WO2012061620A1

WO2012061620A1 - Methods for treating, diagnosing, and monitoring rheumatoid arthritis

Info

Publication number: WO2012061620A1
Application number: PCT/US2011/059195
Authority: WO
Inventors: Glynn Dennis, Jr.; Flavius Martin; Michael J. Townsend
Original assignee: Genentech, Inc.; F. Hoffmann-La Roche Ag
Priority date: 2010-11-04
Filing date: 2011-11-03
Publication date: 2012-05-10

Abstract

Methods of identifying, diagnosing, and prognosing rheumatoid arthritis are provided, as well as methods of treating rheumatoid arthritis. Also provided are methods for identifying effective rheumatoid arthritis therapeutic agents and predicting responsiveness to rheumatoid arthritis therapeutic agents.

Description

METHODS FOR TREATING, DIAGNOSING, AND MONITORING

RHEUMATOID ARTHRITIS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of provisional U.S. Application No. 61/410,325 filed November 4, 2010 which is hereby incorporated by reference in its entirety.

FIELD

[0002] Methods of identifying, diagnosing, and prognosing rheumatoid arthritis are provided, as well as methods of treating rheumatoid arthritis. Also provided are methods for identifying effective rheumatoid arthritis therapeutic agents and predicting responsiveness to rheumatoid arthritis therapeutic agents.

BACKGROUND

[0003] Rheumatoid arthritis (RA) is a clinically important, chronic systemic autoimmune inflammatory disease affecting between 1.3 and 2.1 million persons in the United States (See, e.g., Alamanosa and Drosos, Autoimmun. Rev., 4: 130-136 (2005)). RA is an autoimmune disorder of unknown etiology. Most RA patients suffer a chronic course of disease that, even with currently available therapies, may result in progressive joint destruction, deformity, disability and even premature death. More than 9 million physician visits and more than 250,000 hospitalizations per year result from RA.

[0004] Diagnosis of RA typically relies on clinical and laboratory evaluation of a patient's signs and symptoms. Generally, laboratory evaluation of a patient suspected of having RA may include determination of the level of certain antibodies in serum known as rheumatoid factor (RF) and antibodies to cyclic citrullinated peptide (anti-CCP). (See, e.g., Schellekens et al, Arthritis Rheum., 43: 155-163 (2000); DiFranco et al, Rev. Rheum. Engl. Ed., 66(5):251-255 (1999); Rantapaa-Dahlqvist et al, Arthritis Rheum., 48:2741-2749 (2003); Li et al, Bioinformatics 22(12): 1503-1507 (2006); Russell et al, J. Rheumatol., 33(7): 1240-1242 (2006); Ota, Rinsho byori. Jap. J. Clin. Pathol, 54(8)861-868 (2006);

Avouac et al, Ann. Rheum. Dis., 65(7):845-851 (2006)). While these antibodies are often found in the serum of RA patients, not all RA patients have them. An additional blood test known as the erythrocyte sedimentation rate (ESR) may also be used. An elevated ESR indicates the general presence of an inflammatory process, although not necessarily RA. Further blood tests may be used to assess the level of other factors, such as C-reactive protein (CRP) that have been associated with RA. In addition, radiographic analysis of affected joints may be performed. In sum, such currently available laboratory tests to diagnose RA are imprecise and imperfect.

[0005] In certain instances, diagnosis of RA is made if a patient satisfies certain

American College of Rheumatology (ACR) criteria. Certain such criteria include morning stiffness in and around the joints lasting for at least 1 hour before maximal improvement; arthritis of three or more joint areas: at least three joint areas have simultaneously had soft tissue swelling or fluid (not bony overgrowth alone) observed by a physician; the 14 possible joint areas (right and left) are proximal interphalangeal (PIP), metacarpophalangeal (MCP), wrist, elbow, knee, ankle, and metatarsophalangeal (MTP) joints; arthritis of hand joints: at least one joint area swollen as above in wrist, MCP, or PIP joint; symmetric arthritis:

simultaneous involvement of the same joint areas (as in arthritis of three or more joint areas, above) on both sides of the body (bilateral involvement of PIP, MCP, or MTP joints is acceptable without absolute symmetry); rheumatoid nodules: subcutaneous nodules over bony prominences or extensor surfaces or in juxta-articular regions that are observed by a physician; serum rheumatoid factor: demonstration of abnormal amounts of serum

rheumatoid factor by any method that has been positive in fewer than five percent of normal control patients; radiographic changes: radiographic changes typical of rheumatoid arthritis on posteroanterior hand and wrist X-rays, which must include erosions or unequivocal bony decalcification localized to or most marked adjacent to the involved joints (osteoarthritis changes alone do not qualify). Diagnosis of RA is typically made if a patient satisfies at least four of the above criteria.

[0006] In certain instances, a diagnosis of RA is made if a patient has a particular Disease Activity Score (DAS) (see, e.g., Van der Heijde D. M. et al, J Rheumatol, 1993, 20(3): 579- 81; Prevoo M. L. et al, Arthritis Rheum, 1995, 38: 44-8). The DAS system represents both current state of disease activity and change. The DAS scoring system uses a weighted mathematical formula, derived from clinical trials in RA. For example, the DAS 28 is 0.56( T28)+0.28( SW28)+0.70(Ln ESR)+0.014 GH wherein T represents tender joint number, SW is swollen joint number, ESR is erythrocyte sedimentation rate, and GH is global health. Various values of the DAS represent high or low disease activity as well as remission, and the change and endpoint score result in a categorization of the patient by degree of response (none, moderate, good). [0007] A number of published studies report the attempted identification of reliable biomarkers for diagnostic and prognostic purposes. (See e.g., Rioja et al, Arthritis and Rheum. 58(8):2257-2267 (2008); Pyrpasopoulou et al, Mol. Diagn. Ther. 14(l):43-48 (2010); WO 2004/0009479; WO 2007/0105133; WO 2007/038501; WO 2007/135568; WO

2008/104608; WO 2008/056198; WO 2008/132176; and WO 2008/154423). No clinically validated diagnostic markers, however, e.g., biomarkers, have been identified that enable clinicians or others to accurately define pathophysiological aspects of rheumatoid arthritis, clinical activity, response to therapy, prognosis, or risk of developing the disease.

Accordingly, as RA patients seek treatment, there is considerable trial and error involved in the search for therapeutic agent(s) effective for a particular patient. Such trial and error often involves considerable risk and discomfort the the patient in order to find the most effective therapy. Thus, there is a need for more effective means for determining which patients will respond to which treatment and for incorporating such determinations into more effective treatment regimens for rheumatoid arthritis patients.

[0008] Molecular insight resulting from genome-scale analysis of synovial tissue gene expression is beginning to emerge with potential to assist in dissection of RA disease heterogeneity. Early studies of gene expression in synovial tissues identified genes involved in B and T cell regulation in RA compared to osteoarthritis (OA). Unsupervised clustering of this data revealed considerable molecular heterogeneity that stratified patients into one of two main groups. One group of patients exhibited gene expression patterns consistent with ongoing inflammation and adaptive immunity, evidenced by increased expression of immunoglobulin (Ig) genes and other B cell-specific transcripts. This group was further subdivided based on the complement pathway in patients with a predominant classic complement pathway activition versus alternative complement pathway activation. Similar to OA, gene expression pattern in the second group of patients appeared devoid of

immunoregulatory pathways and instead expressed sets of genes involved in extracellular matrix remodeling. Histological analysis for immune cell types revealed that differences in gene expression patterns might reflect differences in relative cellular composition.

[0009] Another published study employed pathway analysis to demonstrate that lymphoid-follicle containing tissues increased the expression of sets of genes involved in Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) signaling, lymphocyte signaling and interleukin (IL)-7 signal transduction. Timmer et al., Arth. &

Rheum. 56:2492-2502 (2007). In contrast, tissues characterized by diffuse cellular infiltration had reduced expression of immunoregulatory genes and pathways. Based on these and several other early profiling studies, RA synovial tissues can be molecularly stratified into at least two distinct groups based on broad differences in inflammatory transcript abundance. Several explanations have been put forward to account for these observations, some trivial related to technical factors (intra-joint inflammation heterogeneity and synovial biopsy techniques) or disease stage (OA can associate with RA at later stages). However, more recent studies controlled these parameters through multiple biopsies or larger joint samples and patient and joint selection using clinical, preclinical, radiological and histological parameters. As a corollary of these results, profiled joint heterogeneity does not appear to be a simple dichotomy between actively inflamed joints and inactive ones and extends beyond differences in cellularity. Nonetheless, while these studies provide an initial frame, the extent of molecular RA heterogeneity as described through gene profiling, has not been thoroughly determined and validated by independent investigators or in multiple cohorts of patients. This is due in part to difficulties in acquiring large collections of patients with well controlled quality synovium for initial class discovery and replication cohorts as well as to technical complexity of the experimentation (platforms and analysis tools and strategies). Other challenges include the longitudinal stability of a proposed type and correlations with preclinical and clinical disease parameters and in particular disease therapy. Finally, it remains important to determine if molecular heterogeneity in RA reflects a compilation of underlying pathogenic processes, each of which may respond differently to targeted therapy.

[0010] Clinical success in the treatment of moderate to severe RA with targeted blockade of cytokine pathways including TNFa, IL-Ιβ, and IL-6, as well as with therapies targetd to T cells (e.g., CTLA4-Ig) and B cells (e.g., anti-CD20 mAbs) corroborates the important roles of these cytokines and lymphocytes in RA pathogenesis. TNFa blocking agents are widely used in the treatment of RA as first-line biologies with significant response rates characterized by reduced disease activity and delayed bone destruction. However, only a small subset of RA patients achieve major clinical responses with TNFa blockade therapy, and approximately one third fail to respond entirely. Prospective identification of patients with respect to their response to anti-TNFa as well as other targeted therapies represents an important challenge in today's therapeutic management of RA. Despite various post-hoc associations between systemic and local measures of disease activity and response to therapy, predictive relationships have yet to be established. Van Vollenhoven, et al., Arth. & Rheum. 48: 1500-

1503 (2003). As examples, clinical response to TNF blockade appears to be in part associated with the degree of inflammation within synovial tissues (Van der Pouw Kraan, et al., Ann. Rheum. Dis. 67:563-566 [2008]; Wijbrandts, et al., Ann. Rheum. Dis. 67: 1139-1144 [2008]); patients containing lymphocyte aggregates are significantly associated with response

(Klaasen, et al, Arth. & Rheum. 60:3217-24 [2009]; Lindberg et al, PLoS ONE 5(6):el 1310 [2010]). Similarly, TNFa protein expression in the synovium before initiation of treatment associates with response to infliximab (Wijbrandts, et al., Ann. Rheum. Dis. 67: 1139-1144 [2008]). Moreover, inflammatory markers such as ESR, CRP, anti-CCP as well as plasma TNFa levels are marginally associated with response to TNFa blockade. Recent attempts to correlate SNPs in PTPRC and TNFR alleles have shown some success but cannot be used effectively for prediction of response in clinical practice. Conversely, high levels of rheumatoid factor (RF), particularly of the IgA isotype, have been correlated with poor response to anti-TNFa suggesting that subtleties of the inflammation composition will stratify further response to anti-TNFa. In addition, B cell autoimmunity in RA as measured by RF or anti-CCP antibodies can be dissociated from joint lymphoneogensis. Cantaert et al., J. Immunol. 181 :785-94 (2008). Collectively, these reported observations suggest that multiple pathogenetic pathways are acting in concert in RA, which could explain the heterogenous response to targeted therapies.

[0011] International Patent Application No. PCT/US2010/047734 (Intn'l Pub. No. WO 2011/028945) describes a statistically rigorous interrogation of genome-wide transcription in a large set of RA synovial tissues. RA joints were stratified into four molecular phenotypes that differed transcriptionally but not in disease duration, radiographic state or systemic measures of inflammation. Meta-analysis revealed that each phenotype expressed distinct transcriptional programs reflecting biological differences with pathological relevance. Gene expression modules were developed for each phenotype, refined using statistical learning procedures and validated on independent data sets. In addition, phenotype-intrinsic modules were used to identify molecular biomarkers to stratify new patients into subtypes of RA with predictable responses to B cell targeted therapy, such as anti-CD20 monoclonal antibodies.

[0012] It would be highly advantageous to have additional diagnostic methods, including molecular-based diagnostic methods, that can be used to objectively identify the presence of and/or classify the disease in a patient, define pathophysiologic aspects of rheumatoid arthritis, clinical activity, response to therapy, including response to treatment with various RA therapeutic agents, prognosis, and/or risk of developing rheumatoid arthritis. In addition, it would be advantageous to have molecular-based diagnostic markers associated with various clinical and/or pathophysiological and/or other biological indicators of disease. Thus, there is a continuing need to identify new molecular biomarkers associated with rheumatoid arthritis as well as other autoimmune disorders. Such associations would greatly benefit the identification of the presence of rheumatoid arthritis in patients or the determination of susceptibility to develop the disease. Such associations would also benefit the identification of pathophysiologic aspects of RA, clinical activity, response to therapy, or prognosis. In addition, statistically and biologically significant and reproducible information regarding such associations could be utilized as an integral component in efforts to identify specific subsets of patients who would be expected to significantly benefit from treatment with a particular therapeutic agent, for example where the therapeutic agent is or has been shown in clinical studies to be of therapeutic benefit in such specific RA patient subpopulation.

[0013] The invention described herein meets the above-described needs and provides other benefits.

[0014] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety for any purpose.

SUMMARY

[0015] The methods of the invention are based, at least in part, on the use of expression of one or a combination of genes or expression of one or a combination of proteins, which expression is indicative of distinct molecular subtypes (also referred to herein as molecular phenotypes) of rheumatoid arthritis (RA), to predict patient responsiveness to treatment with TNFa inhibitor(s). Certain aspects of the RA molecular subtypes referred to herein were previously described in International Patent Application No. PCT/US2010/047734 (Intn'l Pub. No. WO 2011/028945). The terms "molecular phenotype" and "molecular subtype" are used interchangeably herein.

[0016] Accordingly, in one aspect, methods of predicting the response of a subject to a therapy comprising a TNFa inhibitor are provided. In certain embodiments, the method comprises measuring in a biological sample obtained from the subject expression of one or a combination of genes, or expression of one or a combination of proteins encoded by the one or the combination of genes, wherein the one or the combination of genes is selected from Table 2. In a further embodiment, the expression of the one or the combination of genes, or the expression of the one or the combination of proteins, comprises a gene signature or a protein signature, respectively, which is predictive of response of the subject to therapy comprising the TNFa inhibitor. In certain embodiments, the gene signature or the protein signature is indicative of M subtype. In yet another embodiment, the combination of genes comprises at least five, or at least 10, or at least 20, or at least 30, or at least 40 genes selected from Table 2. In still another embodiment the combination of genes comprises ACTN1, ARL7, ATP6V0D1, ATP6V1A, C5R1, C9orf88, CAPZB, CCL2, CCR1, CTSB, CTSL, CTSZ, CXCL3, EIF4E2, EMILIN2, FAM50B, FLJ11259, FLJ20847, FLNA, FZD4, GSTOl, HCK, ICAM1, KIAA0485, KIAA0582, LACTB, LILRB2, LILRB3, MBD2, MFHAS1, NAGA, NPC1, NRP2, P2RX4, PGD, PLAU, PLAUR, RABGAP1, RAPGEF1, RHOG, SERPINB1, SLC16A3, TCF7L2, TFRC, TM7SF1, TPM4, UBE3A, VEGF, VPS 13 A, VPS37C, and ZYX. In yet another embodiment, the combination of genes further comprises one or more genes selected from CLK1, HSMPP8, MGC2752, and MICAL3. In certain embodiments, the biological sample is synovial tissue, synovial fluid, plasma, or serum.

[0017] In another aspect, methods of predicting response to therapy comprising a TNFa inhibitor comprise measuring in a biological sample obtained from the subject expression of one or a combination of genes, or expression of one or a combination of proteins encoded by the one or the combination of genes, wherein the one or the combination of genes is selected from Table 4 are provided. In certain embodiments, the expression of the one or the combination of genes, or the expression of the one or the combination of proteins, comprises a gene signature or a protein signature, respectively, which is predictive of response of the subject to therapy comprising the TNFa inhibitor. In certain embodiments, the gene signature or the protein signature is indicative of Fl subtype. In another embodiment, the combination of genes comprises at least five, or at least 10, or at least 20, or at least 30, or at least 40 genes selected from Table 4. In still another embodiment the combination of genes comprises ABCA1, ADRBK1, AP1S2, C10orf38, C16orf9, CASK, CD68, CDH11, CDH5, COL18A1, COL4A1, COROIA, CREB3L1, CTSS, CYBB, FBP1, FCGR2C, FCGR3A, FCGR3B, FGL2, FLJ11127, FLJ20364, FLJ22662, FLJ44635, FPRL2, GPR116, GUCY1A3, HAVCR2, HEPH, HEYL, ITGB2, KCTD15, KIAA1374, KYNU, LILRA2, LPIN1, LST1, MAP IB, MAP4K4, MARCO, MFAP2, MGC 17943, MGC48972, MSR1, NXN, PNKP, POSTN, PTPNS1, QARS, RNASET2, SEPT11, SGKL, STAT5A, TPM1, TRIM14, VSIG4, YIF1, and ZNF462. In yet another embodiment, the combination of genes further comprises one or more genes selected from LOC90139 and SLC38A2. In certain embodiments, the protein signature comprises periostin. In certain embodiments, the biological sample is synovial tissue, synovial fluid, plasma, or serum.

[0018] In another aspect, methods of predicting response to therapy comprising a TNFa inhibitor comprise measuring in a biological sample obtained from the subject expression of one or a combination of genes, or expression of one or a combination of proteins encoded by the one or the combination of genes, wherein the one or the combination of genes is selected from Table 3 are provided. In certain embodiments, the expression of the one or the combination of genes, or the expression of the one or the combination of proteins, comprises a gene signature or a protein signature, respectively, which is predictive of response of the subject to therapy comprising the TNFa inhibitor. In certain embodiments, the gene signature or the protein signature is indicative of F2 subtype. In another embodiment, the combination of genes comprises at least five, or at least 10, or at least 20 genes selected from Table 3. In still another embodiment the combination of genes comprises ABTB2, ARGBP2, AUTS2, BBSl, CBX7, CLU, FANCA, FLJ10970, FLJ32803, FZD8, GABARAPLl, GPR64, GULP1, HMGB3, LOC201895, LTBP3, MSL3L1, NDFIP1, NOVA1, NTN4, NTRK2, PCOLCE2, PLEKHA1, POSTN, PTTG1, RNASE4, SCARA3, SLC29A1, and SLC35A1. In yet another embodiment, the combination of genes further comprises one or more genes selected from CHD9, IDH2, IP09, KBTBD9, and LOC283481. In certain embodiments, the protein signature comprises periostin and/or clusterin. In certain embodiments, the biological sample is synovial tissue, synovial fluid, plasma, or serum.

[0019] In one aspect, gene expression is measured by microarray. In another aspect gene expression is measured by real-time quantitative polymerase chain reaction (qPCR). In another aspect, gene expression is measured by multiplex-PCR. According to another embodiment, gene expression is measured by observing protein expression levels of an aforementioned gene. According to another embodiment, expression of a gene of interest is considered elevated when compared to a healthy control if the relative mRNA level of the gene of interest is greater than 2 fold of the level of a control gene mRNA. According to another embodiment, the relative mRNA level of the gene of interest is greater than 3 fold, 5 fold, 10 fold, 15 fold, 20 fold, 25 fold, or 30 fold compared to a healthy control gene expression level. In one aspect, the gene expression level is measured by a method selected from a PCR method, a microarray method, or an immunoassay method. In one embodiment, the microarray method comprises the use of a microarray chip having one or more nucleic acid molecules that can hybridize under stringent conditions to a nucleic acid molecule encoding a gene mentioned above or having one or more polypeptides (such as peptides or antibodies) that can bind to one or more of the proteins encoded by the genes mentioned above. In one embodiment, the PCR method is qPCR. In one embodiment, the PCR method is multiplex -PCR. According to one embodiment, the immunoassay method comprises binding an antibody to protein expressed from a gene mentioned above in a patient sample and determining if the protein level from the patient sample is elevated. In certain

embodiments, the immunoassay method is an enzyme-linked immunosorbent assay (ELISA), electro-chemiluminescence assay (ECLA), or multiplex microsphere-based assay platform, e.g., Luminex® platform.

[0020] In yet another aspect, methods of treating rheumatoid arthritis with a

therapeutically effective amount of a TNFa inhibitor are provided. In certain embodiments, a biological sample obtained from the patient has been shown to possess a M subtype gene signature or a M subtype protein signature, wherein the M subtype gene signature comprises expression of one or a combination of genes, and the M subtype protein signature comprises expression of one or a combination of proteins encoded by the one or the combination of genes, respectively, wherein the one or the combination of genes is selected from Table 2. In a further embodiment, the combination of genes comprises at least five, or at least 10, or at least 20, or at least 30, or at least 40 genes selected from Table 2. In a still further

embodiment, the combination of genes comprises ACTN1, ARL7, ATP6V0D1, ATP6V1A,

C5R1, C9orf88, CAPZB, CCL2, CCR1, CTSB, CTSL, CTSZ, CXCL3, EIF4E2, EMILIN2,

FAM50B, FLJ11259, FLJ20847, FLNA, FZD4, GSTOl, HCK, ICAM1, KIAA0485,

KIAA0582, LACTB, LILRB2, LILRB3, MBD2, MFHAS1, NAGA, NPC1, NRP2, P2RX4,

PGD, PLAU, PLAUR, RABGAP1, RAPGEF1, RHOG, SERPINB1, SLC16A3, TCF7L2,

TFRC, TM7SF1, TPM4, UBE3A, VEGF, VPS 13 A, VPS37C, and ZYX. In yet another embodiment, the combination of genes further comprises one or more genes selected from

CLK1, HSMPP8, MGC2752, and MICAL3. In certain embodiments, the biological sample is synovial tissue, synovial fluid, plasma or serum. In certain embodiments, the TNFa inhibitor is selected from etanercept, infliximab, adalimumab, golimumab, and certolizumab pegol.

[0021] In another aspect, methods of treating rheumatoid arthritis with a therapeutically effective amount of a TNFa inhibitor comprise obtaining a biological sample from the patient and demonstrating that the sample possesses a Fl subtype gene signature or a Fl subtype protein signature, wherein the Fl subtype gene signature comprises expression of one or a combination of genes, and the Fl subtype protein signature comprises expression of one or a combination of proteins encoded by the one or the combination of genes, respectively, wherein the one or the combination of genes is selected from Table 4. In a further

embodiment, the combination of genes comprises at least five, or at least 10, or at least 20, or at least 30, or at least 40 genes selected from Table 4. In a still further embodiment, the combination of genes comprises ABCA1, ADRBK1, AP1S2, C10orO8, C16orf9, CASK, CD68, CDH11, CDH5, COL18A1, COL4A1, COROIA, CREB3L1 , CTSS, CYBB, FBP1, FCGR2C, FCGR3A, FCGR3B, FGL2, FLJ11127, FLJ20364, FLJ22662, FLJ44635, FPRL2, GPR116, GUCY1A3, HAVCR2, HEPH, HEYL, ITGB2, KCTD15, KIAA1374, KYNU, LILRA2, LPIN1, LST1, MAP IB, MAP4K4, MARCO, MFAP2, MGC 17943, MGC48972, MSR1, NXN, PNKP, POSTN, PTPNS1, QARS, RNASET2, SEPT11, SGKL, STAT5A, TPMl, TRIM14, VSIG4, YIF1, and ZNF462. In yet another embodiment, the combination of genes further comprises one or more genes selected from LOC90139 and SLC38A2. In certain embodiments, the protein signature comprises periostin. In certain embodiments, the biological sample is synovial tissue, synovial fluid, plasma, or serum. In certain

embodiments, the TNFa inhibitor is selected from etanercept, infliximab, adalimumab, golimumab, and certolizumab pegol.

[0022] In another aspect, methods of treating rheumatoid arthritis with a therapeutically effective amount of an RA therapeutic agent other than a TNFa inhibitor comprise obtaining a biological sample from the patient and demonstrating that the sample possesses a F2 subtype gene signature or a F2 subtype protein signature, wherein the F2 subtype gene signature comprises expression of one or a combination of genes, and the F2 subtype protein signature comprises expression of one or a combination of proteins encoded by the one or the combination of genes, respectively, wherein the one or the combination of genes is selected from Table 3. In a further embodiment, the combination of genes comprises at least five, or at least 10, or at least 20 genes selected from Table 3. In a still further embodiment, the combination of genes comprises ABTB2, ARGBP2, AUTS2, BBS1, CBX7, CLU, FANCA, FLJ10970, FLJ32803, FZD8, GABARAPL1, GPR64, GULP1, HMGB3, LOC201895, LTBP3, MSL3L1, NDFIP1, NOVA1, NTN4, NTRK2, PCOLCE2, PLEKHA1, POSTN, PTTG1, RNASE4, SCARA3, SLC29A1, and SLC35A1. In yet another embodiment, the combination of genes further comprises one or more genes selected from CHD9, IDH2, IP09, KBTBD9, and LOC283481. In certain embodiments, the protein signature comprises periostin and/or clusterin. In certain embodiments, the biological sample is synovial tissue, synovial fluid, plasma, or serum. [0023] In another aspect, methods of selecting a therapeutic agent for treatment of an RA patient are provided. In certain embodiments, the methods comprise (a) obtaining a biological sample from the patient; (b) measuring expression of one or a combination of genes, or one or a combination of proteins encoded by the one or the combination of genes, wherein expression of the one or the combination of genes, or expression of the one or the combination of proteins, comprises a gene signature or a protein signature, respectively, wherein the one or the combination of genes is selected from Table 2, wherein the gene signature or the protein signature is indicative of M subtype; (c) determining whether the sample is positive or negative for M subtype; (d) measuring expression of one or a combination of genes, or one or a combination of proteins encoded by the one or the combination of genes, wherein expression of the one or the combination of genes, or expression of the one or the combination of proteins, comprises a gene signature or a protein signature, respectively, wherein the one or the combination of genes is selected from Table 4, wherein the gene signature or the protein signature is indicative of Fl subtype; (e) determining whether the sample is positive or negative for Fl subtype; (f) measuring expression of one or a combination of genes, or one or a combination of proteins encoded by the one or the combination of genes, wherein expression of the one or the combination of genes, or expression of the one or the combination of proteins, comprises a gene signature or a protein signature, respectively, wherein the one or the combination of genes is selected from Table 3, wherein the gene signature or the protein signature is indicative of F2 subtype; (g) determining whether the sample is positive or negative for F2 subtype; (h) selecting a TNFa inhibitor as the therapeutic agent provided the sample is positive for at least one of the subtypes selected from M and Fl; and (i) selecting a therapeutic agent other than a TNFa inhibitor provided the sample is negative for at least one of the subtypes selected from M and Fl and positive for F2 subtype. In certain embodiments, the TNFa inhibitor is selected as the therapeutic agent, and the TNFa inhibitor is selected from etanercept, infliximab, adalimumab, golimumab, and certolizumab pegol.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 shows bar-plots depicting the number of RA patients demonstrating a poor (open bar), moderate (striped) or good response (stippled) to anti-TNFa therapeutics in (A) all comers, (B) Fl subtype, (C) F2 subtype, (D) L subtype, and (E) M subtype as described in Example 2. [0025] Figure 2 shows a graphical plot of (A) serum clusterin levels and (B) serum periostin levels in RA patients and in healthy controls as described in Example 3. Serum clusterin levels are plotted on the vertical axis in ng/ml (A); serum periostin levels are plotted on the vertical axis in pg/ml (B); RA patients and healthy controls (HC) are indicated on the horizontal axis in (A) and (B). Each square represents one RA patient or healthy control, as indicated.

DETAILED DESCRIPTION

[0026] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al, Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provide one skilled in the art with a general guide to many of the terms used in the present application.

CERTAIN DEFINITIONS

[0027] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0028] "Rheumatoid arthritis," (RA) refers to a chronic systemic autoimmune

inflammatory disease that mainly involves the synovial membrane of multiple joints with resultant injury to the articular cartilage, resulting in joint destruction. The main presenting symptoms in RA are pain, stiffness, swelling, and/or loss of function of one or more joints.

[0029] The term "polynucleotide" or "nucleic acid," as used interchangeably herein, refers to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA

polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters,

phosphoamidates, cabamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc. ), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping groups moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-2'-0- allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, a- anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S("thioate"), P(S)S ("dithioate"), "(0)NR 2 ("amidate"), P(0)R, P(0)OR^*, CO or CH 2 ("formacetal"), in which each R or R is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (~0~) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0030] "Oligonucleotide," as used herein, refers to short, single stranded polynucleotides that are at least about seven nucleotides in length and less than about 250 nucleotides in length. Oligonucleotides may be synthetic. The terms "oligonucleotide" and

"polynucleotide" are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides. [0031] The term "primer" refers to a single stranded polynucleotide that is capable of hybridizing to a nucleic acid and allowing the polymerization of a complementary nucleic acid, generally by providing a free 3'-OH group.

[0032] The term "array" or "microarray" refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes (e.g., oligonucleotides), on a substrate. The substrate can be a solid substrate, such as a glass slide, or a semi-solid substrate, such as nitrocellulose membrane.

[0033] The term "amplification" refers to the process of producing one or more copies of a reference nucleic acid sequence or its complement. Amplification may be linear or exponential (e.g., PCR). A "copy" does not necessarily mean perfect sequence

complementarity or identity relative to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not fully complementary, to the template), and/or sequence errors that occur during amplification.

[0034] The term "detection" includes any means of detecting, including direct and indirect detection.

[0035] "Elevated expression" or "elevated levels" refers to an increased expression of a mR A or a protein in a patient relative to a control, such as an individual or individuals who are not suffering from RA.

[0036] The term "molecular subtype," used interchangeably with "molecular phenotype," refers to a subtype or phenotype of RA characterized by the expression of one or more particular genes or one or more particular proteins, or a particular pattern of expression of a combination of genes or a combination of proteins. The expression of particular genes, proteins or combinations of genes or proteins may be further associated with certain pathological, histological, and/or clinical features of RA.

[0037] The term "multiplex -PCR" refers to a single PCR reaction carried out on nucleic acid obtained from a single source (e.g., a patient) using more than one primer set for the purpose of amplifying two or more DNA sequences in a single reaction.

[0038] As used herein, "rheumatoid factor," or "RF," refers to IgM, IgG, or IgA isotypes, singly or in any combination, of antibodies detected in patient serum and directed to antigenic determinants present on human and animal IgG. [0039] The term "positive for RF" refers to a result of an assay for RF, e.g., an ELISA assay, where the result is above a threshold or cutoff value for that assay for samples that are considered to reproducibly contain detectable levels of RF.

[0040] The term "negative for RF" refers to a result of an assay for RF, e.g., an ELISA assay, where the result is at or below a threshold or cutoff value for that assay for samples that are considered to reproducibly contain undetectable levels of RF.

[0041] "Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures.

Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

[0042] "Stringent conditions" or "high stringency conditions", as defined herein, can be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%

polyvinylpyrrolidone/5 OmM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42C; or (3) overnight hybridization in a solution that employs 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μ /ιη1), 0.1% SDS, and 10% dextran sulfate at 42C, with a 10 minute wash at 42C in 0.2 x SSC (sodium chloride/sodium citrate) followed by a 10 minute high- stringency wash consisting of 0.1 x SSC containing EDTA at 55C.

[0043] "Moderately stringent conditions" can be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20%

formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

[0044] The term "biomarker" as used herein refers to an indicator of e.g, a pathological state of a patient, which can be detected in a biological sample of the patient. Biomarkers include, but are not limited to, DNA, RNA, protein, carbohydrate, or glycolipid-based molecular markers.

[0045] The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition. For example, "diagnosis" may refer to identification of a particular type of RA. "Diagnosis" may also refer to the classification of a particular subtype of RA, e.g., by histopathological criteria (e.g., lymphoid infiltration or follicle-like lymphoid cluster), or by molecular features (e.g., a subtype characterized by expression of one or a combination of particular genes or proteins encoded by said genes).

[0046] The term "aiding diagnosis" is used herein to refer to methods that assist in making a clinical determination regarding the presence, or nature, of a particular type of symptom or condition of RA. For example, a method of aiding diagnosis of RA can comprise measuring the expression of certain genes in a biological sample from an individual.

[0047] The term "prognosis" is used herein to refer to the prediction of the likelihood of autoimmune disorder-attributable disease symptoms of an autoimmune disease such as RA.

The term "prediction" is used herein to refer to the likelihood that a patient will respond either favorably or unfavorably to a drug or set of drugs. In one embodiment, the prediction relates to the extent of those responses. In one embodiment, the prediction relates to whether and/or the probability that a patient will survive or improve following treatment, for example treatment with a particular therapeutic agent, and for a certain period of time without disease recurrence. The predictive methods of the invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient.

The predictive methods of the present invention are valuable tools in predicting if a patient is likely to respond favorably to a treatment regimen, such as a given therapeutic regimen, including for example, administration of a given therapeutic agent or combination, surgical intervention, steroid treatment, etc., or whether long-term survival of the patient, following a therapeutic regimen is likely.

[0048] As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed before or during the course of clinical pathology. Desirable effects of treatment include preventing the occurrence or recurrence of a disease or a condition or symptom thereof, alleviating a condition or symptom of the disease, diminishing any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, ameliorating or palliating the disease state, and achieving remission or improved prognosis. In some embodiments, methods and compositions of the invention are useful in attempts to delay development of a disease or disorder.

[0049] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A "therapeutically effective amount" of a therapeutic agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent 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 but not necessarily, 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.

[0050] An "individual," "subject" or "patient" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, primates (including human and non-human primates) and rodents (e.g., mice and rats). In certain embodiments, a mammal is a human.

[0051] A "control subject" refers to a healthy subject who has not been diagnosed as having RA and who does not suffer from any sign or symptom associated with RA.

[0052] The term "sample," as used herein, refers to a composition that is obtained or derived from a subject of interest that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics. For example, the phrase "disease sample" and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and/or molecular entity that is to be characterized.

[0053] By "tissue" or "cell sample" is meant a collection of similar cells obtained from a tissue of a subject or patient. The source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a disease tissue/organ. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. A "reference sample", "reference cell", "reference tissue", "control sample", "control cell", or "control tissue", as used herein, refers to a sample, cell or tissue obtained from a source known, or believed, not to be afflicted with the disease or condition for which a method or composition of the invention is being used to identify. In one embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy part of the body of the same subject or patient in whom a disease or condition is being identified using a composition or method of the invention. In one embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy part of the body of an individual who is not the subject or patient in whom a disease or condition is being identified using a composition or method of the invention.

[0054] For the purposes herein a "section" of a tissue sample is meant a single part or piece of a tissue sample, e.g. a thin slice of tissue or cells cut from a tissue sample. It is understood that multiple sections of tissue samples may be taken and subjected to analysis according to the present invention, provided that it is understood that the present invention comprises a method whereby the same section of tissue sample is analyzed at both morphological and molecular levels, or is analyzed with respect to both protein and nucleic acid.

[0055] By "correlate" or "correlating" is meant comparing, in any way, the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocols and/or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to the embodiment of gene expression analysis or protocol, one may use the results of the gene expression analysis or protocol to determine whether a specific therapeutic regimen should be performed.

[0056] A "medicament" is an active drug to treat a disease, disorder, and/or condition. In one embodiment, the disease, disorder, and/or condition is RA or its symptoms or side effects.

[0057] The term "increased resistance" to a particular therapeutic agent or treatment option, when used in accordance with the invention, means decreased response to a standard dose of the drug or to a standard treatment protocol.

[0058] The term "decreased sensitivity" to a particular therapeutic agent or treatment option, when used in accordance with the invention, means decreased response to a standard dose of the agent or to a standard treatment protocol, where decreased response can be compensated for (at least partially) by increasing the dose of agent, or the intensity of treatment.

[0059] "Patient response" or "response" can be assessed using any endpoint indicating a benefit to the patient, including, without limitation, (1) inhibition, to some extent, of disease progression, including slowing down and complete arrest; (2) reduction in the number of disease episodes and/or symptoms; (3) reduction in lesional size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of disease cell infiltration into adjacent peripheral organs and/or tissues; (5) inhibition (i.e. reduction, slowing down or complete stopping) of disease spread; (6) decrease of auto-immune response, which may, but does not have to, result in the regression or ablation of the disease lesion; (7) relief, to some extent, of one or more symptoms associated with the disorder; (8) increase in the length of disease-free presentation following treatment; and/or (9) decreased mortality at a given point of time following treatment.

[0060] The term "gene signature" is used interchangeably with "gene expression signature" and refers to one or a combination of genes whose expression is indicative of a particular subtype of RA characterized by certain molecular, pathological, histological, and/or clinical features. In certain embodiments, the expression of one or more genes comprising the gene signature is elevated compared to that in control subjects. In certain embodiments, the expression of one or more genes comprising the gene signature is decreased compared to that in control subjects. In certain embodiments, the expression of one or more genes comprising the gene signature is differentially regulated in subjects with a particular RA subtype compared to the expression of those gene(s) in control subjects or in subjects identified as possessing a different RA subtype.

[0061] The term "protein signature" is used interchangeably with "protein expression signature" and refers to one or a combination of proteins whose expression is indicative of a particular subtype of RA characterized by certain molecular, pathological, histological, and/or clinical features. In certain embodiments, the expression of one or more proteins comprising the protein signature is elevated compared to that in control subjects. In certain

embodiments, the expression of one or more proteins comprising the protein signature is decreased compared to that in control subjects. In certain embodiments, the expression of one or more proteins comprising the protein signature is differentially regulated in

subjects with a particular RA subtype compared to the expression of those protein(s) in control subjects or in subjects identified as possessing a different RA subtype.

[0062] A "RA therapeutic agent," a "therapeutic agent effective to treat RA," and grammatical variations thereof, as used herein, refer to an agent that when provided in an effective amount is known, clinically shown, or expected by clinicians to provide a therapeutic benefit in a subject who has RA.

[0063] A "B-cell surface marker" or "B-cell surface antigen" herein is an antigen expressed on the surface of a B cell that can be targeted with an antagonist that binds thereto. Exemplary B-cell surface markers include the CD10, CD19, CD20 (MS4A1), CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85, and CD86 leukocyte surface markers (for descriptions, see The Leukocyte Antigen Facts Book, 2nd Edition. 1997, ed. Barclay et al. Academic Press, Harcourt Brace & Co., New York). Other B-cell surface markers include RP105, FcRH2, B-cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, Btig, NAG 14, SLGC16270, FcRHl, IRTA2, ATWD578, FcRFB, IRTA1, FcRH6, BCMA, and 239287. The B-cell surface marker of particular interest is preferentially expressed on B cells compared to other non-B-cell tissues of a mammal and may be expressed on both precursor B cells and mature B cells.

[0064] An "antibody that binds to a B-cell surface marker" is a molecule that, upon binding to a B-cell surface marker, destroys or depletes B cells in a mammal and/or interferes with one or more B-cell functions, e.g. by reducing or preventing a humoral response elicited by the B cell. The antibody in certain instances is able to deplete B cells (i.e. reduce circulating B-cell levels) in a mammal treated therewith. Such depletion may be achieved via various mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), inhibition of B-cell proliferation, and/or induction of B-cell death (e.g. via apoptosis).

[0065] An "antagonist" refers to a molecule capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of a particular or specified protein, including its binding to one or more receptors in the case of a ligand or binding to one or more ligands in case of a receptor. Antagonists include antibodies and antigen-binding fragments thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids,

polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translation control sequences, and the like. Antagonists also include small molecule inhibitors of the protein, and fusion proteins, receptor molecules and derivatives which bind specifically to the protein thereby sequestering its binding to its target, antagonist variants of the protein, antisense molecules directed to the protein, R A aptamers, and ribozymes against the protein.

[0066] A "B-cell antagonist" is a molecule that, upon binding to a B-cell surface marker, destroys or depletes B cells in a mammal and/or interferes with one or more B-cell functions, e.g. by reducing or preventing a humoral response elicited by the B cell. The antagonist in certain instances is able to deplete B cells (i.e. reduce circulating B-cell levels) in a mammal treated therewith. Such depletion may be achieved via various mechanisms such as ADCC and/or CDC, inhibition of B-cell proliferation, and/or induction of B-cell death (e.g. via apoptosis). Exemplary antagonists include synthetic or native-sequence peptides, fusion proteins, and small-molecule antagonists that bind to the B-cell marker, optionally conjugated with or fused to a cytotoxic agent. Examples include but are not limited to, e.g., CD22 antibodies, CD20 antibodies, BR3 antibodies (e.g., WO0224909), and BR3-Fc

immunoadhesin.

[0100] Examples of CD20 antibodies include: "C2B8," which is now called "rituximab" ("RITUXAN^®") (U.S. Pat. No. 5,736,137); the yttrium- [90] -labeled 2B8 murine antibody designated "Y2B8" or "ibritumomab tiuxetan" (ZEVALIN^®) commercially available from IDEC Pharmaceuticals, Inc. (U.S. Pat. No. 5,736,137; 2B8 deposited with ATCC under accession no. HB11388 on Jun. 22, 1993); murine IgG2a "Bl," also called "tositumomab,"

131 131 131

optionally labeled with I to generate the " I-Bl" or "iodine I tositumomab" antibody

(BEXXAR™) commercially available from Corixa (see, also, U.S. Pat. No. 5,595,721); murine monoclonal antibody "1F5" (Press et al. Blood 69(2):584-591 (1987) and variants thereof including "framework-patched" or humanized 1F5 (WO 2003/002607, Leung, S.; ATCC deposit HB-96450); murine 2H7 and chimeric 2H7 antibody (U.S. Pat. No.

5,677,180); humanized 2H7 (see, e.g.. WO04/056312; US20060024295); HUMAX-CD20™ antibodies (Genmab, Denmark); the human monoclonal antibodies set forth in WO

2004/035607 (Teeling et al); AME-133™ antibodies (Applied Molecular Evolution); A20 antibody or variants thereof such as chimeric or humanized A20 antibody (cA20, bA20, respectively) (US 2003/0219433, Immunomedics); and monoclonal antibodies L27, G28-2, 93-1 B3, B-Cl or NU-B2 available from the International Leukocyte Typing Workshop (Valentine et al., In: Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University Press (1987)).

[0101] The terms "BAFF," "BAFF polypeptide," "TALL-1" or "TALL-1 polypeptide," "BLyS", and "THANK" when used herein encompass "native-sequence BAFF polypeptides" and "BAFF variants." "BAFF" is a designation given to those polypeptides that have the human BAFF sequence as set forth in, for example, U.S. Pat. Pub. No. 2006/0110387, and homologs and fragments and variants thereof, which have the biological activity of the native- sequence BAFF. A biological activity of BAFF can be selected from the group consisting of promoting B-cell survival, promoting B-cell maturation, and binding to BR3. The term "BAFF" includes those polypeptides described in Shu et al, J. Leukocyte Biol., 65:680 (1999); GenBank Accession No. AF136293; WO 1998/18921; EP 869,180; WO 1998/27114; WO 1999/12964; WO 1999/33980; Moore et al, Science, 285:260-263 (1999); Schneider et al, J. Exp. Med., 189: 1747-1756 (1999); and Mukhopadhyay et al, J. Biol. Chem.,

274: 15978-15981 (1999).

[0102] The term "BAFF antagonist" as used herein is used in the broadest sense, and includes any molecule that (1) binds a native-sequence BAFF polypeptide or binds a native- sequence BR3 polypeptide to block, partially or fully, BR3 interaction with BAFF

polypeptide, and (2) partially or fully blocks, inhibits, or neutralizes native-sequence BAFF signaling. Native-sequence BAFF polypeptide signaling promotes, among other things, B- cell survival and B-cell maturation. The inhibition, blockage, or neutralization of BAFF signaling results in, inter alia, a reduction in the number of B cells. A BAFF antagonist as defined herein will partially or fully block, inhibit, or neutralize one or more biological activities of a BAFF polypeptide, in vitro or in vivo. In one embodiment, a biologically active BAFF potentiates any one or a combination of the following events in vitro or in vivo: an increased survival of B cells, an increased level of IgG and/or IgM, an increased numbers of plasma cells, and processing of NF-Kb2/100 to p52 NF-κβ in splenic B cells (e.g., Batten et al., J. Exp. Med. 192: 1453-1465 (2000); Moore et al, Science 285:260-263 (1999); and Kayagaki et al., Immunity, 10:515-524 (2002)).

[0103] In some embodiments, a BAFF antagonist as defined herein includes anti-BAFF antibodies, BAFF-binding polypeptides (including immunoadhesins and peptides), and BAFF-binding small molecules. BAFF antagonists include, for example, the BAFF-binding antibodies described in WO 2002/02641 (e.g., antibodies comprising the amino acid sequence of any of SEQ ID NOS: l-46, 321-329, 834-872, 1563-1595, 1881-1905 of Table 1 thereof). In a further embodiment, the immunoadhesin comprises a BAFF-binding region of a BAFF receptor (e.g., an extracellular domain of BR3, BCMA, or TACI). In a still further embodiment, the immunoadhesin is BR3-Fc. Other examples of BAFF-binding Fc proteins can be found in WO 2002/66516, WO 2000/40716, WO 2001/87979, WO 2003/024991, WO 2002/16412, WO 2002/38766, WO 2002/092620, and WO 2001/12812. Methods of making BAFF antagonists are described, for example, in US 2005/0095243 and US 2005/0163775.

[0104] The terms "BR3", "BR3 polypeptide" or "BR3 receptor" when used herein encompass native-sequence BR3 polypeptides and BR3 variants, as defined hereinbelow. "BR3" is a designation given to those polypeptides comprising, for example, the human BR3 sequence set forth in WO 2003/14294 and US 2005/0070689. BR3 polypeptides can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant and/or synthetic methods. The term BR3 includes the BR3 polypeptides described in WO 2002/24909, WO 2003/14294, and US 2005/0070689. Anti- BR3 antibodies can be prepared in accordance with methods set for in, for example, WO 2003/14294 and US 2005/0070689.

[0105] A "native-sequence" BR3 polypeptide or "native BR3" comprises a polypeptide having the same amino acid sequence as the corresponding BR3 polypeptide derived from nature. Such native-sequence BR3 polypeptides can be isolated from nature or can be produced by recombinant and/or synthetic means. The term "native-sequence BR3 polypeptide" specifically encompasses naturally occurring truncated, soluble or secreted forms (e.g., an extracellular domain sequence), naturally occurring variant forms (e.g., alternatively spliced forms) and naturally occurring allelic variants of the polypeptide. The BR3 polypeptides of the invention include the BR3 polypeptide comprising or consisting of the contiguous sequence of amino acid residues 1 to 184 of a human BR3 (see WO

2003/14294 and US 2005/0070689). [0106] A BR3 "extracellular domain" or "ECD" refers to a form of the BR3 polypeptide that is essentially free of the transmembrane and cytoplasmic domains. ECD forms of BR3 include a polypeptide comprising any one of the amino acid sequences selected from the group consisting of amino acids 1-77, 2-62, 2-71, 1-61, 7-71, 23-38 and 2-63 of human BR3. In certain embodiments, BAFF antagonists are polypeptides comprising any one of the above-mentioned ECD forms of human BR3 and variants and fragments thereof that bind a native BAFF.

[0107] "BR3 variant" means a BR3 polypeptide having at least about 80% amino acid sequence identity with the amino acid sequence of a native-sequence, full-length BR3 or BR3 ECD and binds a native-sequence BAFF polypeptide. Optionally, the BR3 variant includes a single cysteine-rich domain. Such BR3 variant polypeptides include, for instance, BR3 polypeptides wherein one or more amino acid residues are added, or deleted, at the N- and/or C-terminus, as well as within one or more internal domains, of the full-length amino acid sequence. Fragments of the BR3 ECD that bind a native sequence BAFF polypeptide are also contemplated.

[0108] The term "APRIL antagonist" as used herein is used in the broadest sense, and includes any molecule that (1) binds a native-sequence APRIL polypeptide or binds a native- sequence ligand to APRIL to block, partially or fully, the ligand's interaction with APRIL polypeptide, and (2) partially or fully blocks, inhibits, or neutralizes native-sequence APRIL signaling. Native-sequence APRIL polypeptide signaling promotes, among other things, B- cell survival and B-cell maturation. APRIL (a proliferation-inducing ligand) is a TNF family member with a shared receptor to BAFF. Examples of APRIL antagonists include but are not limited to atacicept (same as TACI-Ig immunoadhesin) and a BAFF/ APRIL antagonist (soluble BCMA-Fc).

[0109] The term "cytokine" is a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are

lymphokines, monokines; interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15, IL-17A, IL-17F, IL-17A/F; a tumor necrosis factor such as TNF-a or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native-sequence cytokines, including synthetically produced small-molecule entities and pharmaceutically acceptable derivatives and salts thereof. [0110] For the purposes herein, "tumor necrosis factor-alpha (TNF-alpha)" refers to a human TNF-alpha molecule comprising the amino acid sequence as described in Pennica et al, Nature, 312:721 (1984) or Aggarwal et al, JBC, 260:2345 (1985).

[0111] A "TNF-alpha inhibitor" herein is an agent that inhibits, to some extent, a biological function of TNF-alpha, generally through binding to TNF-alpha and neutralizing its activity. Examples of TNF-alpha inhibitors specifically contemplated herein are etanercept (ENBREL^®), infliximab (REMICADE^®), adalimumab (HUMIRA^®), golimumab

(SIMPONI™), and certolizumab pegol (CIMZIA^®).

[0112] An "IL-17A/F binding agent" is an agent, e.g., an antibody, that binds to the cytokine IL-17A/F or an agent that is cross-reactive with IL-17A and IL-17F.

[0113] An "IL-6 binding agent" is an agent, e.g., an antibody, that binds to the cytokine

IL-6.

[0114] A "CD4 binding agent" is an agent, e.g., an antibody, that binds to the surface glycoprotein CD4 expressed on cells of the T lymphocyte lineage.

[0115] Examples of "disease-modifying anti-rheumatic drugs" or "DMARDs" include hydroxycloroquine, sulfasalazine, methotrexate (plus oral and subcutaneous methrotrexate), leflunomide, azathioprine, D-penicillamine, Gold (oral), Gold (intramuscular), minocycline, cyclosporine, Staphylococcal protein A immunoadsorption, including salts and derivatives thereof, etc.

[0116] "CTLA4" is expressed on activated T lymphocytes and is involved in down- regulation of the immune response. Other names for CTLA4 in the literature include cytotoxic T-lymphocyte-associated antigen 4, cytotoxic T-lymphocyte-associated protein 4, cell differentiation antigen CD 152, and cytotoxic T-lymphocyte-associated granule serine protease 4.

[0117] A therapeutic agent that has "marketing approval," or that has been "approved as a therapeutic agent," or grammatical variations thereof of these phrases, as used herein, refer to an agent (e.g., in the form of a drug formulation, medicament) that is approved, licensed, registered or authorized by a relevant governmental entity (e.g., federal, state or local regulatory agency, department, bureau) to be sold by and/or through and/or on behalf of a commercial entity (e.g., a for-profit entity) for the treatment of a particular disorder (e.g., RA) or a patient subpopulation (e.g., patients of a particular ethnicity, gender, lifestyle, disease risk profile, etc.). A relevant governmental entity includes, for example, the Food and Drug Administration (FDA), European Medicines Evaluation Agency (EMEA), and equivalents thereof.

[0118] "Antibodies" (Abs) and "immunoglobulins" (Igs) refer to glycoproteins having similar structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which generally lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.

[0119] The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein). An antibody can be chimeric, human, humanized and/or affinity matured.

[0120] The terms "full length antibody," "intact antibody" and "whole antibody" are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain the Fc region.

[0121] "Antibody fragments" comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')₂, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0122] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')₂ fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0123] "Fv" is a minimum antibody fragment which contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. Collectively, the six CDRs of an Fv confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0124] The Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')₂ antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0125] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0126] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler et al, Nature, 256: 495 (1975); Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring

Harbor Laboratory Press, 2^nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T- Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technologies (see, e.g., Clackson et al, Nature, 352: 624-628 (1991); Marks et al, J. Mol. Biol. 222: 581-597 (1992); Sidhu et al, J. Mol. Biol. 338(2): 299-310 (2004); Lee et al, J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al, J. Immunol. Methods 284(1-2): 119-132(2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., W098/24893; WO96/34096; W096/33735;

WO91/10741; Jakobovits et al, Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al, Nature 362: 255-258 (1993); Bruggemann et al, Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al, Bio. Technology 10: 779-783 (1992); Lonberg et al, Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al, Nature Biotechnol. 14: 845-851 (1996);

Neuberger, Nature Biotechnol. 14: 826 (1996) and Lonberg and Huszar, Intern. Rev.

Immunol. 13: 65-93 (1995).

[0127] The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to

corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81 :6855-9855 (1984)).

[0128] "Humanized" forms of non-human {e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human

immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma &

Immunol. 1 : 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[0129] A "human antibody" is one which comprises an amino acid sequence

corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. Such techniques include screening human-derived combinatorial libraries, such as phage display libraries (see, e.g., Marks et al, J. Mol. Biol, 222: 581-597 (1991) and Hoogenboom et al, Nucl. Acids Res., 19: 4133-4137 (1991)); using human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies (see, e.g., Kozbor J. Immunol, 133: 3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp. 55-93 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J. Immunol, 147: 86 (1991)); and generating monoclonal antibodies in transgenic animals (e.g., mice) that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production (see, e.g., Jakobovits et al, Proc. Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al, Nature, 362: 255 (1993); Bruggermann et al, Year in Immunol, 7: 33 (1993)). This definition of a human antibody specifically excludes a humanized antibody comprising antigen-binding residues from a non-human animal.

[0130] An "affinity matured" antibody is one with one or more alterations in one or more CDRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In one

embodiment, an affinity matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVR and/or framework residues is described by:

Barbas et al. Proc Nat. Acad. Sci. USA 91 :3809-3813 (1994); Schier et al. Gene 169: 147-155 (1995); Yelton et al. J. Immunol. 155: 1994-2004 (1995); Jackson et al, J. Immunol.

154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0131] A "blocking antibody" or an "antagonist antibody" is one which inhibits or reduces a biological activity of the antigen it binds. Certain blocking antibodies or antagonist antibodies partially or completely inhibit the biological activity of the antigen.

[0132] As used herein, "growth-inhibitory" antibodies are those that prevent or reduce proliferation of a cell expressing an antigen to which the antibody binds. For example, the antibody may prevent or reduce proliferation of B cells in vitro and/or in vivo.

[0133] Antibodies that "induce apoptosis" refer to antibodies that induce programmed cell death, e.g. of a B cell, as determined by standard apoptosis assays, such as binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies).

[0134] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native-sequence Fc region or amino-acid-sequence-variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include but are not limited to: Clq binding and complement- dependent cytotoxicity (CDC); Fc-receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell-surface receptors {e.g. B-cell receptor); and B-cell activation.

[0135] The term "Fc region" herein is used to define a C-terminal region of an

immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is typically defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

[0136] Unless indicated otherwise herein, the numbering of the residues in an

immunoglobulin heavy chain is that of the EU index as in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, Ed. 5 (Public Health Service, National Institutes of Health, Bethesda, MD, 1991)). The "EU index as in Kabat" refers to the residue numbering of the human IgGl EU antibody.

[0137] A "functional Fc region" possesses an "effector function" of a native-sequence Fc region. Exemplary "effector functions" include but are not limited to Clq binding; CDC; Fc- receptor binding; ADCC; phagocytosis; down-regulation of cell-surface receptors (e.g. B-cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g. an antibody- variable domain) and can be assessed using various assays as disclosed, for example, herein.

[0138] A "native-sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native-sequence human Fc regions include a native-sequence human IgGl Fc region (non-A and A allotypes); native-sequence human IgG2 Fc region; native-sequence human IgG3 Fc region; and native-sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

[0139] A "variant Fc region" comprises an amino acid sequence which differs from that of a native- sequence Fc region by virtue of at least one amino acid modification, typically one or more amino acid substitution(s).

[0140] The term "Fc-region-comprising antibody" refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by

recombinant engineering the nucleic acid encoding the antibody. Accordingly, a composition comprising an antibody having an Fc region can comprise an antibody with K447, with all K447 removed, or a mixture of antibodies with and without the K447 residue.

[0141] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR is a native-human FcR. In some embodiments, an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of those receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain.

Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457- 92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

[0142] The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al, J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunology Today, 18 (12):592-8 (1997); Ghetie et al, Nature Biotechnology, 15 (7):637-40 (1997); Hinton et al., J. Biol. C/zem.,279(8):6213-6 (2004); WO 2004/92219 (Hinton et al).

[0143] Binding to human FcRn in vivo and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, e.g. , in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides with a variant Fc region are administered. WO 2000/42072 (Presta) describes antibody variants with improved or diminished binding to FcRs. See, also, for example, Shields et al, J. Biol. Chem., 9(2): 6591- 6604 (2001).

[0144] "Human effector cells" are leukocytes which express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least FcyRIII and perform ADCC effector function(s). Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural-killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. The effector cells may be isolated from a native source, e.g. , from blood.

[0145] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells {e.g., NK cells, neutrophils, and macrophages) enables these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII, and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol, 9:457-492 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. 5,500,362 or 5,821,337 or U.S. 6,737,056 (Presta), may be performed.

Useful effector cells for such assays include PBMC and NK cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al, Proc. Natl. Acad. Sci. (USA), 95:652- 656 (1998).

[0146] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass), which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods, 202: 163 (1996), may be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with a variant Fc region) and increased or decreased Clq binding capability are described, e.g., in U.S. 6,194,551 and WO 1999/51642. See, also, e.g., Idusogie et al, J. Immunol. 164:4178-4184 (2000).

[0147] "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g. , an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art.

[0148] The term "substantially similar" or "substantially the same," as used herein, denotes a sufficiently high degree of similarity between two numeric values (for example, one associated with an antibody of the invention and the other associated with a

reference/comparator antibody), such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, less than about 50%, less than about 40%>, less than about 30%, less than about 20%, and/or less than about 10% as a function of the reference/comparator value.

[0149] The phrase "substantially reduced," or "substantially different," as used herein, denotes a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value for the reference/comparator molecule.

[0150] A "small molecule" or "small organic molecule" is defined herein as an organic molecule having a molecular weight below about 500 Daltons.

[0151] The word "label" when used herein refers to a detectable compound or composition. The label is typically conjugated or fused directly or indirectly to a reagent, such as a nucleic acid probe or an antibody, and facilitates detection of the reagent to which it is conjugated or fused. The label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which results in a detectable product.

[0152] An "isolated" biological molecule, such as a nucleic acid, polypeptide, or antibody, is one which has been identified and separated and/or recovered from at least one component of its natural environment.

[0153] Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X."

[0154] The term "pharmaceutical formulation" refers to a sterile preparation that is in such form as to permit the biological activity of the medicament to be effective, and which contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

[0155] A "sterile" formulation is aseptic or free from all living microorganisms and their spores.

[0156] A "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products or medicaments, that contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings concerning the use of such therapeutic products or medicaments and the like.

[0157] A "kit" is any manufacture (e.g a package or container) comprising at least one reagent, e.g., a medicament for treatment of RA or joint damage, or a probe for specifically detecting a biomarker gene or protein of the invention. In certain embodiments, the manufacture is promoted, distributed, or sold as a unit for performing the methods of the present invention.

[0158] A "target audience" is a group of people or an institution to whom or to which a particular medicament is being promoted or intended to be promoted, as by marketing or advertising, especially for particular uses, treatments, or indications, such as individual patients, patient populations, readers of newspapers, medical literature, and magazines, television or internet viewers, radio or internet listeners, physicians, drug companies, etc.

[0159] The term "serum sample" refers to any serum sample obtained from an individual. Methods for obtaining sera from mammals are well known in the art.

[0160] The expression "not responsive to," "non-response" and grammatical variants thereof, as it relates to the reaction of subjects or patients to one or more of the medicaments that were previously administered to them, describes those subjects or patients who, upon administration of such medicament(s), did not exhibit any or adequate signs of treatment of the disorder for which they were being treated, or they exhibited a clinically unacceptably high degree of toxicity to the medicament(s), or they did not maintain the signs of treatment after first being administered such medicament(s), with the word treatment being used in this context as defined herein. The phrase "not responsive" includes a description of those subjects who are resistant and/or refractory to the previously administered medication(s), and includes the situations in which a subject or patient has progressed while receiving the medicament(s) that he or she is being given, and in which a subject or patient has progressed within 12 months (for example, within six months) after completing a regimen involving the medicament(s) to which he or she is no longer responsive. The non-responsiveness to one or more medicaments thus includes subjects who continue to have active disease following previous or current treatment therewith. For instance, a patient may have active disease activity after about one to three months of therapy with the medicament(s) to which they are non-responsive. Such responsiveness may be assessed by a clinician skilled in treating the disorder in question.

[0161] For purposes of non-response to medicament(s), a subject who experiences "a clinically unacceptably high level of toxicity" from previous or current treatment with one or more medicaments experiences one or more negative side-effects or adverse events associated therewith that are considered by an experienced clinician to be significant, such as, for example, serious infections, congestive heart failure, demyelination (leading to multiple sclerosis), significant hypersensitivity, neuropathological events, high degrees of autoimmunity, a cancer such as endometrial cancer, non-Hodgkin's lymphoma, breast cancer, prostate cancer, lung cancer, ovarian cancer, or melanoma, tuberculosis (TB), and the like.

[0162] By "reducing the risk of a negative side effect" is meant reducing the risk of a side effect resulting from treatment with the antagonist herein to a lower extent than the risk observed resulting from treatment of the same patient or another patient with a previously administered medicament. Such side effects include those set forth above regarding toxicity, and are preferably infection, cancer, heart failure, or demyelination.

[0163] The "amount" or "level" of a biomarker associated with an increased clinical benefit to a RA patient or patient with joint damage is a detectable level in a biological sample. These can be measured by methods known to one skilled in the art and also disclosed herein. The expression level or amount of biomarker assessed can be used to determine the response to the treatment.

[0164] The terms "level of expression" or "expression level" in general are used interchangeably and generally refer to the amount of a polynucleotide or an amino acid product or protein in a biological sample. "Expression" generally refers to the process by which gene-encoded information is converted into the structures present and operating in the cell. Therefore, as used herein, "expression" of a gene may refer to transcription into a polynucleotide, translation into a protein, or even posttranslational modification of the protein. Fragments of the transcribed polynucleotide, the translated protein, or the post- translationally modified protein shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a posttranslational processing of the protein, e.g., by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide as mR A and then translated into a protein, and also those that are transcribed into R A but not translated into a protein (for example, transfer and ribosomal RNAs).

RHEUMATOID ARTHRITIS

[0165] Autoimmune diseases remain clinically important diseases in humans. As the name implies, autoimmune diseases act through the body's own immune system. While the pathological mechanisms differ among individual types of autoimmune diseases, one general mechanism involves the generation of antibodies (referred to herein as self-reactive antibodies or autoantibodies) directed against specific endogenous proteins. Physicians and scientists have identified more than 70 clinically distinct autoimmune diseases, including RA, multiple sclerosis (MS), vasculitis, immune-mediated diabetes, and lupus such as systemic lupus erythematosus (SLE). While many autoimmune diseases are rare - affecting fewer than 200,000 individuals - collectively, these diseases afflict millions of Americans, an estimated five percent of the population, with women disproportionately affected by most diseases. The chronic nature of these diseases leads to an immense social and financial burden.

[0166] Inflammatory arthritis is a prominent clinical manifestation in diverse autoimmune disorders including RA, psoriatic arthritis (PsA), SLE, Sjogren's syndrome, and polymyositis. Most of these patients develop joint deformities on physical examination but typically only RA and PsA patients manifest bone erosions on imaging studies.

[0167] RA is a chronic inflammatory disease that affects approximately 0.5 to 1% of the adult population in northern Europe and North America, and a slightly lower proportion in other parts of the world. Alamanos and Drosos, Autoimmun. Rev., 4: 130-136 (2005). It is a systemic inflammatory disease characterized by chronic inflammation in the synovial membrane of affected joints, which ultimately leads to loss of daily function due to chronic pain and fatigue. The majority of patients also experience progressive deterioration of cartilage and bone in the affected joints, which may eventually lead to permanent disability. The long-term prognosis of RA is poor, with approximately 50% of patients experiencing significant functional disability within 10 years from the time of diagnosis. Keystone, Rheumatology, 44 (Suppl. 2): ii8-iil2 (2005). Life expectancy is reduced by an average of 3- 10 years. Alamanos and Drosos, supra. Patients with a high titer of rheumatoid factor (RF) (approximately 80% of patients) have more aggressive disease (Bukhari et al, Arthritis Rheum., 46: 906-912 (2002)), with a worse long-term outcome and increased mortality over those who are RF negative. Heliovaara et al., Ann. Rheum. Dis., 54: 811-814 (1995)).

[0168] The pathogenesis of chronic inflammatory bone diseases, such as RA, is not fully elucidated. Such diseases are accompanied by bone loss around affected joints due to increased osteoclastic resorption. This process is mediated largely by increased local production of pro-inflammatory cytokines. Teitelbaum, Science, 289: 1504-1508 (2000); Goldring and Gravallese, Arthritis Res., 2(l):33-37 (2000). These cytokines can act directly on cells in the osteoclast lineage or indirectly by affecting the production of the essential osteoclast differentiation factor, receptor activator of NFKB ligand (RANKL), and/or its soluble decoy receptor, osteoprotegerin (OPG), by osteoblast/stromal cells. Hossbauer et al.,

J. Bone Min. Res., 15(1):2-12 (2000). Tumor necrosis factor-alpha (TNF-a) is a major mediator of inflammation. Its importance in the pathogenesis of various forms of bone loss is supported by several lines of experimental and clinical evidence. Feldmann et al, Cell, 85(3):307-310 (1996). However, TNF-a is not essential for osteoclastogenesis (Douni et al, J. Inflamm., 47:27-38 (1996)), erosive arthritis (Campbell et al, J. Clin. Invest.,

107(12): 1519-1527 (2001)), or osteolysis (Childs et al, J. Bon. Min. Res., 16:338-347 (2001)), as these can occur in the absence of TNF-a.

[0169] In RA specifically, an immune response is thought to be initiated/perpetuated by one or several antigens presenting in the synovial compartment, producing an influx of acute inflammatory cells and lymphocytes into the joint. Successive waves of inflammation lead to the formation of an invasive and erosive tissue called p annus. This contains proliferating fibroblast-like synoviocytes and macrophages that produce proinflammatory cytokines such as TNF-a and interleukin-1 (IL-1). Local release of proteolytic enzymes, various

inflammatory mediators, and osteoclast activation contributes to much of the tissue damage. There is loss of articular cartilage and the formation of bony erosions. Surrounding tendons and bursa may become affected by the inflammatory process. Ultimately, the integrity of the joint structure is compromised, producing disability.

[0170] The precise contributions of B cells to the immunopathogenesis of RA are not completely characterized. However, there are several possible mechanisms by which B cells may participate in the disease process. Silverman and Carson, Arthritis Res. Ther., 5 Suppl. 4: Sl-6 (2003).

[0171] Historically, B cells were thought to contribute to the disease process in RA predominantly by serving as the precursors of autoantibody-producing cells. A number of autoantibody specificities have been identified including antibodies to Type II collagen, and proteoglycans, as well as RFs. The generation of large quantities of antibody leads to immune complex formation and the activation of the complement cascade. This in turn amplifies the immune response and may culminate in local cell lysis. Increased RF synthesis and complement consumption has been correlated with disease activity. The presence of RF itself is associated with a more severe form of RA and the presence of extra-articular features.

[0172] Evidence exists (Janeway and Katz, J. Immunol, 138: 1051 (1998); Rivera et al, Int. Immunol, 13: 1583-1593 (2001)) showing that B cells are highly efficient antigen- presenting cells (APC). RF-positive B cells may be particularly potent APCs, since their surface immunoglobulin would readily allow capture of any immune complexes regardless of the antigens present within them. Many antigens may thus be processed for presentation to T cells. In addition, it has been recently suggested that this may also allow RF-positive B cells to self-perpetuate. Edwards et al, Immunology, 97: 188-196 (1999).

[0173] For activation of T cells, two signals need to be delivered to the cell; one via the

T-cell receptor (TCR), which recognizes the processed peptide in the presence of major histocompatibility complex (MHC) antigen, and a second, via co-stimulatory molecules.

When activated, B cells express co-stimulatory molecules on their surface and can thus provide the second signal for T-cell activation and the generation of effector cells.

[0174] B cells may promote their own function as well as that of other cells by producing cytokines. Harris et al, Nat. Immunol, 1 : 475-482 (2000). TNF-a, IL-1, lymphotoxin-a, IL-

6, and IL-10 are amongst some of the cytokines that B cells may produce in the RA synovium.

[0175] Although T-cell activation is considered to be a key component in the

pathogenesis of RA, recent work using human synovium explants in severe combined immunodeficiency disorders (SCID) mice has demonstrated that T-cell activation and retention within the joint is critically dependent on the presence of B cells. Takemura et al., J. Immunol., 167: 4710-4718 (2001). The precise role of B cells in this is unclear, since other APCs did not appear to have the same effect on T cells.

[0176] Structural damage to joints is an important consequence of chronic synovial inflammation. Between 60% and 95% of patients with RA develop at least one radiographic erosion within 3-8 years of disease onset. Paulus et ah, J. Rheumatol., 23: 801-805 (1996); Hulsmans et al., Arthritis Rheum., 43: 1927-1940 (2000). In early RA, the correlation between radiographic damage scores and functional capacity is weak, but after 8 years of disease, correlation coefficients can reach as high as 0.68. Scott et al, Rheumatology, 39: 122-132 (2000). In 1,007 patients younger than age 60 years who had RA for at least four years, Wolfe et al. {Arthritis Rheum, 43 Suppl. 9:S403 (2000)) found a significant association among the rate of progression of the Larsen radiographic damage score (Larsen et al, Acta Radiol. Diagn. 18:481-491 (1977)), increasing Social Security disability status, and decreasing family income.

[0177] Diagnosis of RA may be according to current American College of Rheumatology (ACR) criteria and may include include morning stiffness in and around the joints lasting for at least 1 hour before maximal improvement; arthritis of three or more joint areas: at least three joint areas have simultaneously had soft tissue swelling or fluid (not bony overgrowth alone) observed by a physician; the 14 possible joint areas (right and left) are proximal interphalangeal (PIP), metacarpophalangeal (MCP), wrist, elbow, knee, ankle, and

metatarsophalangeal (MTP) joints; arthritis of hand joints: at least one joint area swollen as above in wrist, MCP, or PIP joint; symmetric arthritis: simultaneous involvement of the same joint areas (as in arthritis of three or more joint areas, above) on both sides of the body (bilateral involvement of PIP, MCP, or MTP joints is acceptable without absolute symmetry); rheumatoid nodules: subcutaneous nodules over bony prominences or extensor surfaces or in juxta-articular regions that are observed by a physician; serum rheumatoid factor:

demonstration of abnormal amounts of serum rheumatoid factor by any method that has been positive in fewer than five percent of normal control patients; radiographic changes:

radiographic changes typical of rheumatoid arthritis on posteroanterior hand and wrist X-rays, which must include erosions or unequivocal bony decalcification localized to or most marked adjacent to the involved joints (osteoarthritis changes alone do not qualify). Diagnosis of RA is typically made if a patient satisfies at least four of the above criteria.

[0178] Prevention or retardation of radiographic damage is one of the goals of RA treatment. Edmonds et al., Arthritis Rheum., 36:336-340 (1993). Controlled clinical trials of 6 or 12 months' duration have documented that the progression of radiographic damage scores was more rapid in the placebo group than in groups that received methotrexate (MTX) (Sharp et al., Arthritis Rheum., 43: 495-505 (2000)), leflunomide (Sharp et al., supra), sulfasalazine (SSZ) (Sharp et al, supra), prednisolone (Kirwan et al, N. Engl. J. Med., 333: 142-146 (1995); Wassenburg et al, Arthritis Rheum, 42: Suppl 9:S243 (1999)), interleukin-1 receptor antagonist (Bresnihan et al, Arthritis Rheum, 41 : 2196-2204 (1998)), or an infliximab/MTX combination. Lipsky et al., N. Eng. J. Med., 343: 1594-1604 (2000). Clinical trials have also documented that radiographic progression following treatment with etanercept was less rapid than that following treatment with MTX. Bathon et al, N. Engl. J. Med., 343: 1586-1593 (2000). Other studies have evaluated radiographic progression in patients treated with corticosteroids (Joint Committee of the Medical Research Council and Nuffield Foundation, Ann Rheum. Dis., 19:331-337 (1960); Van Everdingen et al, Ann. Intern. Med., 136: 1-12 (2002)), cyclosporin A (Pasero et al, J. Rheumatol, 24:2113-2118 (1997); Forre, Arthritis Rheum., 37: 1506-1512 (1994)), MTX versus azathioprine (Jeurissen et al, Ann. Intern. Med., 114:999-1004 (1991)), MTX versus auranofm (Weinblatt et al, Arthritis Rheum., 36:613-619 (1993)), MTX (meta-analysis) (Alarcon et al, J. Rheumatol, 19: 1868-1873 (1992)), hydroxychloroquine (HCQ) versus SSZ (Van der Heijde et al, Lancet, 1 : 1036-1038), SSZ

(Hannonen et al, Arthritis Rheum., 36: 1501-1509 (1993)), the COBRA (Combinatietherapei Bij Reumatoide Artritis) combination of prednisolone, MTX, and SSZ (Boers et al., Lancet, 350:309-318 (1997); Landewe et al, Arthritis Rheum., 46: 347-356 (2002)), combinations of MTX, SSZ, and HCQ (O'Dell et al, N. Engl J. Med., 334: 1287-1291 (1996); Mottonen et al, Lancet, 353: 1568-1573 (1999)), the combination of cyclophosphamide, azathioprine, and HCQ (Csuka et al, JAMA, 255:2115-2119 (1986)), and the combination of adalimumab with MTX. Keystone et al, Arthritis Rheum., 46 Suppl. 9:S205 (2002).

[0179] The FDA has now approved labeling claims that certain medications, e.g., leflunomide, etanercept, and infliximab, slow the progression of radiographic joint damage. These claims are based on the statistically significant differences in progression rates observed between randomly assigned treatment groups and control groups. However, the progression rates in individuals within the treatment and control groups overlap to a considerable extent. Therefore, despite significant differences between treatment groups, these data cannot be used to estimate the probability that a patient who is starting a treatment will have a favorable outcome with respect to progression of radiographic damage. Various methods have been suggested to categorize paired radiographs from individual patients as not progressive, e.g., damage scores of 0 at both time points, no increase in damage scores, no new joints with erosions, and a change in score not exceeding the smallest detectable difference {i.e., 95% confidence interval for the difference between repeated readings of the same radiograph). Lassere et al, J. Rheumatol, 26: 731-739 (1999).

[0180] Determining whether there has been increased structural damage in an individual patient during the interval between paired radiographs obtained at the beginning and end of a

6- or 12-month clinical trial has been difficult, for several reasons. The rate of radiographic damage is not uniform within a population of RA patients; a few patients may have rapidly progressing damage, but many may have little or no progression, especially if the tie interval is relatively short. The methods for scoring radiographic damage, e.g., Sharp (Sharp et al,

Arthritis Rheum., 14: 706-720 (1971); Sharp et al, Arthritis Rheum., 28: 1326-1335 (1985)),

Larsen (Larsen et al, Acta Radiol Diagn., 18: 481-491 (1977)), and modifications of these methods (Van der Heijde, J. Rheumatol, 27: 261-263 (2000)), depend on the judgment and the interpretation of the reader as to what is real. Factors to determine are whether an apparent interruption of the subchondral cortical plate is real, or whether a decrease in the distance between the cortices on opposite sides of a joint is real, or is due to a slight change in the position of the joint relative to the film and the radiographic beam, to a change in radiographic exposure, or to some other technical factor. [0181] Therefore, the recorded score is an approximation of the true damage, and for many subjects, the smallest detectable difference between repeat scores of the same radiographs is larger than the actual change that has occurred during the interval between the baseline and final radiographs. If the reader is blinded to the temporal sequence of the films, these unavoidable scoring errors may be in either direction, leading to apparent "healing" when the score decreases or to apparent rapid progression when reading error increases the difference between films. When the study involves a sufficiently large population of patients who have been randomly assigned to receive an effective treatment as compared with placebo, the positive and negative reading errors offset each other, and small but real differences between treatment groups can be detected.

[0182] The imprecision of the clinical measures that are used to quantitate RA disease activity has caused a similar problem. Statistically significant differences between certain outcome measures from clinical trials were not useful for estimating the probability of improvement for an individual who was starting the treatment. Paulus et al, Arthritis Rheum., 33:477-484 (1990). Attribution of individual improvement became practical with the creation of the American College of Rheumatology (ACR) 20% composite criteria for improvement (ACR20), which designated a patient as improved if there was 20%

improvement in the tender and swollen joint counts and 20% improvement in at least three of five additional measures (pain, physical function, patient global health assessment, physician global health assessment, and acute-phase reactant levels). Felson et al, Arthritis Rheum., 38:727-735 (1995). All of these measures have large values for the smallest detectable difference, but by requiring simultaneous improvement in five of the seven aspects of the same process (disease activity), the randomness of the seven measurement errors is constrained, and it is easier to attribute real improvement to the individual.

[0183] In RA, joint damage is a prominent feature. Radiologic parameters of joint destruction are seen as a key outcome measure in descriptions of disease outcome. In the recent OMERACT (Outcome Measures in Rheumatology Clinical Trials) consensus meeting, radiology was chosen as part of the core set of outcome measures for longitudinal

observational studies. Wolfe et al, Arthritis Rheum., 41 Supp 9: S204 (1998) abstract.

Radiology is also part of the WHO/ILAR (World Health Organization/International League of Associations for Rheumatology) required core set of measures for long-term clinical trials. Tugwell and Boers, J. Rheumatol, 20:528-530 (1993). [0184] Available data on the outcome of radiologic damage in RA have been obtained in both short-term and long-term studies. In short-term studies of RA patients with recent-onset disease, radiographs obtained every six months showed that after an initial rapid progression, there was diminution of the progression rate of radiologic damage in the hands and feet after two to three years. Van der Heijde et al, Arthritis Rheum., 35: 26-34 (1992); Fex et al., Br. J. Rheumatol., 35: 1106-1055 (1996). In long-term studies with radiographs taken less frequently, a constant rate of progression was found, with relentless deterioration of damage up to 25 years of disease duration. Wolfe and Sharp, Arthritis Rheum., 41 : 1571-1582 (1998); Graudal et al, Arthritis Rheum., 41 : 1470-1480 (1998); Plant et al, J. Rheumatol, 25:417- 426 (1998); Kaarela and Kautiainen, J. Rheumatol, 24: 1285-1287 (1997). Whether these differences in radiographic progression pattern are due to differences in the scoring techniques is not clear.

[0185] The scoring systems used differ in the number of joints being scored, the presence of independent scores for erosions (ERO) and joint space narrowing (JSN), the maximum score per joint, and the weighing of a radiologic abnormality. As yet, there is no consensus on the scoring method of preference. During the first three years of follow-up in a cohort study of patients with early arthritis, JSN and ERO were found to differ in their contribution to the measured progression in radiologic damage of the hands and feet. Van der Heijde et al, Arthritis Rheum., 35:26-34 (1992). Furthermore, methods that independently score ERO and JSN, such as the Sharp and Kellgren scores, were found to be more sensitive to change in early RA than methods using an overall measure, such as the Larsen score. Plant et al, J. Rheumatol, 21 : 1808-1813 (1994); Cuchacovich et al, Arthritis Rheum., 35:736-739 (1992). The Sharp score is a very labor-intensive method. Van der Heijde, Baillieres Clin.

Rheumatol, 10:435-533 (1996). In late or destructive RA, the Sharp and the Larsen methods were found to provide similar information. However, the sensitivity to change of the various scoring methods late in the disease has not yet been investigated, and it can be argued that the scoring methods that independently measure ERO and JSN provide useful information.

Pincus et al, J. Rheumatol, 24:2106-2122 (1997). See also Drossaers-Bakker et al, Arthritis Rheum., 43: 1465-1472 (2000), which compared the three radiologic scoring systems for the long-term assessment of RA.

[0186] Paulus et al, Arthritis Rheum., 50: 1083-1096 (2004) categorized radiographic joint damage as progressive or non-progressive in individuals with RA participating in clinical trials, and concluded that RA joint damage in an observational cohort can be classified as progressive or non-progressive with the use of a composite definition that includes a number of imprecise and related, but distinct, measures of structural joint damage. It appears that in day-to-day clinical management of an RA patient, an interval change between a pair of radiographs of at least five Sharp radiographic damage score units should be present before one considers the structural change to be real and uses it as the basis for a treatment decision.

CERTAIN RA THERAPEUTIC AGENTS

[0187] Initial therapy of RA typically involves administration of one or more of the following drugs: nonsteroidal antiinflammatory drugs (NSAIDs), e.g., acetylsalicylic acid (e.g., aspirin), ibuprofen (Motrin), naproxen (Naprosyn), indomethacin (Indocin),

nabumetone (Relafen), tolmetin (Tolectin); glucocorticoid (via joint injection); and low-dose prednisone. See "Guidelines for the management of rheumatoid arthritis," Arthritis &

Rheumatism 46(2): 328-346 (February, 2002). The majority of patients with newly diagnosed RA are started with disease-modifying antirheumatic drug (DMARD) therapy within 3 months of diagnosis. DMARDs commonly used in RA are hydroxychloroquine, sulfasalazine, methotrexate (plus oral and subcutaneous methotrexate), leflunomide, azathioprine, D- penicillamine, Gold (oral), Gold (intramuscular), minocycline, cyclosporine, Staphylococcal protein A immunoadsorption. In certain instances, patients are treated with

immunomodulating agents such as azathioprine or cyclophosphamide. Additional RA therapeutic agents include an anti-cytokine agent (e.g., anti-tumor necrosis factor a, anti- interleukin-1 -receptor (e.g., anakinra), anti-interleukin 10, anti-interleukin 6 receptor, anti- interleukin 6, anti-interferon alpha, anti-B-lymphocyte stimulator), an inhibitor of

costimulation (e.g., anti-CD 154, CTLA4-Ig (e.g., abatacept)).

[0188] In certain instances, TNFa inhibitors have been used for therapy of RA.

Exemplary TNFa inhibitors include etanercept (sold under the trade name ENBREL^®), infliximab (sold under the trade name REMICADE^®), adalimumab (sold under the trade name HUMIRA^®), golimumab (sold under the trade name SIMPONI™) and certolizumab pegol (sold under the trade name CIMZIA^®).

[0189] Etanercept (sold under the trade name ENBREL^®) is an injectable drug approved in the U.S. for therapy of active RA. Etanercept binds to TNFa and serves to remove most TNFa from joints and blood, thereby preventing TNFa from promoting inflammation and other symptoms of rheumatoid arthritis. Etanercept is an "immunoadhesin" fusion protein consisting of the extracellular ligand binding portion of the human 75 kD (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of a human IgGl . The drug has been associated with negative side effects including serious infections and sepsis, and nervous system disorders such as multiple sclerosis (MS). See, e.g., www.remicade- infliximab . com/pages/ enbrel_embrel.html.

[0190] Infliximab, sold under the trade name REMICADE^®, is an immune-suppressing drug prescribed to treat RA and Crohn's disease. Infliximab is a chimeric monoclonal antibody that binds to TNFa and reduces inflammation in the body by targeting and binding to TNFa which produces inflammation. Infliximab has been linked to certain fatal reactions such as heart failure and infections including tuberculosis as well as demyelination resulting in MS. See, e.g., www.remicade-infliximab.com.

[0191] In 2002, Abbott Laboratories received FDA approval to market adalimumab (sold under the trade name HUMIRA^®), previously known as D2E7. Adalimumab is a human monoclonal antibody that binds to TNFa and is approved for reducing the signs and symptoms and inhibiting the progression of structural damage in adults with moderately to severely active RA who have had insufficient response to one or more traditional disease modifying DMARDs.

[0192] In April 2009, Centocor Ortho Biotech Inc. received FDA approval to market golimumab (sold under the trade name SIMPONI™) for patients with moderate to severe RA, psoriatic arthritis, and ankylosing spondylitis. Golimumab is a human IgGlK monoclonal antibody specific for human TNFa and which is self-administered by patients subcutaneously once every month. Golimumab binds to both soluble and transmembrane bioactive forms of TNFa. Similar to other agents that inhibit TNFa, golimumab has been associated with certain adverse events such as risk of infection, including serious and life-threatening fungal infections.

[0193] In May 2009, certolizumab pegol (sold under the trade name CIMZIA^®) was approved by the FDA for treatment of patients with RA. It is administered by a healthcare professional by subcutaneous injection every two weeks during induction and then every four weeks during maintenance. Certolizumab pegol is a recombinant, humanized antibody Fab' fragment, with specificity for human TNFa, conjugated to an approximately 40kDa polyethylene glycol (PEG2MAL40K). Certolizumab pegol has also been associated with certain safety risks such as increased risk of serious infection, similar to other TNFa inhibitors.

[0194] In certain instances, the rituximab antibody (sold under the trade name

RITUXAN^®) has been used as a therapy for RA. Rituximab is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen. Rituximab is the antibody called "C2B8" in U.S. Pat. No. 5,736,137 issued Apr. 7, 1998 (Anderson et al).

[0195] Another anti-CD20 antibody is ocrelizumab. Ocrelizumab is a humanized variant of an anti-CD20 antibody, 2H7. Such humanized 2H7 variants are described, for example, in International Publication No. WO 2004/056312 (International Application No.

PCT/US2003/040426).

[0196] RA therapeutic agents having B-cell antagonist activity can be identified, for example, by screening compounds for certain biological properties. For example, a method of screening can be employed as described in Sundberg et al, Cancer Research 66, 1775- 1782 (2006) wherein a compound was screened for inhibition of B-cell proliferation by targeting c-myc protein for rapid and specific degradation. See also Mackay et al., Annual Review of Immunology, 21 : 231-264 (2003) regarding BAFF, APRIL, and a tutorial on B-cell survival and screening, and Thangarajh et al., Scandinavian J. Immunol., 65(1):92 (2007) on B-cell proliferation and APRIL. In addition, Sakurai et al., European J. Immunol., 37(1): 110 (2007) discloses that TACI attenuates antibody production co-stimulated by BAFF-R and CD40. Further, Acosta-Rodriguez et al., European J. Immunol., 37(4):990 (2007) discloses that BAFF and LPS cooperate to induce B cells to become susceptible to CD95/Fas-mediated cell death. Further screening methods can be found in Martin and Chan, "B Cell

Immunobiology in Disease: Evolving Concepts from the Clinic Annual Review of

Immunology," 24:467-496 (2006), Pillai et al, "Marginal Zone B Cells" Annual Review of Immunology, 23: 161-196 (2005), and Hardy and Hayakawa, "B Cell Development Pathways," Annual Review of Immunology, 19:595-621 (2001). From these and other references the skilled artisan can screen for the appropriate antagonists. Microarrays can be used for this purpose (Hagmann, Science, 290:82-83 (2000)), as well as RNA interference (RNAi) (Ngo et al, Nature, 441 : 106-110 (2006)).

[0197] B-cell antagonists included within the scope of the present invention include antibodies, synthetic or native-sequence peptides, immunoadhesins, and small-molecule antagonists that bind to a B-cell surface marker or a B-cell specific survival or proliferation factor, optionally conjugated with or fused to another molecule. In certain embodiments, the antagonist comprises an antibody or immunoadhesin. It includes BLyS antagonists such as immunoadhesins, including, but not limited to, anti-CD23 (e.g., lumiliximab), anti-CD20, anti-CD22, or anti-BR3 antibodies, APRIL antagonists, and/or BLyS immunoadhesins. In certain embodiments, the BLyS immunoadhesin is selected from BR3 immunoadhesin comprising the extracellular domain of BR3, TACI immunoadhesin comprising the extracellular domain of TACI, and BCMA immunoadhesin comprising the extracellular domain of BCMA. Certain embodiments of BR3 immunoadhesin include hBR3-Fc as described in WO 2005/00351, U.S. Pat. Pub. No. 2005/0095243, U.S. Pat. Pub. No.

2005/0163775 and WO 2006/068867. In certain embodiments, the BLyS antagonist is an anti-BLyS antibody, wherein the anti-BLyS antibody binds BLyS within a region of BLyS comprising residues 162-275, or an anti-BR3 antibody, wherein the anti-BR3 antibody binds BR3 in a region comprising residues 23-38 of human BR3. In certain embodiments, the immunoadhesins are selected from TACI-Ig (atacicept) and BR3-Ig. In certain embodiments, the B-cell antagonist is to CD20, CD22, BAFF, or APRIL. In certain such embodiments, the antagonist is an antibody or TACI-Ig.

[0198] The CD22 antigen, or CD22, also known as BL-CAM or Lyb8, is a type 1 integral membrane glycoprotein with molecular weight of about 130 (reduced) to 140kD (unreduced). It is expressed in both the cytoplasm and cell membrane of B-lymphocytes. CD22 antigen appears early in B-cell lymphocyte differentiation at approximately the same stage as the CD 19 antigen. Unlike certain other B-cell markers, CD22 membrane expression is limited to the late differentiation stages comprised between mature B cells (CD22+) and plasma cells (CD22-). The CD22 antigen is described, for example, in Wilson et al, J. Exp. Med., 173: 137 (1991) and Wilson et al., J. Immunol, 150:5013 (1993).

[0199] Certain exemplary anti-CD22 antibodies include those described in EP 1,476,120 (Tedder and Tuscano), EP 1,485,130 (Tedder), and EP 1,504,035 (Popplewell et al), as well as those described in U.S. Pat. Pub. No. 2004/0258682 (Leung et al), U.S. Pat. No.

5,484,892 (Dana-Farber), U.S. Pat. No. 6,183,744 (Immunomedics, epratuzumab), and U.S. Pat. No. 7,074,403 (Goldenberg and Hansen).

[0200] BLyS (also known as BAFF, TALL-1, THANK, TNFSF13B, or zTNF4) is a member of the TNF1 ligand superfamily that is essential for B-cell survival and maturation. BAFF overexpression in transgenic mice leads to B-cell hyperplasia and development of severe autoimmune disease (Mackay et al., J. Exp. Med., 190: 1697-1710 (1999); Gross et al, Nature, 404:995-999 (2000); Khare et al, Proc. Natl Acad. Sci. U.S.A, 97:3370-3375

(2000) ). BAFF levels are elevated in human patients with a variety of autoimmune disorders, such as SLE, RA, and Sjogren's syndrome (Cheema et al, Arthritis Rheum., 44: 1313-1319

(2001) ; Groom et al, J. Clin. Invest., \09 59-68 (2002); Zhang et al, J. Immunol, 166:6-10 (2001)). Furthermore, BAFF levels correlate with disease severity, suggesting that BAFF can play a direct role in the pathogenesis of these illnesses. BAFF acts on B cells by binding to three members of the TNF receptor superfamily, TACI, BCMA, and BR3 (also known as BAFF-R) (Gross et al, supra; Thompson et al, Science, 293:2108-2111 (2001); Yan et al, Curr. Biol. 11 : 1547-1552 (2001); Yan et al, Nat. Immunol, 1 :37-41 (2000); Schiemann et al, Science, 293:2111-2114 (2001)).

[0201] Of the three, only BR3 is specific for BAFF; the other two also bind the related TNF family member, A proliferation-inducing ligand (APRIL). Comparison of the phenotypes of BAFF and receptor knockout or mutant mice indicates that signaling through BR3 mediates the B-cell survival functions of BAFF (Thompson et al, supra; Yan et al, supra, 2001; Schiemann et al, supra). In contrast, TACI ap-pears to act as an inhibitory receptor (Yan, Nat. Immunol, 2:638-643 (2001)), while the role of BCMA is unclear (Schiemann et al, supra). US 2007/0071760 discloses treating B-cell malignancies using a TACI-Ig fusion molecule in an amount sufficient to suppress proliferation-inducing functions of BlyS and APRIL.

[0202] BR3 is a 184-residue type III transmembrane protein expressed on the surface of B cells (Thompson et al, supra; Yan, Nat. Immun., supra). The intracellular region bears no sequence similarity to known structural domains or protein-protein interaction motifs.

Nevertheless, BAFF-induced signaling through BR3 results in processing of the transcription factor NF-B2/p 100 to p52 (Claudio et al, Nat. Immunol, 3:958-965 (2002); Kayagaki et al, Immunity, 10:515-524 (2002)). The extracellular domain (ECD) of BR3 is also divergent. TNFR family members are usually characterized by the presence of multiple cysteine-rich domains (CRDs) in their extracellular region; each CRD is typically composed of about 40 residues stabilized by six cysteines in three disulfide bonds. Conventional members of this family make contacts with ligand through two CRDs interacting with two distinct patches on the ligand surface (Bodmer et al, Trends Biochem. Sci., 27: 19-26 (2002)). However, the BR3 ECD contains only four cysteine residues, capable of forming a partial CRD at most, raising the question of how such a small receptor imparts high-affinity ligand binding. [0203] It has been shown that the BAFF-binding domain of BR3 resides within a 26- residue core region (Kayagaki et al, supra). Six BR3 residues, when structured within a β- hairpin peptide (bhpBR3), were sufficient to confer BAFF binding and block BR3-mediated signaling. Others have reported polypeptides purported to interact with BAFF {e.g., WO 2002/24909, WO 2003/035846, WO 2002/16312, and WO 2002/02641).

[0204] Loss of function and radiographic change occur early in the course of the disease. These changes can be delayed or prevented with the use of certain DMARDs. Although several DMARDs are initially clinically effective and well tolerated, many of these drugs become less effective or exhibit increased toxicity over time. Based on its efficacy and tolerability, MTX has become the standard therapy by which other treatments are measured. Bathon et al, N. Eng. J. Med., 343: 1586-1593 (2000); Albert et al, J. Rheumatol, 27:644- 652 (2000).

[0205] Recent studies have examined radiographic progression in patients with late-stage RA who have taken leflunomide, MTX, or placebo (Strand et al, Arch. Intern. Med., 159:2542-2550 (1999)) as well as patients who have taken infliximab plus MTX or placebo plus MTX following a partial response to MTX. Lipsky et al, N. Engl. J. Med., 343: 1594- 1602 (2000); Maini et al, Lancet, 354: 1932-1939 (1999). In the first year of the ENBREL™ ERA (early RA) trial, etanercept was shown to be significantly more effective than MTX in improving signs and symptoms of disease and in inhibiting radiographic progression. Bathon et al, N. Eng. J. Med., 343: 1586-1593 (2000). Genovese et al, Arthritis Rheum. 46: 1443- 1450 (2002) reports results from the second year of the study, concluding that etanercept as monotherapy was safe and superior to MTX in reducing disease activity, arresting structural damage, and decreasing disability over two years in patients with early aggressive RA. Also studied was the safety and clinical activity of ocrelizumab (a humanized antibody targeting C D20+B cells) in combination with MTX in moderate-to-severe RA patients (Ph I/II ACTION study). Genovese et al, Arthritis Rheum., 54(9):S66-S67 (Sept. 2006).

[0206] Further, reduction in radiographic progression in the hands and feet was observed in patients with early RA after receiving infliximab in combination with MTX. Van der Heijde et al, Annals Rheumatic Diseases, 64:417 (2005). Patients with early RA achieved a clinically meaningful and sustained improvement in physical function after treatment with infliximab. Smolen et al, Annals Rheumatic Diseases, 64:418-419 (2005).

[0207] The effect of infliximab therapy on bone mineral density in patients with ankylosing spondylitis (AS) resulting from a randomized, placebo-controlled trial named ASSERT) is reported by Van der Heijde et al, Annals Rheumatic Diseases, 64:319 (2005). The ASSERT trial showed that infliximab improved fatigue and pain in patients with AS. Van der Heijde et al, Annals Rheumatic Diseases, 64:318-319 (2005). The efficacy and safety of infliximab in AS patients treated according to ASSERT are described by van der Heijde et al., Arthritis Rheum., 5:582-591 (2005). The authors conclude that infliximab was well tolerated and effective in a large cohort of patients with AS during a 24-week study period. In addition, the effect of infliximab therapy on spinal inflammation was assessed by magnetic resonance imaging in a randomized, placebo-controlled trial of 279 patients with AS. Van der Heijde et al, Annals Rheumatic Diseases, 64:317 (2005). The manner in which the treatment effect on spinal radiographic progression in patients with AS should be measured is addressed by van der Heijde et al, Arthritis Rheum. 52: 1979-1985 (2005).

[0208] The results of radiographic analyses of the infliximab multinational PsA controlled trial (IMPACT) after one year are reported by Antoni et al, Annals Rheumatic Diseases 64: 107 (2005). Evidence of radiographic benefit of treatment with infliximab plus MTX in RA patients who had no clinical improvement, with a detailed subanalysis of data from the anti-TNF trial in RA with concomitant therapy study, is reported by Smolen et al, Arthritis Rheum. 52: 1020-1030 (2005). Radiographic progression (as measured by mean change in modified Sharp/van der Heijde score) was much greater in patients receiving MTX plus placebo than in patients receiving infliximab plus MTX. The authors conclude that even in patients without clinical improvement, treatment with infliximab plus MTX provided significant benefit with regard to the destructive process, suggesting that in such patients these two measures of disease are dissociated. The association between baseline radiographic damage and improvement in physical function after treatment of patients having RA with infliximab is described by Breedveld et al, Annals Rheumatic Diseases, 64:52-55 (2005). Structural damage was assessed using the van der Heijde modification of the Sharp score. The authors conclude that greater joint damage at baseline was associated with poorer physical function at baseline and less improvement in physical function after treatment, underlining the importance of early intervention to slow the progression of joint destruction. RHEUMATOID ARTHRITIS MOLECULAR BIOMARKERS

[0209] A number of investigators have carried out microarray gene expression profiling studies of synovial tissue isolated from RA patients. The published studies include van der

Pouw Kraan TC et al., Discovery of distinctive gene expression profiles in rheumatoid synovium using cDNA microarray technology: evidence for the existence of multiple pathways of tissue destruction and repair, Genes Immun Apr;4(3): 187-96 (2003); van der Pouw Kraan TC, et al, Rheumatoid arthritis is a heterogeneous disease: evidence for differences in the activation of the STAT-1 pathway between rheumatoid tissues, Arthritis Rheum Aug;48(8):2132-45 (2003); Finis K et al, Analysis of pigmented villonodular synovitis with genome-wide complementary DNA microarray and tissue array technology reveals insight into potential novel therapeutic approaches, Arthritis Rheum Mar;54(3): 1009- 19 (2006); Lindberg J, et al., Effect of infliximab on mRNA expression profiles in synovial tissue of rheumatoid arthritis patients, Arthritis Res Ther. 8(6):R179 (2006); van der Pouw Kraan TC et al., Responsiveness to anti-tumour necrosis factor alpha therapy is related to pre- treatment tissue inflammation levels in rheumatoid arthritis patients, Ann Rheum Dis.

Apr;67(4):563-6 (2008); Huber R et al, Identification of intra-group, inter-individual, and gene-specific variances in mRNA expression profiles in the rheumatoid arthritis synovial membrane, Arthritis Res Ther 10(4):R98 (2008); Badot V et al, Gene expression profiling in the synovium identifies a predictive signature of absence of response to adalimumab therapy in rheumatoid arthritis, Arthritis Res Ther. 11(2):R57 (2009), Epub 2009 Apr 23.

GENERAL TECHNIQUES

[0210] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al, 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F. M.

Ausubel et al., eds., 1987, and periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et al, eds., 1994).

[0211] Primers, oligonucleotides and polynucleotides employed in the present invention can be generated using standard techniques known in the art.

[0212] Gene expression signatures associated with RA and certain subtypes of RA are provided herein. These signatures constitute biomarkers for RA and/or subtypes of RA, and/or predispose or contribute to development, persistence and/or progression of RA.

Accordingly, the invention disclosed herein is useful in a variety of settings, e.g., in methods and compositions related to RA diagnosis and therapy. Detection of Gene Expression Levels

[0213] Nucleic acid, according to any of the methods described herein may be RNA transcribed from genomic DNA or cDNA generated from RNA. Nucleic acid may be derived from a vertebrate, e.g., a mammal. A nucleic acid is said to be "derived from" a particular source if it is obtained directly from that source or if it is a copy of a nucleic acid found in that source.

[0214] Nucleic acid includes copies of the nucleic acid, e.g., copies that result from amplification. Amplification may be desirable in certain instances, e.g., in order to obtain a desired amount of material for detecting variations. The amplicons may then be subjected to a variation detection method, such as those described below, to determine expression of certain genes.

[0215] A microarray is a multiplex technology that typically uses an arrayed series of thousands of nucleic acid probes to hybridize with, e.g, a cDNA or cRNA sample under high- stringency conditions. Probe-target hybridization is typically detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target. In typical microarrays, the probes are attached to a solid surface by a covalent bond to a chemical matrix (via epoxy-silane, amino- silane, lysine, polyacrylamide or others). The solid surface is for example, glass, a silicon chip, or microscopic beads. Various microarrays are commercially available, including those manufactured, for example, by Affymetrix, Inc. and Illumina, Inc.

[0216] A biological sample may be obtained using certain methods known to those skilled in the art. Biological samples may be obtained from vertebrate animals, and in particular, mammals. In certain instances, a biological sample is synovial tissue, serum or peripheral blood mononuclear cells (PBMC). By screening such body samples, a simple early diagnosis can be achieved for diseases such as RA. In addition, the progress of therapy can be monitored more easily by testing such body samples for variations in expression levels of target nucleic acids (or encoded polypeptides).

[0217] Subsequent to the determination that a subject, or the tissue or cell sample comprises a gene expression signature disclosed herein, it is contemplated that an effective amount of an appropriate RA therapeutic agent may be administered to the subject to treat the RA in the subject. Clinical diagnosis in mammals of the various pathological conditions described herein can be made by the skilled practitioner. Clinical diagnostic techniques are available in the art which allow, e.g., for the diagnosis or detection of RA in a mammal. [0218] A RA therapeutic agent can be administered in accordance with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Optionally, administration may be performed through mini-pump infusion using various commercially available devices. Kits

[0219] For use in the applications described or suggested herein, kits or articles of manufacture are also provided. Such kits may comprise a carrier means being

compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise a probe that is or can be detectably labeled. Such probe may be a polynucleotide specific for a polynucleotide comprising one or more genes of a gene expression signature. Where the kit utilizes nucleic acid hybridization to detect the target nucleic acid, the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label.

[0220] Kits will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and may also indicate directions for either in vivo or in vitro use, such as those described above. Other optional components in the kit include one or more buffers {e.g., block buffer, wash buffer, substrate buffer, etc), other reagents such as substrate {e.g., chromogen) which is chemically altered by an enzymatic label, epitope retrieval solution, control samples (positive and/or negative controls), control slide(s) etc.

Methods of Marketing

[0221] The invention herein also encompasses a method for marketing a RA therapeutic agent or a pharmaceutically acceptable composition thereof comprising promoting to, instructing, and/or specifying to a target audience, the use of the agent or pharmaceutical composition thereof for treating a patient or patient population with RA from which a sample has been obtained showing the presence of a genetic variation as disclosed herein. [0222] Marketing is generally paid communication through a non-personal medium in which the sponsor is identified and the message is controlled. Marketing for purposes herein includes publicity, public relations, product placement, sponsorship, underwriting, and sales promotion. This term also includes sponsored informational public notices appearing in any of the print communications media designed to appeal to a mass audience to persuade, inform, promote, motivate, or otherwise modify behavior toward a favorable pattern of purchasing, supporting, or approving the invention herein.

[0223] The marketing of the diagnostic method herein may be accomplished by any means. Examples of marketing media used to deliver these messages include television, radio, movies, magazines, newspapers, the internet, and billboards, including commercials, which are messages appearing in the broadcast media.

[0224] The type of marketing used will depend on many factors, for example, on the nature of the target audience to be reached, e.g., hospitals, insurance companies, clinics, doctors, nurses, and patients, as well as cost considerations and the relevant jurisdictional laws and regulations governing marketing of medicaments and diagnostics. The marketing may be individualized or customized based on user characterizations defined by service interaction and/or other data such as user demographics and geographical location.

EXAMPLES

[0225] The following are examples of the methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

Example 1

Methods and Subjects

Subjects and Synovial Biopsies

[0226] All procedures involving specimens obtained from human subjects were performed under a protocol approved by the University of Michigan Institutional Review Board. Human synovial tissues were obtained by synovectomy from affected joints in patients diagnosed with RA based upon the presence of at least four of the seven criteria developed by the American College of Rheumatology for RA (Arnett, F. C, et al., Arthritis Rheum., 31 : 315-324 (1988)). Excised tissues were immediately snap-frozen in liquid nitrogen and stored at -80° C. For matched histology sections, samples were brought briefly to -20° C, cryostat sectioned and immediately brought back to -80° C. Frozen samples were homogenized in Qiagen brand RLT and R A was isolated according to the manufacturers recommended protocol (Qiagen, Valencia, CA.)

Methods

Microarray hybridization

[0227] The methods for preparation of cRNA and for array hybridization were provided by Affymetrix, Inc. (Santa Clara, CA). Briefly, 3 μg of total RNA was converted into double- stranded cDNA using a cDNA synthesis kit, Superscript Choice (Invitrogen, Carlsbad, CA) and a T7-(dT)₂4 oligomer primer (Biosearch Technologies, Inc., Novato, CA). Double- stranded cDNA was purified using affinity resin Sample Cleanup Module Kit (Affymetrix, Inc.) and then ethanol precipitated. Labeled cRNA was generated from the cDNA by using a T7 RNA polymerase and biotin-labeled nucleotide in an in vitro transcription reagents (Enzo Diagnostics, Inc., Farmingdale, NY). The labeled cRNA was purified using Affymetrix Sample Cleanup Module Kit. The amount of labeled cRNA was determined by measuring absorbance at 260 nm and using the convention that 1 OD at 260 nm corresponds to 40 μg/ml of RNA. Fifteen micrograms of labeled cRNA was fragmented by incubating at 94°C for 30 min in 40 mM Tris-acetate pH 8.1, 100 mM potassium acetate and 30 mM magnesium acetate. Samples were then hybridized to GeneChip® Human Genome U133 Plus 2.0 Arrays (Affymetrix, Inc.) at 45°C for 19 hours in a rotisserie oven set at 60 rpm. Arrays were washed and stained in the Affymetrix Fluidics station and scanned on GeneChip® scanner 3000. Data analysis was performed using the Affymetrix GeneChip® operating system and analysis software.

Statistical analyses

[0228] Statistical analyses of microarray data was performed with the open-source tools available in the statistical programming environment, R (available at the URL: cran(dot)r- project(dot)org) and the commercially available Spotfire Decision Site (TIBCO Software Inc, Palo Alto, CA). Identification of molecular subtypes was performed by multi-scale bootstrap resampling using the R package, Pvclust (Shimodaira, H and Suzuki, R., Bioinformatics, 22(12), 2006). Heatmap visualizations and identification of differentially expressed genes was performed using analysis of variance provided by Spotfire. Identification of pathways significantly over-represented within each subtype was performed using CoPub, following the developers' recommended protocol (Frijters, R. et al, Nucleic Acids Res. 36:W406-W410 (Web server issue doi: 10.1093/nar/gkn215) (2008)); available at the URL:

services(dot)nbic(dot)nl(slash)cgi-bin(slash)copub(slash)microarray_analysis(dot)pl. Briefly, Affymetrix probeset identifiers that were specifically upregulated within each subtype (-1000 top ranked probesets) were uploaded to the web-server. The GeneChip® Human Genome U133A Plus 2.0 Array (Affymetrix, Inc.) was selected as the background data set, the search category was limited to biological processes and all calculation settings were left at their defaults. The resulting data was saved to a personal computer and formatted for comparative heatmap visualization in Spotfire.

Identification of Classifiers: Molecular Subtype Training and Testing

[0229] As described in International Patent Application No. PCT/US2010/047734 (Intn'l Pub. No. WO 2011/028945), we used the filtered expression data set consisting of 20,776 probes and the molecularly- defined subtype class labels to build a series of two-class and multiclass classification models that could distinguish (i) each putative patient subtype from the other three subtypes or (ii) mutually distinguish all four subclasses from each other, respectively. We refer to such classification models herein as "classifiers." In the case where multiple samples were available from the same patient, one sample from that patient was selected at random to enter into the model. Variable (probe) selection and model training were performed using the CMA package (Slawski et al., BMC Bioinformatics 9:439 (2008)). In the case of the two-class models, variable selection was performed by ranking each probe's association with a given class label according to either the absolute value of its two-sample t- statistic or its robust Wilcoxon statistic. For the multiclass model, each probe was ranked by the values of its one-way F-statistic or its robust Kruskall-Wallis test statistic across all four putative classes. The values of the test statistics were recorded over N=48 rounds of leave- one-out cross-validation (LOOCV), or, when the class sizes were deemed large enough, i.e., for the F2, L and M two-class models, over 100 repeated rounds of 5-fold cross-validation. For each model and choice of test statistic, and at each round of cross-validation, a list of the top 20 probes with the largest, most significant values of their test statistic was retained and the union of these lists of probes make up the subtype-specific classifiers referred to as L subtype classifier (see Table 1 below), M subtype classifier (see Table 2 below), F2 subtype classifier (see Table 3 below), and Fl subtype classifier (see Table 4 below).

[0230] Performing linear discriminant analysis (LDA) in the CMA package, an estimated class label, obtained from using these specific 48 patient samples, was compared to the original estimated labels of the clustering results (see Table 5 for sensitivity and specificity). These subtype-specific classifiers performed significantly better than classifiers developed when all steps were repeated using randomly permuted class labels, which resulted in increased rates of misclassification.

[0231] Both microarray hybridization and real-time qPCR were employed on the same patient samples to evaluate the performance of the classifier genes using microarray probes and qPCR probes as indicated in Tables 1-4. Microarray hybridization is described above. Real-time qPCR was carried out as follows.

[0232] cDNA synthesis was performed using the iScript™ cDNA synthesis kit and protocol (Biorad, Hercules, CA). Two hundred ng of total RNA was added to a 20μ1 cDNA reaction mixture containing 4μ1 5x iScript™ reaction mixture, Ιμΐ iScript™ reverse transcriptase and nuclease-free water. The reverse transcription reaction mixture was incubated at 25°C for 5 minutes, 42°C for 30 minutes and 85°C for 5 minutes.

[0233] A gene specific pre-amplification of cDNA samples was performed using the TaqMan^® PreAmp Master Mix (Applied Biosystems, Foster City, CA). One μΐ of a total of 77 20X TaqMan^® Gene Expression Assays (all assays contained FAM™ dye-labeled MGB probes, Applied Biosystems, Foster City, CA) were pooled and diluted with IX TE buffer for a final concentration of 0.2X per assay. Per sample, 1.25μ1 of cDNA, 1.25μ1 of the pooled assay mix and 2.5μ1 of 2X TaqMan^® PreAmp Master Mix (Applied Biosystems) were mixed. The pre-amplification reactions were done in a GeneAmp^® PCR System 9700 (Applied Biosystems, Foster City, CA) using the protocol, 95°C for 10 minutes, and 14 cycles of 95°C for 15 seconds and 60°C for 4 minutes. After thermal cycling, the pre-amp lifted samples were diluted five times with IX TE buffer.

[0234] Semi-quantitative real-time RT-PCR validation of microarray data for classifier genes and three housekeeping genes (HPRTl, GAPDH and B-Actin) was performed using the BioMark™ 48.48 Dynamic Arrays (Fluidigm Corporation, South San Francisco, CA). A sample mix, containing 2.5μ1 of pre-amplified cDNA, 2.5μ1 of TaqMan^® Universal PCR Master Mix (Applied Biosystems, Foster City, CA) and 0.25μ1 of DA Sample Loading Reagent (Fluidigm Corporation, South San Francisco, CA) and an assay mix containing 2.5μ1 20x TaqMan^® Gene Expression Assay (Applied Biosystems, Foster City, CA) and 2.5 μΐ DA Assay Loading reagent (Fluidigm Corporation, South San Francisco, CA) were prepared. Following priming of the 48.48 Dynamic Array with control line fluid in an IFC controller (Fluidigm Corporation, South San Francisco, CA), 5μ1 sample mix was loaded into each sample inlet and 5μ1 assay mix into the detector inlet of the chip. All samples were loaded in duplicate. The chip was subsequently placed in the IFC Controller for loading and mixing of the samples and assays and then transferred to the BioMark™ Real-Time PCR System. The cycling program consisted of 10 minutes at 95°C followed by 40 cycles of 95°C for 15 seconds and 1 minute at 60°C.

[0235] Data was analyzed using the Fluidigm Gene Expression Data Analysis software (version 2.1.1, Fluidigm Corporation, South San Francisco, CA) to obtain CT values. The relative abundance was calculated using the delta-delta-cycle threshold (ddCT) according to the formula: 2^A(average CT gene A-average CT HPRTl). HPRTl was the most stable house keeping gene.

[0236] The same statistical learning procedures that were applied to the microarray hybridization data (see above and International Patent Application No. PCT/US2010/047734 [Intn'l Pub. No. WO 2011/028945]) were applied to the qPCR-ddCT values using a linear discriminate analysis model and leave-one-out cross validation (LOOCV).

[0237] Table 5 below is a performance evaluation table comparing subtype-specific classifier performance for microarray hybridization assay and qPCR assay. For each of microarray hybridization and qPCR, sensitivity and specificity are indicated for each of the RA subtype classifiers. In Table 5, Sensitivity = TP/(TP+FN) and Specificity = TN/(FP+TN), where TP = true positive, FN = false negative, TN = true negative, and FP = false positive. Table 5. Classifier performance in microarray hybridization and qPCR.

[0238] As indicated by the results shown in Table 5, the subtype-specific classifiers for each of Fl, F2, L, and M subtypes show both good sensitivity, with a range of 60%-82% depending on assay type and classifier, and good specificity, with a range of 86-100% depending on assay type and classifier, for both microarray hybridization and qPCR. These results thus show that biological samples from RA patients may be accurately classified using different types of assays, e.g., microarray hybridization or qPCR, based on the classifier genes described herein. Example 2

[0239] To investigate the ability of each of the four classifiers described above to predict therapeutic response to TNFa blockade, we assessed an independent patient cohort for the presence of the four RA subtypes. The independent patient cohort was described in Lindberg et al., PLoS One 5(6):el 1310 (2010), and the data is publicly available at Gene Expression Omnibus (GEO) (Barrett et al, Nucl. Acids Res. 35:D760-65 (2007) under accession number GSE21537. GEO is accessible on the world-wide web at

ncbi(dot)nlm(dot)nih(dot)gov(slash)geo. Of the 62 patients in this cohort, forty-five were assigned to at least one of the four molecular subtypes, 14 were unassigned and three patients possessed all four subtypes (data not shown). For the group of forty- five patients (allcomers), nine responded poorly to anti-TNFa therapy (20%), 22 responded moderately well (49%), and 14 were identified as good responders (31%). Of 14 patients identified as good responders to anti-TNFa therapy, 12 possessed the Fl and/or M subtypes (two patients possessed both). Importantly, the Fl subtype was absent from all but one of the poor responders and the M subtype was absent from all but two of the poor responders. See Figs. IB and IE. The converse was true for the F2 subtype, where 7/9 poor responders were classified as F2, but only 4/14 good responders were of the F2 subtype. See Fig. 1C. The L subtype was uniformly distributed among the response groups (Fig. ID). These results show that the Fl and M subtypes preferentially classified patients as good responders to anti-TNFa therapy, whereas the F2 subtype preferentially classified poor responders.

[0240] We combined these findings into a patient stratification scheme that posed Fl and M subtypes against the F2 subtype. As discussed above, patients possessing an Fl and/or M subtype, regardless of F2 or L status, classified 12/14 patients as good responders to anti- TNFa therapy. Patients possessing an F2 subtype but not Fl and M subtypes identified 7/9 patients as poor responders to anti-TNFa therapy. These results represent a nearly 3 -fold improvement in identifying patients that respond well to TNFa blockade (86% Fl and/or M versus 31% all-comers). Likewise, this strategy also provided a nearly 4-fold improvement in identifying poor responders (78% F2 versus 20% all-comers). This data suggests that the RA molecular subtypes we have described can be used for identifying patients likely to respond to anti-TNFa therapy as well as those unlikely to respond. Such an ability to identify good responders and poor responders to such therapeutics may well have a significant impact on clinical practice and provide substantial benefit to patients. Example 3

[0241] By analyzing the genes comprising each of the four RA subtype classifiers described above, we can identify genes encoding proteins that are secreted or are processed into soluble forms. Such proteins can potentially be detected systemically in, for example, serum or plasma. Such systemic biomarkers may have utility alone or in combination with other biomarkers, for example, as part of a diagnostic test to select patients for optimal response to therapy.

[0242] The process for selection of candidate genes starts with an analysis to identify genes that are expressed specifically in the pre-defined subtypes using an ANOVA statistical analysis or similar statistical method employing a threshold cutoff for p value and fold change. Gene candidates then undergo analysis for localization of expression, coding for soluble proteins, or coding for membrane proteins that may have soluble variants formed through alternate splicing or post-translational proteolytic cleavage.

[0243] After protein biomarker candidates have been identified, immunoassays (e.g., enzyme-linked immunosorbent assay [ELISA], electro-chemiluminescence assay [ECLA], or Luminex-based platforms) are constructed using capture and detection antibodies together with the respective protein standards. Such assays can be utilized to determine the levels of biomarkers in biological samples such as synovial fluid, plasma, and serum. These biomarkers can be compared against other biomarkers as well as against clinical information such as disease diagnosis, disease activity, and drug response outcome.

[0244] Using the process described above and analyzing the genes of each of the subtype classifiers, we identified as candidate systemic biomarkers clusterin (F2 subtype) and periostin (Fl and F2 subtypes) and assayed serum levels of these proteins in a group of RA patients and in healthy controls. Serum clusterin levels were measured using a commercially available immunoassay (QuantikineR Human Clusterin, R&D systems, cat# DCLU00) and serum periostin levels were measured using an in-house immunoassay. The results are shown in Figs. 2A (clusterin) and 2B (periostin). For each of clusterin and periostin, serum levels were statistically higher in the group of RA patients compared to the group of healthy controls (p<0.0001 as determined by t test). Thus, these data demonstrate that clusterin and periostin serum levels can be used as serum biomarkers of RA and may, for example, be included in biomarker assays designed to identify F2 and Fl subtypes of disease. Table 1. L subtype (phenotype) classifier genes and probes

Table 2. M subtype (phenotype) classifier genes and probes

Table 3. F2 subtype (phenotype) classifier genes and probes

Table 4. Fl subtype (phenotype) classifier genes and probes

Claims

WHAT IS CLAIMED IS:

1. A method of predicting the response of a subject to a therapy comprising a TNFa inhibitor, the method comprising measuring in a biological sample obtained from the subject expression of one or a combination of genes, or expression of one or a combination of proteins encoded by the one or the combination of genes, wherein the one or the combination of genes is selected from Table 2, wherein expression of the one or the combination of genes, or expression of the one or the combination of proteins, comprises a gene signature or a protein signature, respectively, and wherein the gene signature or the protein signature, respectively, is predictive of response of the subject to the therapy comprising the TNFa inhibitor.

2. The method of claim 1, wherein the combination of genes comprises at least five, or at least 10, or at least 20, or at least 30, or at least 40 genes selected from Table 2.

3. The method of claim 2, wherein the combination of genes comprises ACTN1, ARL7, ATP6V0D1, ATP6V1A, C5R1, C9orf88, CAPZB, CCL2, CCR1, CTSB, CTSL, CTSZ, CXCL3, EIF4E2, EMILIN2, FAM50B, FLJ11259, FLJ20847, FLNA, FZD4, GSTOl, HCK, ICAM1, KIAA0485, KIAA0582, LACTB, LILRB2, LILRB3, MBD2, MFHAS1, NAGA, NPC1, NRP2, P2RX4, PGD, PLAU, PLAUR, RABGAP1, RAPGEF1, RHOG, SERPINB1, SLC16A3, TCF7L2, TFRC, TM7SF1, TPM4, UBE3A, VEGF, VPS 13 A, VPS37C, and ZYX.

4. The method of claim 3, wherein the combination of genes further comprises one or more genes selected from CLK1, HSMPP8, MGC2752, and MICAL3.

5. A method of predicting the response of a subject to a therapy comprising a TNFa inhibitor, the method comprising measuring in a biological sample obtained from the subject expression of one or a combination of genes, or expression of one or a combination of proteins encoded by the one or the combination of genes, wherein the one or the combination of genes is selected from Table 4, wherein expression of the one or the combination of genes, or expression of the one or the combination of proteins, comprises a gene signature or a protein signature, respectively, and wherein the gene signature or the protein signature, respectively, is predictive of response of the subject to the therapy comprising the TNFa inhibitor.

6. The method of claim 5, wherein the combination of genes comprises at least five, or at least 10, or at least 20, or at least 30, or at least 40 genes selected from Table 4.

7. The method of claim 6, wherein the combination of genes comprises ABCA1, ADRBKl, AP1S2, C10orf38, C16orf9, CASK, CD68, CDHl l, CDH5, COL18A1, COL4A1, COROIA, CREB3L1, CTSS, CYBB, FBP1, FCGR2C, FCGR3A, FCGR3B, FGL2,

FLJ11127, FLJ20364, FLJ22662, FLJ44635, FPRL2, GPR116, GUCY1A3, HAVCR2, HEPH, HEYL, ITGB2, KCTD15, KIAA1374, KYNU, LILRA2, LPIN1, LST1, MAP IB, MAP4K4, MARCO, MFAP2, MGC17943, MGC48972, MSR1, NXN, PNKP, POSTN, PTPNSl, QARS, RNASET2, SEPTl l, SGKL, STAT5A, TPMl, TRIM14, VSIG4, YIF1, and ZNF462.

8. The method of claim 7, wherein the combination of genes further comprises one or more genes selected from LOC90139 and SLC38A2.

9. A method of predicting the response of a subject to a therapy comprising a TNFa inhibitor, the method comprising measuring in a biological sample obtained from the subject expression of one or a combination of genes, or expression of one or a combination of proteins encoded by the one or the combination of genes, wherein the one or the combination of genes is selected from Table 3, wherein expression of the one or the combination of genes, or expression of the one or the combination of proteins, comprises a gene signature or a protein signature, respectively, and wherein the gene signature or the protein signature, respectively, is predictive of non-response of the subject to the therapy comprising the TNFa inhibitor.

10. The method of claim 9, wherein the combination of genes comprises at least five, or at least 10, or at least 20 genes selected from Table 3.

11. The method of claim 10, wherein the combination of genes comprises ABTB2, ARGBP2, AUTS2, BBS1, CBX7, CLU, FANCA, FLJ10970, FLJ32803, FZD8,

GABARAPL1, GPR64, GULP1, HMGB3, LOC201895, LTBP3, MSL3L1, NDFIP1, NOVA1, NTN4, NTRK2, PCOLCE2, PLEKHA1, POSTN, PTTG1, RNASE4, SCARA3, SLC29Al, and SLC35A1.

12. The method of claim 11, wherein the combination of genes further comprises one or more genes selected from CHD9, IDH2, IP09, KBTBD9, and LOC283481.

13. The method of any one of claim 1, 5, or 9, wherein the biological sample is synovial tissue.

14. The method of any one of claim 1 , 5, or 9, wherein the expression of the one or the combination of genes is measured using a PCR method or a microarray chip.

15. The method of any one of claim 1, 5, or 9, wherein the biological sample is selected from serum, plasma, and synovial fluid.

16. The method of claim 15, wherein the measuring comprises using an immunoassay.

17. A method of treating rheumatoid arthritis in a patient comprising administering a therapeutically effective amount of a TNFa inhibitor to the patient to treat the rheumatoid arthritis, provided that a biological sample obtained from the patient has been shown to possess a M subtype gene signature or a M subtype protein signature, wherein the M subtype gene signature comprises expression of one or a combination of genes, and the M subtype protein signature comprises expression of one or a combination of proteins encoded by the one or the combination of genes, respectively, wherein the one or the combination of genes is selected from Table 2.

18. The method of claim 17, wherein the combination of genes comprises at least five, or at least 10, or at least 20, or at least 30, or at least 40 genes selected from Table 2.

19. The method of claim 18, wherein the combination of genes comprises ACTN1, ARL7, ATP6V0D1, ATP6V1A, C5R1, C9orf88, CAPZB, CCL2, CCR1, CTSB, CTSL, CTSZ, CXCL3, EIF4E2, EMILIN2, FAM50B, FLJ11259, FLJ20847, FLNA, FZD4, GSTOl, HCK, ICAM1, KIAA0485, KIAA0582, LACTB, LILRB2, LILRB3, MBD2, MFHAS1, NAGA, NPC1, NRP2, P2RX4, PGD, PLAU, PLAUR, RABGAP1, RAPGEF1, RHOG, SERPINB1, SLC16A3, TCF7L2, TFRC, TM7SF1, TPM4, UBE3A, VEGF, VPS 13 A, VPS37C, and ZYX.

20. The method of claim 19, wherein the combination of genes further comprises one or more genes selected from CLK1, HSMPP8, MGC2752, and MICAL3.

21. A method of treating rheumatoid arthritis in a patient comprising administering a therapeutically effective amount of a TNFa inhibitor to the patient to treat the rheumatoid arthritis, provided that a biological sample obtained from the patient has been shown to possess a Fl subtype gene signature or a Fl subtype protein signature, wherein the Fl subtype gene signature comprises expression of one or a combination of genes, and the Fl subtype protein signature comprises expression of one or a combination of proteins encoded by the one or the combination of genes, respectively, wherein the one or the combination of genes is selected from Table 4.

22. The method of claim 21, wherein the combination of genes comprises at least five, or at least 10, or at least 20, or at least 30, or at least 40 genes selected from Table 4.

23. The method of claim 22, wherein the combination of genes comprises ABCA1, ADRBKl, AP1S2, C10orf38, C16orf9, CASK, CD68, CDHl l, CDH5, COL18A1, COL4A1, COROIA, CREB3L1, CTSS, CYBB, FBP1, FCGR2C, FCGR3A, FCGR3B, FGL2,

24. The method of claim 23, wherein the combination of genes further comprises one or more genes selected from LOC90139 and SLC38A2.

25. A method of treating rheumatoid arthritis in a patient comprising administering a therapeutically effective amount of an RA therapeutic agent to the patient to treat the rheumatoid arthritis, wherein the RA therapeutic agent is other than a TNFa inhibitor, provided that a biological sample obtained from the patient has been shown to possess a F2 subtype gene signature or a F2 subtype protein signature, wherein the F2 subtype gene signature comprises expression of one or a combination of genes, and the F2 subtype protein signature comprises expression of one or a combination of proteins encoded by the one or the combination of genes, respectively, wherein the one or the combination of genes is selected from Table 3.

26. The method of claim 25, wherein the combination of genes comprises at least five, or at least 10, or at least 20 genes selected from Table 3.

27. The method of claim 26, wherein the combination of genes comprises ABTB2, ARGBP2, AUTS2, BBS1, CBX7, CLU, FANCA, FLJ10970, FLJ32803, FZD8,

28. The method of claim 27, wherein the combination of genes further comprises one or more genes selected from CHD9, IDH2, IP09, KBTBD9, and LOC283481.

29. The method of claim 9 or claim 25, wherein the protein signature comprises clusterin.

30. The method of any one of claims 5, 9, 21, and 25, wherein the protein signature comprises periostin.

31. The method of claim 17 or claim 21 , wherein the TNFa inhibitor is selected from etanercept, infliximab, adalimumab, golimumab, and certolizumab pegol.

A method of selecting a therapeutic agent for treatment of an RA patient comprising: a. obtaining a biological sample from the patient;

b. measuring expression of one or a combination of genes, or one or a

combination of proteins encoded by the one or the combination of genes, wherein expression of the one or the combination of genes, or expression of the one or the combination of proteins, comprises a gene signature or a protein signature, respectively, wherein the one or the combination of genes is selected from Table 2, wherein the gene signature or the protein signature is indicative of M subtype;

c. determining whether the sample is positive or negative for M subtype;

d. measuring expression of one or a combination of genes, or one or a

combination of proteins encoded by the one or the combination of genes, wherein expression of the one or the combination of genes, or expression of the one or the combination of proteins, comprises a gene signature or a protein signature, respectively, wherein the one or the combination of genes is selected from Table 4, wherein the gene signature or the protein signature is indicative of Fl subtype;

e. determining whether the sample is positive or negative for Fl subtype;

f. measuring expression of one or a combination of genes, or one or a

combination of proteins encoded by the one or the combination of genes, wherein expression of the one or the combination of genes, or expression of the one or the combination of proteins, comprises a gene signature or a protein signature, respectively, wherein the one or the combination of genes is selected from Table 3, wherein the gene signature or the protein signature is indicative of F2 subtype;

g. determining whether the sample is positive or negative for F2 subtype;

h. selecting a TNFa inhibitor as the therapeutic agent provided the sample is positive for at least one of the subtypes selected from M and Fl; and i. selecting a therapeutic agent other than a TNFa inhibitor provided the sample is negative for at least one of the subtypes selected from M and Fl and positive for F2 subtype.

33. The method of claim 32, wherein the TNFa inhibitor is selected as the therapeutic agent, and the TNFa inhibitor is selected from etanercept, infliximab, adalimumab, golimumab, and certolizumab pegol.