Following baseline measurements, patients begin receiving treatment. They are randomized and treated with either D2E7 or placebo in a blinded fashion.
Improvements are marked by an increase from predose baseline after study medication in pre-bronchodilator FEV1 and change froin baseline in total score of the St.
George's Respiratory Questionnaire (fortes, P.W., et al. (1991) Resp. Med.
85(suppl):25) which indicates an improvement in the patients' quality of life. Improvements are also seen as an increase from baseline FVC at trough, an increase in time to first COPD
= exacerbation, and a decrease from baseline in post-exercise breathlessness (modified Borg Scale; Stulbarg, M., Adams, L. Dyspnea. In: Murray J, Nadel f, eds.
Textbook of Respiratory Medicine. Philadelphia, PA: WB Saunders, 2000; 541-552). Measures of safety are adverse events, vital signs, electrocardiogram at all double-blind visits, and laboratory assessments.
Example 9: TNFa Inhibitor iu Treatment of IPF
Clinical study of D2E7 in hlillIall subjects with IPF.
A multi-center, double-blind, placebo-controlled study comparing treatment of IPF patients with D2E7 versus treatment with placebo is performed. Patients are eligible for the study if they have histologically verified fPF and have a decline in lung function of at least 10% during the 12 months prior to the beginning of the study, despite continuous or repeated treatment with glucocorticoids or other immunosuppressive agents or both for at least 6 months. The inain histological feature used to identify 11)F is the presence of subpleural and periacima fibrotic lesions with only minor cellular infiltration. The absence of bilateral patchy infiltrates on high-resolution computed tomography and the demonstration of predominantly peripheral distribution of lesions are the radiological criteria for identifying the disease. Patients with a history of exposure to organic or inorganic dust or drugs known to cause pulmonary fibrosis and those with connective-tissue disease or other chronic lung diseases are excluded.
Patients with end-stage IPF as identified on the basis of a total lung capacity of less than 45% of the predicted normal are also excluded. Baseline values for repeat pulmonary function tests, PVC, total lung capacity (TLC), and oxygen saturation are taken.
Following baseline measurements, patients begin receiving treatment. They are randomized and treated with either D2E7 or placebo in a blinded fashion.
Improvements in EPP patients include an increase in the overall survival rate of patients in the study, and improvements in FVC, total lung capacity (TLC) and oxygen saturation. Improvement in puhnonary function is defined as a 10% or greater increase in predicted value of FVC or TLC, or a 3% or greater increase in oxygen saturation with the same fraction of expired air, resting or exertional. A decrease of similar manner for each measure is considered a deterioration. Patients who do not demonstrate improvement or deterioration are considered stable.
Example 10: TIN-Fa Inhibitor In Reducing Inflammation and Restenosis Study of restenosis using mouse carotid arteiy model The following study of restenosis is performed using the mouse carotid artery model (Kumar and Lindner (1997) Arterioscler. Thromb. Vasa Biol. 17:2238; de Waard et al. (2002) Arterioscler. Thromb. Vasc. Biol. 22:1978). Mice, ranging in age from two to four months, are anesthetized by intraperitoneal (i.p.) injection of a solution of xylazine. The left common carotid artery is dissected and ligated near the carotid bifurcation. Mice arc then allowed to recover.

A monoclonal anti-TNFa antibody which is known to bind and neutralize mouse TNFa, e.g., antibody TN3 (TN3-19.12) (see Marzi et al. (1995) Shock 3:27;
Williams et al. (1992) ProcNatl Acad Sci USA. 89:9784; BD Biosciences Pharmingen) is administered to the experimental group. Mice receive daily subcutaneous injections per week of either the anti-TNF antibody or a placebo. At either 2.5 or 4 weeks after the ligation of the carotid artery, mice are sacrificed and subsequently fixed by perfusion with 4% parafonnaldehyde inPBS. The carotid arteries are excised, immersed in '70%
(v/v) ethanol, and enabedded in paraffin. The nonligated right carotid artery serves as an internal control for both the D2E7 injected and placebo injected mice. Serial sections are cut for morphometric analysis, as described in de Waard et al., supra.
Morphometric analysis provides a measurement of the total vessel area for the treated and untreated ligated carotids at certain set distances from a comrnon physical reference point. It has previously been shown that the ligation results in the narrowing of the arteries (constructive remodeling) (Kumar and Lindner, supra; Kumar et al.
(1997) Circa/at/on 96:4333). Cross sections of the carotids are mounted on microscopic slides and stained with hematoxylin and eosin. Images of the carotid arteries are obtained using microscopic digital photography and the cross sectional areas of the intimal and the media are measured for a decrease in arterial narrowing (i.e, larger vessel diameter) as compared to placebo injected mice.
Example 11 : TNFo. Inhibitor in Monkey Model of Atherosclerosis Effect of D2E7 in monkey model of atherosclerosis.
The following study is performed using a diet-induced monkey model of atherosclerosis (Lentz SR et al. (2002) Circulation 106(7):842-6; Sundell CL
et al.
(2003)305(3):1116-23).
Adult cynomolgus monkeys (Macaca fascicularis) are fed an atherogenic diet that contains 0.7% cholesterol and 43% total calories as fat. After 44 1 months on the atherogenic diet, animals are sedated with ketamine hydrochloride (20mg/kb IM) and anesthetized with sodium pentobarbital (20mg/kg IV). A nonobstructive catheter is inserted into an axillmy artery for blood sanapling, and the axillaty vein is cannulated for administration of either D2E7 or placebo and supplemental anesthesia (soditun pentobarbital 5mg/kg per hour). D2E7 has been shown to effectively inhibit TNTicc activity in a variety of species, including cynomolgus monkeys (see U.S.
Patent No.
6,258,562).
Prior to infusion of D2E7 or placebo, blood is collected from the axillary artery catheter directly into a 1/10 volume of 3.8% soditun citrate for hemostatic assaying.
After collection, blood samples are placed immediately on ice, and plasma is isolated by centrifugation at 2500g for 30 minutes at 4 C. Additional blood samples are collected into serum separator tubes for determination of cholesterol or into serum separator tubes prepared with 3.4 nimol/L EDTA for determination of total plasma homocysteine (tHCY).
D2E7 or placebo is infused in laml of saline over 10 minutes through the axillary vein catheter. After infusion, blood samples are collected regularly.
The degree to which the animals axe suffering atherosclerosis after treatment is assessed in various ways. Serum samples are regularly taken from the monkeys and assayed for total cholesterol, BM, cholesterol, LDL cholesterol, tliCY and triglycerides.
Treated monkeys are examined to determine if total cholesterol, and LDL
cholesterol, and tHCY levels are lower as compared to placebo treated monkeys, and whether LIDL
levels are higher.
Example 12: TNFcc Inhibitor on Treating Restenosis in Patients Study of D2E7 in human subjects with restenosis Patients who have undergone balloon angioplasty are chosen for the study, as they have an increased chance of restenosis occurring within the first six months following angioplasty.
Prior to treatment, estimates of vessel and lesion parameters are made with reference to the guiding catheter. Estimates include referencc vessel diameter (RVD), pretreatment minimal luminal dianieter (Iva,D, which is determined by (RVD X
[1 ¨
preprocedural percent diameter stenosisp, postprocedural MID (which is determined by (RVD X [1 ¨ postprocedural percent diameter stenosisp, acute gain (postprocedural MLD ¨ preprocedural NUM), number of diseased vessels and number of traded vessels.
Experimental group of patients are administered either D2E7 in biweekly and weeldy closes of 40 nig or a placebo. Dosages may be adjusted by an ordinarily skilled artisan knowledgeable in restenosis. Patients are following and assessed at six months post-angioplasty to determine whether restenosis has occurred. Patients are also assessed at 9 months and long-term to determine the effect of delayed restenosis in those groups where restenosis was prevented or reduced due to treatment. Estimates of vessel and lesion parameters are recorded following D2E7 treatment. Statistical analysis is performed to compare the extent of restenosis in the patients. (Jackson et al.
(2003) Aln Heart J145:875).
Example 13: TNFa Inhibitor on Treating Heart Failure =
Clinical study ofD2E7 in human subjects with heart failure Patients with stable New Yorlc Association (NYHA) class 11 or IV heart failure and left ventricular ejection fraction of less than 35% are chosen for the study. Under the NYHA standard, class Da patients are defined as those with marked limitation of activity, i.e., they are comfortable only at rest, and class IV patients are defined as those who should be at complete rest, i.e., confined to bed or chair, or where any physical activity brings on discomfort and symptoms occur at rest. As described in Burns et al., left ventricular ejection fraction is associated with six-month mortality (Burns et al.
(2002) .T.4,71 Coll Cardio/. 39:34 Patients receive biweekly doses of D2E7 at 40 mg, or a dosage adjusted by an ordinarily skilled artisan knowledgeable in heart failure. The control group is given a placebo. Patients undergo exarninations at I, 2, 6, 10, 14, 20, and 18 weeks.
At each visit, each patient is examined and given an assessment of their overall heart failure status, relative to their status at the onset of the study, i.e., their NYHA
class is assessed.
At the end of the heart failure study, the patient's final NYHA class is compared to the initial NPIYA class.

Example 14: TNFcc Inhibitor in Mouse Model for Diabetes Study of TNF antibody in NOD mouse model The following study is performed using the nonobese diabetic (NOD) mouse model for type 1 diabetes. At the onset of the study, insulin levels are established by testing glucose levels in the blood of the NOD mice. Baseline insulin levels are established by fasting the mice overnight (17 hours). The blood glucose level is checked, and checked again 4 minutes after administering glucose. Blood glucose is determined with a reflectance meter. Glucose (200 mg/m.1, in 0.85% sodium chloride) in 1 mL syringes were prewarmed to 40 C and mice injected ip at 3 g/kg body weight. The second blood glucose measurement is determined 4 minutes after administering the glucose. Samples of the second blood measurement are used to determine the blood glucose level using the Glucometer Elite. The remaining sample of blood is collected into microfuge tube and used to separate the serum for insulin or C-peptide determination. Insulin levels are determined using a rodent radioinuramassay (1Z1A) kit per manufacturers' instruction or an enzyme-linked irnmiumassay (EL1SA).
Diabetic mice are chosen based on the criteria that they have blood glucose readings greater than 300 ing/aL. Non-diabetic inice are chosen such that their glucose reaclings.are uncler 200 ing/dL by glucose meter. NOD mice (those which displayed the glucose reading described above) are allowed to develop diabetes, and are administered doses of a placebo or a monoclonal anti-TNFcc antibody which is known to bind and neutralize mouse TNFcc, e.g., antibOdy TN3 (TN3-19.12) (see Marzi et al.
(1995) Shock 3:27; Williams et al. (1992) Proc Nall Acad Sci U S A. 89:9784; BD Biosciences Pharmingen). The mice receive daily subcutaneous injections of the TNF
antibody or a placebo. Insulin and glucose levels are measured at weekly increments to determine whether there is a decrease in blood glucose levels.

Example :15: TNrce, Inhibitor in Mouse Model of Diabetes Study of TNF antibody in type-2 diabetic 7nouse niodel The following study is performed using the NSY mouse model (type 2 diabetes) (Ueda et al, Diabetes Vol. 48, May 1999, 1168: 1174). The NSY mouse closely mimics human type 2 diabetes in that the onset is age-dependant, the animals are not severely obese, and both insulin resistance and impaired insulin response to glucose contribute to disease development. This study evaluates a number of phenotypic data, including glucose levels, insulin levels, height, and weight of-the mouse.
Glucose is measuxed in the NSY mouse according to standard techniques, including by an intravenous glucose-tolerance test. Baseline glucose resistance is measured prior to 12 weeks before the initiation of the study, and glucose, insulin, height, and weights are charted accordingly.
NSY mice are administered doses of either a placebo or a :monoclonal anti-TNFa antibody -which is known to bind and neutralize mouse TNFa, e.g., antibody TN3 (TN3-19.12) (see Marzi et al. (1995) Shock 3:27; Williams et al. (1992) PIOC Nall Acad Sci U
SA. 89:9784; BD Biosciences Phamiingen). Mice receive daily subcutaneous injections of the anti-TNF antibody or a placebo. Glucose level measurements are taken minutes after intraperitoneal glucose administration at 0, 12, 24, 36, and 48 weeks following the initiation of the study to examble whether there is a decrease in glucose intolerance.
Example 16: TNEct. Inhibitor in Obese Mouse Model Study of TNF antibody in mouse model for obesity The following study is performed using the obese mice (ob/ob) murine model.
Mice are evaluated for weight loss and a reduction in their body mass index.
Obese mice are characterized by marked obesity, hyperphagia, transient hyperglycemia and markedly elevated plasma insulin concentration associated with an increase in number and size of the beta cells of thc islets of Langerhans (Coleman, supra). Obese mice (ob/ob) are phenotypically distinguished from their lean littennates (ob/1-and +/A-) at about 26 days of age on basis of body weight. Obese mice gain weight rapidly and have marked obesity at 5 weeks of age. Obese mice reach a maximum body weight of 60-grams at an age of 7-8 months, while lean littermates reach their maximal weight of 30-40 grams in 3-4 months (Coleman, supra; Westman (1968) Diabetolog-ia 4:141;
Bray &
York (1971) Physiological reviews. 51:598).
Thirteen (13) week old ob/ob mice and, matched wild-type control mice are weighed to establish a base line weight. The mice are administered doses of either a placebo or a monoclonal anti-TNFa antibody which is known to bind and neutralize mouse TN-Fn, e.g., antibody TN3 (TN3-19.12) (see Marzi et al. (1995) Shock 3:27;
Williams et al. (1992) Proc Nail Acad Sci USA. 89:9784; BD Biosciences Phanningen). Mice receive daily subcutaneous injections of the TNF zintibody or a placebo. All mice are fed a high-fat diet (58% fat, Research Diets D12330) for weeks. Body weights are recorded weekly. Atter 12 weeks, the mice are euthanized, and the fat pads are dissected and weighed, as well as the final weight of the animal to determine the final body mass index (13MI) and occurrence of obesity.
Alternatively, ob/ob mice can be treated with D2E7 beginning at birth, and fed a regular diet, i.e., not low-fat, not high-fat diet. Treated and control mice (ob/ob atm-mates) are weighed weekly. Normally, at five weeks ob/ob mice exhibit a BMI
which indicates that they are obese. Mice are examined at five weeks to determine if they have a lower BA/ft:measurement than the controls.
Example 17: TNFoc Inhibitor in Treating Type 2 Diabetes in Humans =
Study of D2E7 in 1111171a71 subjects with diabetes type 2 Patients who arc diagnosed with type 2 diabetic are selected for the study.
The following inclusion criteria are used: 40-65 years of age, known duration of diabetes >
12 months, stable BMI <35 kg/m2, supine blood pressure< 140/90 m_m/lig, serum creatinine <106 Amo1/1, nì24-h TJAE between 20 and 200 itg/min in samples assessed weekly during the 3 months prior to the first evaluation and in the 15-day placebo run-in period, and no cardiovascular, hepatic, or systemic disease before the beginning of the study. The subjects do not take any additional drugs other than those for the treatment of their diabetes. For three days prior to ancl throughout the duration of the study, the patients follow an isocaloric diet (-0.13 mi x kg -1 X day -1 ; 50%
carbohydrates, 35%
lipids, 15% proteins) with no restriction on sodium intake. Adherences to the dietary recommendations are checked at each visit.
Patients are administered 40 trig of D2E7 in a biweekly dosing regiment, although this dose and the frequency of the dose can be adjusted by an ordinarily skilled artisan with lcuowledge of HCV treatments. Patients are monitored at least every week for twelve weeks, with repeated assays like those which were perfornaed prior to the initiation of the D2E7 treatment and as described below.
For each patient's evaluation throughout the study, the following baseline examinations are performed: supine blood pressure measurements; 13Mi; the mean of three twenty four hour urine samples; blood glucose levels; twenty four hour urffie glucose; serum creatine levels; creatinine clearance; and an electrocardiogram reading.
Furthermore, each subject keeps a daily journal to monitor typical type 2 diabetic symptoms such as fatigue, excessive thirst, frequent urination, blurred vision, a high rate of infections, wounds that heal slowly, mood changes, and sexual problems.
Patients are examined to determine if there is a reduction in blood glucose levels in D2E7, as well as reduction in symptoms typical to typell diabetes such as fatigue, excessive thirst, :freqnent urination, blurred vision, a high rate of infections, wounds that heal slowly, and mood changes. =
Example 18: TNFa Inhibitor in Iron Deficiency Anemia Study of TIVF antibody in rat model of iron deficiency The following study is performed using the rat animal model of iron deficiency anemia (Catani et al (2003) Braz. JZ Med. Biol. Res. 36;693). Male Wistar-EPM
rats (approximately three weeks old) are fed an AIN-93G (American Institute of Nutrition Rodent Diets) iron-free diet for a period of two weeks to induce iron deficiency anemia.
Rats arc administered closes of a placebo or a monoclonal anti-TNFa antibody which is known to bind and neutralize rat TNFa, e.g., antibody TN3 (TN3-19.12) (see Marzi et al. (1995) Shock 3:27; Williams et al. (1992) Proc Natl Acctd Sci U S A.
89:9784; BD
Biosciences Pharmingen). Blood samples are taken pre and post treatment to determine hemoglobin and hematocrit values. For the analysis of hematocrit and hemoglobin concentration, blood is collected and mixed with 5m1 of 0.5 M EDTA.
Hernatocrit is determined by centrifugation of blood in sealed heparinized capillaries.
Hemoglobin concentrations are calculated from the absorbance of cyanmethemoglobin at 546 rim.
Rats are examined to determine if there was an improved hematocrit measurement.
Example 19: TNFa Inhibitor Study of Chronic Disease Anemia Study of TIVF antibody on anenzia associated with chronic inflanzmatory disease The following study is performed using a rat model of anemia of chronic disease (Coccia et al, (2001) Exp. Hematology 29;1201). Eight to ten week old female Lewis rats are inoculated on day 0 with an intraperitoneal (i.p.) injection. of peptidoglycan-polysaccharide polymers (PG-APS) suspended in 0.85% saline equilibrated to a dose of 15 i_tg rharnnose/kg. Blood is collected via tail veins into EDTA-coated Microtainer tubes and complete blood counts (CBC) are performed on an ADVA 120 Hematology Systenr calibrated for rat blood. An additional blood sample is collected and separated on Microtainer serum separator centrifuge tubes and sera are analyzed for iron, bilirubin, and endogenous EPO concentrations. Rats are administered doses of a placebo or a monoclonal anti-TNEa antibody which is known to bind and neutralize mouse 'FNI7a, e.g., antibody TN3 (TN3-19.12) (see Marzi et al. (1995) Shock 3:27; Williams et aL
(1992) P7'OC Nall Acad Sci USA. 89:9784; BD Bioseiences Pharmingen), and examined for improved iron, bilirubin, and EPO concentration measurements.
Example 20: TNFa Inhibitor on Anemia Study of D2E7 antibody in human subjects with anemia Patients who exhibit symptoms commonly associated with anemia are examined and tested to determine if they stiffer from anemia. Symptoms commonly associated with anemia are fatigue, chest pain, shortness of breath, pale complexion, and rapid heart rate. Examples of tests which indicate anemia are the complete blood count (CBC), reticulocyte count, and measurements of iron supply, including the serum iron, total iron-binding capacity, and sertun ferritin. In patients with sever anemia and abnormalities in red blood cell moiphology, a bone marrow aspirate and biopsy are important diagnostic tools. Patients who suffer from anemia are selected for the study.
In the CBC test, automated cell counters measure a number of panuneters as part of the CDC, including the hemoglobin, red blood cell count, red blood cell volume distribution, platelet count, and white blood cell count. The counter also calculates the hematocrit (based on the RBC count and volume), the mean cell volume (MCV) (based on volume distribution), mean cell hemoglobin (MCH)(hemoglobin divided by hematocrit), and the red cell distribution width (RDW). The red cell indices and RDW
are used together with a direct inspection of the Wright-stained blood smear to evaluate red blood cell morphology.
Like the CBC test, an accurate measure of the reticulocyte count is key to the initial classification of any anemia. Reticulocytes are newborn red blood cells that contain sufficient residual RNA that they can be stained with a supravital dye and = counted as a percent of the circulating red cell population. In the basal state, the normal reticulocyte count ranges from 1 to 2 percent according to the counting method. This correlates with the normal daily replacement of approximately I percent of the circulating red blood cell population. Increases in the reticulocyte count provide a reliable measure of the red blood cell production response to anemia.
To use the reticulocyte count as a production measure, it must first be corrected for changes in the patient's hematocrit and for the effect of erythropoietin on the early release of marrow reticulocytes into circulation. The hematocrit (HOT) correction converts the reicultocyte percentage to au absolute niunber:
% Reticulocytes X patient HCT = absolute % reticulocytes 45%
The marrow reticulocyte ("shift") correction involves dividing the absolute percentage by a factor of 1.5 to 2.5 whenever there is prominent polychromasia on the peripheral blood smear. The shift correction should always be applied to any patient with aneinia aud a very high reticulocyte count to provide a true index of effective red blood cell production. A nomml patient will respond to a hernatocrit less than 30 percent with a two-to three-fold increase in the reticulocyte production index. This measure alone, therefore, will confima the fact that the patient has an appropriate erytlu-opoietin response, a noiinal erythroid marrow, and sufficient iron supply to meet the challenge.
When the reticulocyte index falls below 2, a defect in manow proliferation or precursor maturation rnust be present.
Standard naeasures of iron supply include the serum iron, transferring iron-binding capacity (TE3C), and the serum ferritin level. The nonnal senim iron ranges from 9 to 27 pinol/L (50 to 150 Rg/c1L), while the normal TIBC is 54 to 64 mon (300 to 360 p.g/dL). Therefore, in the basal state, only 30 to SO percent of the transferring in circulation is saturated with iron. Important information is provided by each measurement as well as the calculated percent saturation. The serum fenitin is used to evaluate body iron stores. Adult males have serum fenitin levels of between 50 and 150 rrig/L, corresponding to iron stores of from. 600 to 1000 mg. Adult females have lower serwn fenitin levels (15 to 50 mg/L) and smaller iron stores (0 to 300 mg).
Lower senun fen-itin levels are observed as iron stores are depleted; levels below 15mg/L
indicate store exhaustion and iron deficiency.
A sample of bone marrow is readily obtained by needle aspirate or biopsy. It is of greatest value in patients who have a hypoproliferative anemia or a disorder of red blood cell maturation, providing valuable information as to marrow structure and cellularity, as well as precursor proliferation and maturation. The ratio of erythroid to graimlocytic precursors (E/G ration) is used to asses the proliferative capacity of erythorid precursors.
A patient with hypoproliferative anemia and a reticulocyte index <2 will demonstrate an E/G ratio or 1:2. In contrast, the hemolytic anemia patient with a production index to 5 will have an E/G ratio >1:1. Red cell precursor maturation defects are identified Erom the mismatch between the E/G ratio and reticulocyte production index.
These individuals demonstrate and E/G ratio of greater than 1:1 together with a low reticulocyte index, typical of the ineffective erythorpoiesis of a maturation disorder.
Following baseline measurements, patients begin receiving treatment. They are nuidomized and treated with either D2E7 or placebo in a blinded fashion.
Patients' complete blood count (CBC), reticulocyte count, and measurements of iron supply are monitored at least every two weeks.

Example 21: TN1Ta Inhibitor iu Animal Model of Neuropathie Pain l'NF antibody in rat sciatic nerve ligation model 'The following study is performed using the rat sciatic nerve ligation model for neuropathic pain (Bennett and Zie (1938) Pain 33:87). Baseline behavioral measurements (response to mechanical allodynia and heat hyperalgesia, protocols are described below) are made prior to surgery. Heat hyperalgesia refers to the rat heat pain threshold, and mechanical allodynia refers to the response threshold to light tactile stimuli. Male Sprague-Dawley rats, weighing between 120-150 grams, are anesthetized and a sciatic nerve ligation procedure is performed on each. The sciatic nerve ligation procedure involves exposing the common sciatic nerve, which is then tied loosely with 4 ligatures with about 1 inni spacing. Rats are allowed to recover and are administered doses of either a placebo or a monoclonal anti-TNFcf, antibody which is known to bind and neutralize rat TNFo., e.g., antibody TN3 (TN3-19.12) (see Mari et al.
(1995) Shock 3:27; Williams et al. (1992) Proc Natl Acad Sci US A. 89:9784; BD Biosciences Pharmingen). The experimental groups receive daily subcutaneous injections per week of TNF antibody or a placebo.
Following the surgery, mechanical allodynia and heat hyperalgesia are performed on a weekly basis for 10 weeks. Analgesia testing examines responses to noxious heat and is determined by placing the rats in a chamber with a clear glass floor and aiming a radiant heat source from, beneath the floor at the plantar surface of the affected foot.
Withdrawal latency and duration are recorded. Increased latency to withdraw the bind paw after treatment is demonstrative of analgesic activity.
Responses to normally innocuous mechanical stimuli (mechanical allodynia measurement) are deterrnined by placing the rats in a chamber with a screen floor and stimulating the plantar surface of the hind paw with graduated von Frey hairs which arc calibrated by the grams of force required to bend them. Rats with sciatic nerve ligation respond to lower grams of mechanical stimulation by reflexive withdrawal of the foot than unoperated rats. This response to stimuli which arc normally innocuous is termed allodynia. Increases in the grams of mechanical force required to produce foot withdrawal after treatment is demonstrative of autiallodynic activity and a decrease in neuropathic pain.
Example 22: TNEa Inhibitor in Animal Model of Neuropatbic Pain Study of .77VF antibody in rat segmental spinal nerve ligation model The following study is performed using the rat segmental spinal nerve ligation model for neuropathic pain (Kim and Chung, Pain 50 (1992) 355-363.). Male Sprague-Dawley rats, weighing 120-150 grams, are anesthetized, and placed in a prone position.
The left paraspinal muscles are separated from the spinous processes at the L4 levels. The left L5 and L6 nerve roots are exposed and tightly ligated with 6-0 surgical silk suture distal =to the dorsal root ganglion. Rats are allowed to recover and are = adnainistered doses of either a placebo or a monoclonal anti-TNFa antibody which is known to bind and neutralize rat TNFa, e.g., antibody TN3 (TN3-19.12) (see Marzi et al. (1995) Shock- 3:27; Williams et al. (1992) Proc Nati Acad Sci U SA.
89:9784; BD
Biosciences Phanuingen). The experimental groups receive daily subcutaneous injections per week of TNF antibody or a placebo. Baseline behavioral measurements (response to mechanical allodynia and heat hyperalgesia testing, as described above) are made prior to surgery. Following the surgery, Inechanical allodynia and analgesia testing for neuropathic pain are performed on a weekly basis for 10 weeks.
Example 23: TNFa Inhibitor in Treatment of Nettropathic Pain Study examining D2E7 iii hianan subjects with neuropathic pain Patients diagnosed with neuropathic pain are selected for the study. Clinical neuropathic pain is determined based on clinical grounds, including history, physical examination and appropriate investigation of symptoms and signs expressed by the patient. The definitions of diagnostic criteria defined in the International Association for the Study of Pain (IASP) Classification of Chronic Pain are used to support the clinical diagnosis of neuropathic pain. Patients are excluded based on criteria including, but not limited to, another pain problem of equal or greater severity that might impair the assessment of neuropathic pain; significant neurological or psychiatric disorders unrelated to causes of neuropathic pain which might impair the assessment of neuropathic pain; current drug or alcohol abuse; and clinically significant liver, renal or pulmonary disease.
Evaluations of patient neuropathic pain are made using standard pain assessment tools such as the Short Form-McGill Pain Questionnaire (SF-MPQ); a 100-mm vertical Visual Analog Scale (VAS) (0 =no pain, 100 = intolerable pain); and the Clinician Global Impression of Change (CGIC). Patient's may also use a daily diary to score their neuropathic pain. Each evening, patients rate the average intensity of their pain during the preceding 24 hours.
Following a week of baseline measurements, patients begin receiving treatment.
They are randomized and treated with either D2E7 or placebo in a blinded fashion.
Patients are monitored every two weeks, and examined for a reduction in the patient's neuropathic pain assessment and average intensity of pain, as charted in their daily diaries.
Example 24: TNIN Inhibitor in Animal Model for Hepatitis C Infection Study ofD2E7 in HC 17 chimpanzee inodel The following study is performed using the chimpanzee hepatitis C virus (FICV) model (Shimizu et al. (1990) Proc, Natl. Acaa Sci. USA 87:6441).
Chimpanzees are inoculated intravenously (i.v.) with 0.5m1 of undiluted plasma obtained from a patient with posttransfusion acute non-A, non-.B hepatitis.
The inoculum contains, for example, 106'5 chimpanzee 50% infectious doses per nil (CID50/m1) of PICV. Serum samples and liver biopsy specimens are taken before inoculation and weekly alter treatment. After inoculation, chimpanzees are administered doses of D2E7 or a placebo. D2E7 is effective at binding TNF in a high affinity manner across species, see U.S. Patent No. 6,258,562.

HCV levels following innoculation are monitored in a number of ways. Serum samples axe regularly taken from the chimpanzees and assayed for alanine aminotransferase (ALT). The ALT assay is one of a group of tests lcnown as liver function tests (or LF1s), and is used to monitor damage to the liver.
Circulating antibody to IICY (anti-C100-3 antibody) is detected by the BCV antibody ELISA
test system. In addition, HCV is detected in the serum samples, cllNA/PCR assays are performed as described in Weiner et al, (1990) Lancet 335;1. Frozen liver biopsy specimens are also tested for the cytoplasmic antigen by ininnumfluorescent staining with monoclonal antibody 48-1 according to the method described in Shimizu et al (1985) Proc. Natl. Acad. Sci. USA 82;2138. Treated chimpanzees are examined to determine if ALT levels and HCV serum levels are lower as compared to placebo treated chimpanzees.
Example 25: TNFcc Inhibitor in Human HCV Infection Study of D2E7 in treating TICY in hunians Men and women aged 18 to 70 years with compensated chronic HCV infection are selected. To qualify for the study, patients must test positive for anti-HCV (second-generation enzyme inununoassay) and HCV RNA by reverse transcription ¨polymerase chain reaction (RT-PCR). Patients also have a liver biopsy within a year of the study entry showing chronic hepatitis, and have elevated serum alanine transaminase (ALT) levels for at least 6 months before initiation of treatment. Entry leukocyte counts should be least 2,500/pL; the platelet counts shoirld be greater than 70,000/11L.
Exclusion criteria include but are not limited to any other cause of liver disease or other relevant disorders, including human immunodeficiency or hepatitis B virus coinfection;
clinically significant cardiac or cardiovascular abnormalities, organ grafts, systemic bacterial or fungal infection; clinically significant bleeding disorders; alcohol or drug abuse, within the previous year.
Pretreatment and post-treatment serum HCV RNA is quantified by a standardized RT-PCR assay. Qualitative detection of HCV RNA is performed by RT-PCR in serum smnples obtained post treatment. Genotyping of HCV is performed by reverse hybridization assay. Emotional and psychological states are measured using suitable health-related quality of life scales.
Following baseline measurements, patients begin receiving treatment. They are randomized and treated with either D2E7 or placebo in a blinded fashion.
Patients are administered 40 mg of D2E7 in a biweeldy dosing regiment, although this dose and the frequency of the dose can be adjusted by an ordinarily skilled artisan with lcnowledge of HCV treatments. Patients are monitored at least every 4 weeks, with repeated assays like those which were performed prior to the initiation of the D2E7 treatment.
A
decrease in HCV levels relative to those who received only placebo is evidenced by a weaker RT-P CR signal.
Example 26: TNEcc Inhibitor in Mouse Model for Psoriasis Study of TNF antibody in SCID 71101iSe model of psoriasis Severe Combined Immunodeficient (SOD) mice that have undergone transplantation of htunan psoriasis plaques are selected as an animal model to study psoriasis because these mice retain the typical clinical and histological featin-es of psoriasis for a prolonged period (Nickoloff et al. (1995) Aln Pathol 146:580-8).
2-3 month old :female out bred C.E17 scrip mice are obtained from a pathogen-free animal breeding facility. Human skin speciinens are taken from white male patients with chronic plaque psoriasis. The spindle-shaped skin specimens 1 x 3 cm inches in size comprising clinically involved skin are obtained under local anesthesia and are prepared for transplantation by removing 'subcutaneous fat, held in cooled phosphate-buffered saline (PBS). Skin specimens are grafted within 1-2 hours.
The full-thickness skin specimens are dissected into pieces 8-10 mm in diameter and are then transplanted on to the back of the mice, each mouse carrying one transplant.
For the surgical procedure, mice are anesthetized by intraperitoneal injection (i.p.) of a 1:1 mixture of miclazolam and fentanyl dihydrogen citrate. A spindle-shaped piece of full thickness slcin is grafted onto a corresponding excisional full thickness defect of the shaved central dorsum and is fixed by 6-0 atratunatic monofilarnent sutures.
After a sterile Vaseline impregnated gauze is applied, the grant is protected from injury by suturing a sldn pouch over the transplanted area using the adjacent lateral skin. The sutures and over-tied pouch are left in place until they resolve spontaneously after 2-3 weeks.
The SCID-human skin chimeras exhibit symptoms similar to human psoriasis. A
transplanted plaque on the SCID mouse shows clinical features typical of psoriasis including scales, erythema, and thickening. This model also exhibits histological features typical of psoriasis including parakeratosis, acanthosis, elongated rete ridges, supra-papillary thinning, and lymphornononuclear infiltrates in the papillary dermis.
Transplanted SCID mice are are injected subcutaneously at the site of the lesion with either a placebo or a monoclonal anti-TNFa antibody which is known to bind and neutralize mouse TNFcc, e.g., antibody TN3 (TN3-19.12) (see Marzi et al.
(1995) Shock 3:27; Williams et al. (1992) Proc Natl Acad Sci USA. 89:9784; BD Bioseiences Phanningen). The experimental groups receive daily subcutaneous injections per week of TNF antibody or a placebo. Improvement in the TNF antibody treated. SCID
mice is evidenced by a reduction in the symptoms associated with the psoriasis plaques.
Example 27: TNFa Inhibitor in Clinical Studies for Psoriasis D2E7 in lniman subjects with psoriasis Patients with moderate to severe chronic plaque psoriasis are selected for the study. None of the patients will have received any psoriasis treatments for at least 4 weeks or any topical treatments for at least 2 weeks before study entry. Doses of D2E7 begin at 40 mg weekly or 40 nig every other week administered by subcutaneous injection.
Patients arc examined clinically every 2-,4 weeks. Clinical activity of psoriatic skin lesions is evaluated by means of the Psoriasis Area and Severity Index (PAS1) (Fredrilcsson and Pettersson (1978) Dermatologica 157:238-44) and the Physician's Global Assessment by the same investigator to ensure consistent evaluations.
At week 12, the primary end point of proportion of patients achieving at least 75%
reduction in PASI score compared to baseline is determined. Pniritus is assessed by using a validated scale. Quality able assessments arc measured using validated instruments, including, but not Limited to the DLQI, SF-36, and EQ-5D. Full-body photographs excluding the face are taken at scheduled visits throughout the study.
Skin biopsy specimens are obtained at scheduled intervals during the tudy to correlate histology and biomarkers in the skin with treatment. A biopsy of normal skin is obtained at baseline for comparison with psoriatic skin.
Example 28. TNFa 'Inhibitor-In Animal Model for Behcet's Disease Study of TIVF antibody in Behcet's syndrome mouse model The following study is performed using the rnouse HSV model of Behcet's disease (Hirata, Y., et al. (1993)Acta. Otolmyngol. Suppl. 503:79). Earlobes of mice which express InunanTNFcc (see EMBO J(1991) 10:4025-4031 for further description) are scratched with a needle, then inoculated with 1.0 x 106 plaque-forming units (pfu)/naL of Herpes Simplex Virus type 1 (HSV1) (KOS strain) solution, which causes inflammatory cells to accumulate in and around the blood vessels. As a result, intestinal, oral, ear lobe, and genital epithelial lesions occur. A mouse with Behcet's disease-like syndrome is defined as a mouse with two or more symptoms, which are similar to the typical morphological changes seen in human Belicet's disease.
A monoclonal anti-TN-Fa antibody which is lcnown to bind and neutralize mouse TNFcc, e.g., antibody TN3 (TN3-19.12) (see Marzi et al. (1995) Shock 3:27;
Williams et al. (1992) Proc Natl Acad Sci USA.89:9784; BD Biosciences Pharmingen) is adnainistered to the HSV-induced Behcet's syndrome mice in a range of doses both before and after inoculation, or from the day of lesion occurrence.
Appropriate placebo controls are also administered. Hair loss, ulceration of the mouth and genital skin, and eye involvement is monitored, and tissue samples are collected from lesions.
Tissue samples are formalin-fixed and paraffin-embedded for sectional analysis.
Lesion sections are stained with hematoxylin and eosin and examined for the appearmace of inflanamatory cell. As a control, 30 mice are inoculated at the same site with a culture medium. Four weeks later, a second inoculation is performed using the same method, followed by 16 weeks of observation.

Mice are examined for hair regrowth and a decrease in ulcerations in the treated mice as compared to placebo treated mice. Improvements in lesions in treated mice, as determined through visual inspection and histological analysis, noting a decrease in inflarrunation at the site of the ulceration and a decrease in the number of inflarrnuatory cells, T cells, at the site of the ulceration also are further indications of improvements.
Example 29. TNEcy. Inhibitor In Treating Kawasalci's Disease Effect of TNF antibody in Kaivasalci's Disease using L. casei 171011Se model Using the mouse L. casei model of Kawasaki's disease (Lehman, T.J., et al.
(1985) Arthritis Rheum 28:652; along, T.T. (2002)./nt finimmo/ 15:79; Brahn, E., et al.
(1999) Clin Immunol 90:147), the following study is performed. Coronary arteritis is induced in mice expressing human TN-Fa (see above) with a single intraperitoneal (ip) injection ofiactobacillus easel cell fragments. It has been shown that histologic sections of the hearts of mice treated with L. casei, resemble the vasculitis and aneurysms observed in the meditun-sized coronary arteries of children with Kawasaki disease (Lehman et al. (1985) Arthritis Rheum 28:652; Duong (2002) Int Immunol 15:79; Bra.hn et al. (1999) Clin Immimol 90:147).
L. casei injected mice are administered a either control placebo or a monoclonal anti-TNFec antibody which is known to bind and neutralize mouse 'TNFet, e.g., antibody TN3 (TN3-19.12) (see Marzi et al. (1995) Shock 3:27; Williams et al. (1992) PrOC Natl ilcad Sci USA. 89:9784; BD Biosciences Pharrningen) intraperitoneally through standu-d protocols. Hearts from injected mice are harvested ors day 14 (early disease) or at the end of the study (established disease). Histologic sections are scored blindly for vasculitis.
A decrease in corollary arteritis is assessed by determining a reduction in inflammatory lesions of the coronary vessel wall of TNF antibody treated mice as compared to placebo treated animals. A decrease in coronary m-teritis is assessed as a - reduction in inflammatory mononuclear cell infiltrate of the coronary vessel wall accompanied by a reduction in intirnal proliferation and less narrowing of the vessel lumen as compared to placebo treated animals.
Example 30. TNFa Inhibitor In Animal Model for Kawasaki's Disease TNF antibody in Kawasalci's Disease Wing ANCA .tIffouse Model = The following study is performed using the mouse anti-endothelial cell antibodies (ANCA) model of Kawasaki's Disease (Grunebaum et al. (2002) Clin.
Exp.
ionnunoL 130:233; Blank et al. (1995) Clin. Exp. 'minima. 102:120; Tomer et al. (1995) Arthritis Rheum. 38:1375; Damianovich et al. (1996) J. Inununol. 156:4946).
Aninaals are immunized with anti-endothelial cell antibodies (ANCA) containing proteinase 3-specific antibodies derived from a Wegener's granulomatosis patient's plasma.
Mice are immunized with purified ANCA and control mice are injected with normal IgG.
Mice are administered weekly doses of either a placebo or a monoclonal anti-TNFa antibody which is lcnown to bind and neutralize mouse TNFa, e.g., antibody TN3 (TN3-19.12) (see Marzi et al. (1995) Shock 3:27; Williams et al. (1992) PIOC Natl Acad Sei US A. =
=89:9784; BD Biosciences Pharrningen) for upto four months. Three months after-the immunization with the human ANCA, mice develop endogenous antibodies to ANCA.
Mice are euthanized ,and lungs, kidneys, and heart are examined histologically for lymphoid cell infiltration surrounding arterioles and venules, as well as deposition of Igs at the outer part of blood vessel walls like that observed in patients with Kawasaki's Disease (Grunebaurn et al. (2002) Chi?. Exp. Immunol. 130:233; Blank et al.
(1995) Clin. Exp. Immunol. 102:120; Tomer et al. (1995) Arthritis Rheum. 38:1375;
Damianovich et al. (1996)J Immunol. 156:4946). A decrease in lymphoid cell infiltration and IgG deposition in vessel walls and a decrease in antibody titre of ANCA
is indicative of an improvement in Kawasaki's disease.

Example 31. TNEa Inhibitor in Treatment of Kawasaki's Disease Clinical study of D2E7 in human subjects with Kawasaki's disease Patients suffering from Kawasaki's Disease (ICD) are enrolled into the study;
all patients have fever and at least 4 of the 5 clinical criteria published for ICD (Barron, K.S., et al. (1999) J. Rheunzatol. 26:170). Case-controls are also identified.
The diameter of the coronary arteries is measured by echocardiography and corrected for body surface area. Electrocardiograms are screened for typical changes that may be present in KD including prolonged PR or QT interval, abnormal Q waves, ST- and T-wave changes, low voltages or arrhythrnias, ICD patients are administered either D2E7 in biweeldy and weekly doses of 40 mg or a placebo. Dosages may be adjusted by an ordinarily skilled artisan knowledgeable in K.D. Patients are monitored for fever reduction. Adjuvant therapy, e.g, corticosteroids, are administered as needed.
Patients are monitored and follow-up echocardiography is used to determine if coronary artery damage has occurred or whether an improvement in coronary artery lesions, demonstrated through improved echocardiogram results has occtuTed.
Example 32. TNFa Inhibitor in Treatment of Beheees Disease Clinical Study of D2E7 in HUnzait Subjects With Behcet's Disease Patients for the study arc selected because they fulfill International Study Group criteria, which requires the presence of oral ulceration plus any two of genital ulceration, typical defined eye lesions, typical defined skin lesions, or a positive pathergy test (Lancet. (1990) 335:1078; Kaklaniani, V.G. et al. (2001) Senzin. Arthritis Rheum.
30:299) for a mean of 6 years. Behcet's patients are administered either D2E7 in biweekly and weekly doses of 40 mg or a placebo. Dosages may be adjusted by an ordinarily skilled artisan knowledgeable in Behcet's disease. Treated and placebo patients are given a systemic examination and detailed ophthalmological assessment, including visual acuity, measurement of intraocular pressure, slit-lamp biomicroscopy, and indirect oplithalmoscopy of the posterior segnient followed by fundus photography, both before and following the treatment regime. Patients are examined for an improvement in the documented symptoms associated with Behcet's disease, e.g., reduction in eye inflammation and reduction in number or severity of mouth ulcers.
Example 33: TNFcc Inhibitor iii Animal Model for Lupus Study of 'INF antibody in nzouse lupus model The IVIRL/1pr mouse model is chosen to study lupus (Reilly and Gilkeson (2002) Immunologic Research. 25(2):143-153; Mishra et al. (2003) Jain Invest.
111(4):539-552). MRL/1pr mice exhibit the onset of an accelerated autoimmune syndrome with polyelonal B cell activation and hypergammaglobulinemia beginning at about 8 weeks of age. In IvIRL/Ipr mice, there is serologic evidence of an array of autoantibodies, including anti-double- stranded DNA ( anti-dsDNA) autoantibodies and hypocomplementemia by 12-16 weeks of age. MRL/1pr mice exhibit clinical signs of arthritis, massive lymphadenopathy, splenomegaly, vasculitis, and glomerulonephritis (GN) by the age of 16-24 weeks. Approximately 50% of MRL/lpr mice die by 24 weeks of age, primarily from renal failure.
Eight week old female IvIRUIF Take are used in this study. At fourteen weeks, MRL/Ipr mice are injected intraperitoneally (i.p.) with either varying concentrations of a placebo or Rats are allowed to recover and are administered doses of either a placebo or a monoclonal ariti-TNPcc antibody which is known to bind and neutralize mouse TNFcc, e.g., antibody TN3 (TN3-19.12) (seelVlarzi et al.
(1995) Shock 3:27; Williams et al. (1992) Proc Nati Acad Sci U S A. 89:9784; BD Bioscienees Pharmingen). The experimental groups receive daily subcutaneous injections per week of TNF antibody or a placebo.
Some patients with lupus develop lupus nephritis which is defined by persistent inflammation (irritation and swelling) in the kidney. These patients may eventually develop renal failure and require dialysis or kidney transplantation. To examine the progression of renal disease, IvIRL/lpr mice are placed in metabolic cages for 24-hour mine collections after injection with D2E7. Urinary albumin excretion is determined pre and post treatment with D2E7 by ELISA using a standard curve of known - 13"/ -concentrations of mouse albumin (Cappel Research products, Durham, North Carolina, USA, as described in Weinberg et al. (1994)J Exp Med. 179:651). Improvements in early disease manifestations and progression of proteinuria are evidenced by a decrease in mean albumin excretion after treahnent.
Mice are sacrificed at week 19 by cervical dislocation after isoflurane anesthesia and the kidneys are removed. One kidney is fixed with buffered formalin, embedded in paraffin, sectioned and is stained with H&E. Renal pathology is examined and graded by standard methods for glornerular inflammation, proliferation, crescent formation, and necrosis. Interstitial changes and vasculitis are also noted. Scores from 0 to 3 are assigned or each of the features, and then added together to yield a final renal score, as described by Watson et al. (1992)J Exp Med. 17 6:1645-1656. Scores for necrosis and crescent formation are doubled prior to adding. For example, glomerular inflammation is graded as follows: 0, normal; 1, few inflammatory cells; 2, moderate inflammation; and 3, severe inflammation. Improvements are evidenced by minimal signs of inflammation or cellular proliferation (a lower renal pathology index) in the kidney section from the D2E7 treated mouse when compared to the placebo treated mouse.
Spleen weight is also measured to determine the delay or prevention of the progression of lupus activity in the mice. Spleen size is an indicator of lupus activity that reflects the underlying immunopathology of the disease. MRL/lpr mice develop massive splenornegaly and lymphadenopathy with disease progression. To determine spleen size, at age 19 weeks, mice animals from each group (treatment and placebo) are sacrificed and the mean spleen weights determined. A lower niean spleen weight indicates an improvement in lupus.
Example 34: TNEa. Inhibitor Treatment for Lupus Study examining D2E7 in human subjects with lupus Patients with diagnosed lupus are selected for the study based. Patients are selected based on their presentation of symptoms commonly associated with lupus including fever, fatigue, general discomfort, uneasiness or ill feeling (malaise), weight loss, skin rash, "butterfly" rash, sunlight aggravates skin rash, sensitivity to sunlight, joint pain and swelling, arthritis, swollen glands, muscle aches, nausea and vomiting, pleuritic chest pain, seizures, and psychosis. Additional symptoms include blood in the mine, coughing up blood, nosebleed, swallowing difficulty, skin color is patchy, red spots on skin, Engers that change color upon pressure or in the cold (Raynaud's phenomenon), numbness and tingling, mouth sores, hair loss, abdominal pain and visual disturbance. Patients are given a physical examination to determine whether or not they exhibit any of the characteristic symptoms indicative of lupus. The diagnosis of lupus is based upon the presence of at least four out of eleven typical characteristics of the disease.
Tests to determine the presence of these disease manifestations may vary but will include some of the following: antinuclear antibody (ANA) panel including anti-DNA
and anti-Smith antibodies, with the latter two tests generally positive in lupus alone;
characteristic skin rash or lesions; chest X-ray showing pleuritis or pericarditis; listening to the chest with a stethoscope to reveal heart friction rub or pleural friction rub; =
urinalysis to show blood, casts, or protein in the urine; a complete blood cell count showing a decrease in some cell types; kidney biopsy; and neurological examination.
This disease may also alter the results of the following tests: WBC count;
serum globulin electrophoresis; rheumatoid factor; protein, urine; protein electrophoresis -serum;
mononucleosis spot test; erythrocyte sediinentation rate (ESR); cryoglobulins;
direct Coombs' test; complement component 3 (03); complement; antithyroid microsomal antibody; antithyroglobulin-antibody; antimitochondrial antibody; and anti-smooth inusele antibody.
Patients are randomly divided into experimental and placebo oups, and are administered either D2E7 or the placebo. Desage nuigcs are used in the study to determine what dose is most effective for treating lupus. Dosages should begin at 40 mg, which is the D2E7 dose which has been found to be most effective at treating rheumatoid arthritis in patients. Patients are given 4 to 7 infusions of either D2E7 or placebo. Patients are re-examined every other week to determine if lupus symptoms are reduced or treated, determined by a reduction in the ESR and C-reactive protein (CRP) levels.

Example 35: TNFcc Inhibitor on Sjogren's Syndrome Study examining D2E7 in human subjects with SjOgren's syndrome.
Patients who meet the European and the American College of Rheumatology classification for primary Sjogren's disease are selected for the study (see Vitali et a.
(1993) Arthritis Rheum 36:340-7; Fox et al. (1986) Arthritis Rheum. 29:577-85).
Patients are at least 18 years old. At the time of enrollment all patients have active primary Sjogren's disease which is defined as the presence at screening of at least an elevated erythrocyte sedinaentation rate (ESR; >25/mm/hr) or hyperganamaglobulinemia (>1.4 grn/liter). Disease-modifying antirheumatic drugs (DMARDs) and corticosteroids are not allowed during the study and are discontinued at least 4 weeks before baseline.
Exclusion criteria include=serious infection in the previous 3 months, latent tuberculosis, documented human immunodeficiency virus or hepatitis C virus infection, life threatening vasculitis, lmovvn malignancy, concomitant severe or uncontrolled disease, and the presence of any other connective tissue disease.
The study includes administering 3 infusions of D2E7 (at a dosage of about 40 mg) at weeks 0, 2, and 6 and 2 follow-up visits at weeks 10 and 14. Patients are allowed to continue artificial tears, provided that the dosage and schedule are stable throughout the study.
Clinical, ophtbahnologic, and biologic evaluations are performed at baseline and at weeks 2, 4, 6, 10, and 14. Clinical assessments are performed by the same physician.
These include a general physical examination, a dry mouth evaluation (nsing a scale of 0-2 where 0¨ no dryness, 1¨mild-to inoderate dryness, and 2=severe dryness), and a speech test (number of dines the word "puttica" can be repeated during a 2-minute period, a technique presented by P.J. Shirlaw at the conference on New Advances in Basic Science, Diagnosis and Treatment of Sjogren's Syndrome, London, January 1997).
In addition, unstimulatecl whole saliva is collected for 5 minutes using the spitting technique according to established methods, and samples are weighed on an analytical balance to determine the volume of saliva obtained (1 gm----lml) (Navazesh (1993) ylniz /WACO(' SCi 694:72-7). A dry eye evaluation is also performed (scored on a scale of 0-2, where 0¨no symplon-is, 1=--mild-to-moderate symptoms relieved by artiEcial tears (Arts), and 2=- severe symptoms unrelieved by ATs), and the frequency of 11Se of Arts is determined.
Patients are also given a fatigue evaluation. (0-100 nun visual analog scale (VAS)) and answer a fatigue questionnaire (0---no fatigue, 1 = mild fatigue not interfering with daily activities, 2= nioderate fatigue that interferes with daily activities, and 3 --fatigue, with severely reduced activities). The clinical assessment may also include a tender joint count (maximum 64), tender point count (maximum 18), and patient's assessment of pain (0-100-mm VAS). Patient's and physician's global assessments were made using a 0-100 nun VAS.
All ophthalmologic assessments are performed by the same physician and include a fluorescein tear film breakup time (TBDT) test, the Schirmer I
test, and a corneal evaluation performed by lissamine green staining (van Bijsterveld score of 0-9).
Biologic parameters are measured through out the study and include the ESR, C-reactive protein level (CRP), complete blood cell count, renal and liver function tests, creating phospholcinase levels, serum levels of IgA, IgM, IgG, antinuclear antibodies (ANA), and rheumatoid factor(RE, and lymphocyte typing (numbers of CD4--- and CDS=
cells).
Diminishment in the symptoms associated with Sjogren's syndrome symptoms include reduction in the tender points and pain in the peripheral joints.
Example 36: TNFa Inliihitor on Juvenile Rheumatoid Arthritis Study examining D2E7 in children with juvenile rheumatoid arthritis Patients with diagnosed juvenile rheum. atoid arthritis (JRA) are selected for the study. Patients receive D2E7 for 16 weeks and are then randomly divided into experiinental and placebo groups. Patients are then administered either D2E7 or the placebo. Patients are administered a dosage range of between about 20 mg/m2 /BSA
(Body surface area) to a maximum of 40 nag every other week. Patients are given subcutaneous injections of either D2E7 or placebo on every other week for the duration of the treatinent. Patients are re-examined every other week to determine Utile symptoms of JRA are reduced or treated. Improvements in IRA are determined by a decrease in the, clinical symptoms of the disease. Improvement in JRA is determing using criteria defined by Giannini (Giannini et al. (1997) Arthritis &Rheumatism 40:1202). Using this criteria, the definition of improvement is al least a 30%
improvement from baseline in 3 of any 6 variables in the core set, with no more than 1 of the remaining variables worsening by >30%. The variables in the core set consist of =
physician global assessment of disease activity, parent/patient assessment of overall well-being (each scored on a 10-cni Visual Analog Scale), functional ability, nruuaber of joints with active arthritis, number of joints with limited range of motion, and erythrocyte sedimentation rate.
Example 37: Crystallization of D2E7 F(ab)'2 fragment Generation and purification of the .D2E7 Fab) '2 Fragment A D2E7 F(ab)'2 fragment was generated and purified according to the following procedure. Two nil of D2E7 IgG (approximately 63 mg/nil) was dialyzed against 1 liter of Buffer A (20 niMNa0Ac, pH 4) overnight. After dialysis, the protein was diluted to a concentration of 20 ing/ml. Immobilized pepsin (Pierce; 6.7 ml of slurry) was mixed with 27 rril of Buller A, mixed, and centrifuged (Beckman floor centrifuge, 5000 rpm, 10 min). The supernatant was removed, and this washing procedure was repeated twice more. The washed immobilized pepsin was re-suspended in 13.3 nil of Buffer A.

(7.275 ml, 20 ing/ml, 145.5 mg) was mixed with 7.725 ml of Buffer A 13nd 7.5 ml of the washed immobilized pepsin slurry. The D2E7/1)epsin mixture was incubated at 37 C
= for 4.5 hr with shaking (300 rpm). The inunobilized pepsin was then separated by centrifugation. Analysis of the supernatant by SDS-PAGE indicated that the digestion of D2E7 was essentially complete (-115 IcDa band unreduced, ¨30 and ¨32 IcDa bands reduced).
The D2E7 F(ab)'2 fragment was separated from intact D2E7 and Fc fragments using Protein A chromatography. One-half of the above reaction supernatant (10 nil) was diluted with 10 ml of Buffer B (20 traM Na phosphate, pH 7), filtered through a 0.45 p_ni Acroctisk filter, and loaded onto a 5 nil Protein A Sepharose column (Pharrnaciaili-Trap; previously washed with 50 inl of Buffer B). Fractions were collected.
After the protein mixture was loaded, the coltunn was washed with Buffer B until the absorbance at 280 inn re-established a baseline. Bound proteins were eluted with 5 ml of Buffer C
(100 inlvf citric acid, pH 3); these fractions were neutralized by adding 0.2 ml of 2 M
Tris-HCI, pH 8.9. Fractions were analyzed by SDS-PAGE; those that contained the D2E7 F(ab)12 fragment were pooled (-42 m1). Protein concentrations were determined by absorbance at 280 inn in 6 M guanidine-Ha, pH 7 (calculated extinction coefficients:
D2E7, 1.39 (AU-m1)/ing;17(ab)'2, 1.36 (AU-m1)/mg). The flow-though pool contained ¨38.2 mg protein (concentration, 0.91 ing/m1), which represents a 79% yield of F(ab)I2 (theoretical yield is ¨2/3 of starting material, divided by two [only half purified], i.e.
¨48.5 nag).
The D2E7 F(ab)'2 fragment was further purified by size-exclusion chromatography. The pooled Protein A flow-through was concentrated from ¨42 to ¨20 ml, and a portion (5 ml, ¨7.5 mg) was then chromatographed on a Superdex 200 column (26/60, Pharmacia) previously equilibrated (and eluted) with Buffer D (20 m_M
HEPES, pH 7, 150 rnM NaC1, 0.1 InM EDTA). Two peaks were noted by absorbance _at 280 nm:
Peak 1, eluting at 172-200 ml, consisted ofF(ab)'2 (analysis by SDS-PAGE; ¨115 kDa band unreduced, ¨30 and ¨32 lcDa bands reduced); Peak 2, eluting at 236-248 ml, consisted of low molecular weight fragment(s) (-15 IcDa, reduced or unreduced). Peak 1 was concentrated to 5.3 mg/ml for crystallization trials.
Crystallization of the D2E7 F(ab)'2 Fragnzent The D2E7 F(ab)t2 fragment (5.3 ing/m1 in 20 inlYI HUES, pH 7, 150 111M NaC1, 0.1 niM EDTA) was crystallized using the sitting drop vapor diffusion method by mixing equal volumes of F(ab)12 and custallization buffer (approx. 1 p.1 of each) and allowing the mixture to equilibrate against the crystallization Buffer Bt 4 or 18 'C. The crystallization buffers used consisted of the Hampton Research Crystal Screens I
(solutions 1-48) and II (solutions 1-48), Emerald Biostractures Wizard Screens I and 11 (each solutions 1-48), and the Jena Biosciences screens 1-10 (each solutions 1-24).
Crystals were obtained wider many different conditions, as summarized in Table 1.

Table 1. Swrunary of crystallization conditions for the D2E7 F(ab)'2 fragment.

Temp Screen Solution C Condition Result - ______________________________________ ---Hamirton 1 32 4 2.0 M (NII . 4)2304 tiny needle clusters 0.2 M Ca(Oac)2, 0.1 M Na cacodylate pH 6.5, 18% medium sized needle Hampton 1 46 4 __________________ PEG 8K clusters ____ Hampton 1 48 4 0.1 M Tris HCI pH 8.5, 2.0 M
N114112PO4 micro needle clusters 0.01 M hexadecyltrimethylammonhun bromide, 0.5 Hampton 2 2 4 small shard crystals M NaC1, 0.01 M MgC12 0.2 M (NH4)2SO4, 0.1 MNa0Ac pll 4.6, 30% PEG
Hampton 2 13 4 2000 small needle clusters MME
0.5 M (NH4)2SO4, 0.1M Na0Ac pH 5.6, LOM
Hampton 2 15 4 = large needle clusters Li2SO4 0.5M NaCI, 0.1M Na0Ac pH 5.6, 4% Ethylene Hampton 2 16 4 large irregular crystal __________________ 'mine polymer Hampton 1 34 18 0.1 Na0Ac pH 4.6, 2.0 M Na Fonnate needle clusters 0.1M Hepes pH 7.5, 0.8M mono-sodium Hampton 1 35 18 dihydrogen phosphate, 0.8M mouo-potasium needle clusters __________________ dihydrogen phosphate Hampton 2 9 18 0.1M Na0Ac pH 4.6, 2.0M NaC1 dense needle clusters needles & amorphous Hampton 2 12 18 0.1M CdC12, 0.1M Na0Ac pH 4.6, 30% PEG 400 crystals , ____________ 0.5M (NH4)2S,04, 0.1M Na0Ac pH 5.6, 1.0M
Hampton 2 15 18 tiny ueedle clusters Li2SO4 1.21vINaH2PO4, 0.8M K213PO4, 0.1M CAPS pH Medium large needle Wizard I 27 4 10.5, 0.2 M Li250,1 clusters 1.26M (N1-14)2SO4, 0.1 M Na0Ac pH 4.5, 0.2M
Wizard I 30 4 small needle clusters NaC1 Wizard II 8 10% PEG 8K, 0.1M Na/K phosphate pH 6.2, 0.2M Large plate crystals grown 4.
NaCl in clusters Wizard H 43 4 10% PEK 8K, 0.1M Tris pH 7.0, 0.2 M MgC12 micro needle clusters Wizard I 4 18 35% MPD, 0.1M huidazole pH 8.0, 0.2M MgC12 rod shaped crystal 1.2M NaH2PO4, 0.81v1K2IIP04, 0.1M CAPS pH
Wizard I "r7 18 Needle clusters 10.5, 0.2 IvILi2SO4 Wizard II 7 18 30% PEG 3K, 0.1M Tris pH 8.5, 0.2M NaC1 tiny needle clusters 10% 2-propanol, 0.1M cacodylate pH 6.5, 0.2M tiny hexagonal or Wizard TI 11 18 Zn(Oac)2 rhombohedral crystals Wizard H 46 18 1.0M AP, 0.1M Imidazole pH 8.0, 0.2M NaC1 1 irregular crystal J13 1 D6 4 30% PEG 31C, 0.1M Tris HCI pH 8.5, 0.2M Li2SO4 tiny needles in precipitate 20% PEG 4K, 0.1M Tris HCI pH 8.5, 0.2M Na f13 2 B6 4 tiny needle cluster balls Cacodylate .T1.3 3 A1 4 8% PEG 4K, 0.8M LiC1, 0.1M
Tris I-ICI pH 8.5 Large frost-like crystals JI3 3 B1 4 15% PEG 4K, 0.2M (NH4)2SO4 tiny needle clusters 30% PEG 4K, 0.1M Na Citrate pH 5.6, 0.2M
TB 3 D5 4 __ NII40Ac tiny needles in precipitate.
.TI3 4 B1 4 15% PEG 6K, 0.05M KC1, 0.01M MgCl2 needle cluster balls 12% PEG 4K, 0.1M Na0Ac pH 4.6, 0.2M
T 18 needle clusters __________________ NI-1,0Ac needle clusters in JB 3 B1 18 15% PEG 4K, 0.2M (NII.02SO4 _______________________________________________ plecipitate 25% PEG 4K, 0.IM Na Citrate pH 5.6, 0.2M
JD 3 C6 18 __ NR,OAc Jong, thin needles JI3 4 C5 18 8% PEG 8K, 0.2 M L1C1, 0.051\4 MgSO4 frost-like crystals Temp Screen Souk tion Condition Result ________________ C
JB 5 A3 4 15% PEG 8K, 0.2M (NH,1)2SO4 long single needles in _________________________________________________ phase separation IB 5 A4 4 15% PEG 8K, 0.5M Li2SO4 ____ tin needle clusters 15% PEG 8K, 0.1M Na MES pH 6.5, 0.2M
JD 5 A5 4 needle duster balls Ca(0Ac)2 =
J13 6 B2 4 1.6M (NH.1)2SO4, 0.5 LiC1 tiny needle cluster balls JD 6 C2 4 2.0 M (NI14)2S0.), 0.1M Na0Ac pH 4.6 micro needle clusters J13 10 D3 18 2.0M Na Fonnate, 0.1M Na0Ac pH 4.6 needle clusters The following conditions (as described in Table 1) produced crystals which can be used for diffraction quality crystals: Wizard II, 11, 18, 10% 2-propanol, 0.1M
cacodylate pEI 6.5, 0.2M Zn(Oac)2, tiny hexagonal. or rhorn. Xtals; Wizard II, 10% PEG
8K, 0.1M Na/K phosphate pH 6.2, 0.2M NaCI, large plate xtals gown in clusters;
.D3 3, C6, 18, 25% PEG 4K, 0.1M Na Citrate pH 5.6, 0.2M Ammonium Acetate, long, thin needles; Hampton 2, 15, 18, 0.5M AS, 0.1M Na Acetate trihydrate pH 5.6, 1.0M
Li Sulfate monohydrate, tiny needle clusters.
Example 38: Crystallization of D2E7 Fab fragment Generation and purification of the D2E7 Fab Fragnzent A D2E7 Fab fragment was generated and purified according to the following procedure. Four nil of D2E7 IgG (diluted to about 20 ing/m1) was diluted with 4 nal of Buffer E (20 mM Na phosphate, 5 mM cysteine-EICI, 10 mI\4 EDTA, pH7) and mixed with 6.5 inl of a slun-y of immobilized papain (Pierce, 1%; previously washed twice with 26 ml of Buffer E). The D2E7/papain mixture was incubated at 37 C overnight with shaking (300 rpm). The immobilized papain and precipitated protein were separated by centrifugation; analysis of the supernatant by SDS-PAGE indicated that the digestion of D2E7 was partially complete (-55, 50, 34, arid 30 kDa bands unreduced, with some intact and partially digested D2E7 at ¨115 and ¨150 kDa; ¨30 and ¨32 kDa bands reduced, as well as a ¨50 kDa band). Nonetheless, the digestion was halted and subjected to purification.
The D2E7 Fab fragment was purified by Protein A chromatography and Superclex 200 size-exclusion cluoniatography essentially as described above for the F(ab)'.2 fragment. The Protein A column flow-through pool (21 inl) contained ¨9.2 mg (0.44 mg/m1), whereas the Protein A ciliate (4 ml) contained ¨19.5 mg (4.9 mg/ml).
Analysis by SDS-PAGE indicated that the flow-through was essentially pure Fab fragment (-48 and ¨30 kDa unreduced, broad band at ¨30 kDa reduced), whereas the eluate was intact and partially-digested D2E7. The Fab fragment was further purified on a Superdex 200 coltunn, eluting at 216-232 nal, i.e., as expected, after the F(ab)'2 fragment but before the small Fc fragments. The D2E7 Fab fragment concentrated to 12.7 mg/m1 for crystallization trials, as described below.
Oystallization of the D217,7 Fab Frag,inent The D2E7 Fab fragment (12.7 ing/m1 in 20 inlviHEPES, pH 7, 150 mMNaCI, 0.1 inIVI EDTA) was crystallized using the sitting drop vapor diffusion method essentially as described above for the F(ab)', fragment. Crystals were obtained under many different conditions, as summarized in Table 2.
Table 2. Summary of crystallization conditions for the D2E7 Fab fragment.
Temp Screen Solution Conditi C on Result Ilaini;tO-n 1-1. '4 4 0.1M Tris pH 8.5, 2M
(N114)2SO4 wispy needles Hampton 1 10 4 0.2M NI140Ac, 0.1M Na0Ac pH 4.6, 30% PEG wispy needle clusters ___________________ 4K
Hampton 1 18 4 0.2M Mg(0Ac)2, 0.1M Na Cacodylate pH 6.5, needle clusters ___________________ 20% PEG 8K
Hampton 1 20 4 0.2M (NH4)2SO4, 0.1M Na0Ac pH 4.6, 25% PEG tiny needle clusters ___________________ 4K
Hampton 1 32 4 2M (N114)2SO4 long, wispy needles Hampton 1 33 4 4M Na Formate tiny needle clusters Hampton 1 38 4 0.1M Hepes pH 7.5 tidy needle clusters Hampton 1 43 4 30% PEG 1500 tiny needle clusters IIampton 1 46 4 0.2M Ca(0A02, 0.11\4 Na Cacodylate pkI 6.5, 18% large plate clusters ___________________ PEG 8K
Hampton 1 47 4 0.1M Na0Ac pH 4.6, 2M (NI-14)2SO4 long, wispy needles Hampton 2 1 4 2M NaCI, 10% PEG 6K small plate clusters Irampion 2 2 4 0.01M flexadecyltrimethylammoninna bromide, round 8s irregular plates 0.51v1 NaCl, 0.01 M8C12 ______________ Hampton 2_ 5 4 2M (N114)2804, 5% isopropanol long fiber ropes II-aMpton-2 13 4 10.2M (NIT,O2S0.1, 0.1M Na0Ac pH 4.6, 25% PEG tiny, wispy needle clusters ___________________ IMME 2K
Hampton 2 14 4 0.2M K/Na Tatrate, 0.IM Na Citrate pH 5.6, 2M tiny needle clusters (N/14)zSGi Ilampton 2 27 4 0.01M ZuSO4, 0.1 MES pH 6.5, 25% PEG MME tiny needle clusters -Hampton 2 28 4 30% M_PD tiny needle clusters Ilampton 1 4 18 0.1M Tris pH 8.5, 2tril (N11,1)280., needle clusters . Temp Screen Solution o, Condition Result Hampton 1 9 18 0.2M NI-1.40Ae, 0.IM Na Citrate pH 5.6, 30% PEG needle clusters __________________ 4K
Hampton 1 17 18 0.2M Li2SO4, 0.1MTris pH 8.5, 30% PEG 4K long, wispy needles Harff ton 1 32 18 2M (NH4)2SO4 needle clusters Hainptou 1 33 18 4M Na Formate tiny needle clusters Hampton I 38 18 0.1M Repos pH 7.5 fiber bundles Hampton 1 43 18 30% PEG 1500 = tiny needle clusters Harnpton 1 47 18 0.1M Na0Ac pi-14.6, 2M
(NH4)2S0,4 tiny needle clusters Hampton 2 1 18 2M NaCI, 10% PEG 61<. long, wispy needle clusters Hampton 2 5 18 2M (NI-14)2SO4, 5% 2-propanol tiny needle clusters Hampton 2 9 18 0.1M Na0Ac pH 4.6, 2M NaC1 long, wispy needles Hampton 2 13 18 0.2M (NI-14)2SO4, 0.1M Na0Ac pH 4.6, 25% PEG tiny needle clusters __________________ IvEvIE 2K
Hampton 2 14 18 0.2M IC/Na Tartrate, 0.1M Na Citrate pH 5.6, 2M long wispy needles (N1-14)2SO4 Hampton 2 27 18 0.01M ZnSO4, 0.1 MES pH 6.5, 25% PEG MME tiny needle clusters WizardI 20 4 0.4M NaH2PO4/1.6M K2I1PO4, 0.1M Imidazole pH tiny needle clusters 8, 0.2M NaC1 Wizard I 28 4 20% PEG 3K, 0.11\4 Hepcs plI 7.5, 0.2M NaCI large orthorhombic plate clusters Wizard I 31 4 20% PEG SK, 0.1M phosphate citrate pH 4.2, wispy needle clusters 0.2M NaCI ____________________________________________________ Wizard I 39 4 20% PEG 1K, 0.1M phosphate citrate pH 4.2, needle clusters 0.2M Li2SO4 Wizard II 3 4 20% PEG 8K, 0.1M Tris pH 8.5, 0.2M MgC12 large hexagonal or orthorhombic plate cluster in phase sep Wizard 11 4 4 2M (N1-14)2SO4, 0.1M Cacodylate pH 6.5, 0.2 1'IaC1 tiny needle clusters Wizard II 9 4 2M (NH4)2SO4, 0.1M phosphate citrate pH 4.2 tiny, wispy needle clusters Wizard 11 28 4 20% PEG 8K, 0.1M MES p1-1 6, 0.2M Ca(0Ac)2 tiny needle clusters; large _______________________________________________ wispy needle clusters Wizard II 35 4 0.8M Na112PO4/1.2M K2111304, 0.1M Na0Ac pH tiny hber bundles 4.5 Wizard II 38 4 2.5M NaC1, 0.1M Na0Ac pH 4.5, 0.2M Li2SO4 long wispy needles Wizard II 47 4 -2.5M NaC1, 0.1M Inaidazole pH g, 0.2M Zu(OAc)2 tiny needle clusters Wizard I 6 18 20% PEG 3K, 0.1M Citrate pH 5.5 needle clusters Wizard I 20 18 0.4M NalI2PO4/1.6M K2I-IP04, 0.1M Iruidazole pH tiny needle chistcrs __________________ 8, 0.2M NaCl -Wizard I 27 18 1.2M Na112PO4/0.81vi K2111'04, 0.1M CAPS pH 10, wispy needle clusters 0.2M Li2804 Wizard 1 30 18 1.26M (N1-14)2SO4, 0.1M NaO.Ac pH 4.5, 0.2M wispy needles __________________ NaC1 Wizard I 31 18 20% PEG 8K, 0.1M phosphate citrate pH 4.2, tiny needle clusters __________________ 0.2M NaCI
Wizard I 33 18 2M (NI-14)2SO4, 0.1M CAPS pH 10.5, 0.2M Li2SO4 fiber bundles Wizard I 39 18 20% PEG 1TC, 0.1/v1 phosphate citrate pH 4.2, needle clusters __________________ 0.2M Li2SO4 Wizard 11 4 18 2M (N1-14)2SO4, 0.1M Cacodylate pH 6.5, 0.2 NaCI needle clusters Wizard II 9 18 21\4 (NII4)2SO4, 0.1M phosphate ciliate pH 4.2 wispy needles Wizard II 35 18 0.8M NalI2P
04/1.2M K21-004, 0.11v1Na0Ac pH tiny needle clusters L
4.5 Wizarcl 11 38 18 2.5M NaCI, 0.1M Na0Ac pH 4.5, 0.21v[ Li,SO4 tiny needle clusters --The following conditions (as described in Table 2) produced crystals which can be used for diffraction quality crystals: Hampton 2, 1, 4C, 2M NaC1, 10% PEG
6K, small plate clusters; Hampton 1 46, 4C, 0.2lvl Ca Acetate, 0.1M Na Cacodylate, pH 6.5, 1S% PEG 8K, large plate clusters; Wizard I, 28, 4C, 20% PEG 3K, 0.1M Hepes pH
7.5, 0.2M NaCI, large orthorhombic plate clusters; Wizard If 3, 4C, 20% PEG 8K, 0.1M Tris pH 8.5, 0.2M MgC12, lrg hex or orth plate cluster in phase sep.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following = claims.

DENLA.NDES OU BREVETS VOLIINIINEUX
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CECI EST LE TOME 1 DE t3 NOTE: Pour les tomes additioneIs, veillez contacter le Bureau Cana.dien des Brevets.

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Claims

What is claimed:

1. Use of adalimumab for reducing or inhibiting symptoms in a patient with psoriatic arthritis, wherein the adalimumab is for subcutaneous administration at a dose of 40 mg every other week.