NZ614170B2 - Assays for detecting autoantibodies to anti-tnf? drugs - Google Patents

Abstract

Disclosed is a method for detecting the presence or level of an autoantibody to an anti-TNF? drug in a sample without interference from the anti-TNF? drug in the sample, the method comprising: a) contacting the sample with an acid to dissociate preformed complexes of the autoantibody and the anti-TNF? drug, wherein the sample has or is suspected of having an autoantibody to the anti-TNF? drug; b) contacting the sample with a labelled anti-TNF? drug following dissociation of the preformed complexes; c) neutralising the acid in the sample to form labelled complexes of the labelled anti-TNF? drug and the autoantibody; d) subjecting the labelled complexes to size exclusion chromatography to separate the labelled complexes; and e) detecting the labelled complexes, thereby detecting the presence or level of the autoantibody without interference from the anti-TNF? drug in the sample. -TNF? drug, wherein the sample has or is suspected of having an autoantibody to the anti-TNF? drug; b) contacting the sample with a labelled anti-TNF? drug following dissociation of the preformed complexes; c) neutralising the acid in the sample to form labelled complexes of the labelled anti-TNF? drug and the autoantibody; d) subjecting the labelled complexes to size exclusion chromatography to separate the labelled complexes; and e) detecting the labelled complexes, thereby detecting the presence or level of the autoantibody without interference from the anti-TNF? drug in the sample.

Description

ASSAYS FOR DETECTING AUTOANTIBODIES TO ANTI-TNFα DRUGS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 61/444,097, filed February 17, 2011, U.S. Provisional Application No. 61/484,594, filed May 10, 2011, and U.S. Provisional Application No. ,501, filed June 13, 2011, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION [0001a] Any discussion of the prior art hout the ication should in no way be considered as an admission that such prior art is widely known or forms part of common l knowledge in the field.

Autoimmune disorders are a significant and widespread medical problem. For example, rheumatoid arthritis (RA) is an autoimmune disease affecting more than two million people in the United States. RA causes chronic inflammation of the joints and typically is a ssive illness that has the potential to cause joint destruction and functional disability. The cause of rheumatoid arthritis is unknown, although c predisposition, infectious agents and environmental factors have all been implicated in the etiology of the disease. In active RA, symptoms can include fatigue, lack of appetite, low grade fever, muscle and joint aches and stiffness. Also during disease flare ups, joints frequently become red, swollen, painful and tender, due to inflammation of the synovium. Furthermore, since RA is a systemic disease, inflammation can affect organs and areas of the body other than the joints, including glands of the eyes and mouth, the lung lining, the rdium, and blood s.

Traditional treatments for the ment of RA and other autoimmune disorders include fast acting "first line drugs" and slower acting d line drugs." The first line drugs reduce pain and inflammation. Example of such first line drugs include n, naproxen, ibuprofen, etodolac and other eroidal anti-inflammatory drugs (NSAIDs), as well as corticosteroids, given orally or injected directly into tissues and joints. The second line drugs promote disease remission and prevent progressive joint destruction and are also referred to as disease-modifying anti-rheumatic drugs or DMARDs. Examples of second line drugs include gold, hydrochloroquine, azulfidine and immunosuppressive agents, such as rexate, azathioprine, cyclophosphamide, chlorambucil and cyclosporine. Many of these drugs, however, can have detrimental side-effects. Thus, additional therapies for rheumatoid arthritis and other autoimmune disorders have been sought.

Tumor necrosis factor alpha (TNF-u) is a cytokine produced by numerous cell types, including monocytes and macrophages, that was originally identified based on its ability to induce the is of n mouse tumors. Subsequently, a factor termed cachectin, associated with cachexia, was shown to be cal to TNF-u. TNF-u has been ated in the pathophysiology of a variety of other human diseases and disorders, including shock, sepsis, infections, mune diseases, RA, Crohn’s disease, transplant rejection and graft-versus-host disease.

Because of the harmful role of human TNF-u (hTNF-u) in a variety of human disorders, therapeutic strategies have been designed to t or counteract hTNF-u ty.

In particular, dies that bind to, and neutralize, hTNF-(x have been sought as a means to inhibit hTNF-u activity. Some of the earliest of such antibodies were mouse monoclonal antibodies (mAbs), secreted by hybridomas prepared from lymphocytes of mice immunized with hTNF-(x (see, e. g., US. Pat. No. 024 to Moeller et al.). While these mouse anti- hTNF-(x antibodies often displayed high affinity for hTNF-(x and were able to neutralize hTNF-u activity, their use in vivo has been limited by problems associated with the administration of mouse antibodies to humans, such as a short serum half-life, an ity to trigger certain human effector fianctions, and elicitation of an unwanted immune response against the mouse antibody in a human (the “human anti-mouse antibody” (HAMA) reaction).

[0006] More recently, biological therapies have been d to the treatment of autoimmune disorders such as toid arthritis. For example, four TNFOL inhibitors, REMICADETM (infliximab), a chimeric anti-TNFOL mAb, T'V' (etanercept), a TNFR- Ig Fc fusion protein, HUMIRAT'V' (adalimumab), a human anti-TNFOL mAb, and CIMZIA® (certolizumab pegol), a PEGylated Fab fragment, have been approved by the FDA for treatment of rheumatoid arthritis. CIMZIA® is also used for the treatment of moderate to severe Crohn’s disease (CD). While such biologic therapies have demonstrated success in the treatment of rheumatoid arthritis and other autoimmune disorders such as CD, not all subjects treated respond, or respond well, to such therapy. Moreover, administration of TNFu inhibitors can induce an immune response to the drug and lead to the production of autoantibodies such as human himeric antibodies (HACA), human umanized antibodies (HAHA), and human anti-mouse antibodies . Such HACA, HAHA, or HAMA immune responses can be associated with ensitive reactions and dramatic changes in pharmacokinetics and biodistribution of the immunotherapeutic TNFOL inhibitor that preclude further treatment with the drug. Thus, there is a need in the art for assays to detect the presence of autoantibodies to anti-TNFα biologics in a patient sample to monitor TNFα inhibitor therapy and to guide treatment decisions. It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to e a useful alternative.

BRIEF SUMMARY OF THE ION The present invention provides assays for detecting and measuring the presence or level of autoantibodies to anti-TNFα drug therapeutics in a sample. The present invention is useful for optimizing therapy and monitoring patients receiving anti- TNFα drug therapeutics to detect the presence or level of autoantibodies (e.g., HACA and/or HAHA) against the drug. The present ion also provides methods for selecting therapy, optimizing therapy, and/or reducing toxicity in subjects receiving anti- TNFα drugs for the treatment of TNFα-mediated disease or disorders.

] According to a first aspect, the t invention provides a method for detecting the presence or level of an autoantibody to an anti-TNFα drug in a sample without interference from the anti-TNFα drug in the sample, the method comprising: a) contacting the sample with an acid to dissociate med complexes of the autoantibody and the anti-TNFα drug, wherein the sample has or is suspected of having an autoantibody to the anti-TNFα drug; b) contacting the sample with a labeled anti-TNFα drug following dissociation of the med complexes; c) neutralizing the acid in the sample to form labeled complexes of the labeled anti-TNFα drug and the autoantibody; d) subjecting the labeled complexes to size exclusion chromatography to separate the d xes; and e) detecting the labeled complexes, thereby detecting the presence or level of the autoantibody without erence from the anti-TNFα drug in the [0007b] According to a second aspect, the present invention provides a method of making a diagnosis in a t receiving a course of therapy with an anti-TNFα drug for zing therapy and/or reducing toxicity to the anti-TNFα drug, the method comprising: a) determining the presence or level of an autoantibody to the NFα drug in a sample obtained from the subject without interference from the anti-TNFα drug in the sample, the method comprising: (i) contacting the sample with an acid to iate preformed complexes of the autoantibody and the anti-TNFα drug, wherein the sample has or is suspected of having an autoantibody to the anti- TNFα drug; (ii) contacting the sample with a labeled anti-TNFα drug following dissociation of the preformed complexes; (iii) neutralizing the acid in the sample to form labeled complexes of the labeled anti-TNFα drug and the autoantibody; (iv) subjecting the labeled complexes to size exclusion chromatography to separate the labeled complexes; and (v) detecting the labeled complexes to y detect the presence or level of the autoantibody without erence from the anti-TNFα drug in the sample; and b) making a diagnosis that a subsequent dose of the course of therapy be altered or making a diagnosis that a different course of therapy be administered to the subject for which a determination of step (a) is positive. [0007c] Unless the context clearly requires otherwise, throughout the ption and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0008] In another aspect, the present invention es a method for ing the presence or level of an autoantibody to an anti-TNFα drug in a sample without interference from the anti-TNFα drug in the sample, the method comprising: (a) contacting the sample with an acid to dissociate preformed complexes of the tibody and the anti-TNFα drug, wherein the sample has or is suspected of having an autoantibody to the NFα drug; (b) contacting the sample with a labeled NFα drug following dissociation of the preformed complexes; (c) neutralizing the acid in the sample to form labeled complexes (i.e., immunocomplexes or conjugates) of the labeled anti-TNFα drug and the autoantibody (i.e., wherein the labeled anti-TNFα drug and autoantibody are not covalently attached to each other); (d) subjecting the labeled complexes to size exclusion chromatography to separate the labeled complexes (e.g., from free d NFα drug); (e) detecting the labeled complexes, thereby detecting the presence or level of the autoantibody without interference from the anti-TNFα drug in the sample.

In some embodiments, the anti-TNFα drug is selected from the group consisting of REMICADE™ (infliximab), ™ (etanercept), HUMIRA™ mumab), CIMZIA® (certolizumab pegol), SIMPONI® umab; CNTO 148), and combinations thereof.

[0010] In other embodiments, the anti-TNFα drug autoantibody includes, but is not limited to, human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti-mouse antibodies (HAMA), as well as ations thereof.

WO 54253 In certain alternative embodiments, steps (a) and (b) are performed simultaneously, e.g., the sample is contacted with an acid and a labeled anti-TNFOL drug at the same time. In certain other alternative embodiments, step (b) is performed prior to step (a), e.g., the sample is first contacted with a labeled anti-TNFu drug, and then contacted with an acid. In further embodiments, steps (b) and (c) are performed simultaneously, e.g., the sample is contacted with a labeled anti-TNFOL drug and lized (e.g., by contacting the sample with one or more neutralizing agents) at the same time.

In particular embodiments, the sample is contacted with an amount of an acid that is sufficient to dissociate preformed complexes of the autoantibody and the anti-TNFu drug, such that the labeled anti-TNFOL drug, the unlabeled anti-TNFOL drug, and the autoantibody to the anti-TNFu drug can brate and form complexes therebetween.

In another aspect, the present invention es a method for optimizing therapy and/or reducing toxicity to an anti-TNFu drug in a subject receiVing a course of therapy with the anti-TNFu drug, the method comprising: (a) detecting the ce or level of an autoantibody to the NFOL drug in a sample from the subject without interference from the anti-TNFOL drug in the sample, the method sing: (i) contacting the sample with an acid to dissociate preformed complexes of the autoantibody and the anti-TNFOL drug, n the sample has or is suspected of having an autoantibody to the anti-TNFu drug; (ii) contacting the sample with a labeled anti-TNFu drug following dissociation of the preformed complexes; (iii) neutralizing the acid in the sample to form labeled complexes (i.e., immuno-complexes or ates) of the labeled anti-TNFOL drug and the autoantibody (z'.e., n the d anti-TNFOL drug and autoantibody are not covalently attached to each other); (iv) subjecting the labeled complexes to size exclusion chromatography to separate the labeled complexes (e.g., from free labeled anti-TNFu drug); and (v) detecting the labeled complexes (e.g., thereby detecting the presence or level of the tibody without interference from the anti-TNFu drug in the sample); and (b) determining a subsequent dose of the course of therapy for the subject or whether a different course of therapy should be administered to the t based upon the presence or level of the autoantibody, thereby optimizing therapy and/or reducing toxicity to the NFOL drug. s for ing anti-TNFOL drugs and anti-drug antibodies are further described in PCT Publication No. , the sure of which is hereby incorporated by reference in its entirety for all purposes.

In other aspects, the present invention provides a method for selecting a course of therapy (e.g., selecting an appropriate anti-TNFOL drug) for the treatment of a TNFd-mediated disease or disorder in a subject, the method comprising: (a) analyzing a sample obtained from the t to determine the presence, level, or genotype of one or more markers in the sample; (b) applying a statistical algorithm to the presence, level, or genotype of the one or more markers determined in step (a) to generate a disease activity/severity index; and (c) selecting an appropriate course of therapy (e.g., anti-TNFOL therapy) for the subject based upon the disease ty/severity index.

In a related aspect, the present invention provides a method for optimizing y and/or reducing toxicity in a subject receiving a course of therapy for the treatment of a TNFu-mediated disease or disorder, the method comprising: (a) analyzing a sample obtained from the subject to determine the presence, level, or genotype of one or more markers in the sample; (b) applying a statistical algorithm to the presence, level, or genotype of the one or more markers determined in step (a) to generate a disease activity/severity index; and (c) determining a subsequent dose of the course of therapy for the t or whether a different course of y should be administered to the subject based upon the disease activity/severity index.

In particular embodiments, the methods of the t invention se detecting, ing, or determining the presence, level (concentration (e.g., total) and/or activation (e.g., phosphorylation)), or genotype of one or more specific markers in one or more of the following categories of biomarkers: (l) Inflammatory markers (2) Growth factors (3) Serology (e.g., immune markers) (4) Cytokines and chemokines (5) Markers of oxidative stress (6) Cell surface receptors (e.g., CD64, others) (7) Signaling pathways (8) Other s (e.g., genetic markers such as inflammatory pathway genes).

In fithher embodiments, the presence and/or level of one or both of the ing markers can also be detected, measured, or determined in a patient sample (e.g., a serum sample from a patient on anti-TNF drug therapy): (9) anti-TNF drug levels (e.g., levels of free anti-TNFOL therapeutic antibody); and/or (10) anti-drug dy (ADA) levels (e.g., levels of autoantibody to the anti-TNF drug).

In particular ments, a single statistical algorithm or a combination of two or more statistical algorithms can then be applied to the presence, concentration level, activation level, or genotype of the markers detected, measured, or determined in the sample to thereby generate the disease activity/severity index.

[0020] In certain ces, the sample is obtained by isolating PBMCs and/or PMN cells using any technique known in the art. In other embodiments, the sample is a tissue biopsy, e.g., from a site of inflammation such as a portion of the gastrointestinal tract or synovial tissue.

Accordingly, in some aspects, the methods of the invention provide information useful for guiding treatment decisions for patients receiving or about to receive anti-TNFOL drug therapy, e.g., by selecting an appropriate anti-TNFOL y for initial treatment, by determining when or how to adjust or modify (e.g., increase or decrease) the uent dose of an NFOL drug, by determining when or how to combine an anti-TNFOL drug (e.g., at an initial, increased, decreased, or same dose) with one or more immunosuppressive agents such as methotrexate (MTX) and/or azathioprine (AZA), and/or by determining when or how to change the current course of therapy (e.g., switch to a ent anti-TNFOL drug or to a drug that targets a different mechanism such as an IL-6 or-inhibiting monoclonal antibody).

Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows an exemplary embodiment of the assays of the present invention n size exclusion HPLC is used to detect the binding between TNFu-Alexa647 and T'V'.

Figure 2 shows dose response curves of HUMIRAT'V' binding to TNFu-Alexa647.

Figure 3 shows a current ELISA-based method for measuring HACA levels, known as the bridging assay.

Figure 4 illustrates an exemplary outline of the autoantibody detection assays of the present ion for measuring the concentrations of HACA/HAHA generated against REMICADET'V'.

Figure 5 shows a dose response is of anti-human IgG dy binding to REMICADETM-Alexa647.

Figure 6 shows a second dose response analysis of anti-human IgG antibody binding to REMICADETM-Alexa647.

[0029] Figure 7 shows dose response curves of anti-human IgG antibody binding to REMICADETM-Alexa647.

Figure 8 shows REMICADETM-Alexa647 immunocomplex formation in normal human serum and HACA positive serum.

Figure 9 provides a summary ofHACA measurements from 20 patient serum samples that were performed using the bridging assay or the mobility shift assay of the present invention.

Figure 10 provides a summary and ison of current methods for measuring serum trations of HACA to the novel HACA assay of the present invention.

Figure 11 shows SE-HPLC profiles of fluorophore (Fl)-labeled IFX incubated with normal (NHS) or ositive (HPS) serum. The addition of increasing amounts of HACA-positive serum to the incubation mixture dose-dependently shifted the IFX-Fl peak to the higher molecular mass eluting positions, Cl and C2.

Figure 12 shows dose-response curves of the bound and free IFX-Fl generated with increasing dilutions of HACA-positive serum as determined by the mobility shift assay. (A) Increasing dilutions of ositive serum were incubated with 37.5 ng of IFX-Fl. The higher the dilution (less HACA) the more free IFX-Fl was found in the SE-HPLC analysis.

(B) Increasing dilutions of HACA-positive serum were ted with 37.5 ng of IFX-Fl.

The higher the dilution (less HACA) the less HACA bound IFX-Fl was found in the SE- HPLC analysis.

Figure 13 shows SE-HPLC s of TNFu-Fl incubated with normal (NHS) or IFX-spiked serum. The addition of increasing amounts of IFX-spiked serum to the incubation mixture dose-dependently d the fluorescent TNFu peak to the higher molecular mass eluting positions.

Figure 14 shows dose-response curves of the bound and free TNFOL generated with increasing dilutions of IFX-spiked serum as determined by the mobility shift assay.

Increasing concentrations of IFX added to the tion mixture decreases the percentage of free TNFu while increasing the percentage of bound TNFu.

Figure 15 shows the measurement of relative HACA level and IFX tration in IBD patients treated with IFX at ent time points by the mobility shift assay.

[0038] Figure 16 shows patient management- measurement ofHACA level and IFX concentration in the sera of IBD patients treated with IFX at different time points.

Figure 17 shows ary embodiments of the assays of the present invention to detect the presence of (A) utralizing or (B) neutralizing autoantibodies such as HACA.

Figure 18 shows an alternative embodiment of the assays of the present invention to detect the presence of neutralizing autoantibodies such as HACA.

Figure 19 shows mobility shift profiles of Fl-labeled ADL incubated with normal human serum (NHS) in the presence of different amounts of anti-human IgG. The addition of increasing amounts of anti-human IgG to the incubation e dose-dependently shifted the free Fl-ADL peak (FA) to the higher molecular mass eluting positions, Cl and C2, while the internal control (IC) did not change.

Figure 20 shows a dose-response curve of uman IgG on the shift of fiee Fl- ADL. Increasing s of anti-human IgG were incubated with 37.5 ng of Fl-ADL and internal control. The more the antibody was added to the reaction mixture the lower the ratio of free Fl-ADL to internal control.

[0043] Figure 21 shows mobility shift profiles of Fl-labeled TNF-u incubated with normal human serum (NHS) in the presence of different amounts ofADL. Ex = 494 nm; Em = 519 2012/025437 nm. The on of increasing amounts ofADL to the incubation mixture dose-dependently shifted the free TNF-Fl peak (FT) to the higher molecular mass eluting positions, while the internal control (IC) peak did not change.

Figure 22 shows a dose-response curve ofADL on the shift of free TNF-u-Fl.

Increasing amounts ofADL were incubated with 100 ng of TNF-(x-Fl and internal control.

The more the antibody ADL was added to the reaction mixture the lower the ratio of free TNF-(x-Fl to internal control.

Figure 23 shows the mobility shift profiles of Fl-labeled Remicade (IFX) Incubated with Normal (NHS) or Pooled HACA Positive Patient Serum.

[0046] Figure 24 shows the mobility shift profiles of Fl-Labeled HUMIRA (ADL) incubated with normal (NHS) or Mouse Anti-Human IgGl Antibody.

Figure 25 shows the mobility shift es of eled HUMIRA (ADL) incubated with normal (NHS) or pooled HAHA positive patient serum.

Figure 26 shows an illustration of the effect of the acid dissociation step. “A” represents labeled-Remicade, “B” represents HACA, “C” represents Remicade.

Figure 27 shows the percent free labeled-Infliximab as a function of Log Patient Serum percentage without an acid iation step.

Figure 28 shows the percent free labeled-Infliximab as a function of Log Patient Serum percentage with an acid dissocation step.

[0051] Figure 29 shows the serum IFX levels in a t treated with Infliximab as a on of time for the Patient Case 1.

Figure 30 shows the serum IFX levels in a t treated with Infliximab as a function of time for the Patient Case 3.

Figure 31 shows the serum TNFoc levels in a patient treated with Infliximab as a function of time for the Patient Case 3.

Figure 32 shows the ty shift profiles of Fl-Labeled-IFX for Patient Case 1 (A); Patient Case 2 (B, C); and Patient Case 4 (D).

Figure 33 shows the mobility shift profiles of of Fl-Labeled-IFX for Patient Case 5 (A); Patient Case 6 (B, C); and Patient Case 7 (D, E).

[0056] Figure 34 shows cytokine levels in different patient serum groups.

Figure 35 shows the analysis of samples containing exa488 and Remicade by mobility shift assay using a fluorescence detector with gain settings at ent values.

Figure 36 shows isoabsorbance plots taken for normal human serum (top panel) and TNF-Alexa488 (bottom panel) in HPLC mobile phase (1X PBS, 0.1% BSA in water).

Excitation wavelengths are plotted on the Y-axis and emission wavelengths are d on the X-axis.

Figure 37 shows the HPLC analysis of normal human serum (left) and 25ng TNF- Alexa488 (right) ed with indicated settings. The background level of fluorescence from normal human serum is greatly decreased.

[0060] Figure 38 shows the standard curve generated by HPLC is of samples containing a fixed amount of TNF-Alexa488 and titrated with various amounts of Remicade.

Figure 39 shows a comparison of Infliximab determination in clinical s by mobility shift assay and ELISA. Dark grey points are for HACA-positive samples and light grey points are for HACA-negative samples. Dashed lines represent lower limits of quantitations for the respective methods.

Figure 40 shows a ison ofHACA determination in clinical samples by mobility shift assay and ELISA.

Figure 41 shows the cumulative counts of HACA-positive clinical samples as determined by mobility shift assay and ELISA.

DETAILED DESCRIPTION OF THE INVENTION 1. uction The present invention is based in part on the discovery that a neous mobility shift assay using size exclusion chromatography and acid dissociation to enable equilibration of immune complexes is particularly advantageous for measuring the presence or level of autoantibodies (e.g., HACA, HAHA, etc.) that are generated against anti-TNFu drugs. Such autoantibodies are also known as anti-drug antibodies or ADA. As a result, the presence or level of autoantibodies to an anti-TNFu drug stered to a subject in need thereof can be measured without ntial interference from the administered anti-TNFu drug that is also present in the subject’s sample. In particular, a subject’s sample can be incubated with an amount of acid that is sufficient to provide for the measurement of the presence or level of autoantibodies in the presence of the anti-TNFu drug without substantial interference from high anti-TNFu drug levels.

High anti-TNFOL drug levels in a sample (e.g., high infliximab levels) eres with the measumrent of anti-drug antibody levels (e.g, HACA ). Under certain high drug conditions, the anti-drug antibody present in a sample is complexed with the unlabeled drug also present in the sample. When a labeled drug, 6.g. labeled-infliximab, is contacted with the , the anti-drug antibody present in the sample is kinetically trapped from forming a complex with the d drug. In this way, the preformed complexes of anti-drug antibody and the unlabeled drug interfere with the measurement of anti-drug antibody, which depends on the formation of a complex between the anti-drug antibody t and the labeled drug.

The acid dissociation step described herein allows for the anti-drug antibody present in the sample to dissociate from the unlabeled drug and reform complexes with both the d and led drug. By dissociating the anti-drug antibody from the unlabeled drug, the rug antibody present in a sample can equilibrate between the labeled drug and the unlabeled drug.

As shown in Figure 27, high levels of anti-TNFu drug (e.g., infliximab) interfere with the detection of anti-drug antibodies (e.g., antibodies to infliximab or ATI) when the mobility shift assay is performed without an acid dissociation step. However, Figure 28 shows that acid dissociation followed by homogeneous solution phase binding cs to allow the equilibration and reformation of immune xes significantly increased the anti-TNFu drug tolerance such that anti-drug antibodies can be measured in the presence of high levels of anti-TNFu drug (e.g, up to or at least about 60 ug/mL). As such, the assays of the present invention are particularly advantageous over methods tly available because they enable the detection and ement of anti-drug antibodies at any time during therapy with an anti-TNFu drug (e.g, irrespective of low, medium, or high levels of anti-TNFu drug in a sample such as a blood sample), thereby overcoming a major limitation of s in the art which require sample collection at trough concentrations of the drug.

[0067] In certain aspects, the present invention is ageous because it addresses and overcomes current limitations associated with the administration of anti-TNFu drugs such as infliximab, in part, by providing information useful for guiding treatment decisions for those patients receiving or about to receive NFOL drug therapy. In particular, the methods of the present invention find utility for selecting an appropriate anti-TNFu y for initial treatment, for determining when or how to adjust or modify (e.g., increase or decrease) the subsequent dose of an anti-TNFu drug to optimize therapeutic efficacy and/or to reduce ty, for determining when or how to combine an anti-TNFu drug (e.g., at an initial, increased, decreased, or same dose) with one or more immunosuppressive agents such as methotrexate (MTX) or azathioprine (AZA), and/or for determining when or how to change the current course of therapy (e.g, switch to a different anti-TNFu drug or to a drug that targets a different mechanism).

Accordingly, the present invention is particularly useful in the following methods of improving patient management by guiding treatment decisions: 1. Crohn’s disease prognostics: Treat patients most likely to benefit from therapy 2. Anti-therapeutic dy monitoring (ATM) + Biomarker-based disease activity index 3. ATM sub-stratification 4. ATM with pharmacokinetic modeling 5. Monitor response and predict risk of relapse: a. Avoid chronic maintenance therapy in patients with low risk of ence b. Markers of l healing c. Therapy selection: Whether to combine or not to combine anti-TNF drug therapy with an immunosuppressive agent such as MTX or AZA 6. Patient selection for biologics. 11. ions As used herein, the following terms have the meanings ascribed to them unless ed ise.

The terms “anti-TNFOL drug” or “TNFOL inhibitor” as used herein is ed to encompass agents ing proteins, antibodies, antibody fragments, filSlOI‘l proteins (e.g., Ig filsion proteins or PC filSlOI‘l proteins), multivalent binding proteins (e.g., DVD Ig), small molecule TNFOL antagonists and similar naturally- or nonnaturally-occurring molecules, and/or recombinant and/or ered forms thereof, that, directly or indirectly, inhibit TNFOL ty, such as by ting interaction of TNFOL with a cell surface or for TNFOL, inhibiting TNFOL protein production, inhibiting TNFOL gene expression, inhibiting TNFOL secretion from cells, inhibiting TNFOL receptor signaling or any other means resulting in decreased TNFOL activity in a subject. The term “anti-TNFu drug” or “TNFu inhibitor” preferably includes agents which interfere with TNFOL activity. Examples of anti-TNFOL drugs include, without limitation, infliximab (REMICADETM, Johnson and Johnson), human anti- TNF monoclonal antibody adalimumab (D2E7/HUMIRAT'V', Abbott Laboratories), cept (ENBRELT'V', Amgen), certolizumab pegol (CIMZIA®, UCB, Inc.), golimumab (SIMPONI®; CNTO 148), CDP 571 (Celltech), CDP 870 (Celltech), as well as other compounds which inhibit TNFOL activity, such that when administered to a t suffering from or at risk of suffering from a disorder in which TNFu activity is detrimental (e.g., RA), the disorder is treated.

The term “TNFOL” is intended to include a human cytokine that exists as a 17 kDa secreted form and a 26 kDa membrane ated form, the biologically active form of which is composed of a trimer of noncovalently bound 17 kDa molecules. The structure of TNFOL is bed further in, for example, Jones et al., Nature, 338:225-228 (1989). The term TNFOL is intended to e human TNFu, a recombinant human TNFu (rhTNF-(x), or TNFu that is at least about 80% identity to the human TNFu protein. Human TNFu consists of a 35 amino acid (aa) cytoplasmic domain, a 21 aa embrane segment, and a 177 aa extracellular domain (ECD) (Pennica, D. et al. (1984) Nature 312:724). Within the ECD, human TNFu shares 97% aa sequence identity with rhesus TNFOL, and 71% to 92% aa sequence identity with bovine, canine, cotton rat, equine, feline, mouse, e, and rat TNFOL. TNFOL can be prepared by standard recombinant expression methods or purchased commercially (R & D Systems, Catalog No. , Minneapolis, Minn.).

[0072] In certain embodiments, “TNFu” is an “antigen,” which includes a molecule or a n of the molecule capable of being bound by an anti-TNF-(x drug. TNFOL can have one or more than one epitope. In n instances, TNFu will react, in a highly selective manner, with an anti-TNFu antibody. Preferred antigens that bind antibodies, fragments, and regions of anti-TNFu antibodies include at least 5 amino acids of human TNFu. In certain instances, TNFu is a ient length haVing an e of TNFu that is capable of binding anti-TNFu antibodies, fragments, and regions thereof The term “predicting responsiveness to an anti-TNFu drug” is intended to refer to an ability to assess the likelihood that treatment of a subject with an anti-TNFu drug will or will not be effective in (e.g., provide a measurable benefit to) the subject. In particular, such an ability to assess the likelihood that ent will or will not be effective typically is exercised after treatment has begun, and an indicator of effectiveness (e.g., an indicator of measurable benefit) has been observed in the subject. Particularly red anti-TNFu drugs are biologic agents that have been approved by the FDA for use in humans in the treatment of TNFu-mediated diseases or disorders and include those anti-TNFOL drugs bed herein.

[0074] The term “size exclusion chromatography” or “SEC” es a chromatographic method in which molecules in solution are separated based on their size and/or hydrodynamic volume. It is applied to large molecules or macromolecular complexes such as proteins and their conjugates. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel filtration tography.

The terms “complex,3, C"1mmuno-complex,3, “conjugate,” and “immunoconjugate” include, but are not limited to, TNFu bound (e.g, by non-covalent means) to an anti-TNFu drug, an anti-TNFOL drug bound (e.g., by non-covalent means) to an autoantibody against the NFu drug, and an anti-TNFu drug bound (e.g, by non-covalent means) to both TNFu and an autoantibody against the NFOL drug.

As used herein, an entity that is modified by the term “labeled” includes any entity, molecule, protein, enzyme, antibody, antibody fragment, cytokine, or related species that is conjugated with another molecule or chemical entity that is empirically detectable. Chemical species suitable as labels for labeled-entities include, but are not limited to, fluorescent dyes, e. g. Alexa Fluor® dyes such as Alexa Fluor® 647, quantum dots, optical dyes, luminescent dyes, and radionuclides, e.g. 125I.

The term tive amount” includes a dose of a drug that is capable of achieving a therapeutic effect in a subject in need thereof as well as the bioavailable amount of a drug.

The term “bioavailable” includes the fraction of an stered dose of a drug that is available for therapeutic activity. For example, an effective amount of a drug useful for treating diseases and disorders in which TNF-u has been implicated in the pathophysiology can be the amount that is capable of ting or relieving one or more symptoms associated therewith.

The phrase “fluorescence label detection” includes a means for detecting a fluorescent label. Means for detection include, but are not limited to, a spectrometer, a fluorimeter, a photometer, and a detection device commonly orated with a chromatography instrument such as, but not limited to, size exclusion-high performance liquid chromatography, such as, but not limited to, an Agilent-1200 HPLC .

The phrase “optimize therapy” es optimizing the dose (e.g., the effective amount or level) and/or the type of a particular therapy. For e, optimizing the dose of an anti-TNFu drug includes increasing or decreasing the amount of the anti-TNFu drug subsequently administered to a subject. In certain instances, optimizing the type of an anti- TNFOL drug includes ng the administered NFu drug from one drug to a different drug (e.g., a different NFu drug). In other instances, optimizing therapy includes co- administering a dose of an anti-TNFu drug (e.g., at an sed, decreased, or same dose as the previous dose) in combination with an immunosuppressive drug.

The term “co-administer” includes to administer more than one active agent, such that the duration of physiological effect of one active agent overlaps with the physiological effect of a second active agent.

The term “subject,” “patient,” or “individual” typically refers to humans, but also to other s including, e.g., other primates, rodents, canines, felines, equines, , es, and the like.

The term e of y” includes any therapeutic approach taken to relieve or prevent one or more symptoms associated with a TNFu-mediated disease or disorder. The term encompasses administering any compound, drug, procedure, and/or regimen useful for improving the health of an individual with a TNFu-mediated e or disorder and includes any of the therapeutic agents described herein. One d in the art will appreciate that either the course of therapy or the dose of the current course of therapy can be changed (e.g., increased or decreased) based upon the presence or concentration level of TNFu, anti-TNFu drug, and/or anti-drug dy using the methods of the t invention.

[0083] The term “immunosuppressive drug” or “immunosuppressive agent” includes any substance capable of producing an immunosuppressive effect, e.g., the prevention or diminution of the immune response, as by irradiation or by administration of drugs such as anti-metabolites, anti-lymphocyte sera, antibodies, etc. Examples of immunosuppressive drugs include, Without limitation, thiopurine drugs such as azathioprine (AZA) and metabolites thereof; anti-metabolites such as rexate (MTX); sirolimus (rapamycin); temsirolimus; everolimus; tacrolimus (PK-506); FK-778; anti-lymphocyte globulin antibodies, anti-thymocyte globulin antibodies, anti-CD3 antibodies, anti-CD4 antibodies, and dy-toxin conjugates; cyclosporine; mycophenolate; mizoribine monophosphate; scoparone; glatiramer e; metabolites thereof; pharmaceutically acceptable salts thereof; derivatives thereof; gs thereof; and combinations thereof.

The term “thiopurine drug” includes azathioprine (AZA), 6-mercaptopurine (6-MP), or any metabolite thereof that has therapeutic efficacy and includes, Without limitation, 6- thioguanine (6-TG), ylmercaptopurine riboside, 6-thioinosine nucleotides (e.g., 6- thioinosine monophosphate, 6-thioinosine phate, 6-thioinosine triphosphate), 6- thioguanine nucleotides (e.g., 6-thioguanosine monophosphate, 6-thioguanosine diphosphate, 6-thioguanosine triphosphate), xanthosine nucleotides (e.g., 6-thioxanthosine monophosphate, 6-thioxanthosine diphosphate, 6-thioxanthosine triphosphate), derivatives thereof, analogues thereof, and combinations thereof The term “sample” includes any biological specimen obtained from an individual.

Samples include, without tion, whole blood, plasma, serum, red blood cells, white blood cells (e. g., eral blood mononuclear cells (PBMC), polymorphonuclear (PMN) cells), ductal lavage fluid, nipple aspirate, lymph (e.g., inated tumor cells of the lymph node), bone marrow aspirate, saliva, urine, stool (i.e., feces), sputum, bronchial lavage fluid, tears, fine needle aspirate (e.g., harvested by random periareolar fine needle aspiration), any other bodily fluid, a tissue sample such as a biopsy of a site of inflammation (e.g., needle biopsy), cellular extracts thereof, and an immunoglobulin ed on derived from one or more of these bodily fluids or tissues. In some ments, the sample is whole blood, a fractional ent thereof such as plasma, serum, or a cell pellet, or an immunoglobulin enriched fraction thereof . One skilled in the art will appreciate that samples such as serum samples can be diluted prior to the analysis. In certain embodiments, the sample is obtained by isolating PBMCs and/or PMN cells using any technique known in the art. In certain other embodiments, the sample is a tissue biopsy such as, e.g, from a site of inflammation such as a portion of the gastrointestinal tract or al .

The steps of the methods of the t invention do not necessarily have to be performed in the particular order in which they are presented. A person of ordinary skill in the art would understand that other orderings of the steps of the methods of the invention are encompassed within the scope of the present invention.

[0087] Brackets, “[ ]” indicate that the species within the brackets are referred to by their concentration. 111. Description of the Embodiments The present invention provides assays for detecting and measuring the presence or level of autoantibodies to anti-TNFOL drug therapeutics in a sample. The present invention is useful for optimizing therapy and monitoring patients receiving NFOL drug therapeutics to detect the presence or level of autoantibodies (e.g, HACA and/or HAHA) against the drug.

The t invention also provides methods for selecting therapy, optimizing therapy, and/or reducing toxicity in subjects receiving anti-TNFOL drugs for the treatment of TNFu-mediated disease or disorders.

[0089] In one aspect, the present invention provides a method for detecting the presence or level of an autoantibody to an anti-TNFOL drug in a sample without interference from the anti- TNFOL drug in the sample, the method comprising: (a) contacting the sample with an acid to dissociate preformed complexes of the autoantibody and the anti-TNFu drug, wherein the sample has or is suspected of having an autoantibody to the anti-TNFOL drug; (b) ting the sample with a labeled anti-TNFOL drug following dissociation of the preformed complexes; (c) neutralizing the acid in the sample to form labeled complexes (i.e., immunocomplexes or conjugates) of the labeled anti-TNFu drug and the autoantibody (i.e., wherein the labeled NFOL drug and autoantibody are not covalently attached to each other); (d) subjecting the labeled complexes to size exclusion chromatography to separate the labeled complexes (e.g., from free labeled anti-TNFu drug); and (e) detecting the labeled complexes, thereby detecting the presence or level of the autoantibody t interference from the anti-TNFOL drug in the sample.

Without being bound by any particular theory, it is believed that acid dissociation changes the Kd between the tibody (also known as an anti-drug antibody or ADA) and the anti-TNFu drug. In particular, it is theorized that acid dissociation disrupts the bonds n the ADA and the anti-TNFOL drug. These bonds include, but are not limited to, hydrogen bonds, electrostatic bonds, Van der Waals forces, and/or hydrophobic bonds. The addition of acid increases the pH and thus the hydrogen ion concentration increases. The hydrogen ions can now compete for the previously mentioned non-covalent interactions.

This ition lowers the Kd between the ADA and the anti-TNFOL drug.

In some embodiments, the NFOL drug is selected from the group consisting of DET'VI (infliximab), ENBRELT'V' (etanercept), HUMIRATM (adalimumab), CIMZIA® (certolizumab pegol), SIMPONI® (golimumab; CNTO 148), and combinations thereof.

[0092] In other embodiments, the NFOL drug autoantibody includes, but is not limited to, human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti-mouse dies (HAMA), as well as combinations thereof.

In certain alternative embodiments, steps (a) and (b) are performed simultaneously, e.g., the sample is contacted with an acid and a labeled anti-TNFOL drug at the same time. In certain other alternative embodiments, step (b) is performed prior to step (a), e.g., the sample is first contacted with a labeled anti-TNFOL drug, and then contacted with an acid. In r embodiments, steps (b) and (c) are performed aneously, e.g., the sample is contacted with a d anti-TNFOL drug and neutralized (e.g., by ting the sample with one or more neutralizing agents) at the same time.

In particular ments, the sample is ted with an amount of an acid that is sufficient to dissociate preformed complexes of the autoantibody and the anti-TNFu drug, such that the labeled anti-TNFOL drug, the unlabeled anti-TNFOL drug, and the autoantibody to the anti-TNFu drug can equilibrate and form complexes therebetween.

In preferred embodiments, the methods of the invention comprise detecting the presence or level of the autoantibody without substantial erence from the anti-TNFu drug that is also present in the sample. In such embodiments, the sample can be contacted with an amount of an acid that is sufficient to allow for the detection and/or measurement of the autoantibody in the presence of a high level of the anti-TNFOL drug.

In some embodiments, the phrase “high level of an anti-TNFoc drug” includes drug levels of from about 10 to about 100 ug/mL, about 20 to about 80 ug/mL, about 30 to about 70 ug/mL, or about 40 to about 80 ug/mL. In other embodiments, the phrase “high level of an anti-TNFoc drug” includes drug levels greater than or equal to about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ug/mL.

In some embodiments, the acid comprises an organic acid. In other embodiments, the acid comprises an inorganic acid. In fiarther embodiments, the acid comprises a mixture of an organic acid and an inorganic acid. Non-limiting examples of organic acids include citric acid, isocitric acid, glutamic acid, acetic acid, lactic acid, formic acid, oxalic acid, uric acid, roacetic acid, benzene sulfonic acid, aminomethanesulfonic acid, camphor sulfonic acid, chloroacetic acid, bromoacetic acid, iodoacetic acid, propanoic acid, butanoic acid, glyceric acid, succinic acid, malic acid, aspartic acid, and combinations thereof. Non- limiting examples of inorganic acids include hydrochloric acid, nitric acid, oric acid, sulfuric acid, boric acid, hydrofluoric acid, hydrobromic acid, and combinations thereof In certain embodiments, the amount of an acid corresponds to a concentration of from about 0.01M to about 10M, about 0.1M to about 5M, about 0.1M to about 2M, about 0.2M to about 1M, or about 0.25M to about 0.75M of an acid or a mixture of acids. In other ments, the amount of an acid corresponds to a concentration of greater than or equal to about 0.01M, 0.05M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 2M, 3M, 4M, 5M, 6M, 7M, 8M, 9M, or 10M of an acid or a mixture of acids. The pH ofthe acid can be, for example, about 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5.

In some embodiments, the sample is contacted with an acid an amount of time that is sufficient to dissociate preformed complexes of the autoantibody and the anti-TNFu drug.

In certain instances, the sample is contacted (e.g., incubated) with an acid for a period of time ranging from about 0.1 hours to about 24 hours, about 0.2 hours to about 16 hours, about 0.5 hours to about 10 hours, about 0.5 hours to about 5 hours, or about 0.5 hours to about 2 hours.

In other instances, the sample is contacted (e.g., incubated) with an acid for a period of time that is greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, l, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 hours. The sample can be contacted with an acid at 4°C, room temperature (RT), or 37°C.

[0100] In certain embodiments, the step of neutralizing the acid comprises raising the pH of the sample to allow the formation of complexes between the labeled anti-TNFu drug and the autoantibody to the anti-TNFu drug as well as xes n unlabeled anti-TNFOL drug and the autoantibody. In some embodiments, the acid is neutralized by the addition of one or more neutralizing agents such as, for example, strong bases, weak bases, buffer solutions, and combinations thereof. One d in the art will appreciate that neutralization reactions do not necessarily imply a ant pH of 7. In some instances, acid neutralization results in a sample that is basic. In other instances, acid neutralization results in a sample that is acidic (but higher than the pH of the sample prior to adding the lizing agent). In particular embodiments, the neutralizing agent comprises a buffer such as phosphate buffered saline (e.g., 10x PBS) at a pH of about 7.3.

In some embodiments, step (b) further comprises ting an internal control with the sample together with a labeled NFOL drug (e.g., before, during, or after dissociation of the preformed complexes). In certain instances, the internal l comprises a labeled internal control such as, e.g., Biocytin-Alexa 488. In certain other ces, the amount of the labeled internal control ranges from about 1 ng to about 25 ng, about 5 ng to about 25 ng, about 5 ng to about 20 ng, about 1 ng to about 20 ng, about 1 ng to about 10 ng, or about 1 ng to about 5 ng per 100 uL of sample analyzed. In further instances, the amount of the labeled al control is greater than or equal to about 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, or 25 ng per 100 uL of sample analyzed.

[0102] As one non-limiting example of the methods of the present invention, samples such as serum samples (e.g., serum from subjects receiVing therapy with an anti-TNFu drug such as Remicade (IFX)) can be incubated with 0.5M citric acid, pH 3.0 for one hour at room temperature. Following the dissociation of preformed complexes between (unlabeled) anti- TNFOL drug and autoantibodies to the anti-TNFu drug (e.g., anti-drug antibodies such as anti- 2012/025437 IFX antibodies (AT1)), labeled anti-TNFOL drug (e.g., IFX-Alexa 488) and an internal control can be added and the reaction mixture and (e.g., immediately) neutralized with a neutralizing agens such as le PBS, pH 7.3. After neutralization, the on mixture can be incubated for another hour at room ature (e.g., on a plate shaker) to allow equilibration and to complete the ation of immune complexes between either the labeled or unlabeled anti- TNFOL drug and the anti-drug antibody. The samples can then be filtered and analyzed by SEC-HPLC as described herein.

In particular embodiments, the methods of the present invention (e.g., comprising acid dissociation followed by homogeneous solution phase binding kinetics) significantly increases the IFX drug tolerance such that the ATI can be measured in the presence of IFX up to about 60 ug/mL. See, Example 14 and Figures 27-28. In other words, the methods of the invention can detect the presence or level of autoantibodies to anti-TNFOL drugs such as ATI as well as autoantibodies to other anti-TNFu drugs in the presence of high levels of anti- TNFOL drugs (e.g., IFX), but without substantial interference therefrom.

[0104] In another aspect, the present invention provides a method for zing therapy and/or reducing toxicity to an anti-TNFu drug in a subject ing a course of therapy with the NFu drug, the method comprising: (a) detecting the ce or level of an autoantibody to the anti-TNFOL drug in a sample from the subject t interference from the anti-TNFOL drug in the sample, the method comprising: (i) contacting the sample with an acid to dissociate preformed complexes of the autoantibody and the anti-TNFOL drug, wherein the sample has or is suspected of having an autoantibody to the anti-TNFu drug; (ii) contacting the sample with a d anti-TNFu drug following dissociation of the preformed complexes; (iii) neutralizing the acid in the sample to form labeled complexes (i.e., immuno-complexes or ates) of the labeled anti-TNFOL drug and the autoantibody (z'.e., wherein the labeled anti-TNFOL drug and autoantibody are not covalently attached to each other); (iv) subjecting the labeled complexes to size exclusion chromatography to separate the labeled complexes (e. g., from free labeled anti-TNFOL drug); and (V) detecting the labeled complexes (e.g., thereby detecting the presence or level of the autoantibody without interference from the anti-TNFOL drug in the sample); and (b) determining a subsequent dose of the course of therapy for the subject or whether a different course of y should be administered to the subject based upon the presence or level of the autoantibody, thereby optimizing therapy and/or reducing toxicity to the anti-TNFOL drug.

In certain embodiments, the subsequent dose of the course of therapy is increased, decreased, or maintained based upon the presence or level of the autoantibody. As a non- limiting example, a subsequent dose of the course of therapy is sed when a high level of the tibody is detected in the . In other embodiments, the different course of therapy comprises a different anti-TNFOL drug, the current course of therapy along with an immunosuppressive agent, or switching to a course of therapy that is not an anti-TNFOL drug (e.g, discontinuing use of an anti-TNFOL therapeutic antibody). As a non-limiting example, a different course of therapy is administered when a high level of the autoantibody is detected in the sample.

In certain alternative embodiments, steps (i) and (ii) are performed simultaneously, e.g, the sample is ted with an acid and a d anti-TNFOL drug at the same time. In certain other alternative embodiments, step (ii) is performed prior to step (i), e.g, the sample is first ted with a d anti-TNFOL drug, and then contacted with an acid. In further embodiments, steps (ii) and (iii) are performed simultaneously, e.g., the sample is ted with a labeled anti-TNFOL drug and neutralized (e.g, by contacting the sample with one or more neutralizing agents) at the same time.

An anti-TNFu drug can be labeled with any of a y of detectable group(s). In preferred embodiments, an anti-TNFu drug is labeled with a fluorophore or a fluorescent dye.

Non-limiting examples of fluorophores or fluorescent dyes include those listed in the Molecular Probes Catalogue, which is herein incorporated by reference (see, R. Haugland, The Handbook-A Guide to Fluorescent Probes and Labeling Technologies, 10th Edition, Molecular probes, Inc. (2005)). Such exemplary fluorophores or fluorescent dyes e, but are not limited to, Alexa Fluor® dyes such as Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 5 l4, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 635, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, and/or Alexa Fluor® 790, as well as other fluorophores including, but not limited to, Dansyl de (DNS-Cl), 5-(iodoacetamida)fluoroscein (S-IAF), fluoroscein 5- isothiocyanate (FITC), tetramethylrhodamine 5- (and thiocyanate (TRITC), 6-acryloyl- thylaminonaphthalene (acrylodan), obenzooxa-l,3,-diazolyl chloride (NBD-Cl), ethidium bromide, Lucifer Yellow, 5-carboxyrhodamine 6G hydrochloride, Lissamine rhodamine B sulfonyl chloride, Texas RedTM sulfonyl chloride, BODIPYT'V', naphthalamine sulfonic acids (e.g., l-anilinonaphthalenesulfonic acid (ANS), 6-(p- inyl)naphthalen-esulfonic acid (TNS), and the like), Anthroyl fatty acid, DPH, Parinaric acid, TMA-DPH, Fluorenyl fatty acid, fluorescein-phosphatidylethanolamine, Texas Red-phosphatidylethanolamine, Pyrenyl-phophatidylcholine, Fluorenyl- otidylcholine, Merocyanine 540,l-(3-sulfonatopropyl)[B-[Z[(di-n-butylamino)-6 naphthyl]vinyl]pyridinium betaine (Naphtyl Styryl), 3,3’dipropylthiadicarbocyanine (diS-Cg- (5)), 4-(p-dipentyl aminostyryl)-l-methylpyridinium (diASP), Cy-3 Iodo Acetamide, Cy-S- N—Hydroxysuccinimide, CyIsothiocyanate, rhodamine 800, IR-l25, Thiazole Orange, Azure B, Nile Blue, Al Phthalocyanine, Oxaxine l, 4’, idinophenylindole (DAPI), Hoechst 33342, TOTO, Acridine Orange, Ethidium Homodimer, N(ethoxycarbonylmethyl)- 6-methoxyquinolinium (MQAE), Fura-2, Calcium Green, Carboxy SNARF-6, BAPTA, coumarin, phytofluors, Coronene, metal-ligand complexes, IRDye® 700DX, IRDye® 700, IRDye® 800RS, IRDye® 800CW, IRDye® 800, Cy5, Cy5.5, Cy7, DY 676, DY680, DY682, DY780, and mixtures thereof. Additional suitable fluorophores include enzyme-cofactors; lanthanide, green fluorescent protein, yellow fluorescent protein, red fluorescent n, or mutants and tes thereof In one embodiment of the invention, the second member of the specific g pair has a detectable group attached thereto.

Typically, the fluorescent group is a hore ed from the category of dyes comprising polymethines, pthalocyanines, cyanines, xanthenes, fluorenes, rhodamines, coumarins, fluoresceins and BODIPYT'V'.

In one embodiment, the fluorescent group is a near-infrared (NIR) fluorophore that emits in the range of between about 650 to about 900 nm. Use of near infrared fluorescence technology is advantageous in biological assays as it substantially ates or reduces background from auto fluorescence of biosubstrates. Another benefit to the near-IR fluorescent technology is that the scattered light from the excitation source is y reduced since the scattering intensity is proportional to the inverse fourth power of the wavelength.

Low background fluorescence and low scattering result in a high signal to noise ratio, which is essential for highly sensitive ion. Furthermore, the optically transparent window in the near-IR region (650 nm to 900 nm) in biological tissue makes NIR fluorescence a WO 54253 valuable technology for in viva imaging and subcellular detection applications that require the transmission of light through biological components. Within aspects of this ment, the cent group is preferably selected form the group consisting of IRDye® 700DX, IRDye® 700, IRDye® 800RS, IRDye® 800CW, IRDye® 800, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, Alexa Fluor® 790, Cy5, Cy5.5, Cy7, DY 676, DY680, DY682, and DY780. In n embodiments, the near infrared group is IRDye® 800CW, IRDye® 800, IRDye® 700DX, IRDye® 700, or c DY676.

Fluorescent labeling is accomplished using a chemically reactive derivative of a fluorophore. Common ve groups include amine reactive isothiocyanate derivatives such as FITC and TRITC (derivatives of fluorescein and rhodamine), amine reactive imidyl esters such as NHS-fluorescein, and sulfllydryl reactive maleimide activated fluors such as fluorescein-S-maleimide, many of which are commercially available. on of any of these reactive dyes with an NFu drug results in a stable covalent bond formed between a fluorophore and an anti-TNFu drug.

[0111] In certain instances, following a fluorescent labeling reaction, it is often necessary to remove any nonreacted fluorophore from the labeled target molecule. This is often accomplished by size exclusion chromatography, taking advantage of the size difference between fluorophore and labeled protein.

Reactive fluorescent dyes are ble from many sources. They can be obtained with different reactive groups for attachment to various functional groups within the target molecule. They are also available in labeling kits that contain all the components to carry out a labeling reaction. In one preferred aspect, Alexa Fluor® 647 C2 maleimide is used from Invitrogen (Cat. No. A-20347).

Specific immunological binding of an anti-drug antibody (ADA) to an anti-TNFOL drug can be detected ly or ctly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. In certain instances, an anti-TNFOL drug that is labeled with iodine-125 (1251) can be used for determining the concentration levels ofADA in a . In other instances, a chemiluminescence assay using a chemiluminescent anti-TNFOL drug that is ic for ADA in a sample is suitable for sensitive, non-radioactive detection ofADA concentration levels. In particular instances, an anti-TNFu drug that is labeled with a fluorochrome is also le for determining the concentration levels ofADA in a sample. Examples of fluorochromes include, without limitation, Alexa Fluor® dyes, DAPI, fluorescein, Hoechst 33258, R—phycocyanin, B- phycoerythrin, R—phycoerythrin, rhodamine, Texas red, and lissamine. Secondary antibodies linked to fluorochromes can be obtained commercially, e.g, goat F(ab’)2 anti-human IgG- FITC is available from Tago Immunologicals (Burlingame, CA).

Indirect labels include various enzymes well-known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), B-galactosidase, urease, and the like. A horseradish-peroxidase ion system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a e product in the presence of hydrogen peroxide that is detectable at 450 nm. An alkaline phosphatase ion system can be used with the chromogenic substrate p-nitrophenyl phosphate, for example, which yields a soluble product readily able at 405 nm. rly, a B-galactosidase detection system can be used with the chromogenic substrate ophenyl-B-D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm. An urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals; St.

Louis, MO). A useful secondary antibody linked to an enzyme can be obtained from a number of commercial sources, e.g, goat F(ab’)2 anti-human IgG-alkaline phosphatase can be purchased from Jackson ImmunoResearch (West Grove, PA.).

A signal from the direct or indirect label can be analyzed, for example, using a ophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of 1251; or a eter to detect fluorescence in the presence of light of a certain wavelength. For detection of enzyme-linked antibodies, a quantitative analysis ofADA levels can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, CA) in accordance with the manufacturer’s instructions. If desired, the assays of the t ion can be automated or performed robotically, and the signal from multiple s can be ed simultaneously.

In certain embodiments, size exclusion chromatography is used. The underlying principle of SEC is that particles of different sizes will elute (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size.

Provided that all the particles are loaded simultaneously or near simultaneously, particles of the same size elute er. Each size exclusion column has a range of lar weights that can be separated. The ion limit defines the molecular weight at the upper end of this range and is where molecules are too large to be trapped in the stationary phase. The permeation limit defines the molecular weight at the lower end of the range of separation and is where molecules of a small enough size can penetrate into the pores of the stationary phase completely and all molecules below this molecular mass are so small that they elute as a single band.

In certain aspects, the eluent is collected in nt s, or fractions. The more similar the particles are in size, the more likely they will be in the same fraction and not detected separately. Preferably, the collected fractions are examined by spectroscopic techniques to determine the concentration of the les eluted. Typically, the spectroscopy detection techniques useful in the present invention e, but are not limited to, fluorometry, refractive index (RI), and ultraviolet (UV). In certain instances, the n volume decreases roughly linearly with the logarithm of the molecular hydrodynamic volume (i.e., heaver moieties come off .

The present invention further provides a kit for detecting the presence or level of an autoantibody to an anti-TNFu drug in a sample. In particular embodiments, the kit comprises one or more of the following components: an acid (or mixture of acids), a labeled anti-TNFu drug (e.g., a labeled anti-TNFu antibody), a labeled al control, a neutralizing agent (or mixtures thereof), means for ion (e.g., a cence detector), a size exclusion-high performance liquid chromatography (SE-HPLC) instrument, and/or instructions for using the kit.

In other aspects, the present ion provides a method for selecting a course of therapy (e.g., selecting an riate anti-TNFu drug) for the treatment of a TNFu-mediated disease or disorder in a subject, the method comprising: (a) analyzing a sample obtained from the subject to determine the presence, level, or genotype of one or more markers in the sample; (b) applying a statistical algorithm to the presence, level, or genotype of the one or more markers determined in step (a) to generate a disease activity/severity index; and (c) selecting an appropriate course of therapy (e.g., anti-TNFu therapy) for the t based upon the disease activity/severity index.

In a related aspect, the present invention provides a method for optimizing therapy and/or reducing toxicity in a subject receiving a course of therapy for the treatment of a TNFu-mediated disease or disorder, the method comprising: (a) analyzing a sample ed from the subject to ine the presence, level, or genotype of one or more markers in the sample; (b) applying a statistical algorithm to the presence, level, or genotype of the one or more markers determined in step (a) to generate a disease activity/severity index; and (c) determining a subsequent dose of the course of therapy for the subject or r a different course of therapy should be administered to the subject based upon the disease activity/severity index.

In some embodiments, the course oftherapy ses an anti-TNFOL antibody. In certain instances, the anti-TNFOL antibody is a member selected from the group consisting of REMICADETM imab), ENBRELT'V' (etanercept), HUMIRAT'V' (adalimumab), CIMZIA® (certolizumab pegol), SIMPONI® (golimumab; CNTO 148), and combinations thereof. In other embodiments, the course of therapy comprises an anti-TNFOL dy along with an suppressive agent.

[0122] In certain embodiments, the level of one or more markers ses a total level, an activation level, or combinations thereof. In particular instances, the one or more markers is a member selected from the group consisting of an inflammatory marker, a growth factor, a serology marker, a cytokine and/or chemokine, a marker of oxidative stress, a cell surface receptor, a signaling pathway , a genetic marker, an anti-TNFOL antibody, an anti-drug antibody (ADA), and combinations thereof.

In some instances, the inflammatory marker is a member selected from the group ting of CRP, SAA, VCAM, ICAM, calprotectin, lactoferrin, IL-8, Rantes, TNFOL, IL-6, IL-l B, SIGGAIZ, M2~pymvate kinase (PK), IEE-‘N, 11.92, TGF, IL-l3, IL-lS, IL-12, and combinations thereof. In other instances, the growth factor is a member selected from the group consisting of GM-CSF, VEGF, EGF, keratinocyte growth factor (KGF; FGF7), and combinations thereof. In yet other instances, the serology marker is a member ed from the group consisting of an eutrophil antibody, an anti-microbial antibody, an anti- Saccharomyces siae antibody, and combinations thereof. In further instances, the cytokine is a member selected from the group consisting of TNFu, IL-6, IL-lB, IFN—y, IL-lO, and combinations thereof In other instances, the cell surface receptor is CD64. In yet other instances, the signaling pathway marker is a signal transduction molecule. In other instances, the genetic marker is a on in an inflammatory pathway gene.

In certain embodiments, step (a) comprises determining the presence, level, and/or genotype of at least two, three, four, five, six, seven, eight, nine, ten, fifteen, , thirty, forty, fifty, or more markers in the sample. In certain ces, the sample is selected from the group consisting of serum, plasma, whole blood, stool, peripheral blood mononuclear cells (PBMC), rphonuclear (PMN) cells, and a tissue biopsy.

In other embodiments, the statistical algorithm comprises a learning statistical classifier system. In some instances, the learning statistical classifier system is selected from the group consisting of a random forest, classification and regression tree, boosted tree, neural network, support vector machine, general chi-squared automatic ction detector model, interactive tree, multiadaptive regression spline, e learning classifier, and combinations thereof. In certain instances, the statistical algorithm comprises a single learning statistical classifier system. In certain other instances, the tical algorithm comprises a combination of at least two learning statistical classifier systems. In some instances, the at least two learning statistical classifier s are d in tandem. Non- limiting examples of statistical algorithms and analysis le for use in the invention are described in International Application No. PCT/U8201 1/056777, filed October 18, 2011, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

In some embodiments, the method further comprises sending the s from the selection or determination of step (c) to a clinician. In other embodiments, step (c) ses selecting an initial course of therapy for the subject.

In other embodiments, step (b) further comprises applying a tical algorithm to the presence, level, or genotype of one or more markers determined at an earlier time during the course of therapy to generate an earlier disease activity/severity index. In some instances, the earlier disease activity/severity index is compared to the disease activity/severity index generated in step (b) to determine a uent dose of the course of therapy or whether a different course of therapy should be administered. In certain embodiments, the uent dose of the course of therapy is increased, decreased, or maintained based upon the disease activity/severity index generated in step (b). In some instances, the different course of y comprises a different anti-TNFOL antibody. In other instances, the different course of therapy comprises the current course of therapy along with an immunosuppressive agent. s for detecting anti-TNFOL antibodies and anti-drug dies (ADA) are described herein and in PCT Publication No. , the disclosure of which is hereby incorporated by reference in its entirety for all purposes. In ular embodiments, the presence or level of anti-drug antibodies is determined in accordance with the methods of the invention comprising an acid dissociation step by contacting a sample with an acid prior to, during, and/or after ting the sample with a d anti-TNFOL drug.

In another aspect, the present ion provides a method for predicting the course of a TNFu-mediated disease or disorder in a t, the method comprising: WO 54253 (a) analyzing a sample obtained from the subject to determine the presence, level, or pe of one or more markers in the sample; (b) applying a statistical algorithm to the presence, level, or genotype of the one or more markers determined in step (a) to generate a disease activity/severity index; and (c) predicting the course of the TNFu-mediated disease or disorder based upon the disease activity/severity index generated in step (b).

In some embodiments, step (b) further comprises applying a statistical algorithm to the presence, level, or genotype of one or more of the markers determined at an earlier time to generate an earlier disease activity/severity index. In certain instances, the earlier disease activity/severity index is compared to the disease activity/severity index ted in step (b) to predict the course of the TNFu-mediated disease or disorder.

Once the diagnosis or prognosis of a subject receiving anti-TNFu drug therapy has been determined or the likelihood of response to an anti-TNFu drug has been predicted in a subject diagnosed with a disease and disorder in which TNFu has been implicated in the pathophysiology, e.g, but not limited to, shock, sepsis, infections, mune diseases, RA, Crohn’s disease, lant rejection and graft-versus-host disease, ing to the methods described herein, the present invention may fiarther comprise ending a course of therapy based upon the diagnosis, prognosis, or tion. In certain instances, the present invention may r comprise administering to a subject a therapeutically effective amount of an NFOL drug useful for treating one or more symptoms associated with the TNFOL- mediated disease or disorder. For therapeutic applications, the anti-TNFu drug can be administered alone or co-administered in combination with one or more additional anti-TNFOL drugs and/or one or more drugs that reduce the side-effects associated with the anti-TNFu drug (e. g., an immunosuppressive . As such, the t invention advantageously enables a clinician to practice “personalized medicine” by guiding treatment decisions and informing therapy selection and optimization for anti-TNFOL drugs such that the right drug is given to the right patient at the right time.

IV. Disease Activity/Severity Index In certain aspects, the present invention provides an algorithmic-based analysis of one or a plurality of (e.g, two, three, four, five, six, seven, or more) kers to improve the accuracy of selecting therapy, optimizing therapy, reducing ty, and/or monitoring the efficacy of eutic treatment to anti-TNFOL drug therapy.

As a non-limiting e, the disease activity/severity index in one embodiment comprises detecting, measuring, or determining the presence, level ntration (e.g, total) and/or tion (e.g., phosphorylation)), or genotype of one or more specific biomarkers in one or more of the following categories of biomarkers: (l) Inflammatory markers (2) Growth factors (3) Serology (e.g., immune markers) (4) Cytokines and ines (5) Markers of ive stress (6) Cell surface receptors (e.g., CD64, ) (7) Signaling pathways (8) Other markers (e.g., genetic markers such as inflammatory pathway genes).

In fiarther embodiments, the presence and/or level of one or both of the following markers can also be detected, measured, or determined in a patient sample (e.g., a serum sample from a t on anti-TNFu drug y): (9) anti-TNFu drug levels (e.g., levels of free anti-TNFOL therapeutic antibody); and/or (10) rug antibody (ADA) levels (e.g., levels of autoantibody to the anti-TNFu drug).

A single statistical algorithm or a combination of two or more statistical algorithms described herein can then be applied to the presence, concentration level, activation level, or pe of the markers detected, measured, or determined in the sample to thereby select therapy, optimize therapy, reduce ty, or monitor the efficacy of therapeutic treatment with an anti-TNFOL drug. As such, the methods of the invention flnd utility in determining patient management by determining t immune status. tanding the clinical course of disease will enable physicians to make better informed ent decisions for their inflammatory disease patients(e.g., IBD (e.g., s disease), rheumatoid arthritis (RA), others) and may help to direct new drug development in the fiature. The ideal biomarker(s) for use in the disease activity/severity index described herein should be able to identify individuals at risk for the disease and should be disease- specific. Moreover, the biomarker(s) should be able to detect disease activity and r the effect of treatment; and should have a predictive value towards relapse or recurrence of the disease. Predicting disease course, however, has now been expanded beyond just disease recurrence, but perhaps more importantly to include predictors of disease complications including surgery. The present invention is particularly advantageous because it provides indicators of disease activity and/or severity and enables a prediction of the risk of relapse in those patients in remission. In addition, the biomarkers and disease activity/severity index of present invention have enormous implications for patient management as well as therapeutic decision-making and would aid or assist in directing the appropriate y to those patients who would most likely benefit from it and avoid the expense and potential toxicity of chronic maintenance y in those who have a low risk of recurrence.

A. Inflammatory Markers Although disease course of an inflammatory disease is typically ed in terms of inflammatory activity by noninvasive tests using white blood cell count, this method has a low specificity and shows d correlation with disease activity.

[0138] As such, in certain embodiments, a variety of inflammatory markers, ing biochemical markers, serological markers, protein markers, c markers, and other clinical or echographic teristics, are particularly useful in the methods of the present invention for selecting therapy, optimizing therapy, reducing toxicity, and/or ring the efficacy of therapeutic treatment with one or more therapeutic agents such as biologics (e.g., NFOL drugs). In certain aspects, the methods described herein utilize the application of an algorithm (e.g, statistical analysis) to the presence, concentration level, and/or genotype determined for one or more inflammatory markers (e.g., alone or in combination with biomarkers from other categories) to aid or assist in predicting disease course, selecting an appropriate anti-TNFOL drug therapy, optimizing anti-TNFOL drug therapy, reducing toxicity associated with anti-TNFOL drug therapy, or monitoring the efficacy of therapeutic treatment with an anti-TNFOL drug.

Non-limiting examples of inflammatory markers suitable for use in the present invention e biochemical, serological, and protein markers such as, e.g., nes, Chemokines, acute phase proteins, cellular adhesion molecules, SlOO ns, and/or other inflammatory markers. 1. Cytokines and Chemokines The determination of the presence or level of at least one cytokine or chemokine in a sample is particularly useful in the present invention. As used , the term “cytokine” includes any of a variety of polypeptides or proteins secreted by immune cells that regulate a range of immune system fianctions and encompasses small cytokines such as ines.

The term “cytokine” also includes adipocytokines, which comprise a group of cytokines secreted by ytes that fianction, for e, in the regulation of body weight, hematopoiesis, angiogenesis, wound healing, insulin resistance, the immune response, and the inflammatory response.

In certain aspects, the presence or level of at least one cytokine ing, but not limited to, TNFu, lated weak inducer of apoptosis (TWEAK), osteoprotegerin (OPG), IFN-0L, IFN-B, IFN-y, IL- 1 0L, IL- 1 [3, IL-1 receptor antagonist (IL- lra), IL-2, IL-4, IL-5 , IL-6, soluble IL-6 receptor (sIL-6R), IL-7, IL-8, IL-9, IL-10, IL-12, IL-l3, IL-lS, IL-l7, IL-23, and IL-27 is determined in a sample. In n other aspects, the ce or level of at least one chemokine such as, for example, CXCLl/GROl/GROOL, CXCLZ/GROZ, CXCL3/GRO3, CXCL4/PF-4, CXCLS/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, CXCL9/MIG, CXCLlO/IP-lO, CXCLl l/I-TAC, CXCLlZ/SDF-l, CXCLl3/BCA-l, CXCLl4/BRAK, CXCLlS, CXCL l 6, CXCLl7/DMC, CCLl, CCL2/MCP- l, CCL3/MIP-l 0L, CCL4/MIP-l B, CCLS/RANTES, CCL6/ClO, CCL7/MCP-3, CCL8/MCP-2, CCL9/CCLlO, CCLl l/Eotaxin, CCLlZ/MCP-S, CCLl3/MCP-4, CCLl4/HCC-l, CCLlS/MIP-S, CCLl6/LEC, TARC, CCL18/MIP-4, CCLl9/MIP-3B, CCL20/MIP-30L, CCLZl/SLC, CCL22/MDC, CCL23/MPIFl, CCL24/Eotaxin-2, CCL25/TECK, Eotaxin-3, CCL27/CTACK, CCL28/MEC, CLl, CL2, and CX3CL1 is determined in a sample. In certain fiarther aspects, the presence or level of at least one ytokine ing, but not limited to, leptin, adiponectin, resistin, active or total plasminogen activator inhibitor-1 (PAI- l), visfatin, and retinol binding protein 4 (RBP4) is determined in a sample. Preferably, the ce or level of TNFu, IL-6, IL-8, IL- 1 [3, IL-2, IL- 1 2, IL- 1 3 , IL- 1 5 , IFN (e.g., IFN-0L, IFN-B, IFN-y), IL-lO, CCLS/RANTES, and/or other cytokines or chemokines is determined.

In certain instances, the presence or level of a particular cytokine or ine is detected at the level ofmRNA expression with an assay such as, for example, a hybridization assay or an amplification-based assay. In certain other instances, the presence or level of a particular cytokine or chemokine is detected at the level of protein expression using, for e, an immunoassay (e.g., ELISA) or an immunohistochemical assay. Suitable ELISA kits for determining the presence or level of a cytokine or chemokine of interest in a serum, plasma, saliva, or urine sample are available from, e.g., R&D Systems, Inc. apolis, MN), Neogen Corp. (Lexington, KY), Alpco Diagnostics (Salem, NH), Assay Designs, Inc.

(Ann Arbor, MI), BD Biosciences Pharmingen (San Diego, CA), ogen (Camarillo, CA), Calbiochem (San Diego, CA), CHEMICON International, Inc. (Temecula, CA), Antigenix America Inc. (Huntington Station, NY), QIAGEN Inc. cia, CA), Bio-Rad Laboratories, Inc. (Hercules, CA), and/or Bender tems Inc. (Burlingame, CA).

The human IL-6 polypeptide sequence is set forth in, e.g., Genbank Accession No.

NP_00059l. The human IL-6 mRNA (coding) sequence is set forth in, e.g., Genbank Accession No. NM_000600. One skilled in the art will appreciate that IL-6 is also known as interferon beta 2 (IFNB2), HGF, HSF, and BSF2.

The human IL-lB polypeptide sequence is set forth in, e.g., Genbank ion No.

NP_000567. The human IL-lB mRNA (coding) sequence is set forth in, e.g., k ion No. 576. One skilled in the art will appreciate that IL-lB is also known as ILlF2 and IL-lbeta.

The human IL-8 polypeptide sequence is set forth in, e.g., Genbank Accession No.

NP_000575 (SEQ ID NO: 1). The human IL-8 mRNA (coding) sequence is set forth in, e.g., Genbank Accession No. NM_0005 84 (SEQ ID NO:2). One skilled in the art will appreciate that IL-8 is also known as CXCL8, K60, NAF, GCPl, LECT, LUCT, NAPl, 3-lOC, GCP-l, LYNAP, MDNCF, MONAP, NAP-l, SCYB8, TSG-l, AMCF-I, and b-ENAP.

The human TWEAK ptide sequence is set forth in, e.g., Genbank Accession Nos. NP_003800 and AAC5 1923. The human TWEAK mRNA (coding) ce is set forth in, e.g., Genbank Accession Nos. NM_003 809 and BClO4420. One skilled in the art will appreciate that TWEAK is also known as tumor necrosis factor ligand superfamily member 12 (TNFSFl2), APO3 ligand (APO3L), CD255, DR3 ligand, growth factor- inducible l4 (Fnl4) , and UNQ18 l/PRO207. 2. Acute Phase Proteins The determination of the presence or level of one or more acute-phase proteins in a sample is also useful in the present invention. Acute-phase ns are a class of proteins whose plasma concentrations increase (positive acute-phase proteins) or decrease (negative acute-phase proteins) in response to inflammation. This response is called the acute-phase reaction (also called acute-phase response). Examples of positive acute-phase ns include, but are not limited to, C-reactive protein (CRP), D-dimer protein, mannose-binding protein, alpha l-antitrypsin, alpha l-antichymotrypsin, alpha 2-macroglobulin, fibrinogen, prothrombin, factor VIII, von Willebrand factor, nogen, complement factors, ferritin, serum amyloid P component, serum amyloid A (SAA), ucoid (alpha l-acid rotein, AGP), ceruloplasmin, haptoglobin, and combinations thereof. Non-limiting examples of negative acute-phase proteins include n, transferrin, transthyretin, transcortin, retinol-binding protein, and combinations thereof Preferably, the presence or level of CRP and/or SAA is determined.

In n ces, the presence or level of a particular acute-phase protein is detected at the level ofmRNA expression with an assay such as, for example, a hybridization assay or an amplification-based assay. In n other instances, the presence or level of a particular phase protein is detected at the level of protein expression using, for example, an immunoassay (e.g., ELISA) or an immunohistochemical assay. For example, a sandwich colorimetric ELISA assay available from Alpco Diagnostics (Salem, NH) can be used to determine the level of CRP in a serum, plasma, urine, or stool sample. Similarly, an ELISA kit available from Biomeda Corporation (Foster City, CA) can be used to detect CRP levels in a sample. Other s for determining CRP levels in a sample are described in, e. g., US. Patent Nos. 6,838,250 and 6,406,862; and US. Patent Publication Nos. 20060024682 and 20060019410. Additional methods for determining CRP levels include, e.g., immunoturbidimetry , rapid immunodiffusion assays, and visual agglutination assays. Suitable ELISA kits for determining the presence or level of SAA in a sample such as serum, plasma, saliva, urine, or stool are available from, e.g., Antigenix America Inc.

(Huntington Station, NY), Abazyme (Needham, MA), USCN Life (Missouri City, TX), and/or U.S. Biological (Swampscott, MA).

C-reactive n (CRP) is a protein found in the blood in response to ation (an acute-phase protein). CRP is typically produced by the liver and by fat cells (adipocytes).

It is a member of the pentraxin family of ns. The human CRP polypeptide sequence is set forth in, e.g., Genbank Accession No. NP_000558. The human CRP mRNA (coding) sequence is set forth in, e.g., Genbank Accession No. NM_000567. One skilled in the art will appreciate that CRP is also known as PTXl, MGC88244, and MGC149895.

Serum amyloid A (SAA) proteins are a family of apolipoproteins associated with high-density lipoprotein (HDL) in plasma. Different isoforms of SAA are expressed constitutively (constitutive SAAs) at ent levels or in response to inflammatory stimuli (acute phase SAAs). These proteins are predominantly produced by the liver. The conservation of these proteins throughout invertebrates and vertebrates suggests SAAs play a highly ial role in all animals. Acute phase serum amyloid A proteins (A-SAAs) are secreted during the acute phase of inflammation. The human SAA polypeptide sequence is set forth in, e.g., Genbank Accession No. NP_000322. The human SAA mRNA (coding) ce is set forth in, e.g., Genbank Accession No. NM_000331. One skilled in the art will appreciate that SAA is also known as PIG4, TP53I4, MGC111216, and SAA1. 3. Cellular Adhesion Molecules (IgSF CAMs) The determination of the presence or level of one or more immunoglobulin amily cellular adhesion molecules in a sample is also useful in the present invention.

As used herein, the term “immunoglobulin superfamily cellular adhesion molecule” (lgSF CAM) includes any of a y of polypeptides or proteins located on the surface of a cell that have one or more immunoglobulin-like fold s, and which fianction in intercellular adhesion and/0r signal transduction. In many cases, lgSF CAMS are transmembrane proteins.

Non-limiting examples of IgSF CAMS include Neural Cell Adhesion Molecules (NCAMs; e.g.,NCAM-l20,NCAM-l25,NCAM-l40,NCAM-l45,NCAM-180,NCAM-185, eta), lntercellular Adhesion les (lCAMs, e.g. and , lCAM-l, lCAM-2, lCAM-3, lCAM-4, ICAM-S), Vascular Cell Adhesion Molecule-l (VCAM-l), Platelet-Endothelial Cell Adhesion Molecule-l (PECAM-l), Ll Cell Adhesion Molecule (LlCAM), cell adhesion le With gy to LlCAM (close homolog of L1) (CHLl), sialic acid binding lg- like lectins (SlGLECs; e.g., SIGLEC-l, SIGLEC-2, SIGLEC-3, SlGLEC-4, eta), s (e.g., -l, Nectin-2, Nectin-3, etc), and Nectin-like molecules (e.g., Necl-l, , Neel-3, Neel-4, and Neel-5). Preferably, the presence or level of ICAM-1 and/or VCAM-l is determined. lCAM-l is a transmembrane cellular adhesion protein that is continuously present in low concentrations in the membranes of leukocytes and endothelial cells. Upon cytokine stimulation, the concentrations greatly increase. lCAM-l can be induced by lL-l and TNFOL and is expressed by the vascular endothelium, macrophages, and cytes. In IBD, proinflammatory cytokines cause inflammation by upregulating expression of adhesion molecules such as ICAM-1 and VCAM-l. The increased expression of adhesion molecules recruit more lymphocytes to the infected , resulting in tissue inflammation (see, Goke et al., J., Gastroenterol., 32:480 (1997); and Rijcken et al., Gut, 51:529 (2002)). ICAM-l is encoded by the intercellular adhesion molecule 1 gene (ICAMl; Entrez GenelD:3383; k Accession No. 201) and is produced after processing of the intercellular on molecule 1 precursor polypeptide (Genbank ion No. NP_000192).

VCAM-l is a transmembrane cellular adhesion protein that mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular elium.

Upregulation ofVCAM-1 in endothelial cells by cytokines occurs as a result of increased gene transcription (e.g., in response to Tumor necrosis factor-alpha (TNFOL) and Interleukin-l ). VCAM-l is encoded by the vascular cell adhesion molecule 1 gene (VCAMl; Entrez GeneID:7412) and is produced after differential ng of the transcript (Genbank Accession No. NM_001078 (variant 1) or NM_080682 (variant 2)), and processing of the sor polypeptide splice isoform nk Accession No. NP_001069 rm a) or NP_542413 (isoform b)).

In certain instances, the presence or level of an IgSF CAM is detected at the level of mRNA expression with an assay such as, for example, a hybridization assay or an amplification-based assay. In certain other instances, the presence or level of an IgSF CAM is detected at the level of protein expression using, for e, an immunoassay (e.g., ELISA) or an histochemical assay. Suitable antibodies and/or ELISA kits for ining the presence or level of ICAM-1 and/or VCAM-1 in a sample such as a tissue , , serum, plasma, , urine, or stool are available from, e.g., Invitrogen (Camarillo, CA), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and/or Abcam Inc.

(Cambridge, MA). 4. S100 Proteins

[0155] The determination of the ce or level of at least one S100 protein in a sample is also useful in the present invention. As used herein, the term "S100 protein" includes any member of a family of low molecular mass acidic proteins characterized by cell-type-speciflc expression and the presence of 2 EF-hand calcium-binding domains. There are at least 21 different types of S100 proteins in humans. The name is derived from the fact that S100 proteins are 100% soluble in ammonium sulfate at neutral pH. Most S100 proteins are homodimeric, consisting of two identical polypeptides held together by non-covalent bonds.

Although S100 proteins are structurally similar to calmodulin, they differ in that they are cell- specif1c, expressed in particular cells at different levels depending on environmental s.

S-100 proteins are normally present in cells derived from the neural crest (e.g., Schwann cells, melanocytes, glial cells), chondrocytes, adipocytes, myoepithelial cells, macrophages, Langerhans cells, dendritic cells, and keratinocytes. S100 proteins have been implicated in a variety of intracellular and extracellular fianctions such as the regulation of protein orylation, transcription factors, Ca2+ homeostasis, the dynamics of cytoskeleton constituents, enzyme activities, cell growth and differentiation, and the inflammatory I'GSpOIlSG.

Calgranulin is an S100 protein that is sed in multiple cell types, including renal epithelial cells and neutrophils, and are abundant in infiltrating monocytes and granulocytes under conditions of chronic inflammation. Examples of calgranulins include, 2012/025437 without limitation, calgranulin A (also known as S100A8 or MRP-8), nulin B (also known as S100A9 or ), and calgranulin C (also known as S100A12).

In certain instances, the presence or level of a particular S100 protein is detected at the level ofmRNA expression with an assay such as, for e, a hybridization assay or an amplification-based assay. In certain other instances, the presence or level of a particular S100 protein is detected at the level of protein expression using, for example, an immunoassay (e.g., ELISA) or an immunohistochemical assay. Suitable ELISA kits for determining the presence or level of an S100 protein such as calgranulin A (S100A8), calgranulin B (S100A9), or calgranulin C (S100A12) in a serum, plasma, or urine sample are available from, e.g., Peninsula Laboratories Inc. (San Carlos, CA) and Hycult biotechnology b.v. (Uden, The Netherlands).

Calprotectin, the complex of S100A8 and S100A9, is a calcium- and zinc-binding protein in the cytosol of neutrophils, tes, and nocytes. Calprotectin is a major protein in neutrophilic granulocytes and macrophages and ts for as much as 60% of the total protein in the cytosol fraction in these cells. It is therefore a surrogate marker of neutrophil turnover. Its concentration in stool correlates with the intensity of neutrophil infiltration of the intestinal mucosa and with the severity of ation. In some instances, calprotectin can be measured with an ELISA using small (50-100 mg) fecal samples (see, e.g., Johne et al., ScandJ Gastroenterol., 36:291-296 (2001)). 5. Other atory Markers The determination of the presence or level of errin in a sample is also useful in the present invention. In certain instances, the presence or level of lactoferrin is detected at the level ofmRNA expression with an assay such as, for example, a hybridization assay or an amplification-based assay. In certain other instances, the presence or level of lactoferrin is detected at the level of protein expression using, for example, an immunoassay (e.g., ELISA) or an immunohistochemical assay. A errin ELISA kit available from Calbiochem (San Diego, CA) can be used to detect human lactoferrin in a plasma, urine, bronchoalveolar lavage, or cerebrospinal fluid sample. Similarly, an ELISA kit available from US. Biological (Swampscott, MA) can be used to determine the level of lactoferrin in a plasma sample. US.

Patent ation No. 20040137536 describes an ELISA assay for determining the presence of elevated lactoferrin levels in a stool sample. Likewise, US. Patent Publication No. 20040033537 describes an ELISA assay for determining the concentration of endogenous lactoferrin in a stool, mucus, or bile sample. In some embodiments, then presence or level of 2012/025437 anti-lactoferrin antibodies can be detected in a sample using, e.g., lactoferrin protein or a fragment thereof.

The determination of the presence or level of one or more te kinase isozymes such as Ml-PK and M2-PK in a sample is also useful in the present invention. In certain instances, the presence or level of Ml-PK and/or M2-PK is detected at the level ofmRNA expression with an assay such as, for example, a hybridization assay or an amplification- based assay. In certain other ces, the presence or level of Ml-PK and/or M2-PK is detected at the level of protein expression using, for example, an assay (e.g., ELISA) or an immunohistochemical assay. Pyruvate kinase isozymes Ml/M2 are also known as te kinase muscle isozyme (PKM), pyruvate kinase type K, cytosolic thyroid hormone- binding protein (CTHBP), thyroid hormone-binding protein 1 (THBPl), or opa-interacting protein 3 (OIP3).

In fiarther embodiments, the determination of the presence or level of one or more growth factors in a sample is also useful in the present invention. Non-limiting examples of growth factors include transforming growth factors (TGF) such as TGF-u, TGF-B, TGF-BZ, , etc, which are described in detail below. 6. Exemplary Set of Inflammatory Markers In particular embodiments, at least one or a plurality (e.g., two, three, four, five, six, seven, eight, nine, ten, or more such as, e.g., a panel) of the following inflammatory markers can be detected (e.g., alone or in combination with biomarkers from other categories) to aid or assist in predicting disease course, and/or to e the cy of selecting y, optimizing therapy, reducing toxicity, and/or monitoring the efficacy of therapeutic treatment to anti-TNFu drug therapy: .9” CRP F7 SAA .0 VCAM .9— ICAM Calprotectin Lactoferrin :qorbo 1L8 Rantes H- TNFalpha j. IL-6 k. IL-lbeta l. S 10013112 m. MZ~pymvate kinase (PK) 11. IFN 0. 11.2 p. TGF q. IL-l3 r. IL-15 s. IL12 t. Other chemokines and cytokines.

B. Growth Factors A variety of growth factors, including biochemical markers, serological markers, protein markers, genetic markers, and other clinical or echographic teristics, are suitable for use in the methods of the present invention for selecting therapy, optimizing therapy, reducing ty, and/0r monitoring the efficacy of therapeutic treatment with one or more therapeutic agents such as biologics (e.g, anti-TNFu drugs). In certain aspects, the methods described herein utilize the application of an algorithm (e.g., statistical analysis) to the presence, concentration level, and/or genotype ined for one or more growth factors (e.g., alone or in combination with biomarkers from other categories) to aid or assist in ting disease course, selecting an appropriate NFu drug therapy, optimizing anti- TNFOL drug therapy, reducing toxicity associated with anti-TNFOL drug therapy, or ring the efficacy of therapeutic treatment with an anti-TNFOL drug.

As such, in certain ments, the determination of the presence or level of one or more growth factors in a sample is useful in the present invention. As used herein, the term "growth factor" includes any of a variety of es, polypeptides, or proteins that are e of stimulating cellular proliferation and/0r cellular differentiation.

In certain s, the presence or level of at least one growth factor including, but not limited to, epidermal growth factor (EGF), heparin-binding epidermal growth factor (HB- EGF), vascular endothelial growth factor (VEGF), t epithelium-derived factor (PEDF; also known as SERPINFl), amphiregulin (AREG; also known as schwannoma-derived growth factor (SDGF)), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), transforming growth factor-0t (TGF-0t), transforming growth factor-B (TGF-Bl, TGF- BZ, TGF-B3, eta), endothelin-1 , keratinocyte growth factor (KGF; also known as FGF7), bone morphogenetic proteins (e.g, BMPl-BMPlS), platelet-derived growth factor (PDGF), nerve growth factor (NGF), B-nerve growth factor (B-NGF), neurotrophic factors (e.g., brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), neurotrophin 4 (NT4), eta), growth differentiation factor-9 (GDF-9), ocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), myostatin (GDP-8), erythropoietin (EPO), and thrombopoietin (TPO) is determined in a sample. In particular embodiments, the presence or level of at least one , EGF, bFGF, ET-l, TGF-BZ and/or TGF-B3 is determined. These markers have been found to be significantly higher in active IBD than in controls, indicating that they may play a role in promoting healing after mucosal injury of the l surface of the intestine in IBD.

In n instances, the ce or level of a particular growth factor is detected at the level ofmRNA expression with an assay such as, for example, a hybridization assay or an cation-based assay. In certain other instances, the presence or level of a particular growth factor is detected at the level of protein expression using, for example, an immunoassay (e.g., ELISA) or an immunohistochemical assay. le ELISA kits for determining the presence or level of a growth factor in a serum, plasma, saliva, or urine sample are available from, e.g., Antigenix America Inc. (Huntington Station, NY), Promega (Madison, WI), R&D Systems, Inc. (Minneapolis, MN), InVitrogen (Camarillo, CA), CHEMICON International, Inc. (Temecula, CA), Neogen Corp. (Lexington, KY), PeproTech (Rocky Hill, NJ), Alpco Diagnostics (Salem, NH), Pierce Biotechnology, Inc. (Rockford, IL), and/or Abazyme (Needham, MA).

The human epidermal growth factor (EGF) ptide sequence is set forth in, e.g., Genbank ion No. NP_001954 (SEQ ID NO: 19). The human EGF mRNA g) sequence is set forth in, e.g., k Accession No. NM_001963 (SEQ ID NO:20). One skilled in the art will appreciate that EGF is also known as beta-urogastrone, URG, and HOMG4.

The human vascular endothelial growth factor (VEGF) polypeptide sequence is set forth in, e.g., Genbank Accession Nos. NP_001020537 (SEQ ID NO :21), NP_001020538, NP_001020539, NP_001020540, NP_001020541, NP_001028928, and 367. The human VEGF mRNA (coding) sequence is set forth in, e.g., Genbank Accession No.

NM_001025366 (SEQ ID , NM_001025367, NM_001025368, NM_001025369, NM_001025370, NM_001033756, and NM_003376. One skilled in the art will appreciate that VEGF is also known as VPF, VEGFA, VEGF-A, and MGC70609.

In particular embodiments, at least one or a plurality (e.g., two, three, four, five, six, seven, eight, nine, ten, or more such as, e.g., a panel) of the following growth factors can be detected (e.g., alone or in combination with biomarkers from other ries) to aid or assist in ting disease course, and/or to improve the accuracy of selecting therapy, optimizing therapy, reducing toxicity, and/or ring the efficacy of therapeutic treatment to anti- TNFOL drug therapy: ; VEGF; EGF; Keratinocyte growth factor (KGF; FGF7); and other growth factors.

C. Serology (Immune Markers) The determination of serological or immune markers such as autoantibodies in a sample (e.g., serum sample) is also useful in the present invention. Antibodies against anti- inflammatory molecules such as IL-lO, TGF-B, and others might suppress the body’s ability to control inflammation and the presence or level of these antibodies in the patient indicates the use of powerful immunosuppressive medications such as anti-TNFOL drugs. Mucosal healing might result in a decrease in the antibody titre of dies to bacterial antigens such as, e.g., OmpC, flagellins (cBir—l, Fla-A, Fla-X, etc), 12, and others , ASCA, etc.) As such, in certain aspects, the methods described herein utilize the ation of an algorithm (e.g, statistical analysis) to the presence, concentration level, and/or genotype determined for one or more immune markers (e.g., alone or in combination with biomarkers from other categories) to aid or assist in predicting e course, selecting an appropriate anti-TNFOL drug therapy, optimizing anti-TNFOL drug therapy, reducing toxicity associated with anti-TNFOL drug therapy, or monitoring the efficacy of therapeutic treatment with an anti-TNFOL drug.

Non-limiting examples of serological immune markers suitable for use in the present invention include anti-neutrophil antibodies, anti-Saccharomyces cerevisiae antibodies, and/or other anti-microbial antibodies. 1. Anti-Neutrophil Antibodies The determination ofANCA levels and/or the presence or absence ofpANCA in a sample is useful in the methods of the t invention. As used herein, the term "anti- phil cytoplasmic antibody" or "ANCA" includes antibodies directed to cytoplasmic and/or r components of neutrophils. ANCA activity can be divided into several broad categories based upon the ANCA staining n in phils: (l) cytoplasmic neutrophil staining without perinuclear highlighting (cANCA); (2) perinuclear staining around the outside edge of the nucleus (pANCA); (3) perinuclear staining around the inside edge of the nucleus (NSNA); and (4) diffuse staining with speckling across the entire neutrophil (SAPPA). In certain ces, pANCA ng is sensitive to DNase treatment. The term ANCA encompasses all varieties of anti-neutrophil reactivity, including, but not limited to, cANCA, pANCA, NSNA, and SAPPA. Similarly, the term ANCA encompasses all immunoglobulin isotypes including, without limitation, immunoglobulin A and G.

ANCA levels in a sample from an individual can be determined, for example, using an immunoassay such as an -linked immunosorbent assay (ELISA) with alcohol-fixed neutrophils. The presence or absence of a particular category ofANCA such as pANCA can be determined, for example, using an immunohistochemical assay such as an indirect cent antibody (IFA) assay. Preferably, the presence or absence A in a sample is determined using an immunofluorescence assay with DNase-treated, fixed neutrophils. In addition to fixed neutrophils, antigens specific for ANCA that are suitable for determining ANCA levels include, t limitation, f1ed or partially purified neutrophil extracts; purified proteins, protein fragments, or synthetic peptides such as histone H1 or ANCA- reactive fragments thereof (see, e.g., US. Patent No. 6,074,835); histone H1-like ns, porin antigens, oides antigens, or ANCA-reactive fragments thereof (see, e.g., US.

Patent No. 6,033,864); secretory vesicle antigens or ANCA-reactive fragments thereof (see, e. g., US. Patent Application No. 08/804,106); and anti-ANCA idiotypic antibodies. One skilled in the art will appreciate that the use of additional antigens specific for ANCA is within the scope of the present invention. 2. Anti-Saccharomyces cerevisiae Antibodies The determination ofASCA (e.g., ASCA-IgA and/or ASCA-IgG) levels in a sample is useful in the present invention. As used herein, the term "anti-Saccharomyces cerevz’sz’ae immunoglobulin A" or "ASCA-IgA" includes antibodies of the globulin A isotype that react specifically with S. cerevisiae. Similarly, the term "anti-Saccharomyces siae globulin G" or "ASCA-IgG" es antibodies of the immunoglobulin G isotype that react specifically with S. cerevisiae.

The ination of whether a sample is positive for ASCA-IgA or gG is made using an antigen specific for ASCA. Such an antigen can be any antigen or mixture of antigens that is bound cally by ASCA-IgA and/or ASCA-IgG. Although ASCA antibodies were initially characterized by their ability to bind S. cerevisiae, those of skill in the art will understand that an antigen that is bound specifically by ASCA can be obtained from S. cerevisiae or from a variety of other sources so long as the antigen is capable of g specifically to ASCA antibodies. Accordingly, exemplary sources of an antigen specific for ASCA, which can be used to determine the levels ofASCA-IgA and/or ASCA- IgG in a , include, without tion, whole killed yeast cells such as Saccharomyces or Candida cells; yeast cell wall mannan such as phosphopeptidomannan (PPM); oligosachharides such as oligomannosides; neoglycolipids; anti-ASCA idiotypic antibodies; and the like. Different species and strains of yeast, such as S. cerevisiae strain Sul, Su2, CBS 1315, or BM 156, or Candida albicans strain VW32, are suitable for use as an antigen specific for ASCA-IgA and/or ASCA-IgG. Purified and synthetic antigens specific for ASCA are also suitable for use in determining the levels -IgA and/or ASCA-IgG in a sample. Examples of purified antigens include, without limitation, purified oligosaccharide antigens such as oligomannosides. Examples of synthetic antigens include, without limitation, synthetic oligomannosides such as those described in US. Patent Publication No. 20030105060, e.g., D-Man [3(1-2) D-Man ) D-Man [3(1-2) D-Man-OR, D-Man ) D-Man ) D-Man a(1-2) D-Man-OR, and D-Man a(1-3) D-Man 0L(1-2) D-Man a(1-2) D- Man-OR, wherein R is a hydrogen atom, a C1 to C20 alkyl, or an optionally labeled connector group . ations of yeast cell wall mannans, e.g., PPM, can be used in ining the levels ofASCA-IgA and/or ASCA-IgG in a sample. Such water-soluble surface antigens can be prepared by any appropriate extraction technique known in the art, including, for example, by autoclaving, or can be obtained commercially (see, e. g., Lindberg et al., Gut, 33:909-913 (1992)). The acid-stable fraction of PPM is also useful in the statistical algorithms of the present invention d et al., Clin. Diag. Lab. Immanol., 3:219-226 (1996)). An exemplary PPM that is useful in determining ASCA levels in a sample is derived from S. avaram strain ATCC .

[0178] Purified oligosaccharide antigens such as oligomannosides can also be useful in determining the levels ofASCA-IgA and/or ASCA-IgG in a sample. The purified oligomannoside antigens are preferably converted into neoglycolipids as described in, for example, Faille et al., Eur. J. Microbiol. Infect. Dis., 11:438-446 . One skilled in the art understands that the reactivity of such an oligomannoside antigen with ASCA can be optimized by varying the mannosyl chain length (Frosh et al. , Proc Natl. Acad. Sci. USA, 82: 1 194-1 198 (1985)); the anomeric configuration awa et al. In ology of Fungal Disease," E. Kurstak (ed.), Marcel Dekker Inc., New York, pp. 37-62 (1989); Nishikawa et al., Microbiol. Immanol., 34:825-840 (1990); Poulain et al., Eur. J. Clin.

Microbiol., 23:46-52 (1993); Shibata et al., Arch. Biochem. Biophys, 8-348 (1985); Trinel et al., Infect. Immun, 60:3845-3851 (1992)); or the position of the e (Kikuchi et al., Planta, 190:525-535 (1993)).

Suitable oligomannosides for use in the methods of the present invention e, without limitation, an oligomannoside having the mannotetraose Man(1-3) Man(1-2) Man(1- 2) Man. Such an oligomannoside can be purified from PPM as described in, e.g., Faille et al., supra. An ary neoglycolipid specific for ASCA can be constructed by releasing the annoside from its respective PPM and subsequently coupling the released oligomannoside to 4-hexadecylaniline or the like. 3. Anti-Microbial Antibodies

[0180] The determination of anti-OmpC antibody levels in a sample is also useful in the present invention. As used herein, the term "anti-outer ne n C antibody" or "anti-OmpC antibody" includes antibodies directed to a bacterial outer membrane porin as described in, e. g., PCT Patent Publication No. W0 61. The term "outer membrane protein C" or "OmpC" refers to a bacterial porin that is immunoreactive with an anti-OmpC antibody.

The level of anti-OmpC antibody present in a sample from an individual can be determined using an OmpC protein or a fragment thereof such as an immunoreactive fragment thereof. Suitable OmpC ns useful in determining anti-OmpC antibody levels in a sample include, without limitation, an OmpC protein, an OmpC polypeptide having substantially the same amino acid ce as the OmpC protein, or a fragment thereof such as an immunoreactive fragment thereof As used herein, an OmpC polypeptide generally describes polypeptides having an amino acid sequence with r than about 50% identity, preferably greater than about 60% identity, more preferably greater than about 70% identity, still more preferably greater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with an OmpC protein, with the amino acid identity determined using a sequence alignment m such as CLUSTALW. Such antigens can be prepared, for example, by purification from enteric bacteria such as E. 0011', by recombinant expression of a c acid such as Genbank Accession No. K00541, by synthetic means such as solution or solid phase peptide synthesis, or by using phage display.

[0182] The determination of anti-I2 antibody levels in a sample is also useful in the present ion. As used herein, the term "anti-I2 dy" includes antibodies directed to a microbial antigen sharing gy to bacterial transcriptional regulators as described in, e.g., US. Patent No. 6,309,643. The term "12" refers to a microbial antigen that is immunoreactive with an anti-12 antibody. The microbial 12 protein is a polypeptide of 100 amino acids sharing some similarity weak homology with the predicted protein 4 from C. pasteurianum, RV3557c from Mycobacterium tuberculosis, and a transcriptional regulator from Aquz’fex us. The nucleic acid and protein sequences for the 12 protein are described in, e. g., US. Patent No. 6,309,643.

The level of anti-12 dy present in a sample from an indiVidual can be determined using an 12 n or a fragment thereof such as an immunoreactive fragment thereof. Suitable 12 antigens useful in determining anti-12 antibody levels in a sample e, without limitation, an 12 protein, an 12 polypeptide haVing substantially the same amino acid sequence as the 12 n, or a fragment thereof such as an immunoreactive fragment thereof Such 12 polypeptides t greater sequence similarity to the 12 protein than to the C. pasteurianum protein 4 and include isotype variants and homologs thereof. As used herein, an 12 polypeptide generally describes polypeptides haVing an amino acid sequence with greater than about 50% identity, preferably greater than about 60% ty, more preferably greater than about 70% ty, still more preferably greater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with a naturally-occurring 12 protein, with the amino acid identity ined using a sequence alignment program such as CLUSTALW. Such 12 ns can be prepared, for example, by purification from microbes, by recombinant sion of a c acid encoding an 12 antigen, by synthetic means such as solution or solid phase peptide sis, or by using phage display.

The determination of anti-flagellin antibody levels in a sample is also useful in the present invention. As used herein, the term "anti-flagellin antibody" includes antibodies ed to a protein component of bacterial flagella as described in, e.g., PCT Patent Publication No. WO 03/053220 and US. Patent Publication No. 20040043931. The term "flagellin" refers to a bacterial flagellum protein that is immunoreactive with an anti-flagellin antibody. Microbial flagellins are proteins found in bacterial flagellum that arrange lves in a hollow cylinder to form the filament.

The level of agellin antibody present in a sample from an indiVidual can be determined using a flagellin protein or a fragment thereof such as an immunoreactive fragment thereof. Suitable in antigens useful in determining anti-flagellin antibody levels in a sample include, without tion, a flagellin protein such as Cbir-l flagellin, flagellin X, flagellin A, flagellin B, fragments thereof, and combinations thereof, a flagellin polypeptide haVing substantially the same amino acid sequence as the flagellin protein, or a fragment thereof such as an immunoreactive fragment thereof. As used herein, a flagellin ptide generally describes polypeptides having an amino acid sequence with greater than about 50% identity, preferably greater than about 60% ty, more preferably greater than about 70% identity, still more preferably greater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with a naturally-occurring flagellin protein, with the amino acid identity determined using a ce alignment program such as LW. Such flagellin antigens can be prepared, e.g, by purification from bacterium such as Helicobacter Bill's, Helicobacter mustelae, Helicobacter pylori, Butyrz’vz’brz’o fibrisolvens, and bacterium found in the cecum, by recombinant expression of a nucleic acid ng a flagellin antigen, by synthetic means such as solution or solid phase peptide sis, or by using phage display.

D. Oxidative Stress Markers The determination of markers of oxidative stress in a sample is also useful in the present invention. miting examples of markers of oxidative stress include those that are protein-based or DNA-based, which can be detected by measuring protein oxidation and DNA ntation, respectively. Other examples of s of oxidative stress include organic compounds such as malondialdehyde.

Oxidative stress ents an imbalance between the production and manifestation of reactive oxygen species and a ical system's ability to readily fy the reactive intermediates or to repair the resulting damage. Disturbances in the normal redox state of s can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA. Some reactive oxidative species can even act as messengers through a phenomenon called redox signaling.

In certain embodiments, tives of reactive oxidative metabolites (DROMs), ratios of oxidized to reduced glutathione (Eh GSH), and/or ratios of oxidized to reduced cysteine (Eh CySH) can be used to quantify oxidative stress. See, e. g., Neuman et al., Clin.

Chem, 2-1657 (2007). Oxidative modifications of highly reactive cysteine residues in proteins such as tyrosine phosphatases and thioredoxin-related proteins can also be detected or measured using a technique such as, e.g., mass spectrometry (MS). See, e.g., Naito et al., Anti-Aging Medicine, 7 (5):36-44 (2010). Other markers of oxidative stress include protein- bound acrolein as described, e.g., in Uchida et al., PNAS, 95 (9) 4882-4887 (1998), the free oxygen radical test (FORT), which reflects levels of organic hydroperoxides, and the redox potential of the reduced hione/glutathione disulfide couple, (Eh) GSH/GSSG. See, e. g., on et al., Atherosclerosis, 178(1): 1 15-21 (2005).

E. Cell Surface Receptors The determination of cell surface receptors in a sample is also useful in the present invention. The half-life of anti-TNFu drugs such as Remicade and Humira is significantly decreased in patients with a high level of inflammation. CD64, the high-affinity receptor for immunoglobulin (Ig) G1 and IgG3, is predominantly sed by mononuclear phagocytes.

Resting polymorphonuclear (PMN) cells scarcely express CD64, but the expression of this marker is upregulated by interferon and granulocyte-colony-stimulating factor acting on myeloid precursors in the bone marrow. Crosslinking of CD64 with IgG complexes exerts a number of cellular ses, including the internalization of immune complexes by endocytosis, ytosis of opsonized particles, degranulation, tion of the oxidative burst, and the release of cytokines.

As such, in certain aspects, the methods described herein utilize the application of an algorithm (e.g, statistical analysis) to the presence, concentration level, and/or genotype determined for one or more cell surface receptors such as CD64 (e.g, alone or in combination with biomarkers from other categories) to aid or assist in predicting disease course, selecting an appropriate NFu drug therapy, zing anti-TNFOL drug therapy, reducing toxicity associated with anti-TNFOL drug therapy, or monitoring the y of therapeutic treatment with an anti-TNFu drug.

F. Signaling Pathways The determination of signaling pathways in a sample is also useful in the present ion. Polymorphonuclear (PMN) cell activation, followed by infltration into the intestinal mucosa (synovium for RA) and migration across the crypt epithelium is regarded as a key feature of IBD. It has been estimated by fecal indium-l l l-labeled leukocyte excretion that migration ofPMN cells from the circulation to the diseased section of the intestine is sed by 10-fold or more in IBD patients. Thus, in certain aspects, measuring activation ofPMN cells from blood or tissue ation by measuring signaling pathways using an assay such as the Collaborative Enzyme Enhanced Reactive ImmunoAssay (CEER) is an ideal way to understand inflammatory disease. The CEER technology is described in the following patent documents, which are each herein incorporated by reference in their entirety for all es: PCT Publication Nos. , , W0 2009/108637, , , and ; and PCT Application No. PCT/USZOl 1/066624.

As such, in certain aspects, the methods described herein utilize the application of an algorithm (e.g, statistical analysis) to the ce, concentration level, and/or genotype determined for one or more signal transduction molecules in one or more signaling pathways (e.g., alone or in combination with biomarkers from other categories) to aid or assist in predicting disease course, selecting an appropriate anti-TNFu drug therapy, zing anti- TNFOL drug therapy, reducing toxicity associated with NFOL drug therapy, or monitoring the efficacy of therapeutic treatment with an anti-TNFOL drug. In preferred embodiments, the total level and/or activation (e.g., phosphorylation) level of one or more signal transduction molecules in one or more signaling pathways is measured.

The term “signal transduction molecule” or l transducer” includes proteins and other molecules that carry out the process by which a cell converts an ellular signal or stimulus into a se, typically involving ordered sequences of biochemical reactions inside the cell. Examples of signal transduction molecules include, but are not limited to, receptor tyrosine kinases such as EGFR (e.g, ERl/ErbBl, HER2/Neu/ErbB2, HER3/ErbB3, HER4/ErbB4), VEGFRl/FLTl, VEGFRZ/FLKl/KDR, VEGFR3/FLT4, LK2, PDGFR (e.g., PDGFRA, PDGFRB), c-KIT/SCFR, INSR (insulin receptor), IGF-IR, IGF-HR, IRR (insulin receptor-related receptor), CSF-lR, FGFR l-4, HGFR 1-2, CCK4, TRK A-C, c-MET, RON, EPHA 1-8, EPHB 1-6, AXL, MER, TYRO3, TIE 1-2, TEK, RYK, DDR 1-2, RET, c-ROS, V-cadherin, LTK (leukocyte tyrosine kinase), ALK (anaplastic lymphoma kinase), ROR 1-2, MUSK, AATYK 1-3, and RTK 106; truncated forms of receptor ne kinases such as truncated HER2 receptors with missing amino- terminal extracellular domains (e.g, B2 (p95m), pl 10, p95c, p95n, eta), ted cMET receptors with missing amino-terminal extracellular domains, and truncated HER3 receptors with missing amino-terminal extracellular domains; receptor ne kinase dimers (e.g., p95HER2/HER3; p95HER2/HER2; truncated HER3 receptor with HERl , HER2, HER3, or HER4; HER2/HER2; HER3/HER3; HER2/HER3; HERl/HERZ; HERl/HER3; HER2/HER4; HER3/HER4; eta); non-receptor tyrosine kinases such as BCR—ABL, Src, Frk, Btk, Csk, Abl, Zap70, Fes/Fps, Fak, Jak, Ack, and LIMK; tyrosine kinase signaling e components such as AKT (e.g, AKTl, AKTZ, AKT3), MEK(MAP2K1), ERK2 (MAPKl), ERKl (MAPK3), PI3K (e.g., PIK3CA (pl 10), PIK3Rl (p85)), PDKl, PDK2, phosphatase and tensin g (PTEN), SGK3, 4E-BPl, P7OS6K (e.g., p70 S6 kinase splice variant alpha 1), protein tyrosine atases (e.g., PTPlB, PTPNl3, BDPl, eta), RAF, PLA2, MEKK, JNKK, JNK, p38, Shc (p66), Ras (e.g., K-Ras, N—Ras, H-Ras), Rho, Racl, Cdc42, PLC, PKC, p53, cyclin D1, STATl, STAT3, phosphatidylinositol 4,5-bisphosphate (PIP2), atidylinositol 3,4,5-trisphosphate (PIP3), mTOR, BAD, p21, p27, ROCK, 1P3, TSP-l, NOS, , RSK 1-3, JNK, c-Jun, Rb, CREB, Ki67, in, NF-kB, and IKK; nuclear e receptors such as estrogen receptor (ER), progesterone receptor (PR), en receptor, glucocorticoid or, mineralocorticoid receptor, vitamin A receptor, vitamin D receptor, retinoid receptor, thyroid hormone receptor, and orphan receptors; nuclear receptor coactivators and repressors such as amplified in breast cancer-l (AIBl) and nuclear or corepressor l (NCOR), respectively; and combinations thereof.

[0194] The term “activation state” refers to whether a particular signal transduction molecule is activated. Similarly, the term “activation level” refers to what extent a particular signal transduction molecule is activated. The activation state typically corresponds to the phosphorylation, ubiquitination, and/or complexation status of one or more signal transduction molecules. Non-limiting examples of activation states (listed in parentheses) include: HERl/EGFR (EGFRvIII, phosphorylated (p-) EGFR, EGFR:Shc, ubiquitinated (u-) EGFR, p-EGFRvIII); ErbB2 (p-ErbB2, p95HER2 ated ErbB2), p-p95HER2, ErbB2:Shc, ErbB2:PI3K, ErbB2:EGFR, ErbB2:ErbB3, ErbB2:ErbB4); ErbB3 (p-ErbB3, truncated ErbB3, ErbB3:PI3K, 3:PI3K, ErbB3:Shc); ErbB4 (p-ErbB4, ErbB4:Shc); c- MET (p-c-MET, truncated c-MET, c-Met:HGF complex); AKTl (p-AKTl); AKT2 (p- AKT2); AKT3 (p-AKT3); PTEN (p-PTEN); P7086K (p-P7OS6K); MEK (p-MEK); ERKl (p-ERKl); ERK2 2); PDKl (p-PDKl); PDK2 2); SGK3 (p-SGK3); 4E-BPl BPl); PIK3Rl (p-PIK3Rl); c-KIT (p-c-KIT); ER (p-ER); IGF-lR (p-IGF-lR, IGF- lR:IRS, IRS:PI3K, p-IRS, IGF-lR:PI3K); INSR (p-INSR); FLT3 (p-FLT3); HGFRl (p- HGFRl); HGFR2 (p-HGFR2); RET ); PDGFRA (p-PDGFRA); PDGFRB (p- PDGFRB); VEGFRl (p-VEGFRl, VEGFRl :PLCy, VEGFRl :Src); VEGFR2 (p-VEGFR2, VEGFR2:PLCy, VEGFR2:Src, VEGFR2:heparin sulphate, VEGFR2:VE-cadherin); VEGFR3 (p-VEGFR3); FGFRl (p-FGFRl); FGFR2 (p-FGFR2); FGFR3 (p-FGFR3); FGFR4 (p-FGFR4); TIEl (p-TIEl); TIE2 (p-TIE2); EPHA (p-EPHA); EPHB B); GSK-3B (p-GSK-3B); NF-kB (p-NF-kB, NF-kB-IkB alpha x and ), IkB (p-IkB, p-P65:IkB); IKK (phospho IKK); BAD (p-BAD, BAD:l43); mTOR (p-mTOR); Rsk-l (p- Rsk-l); Jnk (p-Jnk); P38 (p-P38); STATl (p-STATl); STAT3 T3); FAK (p-FAK); RB (p-RB); Ki67; p53 (p-p53); CREB (p-CREB); c-Jun (p-c-Jun); c-Src (p-c-Src); paxillin (p-paxillin); GRB2 (p-GRB2), Shc (p-Shc), Ras (p-Ras), GABl (p-GABl), SHP2 (p-SHP2), GRB2 (p-GRBZ), CRKL (p-CRKL), PLCy (p-PLCy), PKC (e.g., L, , p- PKCS), adducin (p-adducin), RBl (p-RBl), and PYK2 (p-PYKZ).

The following tables provide additional examples of signal transduction molecules for which total levels and/or activation (e.g., phosphorylation) levels can be determined in a sample (e.g., alone or in combination with biomarkers from other categories) to aid or assist in predicting disease course, selecting an appropriate anti-TNFu drug therapy, optimizing anti-TNFu drug therapy, reducing toxicity associated with anti-TNFu drug y, or ring the efficacy of therapeutic treatment with an anti-TNFu drug.

Table 1 Table 2 G. Genetic Markers The determination of the ce or absence of allelic variants (e.g, SNPs) in one or more genetic markers in a sample (e.g., alone or in combination with biomarkers from other categories) is also useful in the methods of the present invention to aid or assist in predicting disease course, selecting an appropriate anti-TNFu drug y, optimizing anti- TNFOL drug therapy, reducing toxicity associated with anti-TNFu drug therapy, or monitoring the efficacy of therapeutic treatment with an NFu drug.

Non-limiting examples of genetic markers include, but are not limited to, any of the inflammatory pathway genes and corresponding SNPs that can be genotyped as set forth in Table 3 (e.g., a NOD2/CARD15 gene, an ILl2/IL23 pathway gene, eta). Preferably, the presence or absence of at least one allelic variant, e.g, a single nucleotide polymorphism (SNP), in the NODZ/CARDlS gene and/or one or more genes in the ILl2/IL23 pathway is determined. See, e.g, Barrett et al., Nat. Genet., 40:955-62 (2008) and Wang et al., Amer. J.

Hum. Genet., 84:399-405 (2009).

Table 3 BTNLZ, SLC26A3, HLA-DRBl, 3 l3 PUSlO rsl3003464 CCLZ, CCL7 rs991804 LYRM4 rs12529198 SLC22A23 rs17309827 IL18RAP rs917997 IL12RB2 rs7546245 IL12RB1 rs374326 CD3D rs3212262 CD3G rs3212262 CD247 rs704853 rs6661505 CD3E 334 IL1 8R1 rs1035127 CCR5 MAPK14 rs2237093 IL 1 8 rs11214108 IFNG rs10878698 MAP2K6 rs2905443 STAT4 rs1584945 IL12A rs6800657 TYKZ 0356 ETV5 rs9867846 MAPK8 rs17697885 IRGM rs13361189 IRGM rs4958847 IRGM rs1000113 IRGM rs11747270 TL1A/TNFSF 1 5 rs6478109 NFSF 1 5 rs6478108 TL1A/TNFSF 1 5 rs4263 839 PTN22 rs2476601 CCR6 rs1456893 CCR6 rs2301436 TGER4 692 5p13/PTGER4 rs4495224 5p13/PTGER4 rs7720838 5p13/PTGER4 rs4613763 ITLN1 rs2274910 ITLN1 rs9286879 ITLN1 rsl 15843 83 IBD5/5q3 1 rs2188962 IBD5/5q31 rs252057 IBD5/5q31 rs10067603 GCKR rs780094 TNFRSF6B 135 ZNF365 rs224136 ZNF365 rs10995271 C1 10rf30 rs7927894 LRRK2;MUC19 rs1175593 IL-27 rs8049439 TLRZ rs4696480 TLRZ 099 TLRZ rs3804100 TLRZ rs5743704 TLRZ rs2405432 TLR4 (D299G) rs4986790 TLR4 (T3991) rs4986791 TLR4 (S360N) rs4987233 TLR9 rs187084 TLR9 rs352140 NFC4 rs4821544 KIF21B rs1 15843 83 IKZF1 rs1456893 C1 10rf30 rs7927894 CL7 rs991804 ICOSLG rs762421 TNFAIP3 rs7753394 FLJ45139 754 PTGER4 rs4613763 ECM1 rs7511649 ECM1 (T130M) rs3737240 ECM1 (G29OS) 4 GL11 (G933D) rs2228224 GL11 (Q1100E) rs2228226 MDR1 (3435C>T) rs1045642 MDR1 (A893 S/T) rs2032582 MAGIZ rs6962966 MAGIZ rs2160322 IL26 rs12815372 IFNG,IL26 rs1558744 IFNG,IL26 rs971545 IL26 rs2870946 ARPCZ rs12612347 IL10,IL19 rs3024493 IL 1 0,IL19 rs3024505 IL23R rs1004819 IL23R rs2201841 IL23R rs11465804 IL23R rs10889677 BTLNZ 480 HLA-DRB1 rs660895 MEP1 rs6920863 MEP1 658 MEP1 rs4714952 MEP1 rs1059276 PUS 1 0 rs13003464 PUS 1 0 689 RNF186 rs3806308 RNF186 rs1317209 RNF186 rs6426833 FCGRZA,C rs10800309 CEP72 rs4957048 DLD,LAMB1 rs4598195 CAPN1 0,KIF 1A rs4676410 IL23R rs11805303 IL23R 847 IL12B/p40 rs1368438 IL12B/p40 rs10045431 IL12B/p40 rs6556416 IL12B/p40 rs6887695 IL12B/p40 rs3212227 STAT3 rs744166 JAKZ rs10974914 JAKZ rs10758669 NKX2-3 rs6584283 NKX2-3 rs10883365 NKX2-3 0140 1L1 8RAP rs917997 LYRM4 rs12529198 CDKAL1 rs6908425 MAGIZ rs2160322 Additional SNPs useful in the present invention include, e.g., 962, rs9286879, rsl 1584383, rs7746082, rsl456893, rslSSl398, rsl7582416, rs3764l47, rsl736l35, rs4807569, rs7758080, and rs8098673. See, e.g., Barrett et al., Nat. Genet, 40:955-62 (2008). 1. NOD2/CARD15 The determination of the presence or absence of allelic variants such as SNPs in the NOD2/CARD15 gene is particularly useful in the present ion. As used herein, the term “NOD2/CARD15 variant” or “NOD2 variant” includes a nucleotide sequence of a NOD2 gene containing one or more changes as compared to the wild-type NOD2 gene or an amino acid sequence of a NOD2 polypeptide containing one or more changes as ed to the wild-type NOD2 polypeptide sequence. NOD2, also known as , has been localized to the IBDl locus on chromosome 16 and identified by positional-cloning (Hugot et al., Nature, 411:599-603 (2001)) as well as a onal candidate gene strategy (Ogura et al., Nature, 411:603-606 (2001); Hampe et al., Lancet, 357: 1925-1928 ). The IBDl locus has a high multipoint linkage score (MLS) for inflammatory bowel disease (MLS=5.7 at marker Dl6S4ll in l6q12). See, e.g., Cho et al., Inflamm. Bowel Dis, 190 (1997); r et al., Am. J. enterol., 96: 1 127-1 132 (2001); Ohmen et al., Hum. M01. Genet., :1679-1683 (1996); Parkes et al., Lancet, 348:1588 (1996); Cavanaugh et al., Ann. Hum.

Genet., 62:291-8 (1998); Brant et al., Gastroenterology, 115:1056-1061 (1998); Curran et al., Gastroenterology, 115:1066-1071 (1998); Hampe et al., Am. J. Hum. Genet., 64:808-816 ; and Annese et al., Eur. J. Hum. Genet., 7:567-573 (1999).

The mRNA (coding) and polypeptide sequences of human NOD2 are set forth in, e. g., Genbank Accession Nos. NM_022162 and NP_071445, respectively. In addition, the complete sequence of human chromosome 16 clone RP11-327F22, which includes NOD2, is set forth in, e.g., Genbank ion No. AC007728. Furthermore, the sequence ofNOD2 from other species can be found in the GenBank database.

The NOD2 n contains terminal caspase recruitment domains (CARDs), which can activate NF-kappa B (NF-kB), and several carboxy-terminal leucine-rich repeat domains (Ogura et al., J. Biol. Chem, 276:4812-4818 (2001)). NOD2 has structural homology with the apoptosis regulator Apaf—l/CED-4 and a class of plant disease ant gene products (Ogura et al., supra). Similar to plant disease resistant gene products, NOD2 has an amino-terminal effector domain, a nucleotide-binding domain and e rich repeats (LRRs). Wild-type NOD2 activates nuclear factor NF-kappa B, making it responsive to bacterial lipopolysaccharides (LPS; Ogura et al., supra; Inohara et al., J. Biol. Chem, 276:2551-2554 (2001). NOD2 can function as an intercellular or for LPS, with the leucine rich repeats required for responsiveness.

Variations at three single nucleotide polymorphisms in the coding region ofNOD2 have been previously described. These three SNPs, designated R702W (“SNP 8”), G908R (“SNP 12”), and 1007fs (“SNP 13”), are located in the carboxy-terminal region of the NOD2 gene (Hugot et al., supra). A fiarther ption of SNP 8, SNP 12, and SNP 13, as well as additional SNPs in the NOD2 gene suitable for use in the invention, can be found in, e.g., US. Patent Nos. 6,835,815; 6,858,391; and 7,592,437; and US. Patent ation Nos. 90639, 20050054021, and 20070072180.

In some embodiments, a NOD2 variant is located in a coding region of the NOD2 locus, for example, within a region encoding several leucine-rich repeats in the carboxy- terminal portion of the NOD2 polypeptide. Such NOD2 variants located in the leucine-rich repeat region ofNOD2 include, without limitation, R702W (“SNP 8”) and G908R (“SNP 12”). A NOD2 t useful in the invention can also encode a NOD2 polypeptide with reduced ability to activate NF-kappa B as compared to NF-kappa B activation by a wild-type 2012/025437 NOD2 polypeptide. As a non-limiting example, the NOD2 t 1007fs (“SNP 13”) results in a truncated NOD2 polypeptide which has d ability to induce NF-kappa B in response to LPS stimulation (Ogura et al., Nature, 411:603-606 (2001)).

A NOD2 variant useful in the invention can be, for example, R702W, G908R, or 1007fs. R702W, G908R, and 1007fs are located within the coding region . In one embodiment, a method of the invention is practiced with the R702W NOD2 variant. As used herein, the term “R702W” includes a single nucleotide polymorphism within exon 4 of the NOD2 gene, which occurs within a t encoding amino acid 702 of the NOD2 protein.

The wild-type NOD2 allele contains a cytosine (c) residue at position 138,991 of the AC007728 sequence, which occurs within a triplet ng an arginine at amino acid702.

The R702W NOD2 variant contains a thymine (t) e at position 1 of the AC007728 sequence, resulting in an arginine (R) to phan (W) substitution at amino acid 702 of the NOD2 protein. Accordingly, this NOD2 variant is denoted “R702W” or “702W” and can also be denoted “R675W” based on the earlier numbering system of Hugot et al., supra. In addition, the R702W variant is also known as the “SNP 8” allele or a “2” allele at SNP 8. The NCBI SNP ID number for R702W or SNP 8 is rs2066844. The presence of the R702W NOD2 variant and other NOD2 variants can be conveniently detected, for example, by allelic discrimination assays or sequence analysis.

A method of the invention can also be practiced with the G908R NOD2 variant. As used herein, the term “G908R” includes a single nucleotide polymorphism within exon 8 of the NOD2 gene, which occurs within a triplet encoding amino acid 908 of the NOD2 n.

Amino acid 908 is located within the leucine rich repeat region of the NOD2 gene. The wild- type NOD2 allele contains a guanine (g) residue at position 128,377 of the AC007728 sequence, which occurs within a triplet encoding glycine at amino acid 908. The G908R NOD2 variant contains a cytosine (c) residue at position 128,377 of the AC007728 sequence, resulting in a glycine (G) to arginine (R) tution at amino acid 908 of the NOD2 n.

Accordingly, this NOD2 variant is denoted “G908R” or “908R” and can also be denoted “G88 1R” based on the earlier numbering system of Hugot et al., supra. In addition, the G908R variant is also known as the “SNP 12” allele or a “2” allele at SNP 12. The NCBI SNP ID number for G908R SNP 12 is 845.

A method of the invention can also be practiced with the 1007fs NOD2 variant.

This variant is an insertion of a single nucleotide that results in a frame shift in the tenth e-rich repeat of the NOD2 protein and is followed by a premature stop codon. The resulting truncation of the NOD2 protein appears to prevent activation ofNF-kappaB in WO 54253 response to ial lipopolysaccharides (Ogura et al., supra). As used herein, the term “1007fs” includes a single nucleotide polymorphism within exon 11 of the NOD2 gene, which occurs in a t encoding amino acid 1007 of the NOD2 protein. The 1007fs variant contains a ne which has been added at position 121,139 of the AC007728 sequence, resulting in a frame shift mutation at amino acid 1007. Accordingly, this NOD2 variant is denoted “1007fs” and can also be denoted “3020insC” or “980fs” based on the r numbering system of Hugot et al., supra. In addition, the 1007fs NOD2 variant is also known as the “SNP 13” allele or a “2” allele at SNP 13. The NCBI SNP ID number for 1007fs or SNP 13 is rs2066847.

[0207] One skilled in the art recognizes that a particular NOD2 variant allele or other polymorphic allele can be conveniently defined, for example, in comparison to a Centre d’Etude du Polymorphisme Humain (CEPH) reference individual such as the individual ated 2 (Dib et al., Nature, 380: 152-154 (1996)), using commercially available nce DNA obtained, for example, from PE Biosystems (Foster City, CA). In addition, specific information on SNPs can be obtained from the dbSNP of the National Center for Biotechnology Information (NCBI).

A NOD2 variant can also be located in a non-coding region of the NOD2 locus. ding regions include, for example, intron sequences as well as 5’ and 3’ untranslated ces. A non-limiting example of a NOD2 variant allele located in a non-coding region of the NOD2 gene is the JWl variant, which is described in Sugimura et al., Am. J. Hum.

Genet., 72:509-518 (2003) and US. Patent Publication No. 20070072180. Examples of NOD2 variant s located in the 3 ’ untranslated region of the NOD2 gene include, without limitation, the JW15 and JW16 variant s, which are described in US. Patent Publication No. 20070072180. Examples ofNOD2 variant s located in the 5’ untranslated region (e.g., promoter region) of the NOD2 gene include, without limitation, the JW17 and JW18 variant alleles, which are described in US. Patent Publication No. 20070072180.

As used herein, the term “JWl variant allele” includes a genetic variation at nucleotide 158 of intervening sequence 8 (intron 8) of the NOD2 gene. In relation to the AC007728 sequence, the JWl variant allele is located at position 128,143. The genetic variation at nucleotide 158 of intron 8 can be, but is not limited to, a single tide substitution, multiple nucleotide substitutions, or a deletion or insertion of one or more tides. The wild-type sequence of intron 8 has a cytosine at position 158. As non- ng examples, a JWl variant allele can have a cytosine (c) to adenine (a), cytosine (c) to guanine (g), or cytosine (c) to thymine (t) substitution at nucleotide 158 of intron 8. In one WO 54253 embodiment, the JWl variant allele is a change from a cytosine (c) to a thymine (t) at nucleotide 158 ofNOD2 intron 8.

The term “JW15 variant allele” includes a genetic variation in the 3’ untranslated region ofNOD2 at nucleotide position 118,790 of the AC007728 sequence. The c variation at nucleotide 118,790 can be, but is not limited to, a single nucleotide tution, multiple nucleotide tutions, or a deletion or insertion of one or more tides. The ype sequence has an adenine (a) at position 118,790. As non-limiting examples, a JW15 variant allele can have an adenine (a) to cytosine (c), adenine (a) to guanine (g), or adenine (a) to thymine (t) tution at nucleotide 118,790. In one embodiment, the JW15 variant allele is a change from an adenine (a) to a cytosine (c) at nucleotide 118,790.

As used herein, the term “JW16 variant allele” includes a genetic variation in the 3 ’ slated region ofNOD2 at nucleotide position 118,031 of the 28 sequence. The genetic variation at nucleotide 118,031 can be, but is not limited to, a single nucleotide substitution, multiple nucleotide substitutions, or a deletion or insertion of one or more nucleotides. The Wild-type sequence has a guanine (g) at position 118,031. As non-limiting examples, a JW16 variant allele can have a guanine (g) to cytosine (c), guanine (g) to adenine (a), or guanine (g) to thymine (t) tution at nucleotide 118,031. In one embodiment, the JW16 variant allele is a change from a guanine (g) to an e (a) at tide 118,031.

The term “JW17 variant allele” includes a genetic variation in the 5 ’ untranslated region ofNOD2 at tide position 154,688 of the AC007728 sequence. The genetic variation at tide 154,688 can be, but is not limited to, a single nucleotide substitution, multiple nucleotide substitutions, or a deletion or insertion of one or more nucleotides. The Wild-type sequence has a cytosine (c) at position 154,688. As non-limiting examples, a JW17 variant allele can have a ne (c) to guanine (g), cytosine (c) to adenine (a), or cytosine (c) to thymine (t) substitution at nucleotide 154,688. In one embodiment, the JW17 variant allele is a change from a cytosine (c) to a thymine (t) at nucleotide 154,688.

As used herein, the term “JWl 8 variant allele” includes a genetic variation in the 5 ’ slated region ofNOD2 at nucleotide on 154,471 of the AC007728 sequence. The genetic variation at nucleotide 1 can be, but is not limited to, a single nucleotide substitution, multiple nucleotide substitutions, or a deletion or insertion of one or more nucleotides. The Wild-type sequence has a cytosine (c) at position 154,471. As non-limiting examples, a JW18 variant allele can have a cytosine (c) to guanine (g), cytosine (c) to adenine (a), or cytosine (c) to thymine (t) substitution at nucleotide 154,471. In one embodiment, the JW18 variant allele is a change from a ne (c) to a thymine (t) at nucleotide 154,471.

It is understood that the methods of the ion can be practiced with these or other NOD2 variant alleles located in a coding region or non-coding region (e.g., intron or promoter region) of the NOD2 locus. It is further understood that the s of the invention can involve determining the presence of one, two, three, four, or more NOD2 variants, including, but not limited to, the SNP 8, SNP 12, and SNP 13 alleles, and other coding as well as ding region variants.

V. Examples

[0215] The present invention will be described in greater detail by way of specific examples. The following es are offered for rative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.

Example 1. Novel Mobility Shift Assay for Measuring Levels of anti-TNFa Biologics.

This example illustrates a novel homogeneous assay for measuring anti-TNFOL drug concentration in a patient sample (e.g., serum) using size exclusion chromatography to detect the g of the anti-TNFu drug to fluorescently labeled TNFOL. The assay is advantageous because it obviates the need for wash steps, uses fluorophores that allow for detection on the visible and/or IR spectra which decreases background and serum interference , increases the ability to detect anti-TNFOL drugs in patients with a low titer due to the high sensitivity of cent label detection, and occurs as a liquid phase reaction, thereby reducing the chance of any changes in the epitope by attachment to a solid surface such as an ELISA plate.

[0217] In one exemplary embodiment, TNFu is labeled with a fluorophore (e.g., Alexa647), wherein the fluorophore can be detected on either or both the visible and IR spectra. The labeled TNFOL is incubated with human serum in a liquid phase reaction to allow the anti- TNFOL drug t in the serum to bind. The labeled TNFOL can also be incubated with known amounts of the anti-TNFu drug in a liquid phase reaction to create a standard curve.

Following incubation, the samples are loaded directly onto a size exclusion column. Binding of the anti-TNFOL drug to the labeled TNFu results in a leftward shift of the peak compared to labeled TNFu alone. The concentration of the NFOL drug present in the serum sample can then be compared to the standard curve and controls.

Figure 1 shows an example of the assay of the present invention n size exclusion HPLC is used to detect the binding n TNFu-Alexa647 and HUMIRATM mumab). As shown in Figure l, the binding of HUMIRATM to TNFu-Alexa647 caused a shift of the TNFu-Alexa647 peak to the left.

Figure 2 shows dose se curves of HUMIRATM g to TNFu-Alexa647. In ular, Figure 2A shows that HUMIRATM dose-dependently increased the shift of TNFOL- Alexa647 in the size exclusion chromatography assay. Figure 2B shows that the presence of 1% human serum did not have a significant effect on the shift of TNFu-Alexa647 in the size exclusion chromatography assay. Figure 2C shows that the presence of pooled RF-positive serum did not have a significant effect on the shift of TNFu-Alexa647 in the size exclusion chromatography assay.

As such, this example demonstrates the utility of the present invention in ring ts receiVing an anti-TNFu drug such as HUMIRAT'V': (l) to guide in the determination of the appropriate drug dosage; (2) to evaluate drug pharmacokinetics, e.g., to determine whether the drug is being cleared from the body too quickly; and (3) to guide treatment decisions, e. g., whether to switch from the t anti-TNFu drug to a different TNFu inhibitor or to r type of therapy.

Example 2. Novel Mobility Shift Assay for Measuring HACA and HAHA Levels.

This example illustrates a novel homogeneous assay for measuring autoantibody (e.g., HACA and/or HAHA) concentrations in a patient sample (e.g., serum) using size exclusion chromatography to detect the binding of these autoantibodies to fluorescently labeled anti-TNFu drug. The assay is advantageous because it obviates the need for wash steps which remove low affinity HACA and HAHA, uses fluorophores that allow for detection on the visible and/or IR spectra which decreases background and serum interference issues, increases the ability to detect HACA and HAHA in patients with a low titer due to the high sensitiVity of fluorescent label detection, and occurs as a liquid phase reaction, thereby reducing the chance of any changes in the epitope by ment to a solid surface such as an ELISA plate.

The clinical utility of measuring tibodies (e.g., HACA, HAHA, etc.) that are generated against TNFu inhibitors is illustrated by the fact that HACAs were detected in 53%, 21%, and 7% of rheumatoid arthritis patients treated with 1 mg/kg, 3 mg/kg, and 10 mg/kg infliximab. When infliximab was combined with methotrexate, the incidence of antibodies was lower 15%, 7%, and 0%, which indicates that concurrent immunosuppressive therapy is effective in lowering rug ses, but also indicates that a high dose of anti-TNFOL antibody might lead to tolerance. In Crohn’s disease, a much higher incidence was reported; after the fifth infusion, 61% of patients had HACA. The clinical response was shortened when HACAs were present. See, Rutgeerts, N. Engl. J. Med, 348:601-608 (2003).

A pective study of infliximab and HACA levels measured over a 3 year period from 2005 to 2008 in 155 patients demonstrated that HACAs were detected in 22.6% (N = 35) of patients with inflammatory bowel disease. See, Af1f et al. , “Clinical Utility of Measuring Infliximab and Human Anti-Chimeric dy Levels in Patients with Inflammatory Bowel Disease”; paper presented at Digestive Disease Week; May 30-June 3, 2009; Chicago, IL.

The authors concluded that changing ent based on clinical symptoms alone may lead to inappropriate management.

The homogeneous mobility shift assay is advantageous over t methods such as the bridging assay shown in Figure 3 for measuring autoantibody (e.g., HACA and/or HAHA) concentrations in a patient sample e the inventive method is capable of ing the concentration of autoantibodies such as HACA without non-specific binding and solid phase interference from the ELISA plate, without erence from the NFOL drug (e.g., with the bridging assay, HACA measurements must be taken at anti-TNFOL drug trough levels), and without any dependency on the multivalency of the antibody (e.g., IgG4 antibodies are not detected using the bridging assay because IgG4 antibodies are bispecif1c and cannot cross-link the same antigen). As such, the present invention has at least the ing advantages over current methods: avoids attachment of antigens to solid surfaces (denaturation avoided); eliminates the IgG4 effect; overcomes therapeutic antibody trough issues; detects antibodies with weak affinities; eliminates non-specific binding of irrelevant IgGs; and increases the sensitivity and specificity of detection.

In one exemplary ment, an anti-TNFOL drug (e.g., DET'V') is labeled with a fluorophore (e.g, Alexa647), wherein the fluorophore can be detected on either or both the visible and IR spectra. The labeled anti-TNFOL drug is incubated with human serum in a liquid phase reaction to allow HACA and HAHA t in the serum to bind. The labeled anti-TNFOL drug can also be incubated with known amounts of an anti-IgG antibody in a liquid phase reaction to create a standard curve. Following incubation, the s are loaded directly onto a size exclusion column. Binding of the autoantibodies to the labeled anti-TNFu drug results in a leftward shift of the peak compared to labeled drug alone. The concentration of HACA and HAHA present in the serum sample can then be compared to the standard curve and controls. Figure 4 illustrates an exemplary outline of the autoantibody detection assays of the present invention for measuring the concentrations of HACA/HAHA generated against REMICADETM. In n instances, high HACA/HAHA levels indicate that the current therapy with REMICADET'V' should be switched to another anti-TNFOL drug such as HUMIRAT'V'.

The principle of this assay is based on the mobility shift of the antibody bound Alexa647-labeled Remicade complex versus free Alexa647-labeled de on size exclusion- high performance liquid chromatography (SE-HPLC) due to the increase in molecular weight of the x.

[0226] The chromatography in this example was performed on an Agilent-1200 HPLC System, using a Bio-Sep 300x7.8 mm SEC-3000 column (Phenomenex) with a molecular weight fractionating range of 5,000 — 700,000 and a mobile phase of IX PBS, pH 7.4, at a flow-rate of 0.5 mL/min with UV detection at 650 nm. A 100 uL sample volume is loaded onto the column for each analysis.

[0227] The antibody bound 47-labeled de complex is formed by incubating a known amount of the antibody and Alexa647-labeled Remicade in the 1X PBS, pH 7.3, elution buffer at room temperature for 1 hour before SE-HPLC is.

Figure 5 shows a dose response analysis of anti-human IgG antibody binding to REMICADETM-Alexa647 as detected using the size exclusion chromatography assay of the present invention. The binding of anti-IgG antibody to REMICADETM-Alexa647 caused a shift of the REMICADETM-Alexa647 peak to the left. Figure 6 shows a second dose response analysis of anti-human IgG dy binding to DETM-Alexa647 as detected using the size exclusion chromatography assay of the present invention. Higher amounts of anti-IgG antibody resulted in a ependent increase in the formation of anti-IgG/REMICADETM- Alexa647 complexes, as ted by a shift of the REMICADETM-Alexa647 peak to the left.

Figure 7 shows dose response curves of anti-IgG dy binding to REMICADETM- Alexa647.

Figure 8 shows REMICADETM-Alexa647 immunocomplex formation in normal human serum and HACA positive serum as detected using the size exclusion chromatography assay of the present invention with 100 ul of injected . As shown in Figure 8, the binding ofHACA present in patient samples to REMICADETM-Alexa647 caused a shift of the REMICADETM-Alexa647 peak to the left. As such, the size exclusion chromatography assay of the invention is particularly advantageous because it es HACA in the presence of REMICADETM, can be utilized while the patient is on therapy, measures both weak and strong HACA binding, is a mix and read mobility shift assay, and can be extended to other approaches which use labeled REMICADET'V' to equilibrate with HACA and REMICADETM.

Figure 9 provides a summary ofHACA measurements from 20 patient serum samples that were performed using the bridging assay or the mobility shift assay of the present invention. This comparative study demonstrates that the present methods have increased sensitivity over current methods because 3 samples that were negative for HACA as measured using the bridging assay were actually HACA positive when measured using the mobility shift assay of the present invention (see, t # 0305, SK07070595, and SK07l 10035).

As such, this example trates the utility of the present invention in monitoring patients receiving an NFu drug (e.g, REMICADET'V') to detect the presence or level of tibodies (e.g, HACA and/or HAHA) against the drug, because such immune ses can be associated with hypersensitive reactions and dramatic s in pharmacokinetics and tribution of the anti-TNFu drug that preclude r treatment with the drug.

In conclusion, Examples 1 and 2 demonstrate that TNFu and anti-TNFu antibodies can be ently labeled with Alexa647. When labeled TNFu-Alexa647 was incubated with anti-TNFu antibodies, the retention time of the labeled TNFu/anti-TNFOL antibody complex was shifted, and the amount of anti-TNFu antibody that caused the shift could be quantitated with HPLC. Furthermore, when labeled anti-TNFor antibody was incubated with anti-human IgG antibody, the retention time of the labeled anti-TNFu antibody/anti-IgG antibody complex was shifted, and the amount of anti-IgG antibody that caused the shift could be quantitated with HPLC. Moreover, low serum content in the assay system was shown to have little effect on HPLC analysis. Finally, a standard curve could be generated for the anti- TNFOL dy and HACA/HAHA assays and could be used to quantitate patient serum anti- TNFu antibody or HACA/HAHA levels. Advantageously, the present invention es in certain aspects a mobility shift assay, such as a homogeneous mix and read assay developed to measure both drug and antibodies against the drug. A standard curve was generated for the anti-TNFu biologic Remicade and Humira and also for the HACA antibodies against Remicade. The mobility shift assay format, unlike ELISA, eliminates coating of antigens to solid surface and is not affected by non-specif1c binding of irrelevant IgGs. The assay format is simple, but very ive and can be used to detect all anti-TNFOL biologic drugs (e.g., Remicade, Humira, Enbrel and Cimzia) as well as the neutralizing antibody (anti- RemicadeTM) in patient serum.

Example 3. Measurement of Human Anti-Chimeric Antibodies (HACA) and Infliximab (IFX) Levels in t Serum Using A Novel Mobility Shift Assay.

ABSTRACT ound: Infliximab (IFX) is a chimeric monoclonal antibody therapeutic against TNFu that has been shown to be effective in treating autoimmune es such as rheumatoid arthritis (RA) and atory bowel disease (IBD). However, antibodies against IFX were found in some IFX-treated patients through the detection of human anti- chimeric antibodies (HACA), which may reduce the drug’s efficacy or induce adverse s. Monitoring ofHACA and IFX levels in individual patients may help to optimize the dosing and treatment with IFX. Current methods for detecting HACA are based on solid- phase assays, which are limited by the fact that the presence of IFX in the circulation may mask the presence ofHACA and, therefore, ement can only be done at least 8 weeks following a dose of IFX. Moreover, this time-lapse fiarther confounds the assays because of the rapid nce of the high molecular weight immune complexes in the blood circulation.

To overcome these drawbacks, we have developed and evaluated a new method to measure serum IFX and HACA levels in patients treated with IFX.

Methods: A novel non-radiolabeled, liquid-phase, size-exclusion (SE)—HPLC mobility shift assay was developed to measure the HACA and IFX levels in serum from patients treated with IFX. The immuno-complex (e.g., TNFu/IFX or IFX/HACA), free TNFOL or IFX, and the ratio of bound/free can be resolved and calculated with high sensitivity.

Serum concentrations of IFX or HACA were determined with standard curves generated by incubating with different trations of IFX or pooled ositive serum. Using this novel assay, we have measured IFX and HACA levels in sera ted from IBD patients treated with IFX who had relapsed and compared the results with those obtained by the ional Bridge ELISA assay.

Results: Dose-response curves were generated from the novel assay with high ivity. Detection ofHACA was demonstrated in the presence of excess IFX. In the 117 serum samples from patients treated with IFX, 65 samples were found to have IFX levels above the detection limit and the average was ll.0+6.9 mg/mL. For HACA levels, 33 (28.2%) samples were found to be positive while the Bridge ELISA assay detected only 24 positive samples. We also identified 9 false negatives and 9 false positives from the samples determined by the Bridge assay. HACA levels were found to be increased in 11 patients during the course of IFX treatment while the IFX levels were found to be significantly decreased .

Conclusions: A novel diolabeled, liquid-phase, ty shift assay has been developed to measure the IFX and HACA levels in serum from patients treated with IFX.

The assay has high sensitivity and accuracy, and the obtained results were reproducible. This novel assay can ageously be used to measure HACA and IFX levels while patients are on y.

INTRODUCTION

[0237] Tumor necrosis factor-alpha (TNFu) plays a pivotal role in the pathogenesis of autoimmune diseases such as Crohn’s disease (CD) and rheumatoid arthritis (RA). It is well documented that blocking TNFOL with therapeutic antibodies such as Infliximab (human- murine chimeric monoclonal IgGlK) or adalimumab (fillly human monoclonal antibody) reduces disease activity in CD and RA. However, about 30-40% of the patients do not respond to anti-TNFu therapy and some patients need higher doses or dosing frequency ments due to lack of sufficient response. Differences of drug bioavailability and pharmacokinetics in individual patients may contribute to the failure of the treatment. genicity of the drugs, which causes patients to p HACA/HAHA, could result in a range of e reactions from mild allergic se to anaphylactic shock. These problems are now recognized by many igators, drug-controlling agencies, health insurance companies, and drug manufacturers. Furthermore, many patients with secondary response failure to one anti-TNFOL drug t from a switch to other anti-TNFOL drugs, suggesting a role of neutralizing antibodies directed specifically against the protein used for treatment (Radstake et al., Ann. Rheum. Dis., 68(11):l739-45 (2009)). Monitoring of patients for drug and HACA/HAHA levels is therefore warranted so that drug administration can be tailored to the dual t and prolonged ies can be given effectively and economically with little or no risk to patients (Bendtzen et (1]., Scand. J. Gastroenter01., 44(7):774-81 (2009)).

Several enzyme-linked immunoassays have been used to assess the circulating levels of drugs and HACA/HAHA. Figure 10 provides a summary of the current assays available for the measurement of HACA in comparison to the novel HACA assay of the present invention. One of the limitations of current methodologies is that antibody levels are difficult to measure when there is a measurable amount of drug in the circulation. In contrast to current solid-phase methods for detecting HACA in which measurements can only be performed at least 8 weeks following a dose of IFX, the novel assay of the present invention is a non-radiolabeled, liquid-phase, size-exclusion (SE)-HPLC assay that is e of measuring HACA and IFX levels in serum from patients while being treated with IFX.

The following are rationales for measuring the serum concentrations of anti-TNFOL biologic drugs and antibodies against TNFu biologic drugs in patients: (1) for PK studies in clinical trials; (2) it may be required by the FDA during clinical trials to monitor a t’s immune response to the biologic drug; (3) to monitor a patient’s response to the biologic drug by ing HACA or HAHA to guide the drug dosage for each patient; and (4) for use as a guide for ing to a different biologic drug when the initial drug fails.

METHODS SE—HPLC analysis iximab (IFX) levels in patient serum. Human inant TNFu was labeled with a fluorophore (“Fl” such as, e.g, Alexa Fluor® 488) according to the manufacture’s instructions. d TNFu was incubated with different amounts of IFX or patient serum for one hour at room ature. Samples of 100 [LL volume were analyzed by size-exclusion chromatography on an HPLC system. FLD was used to monitor the free TNFu-Fl and the bound TNFu-Fl immuno-complex based on their retention times. Serum IFX levels were calculated from the standard curve.

SE—HPLC analysis ofHACA levels in patient serum. Purified IFX was labeled with Fl. Labeled IFX was incubated with different dilutions of pooled HACA-positive serum or diluted patient serum for one hour at room temperature. Samples of 100 uL volume were analyzed by size-exclusion chromatography on an HPLC system. FLD was used to monitor the free IFX-Fl and the bound IFX-Fl immuno-complex based on their retention times. The ratio of bound and free IFX- F1 was used to ine the HACA level.

[0242] Mobility Shift Assay Procedure to Measure HACA in Serum. The principle of this assay is based on the mobility shift of the HACA bound Fl-labeled Infliximab (IFX) x versus free Fl-labeled IFX on size exclusion-high performance liquid tography (SE- HPLC) due to the increase in molecular weight of the complex. The chromatography is performed in an Agilent-1200 HPLC System, using a Bio-Sep 300x7.8 mm SEC-3000 column (Phenomenex) with a molecular weight fractionating range of 5,000-700,000 and a mobile phase of IX PBS, pH 7.3, at a flow-rate of 0.5-1 .0 mL/min with FLD detection. A 100 uL sample volume is loaded onto the column for each analysis. The HACA bound Fl- labeled IFX complex is formed by incubating serum from IFX treated patient and Fl-labeled IFX in the 1X PBS, pH 7.4, elution buffer at room temperature for 1 hour before SE-HPLC analysis. The assay was also run in the presence of varying interference agents, such as rheumatoid factor and TNF-B, in order to validate the assay.

Figure 11 shows the separation of the HACA bound IFX-Fl complex from the free IFX-Fl due to the mobility shift of the high molecular weight x. As seen in panels 0 and d, the retention time of the fluorescent peak shifted from 21.8 min to 15.5-19.0 min. The more the HACA is present in the reaction e, the less the free IFX-Fl remains in the chromatogram and the more the -complex is formed. Figure 12 shows the dose- se curves of the cent peak shift caused by the addition of HACA. Using the HACA positive sample, we could detect the peak shift with 1:1000 dilutions of the serum.

Figure 13 shows the separation of the IFX bound TNFu-Fl complex from the free TNFu-Fl due to the mobility shift of the high molecular weight complex. As seen in panels 0 and d, the ion time of the fluorescent peak shifted from 24 min to l3-l9.5 min. The more the IFX is present in the reaction mixture, the less the free TNFu-Fl remains in the chromatogram and the more the immuno-complex is formed. Figure 14 shows the dose- response curves of the TNFu-Fl peak shift caused by the addition of IFX. Based on the added IFX, the detection limit is 10 ng/mL of IFX in serum.

[0245] The novel mobility shift assay of the present invention was validated by testing serum samples from HACA positive and negative patients measured by the Bridge assay (Table 4). Using this assay, we have analyzed serum samples from 50 healthy subjects and 117 IBD patients treated with IFX. All 50 healthy t samples have an IFX level below the limit of detection, whereas 65 of the patient s have an e IFX concentration of l l .0 ug/ml. Table 5 shows the HACA levels in the serum of healthy controls and IBD patients treated with IFX measured by the Bridge assay and the mobility shift assay. The Bridge assay detected less HACA-positive patients than the mobility shift assay and more false negatives as well as more false positives.

Table 4. Correlation of Relative HACA Levels in Patient Serum from Stron Positive and Negative on Bridge Assay to SE-HPLC Assay.

Bridge assay HPLC shift Correlation assay 100% Table 5. Patient Sam le Anal sis on Serum Levels of HACA with Brid e Assa Cut Off 1.69 /ml and HPLC Shift Assa Cut Off 0.19 Ratio of Bound and Free IFX .

Subjects HACA Positive Bridge Assay Bridge Assay HPLC False Negative False Positive Assay y Control N/A n N/A N/A Patient treated with 117 24 (20.5%) 33 (28.2%) 9 IFX (High IFX) False negative results are caused by patient serum containing high levels of IFX which interferes with the Bridge assay on HACA ination while the SE-HPLC assay is not affected. False positive results are caused by t serum containing high levels of non-specific interference substance which may interfere with the Bridge assay.

[0246] Figure 15 shows the relationship of the HACA level and IFX concentration in IBD patients during the course of IFX treatment. HACA could be detected as early as V10 (30 Weeks) and continued to increase in some patients during IFX treatment. Figure 16 shows that HACA can be detected in the ce of IFX using the assay of the present invention.

A higher level ofHACA in the serum was associated with a lower level of IFX that could be detected (e. g., reduced the ilability). As such, early detection ofHACA while on treatment with IFX can guide the physician and/or patient to switch to other anti-TNF drugs or increase the dose of IFX.

The assays were validated in terms of intra-and inter-assay ion (based on the CV parameter) and susceptibility to erence agents. This analysis is presented below: Infliximab assay HACA assay Parameter CV% Parameter CV% Inter-assay Precision inter-assay ion Analyst to Analyst to Analyst Analyst Infliximab assay Interference Typical Range Concentration Interference Agent tested 3.71—150 U/mL 100 U/mL Interferes with (0—60 pig/m L) (~ 55 ug/mL) ion of Iow concentration IFX samples (<5 ug/mL) TNF—ot 6.2-6.6 pg/mL 0.0125 ng/mL — 100 ng/mL 40 ug/mL Hemolyzed >20 HI 100—300 HI Serum HACA assay Interference Typical Range Concentration erence Agent tested Infliximab 0-100 ug/mL 0.78-100 NA ug/mL TNF-a 6.2-6.5 pg/mL 0.0125 ng/mL— 250 ng/mL 4O ug/mL Hemolyzed >20 HI 100-300 HI NA Serum CONCLUSION Anti-TNFOL biologic drugs can be readily labeled with a fluorophore (“F1”) and the mobility shift assay format used for measuring HACA/HAHA is a homogeneous assay without the coating of antigens to a solid surface and multiple washing and incubation steps like a typical ELISA. Incubation of eled IFX with HACA-positive serum results in the formation of an immune complex which elutes at a different on compared to free Fl- d IFX in SE-HPLC and thus the amount ofHACA can be quantitated. The presence of 2012/025437 other serum ents has little effect on the mobility shift assay. The mobility shift assay format, unlike ELISA, is not affected by non-specific binding of irrelevant IgGs and detects the IgG4 isotype. Healthy serum samples do not cause mobility shift of the Fl-labeled IFX and 28.2% of the patients treated with IFX were found to have HACA by the assay of the present invention. As such, the assay format described herein is very ive and can be d to detect all biologic drugs (e.g, Remicade, Humira, Enbrel and Cimzia) as well as their antibodies (e.g., anti-Remicade, anti-Humira, anti-Enbrel and anti-Cimzia) in patient serum. Notably, since HACA can be detected in the presence of IFX using the mobility shift assay of the invention, early detection ofHACA while on treatment with IFX can guide the physician and/or patient to switch to other anti-TNF drugs or increase the subsequent dose of IFX.

We have developed a novel non-radiolabeled, -phase, C assay to measure the IFX and HACA levels in serum s obtained from patients treated with IFX.

The novel assay has high sensitivity, accuracy, and precision, and the results are highly reproducible, which makes this assay le for routine testing of a large number of human serum samples. The new assay format, unlike ELISA, eliminates coating of antigens to solid surfaces and is not affected by non-specific binding of irrelevant IgGs. These advantages of the assay format described herein reduce the false negative and false positive s of the test. Advantageously, the assay format of the present invention is very sensitive and can be used to detect all biologic drugs as well as their antibodies t in the serum while the patient is on therapy.

Example 4. Differentiation Between Neutralizing and Non-Neutralizing Human Anti- Chimeric Antibodies (HACA) in Patient Serum Using Novel Mobility Shift Assays.

This example illustrates novel homogeneous assays for measuring autoantibody (e.g., HACA) concentrations in a patient sample (e.g., serum) and for determining whether such autoantibodies are neutralizing or non-neutralizing autoantibodies using size ion chromatography to detect the binding of these autoantibodies to fluorescently labeled anti- TNFOL drug in the presence of fluorescently labeled TNFu. These assays are advantageous because they obviate the need for wash steps which remove low affinity HACA, use distinct fluorophores that allow for ion on the visible and/or IR spectra which decreases background and serum interference , increase the ability to detect neutralizing or non- neutralizing HACA in patients with a low titer due to the high sensitivity of cent label detection, and occur as a liquid phase reaction, thereby reducing the chance of any changes in the epitope by attachment to a solid surface such as an ELISA plate. 2012/025437 In one exemplary embodiment, an anti-TNFOL drug (e.g., REMICADET'V') is labeled with a fluorophore “Fl” (see, e.g, Figure 17A), n the fluorophore can be detected on either or both the visible and IR spectra. Similarly, TNFOL is labeled with a fluorophore “F2” (see, e.g., Figure 17A), wherein the fluorophore can also be detected on either or both the visible and IR spectra, and wherein “Fl” and “F2” are different fluorophores. The labeled anti-TNFOL drug is incubated with human serum in a liquid phase on and the d TNFu is added to the on to allow the formation of complexes (i.e., immuno-complexes) between the labeled anti-TNFu drug, labeled TNFu, and/or HACA present in the serum.

Following incubation, the samples are loaded directly onto a size exclusion . Binding of both the autoantibody (e.g., HACA) and the labeled TNFOL to the labeled anti-TNFOL drug results in a leftward shift of the peak (e.g., “Immuno-Complex l” in Figure 17A) compared to a binary complex between the autoantibody and the labeled anti-TNFOL drug (e.g., “Immuno- Complex 2” in Figure 17A), the labeled drug alone, or the labeled TNFu alone. The presence of this ternary complex of tibody (e.g., HACA), labeled TNFOL, and labeled anti-TNFOL drug indicates that the autoantibody present in the serum sample is a non-neutralizing form of the autoantibody (e.g., HACA), such that the autoantibody does not interfere with the binding between the anti-TNFOL antibody and TNFOL. In one particular embodiment, as shown in Figure 17A, if non-neutralizing HACA is present in the serum, a shift will be observed for both Fl-REMICADETM and F2-TNF0L, resulting in an increase in both the Immuno-Complex l and Immuno-Complex 2 peaks and a decrease in the free Fl-REMICADET'V' and free F2- TNFu peaks. However, the presence of the binary complex between the autoantibody (e.g., HACA) and the labeled anti-TNFOL drug (e.g., “Immuno-Complex 2” in Figure 17B) in the absence of the ternary complex of autoantibody (e.g., HACA), d TNFu, and labeled anti-TNFOL drug indicates that the autoantibody present in the serum sample is a neutralizing form of the autoantibody (e.g., HACA), such that the tibody interferes with the binding between the anti-TNFOL antibody and TNFOL. In one particular embodiment, as shown in Figure 17B, if neutralizing HACA is present in the serum, a shift will be observed for Fl - REMICADETM, ing in an increase in the Immuno-Complex 2 peak, a se in the free Fl-REMICADETM peak, and no change in the free 0L peak. In certain instances, the ce of neutralizing HACA indicates that the current therapy with REMICADETM should be switched to another anti-TNFu drug such as HUMIRAT'V'.

In an alternative embodiment, the labeled anti-TNFOL drug is first incubated with human serum in a liquid phase reaction to allow the ion of complexes (i.e., immuno- complexes) between the labeled anti-TNFu drug and HACA present in the serum. Following incubation, the samples are loaded directly onto a first size exclusion column. Binding of the autoantibody (e.g., HACA) to the labeled anti-TNFu drug results in a leftward shift of the peak (e.g, “Immuno-Complex 2” in Figure 18) compared to the d drug alone. The labeled TNFOL is then added to the reaction to determine whether it is capable of displacing (e.g., competing with) the autoantibody (e.g., HACA) for binding to the d anti-TNFu drug, to thereby allow the formation of complexes (i.e., immuno-complexes) between the labeled anti-TNFu drug and the labeled TNFu. Following incubation, the samples are loaded directly onto a second size exclusion column. g of the labeled anti-TNFu drug to the labeled TNFu s in a rd shift of the peak (e.g., “Immuno-Complex 3” in Figure 18) compared to the labeled TNFu alone. Disruption of the binding between the autoantibody (e.g., HACA) and the labeled anti-TNFu drug by the addition of the labeled TNFu indicates that the autoantibody present in the serum sample is a neutralizing form of the autoantibody (e.g., HACA), such that the tibody interferes with the binding between the anti-TNFu antibody and TNFu. In certain ces, the presence of neutralizing HACA indicates that the current therapy with REMICADETM should be switched to r anti-TNFu drug such as HUMIRAT'V'.

Example 5. Analysis of Human Anti-Drug Antibodies (ADA) to Adalimumab in Patient Serum Using a Novel neous Mobility Shift Assay.

Background and Aim: Monoclonal antibodies against TNF-(x such as infliximab (IFX), adalimumab (HUMIRAT'V'), and certolizumab have been shown to be effective in treating inflammatory bowel disease (IBD) and other inflammatory disorders. rug antibodies (ADA) may reduce the drug’s eff1cacy and/or induce adverse effects. However, ADAs have been found not only in patients d with the chimeric antibody infliximab, but also in patients treated with the humanized antibody adalimumab. Monitoring ofADA and drug levels in individual patients may help optimize treatment and dosing of the patient. We have ped a non-radio labeled liquid-phase homogeneous mobility shift assay to accurately measure in the serum both HACA (Human Anti-Chimeric dy) and IFX from patients. This assay method overcomes a major limitation of the t solid-phase assays for detecting HACA, namely the inability to accurately detect HACA in the presence of IFX in circulation. In the t study, we have evaluated this new method for measuring serum ADA and drug levels in patients treated with the humanized antibody drug, adalimumab.

Methods: The ty shift assay was based on the shift in retention time of a free antigen versus n-antibody immunocomplex on size-exclusion separation. Fluorophore- labeled adalimumab or TNF-(x and internal control were mixed with serum samples to measure the ty shift of free umab and TNF-(x in the presence ofADA or drug.

The changes in the ratio of free adalimumab or TNF-(x to internal control are indicators of immunocomplex formation. Serum concentrations ofADA or adalimumab were determined with standard curves generated by incubating with different trations of anti-human IgG antibody or purified adalimumab. Using the mobility shift assay, we measured umab and ADA levels in sera collected from IBD patients treated with adalimumab who had lost response.

Results: Dose-response curves were generated with anti-human IgG antibody for the measurement of mobility shift of labeled adalimumab. The detection limit of the assay was 1 ng of anti-human IgG. Sera from fifty healthy controls were tested for ADA and all of the samples had ADA levels below the ion limit (i.e., no shift of the free labeled- adalimumab). Detection ofADA was also demonstrated in the presence of exogenously added adalimumab. To measure the drug concentration in patients treated with adalimumab, we generated a standard curve with different amounts of adalimumab on the mobility shift of labeled TNF-u, and the detection limit of adalimumab was 10 ng.

Conclusions: The non-radio labeled liquid-phase homogeneous mobility shift assay of the present invention has been d to measure ADA and adalimumab levels in serum samples from patients treated with adalimumab. The assay is found to be reproducible with high sensitivity and accuracy, and can be used to evaluate ADA levels in serum samples from patients treated with adalimumab. e 6. Analysis of rug Antibodies (ADA) to umab in t Serum Using A Novel Proprietary Mobility Shift Assay.

ABSTRACT Background: Anti-TNF-(x drugs such as infliximab (IFX) and adalimumab (ADL) have been shown to be effective in treating inflammatory bowel disease (IBD). However, induction ofADA in the treated patients may reduce the drug’s efficacy and/or induce adverse effects. Indeed, ADAs have been found not only in patients treated with IFX, but also in ts treated with ADL. Monitoring ofADA and drug levels in individual patients may help to optimize treatment and dosing of the patient. We have developed a proprietary ty shift assay to accurately measure in the serum both HACA (Human Anti-Chimeric Antibody) and IFX from IFX-treated patients. This assay overcomes the major limitation of the t solid-phase assays for detecting HACA, namely the inability to accurately detect HACA in the presence of IFX in circulation. In the present study, we have evaluated this new assay to measure serum ADA and drug levels in patients treated with the fully human antibody drug, ADL.

Methods: The mobility shift assay was based on the shift in retention time of the antigen-antibody immunocomplex versus free antigen on size-exclusion chromatography.

Fluorophore-labeled ADL or TNF-u and internal control were mixed with serum samples to measure the mobility shift of labeled ADL and TNF-(x in the presence ofADA or drug. The changes in the ratio of free ADL or TNF-u to al control are the indicators of the immunocomplex formation. Serum concentrations ofADA or ADL were determined with rd curves generated by incubating with ent concentrations of anti-human IgG antibody or purified ADL. Using this assay, we measured ADL and ADA levels in sera collected from IBD ts treated with ADL.

[0259] Results: Dose-response curves were generated with anti-human IgG antibody for the measurement of mobility shift of labeled ADL. The detection limit of the assay was 10 ng of anti-human IgG. Sera from 100 healthy controls were tested for the ADA and all of the samples had an ADA level below detection limit (no shift of free labeled ADL). Detection of ADA was demonstrated in five out of l 14 IBD patient samples treated with ADL. To measure the drug concentration in patients treated with ADL, we generated a standard curve with ent amounts ofADL on the shift of labeled TNF-(x with the ion limit of 10 Conclusions: We have applied our etary non-radio labeled liquid-phase homogeneous ty shift assay to measure the ADA and ADL levels in serum from patients treated with ADL. The assays are ucible with high sensitivity and accuracy, and are useful for evaluating ADA levels in serum samples from patients treated with ADL.

INTRODUCTION Anti-tumor necrosis factor-alpha (TNF-(x) biologics such as infliximab (IFX), etanercept, adalimumab (ADL) and certolizumab pegol have been shown to reduce disease activity in a number of mune diseases, including Crohn’s Disease (CD) and rheumatoid arthritis (RA). However, some ts do not respond to NF-(x therapy, while others need higher or more frequent dosage due to lack of sufficient response, or develop infiJsion reactions.

Immunogenicity of therapeutic antibodies which causes the patients to develop antibodies t the drugs may contribute to the failure of the treatments and on reactions. ic antibodies like IFX have a higher potential of inducing antibody generation compared to fully zed antibodies such as ADL. The prevalence of antibodies to IFX (HACA) in RA patients varies from 12% to 44% and seems to be inversely proportional to the level of IFX in patient serum and therapeutic response. While the fully humanized ADL is supposed to be less immunogenic than murine or chimeric antibodies, several studies have reported the formation of human anti-humanized dies (HAHA) and showed the prevalence of antibody generation from 1% to 87% in RA and CD patients (Aikawa et al., Immunogenicity of Anti-TNF-alpha agents in autoimmune diseases. Clin.

Rev. Allergy Immun01., 38(2-3):82-9 (2010)).

Many ts with secondary response failure to one anti-TNF-(x drug may benefit from switching to another anti-TNF-u drug or increasing dosage and/or dosing ncy.

Monitoring of patients for drug and anti-drug antibody (ADA) levels is therefore warranted so that drug administration can be tailored to the individual patient. This approach allows dose adjustment when ted or cessation of medication when ADA levels are present.

(Bendtzen et (11., Individual medicine in inflammatory bowel disease: ring bioavailability, pharmacokinetics and immunogenicity of anti-tumour necrosis factor-alpha antibodies. Scand. J. Gastroenterol., 44(7):774-81 (2009); Aflf et al., Clinical utility of measuring infliximab and human anti-chimeric dy concentrations in patients with inflammatory bowel disease. Am. J. enterol., 105(5):1133-9 (2010)).

A number of assays have been developed to measure HACA and HAHA. One of the limitations of the current ologies is that ADA levels cannot be reliably measured when there is a high level of drugs in the circulation.

[0265] We have developed a proprietary non-radiolabeled, liquid-phase, mobility shift assay to measure the ADA and ADL levels in serum from patients d with ADL which is not affected by the presence of the drug in the serum.

METHODS Fluorophore (Fl)-labeled ADL was incubated with patient serum to form the immunocomplex. A Fl-labeled small peptide was included as an internal control in each reaction. Different amounts of anti-human IgG were used to generate a standard curve to determine the serum ADA level. Free Fl-labeled ADL was ted from the antibody bound complex based on its molecular weight by size-exclusion chromatography. The ratio of free Fl-labeled ADL to internal control from each sample was used to extrapolate the HAHA concentration from the standard curve. A similar methodology was used to e ADL levels in patient serum samples with Fl-labeled TNF-u.

RESULTS Figure 19 shows the separation of the uman IgG bound Fl-ADL complex from the free Fl-ADL due to the mobility shift of the high molecular weight complex. As seen in panels 0 to h, the retention time of the fluorescent peak shifted from 10.1 min to 7.3-9.5 min.

The more the anti-human IgG is added in the reaction mixture, the less the free ADL s in the chromatogram and the more the immunocomplex is formed (h to c). The retention time for the internal l is 13.5 min.

Figure 20 shows the dose-response curve of the fluorescent peak shift caused by the on of anti-human IgG. Increasing the concentration of anti-human IgG reduces the ratio of free ADL to internal control due to the formation of the immunocomplex. The assay sensitivity is 10ng/ml of anti-human IgG. The internal control “Fl-BioCyt” ponds to an Alexa Fluor® 488-biocytin (BioCyt) which combines the green-fluorescent Alexa Fluor® 488 fluorophore with biotin and an aldehyde-f1xable primary amine (lysine) rogen Corp.; Carlsbad, CA).

Figure 21 shows the separation of the ADL bound TNF-u-Fl complex from the free TNF-u-Fl due to the mobility shift of the high molecular weight complex. As seen in panels 0 and j, the retention time of the cent peak shifted from 11.9 min to 6.5- 10.5 min. The more the ADL is added in the reaction mixture, the less the free TNF-u-Fl peak remains in the chromatogram and the more the -complex is formed.

Figure 22 shows the dose-response curves of the TNF-u-Fl peak shift caused by the addition of ADL. Based on the added ADL, the detection limit is 10 ng/mL ofADL in serum.

Table 6 shows that serum samples from 100 healthy subjects and 114 IBD patients treated with ADL were analyzed for ADA and ADL levels using the mobility shift assay of the present invention. All 100 healthy subject samples had ADA levels below the limit of ion (no shift of the free Fl-ADL), whereas 5 out of the 114 patient samples had an ADA concentration of 0.012 to >20 ug/ml. The mean ofADL levels in 100 healthy subject samples was 0.76:1.0 ug/ml (range 0 to 9.4 . The mean ofADL levels in 114 serum samples from patients treated with ADL was 10.8+17.8 ug/ml (range 0 — 139 ug/ml ). Four out of five ADA positive samples had undetectable levels ofADL. 2012/025437 Table 6. Patient Serum Levels ofADA and ADL Measured b the Mobilit Shift Assa _-Subjects (n) Age (Years) ADA ADL level sex (MIF) (mean) POSitive (”g/ml) Hea'thy 38/62 1 8-62 (37.1) 0.76+1 .00 Control — IBD Patient Treated with 51/63 20-69 (39.9) 5 (4.3°/o) 10.80117.80 SIONS The ty shift assay format used for ing HACA/IFX is a homogeneous assay without the coating of antigens to a solid surface, and without multiple washing and incubation steps like a typical ELISA. This assay can be applied to measure ADA and anti- TNF drugs. The sensitivity of the assay (in ug/ml range) is higher for both ADA and ADL ement with patient serum compared to ELISA methods (in mg/ml range). Healthy control serum samples did not cause mobility shift of the Fl-labeled ADL, and 4.3% of the patients treated with ADL were found to have ADA by this assay. Although healthy control serum samples caused mobility shift of the Fl-labeled TNF-u, which may have been due to the presence of soluble free receptor of TNF-u, the average ofADL in patients treated with ADL was much higher (10.8 vs. 0.76 mg/ml). Early detection ofADA and monitoring of ADL drug level while the patient is receiving ADL treatment will allow the physician to optimize the dosing ofADL or switch to another anti-TNF-(x drug when appropriate and, thereby, optimizing the overall management of the patient’s disease.

Table 7. Patient Serum Levels ofADA and ADL Measured b the Mobilit Shift Assa _Subjects (11) Sex (MIF) Age (Mean) ADL Level (pg/ml) ADA Positive Healthy 100 38/62 18-62 (37.1) 0.76 i 1.00 Control 51/63 2069 (39. 9) 10.80 + 17.80 94 pg/ml ADL: 4 of 42 (9.52%) 13 113.11my?1:13 111311111131 31311533311 1113“miner 31-1111 111 3311111193 111111 103113311113‘131113.1:111'1'1111111 113913133311 1111111 11111. 11111111113311.3111.13111113.. 13111 1051 1133111131 “ ‘ ~ “.1 1:311 113511911313 113113111113???11119133199111111 {119:311111 (11 1111: 111113 {5131-9101.}. 1111113192112 £1 11111 911.111; : 1131111111: 124111111 2 131111 {1-3111311111111. 1181. 111111 111‘. 1111: 11 1111?? Max 111 103 11311111131 3111119111 3313111113 11.-33 113113 $111.- 3131 1111111111}. 11113111”1 11011111113111 1‘: “ 161113 3311131193 115111 111 ~31" 113111.311 111111 91131. “1:13 10S111 “1.111111111111311«1391,11. 511111.;"(11111‘1 9919311911 131 1511111131111; 1 1'11 1 - -“= 1 . 13 91' 31011111111 1 11111111 111' .. 1131131113 13-11111 1111131911111311‘.11111111? 111.911)1.1‘1111-16‘11111-111111 3111111313119111311111114113 331111333 1119 13911131311111111 131 12119111111111§ .3131. 31-111: 1911‘; Healthy l serum samples do not cause mobility shift of the Fl-labeled ADL.

In a preliminary study, 9.52% of patients with 0.4 [Lg/ml ADL were found to have ADA in this assay.

Example 7: ining the Concentration Levels of REMICADETMand Human Anti- Drug Antibodies.

This example describes a method for determining the levels of Anti-TNFoc Drugs, 6. g. REMICADETM (infliximab), in a serum sample as well as for determining the levels of a human anti-drug dy, 6.g. a human anti-chimeric antibody (HACA) to REMICADETM imab).

[0275] Step 1: Determining concentration level of REMICADETM (infliximab) in a In one exemplary embodiment, TNFu is labeled with a fluorophore (e.g. Alexa647), wherein the fluorophore can be detected by, either or both of, the visible and fluorescent spectra. The labeled TNFu is incubated with human serum in a liquid phase reaction to allow the anti-TNFu drug present in the serum to bind. The labeled TNFOL can also be incubated with known s of the anti-TNFOL drug in a liquid phase reaction to create a standard curve. Following incubation, the samples are loaded ly onto a size exclusion column.

Binding of the NFu drug to the labeled TNFu results in a leftward shift of the peak compared to labeled TNFu alone. The concentration of the anti-TNFu drug present in the serum sample can then be compared to the standard curve and controls.

SE-HPLC analysis of REMICADETM (infliximab) levels in patient serum.

Human recombinant TNFu was labeled with a fluorophore, Alexa Fluor® 488, according to the manufactureR’s instructions. Labeled TNFu was incubated with different amounts of DETM or patient serum for one hour at room temperature. Samples of 100 [LL volume were analyzed by size-exclusion chromatography on an HPLC system. Fluorescence label detection was used to monitor the free labeled TNFOL and the bound labeled TNFOL immuno-complex based on their retention times. Serum REMICADETM levels were ated from the standard curve.

The following equations are relevant to this assay: Equation 1: d-TNFu + REMICADETM a(labeled-TNFdoREMICADETM)cmnp1ex Equation 11: [REMICADETM]without-1abeled-TNm-pmsem= [(labeled- TNFuOREMICADETM)comp1ex] Equation 111: [REMICADETM] = [(labeled-TNFuoREMICADETM)compleX]/[labeled-TNF0L] x [labeled-TNFOL] In Step 1, a known amount of the labeled-TNFu is contacted with a REMICADETM- containing serum sample. The d-TNFu and the REMICADETM form a complex, ed-TNF(XOREMICADETMLOIHPEX, See on 1. Because almost all of the REMICADETM will form a complex with the labeled-TNFu, the concentration of REMICADETM present before introduction of the labeled-TNFOL is equal to the measured concentration of labeled-TNF(XOREMICADETMcompleX, See Equation 11. The concentration level of REMICADETM is calculated by multiplying the ratio of [(label- TNFdoREMICADETM)complex]/[labeled-TNFu] by [labeled-TNFu], See Equation 111. The ratio, [(label-TNF0L.REMICADETM)compleX]/[labeled-TNFu], is obtained by integrating the area-under-the curve for the (label-TNFOLOREMICADETM)cmnp1ex peak, from a plot of signal intensity as a function of elution time from the size exclusion HPLC, and ng this number by the resultant integration of the area-under-the-curve for the labeled-TNFOL peak from the plot. The [labeled-TNFOL] is known a priori.

Step 2: Determining level of human anti-chimeric antibody, HACA.

In one exemplary ment, an anti-TNFu drug, e.g., REMICADETM, is labeled with a fluorophore, e.g, Alexa647, wherein the fluorophore can be ed by, either or both of, the e and fluorescent spectra. The labeled anti-TNFu drug is incubated with human serum in a liquid phase reaction to allow any HACA present in the serum to bind. The labeled anti-TNFu drug can also be incubated with known amounts of an anti-IgG antibody or pooled positive patient serum in a liquid phase reaction to create a standard curve. ing incubation, the samples are loaded directly onto a size exclusion column. Binding of the autoantibodies to the labeled anti-TNFu drug results in a leftward shift of the peak compared to labeled drug alone. The concentration ofHACA t in the serum sample can then be ed to the standard curve and controls.

SE-HPLC analysis of HACA levels in patient serum. Purified REMICADETM was labeled with a hore. Labeled REMICADETM was incubated with different dilutions of pooled HACA-positive serum or diluted patient serum for one hour at room temperature. Samples of 100 uL volume were analyzed by size-exclusion chromatography on an HPLC system. Fluorescence label detection was used to r the free d REMICADETM and the bound labeled REMICADETM immuno-complex based on their retention times. The ratio of bound and free labeled REMICADETM was used to determine the HACA level as described below.

Mobility Shift Assay Procedure to Measure HACA in Serum. The principle of this assay is based on the mobility shift of the complex of an anti-drug antibody, 6.g. HACA, with Alexa647-labeled REMICADETM relative to free Alexa647-labeled REMICADETM, on size exclusion-high performance liquid chromatography (SE-HPLC) due to the increase in molecular weight of the complex. The chromatography is performed in an Agilent-1200 HPLC System, using a Bio-Sep 300x7.8 mm SEC-3000 column (Phenomenex) with a molecular weight fractionating range of 700,000 and a mobile phase of lX PBS, pH 7.3, at a flow-rate of 0.5 — 1.0 mL/min with fluorescence label detection, e.g. UV detection at 650 nm. In front of the Agilent-1200 HPLC System with a p 300x7.8 mm SEC-3000 column is a analytical lumn which is a BioSep 75x7.8 mm SEC-3000. A 100 uL sample volume is loaded onto the column for each analysis. The complex ofHACA and d REMICADETM complex is formed by incubating serum from a REMICADETM- treated patient and labeled REMICADETM in the 1X PBS, pH 7.3, elution buffer at room temperature for 1 hour before SE-HPLC analysis.

The ing equations are nt to this assay: Equation 1v: REMICADETM + labeled-REMICADETM + HACA a (REMICADETMOHACA)compleX + (Labeled-REMICADETMOHACA)compleX Equation v: [REMICADETM]/[REMICADETMOHACAcompleX] = [labeled-REMICADETM]/[ Labeled-REMICADETMOHACAcompleX] on v1: [HACA] = [REMICADETMOHACALompleX + [labeled- REMICADETMOHACALompiex Equation v11: ADETMOHACAcompleX] = [REMICADETM] x ed- REMICADETMOHACAcomplexy[labeled-REMICADETM] Equation VIII: [labeled-REMICADETMOHACAcomplex] = [labeled-REMICADETM] x [labeled-REMICADETMoHACAcomplexy[labeled-REMICADETM] Equation 1x: [REMICADETM]effective_amoum = [REMICADETM] — [HACA] Determining the concentration levels of human anti-TNFoc drug antibodies, e.g.

HACA. A known concentration of Labeled-REMICADETM is added to a serum sample.

HACA forms a x with either REMICADETM or Labeled-REMICADETM, See Equation IV. The [REMICADETM] is determined in Step 1 above. By integrating the area- under-the-curve for the labeled-REMICADETMOHACAcomplex and dividing this number by the resultant ation for the the nder-the-curve for the free Labeled-REMICADETM, the ratio of ed-REMICADETMOHACAcompleX] to [labeled-REMICADETM] is obtained. The ratio of [REMICADETM] to [REMICADETMOHACAcompleX] is equal to the ratio of [labeled- REMICADETM] to [labeled-REMICADETMOHACA com ex], See Equation V. BecauseP HACA equilibrates and forms a complex with both REMICADETM and Labeled- REMICADETM, the total amount of HACA equals the sum of the amount of REMICADETMOHACAcompleX and the amount of labeled—REMICADETMoHACAmmpr, See Equation VI. Because the ratio of [REMICADETM] to [REMICADETMOHACAcomplex] is equal to the ratio of [labeled-REMICADETM] to [labeled-REMICADETMOHACAcompleX], both the [REMICADETM-HACALompleX and the [labeled-REMICADETM-HACA complex] are determined by lying the ratio ofthe [labeled-REMICADETMOHACA cowboy [labeled- REMICADETM] by, respectively, the concentration amount of DETM, determined in Step 1, and the concentration amount of labeled-REMICADETM, known a priori, See Equations VII and VIII. Therefore, the total amount ofHACA equals the sum of (l) the [REMICADETM], from step 1, multipled by [labeled- DETMOHACA)complex]/[labeled-REMICADETM], and (2) the [labeled REMICADETM], known a priori, multipled by [labeled-REMICADETMOHACA)complex]/ [labeled-REMICADETM]. ining the effective concentration levels of REMICADETM. Because HACA xes with REMICADETM, the effective amount ofREMICADETM available in a serum sample is the amount of REMICADETM, measured from Step 1, minus the amount of HACA, measured from Step 2, See Equation IX.

[0287] Exemplary calculation. In patient JAG on V10, the [REMICADETM] was determined to be 7.5 ug/ml, See Figure 160. This result was obtained by following Step 1 and using Equtions 1-11]. 7.5 ug/ml equals 30 ng/ 4 uL. Since 4 uL of sample was used in the measurement in Step 2, a total of 30.0 ng of REMICADETM was present in the sample analyzed. The ratio of [labeled-REMICADETMOHACA]complex/[labeled-REMICADETM] for patient JAG on V10 was 0.25, See Figure 16b. The [labeled-REMICADETM] introduced into the sample was 37.5 ng/ 100uL. Since lOOuL of the labeled-REMICADETM was used in the measurement in Step 2, a total of 37.5 ng of d-REMICADETM was present in the sample analyzed. Using Equation VII, the total amount of REMICADETMOHACAcomplex 2012/025437 was 30 ng multiplied by 0.25, which is equal to 7.5 ng d-REMICADETMOHACAcompelx.

Using Equation VIII, the total amount of labeled-REMICADETMOHACAcomplex was 37.5 ng multiplied by 0.25, which is equal to 9.4 ng d-REMICADETMOHACAcompelx. Using Equation VI, the total amount ofHACA equals the sum of 9.4 ng and 7.5 ng, which equals 16.9 ng HACA. The 16.9 ng HACA was present in 4 uL of sample. The [HACA] was 16.9 ng/4uL, which equals 4.23 ug/ml. Using Equation IX, the effective amount of REMICADETM is equal to 7.5 ug/ml REMICADETM, determined from Step 1, minus 4.23 ug/ml HACA, determined from Step 2. In this exemplary calculation, the effective [REMICADETM] was equal to 3.27ug/ml.

Example 8: Determining the Concentration Levels of TM and Human Anti- Drug Antibodies.

This e describes a method for determining the levels of HUMIRATM in a serum sample as well as for determining the levels of human anti-human antibodies (HAHA).

Step 1: Determining concentration level of HUMIRATM in a sample.

[0290] In one exemplary embodiment, TNFOL is labeled with a fluorophore (e.g. Alexa647), n the fluorophore can be detected by, either or both of, the visible and fluorescent spectra. The labeled TNFOL is incubated with human serum in a liquid phase reaction to allow the anti-TNFu drug present in the serum to bind. The labeled TNFu can also be incubated with known amounts of the anti-TNFOL drug in a liquid phase reaction to create a standard curve. Following incubation, the samples are loaded directly onto a size exclusion column. g of the anti-TNFOL drug to the labeled TNFOL results in a leftward shift of the peak compared to labeled TNFOL alone. The concentration of the anti-TNFOL drug present in the serum sample can then be compared to the standard curve and controls.

SE-HPLC analysis of TM levels in t serum. Human recombinant TNFOL was labeled with a hore, Alexa Fluor® 488, according to the manufacturer’s instructions. Labeled TNFu was incubated with different amounts of TM or patient serum for one hour at room temperature. Samples of 100 [LL volume were ed by size- exclusion chromatography on an HPLC system. Fluorescence label detection was used to monitor the free labeled TNFu and the bound labeled TNFu immuno-complex based on their retention times. Serum HUMIRATM levels were calculated from the standard curve.

The following equations are relevant to this assay: Equation x: labeled-TNFu + HUMIRATM a (labeled-TNFu0HUMIRATM)compleX Equation x1: ATM] =[(labeled-TNFdoHUMIRA)comp1eX] Equation x11: [HUMIRATM] = [(label-TNFdoHUMIRATM)compleX]/[labeled-TNF0i] x [labeled-TNFOL] In Step 1, a known amount of the labeled-TNFOL is contacted with a HUMIRATM- containing serum sample. The labeled-TNFOL and the HUMIRATM form a complex, (labeled- TNFQOHUMIRATM)compleX, See Equation X. Because almost all of the HUMIRATM will form a complex with the labeled-TNFOL, the [HUMIRATM] present before introduction of the labeled-TNFOL is equal to the measured [(labeled-TNFd0HUMIRATM)compleX], See Equation XI. The [HUMIRATM] is ated by multiplying the ratio of [(label- TNFdoHUMIRATM)compieX]/[Labeled-TNFOL] by [labeled-TNFd], See Equation x11. By integrating the area-under-the-curve for the labeled-TNFd and the area-under-the-curve for the (labeled-TNFdoHUMIRATM)COHIP1ex and dividing the resultant integration for (labeled- TNFOLOHUMIRATM)00mp1ex by the resultant integration for the labeled-TNFOL, the ratio of [(label-TNFQOHUMIRATM)compleX] to [labeled-TNFu] is obtained. The [labeled-TNFd] is known a priori.

Step 2: Determining level of human uman antibody, e.g. HAHA. In one exemplary ment, an NFOL drug, e.g., HUMIRATM, is labeled with a fluorophore, e. g., Alexa647, wherein the fluorophore can be ed by, either or both of, the visible and fluorescent spectra. The labeled NFd drug is incubated with human serum in a liquid phase reaction to allow any HAHA present in the serum to bind. The labeled anti-TNFOL drug can also be incubated with known amounts of an anti-IgG antibody or pooled positive patient serum in a liquid phase reaction to create a standard curve. ing incubation, the samples are loaded directly onto a size exclusion column. Binding of the autoantibodies to the labeled anti-TNFd drug results in a leftward shift of the peak compared to labeled drug alone. The concentration of HAHA t in the serum sample can then be compared to the standard curve and ls.

[0295] SE-HPLC analysis of HAHA levels in patient serum. Purified HUMIRATM was labeled with a fluorophore. Labeled HUMIRATM was incubated with ent dilutions of pooled HAHA-positive serum or diluted t serum for one hour at room temperature.

Samples of 100 [LL volume were analyzed by size-exclusion chromatography on an HPLC system. Fluorescence label detection was used to r the free labeled HUMIRATM and the bound labeled HUMIRATM immuno-complex based on their retention times. The ratio of bound and free labeled HUMIRATM was used to determine the HAHA level as described below.

Mobility Shift Assay Procedure to Measure HAHA in Serum. The principle of this assay is based on the mobility shift of the antibody, 6.g. HAHA, bound Alexa647-labeled HUMIRATM complex versus free Alexa647-labeled HUMIRATM on size exclusion-high performance liquid chromatography (SE-HPLC) due to the increase in molecular weight of the complex. The chromatography is performed in an Agilent-1200 HPLC System, using a p 8 mm SEC-3000 column (Phenomenex) with a molecular weight fractionating range of 5,000-700,000 and a mobile phase of lX PBS, pH 7.3, at a flow-rate of 0.5-1.0 mL/min with fluorescence label detection, e.g. UV ion at 650 nm. In front of the Agilent-1200 HPLC System with a Bio-Sep 300x7.8 mm SEC-3000 column is a analytical pre-column which is a BioSep 75x7.8 mm SEC-3000. A 100 uL sample volume is loaded onto the column for each analysis. A 100 uL sample volume is loaded onto the column for each analysis. The HAHA bound labeled HUMIRATM complex is formed by incubating serum from a -treated t and labeled TM in the 1X PBS, pH 7.3, elution buffer at room temperature for 1 hour before SE-HPLC analysis.

Equation x111: HUMIRATM + labeled-HUMIRATM + HAHA a (HUMIRATMOHAHA)compleX + (labeled-HUMIRATMOHAHA)complex Equation x1v: [HUMIRATM]/[HUMIRATM0HAHAcompleX] = ed- HUMIRATM]/[labeled-HUMIRAOHAHAcompleX] Equation xv: [HAHA] = [HUMIRATMOHAHAcompleX] + [labeled- HUMIRATMOHAHAcomplex] on xv1: [HUMIRATMOHAHAcompleX] = [HUMIRATM] x [labeled- HUMIRATM-HAHAcomplexy[labeled-HUMIRATM] Equation xv11: [labeled-HUMIRATMOHAHAcompleX] = [labeled-HUMIRATM] x [labeled- HUMIRATMOHAHAcomplexy[labeled-HUMIRATM] Equation XVIII:[HUMIRATM]effective_am0um = [HUMIRATM] — [HAHA] Calculation for Step 2: A known tration of labeled-HUMIRATM is added to a serum sample. HAHA forms a complex with either HUMIRATM or Labeled-HUMIRATM, See Equation XIII. The [HUMIRATM] is determined in Step 1 as described above. By integrating the area-under-the-curve for the Labeled-HUMIRATMOHAHAcomplex and the area- under-the-curve for the d-HUMIRATM and dividing the ant integration for the Labeled-HUMIRATMOHAHACOmPlex by the resultant integration for the Labeled-HUMIRATM, the ratio of the [Labeled-HUMIRATMOHAHAcompleX] to [Labeled-HUMIRATM] is obtained.

The ratio of the [HUMIRATM] to the [HUMIRATMOHAHAcompleX] is equal to the ratio of the [Labeled-HUMIRATM] to the [Labeled-HUMIRATMOHAHAcomplex], See Equation XIV.

Because HAHA equilibrates and forms a complex with both HUMIRA and Labeled- HUMIRATM, the total amount of HAHA equals the sum of the amount of HUMIRATMoHAHAcomplex and the d-HUMIRATMOHAHAcomplex, See Equation xv.

Because the ratio of [HUMIRATM] to ATMOHAHAcomplex] is equal to the ratio of [Labeled-HUMIRA] to [Labeled-HUMIRATMOHAHAcompleX], the concentration of both the [HUMIRATM-HAHAcomplex] and the [Labeled-HUMIRATM-HAHAcomplex] are determined by multiplying the ratio of the [Labeled-HUMIRAOHAHAcomplexy [Labeled-HUMIRA] by the [HUMIRATM], determined in Step 1, and the [Labeled-HUMIRATM], known a priori, respectively, See ons XVI and XVII. Because HAHA complexes with TM, the ive amount of HUMIRATM available in a serum sample is the amount of HUMIRA, measured from Step 1, minus the amount of HAHA, measured from Step 2, See Equation XVIII.

Exemplary calculation. In patient SL03246013, see Figure 25, the [HUMIRATM] was determined to be 16.9 ug/ml, see Figure 25. This result was ed by following Step 1 and using Equtions X-XII. 16.9 ug/ml equals 67.6 ng/ 4 uL. Since 4 uL of sample was used in the measurement in Step 2, a total of 67.6 ng of HUMIRATM was present in the sample analyzed. The ratio of [labeled-HUMIRATMOHAHA]complex/[labeled-HUMIRATM] for patient SL03246013 was 0.055, see Figure 25. The [labeled-HUMIRATM] introduced into the sample was 37.5 ng/ 100uL. Since 100uL of the labeled-HUMIRATM was used in the measurement in Step 2, a total of 37.5 ng of labeled-HUMIRATM was present in the sample analyzed. Using Equation XVI, the total amount of HUMIRATMOHAHAcomplex was 67.6 ng multiplied by 0.055, which is equal to 3.71 ng labeled-HUMIRATMOHAHAcompelx. Using Equation XVII, the total amount of labeled-HUMIRATMOHAHA00mph,X was 37.5 ng lied by 0.055, which is equal to 2.06 ng labeled-HUMIRATMOHAHAcompelx. Using Equation XV, the total amount ofHAHA equals the sum of 3.71 ng and 2.06 ng, which equals 5.77 ng HAHA. The 5.77 ng HAHA was present in 4 uL of sample. The [HAHA] was 5.77 ng/4uL, which equals 1.44 ug/ml. Using on XVIII, the effective amount of HUMIRATM is equal to 16.99 ug/ml HUMIRATM, determined from Step 1, minus 1.44 ug/ml HAHA, determined from Step 2. In this ary calculation, the effective [HUMIRATM] was equal to 15.46 ug/ml.

Example 9: Determining the Amount of a Complex of HACA or HAHA with Either REMICADETM, Labeled-REMICADETM, HUMIRA, or Labeled-HUMIRA.

This example describes a method for determining the amount of a complex of HACA or HAHA with either REMICADETM, Labeled-REMICADETM, , or d-HUMIRATM with reference to an internal standard.

By using an internal control, 6.g. Biocytin-Alexa 488, serum cts and variations from one experiment to another experiment can be identified and properly analyzed. The amount of internal control, e.g. Biocytin-Alexa 488, is from about 50 to about 200 pg per 100 [LL analyzed.

[0301] Fluorophore (Fl)-labeled HUMIRATM was incubated with patient serum to form the immunocomplex. A Fl-labeled small peptide, 6.g. Biocytin-Alexa 488, was included as an internal control in each reaction. In one instance, different s of anti-human IgG were used to generate a standard curve to determine the serum HAHA levels. In another instance, titrated pooled positive patient serum that has been calibrated with purified HAHA was used to generate a standard curve to determine the serum HAHA levels. In yet another instance, the method described in Example 7 was used to te a rd curve to determine the serum HAHA levels. Free d HUMIRA was separated from the antibody bound x based on its molecular weight by size-exclusion chromatography. The ratio of free d HUMIRA to an internal control from each sample was used to extrapolate the HAHA concentration from the standard curve. A similar methodology was used to e HUMIRA levels in patient serum samples with labeled TNF-(x.

The initial ratio of the Labeled-Drug, z'.e. Labeled-REMICADETM 0r Labeled- HUMIRA, to the internal control is equal to 100. As depicted in Figures 23 and 24, when the ratio of the d-Drug to the internal control falls below 95, the labeled-drug is inferred to be complexed with an anti-Drug binding nd, 6.g. HACA, HAHA. The ratio of the [Labeled-drug] to [internal control] is obtained by integrating the areas-under-the-curve for the Labeled-Drug and for the internal control and then dividing the resultant integration for the Labeled-Drug by the resultant integration for the internal control.

Example 10: Determining the Ratio of Complexed Anti-TNFoL Drugs to lexed Anti-TNFoL Drugs.

The ratio of the complexed anti-TNFu drug to uncomplexed NFu drug is obtained by integrating the areas-under-the-curve for both the complexed anti-TNFOL drug and the uncomplexed anti-TNFu drug and then dividing the resultant ation for the complexed anti-TNFu drug by the resultant integration for the uncomplexed anti-TNFOL drug.

In one embodiment, the uncomplexed NFu drug is DETM having levels between about 0 ng and 100 ng in a sample. The amount of labeled-REMICADETM is about 37.5 ng.

By using an internal control, 6.g. in-Alexa 488, serum artifacts and variations from one experiment to another experiment can be identified and properly analyzed. The amount of internal control, e.g. Biocytin-Alexa 488, is from about 50 to about 200 pg per 100 [LL analyzed.

[0306] The ratio of the labeled anti-TNFoc drug, 6.g. REMICADETM or HUMIRATM, to the labeled internal control is obtained by integrating the the areas-under-the-curve for both the labeled anti-TNFoc drug and the labeled internal control and then dividing the resultant ation for the d anti-TNFoc drug by the resultant integration for the labeled internal control.

[0307] The ratio of [(labeled-anti-TNFu utoantibody)complex]/[internal control] is obtained by integrating the area-under-the curve for the ed-anti-TNFOL drug-Autoantibody)00mp1ex peak from a plot of signal intensity as a fianction of elution time from the size exclusion HPLC, and dividing this number by the resultant integration of the area-under-the-curve for the internal control peak from the plot. In some embodiments, the labeled anti-TNFoc drug is labeled REMICADETM. In some other embodiments, the d NFoc drug is labeled HUMIRATM.

Example 11: Determining the Ratio of free and complexed labeled TNFoc.

This example describes a method for determining the amount of a complex of labeled-TNFoc with either REMICADETM or HUMIRATM with reference to an internal standard.

By using an internal control, 6.g. Biocytin-Alexa 488, serum artifacts and variations from one experiment to another experiment can be identified and properly analyzed. The amount of internal control, 6.g. Biocytin-Alexa 488, is from about I to about 25 ng per 100 [LL analyzed.

[0310] In one embodiment, the uncomplexed labeled TNFOL has levels between about 50 ng and 150 ng in a . In certain instances, the amount of labeled-TNFOL is about 100.0 ng.

Fluorophore (Fl)-labeled TNFoc was incubated with patient serum to form the immunocomplex. A Fl-labeled small e, 6.g. in-Alexa 488, was included as an internal control in each reaction. A standard curve was created by spiking in known concentrations of purified anti-TNFu drug and then extrapolating from the curve to determine the concentration in units of ug/mL.

The initial ratio of the Labeled-TNFoc to the internal control is equal to 100. When the ratio of the Labeled-TNFoc to the internal control falls below 95, the labeled-TNFOL is inferred to be complexed with an anti-TNFoc drug, 6.g. RemicadeTM, HumiraTM. The ratio of the [Labeled-TNFoc] to [internal control] is obtained by integrating the areas-under-the-curve for the Labeled-TNFoc and for the internal control and then dividing the resultant integration for the Labeled-TNFoc by the resultant integration for the internal control.

Example 12: Optimizing Anti-TNFoc Drug Therapy by Measuring Anti-TNFoc Drug and/0r Anti-Drug Antibody (ADA) Levels.

This example bes methods for optimizing anti-TNFoc drug therapy, ng ty associated with anti-TNFoc drug therapy, and/or monitoring the efficacy of therapeutic treatment with an anti-TNFoc drug by ing the amount (e.g., tration level) of anti-TNFoc drug (e.g., level of free anti-TNFu therapeutic antibody) and/or rug antibody (ADA) (e.g., level of autoantibody to the anti-TNFoc drug) in a sample from a subject receiving anti-TNFOL drug therapy. Accordingly, the methods set forth in the present example provide information useful for guiding treatment decisions, e.g., by ining when or how to adjust or modify (e.g., increase or decrease) the subsequent dose of an anti- TNFoc drug, by determining when or how to e an anti-TNFoc drug (e.g, at an increased, decreased, or same dose) with one or more immunosuppressive agents such as methotrexate (MTX) or azathioprine, and/or by determining when or how to change the current course of therapy (e.g., switch to a different anti-TNFoc drug).

For purposes of illustration only, the following scenarios e a demonstration of how the methods of the t invention advantageously enable therapy to be zed and toxicity (e. g., ffects) to be minimized or reduced based upon the level of anti-TNFoc drug (e. g., level of free anti-TNFu therapeutic antibody) and/or ADA (e.g, level of autoantibody to the anti-TNFoc drug) in a sample from a subject receiving anti-TNFoc drug therapy. The levels of the anti-TNFoc drug and ADA can be measured with the novel assays described herein. |0315| Scenario #1: High level of anti-TNFoc drug with low level of anti-drug antibody ADA .

Drug levels = 10-50 ng/10ul; ADA levels = 0.1-2 l. Patient samples having this profile include samples from ts BAB and JAA on visit 10 (“V10”). See, Figure 16b.

Patients receiving anti-TNFoc drug therapy and having this particular profile should be treated with immunosuppressive drugs like azathioprine (AZA) along with the anti-TNFoc drug (e. g., infliximab). 0318 Scenario #2: Medium level of anti-TNFoc dru with low level ofADA.

[0319] Drug levels = 5-20 ng/ 10ul; ADA levels = 0.1-2 ng/10ul. t samples having this profile include samples from patients DGO, JAG, and JJH on V10. See, Figure 16b.

Patients receiving anti-TNFoc drug therapy and having this particular profile should be treated with immunosuppressive drugs like azathioprine (AZA) along with a higher dose of the anti-TNFoc drug (e.g., infliximab). One skilled in the art will know of suitable higher or lower doses to which the current course of y can be adjusted such that drug therapy is optimized, e.g., a subsequent dose that is at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or ld higher or lower than the current dose. 0321 io #3: Medium level of anti-TNFoc dru with medium level ofADA.

[0322] Drug levels = 5-20 ng/ 10ul; ADA levels = 05-10 ng/10 ul. Patient samples having this profile include samples from patient JMM on visit 10 (“V10”) and patient J-L on visit 14 (“V14”). See, Figure 16b.

Patients receiving anti-TNFoc drug therapy and having this ular profile should be treated with a different drug. As a non-limiting example, a t on infliximab (IFX) therapy and having medium levels of IFX and ADA (i.e., HACA) should be switched to therapy with adalimumab (HUMIRAT'V'). 0324 Scenario #4: Low level of anti-TNFoc dru with hi h level ofADA.

Drug levels = 0-5 ng/10 ul; ADA levels = 30-50 ng/10 ul. Patient samples having this profile include samples from all patients on V14 in Figure 16b.

Patients receiving anti-TNFoc drug therapy and having this particular profile should be treated with a different drug. As a non-limiting example, a t on infliximab (IFX) therapy and having a low level of IFX with a high level ofADA (i.e., HACA) should be switched to therapy with adalimumab (HUMIRAT'V'). e 13. Measurement of Human Anti-Chimeric Antibody (HACA) in t Serum Samples by HPLC Mobility Shift Assay.

This example describes a High Performance Liquid Chromatography (HPLC) procedure intended to quantify the level of Antibodies against Remicade in patient serum samples.

[0328] The principle of the HPLC mobility assay is based on the shift in retention time of the antigen-antibody immune complex verses the free antigen in xclusion HPLC chromatography. Standards, controls and patient samples are acid dissociated for one hour, prior to the addition of fluorescent-labeled Remicade and a cent-labeled internal control, to reduce the effect of circulating de. All reactions are then lized and incubated for one hour to allow for formation of immune complexes. Prior to being injected over a size exclusion column, all reactions are filtered and loaded onto the HPLC system with a storage temperature of 4°C. HACA bound to Remicade is separated from free Remicade by size-exclusion chromatography. The amount ofHACA is ined by the ratio of the area of free labeled Remicade peak over the area of the labeled internal control peak.

[0329] Blood can be collected by venipuncture from patients. The following additional materials can be employed: Chromasolv HPLC Water; l.2mL Micro Titer tubes; Nunc 96 Well Sample Plate; lOXPBS pH 7.4; Remicade-AlexaFluor 488/Biocytin-AlexaFluor 488; lL Sterile Filter Systems; Multiscreen HTS, GV 96-well Filter Plates; BioSep-SEC-S 3000 Guard Column, 75 x 7.8mm; BioSep-SEC-S 3000 Analytical , 300 x 7.8mm; 0.05% Na Azide/HPLC Water; Detector Waste Capillary; HPLC vials; HPLC sample inserts; creen HTS Vacuum Manifold; Agilent1200 HPLC .

An HPLC Mobile Phase (1X solution of PBS pH 7.3 ::0.1) is prepared. 200mL of 10X PBS pH 7.4 is combined with 1750 ml of HPLC water in a graduated cylinder. The pH of the resultant is determined and adjusted with 1N HCl. The total volume is increased to 2000mL with HPLC water. The resultant is filtered through a 0.22uM membrane. A Phenomenex BioSep-SEC-S 3000 guard column and BioSep-SEC-S 3000 analytical column for a HPLC system are used. UV detectors are set to record at 280nm and 210nm. 2012/025437 Standards, ls and patient samples are prepared. Standards, controls and patient serum samples are diluted. Serum samples, standards and controls are prepared on ice in a 0.5mL welled Nunc 96 well plate. Serum sample should be added first, followed by 0.5M Citric Acid pH 3.0, and lastly HPLC water. Standards, controls and samples are incubated for one hour at room temperature on plate shaker to allow for complete iation of samples. The plate is covered with foil during incubation. Remicade- AlexaFluor488/Biocytin-AlexaFluor488 is added. Specified s of de- AlexaFluor488/Biocytin-AlexaFluor488 in HPLC water are prepared. 6 [LL of HPLC water is added to appropriate wells. Remciade-AlexaFluor488/Biocytin- AlexaFluor488 is added to appropriate wells.

Other organic acids may be suitable for use with this assay including, but not limited to, ascorbic acid or acetic acid.

Neutralize Samples. Specified volume of 10X PBS pH 7.4 is added to appropriate wells. Samples are mixed by pipeting up and down six times. Standards, controls and samples are incubated for one hour at room temperature on a plate shaker to allow for complete formation of immuno-complexes. Plate is covered with foil during incubation. The ted mixture is transferred to a 4°C refrigerator if not immediately transferring to HPLC vials.

Column Standard is prepared in new sample plate with 15 [LL of Column Standard and 285 uL of Mobile Phase added to a same given well. Standards, Controls and Samples are diluted to 2% Serum. The specified volume of each standard, l and sample is transferred into the appropriate wells of a new sample plate. To the same sample plate is added the column standard it was prepared in. ed volume of 10X PBS pH 7.4 is added to riate wells. Specified volume of HPLC water is added to appropriate wells.

Samples are mixed by petting up and down six times. Samples are filtered through a 0.2um Multiscreen filter plate. The collection plate is added under filter plate. 295uL of sample is erred to the tive position on filter plate. The attached filter plate is added with sample and collection plate to the vacuum manifold. Sample are filtered through into the collection plate. Standards, controls and samples are transferred into HPLC vials.

[0335] A pipet is used to transfer 250 uL of standards, controls and samples into labeled HPLC insert vials. Standards, controls and samples are loaded onto an HPLC. HPLC Parameters may include the following: Injection volume: 100 uL; Flow Rate: 1.0 mL/min of Elution Buffer A; Stop time: 20min; Post time: Off; Minimum Pressure: 0 Bar; Maximum Pressure: 400 Bar; Thermostat: Off; DAD parameters are 210nm and 280nm with 4nm and Reference Off; Peak width (Response time): n (2s); Slit: 4nm; FLD parameters Excitation: 494nm, Emission: 519nm; One injection per vial; 100ul injection volume for each sample.

Example 14. HACA Acid Dissociation Assay.

As rated in Figure 26, an acid dissociation step allows for the proper equilibration of the complexed species prior to ing the concentration levels of the constituent species. High drug levels can interfere with the ion of anti-drug antibodies such as HACA. As ented in Figure 26, the acid dissociation step allows for the equilibration of the complexes of either the labeled-drug “A” or unlabeled-drug “C” with the anti-drug antibody HACA, “B.” After the introduction of the acid to dissociate the BC complex, high levels ofA may be added. Afterwards, the sample may be diluted and the concentration of “AB” may be measured. The concentration of “BC” after the acid dissociation step can be calculated based on the known or measured s of “A” and “B.” Figures 27 and 28 rate the percent free labeled-Infliximab as a function of Log Patient Serum percentage with and without the acid dissociation step, respectively.

The following materials can be employed in this assay: Remicade- Alexa488/Biocytin—Alexa488; Normal Human Serum; HACA Positive Control (HPC); Column Standard; 10X PBS; lXPBS pH 7.3; Multiscreen Filter Plate; Sample Plate; lN HCl; 0.5M Citric Acid. HPC Titrations in NHS are prepared. Two fold serial dilutions are prepared by transfering 35uls of a sample into 35ul ofNHS. The following solutions can be prepared for use with this example: Solution 1: 90ul of 25% HPC/75%NHS; on 2: 90ul of 12.5% HPC/87.5%NHS; Solution 3: 90ul of 6.25%HPC/93.75%NHS.

Samples may be kept on ice before, during, and after the analysis described herein.

The following ons are prepared: Solution 4: A buffer solution; Solution 5: A column standard solution; Solution 6: 2% NHS; Solution 7: 2% NHS + 37.5 de-Alexa-488/Biocytin-Alexa488.

To these solutions are added serum samples, citric acid, HPLC water in a 96 well sample plate. Serum samples are added to respective wells. 0.5M Citric Acid pH 3.0 is added to respective wells. HPLC Water is added to respective wells.

A series of samples are prepared including the following: Solution 8: buffer; Solution 9: 15 uL column standard and 285 uL 1X PBS pH 7.3; Solution 10: 2% NHS; Solution 11: 2% NHS + 37.5 Remicade-Alexa-488/Biocytin-Alexa488; Solution 12: 2%HPC -- 0% NHS -- 37.5 Remicade-Alexa-488/Biocytin-Alexa488; Solution 13: l%HPC -- 1% NHS -- 37.5 Remicade-Alexa-488/Biocytin-Alexa488; Solution 14: 0.5%HPC + 1.5% NHS + 37.5 Remicade-Alexa-488/Biocytin-Alexa488; Solution 15: 0.25%HPC + 1.75% NHS + 37.5 Remicade-Alexa-488/Biocytin-Alexa488; Solution 16: HPC -- 1.875% NHS -- 37.5 Remicade-Alexa-488/Biocytin-Alexa488; Solution 17: 0.063%HPC -- 1.937% NHS -- 37.5 Remicade-Alexa-488/Biocytin-Alexa488; Solution 18: 0.03l%HPC -- 1.969% NHS -- 37.5 Remicade-Alexa-488/Biocytin-Alexa488; Solution 19: 0.016%HPC -- 1.984% NHS -- 37.5 Remicade-Alexa-488/Biocytin-Alexa488; Solution 20: 2%HPC + 0% NHS + 37.5 Remicade-Alexa-488/Biocytin-Alexa488; on 21: high control; Solution 22: medium control; Solution 23: low control, Solution 24: 2% NHS; Solution 25: 2% NHS + 37.5 Remicade-Alexa-488/Biocytin-Alexa488.

All samples had 5.5 uL 0.5M pH 3 Citric Acid and 10.9 uL HPLC water added to them. 450uL of 0.074 mg/mL Remicade-Alexa488/Biocytin-Alexa488 are prepared. 6[LL ofHPLC water is added to three separate wells. 6[LL of 0.074mg/mL de- AlexaFlour488/Biocytin-AlexaFluor488 is added to remaining wells.

Neutralize samples. 27.6uL of lOXPBS pH 7.3 is added to all wells except one of the wells. Samples are mixed by pipeting up and down 6X. s are incubated for 1 hour at Room Temperature in the dark on plate shaker. 15 [LL of column standard is added the well to which the 27.6uL of lOXPBS pH 7.3 is not added. 285uL of lXPBS pH 7.3 is added the well to which the 27.6uL of lOXPBS pH 7.3 is not added. Samples are diluted to 2% Serum. 18.4uL of each sample is transferred to corresponding wells ofnew sample plate.

Using the same sample plate the standard was made in, 22.6uL of 10X PBS is added to all wells except the well to which the 27.6uL of lOXPBS pH 7.3 is not added. 254uL of HPLC water is added to all wells except the well to which the 27.6uL of lOXPBS pH 7.3 is not added. Samples are mixed by pipetting up and down. 295uL of standards, controls and samples are transferred to a 96 well filter plate. Using a pipet, 250uL of standards, controls and samples are transferred into HPLC insert vials.

Example 15. Patient Case 1 of Patient Who Relapsed with Anti-TNFa Therapy.

Initial testing indicated no HACA in serum and rapidly clearing IFX levels. Half life for IFX was calculated to be 46.9 hours. Dose and Frequency of IFX was increased. The t responded. See Figure 29 for a description of the levels of IFX as a function of time.

[0345] Three months later, the patient relapsed, t was retested and found to have low HACA and no detectable IFX. All cytokines tested were within normal range.

HACA* IFX lFN-y lL-1B lL-6 TNF-a (Hg/lell-lg/ml-l(pg/mL)(pg/mL)(Pg/mL)(pg/mL) The suggested treatment is Azathioprine and optionally ng to an alternative anti-TNF drug y. Also, continue monitoring patient to see if other anti-drug dies (ADA) are formed.

Example 16. Patient Case 2 of Patient who Relapased with Anti-TNFa Therapy.

Four months following l testing two samples, collected 8 days apart, were tested. HACA levels were high and IFX levels were not detectable. The recommendation is that the patient should be switched to an ative anti-TNF therapeutic. 2012/025437 I'atient CollectionHACA IFX IFN-y IL-1|3 IL-6 TNF-a Date (Hg/mL)(Hg/mL)(pg/mL)(pg/mL)(pg/mL)(pg/mL) e 17. Patient Case 3 of Patient Who Relapsed with Anti-TNFa Therapy.

IFX concentration was calculated with a standard curve generated by reaction of different concentrations of IFX to labeled TNF-oc. Sample from 11 days was 3.8 ug/ml on 1:25 dilutions. (At least 3 half-lifes). See Figure 30 for a description of the serum levels of Infliximab as a function of time. See Figure 31 for a description of the serum levels of TNF- 06 as a on of time. The recommended treatment is to combine IFX with an immunosuppressive drug or, optionally, switch to an alternative anti-TNF drug.

Example 18. Patient Case 4 of Patient Who Relapsed with Anti-TNFa Therapy.

[0349] Patient was found to have high HACA and no able IFX. TNF-u levels were elevated; all other cytokines tested were within normal range. ted treatement is to switch to an alternative anti-TNF therapeutic.

HACA IFX IFN-y IL-1|3 IL-6 TNF-a I'atient (Hg/mL)(Hg/mL)(pg/mL)(pg/mL)(pg/mL)(pg/mL) ammwmwmw Figure 32 shows the mobility shift profiles of Fl-Labeled-IFX for Patient Case 1 (A); Patient Case 2 (B, C); and Patient Case 4 (D). e 19. Patient Case 5 of Patient Who Relapsed with Anti-TNFa Therapy.

Patient was found to have low HACA and no detectable IFX level. TNF-u levels were very high; all other cytokine levels tested were within normal range. Suggested therapy is to increase dose or dosing frequency of IFX or switch to an alternative anti-TNF drug along with the on of an immunosuppressive drug. Also a suggested therapy is to continue monitoring patient to see if DA levels increase.

HACA IFX IFN-y IL-1|3 IL-6 TNF-a Patient )(Hg/mL)(pg/mL)(pg/mL)(pg/mL)(pg/mL) Example 20. Patient Case 6 of Patient Who Relapsed with Anti-TNFa y.

Patient was found to have medium HACA levels and low IFX levels. lL-lB and IL- 6 levels were very high. IFN—y was slightly elevated and TNF-u was within normal range.

Suggested treatment is to switch to a different anti-TNFu drug or to therapy with a drug that targets a ent ism (e.g., an lL-6 receptor-inhibiting monoclonal antibody such as Actemra (tocilizumab)) along with the addition of an immunosuppressive drug.

HACA IFX IFN-y IL-1|3 IL-6 TNF-a Patient (Hg/mL)(Hg/mL)(pg/mL)(pg/mL)(pg/mL)(pg/mL) SK07160939-11.06 13.31 366.11 2302.41- Example 21. Patient Case 7 of Patient Who Relapsed with Anti-TNFa Therapy.

[0353] Patient was found to have low HACA levels . Low levels of IFX were detected.

IFN-y levels were high; all other ne levels tested were within normal range. Suggested treatment is to increase dose of IFX or to switch to therapy with a drug that targets a different ism (e.g., an anti-lNFy antibody such as fontolizumab). Alternatively, suggested treatment may be to add an immunosuppressive drug.

HACA IFX IFN-y IL-1|3 IL-6 TNF-a Patient (Hg/mL)(Hg/mL)(pg/mL)(pg/mL)(pg/mL)(pg/mL)

[0354] Figure 33 shows the mobility shift profiles of of Fl-Labeled-IFX for Patient Case 5 (A); Patient Case 6 (B, C); and Patient Case 7 (D, E).

Example 22. Cytokine Levels in Different t Serum Groups.

This example describes the levels of cytokines, such as, but not limited to, IFN—y, ll- , lL-6, and TNFOL, in normal control, infliximab treated UC, humira treated CD, and HACA positive serum samples. As illustrated in Figure 34, HACA-positive patient serum typically had higher levels of all nes tested (6.g. IFN—y, Il-lB, IL-6, and TNFOL). Based upon the presence of tibodies against IFX (z'.e., HACA) and high levels of cytokines, these patients should be ed to an alternative anti-TNF drug, optionally in combination with an immunosuppressive drug.

Example 23: Quantification of HACA rds by Acid Dissociation Assay.

This example describes the quantification ofHACA in standard samples using the acid dissociation assay described in Example 14 with a fixed amount of RemicadeTM- AlexaFluor488 and varying amounts of unlabeled RemicadeTM. In particular, HACA concentrations ranging from 25 U/mL to 100 U/mL can be ined in the presence of led RemicadeTM ranging over several orders of magnitude. Data for ination of HACA in a low-concentration standard (25 U/mL), a medium-concentration rd (50 U/mL), and a high-concentration standard (100 U/mL), are presented in Tables 8, 9, and 10, respectively. The concentration of unlabeled RemicadeTM in each sample was determined using the mobility shift assay described in Example 1. Following acid dissociation and equilibration, the resulting HACA/RemicadeTM-AlexaFluor488 complex in a given sample was ined by SE-HPLC and total HACA was calculated according to the calculations presented in Example 7. The percent recovery of HACA in each analysis (based on the known concentration ofHACA in the standard) is presented.

Table 8. uantification of Low-Concentration HACA Standard 25 U/mL with Va in Remicade Concentration.

Mohili Shift Result Final Conoentration H4048 dt D RemicadeTM meemge Recovery unlabuelllgd Total Recovery %Change (HE-Km Ll l UEm L :' I: 9"D) H.404. I:9"o] FternioalzdeTM 0 22.30 N4. 109.19 N4 2?.8 109.19 00 5.25 55.55 15.52 22.35 25.55 155.21 0 6.94 1.6? 24.00 -_.I"4.56 27".?8 18.46 25.40 101.61 2 5 9.8? 1.28 12.98 -68.86 39.4? 13.11 22.98 91.91 8-45 21-16 83-65 -21 20-83 83-52 2-99 21-32 84-39 1-73 22-21 93-85 23.35 m 2.53 1155 33.13 5.3? 25.25 37.55 2012/025437 Table 9. uantification of Medium-Concentration HACA Standard 50 U/mL with Va in Remicade Concentration.

Mohili Shift Result Final Conoentration H 12 Hemioade‘“ 8verage ry Total Recovery 80 081%] IIIEthange [11 L) [UK L) (98) tFlieuniISEEnieéehEl H (3.8 [231 888 188-88 0 . 8881 50 12.22 0 51 8.18 -22.85 28 £13 32.88 88.88 89.38 19.15 0.19 1.00 -88.85 38.29 25.88 88.59 89.12 88-88 88-88 1.58 82.32 0.02 0 05 -21.82 8883 3.51 85.83 91.85 0.28 89.19 0.85 1.23 -9.19 98.32 2.08 51.23 102.85 Table 10. uantification of Hi h-Concentration HACA Standard 100 U/mL with Va in Remicade Concentration.

Mohili Shift Result Final Conoentration H (2230212 Hemioade‘“ 8verage III Recovery Total Recovery 80 081%] Ethange [11 L) [UK L) (98) tFlieuniISEEnieéehEl H (3.8 [231 0 188 81. 188-81 00 . 88-88 £10.50 1.82 3.50 -81 28 80.50 53.82 98.32 98.32 88-88 —W181-88 18888 1.58 98.38 0.22 0.28 -9.28 98.38 2.83 102.21 102.21 0.28 108.80 1.28 1.20 0.18 108.80 8.35 109.15 109.15 Example 24: A New Paradigm for Anti-TNF Drug Therapy.

[0357] The existing paradigm for anti-TNF drug therapy, based on the drug level and the HACA level determined in a patient , is outlined in the following Table 11: Table 11 Existing Paradigm.

BRUG Milan V "ACE. mqifirw LQW Infirease base MIG LBW se lime HI5H: Lflw Switch Therapy HEW MII‘J cmtfinue Mil) Mist) Indeterminate HIGH: MID Switch Therapy row HIGH fi-ontinue Mist) HIGH matinee HIfiH HIG H Switch Therapy This paradigm is confounded, however, by the high variability in drug levels in ndeterminant patients.

The therapeutic paradigm of the present invention utilizes a disease activity/severity index d from an thmic-based analysis of one or more biomarkers to select therapy, optimize therapy, reduce toxicity, monitor the efficacy of therapeutic treatment, or a combination thereof, with an anti-TNF drug. In certain aspects, the s to be taken based on this new paradigm are outline for various illustrative scenarios in the ing Table 12: Table 12. Paradigm of the Present Invention :4me DRUG H HACA . ._ Index _ . ._ _ (action ' ' ' ' cuminue' ' LOW LOW LOW LOW LOW MID increase Dose LOW LOW HIGH Increase Dose LOW MID LOW Continue LQW MID MIE} Increase Dose LBW MID HIGH Increase Dose LBW HIGH {0W Continue or Decrease Dose In avoid {mick}: LOW HIGH MIG Continue LOW Switch Theraw _ _ nflIfiI—jun _____I_~:H_t;§!_~§ __ MED LGW LBW Continue MID LBW MID increase Dose MID LBW HIGH Increase Dose or Change Therapy MID MID £33?st Continue MED MID MID Continue MI!) MID HIGH Switch Therapy MID HIGH LEW Continue or Decrease “Base to avoid toxicity MID HIGH MID Continue Miflwh £5253? _méflfllfiififlmmmemmmmmmm 11E“ * Low“ L-ow Switch Therapy HIGH LOW MID Switch Therapy HIGH LOW HIGH Switch Therapy HIGH MID LOW Switch Therapy HIGfi MID Min Switch Therapy HIGH MED Switch Therapy V HIGH HIGH HIGH Low switch‘rherapy HIGH HIGH MID Swltth y. men men HIGH S‘wltch Therapy 2012/025437 It is noted that therapeutic s for patients with mid-range HACA levels can be followed with monitoring changes in disease activity. In certain instances, high HACA levels can r a change in therapy despite other parameters, due to the immunological nature of the condition.

Example 25: Detection of Low Levels of de in Tissue Samples.

Patients with Rheumatoid Arthritis (RA) have been shown to have a response to less than 100 ng/mL of Remicade during the course of treatment. A Remicade HPLC mobility shift assay has been developed as discussed herein that detects the presence of Remicade in patient serum avoiding many of the issues with an ELISA format. In certain aspects, the current lower limit of quantitation (LLOQ) for this inventive assay is about 0.49 ug/mL, allowing analysis of most patients. Our current research tes that by adjusting various parameters of the fluorescence detector (shifting the emission wavelength to 525 nm and increasing the PMTGain to 16), the Remicade HPLC mobility shift assay can quantitatively detect as little as 50 ng/mL of Remicade in serum with high reproducibility. In fact, this level of sensitiVity makes analysis of Remicade levels in small ) tissue samples possible. Detection of Remicade within tissues enhances our knowledge of the amount of de that has reached the site of inflammation, yielding more information on pharmacokinetic and mechanistic details of the drug.

Methods

[0362] Isolation of protein from patient tissue is achieved by whole cell extraction. 1-10 mg slices of tissue are placed in a tube and then frozen in a cryo-enVironment. The cryogenic sample is then homogenized using the Covaris ep mechanical tissue disruptor. After pulverization, the sample is transferred to a tube containing ~300 uL tion buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.25% deoxycholate, 1 mM EDTA) containing a ian protease inhibitor il (Sigma, St Louis, Mo). Samples are then immediately transferred to the acoustic portion of the CryoPrep instrument for further disruption by sonication. Samples are then incubated for 45 min on ice to allow filll dissociation of cellular components. Extracts are centrifuged at 4°C for 15 min at high speed.

Supematants are aliquoted and frozen at —80°C. Protein concentrations are quantified using the Lowry protein assay (Bio-Rad). A 200 uL aliquot is thawed and then 5.0 ng of fluorescently labeled inant TNF-u (TNF-Alexa488) is added. After incubation at room temperature for 1 hour, the on is at equilibrium and various TNF- Alexa488/Remicade complexes of increasing molecular weight have formed. After filtering, the sample is injected on a Phenomenex BioSep S-3000 HPLC size exclusion column. This real time, liquid phase assay resolves Remicade-TNF complexes from free TNF based on the size of the complexes formed.

While the current lower limit of quantitation is suitable for the majority of patients, there is a need to se the sensitivity for use in RA patients (see above). In one aspect, the assay relies on detection of 25ng of exa488 in a lOOuL injection on the HPLC size ion column. The use of fluorescence as the method of detection provides flexibility for optimization of excitation and emission wavelengths as well as the ability to se the gain of the photomultiplier tube (PMT). The current settings used for validation of the Remicade assay are: FLD Max:494, kEm2519 PMTGain = 12 These settings were chosen based on published wavelengths for the AlexaFluor 488 group as well as normal PMTGain settings for the Agilent 1200 series FLD. sing the PMTGain increases the signal and the noise, but up to a certain factor the increase in signal is higher than the se in the noise. The step from gain to gain is equal to a factor of 2. The most important parameters to optimize are the excitation and emission wavelengths and while the published maximums are a useful staring point, it is often necessary to optimize them because the excitation depends on the compounds themselves as well as the specific instrument characteristics.

[0364] When detecting low amounts of Remicade, a specific peak ing a complex of exa488 and Remicade arises at a retention time of 9.2 minutes. In one aspect, it is ant for the height of this peak to be at least 3 times over background and that the calculated serum concentration to over multiple replicates to have a ient of variance less than 20%. In certain embodiments, the signal to noise of this specific Remicade- TNFAlexa488 peak to normal human serum background is thus the starting point for increasing the sensitivity of the assay.

To increase the sensitivity, the PMTGain as well as the excitation and emission wavelengths were optimized based on the results of amplification plots and isoabsorbance plots. Remicade was ed in the presence of dilutions of TNF-Alexa488 at different PMTGain levels ranging from 12-18, using the current tion and emission wavelengths of 494 and 519 nm, respectively.

Figure 35 shows a standard amount of TNF-Alexa488 as well as the small peak at Rt=9.2 minutes reflecting a Remicade-TNF complex (top panel). Upon decreasing the amount of exa488 to 2.5 ng, it is clear that the background from 4% Normal Human serum begins to interfere with the resolution of the free TNF peak as well as the peak at 9.2 minutes ing a Remicade-TNF complex (middle panel). sing the PMTGain to 18 (lower panel) increases the signal and noise equally (data is similar for all PMT levels).

It is clear from the data that the ound fluorescence from normal human serum interferes with quantitation of low levels of Remicade using the current settings. To increase the sensitivity of the assay, r modifications of the FLD settings are necessary to decrease the serum background . To investigate this, experiments were performed at different excitation and emission wavelengths based on results from isoabsorbance plots.

The isoabsorbance plots were taken of normal human serum, TNF-Alexa488, mobile phase (lX PBS/0.1%BSA), and water.

Figure 36 shows excitation wavelengths plotted on the Y-axis and emission ngths d on the X-axis. Comparing the plots for normal human serum (top panel) and TNF-Alexa488 (bottom panel) shows significant overlap in both excitation and emission maximums (vertex of the v-shaped region in the plots). Shifting the emission wavelength to at least 525nm will likely maintain high ivity for TNF-Alexa488 while decreasing the normal serum background. The emission wavelength was set to 525 nm and then experiments repeated looking at TNF-Alexa488 as well as normal human serum background.

TNF-Alexa488 was injected in the ce of 4% NHS and the signal-to-noise evaluated.

Figure 37 shows the analysis of normal human serum (left panel) and 25ng TNF- Alexa488 (right panel) by HPLC using the indicated settings. The background level of fluorescence from normal human serum is greatly decreased. After demonstrating the level of background cence from serum was decreased, the signal to noise of the assay was evaluated at several different PMTGain levels ranging from 12-18. The results of the analysis, presented in the Table 13 below, establish that a PMTGain of 16 provides significant benefit.

Table 13 Average Average Area NHS Area TNF Emission Si nal/Ng 0 sei Background Alexa488 ————— The sensitivity of the assay was then probed by generating standard curves such as the plot shown in Figure 38. 2.5 ng TNF-Alexa488 per injection was used Remicade was titrated in the range of 50 ng/mL-5.86 ug/mL to establish the limit of detection. The peak at retention time of 9.2 was again monitored as a judge of signal-to-noise and the lowest concentration that repeatedly (n=20) gave rise to a 3:1 peak height was used to calculate the LOQ. The results of this kind of analysis are presented in the following table.

Table 14 Experimental gs: PMTGain = 16 XEX = 494 nm, kEm = 525 nm 2.5 n Alexa488/lOO L In'ection 0.044 13.00n-/mL 51.02n-/mL Accuracy= 111.40%

[0371] By shifting the Emission wavelength to 525 nm and increasing the PMT gain to 16, the Remicade HPLC mobility shift assay can now quantitatively detect as little as 50 ng/mL of Remicade in serum with high reproducibility. Further optimization may increase the ivity to a greater extent, but the new format should allow analysis of RA patients that show response even at very low Remicade serum trations. ation of low Remicade levels with t response, clinical outcome, and related biomarkers make ons for a more personalized approach to treatment.

Example 26: Clinical study analysis of Mobility Shift Assay vs. ELISA.

Initial studies were performed as above using samples from active CD patients (N = 117) and UC patients (N = 10) treated with infliximab over several weeks. Mobility shift assay data were ed with ELISA results.

As shown in Figure 39, both s correlated lation coefficient = 0.812, p < 2.2 X 10'16 for data collected above the lower limits of quantitation) for determination of infliximab in the samples. 6% of samples determined to be infliximab-negative by ELISA 2012/025437 were shown to be infliximab-positive by the mobility shift assay. None of the samples determined to be infliximab-negative by the mobility shift assay were determined to be infliximab-positive by ELISA. As determined by mobility shift assay, four infliximab- negative samples were found to be HACA-positive. ELISA and mobility shift assay data were also ated for determination of HACA, as shown in Figure 40. 37 of the samples determined as HACA-negative by ELISA were found to be HACA-positive by the mobility shift assay.

Cumulative counts per week of HACA-positive samples were ted over time as shown in Figure 41. While the data for the mobility shift assay (Figure 41, top trace) and ELISA (Figure 41, bottom trace) begin to converge after 60 weeks, the ty shift assay resulted in higher count of HACA-positive specimens at earlier time points. Fisher’s exact test was applied to the data collected at various time points. The p-values as determined by the test were , , and 0.6791 at 46 weeks, 50 weeks, and 66 weeks, respectively.

Taken together, the clinical s indicate that the mobility shift assay overcomes ility and interference limitations in the ELISA. The technology is also applicable to a broad spectrum of protein therapeutics for conditions such as rheumatoid arthritis and inflammatory bowel disease. Given the critical need for precise detection of drug levels and anti-drug antibodies in developing therapeutic strategies, the mobility shift assay allows for better management of patient treatment.

Example 27: Evaluation of A Novel Homogeneous ty Shift Assay For The Measurement of Human himeric Antibodies (HACA) and Infliximab (IFX) Levels in Patient Serum.

Background: The list of antibody-based rapeutics available for the treatment of inflammatory diseases such as inflammatory bowel disease (IBD) and rheumatoid arthritis (RA) is steadily increasing. However, certain patients will generate anti-drug antibodies (ADA) that can cause a range of consequences, including alteration of the drug pharmacokinetics, reduction/loss of drug efficacy, and adverse drug reactions. ring of patients for antibody drug and ADA levels is not only required by the FDA during the drug pment process, but is also very important for appropriate patient management during treatment with these drugs. Different methods are available for the assessment ofADA and drug levels, which include solid phase immunoassay, mmunoprecipitation (RIPA) and Surface Plasmon Resonance (SPR). However, many disadvantages are observed in these methods, including masked/altered epitopes by antigen immobilization or labeling, inability to define species specificity and isotype detection, failure to detect low affinity antibodies, ement for dedicated instruments or radiolabeled reagent, and low drug tolerance in the sample. We have developed a non-radio labeled -phase homogeneous mobility shift assay to measure the HACA and drug levels in serum from patients treated with IFX. This method overcomes many of the limitations of the current methods for measuring HACA and drug level.

Methods: To perform the mobility shift HACA assay, Alexa Fluor 488 (Alexa488) labeled Infliximab (IFX) containing an Alexa488 loading control is incubated with HACA positive serum and allowed to reach equilibrium. After equilibration, the reaction mixture is then injected onto a HPLC column. The free Alexa488-IFX and immune complexes are resolved by size exclusion chromatography (SEC) HPLC and the intensity of the fluorescence in each resolved peak is measured by a fluorescent detector (FLD). The changes in the ratio of the free Alexa488 IFX peak area to the Alexa488 internal l peak area indicate the amount of the immune complexes formed. Different dilutions ofHACA ve serum are used to generate a rd curve, which is fitted with a 5-parameter logistic model to account for asymmetry. The amount ofHACA in the samples is calculated from the standard curve. Similar methodology and analysis are used to measure the IFX level in the serum, except that 88 labeled TNF-0c is utilized to bind IFX and purified IFX is used as the standard. We have performed a full method tion on both HACA and IFX assays, and ed the clinical sample test results with those obtained from ELISA methods.

[0377] Results: Validation of the ty shift HACA assay revealed a lower limit of quantitation of 6.75U/ml in serum samples, which is equivalent to 35.4ng/ml, and this value is lower than the industry requirement (250-500 ng/ml). The linear range of quantitation is 6.75-150 U/ml. The intra-assay and inter-assay precision determination yielded a coefficient of variation of less than 15%, and the accuracy of the assay is within 20%. IFX drug tolerance in the assay is up to 100 ug/ml in the test serum. eutic levels of oprine (AZA) and rexate (MTX), presence of rheumatoid factor (774 IU/ml), normal levels of immunoglobulins, TNFs and soluble TNF receptors have no significant interference in the assay. Serum samples from 100 drug naive healthy ts were tested to set up the cutoff point of 6.75U/ml (Mean+1 .65 SD). One hundred HACA positive serum samples analyzed by bridge ELISA were also evaluated by the mobility shift assay. Overall, there is a strong ation between the two methods on HACA levels (Spearman’s Rho = 0.337, p = 0.0196). However, the new method was able to identify 23 false ve samples from the bridge ELISA. Similar results were obtained from the validation of the mobility shift IFX assay.

Conclusions: Results from this study demonstrated the superiority of the mobility shift assay in measuring HACA and IFX in patient serum samples. This method can also be applied to detect other biopharmaceuticals and ADA in t serum samples such as those d with adalimumab.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually orated by reference.

Claims (28)

WHAT IS CLAIMED IS

1. A method for detecting the ce or level of an autoantibody to an anti-TNFα drug in a sample without interference from the NFα drug in the sample, the method comprising: 5 a) contacting the sample with an acid to dissociate preformed complexes of the autoantibody and the anti-TNFα drug, wherein the sample has or is suspected of having an autoantibody to the anti-TNFα drug; b) contacting the sample with a labeled anti-TNFα drug following dissociation of the preformed complexes; 10 c) neutralizing the acid in the sample to form labeled complexes of the labeled anti-TNFα drug and the autoantibody; d) subjecting the labeled complexes to size exclusion chromatography to separate the labeled complexes; and e) detecting the labeled xes, y detecting the presence or level 15 of the autoantibody without interference from the anti-TNFα drug in the

2. The method of claim 1, n the anti-TNFα drug is selected from the group consisting of infliximab, etanercept, adalimumab, certolizumab pegol, golimumab (CNTO 148) and combinations thereof. 20

3. The method of claim 1 or claim 2, wherein the autoantibody to the anti- TNFα drug is selected from the group consisting of a human anti-chimeric antibody (HACA), a human anti-humanized antibody (HAHA), a human anti-mouse antibody (HAMA), and combinations thereof.

4. The method of any one of claims 1 to 3, wherein the acid comprises an 25 organic acid, an inorganic acid, or a mixture f.

5. The method of claim 4, wherein the organic acid comprises citric acid.

6. The method of any one of claims 1 to 5, n the sample is contacted with an acid at a concentration of from about 0.1M to about 5M.

7. The method of any one of claims 1 to 6, wherein the acid is neutralized by adding one or more neutralizing agents to the sample.

8. The method of any one of claims 1 to 7, wherein step (b) further comprises contacting a labeled internal control with the sample. 5

9. The method of any one of claims 1 to 8, wherein the presence or level of the tibody is detected in the presence of a high level of the anti-TNFα drug, wherein the high level of the anti-TNFα drug corresponds to an anti-TNFα drug level greater than or equal to about 10 g/mL.

10. The method of claim 9, n the high level of the anti-TNFα drug 10 corresponds to an anti-TNFα drug level of from about 10 μg/mL to about 100 μg/mL.

11. The method of any one of claims 1 to 10, wherein the size ion chromatography is size exclusion-high performance liquid chromatography (SE-HPLC).

12. The method of any one of claims 1 to 11, n the sample is serum.

13. The method of any one of claims 1 to 12, wherein the sample is obtained 15 from a subject receiving y with the anti-TNFα drug.

14. The method of any one of claims 1 to 13, wherein the complexes are eluted first, followed by free labeled anti-TNFα drug.

15. The method of any one of claims 1 to 14, wherein the anti-TNFα drug is labeled with a fluorophore or a fluorescent dye. 20

16. A method of making a diagnosis in a subject receiving a course of therapy with an anti-TNFα drug for optimizing therapy and/or ng toxicity to the anti-TNFα drug, the method comprising: a) determining the presence or level of an autoantibody to the anti-TNFα drug in a sample obtained from the subject t interference from the 25 anti-TNFα drug in the sample, the method comprising: (i) contacting the sample with an acid to dissociate preformed complexes of the autoantibody and the NFα drug, wherein the sample has or is suspected of having an autoantibody to the anti- TNFα drug; 5 (ii) contacting the sample with a labeled anti-TNFα drug following dissociation of the preformed complexes; (iii) neutralizing the acid in the sample to form labeled complexes of the d anti-TNFα drug and the autoantibody; (iv) subjecting the labeled complexes to size exclusion chromatography to 10 separate the labeled complexes; and (v) detecting the labeled complexes to thereby detect the ce or level of the autoantibody t interference from the NFα drug in the sample; and b) making a diagnosis that a subsequent dose of the course of therapy be 15 altered or making a diagnosis that a different course of therapy be administered to the subject for which a ination of step (a) is positive.

17. The method of claim 16, wherein the NFα drug is selected from the group consisting of infliximab, etanercept, adalimumab, izumab pegol, golimumab 20 (CNTO 148), and combinations thereof.

18. The method of claim 16 or claim 17, wherein the autoantibody to the anti- TNFα drug is selected from the group consisting of a human anti-chimeric antibody (HACA), a human anti-humanized antibody (HAHA), a human anti-mouse antibody (HAMA), and combinations thereof. 25

19. The method of any one of claims 16 to 18, wherein the diagnosis is that the subsequent dose of the course of therapy is increased, decreased, or maintained based upon the presence or level of the tibody.

20. The method of any one of claims 16 to 18, wherein the different course of therapy comprises a different anti-TNFα drug.

21. The method of any one of claims 16 to 18, wherein the different course of y comprises the current course of therapy along with an immunosuppressive agent.

22. The method of any one of claims 16 to 18, wherein the different course of therapy comprises switching to a course of therapy that is not an anti-TNFα drug. 5

23. The method of any one of claims 16 to 22, wherein the acid comprises an organic acid, an inorganic acid, or a mixture thereof.

24. The method of any one of claims 16 to 23, wherein the presence or level of the autoantibody is detected in the presence of a high level of the anti-TNFα drug, wherein the high level of the anti-TNFα drug corresponds to an NFα drug level 10 greater than or equal to about 10 μg/mL.

25. The method of any one of claims 16 to 24, wherein the size ion chromatography is size exclusion-high mance liquid chromatography (SE-HPLC).

26. The method of any one of claims 16 to 25, wherein the sample is serum.

27. The method of claim 9 or claim 24 wherein the high level of the anti- 15 TNFα drug corresponds to an anti-TNFα drug level from about 10 μg/mL to about 100 μg/mL.

28. The method according to claim 1 or claim 16, substantially as herein described with nce to any one or more of the examples but excluding comparative examples.