KR20150088890A - Assays for detecting neutralizing autoantibodies to biologic therapy - Google Patents

Abstract

The present invention provides assays for detecting and measuring the presence or level of neutralizing and non-neutralizing autoantibodies to biologics such as anti-TNFa drug therapy in a sample. The present invention is useful for monitoring the formation of neutralizing and / or non-neutralizing anti-drug antibodies over time while subjects are being treated with biologics. The invention is also useful for predicting and / or determining the cross-reactivity of neutralizing anti-drug antibodies in a sample of a subject receiving an alternative biologic therapy. Thus, the present invention provides therapeutic decision guidance information for a subject being treated with a biological agent, optimizing therapy therapy, alleviating toxicity and / or monitoring the efficacy of therapeutic treatment for biological therapy regimens To improve accuracy.

Description

ASSAYS FOR DETECTION NEUTRALIZING AUTOANTIBODIES TO BIOLOGIC THERAPY BACKGROUND OF THE INVENTION [0001]

Cross reference of related application

This application claims the benefit of U.S. Provisional Application No. 61 / 505,031, filed July 6, 2011, U.S. Provisional Application No. 61 / 528,072, filed August 26, 2011, and U.S. Provisional Application No. 61 / 535,884, filed September 16, 13 / 802,117, filed March 13, 2013, which is a continuation-in-part of PCT Application No. PCT / US2012 / 045794, filed July 6, 2012, This application also claims priority to U.S. Provisional Application No. 61 / 732,251 filed on November 30, 2012. The disclosures of each of these applications are incorporated herein by reference in their entirety for any purpose.

Autoimmune disorders are a serious and rampant medical challenge. For example, rheumatoid arthritis (RA) is an autoimmune disease affecting over two million Americans. RA is a progressive disease that causes chronic inflammation of the joints and typically has the potential to cause joint destruction and functional disability. Although genetic predisposition, infectious agents, and environmental factors all contribute to the pathogenesis of the disease, the cause of rheumatoid arthritis is unknown. In active RA, symptoms may include fatigue, anorexia, mild fever, muscle and joint pain and stiffness. During the onset of the disease, joints become inflamed, often reddened, inflamed, and painful, due to inflammation of the synovium. In addition, since RA is a systemic disease, inflammation can affect the organs and areas of the body other than the joints, including the eyes and mouth fissure, the lung lining, the pericardium, and the blood vessels.

Conventional treatments for managing RA and other autoimmune disorders include taking the first action with a "first drug" and taking follow-up actions with a "second drug". Primary drugs relieve pain and inflammation. Examples of such primary drugs include aspirin, naproxen, ibuprofen, etodolac, and other non-steroidal anti-inflammatory drugs (NSAIDs) as well as corticosteroids, which are administered either orally or directly into tissues and joints . The secondary drug alleviates the disease and prevents progressive joint destruction and is also referred to as a disease-modified anti-rheumatic drug or DMARD. Examples of such secondary drugs include gold, hydrochlorocholine, azulpidine and immunosuppressants such as methotrexate, azathioprine, cyclophosphamide, chlorambucil and cyclosporin. However, most of the drugs may have fatal side effects. Thus, additional therapies for rheumatoid arthritis and other autoimmune disorders are being sought.

Tumor necrosis factor alpha (TNF-a) is a cytokine produced by many cell types including monocytes and macrophages, originally based on the ability to induce necrosis of certain mouse tumors. Next, factors called cachectin associated with cachexia were found to be identical to TNF-α. TNF-a is involved in the pathophysiology of a variety of human diseases and disorders, including shock, sepsis, infection, autoimmune disease, RA, Crohn's disease, transplant rejection and graft versus host disease.

Due to the detrimental role of human TNF-a (hTNF-a) in various human disorders, therapeutic strategies have been devised to inhibit or neutralize hTNF-a activity. In particular, antibodies that bind to and neutralize hTNF-a have been sought as means for inhibiting hTNF-a activity. Some of the earliest such antibodies were murine monoclonal antibodies (mAbs) secreted by hybridomas prepared from lymphocytes of mice immunized with hTNF-α (see, for example, 5,231,024). These mouse anti-hTNF-a antibodies exhibit high affinity for hTNF-a and neutralize hTNF-a activity, but their use in vivo is associated with a short serum half-life, inability to induce specific human effector function, ("Human anti-mouse antibody" (HAMA) response) to the mouse antibody in mice.

More recently, biological therapies have been applied to treat autoimmune disorders such as rheumatoid arthritis. For example, four TNFa inhibitors, REMICADE ™ (infliximab), chimeric anti-TNFα mAb, ENBREL ™ (etanercept; etanercept), TNFR-Ig Fc fusion protein, HUMIRA ™ ; adalimumab), human anti-TNFα mAb, and CIMZIA® (certolizumab pegol), PEGylated Fab fragments have been approved by the FDA for the treatment of rheumatoid arthritis. CIMZIA® is also used for the treatment of mild to severe Crohn's disease (CD). Although such biological therapies have proven successful in the treatment of other autoimmune disorders such as rheumatoid arthritis and CD, not all treated patients respond to or respond well to such treatment. In addition, administration of a TNFa inhibitor may induce an immune response to the drug and may include administration of an autoantibody such as a human anti-chimeric antibody (HACA), human anti-humanized antibody (HAHA), and human anti-mouse antibody Can be produced. Such HACA, HAHA, or HAMA immune response responses may be associated with rapid changes in pharmacokinetics and bioequivalence of immunotherapeutic TNF [alpha] inhibitors that inhibit hypersensitivity reactions and additional treatment with the drug. Thus, there is a need in the art for an assay to detect the presence of autoantibodies to biological agents, such as anti-TNFa drugs, in patient samples in order to monitor biotherapeutic regimens and guide therapeutic decisions. The present invention fulfills such needs and provides related advantages.

The present invention provides assays for detecting and measuring the presence or level of neutralizing and non-neutralizing autoantibodies to biologics such as anti-TNFa drug therapy in a sample. The present invention is useful for monitoring the formation of neutralizing and / or non-neutralizing anti-drug antibodies over time, while the subject is undergoing biotherapeutic therapy (e. G. Anti-TNFa drug therapy). The present invention is also useful for predicting and / or measuring the cross-reactivity of neutralizing anti-drug antibodies in a sample of a subject receiving an alternative biological therapy (e. G., Alternative anti-TNFa therapy). Accordingly, the present invention provides a method of treating a subject suffering from a biological agent, comprising providing a guide to treatment decisions for a subject to be treated with a biological agent, optimizing therapeutic regimens, alleviating toxicity and / or monitoring the efficacy of treatment regimens with biological therapies Improves accuracy.

In one aspect, the invention provides a method of detecting the presence of an autoantibody in the form of a neutralized and / or non-neutralized form of a biological agent in a sample, comprising:

(a) A sample is contacted with a labeled biologic agent and a labeled biological binding moiety to form:

(i) The first labeled conjugate of the labeled biologic agent and autoantibody (i.e., the immunoconjugate or conjugate) (i.e., the components of the first labeled conjugate are not covalently bonded to each other); And / or

(ii) the labeled biological agent, a labeled biological binding portion and a self-labeled complex of a second antibody (that is, immune-complex or conjugate) (in other words, the component of the labeled conjugate of the second are not covalently bonded to each other );

(b) The first labeled complex and / or the second labeled complex can be applied to size exclusion chromatography to convert them into free (i.e., non-bound) labeled biological binding moieties, free labeled biologics, and / Or a complex of a labeled biological agent and a labeled biological binding moiety;

(c) Levels of free labeled biological binding moieties are measured after size exclusion chromatography (e.g., by measuring the area under the curve (AUC) of free labeled biologically bound partial peaks after size exclusion chromatography (SEC)); And

(d) The level of the free labeled biological binding moiety measured in step (c) can be compared to the level of the free labeled biological binding moiety in the control sample (e. G., The SEC of the reference sample containing only the free labeled biologically binding moiety Followed by measuring the AUC of the free labeled biologically binding partial peak), thereby detecting the presence of autoantibodies in the neutralized and / or non-neutralized form.

In certain embodiments, the autoantibody in neutralized form is detected when the level of the free labeled biological binding moiety measured in step (c) is the same or substantially the same as the level of the free labeled biological binding moiety in the control sample. In certain other embodiments, the autoantibody of the non-neutralizing form is characterized in that the level of the free labeled biological binding moiety measured in step (c) is reduced in comparison to the level of the free labeled biological binding moiety in the control sample (E.g., substantially reduced) or absent (e.g., not detected).

In another aspect, the invention provides a method for determining the level or percentage of autoantibodies in neutralized form to a biological agent in a sample, comprising:

(a) A sample is contacted with a labeled biologic agent and a labeled biological binding moiety to form:

(i) The first labeled conjugate of the labeled biologic agent and autoantibody (i.e., the immunoconjugate or conjugate) (i.e., the components of the first labeled conjugate are not covalently bonded to each other); And / or

(ii) (I.e., the immunoconjugate or conjugate) of the labeled biologic agent, the labeled biological binding moiety and the autoantibody (i.e., the components of the second labeled conjugate are not covalently bonded to each other);

(b) The first labeled complex and / or the second labeled complex may be subjected to size exclusion chromatography to convert them into a free (i.e., unbound) labeled biological binding moiety, a free labeled biologic, and / or a labeled Separating from the complex of the biological agent and the labeled biological binding moiety;

(c) After the size exclusion chromatography, the level of the free labeled biological binding moiety is measured (e.g., by measuring the area under the curve (AUC) of the free labeled biologically bound partial peak after size exclusion chromatography (SEC)); And

(d) The level of the free labeled biological binding moiety measured in step (c) is compared to the normalized level or percentage of the free labeled biological binding moiety in the control sample (e. G., Only the free labeled biologically binding moiety Measuring and normalizing the AUC of the free labeled biologically binding moiety peak after SEC of the reference sample and calculating the level or percentage of the free labeled biologically binding moiety), wherein the normalized The level or percentage corresponds to the level or percentage of autoantibodies in neutralization form.

In another embodiment, the difference between the normalized level or percentage of the free labeled biological binding moiety in the control sample and the level of the free labeled biological binding moiety measured in step (c) is greater than the level of the non-neutralizing form of the autoantibody Or percentage.

In another aspect, the invention provides a method for determining the percentage or level of autoantibody in neutralized form to a biological agent in a sample, comprising:

(a) A sample is contacted with a labeled biologic agent and a labeled biological binding moiety to form:

(i) The first labeled conjugate of the labeled biologic agent and autoantibody (i.e., the immunoconjugate or conjugate) (i.e., the components of the first labeled conjugate are not covalently bonded to each other); And / or

(ii) (I.e., the immunoconjugate or conjugate) of the labeled biologic agent, the labeled biological binding moiety and the autoantibody (i.e., the components of the second labeled conjugate are not covalently bonded to each other);

(b) The first labeled complex and / or the second labeled complex may be subjected to size exclusion chromatography to convert them into a free (i.e., unbound) labeled biological binding moiety, a free labeled biologic, and / or a labeled Separating from the complex of the biological agent and the labeled biological binding moiety;

(c) After the size exclusion chromatography, the level of the free labeled biological binding moiety is measured (e.g., by measuring the area under the curve (AUC) of the free labeled biologically bound partial peak after size exclusion chromatography (SEC)); And

(d) The level of the free labeled biological binding moiety measured in step (c) is compared to the level of the free labeled biological binding moiety in the control sample (e. G., After SEC of the reference sample, AUC), thereby measuring the percentage or level of autoantibodies in neutralized form.

In yet another aspect, the invention provides a method for determining whether a neutralizing form of an autoantibody to a first biologic agent is cross-reactive with a second (i.e., different) biological agent, comprising :

(a) Detect or measure the presence, level or percentage of autoantibody in neutralization form in the sample according to the assays described herein to determine whether the sample is positive or negative for neutralizing forms of autoantibodies; And

If the sample is positive for neutralizing autoantibodies:

(b) The sample is contacted with a labeled second biocide to form a labeled second biochemical and a neutralized form of the labeled complex of the autologous form (i.e., the components of the labeled complex are not covalently bound to each other);

(c) The labeled complex is applied to size exclusion chromatography to separate the labeled complexes (e.g., from a second biologically labeled free biocide); And

(d) Thereby detecting whether the neutralizing form of the autoantibody to the first biologic agent is cross-reactive with the second biological agent.

In certain embodiments, the presence of the labeled conjugate indicates that the neutralizing autoantibody against the first biological preparation is cross-reactive with the second biological agent, i.e., the neutralizing autoantibody is conjugated to the first biological drug Will inhibit the activity of all of the second biological drugs.

In certain other embodiments, the absence of the labeled conjugate indicates that the neutralizing antibody against the first biological agent is not cross-reactive with the second biological agent, i.e., the neutralizing autoantibody is a second biological agent Lt; / RTI >

In some embodiments, the biological agent is selected from the group consisting of an antibody (e.g., an anti-TNFa monoclonal antibody), an antibody fragment, a protein (e.g., a cytokine such as an interleukin), a polypeptide, a peptide, Conjugates, vaccines, nucleic acids, sugars, recombinant forms thereof, engineered forms thereof, and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasma sample, for example, a whole blood, serum, or plasma sample of a subject who has received a biologic therapy. In a preferred embodiment, the sample is serum. In certain embodiments, the subject is a subject suffering from a disease such as autoimmune disease (e.g., rheumatoid arthritis), inflammatory disease (such as inflammatory bowel disease (IBD) such as Crohn's disease (CD) or ulcerative colitis (UC) Or disorder.

In certain embodiments, the sample is suspected to have or have an autoantibody to the biological agent. In other embodiments, biological autologous antibodies include but are not limited to human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti-mouse antibodies (HAMA) as well as combinations thereof .

In certain embodiments, the assay method of the present invention further comprises an acid dissociation step comprising contacting the sample with an acid prior to, during, and / or after contacting the labeled biological agent and the labeled biological binding moiety .

In certain other aspects, the assay method of the present invention comprises detecting the presence or level of one or more isotypes of autoantibodies in neutralized and / or non-neutralized form to a biological agent in a sample.

In one particular aspect, the invention provides a method for detecting the presence of an autoantibody in the form of a neutralized and / or non-neutralized form of an anti-TNF [alpha] drug in a sample,

(a) A sample is contacted with a labeled anti-TNF [alpha] drug and labeled TNF [alpha] to form:

(i) (I.e., the components of the first labeled complex are not covalently bonded to each other); the first labeled complex (i.e., the immunoconjugate or conjugate) of the labeled anti-TNF [alpha] drug and autoantibody; And / or

(ii) (I.e., the immunoconjugate or conjugate) of the labeled anti-TNF [alpha] drug, the labeled TNF [alpha], and the autoantibody (i.e., the components of the second labeled conjugate are not covalently bonded to each other);

(b) The first labeled complex and / or the second labeled complex can be applied to size exclusion chromatography to convert them to free (i.e., unbound) labeled TNF ?, free labeled anti-TNF? Drug and / or labeled anti -TNFa < / RTI > drug and a labeled TNFa complex;

(c) After the size exclusion chromatography, the level of the free labeled TNF [alpha] is measured (for example, by measuring the area under the curve (TNF [alpha]) of the free labeled TNF [alpha] peak after size exclusion chromatography (SEC); And

(d) The free labeled TNF [alpha] measured in step (c) is compared to the level of the free labeled TNF [alpha] in the control sample (e.g., the AUC of the free labeled TNF [alpha] peak after SEC of a reference sample containing only free TNFa , Thereby detecting the presence of autoantibodies in neutralized and / or non-neutralized form.

In certain embodiments, the autoantibody in neutralized form is detected when the level of free labeled TNF [alpha] measured in step (c) is the same or substantially the same as the level of free labeled TNF [alpha] in the control sample. In certain other embodiments, the autoantibody of the non-neutralizing form is characterized in that the level of the free labeled TNF [alpha] measured in step (c) is reduced (e.g., substantially reduced compared to the level of free labeled TNF [ Reduced) or absent (e.g., undetected).

In another particular aspect, the invention provides a method of determining the level or percentage of autoantibodies in neutralization form to an anti-TNF [alpha] drug in a sample, comprising:

(a) A sample is contacted with a labeled anti-TNF [alpha] drug and labeled TNF [alpha] to form:

(i) The first labeled complex (i.e., immunoconjugate or conjugate) of the labeled anti-TNF [alpha] drug and autoantibody (i.e., the components of the first labeled conjugate are not covalently bonded to each other); And / or

(ii) (I.e., the immunoconjugate or conjugate) of the labeled anti-TNF [alpha] drug, the labeled TNF [alpha], and the autoantibody (i.e., the components of the second labeled conjugate are not covalently bonded to each other);

(b) The first labeled complex and / or the second labeled complex can be applied to size exclusion chromatography to convert them to free (i.e., unbound) labeled TNF ?, free labeled anti-TNF? Drug, and / Separating from the complex of anti-TNF [alpha] drug and labeled TNF [alpha];

(c) After the size exclusion chromatography, the level of the free labeled TNF [alpha] is measured (for example, by measuring the area under the curve (TNF [alpha]) of the free labeled TNF [alpha] peak after size exclusion chromatography (SEC); And

(d) The level of free labeled TNF [alpha] measured in step (c) is compared to the normalized level or percentage of free labeled TNF [alpha] in the control sample (e.g., after SEC of a reference sample containing only free TNF [alpha] Measuring and normalizing the AUC of the labeled TNFa peak to yield the level or percentage of free labeled TNFa), wherein the normalized level or percentage of free labeled TNFa in the control sample is the level of autoantibody in neutralized form or Corresponds to the percentage.

In some embodiments, the difference between the normalized level or percentage of free labeled TNF [alpha] in the control sample and the level of free labeled TNF [alpha] measured in step (c) corresponds to the level or percentage of the autoantibody in the non-neutralized form.

In another particular aspect, the invention provides a method of determining the level or percentage of autoantibodies in neutralization form to an anti-TNF [alpha] drug in a sample, comprising:

(a) A sample is contacted with a labeled anti-TNF [alpha] drug and labeled TNF [alpha] to form:

(i) The first labeled complex (i.e., immunoconjugate or conjugate) of the labeled anti-TNF [alpha] drug and autoantibody (i.e., the components of the first labeled conjugate are not covalently bonded to each other); And / or

(ii) (I.e., the immunoconjugate or conjugate) of the labeled anti-TNF [alpha] drug, the labeled TNF [alpha], and the autoantibody (i.e., the components of the second labeled conjugate are not covalently bonded to each other);

(b) The first labeled complex and / or the second labeled complex can be applied to size exclusion chromatography to convert them to free (i.e., unbound) labeled TNF ?, free labeled anti-TNF? Drug, and / Separating from the complex of anti-TNF [alpha] drug and labeled TNF [alpha];

(c) After the size exclusion chromatography, the level of the free labeled TNF [alpha] is measured (for example, by measuring the area under the curve (TNF [alpha]) of the free labeled TNF [alpha] peak after size exclusion chromatography (SEC); And

(d) The level of the free labeled TNF [alpha] measured in step (c) is compared to the level of the free labeled TNF [alpha] in the control sample (for example, by measuring the AUC of free labeled TNF [alpha] after SEC of the reference sample) This measures the percentage or level of neutralizing autoantibodies.

In another particular aspect, the invention provides a method for determining whether an autoantibody in neutralization form to a first anti-TNF alpha drug is cross-reactive with a second (i.e., different) anti-TNF alpha drug, It provides a way to:

(a) Detect or measure the presence, level or percentage of autoantibody in neutralization form in a sample according to the assays described herein to determine whether the sample is positive or negative for autoantibodies in neutralized form; And

If the sample is positive for neutralizing autoantibodies, then:

(b) A sample is contacted with a labeled second anti-TNFα drug to form a labeled conjugate of the labeled second anti-TNFα drug and neutralizing form of the autoantibody (ie, the components of the labeled conjugate Unbound);

(c) The labeled complex is applied to size exclusion chromatography to separate the labeled complexes (e.g., from a second labeled anti-TNFα drug); And

(d) Labeled complex to thereby determine whether the neutralizing form of the autoantibody to the first anti-TNF [alpha] drug is cross-reactive with the second anti-TNF [alpha] drug.

In certain embodiments, the presence of the labeled complex indicates that the neutralizing autoantibody against the first anti-TNF alpha drug is cross-reactive with the second anti-TNF alpha drug, i.e., the neutralizing autoantibody is the first TNF < alpha > drug and the second anti-TNF alpha drug.

In certain other embodiments, the absence of the labeled complex indicates that the neutralizing autoantibody against the first anti-TNF alpha drug is not cross-reactive with the second anti-TNF alpha drug, i.e., the neutralizing autoantibody Will inhibit the activity of the second anti-TNF [alpha] drug.

In some embodiments, the anti-TNFa drug is selected from the group consisting of REMICADE ™ (infliximab), ENBREL ™ (etanercept), HUMIRA ™ (adalimumab), CIMZIA® (Sertoli marbella), SIMPONI® (golimumab) (CNTO 148), and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasma sample, e. G., A whole blood, serum, or plasma sample from a subject undergoing anti-TNFa drug therapy. In a preferred embodiment, the sample is serum. In certain embodiments, the subject is a subject having a TNFa-mediated disease or disorder, such as an autoimmune disease (e.g., rheumatoid arthritis) or an inflammatory disease (such as inflammatory bowel disease (IBD) Colitis (UC)).

In certain embodiments, the sample is suspected to have or have an autoantibody to the anti-TNF [alpha] drug. In other embodiments, anti-TNF alpha drug autoantibodies include, but are not limited to, human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti- Combinations are included.

In a particular aspect, the assay method of the present invention further comprises an acid dissociation step comprising contacting the sample with an acid before, during and / or after contacting the sample with labeled anti-TNF [alpha] drug and labeled TNF [alpha] .

In certain other aspects, the assay methods of the invention include detecting the presence or level of one or more subtypes of autoantibodies in neutralized and / or non-neutralized form for the anti-TNF [alpha] drug in a sample.

In a further aspect, the present invention provides a method for monitoring and / or optimizing therapeutic regimens for a biological agent, in a subject undergoing a biotherapeutic treatment regimen, comprising:

(a) The presence, level or percentage of neutralizing forms of autoantibodies to biologics in accordance with the assays described herein are detected or measured during the therapeutic regimen at a plurality of time points;

(b) Detecting the presence, level, or percentage change of neutralizing form of autoantibodies over time; And

(c) Based on changes in the presence, level or percentage of neutralizing autoantibodies over time, determine whether subsequent doses of the therapeutic regimen for the subject or different therapeutic regimens should be applied to the subject.

In one particular aspect, the invention provides a method for monitoring and / or optimizing therapy for a biological agent in a subject undergoing a biotherapeutic treatment regimen, comprising:

(a) measuring, at a time point t ₀ , the level or percentage of autoantibody in neutralized form to a biological agent in a first sample from a subject as described herein;

(b) measuring, at time point t ₁ , the level or percentage of autoantibody in neutralization form in a second sample from a subject as described herein;

(c) optionally repeating step (b) from the subject to n additional samples at time point t _{n + 1} , wherein n is an integer from 1 to about 25 (e.g., n is 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, Internal range).

(d) detecting a change in the level or percentage of autoantibodies in neutralization form at time points t ₀ to t ₁ or at time points t ₀ to t _{n + 1} ; And

(e) Based on changes in the level or percentage of neutralizing autoantibodies over time, a determination is made as to whether subsequent doses of the therapeutic regimen for the subject or different therapeutic regimens should be applied to the subject.

In a further aspect, the present invention provides a method for optimizing therapy and / or alleviating toxicity in a subject undergoing a therapy with a first biologic agent, comprising:

(a) Whether the neutralizing form of an autoantibody to a first biologic agent is cross-reactive with a second (i.e., different) biological agent is determined by the presence of an autoantibody in neutralized form in the sample from the subject according to the assays described herein, By detecting or measuring a level or a percentage; And

(b) If the neutralizing form of the autoantibody is cross-reactive with the second biological agent, determine whether different therapeutic regimens should be applied to the subject.

In one particular aspect, the invention provides a method of monitoring and / or optimizing therapeutic regimens for an anti-TNF [alpha] drug in a subject undergoing a therapeutic regimen with an anti-TNF [alpha] drug,

(a) Detecting or measuring the presence, level or percentage of autoantibodies in the form of neutralization to an anti-TNF alpha drug according to the assays described herein at multiple time points throughout the course of therapy;

(b) Detecting the presence, level or percentage change of neutralizing form of autoantibodies over time; And

(c) Based on changes in the presence, level, or percentage of neutralizing autoantibodies over time, determine whether different therapeutic regimens should be applied to the subject, or determine the follow-up dose of the therapeutic regimen for the subject.

In another particular aspect, the invention provides a method of optimizing and / or monitoring therapeutic therapy for an anti-TNF [alpha] drug in a subject undergoing a therapeutic regimen with an anti-TNF [alpha] drug,

(a) measuring the level or percentage of autoantibody in neutralized form to an anti-TNFα drug in a first sample from a subject as described herein at time point t ₀ ;

(b) measuring the level or percentage of autoantibody in neutralized form in a second sample from a subject as described herein at time point t ₁ ;

(c) optionally repeating step (b) with n additional samples from the subject at time point t _{n + 1} , wherein n is an integer from 1 to about 25 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, ); ≪ / RTI >

(d) detecting a change in the level or percentage of autoantibodies in neutralization form at time points t ₀ to t ₁ or at time points t ₀ to t _{n + 1} ; and

(e) Based on changes in the level or percentage of neutralizing autoantibodies over time, determine whether subsequent doses of the therapeutic regimen for the subject or different therapeutic regimens should be applied to the subject.

In another particular aspect, the invention provides a method of optimizing treatment and / or alleviating toxicity in a subject undergoing a therapeutic regimen with a first anti-TNF alpha drug, comprising:

(a) By detecting or measuring the presence, level or percentage of autoantibodies in neutralization form in the sample from a subject according to the assays described herein, the neutralizing form of the autoantibody to the first anti-TNFa drug is detected or measured in a second (i. Determining whether the compound is cross-reactive with an anti-TNF [alpha] drug; And

(b) If the neutralizing form of the autoantibody is cross-reactive with the second anti-TNFα drug, determine if a different therapeutic regimen should be applied to the subject.

Other objects, features and advantages of the present invention will be apparent to those skilled in the art from the following detailed description and drawings.

Figure 1 shows that there is a clear correlation between the NAb percent (y-axis) and ATI levels.
Figure 2 shows that ATI concentration ≥ 60 U / ml predicts NAb +.
Figure 3 shows that ATI predicts NAb with ROC AUC of 0.931.
Figure 4 shows the detection of ATI by the fluid phase mobility shift assay of the present invention.
Figure 5 illustrates an exemplary ATI / IFX fluid phase mobility variation assay of the present invention.
Figure 6 shows a non-neutralizing anti-drug antibody (ADA) assay of the invention.
Figure 7 shows the neutralization ADA assay of the present invention.
Figure 8 shows the levels of IFX and AIT over time for five samples from UC patients taken at 1, 2, or 3 months.
Figure 9 shows a peak analysis for determining the percentage of free TNF [alpha] over time in UC patients.
Figure 10 shows the presence variation from non-neutralizing autoantibodies to neutralizing autoantibodies over time as illustrated in the three samples spiked with IFX and taken at 2 or 3 months from UC patients.
Figure 11 shows a peak analysis that measures the percentage of free TNF [alpha] over time in IFX spiked UC patient samples.
Figure 12 shows the use of rabbit anti-human IgGl Fc as a non-neutralizing antibody (Ab) control.
Figure 13 shows the use of ATI positive sera as a mixed neutralizing antibody (NAb) / non-neutralizing antibody (Ab) control.
Figure 14 shows that ATI purification from ATI positive sera results in loss of weak affinity NAb.
Figure 15 shows a peak analysis from a UC patient case study to determine the percentage of free TNF [alpha] in these various control groups.
Figure 16 shows a peak analysis from a CD patient case study to determine the percentage of free TNF [alpha] over time for four samples taken at 7 or 8 weeks for a 30 week period.
Figure 17 shows a peak analysis from another CD patient case study to determine the percentage of free TNFa over time for three samples taken over a 50 week period.
Figure 18 shows a peak analysis from an additional four CD patient case studies for determining the percentage of free TNF [alpha] in a sample during or after treatment regimen induction or maintenance.
Figure 19 shows the detection of non-neutralizing antibody activity through mobility variation assays.
Figure 20 shows the cross-reactivity of ADA to both IFX and ADL, where the binding site of ADA mimics the binding site of TNFa and thus can bind to multiple anti-TNF drugs.
Figure 21 shows examples of two patients (Patients 1 and 2) that measure the cross-reactivity of NAb produced in response to one anti-TNF drug to another anti-TNF drug. In particular, the NAb developed when Humans were being treated for Remicade (IFX) was tested against Humira (ADL).
Figure 22 shows an exemplary embodiment of the assay of the invention for detecting the presence of a non-neutralizing antibody (non-NAb) (top) or neutralizing antibody (NAb) (bottom) against a drug such as IFX or ADL.
Figure 23 shows the fabrication and use of the NAb standard curve.
Figure 24 provides the results of a case study of patient 3 who was treated with IFX but lost responsiveness to IFX to measure the cross-reactivity of NAb produced against IFX against ADL.
Figure 25 provides case study results of patient 4 who had been treated with IFX but lost responsiveness to IFX to measure cross-reactivity to ADL of NAb produced against IFX.
Figure 26 shows a non-limiting example of a patient study demonstrating the development of cross-reactive ATI and ATI-affinity maturation.
Figure 27 shows one embodiment of the competitive ligand binding NAb assay of the present invention. Free TNF-532 represents the maximum black signal. A TNF-532 / IFX-488 negative control without ATI represents the minimum assay signal. (A) Combined ATI that does not affect the test signal. (B) neutralized ATI patient serum to restore the test signal.
Figure 28 shows the clinical utility of the neutralization assay of the present invention.
Figure 29 shows the performance characteristics of the neutralization assay of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

I. INTRODUCTION

The present invention is based in part on the use of size exclusion chromatography, which allows equilibration of immune complexes, and, optionally, homogeneous mobility shift assays using acid dissociation, as the neutralizing and non-neutralizing forms of the neutralizing and non-neutralizing forms produced for biologics such as anti- Is particularly advantageous for determining the presence or level of an antibody (e. G., HACA, HAHA, etc.). Such autoantibodies are also known as anti-drug antibodies or ADA, and their neutralized and non-neutralized forms are also known as NAb and non-NAb, respectively.

In certain embodiments, the homogeneous mobility shift assay of the present invention can be used to detect a biological agent (e. G., An anti-TNFa drug) and a biological binding moiety (e. G. ) And the sample of the object. The assays described herein are advantageous for at least the following reasons: the need for a cleaning step to remove low affinity ADA is eliminated; Using distinct markers such as fluorophore to allow detection in the visible, IR and / or near IR (NIR) spectrum, reducing background and serum interference problems; Increase the ability to detect neutralized and / or non-neutralized ADA in subjects with low titers due to the sensitive sensitivity of fluorescent label detection; And since it is a liquid phase reaction, it reduces the possibility of any change in the epitope by attachment to a solid surface such as an ELISA plate.

In an exemplary embodiment, the assays of the present invention include time-course cases of IBD patients undergoing anti-TNFa drug therapy for monitoring the formation of multiple neutralizing and / or non-neutralizing anti-drug antibodies in samples at different time points It is advantageous because it enables research. The assays of the present invention are also advantageous because the subject is able to determine whether there is a change from non-neutralization to neutralizing anti-drug antibody over time during the anti-TNFa drug therapy. Regardless of any particular theory, neutralizing anti-drug antibodies can result in significant negative clinical outcomes, since this interferes with the binding of the anti-TNFα drug to TNFα, thus leading to loss of efficacy.

In a further exemplary embodiment, the assay of the present invention is found to be useful in predicting and / or measuring the cross-reactivity of neutralizing anti-drug antibodies in a sample of an object with an alternative biological drug, such as other anti-TNF drugs. For illustrative purposes only, if the sample contains neutralizing ADA for one anti-TNF [alpha] drug, then these neutralizing ADAs are cross-reactive against other anti-TNF [alpha] drugs and are likely to be neutralized against them, The recommended treatment modality will be converted to a drug with a different mechanism of action (e.g., a non-anti-TNF agent). However, if the sample contains a non-neutralizing ADA for one anti-TNF alpha drug, the therapeutic adjustments recommended for the subject may be converted to another anti-TNF alpha drug.

Accordingly, the present invention is directed to the use of an anti-TNF alpha drug such as infliximab and adalimumab in the treatment of an anti-TNF alpha drug therapy by providing information useful for guiding a therapeutic decision in a subject being provided anti- And overcomes the current constraints associated with. The methods of the present invention are particularly useful for monitoring the subject receiving the anti-TNF [alpha] drug to detect or measure the formation and / or development of neutralizing ADA (e.g., over time during the anti-TNFa drug therapy regimen) And is also useful for detecting or measuring a change (e.g., an increase) in the amount, percentage, or ratio of neutralizing ADA compared to non-neutralizing ADA over time while the subject is on anti-TNFa drug therapy.

Thus, the present invention provides a method for the treatment or prophylaxis of (1) modulating or modifying (e.g., increasing or decreasing) a subsequent dose of an anti-TNFa drug in terms of the presence, level or percentage of neutralizing ADA to optimize therapeutic efficacy and / (MTX) or azathioprine (AZA) with one or more immunosuppressive agents, such as methotrexate (MTX) or azathioprine (AZA), with a neutralizing ADA Level or percentage of neutralizing ADA, and / or (3) altering the current therapeutic regimens in terms of the presence, level or percentage of neutralizing ADA (e.g., different anti-TNFa drugs or different mechanisms ≪ / RTI > to a drug that targets < RTI ID = 0.0 > and / or < / RTI > This method can be used to determine whether the subject receiving the anti-TNFa drug is taking the correct dose, does not cause an immune response to the drug, should switch to a different drug due to failure at the time of initial drug use (e.g., The development of cross-reactive neutralizing ADA).

II. Justice

As used in the specification, the following terms have their original meanings unless otherwise specified.

The term "biologic" or "biologic agent ", or" biologic drug ", as used herein, refers to a product or substance that is produced or extracted from a biological system (e. . Non-limiting examples of biological agents include antibodies, antibody fragments, proteins, polypeptides, peptides, fusion proteins (e.g., Ig fusion proteins or Fc fusion proteins), polyvalent binding proteins (e.g. DVD Ig), antibody- Gates, vaccines, nucleic acids, sugars, recombinant forms thereof, processed forms thereof, and combinations thereof.

The term "biological binding moiety" includes any molecule, agent or substance that (specifically) binds or interacts with a biological agent. In certain instances, autoantibodies in neutralized form interfere with binding between the biological binding moiety and the biological agent. In certain other instances, autoantibodies in the non-neutralized form do not interfere with the binding between the biological binding moiety and the biological agent. As one non-limiting example, the biological binding moiety comprises TNFa when the biological agent comprises an anti-TNF [alpha] drug. In another non-limiting example, the biological binding moiety comprises an interleukin receptor (e. G., A soluble extracellular fragment of an interleukin receptor) when the biological agent comprises an interleukin such as IL-2.

As used herein, the term "anti-TNFα drug" or "TNFα inhibitor" refers to a protein, antibody, antibody fragment, fusion protein (eg, Ig fusion protein or Fc fusion protein) , Directly or indirectly, such as, for example, a multivalent binding protein (e. G. DVD Ig), a small molecule TNF alpha antagonist and similar naturally occurring or non-naturally occurring molecules, and / or recombinants and / By inhibiting the TNF alpha gene expression by inhibiting TNF alpha protein production by inhibiting TNF alpha and TNF alpha interaction with cell surface receptors, TNF alpha secretion from the cells is suppressed by inhibiting TNF alpha gene expression, Is intended to include agents that inhibit TNF [alpha] activity by inhibiting TNF [alpha] receptor signaling or any other means that decrease activity. The term " anti-TNFa drug "or" TNFa inhibitor "preferably includes an agent that interferes with TNFa activity. Examples of TNFa inhibitors include, but are not limited to, infliximab (REMICADE ™, Johnson and Johnson), human anti-TNF monoclonal antibody adalimumab (D2E7 / HUMIRA ™, Abbott Laboratories), etanercept (ENBREL ™, Amgen, CIMZIA®, UCB, Inc., SIMPONI® CNT0148, CDP 571 (Celltech), and CDP 870 (Celltech), as well as TNFα activity with deleterious disorders And other compounds that inhibit TNF [alpha] activity, such that the disorder is treated upon administration to a subject at risk of suffering or suffering from RA.

The term "TNFa" is intended to include human cytokines, which are a biologically active form of a 17 kDa secreted form and a trimer of noncovalently associated 17 kDa molecules present as a 26 kDa membrane bound form. The structure of TNFa is described, for example, in Jones et al. , Nature, 338: 225-228 (1989). The term TNF [alpha] is intended to include human TNF [alpha], recombinant human TNF [alpha] (rhTNF-a), or TNF [alpha] that is at least about 80% identical to human TNF [alpha] protein. Human TNFa consists of a 35 amino acid (aa) cytosolic domain, 21 aa transmembrane segment, and 177 aa extracellular domain (ECD) (Pennica, D. et al. (1984) Nature 312: 724). In ECD, human TNFα shares 97% to 99% identity with rhesus monkey TNFα, and 71% to 92% identity with cow, dog, cotton rat, horse, cat, mouse, pig and rat TNFα. TNFa can be produced by standard recombinant expression methods or commercially available (R & D Systems, catalog number 210-TA, Minneapolis, Minn.).

In certain embodiments, "TNFa" is an "antigen ", which comprises a molecule or a portion of the molecule to which an anti-TNF-alpha drug can be bound. TNFa may have one or more epitopes. In a particular example, TNF [alpha] reacts with anti-TNF [alpha] antibodies in a highly selective manner. Preferred antigens that bind to antibodies, fragments and regions of anti-TNF [alpha] antibodies comprise at least five amino acids of human TNF [alpha]. In a particular example, TNF [alpha] is of sufficient length to have an epitope of TNF [alpha] that is capable of binding to the anti-TNF [alpha] antibody, its fragments and regions.

 The term " size exclusion chromatography "or" SEC "includes chromatographic methods in which molecules in a 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 the aqueous solution is used for the transfer of the sample through the column, the technique is known as gel filtration chromatography.

The terms "complex," "immuno-complex," "conjugate," and "immunoconjugate" refer to the administration of an anti-TNFα drug (eg, (E.g., by means of non-covalent attachment) to an autoantibody (e. G., A neutralized or non-neutralized anti-drug antibody) to an anti-TNFa drug, TNFa drugs conjugated (e.g., by non-covalent means) to an autoantibody (e. G., A neutralized or non-neutralized anti-drug antibody) to both TNFa and an anti-TNFa drug, But is not limited to.

As used herein, an entity modified by the term "labeled" refers to any entity, molecule, protein, enzyme, antibody, antibody fragment, cytokine, or other molecule or chemical that is empirically detectable Lt; / RTI > and related species that are conjugated with the wild type. The cover - species suitable as a marker of the object is a fluorescent dye, e.g., Alexa Fluor® dye, such as Alexa Fluor® 647, a quantum dot, an optical dye, a luminescent dye, and radiation, including, for example ¹²⁵ I, but this But is not limited to.

The phrase "fluorescence label detection" includes means for detecting a fluorescent labeling substance. Detecting means may be, for example, an Agilent-1200 HPLC System, including, but not limited to, size exclusion-high performance liquid chromatography, including, but not limited to, a spectrophotometer, Fluorescence spectrometers, photometers, and detection devices.

The phrase "optimize therapy" includes optimizing the dosage (e.g., an effective amount or level) and / or type of a particular therapy. For example, optimization of the dose of anti-TNF [alpha] drug includes increasing or decreasing the amount of anti-TNF [alpha] drug that is subsequently administered to the subject. In certain instances, optimization of the type of anti-TNF [alpha] drug involves converting from one drug to a different drug (e.g., a drug that targets a different anti-TNF [alpha] drug or a different mechanism). In another example, the optimization of treatment regimens may be combined with one or more immunosuppressive drugs in combination with a dose of an anti-TNF [alpha] drug (e.g., increased, decreased, or the same dose relative to a previous dose) .

The term " co-administer "includes administration of more than one active agent such that the duration of the physiological effect of one active agent overlaps with the duration of the physiological effect of the second active agent .

The term "subject," " patient, "or" individual "means typically a human, but may also refer to other animals such as other primates, rodents, canines, , Pigs, and the like.

The term " course of therapy "includes any therapeutic approach taken to alleviate or prevent one or more symptoms associated with a disease or disorder. The term encompasses any of the therapeutic agents described herein, including the application of any compound, drug, procedure, and / or therapy useful for improving the health of an individual having the disease or disorder. As a non-limiting example, the dosages of the therapeutic regimens or current therapeutic regimens are determined by the presence or concentration level of the TNFα, anti-TNFα drug and / or anti-drug antibody (eg, Level or percentage of neutralizing and / or non-neutralizing anti-drug antibodies) can be altered (e.g., increased or decreased).

The term "immunosuppressive drug" or "immunosuppressive agent" can be used to achieve the prevention or amelioration of an immune response by the administration of a medicament such as an immunosuppressive effect, e.g., radiation or anti-metabolites, anti-lymphocyte serum, ≪ / RTI > Examples of immunosuppressive drugs include thiopurine drugs such as azathioprine (AZA) and its metabolites; Anti-metabolites such as methotrexate (MTX); Sirolimus (rapamycin); Temsirolimus; Everolimus; Tacrolimus (FK-506); FK-778; Anti-lymphocyte globulin antibody, anti-thymocyte globulin antibody, anti-CD3 antibody, anti-CD4 antibody, and antibody-toxic conjugate; Cyclosporine; Mycophenolate; Mizoribine monophosphate; Scoparone; Glatiramer acetate; Its metabolites; A pharmaceutically acceptable salt thereof; Its derivatives; Its prodrug drugs; ≪ / RTI > and combinations thereof.

The term "thiopurine dug" refers to azathioprine (AZA), 6-mercaptopurine (6-MP), or 6-thioguanine (6-TG) as any metabolite with therapeutic efficacy, (E.g., 6-thioinosine monophosphate, 6-thioinosine diphosphate, 6-thioinosine triphosphate), 6-thioguanine nucleotides For example, 6-thioguanosine monophosphate, 6-thioguanosine triphosphate, 6-thioguanosine triphosphate), 6-thioantosine nucleotides But are not limited to, thiophosphate, neodiphosphate, 6-thioctosine triphosphate), derivatives thereof, analogs thereof, and combinations thereof.

The term "sample" includes any biological specimen obtained from an individual. The samples may include whole blood, serum, plasma, red blood cells, leukocytes (e.g., peripheral blood mononuclear cells (PBMC), polymorphonuclear (PMN) cells), catheter rinses, nipple inhalants, lymph (e.g., Tumor cells), bone marrow aspirates, saliva, urine, feces (e.g., feces), feces, bronchial rinses, tears, fine needle aspirates (e.g., random mastitis fine needle aspirates) But are not limited to, a sample, for example, a biopsy (e.g., a puncture biopsy) of an inflammatory site, an extract thereof, and an immunoglobulin-enriched fraction derived from one or more of said body fluids or tissues. In some embodiments, the sample is whole blood, a portion thereof, such as plasma, serum or cell pellet or an immunoglobulin rich fraction thereof. Those skilled in the art will appreciate that suitable samples such as serum samples may be diluted prior to analysis. In certain embodiments, the sample is obtained by isolating PBMC and / or PMN cells using any technique known in the art. In certain other embodiments, the sample is a tissue biopsy, such as, for example, from an inflammatory site such as the gastrointestinal tract or a portion of a lubrication tissue.

The steps of the method of the present invention need not be performed in the specific order in which they are presented. Those skilled in the art will appreciate that other sequence arrangements of steps of the method of the present invention are also included within the scope of the present invention.

Parentheses "[]" refer to their concentration in the type inside parentheses.

III. Description of Implementation Example

The present invention provides assays for detecting and measuring the presence or level of neutralizing and non-neutralizing autoantibodies to biologics such as anti-TNF alpha drug therapies in a sample. The present invention is useful for monitoring the formation of neutralizing and / or non-neutralizing anti-drug antibodies over time during which the subject is being biologically treated (e. G., Anti-TNFa drug therapy). The present invention is also useful for predicting and / or determining the cross-reactivity of neutralizing anti-drug antibodies in a sample of an object with alternative biological therapy (e. G., Alternative anti-TNFa therapy). Thus, the present invention provides guidance information on treatment decisions for subjects receiving therapeutic treatment with a biological agent, optimizing treatment regimens, alleviating toxicity and / or improving the efficacy of therapeutic regimens for biologic therapy Improves accuracy in monitoring.

In one aspect, the invention provides a method of detecting the presence of a neutralizing and / or non-neutralizing form of an autoantibody to a biological agent in a sample, comprising:

(a) A sample is contacted with a labeled biologic agent and a labeled biological binding moiety to form:

(i) The first labeled conjugate of the labeled biologic agent and autoantibody (i.e., the immunoconjugate or conjugate) (i.e., the components of the first labeled conjugate are not covalently bonded to each other); And / or

(ii) (I.e., the immunoconjugate or conjugate) of the labeled biologic agent, the labeled biological binding moiety and the autoantibody (i.e., the components of the second labeled conjugate are not covalently bonded to each other);

(b) The first labeled complex and / or the second labeled complex can be applied to size exclusion chromatography to convert them into free (i.e., unbound) labeled biological binding moieties, free labeled biologics, and / Separation from the complex of the agent and the labeled biological binding moiety;

(c) After the size exclusion chromatography, the level of the free labeled biological binding moiety is measured (e.g., by measuring the area under the curve (AUC) of the free labeled biologically bound partial peak after size exclusion chromatography (SEC)); And

(d) The level of the free labeled biological binding moiety measured in step (c) is compared to the level of the free labeled biological binding moiety in the control sample (e. G., The SEC of the reference sample containing only the free labeled biologically binding moiety) Followed by measuring the AUC of the free labeled biologically binding partial peak), thereby detecting the presence of autoantibodies in the neutralized and / or non-neutralized form.

In some embodiments, autoantibodies in neutralized form interfere with binding between the biological agent and the biological binding moiety. In other embodiments, the autoantibody of the non-neutralizing form does not interfere with the binding between the biological agent and the biological binding moiety.

In some instances, a free labeled biological binding moiety consists of a labeled biological binding moiety that is substantially free of the bound biological agent (e.g., labeled and / or unlabeled biological agent).

In certain embodiments, the autoantibody in neutralized form is detected when the level of the free labeled biological binding moiety measured in step (c) is the same or substantially the same as the level of the free labeled biological binding moiety in the control sample. In certain other embodiments, the autoantibody of the non-neutralizing form is characterized in that the free labeled biological binding moiety measured in step (c) is reduced in comparison to the level of the free labeled biological binding moiety in the control sample (e. G. Substantially reduced) or absent (e.g., not detected).

In certain embodiments, the level of the free labeled biological binding moiety measured in step (c) is at least about 70%, 75%, 80%, 81%, or 80% of the level of the free labeled biological binding moiety measured in the control sample , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% %, Or 99%, is considered to be substantially equal to the level of the free labeled biological binding moiety in the control sample. In certain embodiments, the level of the free labeled biological binding moiety measured in step (c) is at least about 50%, 55%, 60%, 65% or more than the level of the free labeled biological binding moiety measured in the control sample. , 70%, 75%, 80%, 85%, 90%, or 95% of the free labeled biological binding moiety in the control sample.

In some embodiments, the level of the free labeled biological binding moiety is determined from a plot of signal intensity, as a function of elution time from size exclusion chromatography (e.g., SEC-HPLC), of a free labeled biological binding moiety peak Is measured by integrating the area under the curve (AUC).

In some embodiments, the biological agent is selected from the group consisting of an antibody (e.g., an anti-TNFa monoclonal antibody), an antibody fragment, a protein (e.g., a cytokine such as an interleukin), a polypeptide, a peptide, Antibody-drug conjugates, vaccines, nucleic acids, sugars, recombinant forms thereof, processed forms thereof, and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasma sample, for example, a whole blood, serum, or plasma sample of a subject undergoing biotherapeutic therapy. In a preferred embodiment, the sample is serum. In certain embodiments, the subject is an autoimmune disease (e.g., rheumatoid arthritis), an inflammatory disease (e.g., inflammatory bowel disease (IBD) such as Crohn's disease (CD) or ulcerative colitis (UC) Of the disease or disorder.

In certain embodiments, the sample is suspected of having or having an autoantibody to the biological agent. In other embodiments, the biological autologous antibody includes but is not limited to human anti-chimeric antibody (HACA), human anti-humanized antibody (HAHA), and human anti-mouse antibody (HAMA) do.

In another aspect, the invention provides a method for determining the level or percentage of autoantibodies in neutralized form to a biological agent in a sample, comprising:

(a) A sample is contacted with a labeled biologic agent and a labeled biological binding moiety to form:

(i) The first labeled conjugate of the labeled biologic agent and autoantibody (i.e., the immunoconjugate or conjugate) (i.e., the components of the first labeled conjugate are not covalently bonded to each other); And / or

(ii) (I.e., the immunoconjugate or conjugate) of the labeled biologic agent, the labeled biological binding moiety and the autoantibody (i.e., the components of the second labeled conjugate are not covalently bonded to each other);

(b) The first labeled complex and / or the second labeled complex can be applied to size exclusion chromatography to convert them into free (i.e., unbound) labeled biological binding moieties, free labeled biologics, and / Separation from the complex of the agent and the labeled biological binding moiety;

(c) After the size exclusion chromatography, the level of the free labeled biological binding moiety is measured (e.g., by measuring the area under the curve (AUC) of the free labeled biologically bound partial peak after size exclusion chromatography (SEC)); And

(d) The level of the free labeled biological binding moiety measured in step (c) is compared to the normalized level or percentage of the free labeled biological binding moiety in the control sample (e. G., Only the free labeled biologically binding moiety Measuring and normalizing the AUC of the free labeled biologically binding moiety peak after SEC of the reference sample and calculating the level or percentage of the free labeled biologically binding moiety), wherein the normalized The level or percentage corresponds to the level or percentage of autoantibodies in neutralization form.

In some embodiments, the difference between the normalized level or percentage of the free labeled biological binding moiety in the control sample and the level of the free labeled biological binding moiety measured in step (c) is greater than the level or percentage of the non- ≪ / RTI >

In some instances, a free labeled biological binding moiety consists of a labeled biological binding moiety that is substantially absent of the bound biological agent (e.g., a label and / or unlabeled biological agent).

In certain embodiments, the level or percentage of the free labeled biological binding moiety in the control sample is determined by measuring the peak area of the complex formed (e. G., "Labeled complex") between the labeled biological agent and the labeled biological binding moiety (For example, by measuring the AUC), the measured peak area of the labeled complex is then compared to the peak area of the free labeled biological binding moiety (e. G., By measuring the AUC of the free labeled biologically binding partial peak Quot;). ≪ / RTI >

In certain embodiments, the level of the free labeled biological binding moiety is determined from a plot of signal intensity as a function of elution time from size exclusion chromatography (e.g., SEC-HPLC) under the curve area of the free labeled biologically binding moiety peak (AUC). In other embodiments, the level of complexes formed between labeled biologics and labeled biologically binding moieties can be determined from a plot of signal intensity as a function of elution time from size exclusion chromatography (e. G., SEC-HPLC) Is determined by integrating the AUC of the peak of the biological binding moiety.

In certain embodiments, the partial population of autoantibodies (e.g., ADA) to a biological agent is an autoantibody in neutralization form (e.g., NAb). In some embodiments, the total level of autoantibody to a biological agent in the sample is calculated by adding both the level of autoantibody in neutralized form and the level of autoantibody in non-neutralized form, measured according to the methods of the invention .

In some embodiments, the level of the free labeled biological binding moiety measured in step (c) is further compared with a negative control, a positive control, or a combination thereof. In a further embodiment, the percentage of neutralizing forms of autoantibodies (e.g., NAb) determined in step (d) is greater than the cutoff value or reference range established from a healthy control (e. G., Normal human serum) . In some embodiments, the cutoff value or reference range is expressed as a percentage threshold of NAb that the sample must have to be considered positive for NAb. In this embodiment, the sample is positive for NAb if the percentage of NAb measured in step (d) is greater than or equal to the cutoff value established in a healthy control or reference range. In another embodiment, the sample is negative for NAb when the percentage of NAb measured in step (d) is less than the cutoff value or reference range established in a healthy control. Non-limiting examples of cutoff values or reference ranges include at least about 0.25%, 0.50%, 0.75%, 1.00%, 1.50%, 2.00%, 2.50%, 2.60%, 2.70%, 2.80%, 2.90% 3.0%, 3.02%, 3.03%, 3.04%, 3.05%, 3.06%, 3.07%, 3.08%, 3.09%, 3.10%, 3.20%, 3.30%, 3.40%, 3.50%, 4.00%, 4.50% 5.00%, 5.50%, 6.00%, 6.50%, 7.00%, 7.50%, 8.00%, 8.50%, 9.00%, 9.50%, 10.00% NAb, or any range therein.

In some embodiments, the autoantibodies to the biological agent are all neutralizing antibodies and the sample is defined as having 100% neutralizing anti-drug antibody (NAb) and / or 0% non-neutralizing anti-drug antibody (non- do. In some embodiments, the level of the free labeled biological binding moiety measured in step (c) is generally equal to the level of the free labeled biological binding moiety in the control sample, and the autoantibody is bound to the biological agent and the biological binding moiety Lt; RTI ID = 0.0 > and / or < / RTI >

In other embodiments, none of the autoantibodies to the biological agent are neutralizing antibodies, and the sample is defined as having 100% non-NAb and / or 0% NAb. In these embodiments, the level of the free labeled biological binding moiety measured in step (c) is generally non-existent (e. G., Undetectable) compared to the level of the free labeled biological binding moiety in the control sample, Antibodies are not expected to completely block or interfere with binding between biologics and biological binding moieties.

In a further embodiment, when both the neutralizing and non-neutralizing forms of the autoantibody are present in the sample, the percentages of each species may be measured by itself (e.g., 50% NAb or 50% non- (Defined as an iso-ratio of non-NAb) or as a ratio. In a particular example, the ratio is calculated by dividing the percentage of NAb by the percentage of non-NAb or vice versa. In other examples, the ratio is calculated by dividing the level of NAb by the level of non-NAb or vice versa.

In some embodiments, the biologics may include antibodies (e.g., anti-TNFa monoclonal antibodies), antibody fragments, proteins (e.g., cytokines such as interleukins), polypeptides, peptides, fusion proteins, Antibody-drug conjugates, vaccines, nucleic acids, sugars, recombinant forms thereof, processed forms thereof, and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasma sample, for example, a whole blood, serum, or plasma sample of a subject undergoing biotherapeutic therapy. In a preferred embodiment, the sample is serum. In certain embodiments, the subject is an autoimmune disease (e.g., rheumatoid arthritis), an inflammatory disease (e.g., inflammatory bowel disease (IBD) such as Crohn's disease (CD) or ulcerative colitis (UC) Of the disease or disorder.

In certain embodiments, the sample is suspected to have or have an autoantibody to the biological agent. In other embodiments, biological autologous antibodies include but are not limited to human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti-mouse antibodies (HAMA) as well as combinations thereof .

In another aspect, the invention provides a method for determining the percentage or level of autoantibody in neutralized form to a biological agent in a sample, comprising:

(a) A sample is contacted with a labeled biologic agent and a labeled biological binding moiety to form:

(i) The first labeled conjugate of the labeled biologic agent and autoantibody (i.e., the immunoconjugate or conjugate) (i.e., the components of the first labeled conjugate are not covalently bonded to each other); And / or

(ii) (I.e., the immunoconjugate or conjugate) of the labeled biologic agent, the labeled biological binding moiety and the autoantibody (i.e., the components of the second labeled conjugate are not covalently bonded to each other);

(b) The first labeled complex and / or the second labeled complex can be applied to size exclusion chromatography to convert them into free (i.e., unbound) labeled biological binding moieties, free labeled biologics, and / Separation from the complex of the agent and the labeled biological binding moiety;

(c) After the size exclusion chromatography, the level of the free labeled biological binding moiety is measured (e.g., by measuring the area under the curve (AUC) of the free labeled biologically bound partial peak after size exclusion chromatography (SEC)); And

(d) The level of the free labeled biological binding moiety measured in step (c) is compared with the level of the free labeled biological binding moiety in the control sample (e. G., The AUC of the free labeled biologically binding partial peak after SEC of the reference sample , Thereby measuring the percentage or level of autoantibodies in neutralization form.

In some embodiments, the under-curve area (AUC) of the free labeled biological binding moiety peak is calculated for a sample (e.g., from a patient) and a control sample. In certain embodiments, the labeled biologic agent binds to the labeled biological binding moiety and reduces the assay signal (e. G., The free labeled biological binding of the sample as compared to the AUC of the free labeled biological binding moiety peak of the control sample Reducing the AUC of the partial peak). In another embodiment, the neutralizing form of the autoantibody (NAb) neutralizes the labeled biochemical agent and restores the assay signal (e.g., the AUC of the free labeled biologically bound portion peak of the sample is determined by the free labeled biological Restoring or recovering to a level comparable to the AUC of the binding site peak). In some embodiments, the NAb activity is directly proportional to the measured assay signal. In certain embodiments, the percentage of NAb in the sample is equivalent to the recovery percentage of the black signal. In a specific example, the percentage of NAb is calculated as the ratio of the AUC of the free labeled biologically binding portion peak of the sample to the AUC of the free labeled biologically binding portion peak of the control sample. As a non-limiting example, the percentage of NAb is calculated according to the following equation:% recovery = (free labeled biologically binding portion of sample _AUC -BKGD) / (free labeled biologically binding portion of control sample _AUC -BKGD) * 100.

In some instances, the free labeled biological binding moiety consists of a labeled biological binding moiety that is substantially free of the bound biological agent (e.g., a label and / or unlabeled biological agent).

In certain embodiments, the level of the free labeled biological binding moiety is determined from a plot of signal intensity as a function of elution time from size exclusion chromatography (e. G., SEC-HPLC) to a curve of the free labeled biologically- Is measured by integrating the area (AUC). In other embodiments, the level of complexes formed between labeled biologics and labeled biologically binding moieties can be determined from a plot of signal intensity as a function of elution time from size exclusion chromatography (e. G., SEC-HPLC) Is determined by integrating the AUC of the peak of the biological binding moiety.

In certain embodiments, the partial population of autoantibodies (e.g., ADA) to a biological agent is an autoantibody in neutralization form (e.g., NAb). In some embodiments, the total level of autoantibody to a biological agent in the sample is calculated by adding both the level of autoantibody in neutralized form and the level of autoantibody in non-neutralized form, measured according to the methods of the invention . In certain embodiments, autoantibodies in neutralized form are present in a population of autoantibodies in which both neutralizing autoantibodies and non-neutralizing autoantibodies are included.

In some embodiments, the level of the free labeled biological binding moiety measured in step (c) is further compared with a negative control, a positive control, or a combination thereof. In a further embodiment, the percentage of neutralizing forms of autoantibodies (e.g., NAb) determined in step (d) is compared to a cutoff value or reference range established, for example, from a sample having a low positive for autoantibodies . In some embodiments, the cutoff value or reference range is expressed as a percentage threshold of NAb that the sample must have to be considered positive for NAb. In this embodiment, the sample is positive for NAb if the percentage of NAb measured in step (d) is greater than the established cutoff value or reference range (or the same). In other embodiments, the sample is negative for NAb if the percentage of NAb measured in step (d) is less than the established cutoff value or reference range (or the same). Non-limiting examples of cutoff values or reference ranges include at least about 0.10%, 0.20%, 0.30%, 0.40%, 0.50%, 0.60%, 0.70%, 0.80%, 0.90%, 1.00%, 1.10% 1.20%, 1.21%, 1.22%, 1.23%, 1.24%, 1.25%, 1.26%, 1.27%, 1.28%, 1.29%, 1.30%, 1.40%, 1.50%, 2.00%, 2.50%, 2.60% 3.02%, 3.03%, 3.04%, 3.05%, 3.06%, 3.07%, 3.08%, 3.09%, 3.10%, 3.20%, 3.30%, 3.40%, 2.70%, 2.80%, 2.90%, 3.00% , 3.50%, 4.00%, 4.50%, 5.00%, 5.50%, 6.00%, 6.50%, 7.00%, 7.50%, 8.00%, 8.50%, 9.00%, 9.50%, 10.00% NAb, Range.

In some embodiments, the autoantibodies to the biological agent are all neutralizing antibodies and the sample is defined as having 100% neutralizing anti-drug antibody (NAb) and / or 0% non-neutralizing anti-drug antibody (non- do. In some embodiments, the level of the free labeled biological binding moiety measured in step (c) is generally equal to the level of the free labeled biological binding moiety in the control sample, and the autoantibody is bound to the biological agent and the biological binding moiety Lt; RTI ID = 0.0 > and / or < / RTI >

In other embodiments, none of the autoantibodies to the biological agent are neutralizing antibodies, and the sample is defined as having 100% non-NAb and / or 0% NAb. In these embodiments, the level of the free labeled biological binding moiety measured in step (c) is generally non-existent (e. G., Undetectable) compared to the level of the free labeled biological binding moiety in the control sample, Antibodies are not expected to completely block or interfere with binding between biologics and biological binding moieties.

In a further embodiment, when both the neutralizing and non-neutralizing forms of the autoantibody are present in the sample, the percentages of each species may be measured by itself (e.g., 50% NAb or 50% non- (Defined as an iso-ratio of non-NAb) or as a ratio. In a particular example, the ratio is calculated by dividing the percentage of NAb by the percentage of non-NAb or vice versa. In other examples, the ratio is calculated by dividing the level of NAb by the level of non-NAb or vice versa.

In some embodiments, the biologics include antibodies (e.g., anti-TNFa monoclonal antibodies), antibody fragments, proteins (e.g., cytokines such as interleukins), polypeptides, peptides, fusion proteins, Antibody-drug conjugates, vaccines, nucleic acids, sugars, recombinant forms thereof, processed forms thereof, and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasma sample, for example, a whole blood, serum, or plasma sample of a subject undergoing biotherapeutic therapy. In a preferred embodiment, the sample is serum. In certain embodiments, the subject is an autoimmune disease (e.g., rheumatoid arthritis), an inflammatory disease (e.g., inflammatory bowel disease (IBD) such as Crohn's disease (CD) or ulcerative colitis (UC) Of the disease or disorder.

In certain embodiments, the sample is suspected to have or have an autoantibody to the biological agent. In other embodiments, biological autologous antibodies include but are not limited to human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti-mouse antibodies (HAMA) as well as combinations thereof .

In another aspect, the invention provides a method for determining whether a neutralizing form of an autoantibody to a first biologic agent is cross-reactive with a second (i.e., different) biologic agent, comprising:

(a) Detect or measure the presence, level or percentage of autoantibody in neutralization form in the sample according to the assays described herein to determine whether the sample is positive or negative for neutralizing forms of autoantibodies; And

If the sample is positive for neutralizing autoantibodies, then:

(b) A sample is contacted with a labeled second biocide to form a labeled second biochemical and a labeled conjugate of an autoantibody in neutralized form (i.e., the components of the labeled conjugate are not covalently bound to each other);

(c) The labeled complex is applied to size exclusion chromatography to separate the labeled complexes (e.g., from a second biologically labeled free biocide); And

(d) Thereby detecting whether the neutralizing form of the autoantibody to the first biologic agent is cross-reactive with the second biological agent.

In certain embodiments, the presence of the labeled complex indicates that the neutralizing autoantibody against the first biological preparation is cross-reactive with the second biological agent, i.e., the neutralizing autoantibody is a first biological agent and Will inhibit the activity of both the second biological drug.

In certain other embodiments, the absence of the labeled conjugate indicates that the neutralizing antibody against the first biological agent is not cross-reactive with the second biological agent, i.e., the neutralizing autoantibody is a second biological agent Lt; / RTI >

In some embodiments, the first and second biological agents are independently selected from the group consisting of an antibody (e.g., an anti-TNFa monoclonal antibody), an antibody fragment, a protein (e. G., A cytokine such as an interleukin), a polypeptide, , A fusion protein, a polyvalent binding protein, an antibody-drug conjugate, a vaccine, a nucleic acid, a sugar, a recombinant form thereof, a processed form thereof, and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasma sample, for example, a whole blood, serum, or plasma sample of a subject undergoing biotherapeutic therapy. In a preferred embodiment, the sample is serum. In certain embodiments, the subject is an autoimmune disease (e.g., rheumatoid arthritis), an inflammatory disease (e.g., inflammatory bowel disease (IBD) such as Crohn's disease (CD) or ulcerative colitis (UC) Of the disease or disorder.

In certain embodiments, the sample is suspected to have or have an autoantibody to the biological agent. In other embodiments, biological autologous antibodies include but are not limited to human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti-mouse antibodies (HAMA) as well as combinations thereof .

In certain aspects, the assay of the present invention further comprises an acid dissociation step comprising contacting the sample with an acid before, during and / or after contacting the labeled biological agent and the labeled biological binding moiety .

In certain other aspects, the assays of the invention include detecting the presence or level of one or more subtypes of autoantibodies in the neutralized and / or non-neutralized form of the biological agent in the sample.

In one aspect, the invention provides a method of detecting the presence of an autoantibody in the form of a neutralized and / or non-neutralized form of an anti-TNF [alpha] drug in a sample,

(a) A sample is contacted with a labeled anti-TNF [alpha] drug and labeled TNF [alpha] to form:

(i) (I.e., the components of the first labeled complex are not covalently bonded to each other); the first labeled complex (i.e., the immunoconjugate or conjugate) of the labeled anti-TNF [alpha] drug and autoantibody; And / or

(ii) a labeled anti-drug -TNFα, a labeled complex of the labeled second and TNFα autoantibodies (i. e., immune-complex or conjugate) (in other words, the component of the labeled conjugate of the second share is not in combination with each other );

(b) The first labeled complex and / or the second labeled complex can be applied to size exclusion chromatography to convert them to free (i.e., unbound) labeled TNF ?, free labeled anti-TNF? Drug and / or labeled anti -TNFa < / RTI > drug and a labeled TNFa complex;

(c) The level of free labeled TNF [alpha] was measured after size exclusion chromatography (for example, by measuring the area under the curve of the free labeled TNF [alpha] peak after size exclusion chromatography (SEC) (AUC)); And

(d) The level of the free labeled TNF [alpha] measured in step (c) is compared to the level of free labeled TNF [alpha] in the control sample (e.g., after SEC of a reference sample containing only free TNFa, , Thereby detecting the presence of autoantibodies in the neutralized and / or non-neutralized form.

In some embodiments, autoantibodies in neutralized form inhibit binding between anti-TNF [alpha] drug and TNF [alpha]. In other embodiments, autoantibodies in the non-neutralizing form do not interfere with the binding between the anti-TNF [alpha] drug and TNF [alpha].

In some instances, the free labeled TNFa consists of a labeled TNFa substantially absent of the bound anti-TNF [alpha] drug (e.g., labeled and / or unlabeled anti-TNFa drug).

In certain embodiments, the autoantibody in neutralized form is detected when the level of free labeled TNF [alpha] measured in step (c) is the same or substantially the same as the level of free labeled TNF [alpha] in the control sample. In certain other embodiments, the autoantibody of the non-neutralizing form has a reduced (e.g., substantially reduced) level of free labeled TNF [alpha] measured in step (c) compared to the level of free labeled TNF [ Or is absent (e.g., not detected).

In certain embodiments, the level of free labeled TNF [alpha] measured in step (c) is at least about 70%, 75%, 80%, 81%, 82%, 83% 97%, 98%, 99%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , It is considered to be substantially equal to the level of free labeled TNF [alpha] in the control sample. In certain embodiments, the level of the free labeled TNF [alpha] measured in step (c) is at least about 50%, 55%, 60%, 65%, 70%, or more than the level of the free labeled TNF [ , 75%, 80%, 85%, 90%, or 95%, is considered to be substantially reduced when compared to the level of free labeled TNF [alpha] in the control sample.

In certain embodiments, the level of free labeled TNF [alpha] is determined by integrating the area under the curve (AUC) of the free labeled TNF [alpha] peak from a plot of signal intensity as a function of elution time from size exclusion chromatography (e.g., SEC- .

In some embodiments, the anti-TNF [alpha] drug is selected from the group consisting of REMICADE ™ (Infliximab), ENBREL ™ (etanercept), HUMIRA ™ (adalimumab), CIMZIA® (sertoli marbella), SIMPONI Muramov; CNTO 148), and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasma sample of a subject receiving a whole blood, serum, or plasma sample, for example, an anti-TNFa drug therapy. In a preferred embodiment, the sample is serum. In certain embodiments, the subject is a mammal such as a human, a human, a human, a human, a human, a human, a human, a human, a human, a human, Mediated diseases or disorders.

In certain embodiments, the sample is suspected to have or have an autoantibody to the anti-TNF [alpha] drug. In other embodiments, anti-TNF alpha drug autoantibodies include but are not limited to human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti-mouse antibodies .

In another particular aspect, the invention provides a method for determining the level or percentage of autoantibodies in neutralization form to an anti-TNF [alpha] drug in a sample, comprising:

(a) A sample is contacted with a labeled anti-TNF [alpha] drug and labeled TNF [alpha] to form:

(i) (I.e., the components of the first labeled complex are not covalently bonded to each other); the first labeled complex (i.e., the immunoconjugate or conjugate) of the labeled anti-TNF [alpha] drug and autoantibody; And / or

(ii) (I.e., the components of the second labeled complex are not covalently linked to each other); the second labeled complex of the labeled anti-TNF [alpha] drug, labeled TNF [alpha] and autoantibody (i.e., the immunoconjugate or conjugate);

(b) The first labeled complex and / or the second labeled complex can be applied to size exclusion chromatography to convert them into free (i.e., unbound) labeled TNF ?, free labeled anti-TNF? Drug, and / or labeled anti -TNFa < / RTI > drug and a labeled TNFa complex;

(c) Following the size exclusion chromatography, the level of free labeled TNF [alpha] is determined (e.g., by measuring the area under the curve (TNF [alpha]) of the free labeled TNF [alpha] peak after size exclusion chromatography (SEC); And

(d) The level of free labeled TNF [alpha] measured in step (c) is compared to the normalized level or percentage of free labeled TNF [alpha] in the control sample (e.g., after SEC of a reference sample containing only free TNF [alpha] Measuring or normalizing the AUC of the labeled TNF [alpha] peak to yield the level or percentage of free labeled TNF [alpha]), wherein the normalized level or percentage of free labeled TNF [alpha] in the control sample is the level of autoantibody in neutralization form Corresponds to the percentage.

In some embodiments, the difference between the normalized level or percentage of free labeled TNF [alpha] in the control sample and the level of free labeled TNF [alpha] measured in step (c) corresponds to the level or percentage of the autoantibody in the non-neutralized form.

In some instances, the free labeled TNFa consists of a labeled TNFa substantially absent of the bound anti-TNF [alpha] drug (e.g., labeled and / or unlabeled anti-TNFa drug).

In certain embodiments, the level or percentage of free labeled TNF [alpha] in the control sample is determined by measuring the peak area of the complex formed (e. G., The labeled complex) between the labeled anti-TNFa drug and the labeled TNFa (For example, by measuring the AUC of the free labeled TNF [alpha] peak), and then subtracting the measured peak area of the labeled complexes from the peak area of the free labeled TNF [alpha] do.

In certain embodiments, the level of the free labeled TNF [alpha] labeled is determined from a plot of signal intensity as a function of elution time from size exclusion chromatography (e.g., SEC-HPLC), the area under the curve of the free labeled TNF [ AUC). In other embodiments, the level of complex formed between the labeled anti-TNF [alpha] drug and the labeled TNF [alpha] is determined from a plot of signal intensity as a function of elution time from size exclusion chromatography (e.g., SEC- RTI ID = 0.0 > TNFa < / RTI >

In certain embodiments, the partial population of autoantibodies (e.g., ADA) to an anti-TNF [alpha] drug is an autoantibody in neutralization form (e.g., NAb). In some embodiments, the total level of autoantibody to the anti-TNF [alpha] drug in the sample is increased by adding both the level of autoantibody in neutralized form and the level of autoantibody in non-neutralized form, measured according to the methods of the invention Can be calculated.

In some embodiments, the level of free labeled TNF [alpha] measured in step (c) is further compared with a negative control, a positive control, or a combination thereof. Non-limiting examples of negative controls include mouse monoclonal anti-human IgG ₁ Fc samples and / or rabbit monoclonal anti-human IgG ₁ Fc samples. Non-limiting examples of positive controls include samples of rabbit polyclonal antibody to the F (ab ') ₂ fragment of the anti-TNFa drug and / or serum samples of pooled ADA-positive patients.

In a further embodiment, the percentage of neutralizing form of autoantibodies (e.g., NAb) determined in step (d) is compared to a cutoff value or reference range established from a healthy control (e.g., normal human serum). In certain embodiments, the cutoff value or reference range is expressed as a percentage threshold of NAb that the sample must have to be considered positive for NAb. In this embodiment, the sample is positive for NAb when the percentage of NAb measured in step (d) is greater than or equal to a cutoff value established in a healthy control. In another embodiment, the sample is negative for NAb when the percentage of NAb measured in step (d) is below the cutoff value or reference range established in a healthy control. Non-limiting examples of cutoff values or reference ranges include, for example, at least about 0.25%, 0.50%, 0.75%, 1.00%, 1.50%, 2.00%, 2.50%, 2.60%, 2.70%, 2.80% 3.00, 3.01, 3.02, 3.03, 3.04, 3.05, 3.06, 3.07, 3.08, 3.09, 3.10, 3.20, 3.30, 3.40, 3.50, 4.00, 4.50% , 5.00%, 5.50%, 6.00%, 6.50%, 7.00%, 7.50%, 8.00%, 8.50%, 9.00%, 9.50%, 10.00% NAb or any range therein. In some embodiments, the cutoff value or reference range is between about 3.00% NAb or about 3.06% NAb or between about 3.00% and -3.10% NAb.

In some embodiments, all autoantibodies to the anti-TNF [alpha] drug are neutralizing antibodies, and the sample comprises 100% neutralizing anti-drug antibody (NAb) and / or 0% non-neutralizing anti- drug antibody . In these embodiments, the level of free labeled TNF [alpha] measured in step (c) is generally equal to the level of the free labeled TNF [alpha] in the control sample, and the autoantibody completely blocks or inhibits binding between the anti- TNF [alpha] It is predicted to interfere.

In certain other embodiments, none of the autoantibodies to the anti-TNFa drug are neutralizing antibodies, and the sample is defined as having 100% non-NAb and / or 0% NAb. In these embodiments, the level of free labeled TNF [alpha] measured in step (c) is generally non-existent (e.g., undetectable) compared to the level of free labeled TNF [alpha] in the control sample, It is not expected to completely block or interfere with the binding between the TNF [alpha] drug and TNF [alpha].

In a further embodiment, if both the neutralizing and non-neutralizing forms of the autoantibody are present in the sample, the percentages of each species may be used alone (e.g., 50% NAb or 50% non- NAb and non-NAb) or as a ratio. In a particular example, the ratio is calculated by dividing the percentage of NAb by the percentage of non-NAb or vice versa. In other examples, the ratio is calculated by dividing the level of NAb by the level of non-NAb or vice versa.

In some embodiments, the anti-TNF [alpha] drug is selected from the group consisting of REMICADE ™ (Infliximab), ENBREL ™ (etanercept), HUMIRA ™ (adalimumab), CIMZIA® (sertoli marbella), SIMPONI Muramov; CNTO 148), and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasma sample of a subject receiving a whole blood, serum, or plasma sample, for example, an anti-TNFa drug therapy. In a preferred embodiment, the sample is serum. In certain embodiments, the subject is a mammal such as a human, a human, a human, a human, a human, a human, a human, a human, a human, a human, Mediated diseases or disorders.

In certain embodiments, the sample is suspected to have or have an autoantibody to the anti-TNF [alpha] drug. In other embodiments, anti-TNF alpha drug autoantibodies include but are not limited to human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti-mouse antibodies .

In another particular aspect, the invention provides a method of determining the percentage or level of autoantibody in neutralized form to an anti-TNF [alpha] drug in a sample, comprising:

(a) A sample is contacted with a labeled anti-TNF [alpha] drug and labeled TNF [alpha] to form:

(i) (I.e., the components of the first labeled complex are not covalently bonded to each other); the first labeled complex (i.e., the immunoconjugate or conjugate) of the labeled anti-TNF [alpha] drug and autoantibody; And / or

(ii) a labeled anti-drug -TNFα, a labeled complex of the labeled second and TNFα autoantibodies (i. e., immune-complex or conjugate) (in other words, the component of the labeled conjugate of the second share is not in combination with each other );

(b) The first labeled complex and / or the second labeled complex can be applied to size exclusion chromatography to convert them to free (i.e., unbound) labeled TNF ?, free labeled anti-TNF? Drug and / or labeled anti -TNFa < / RTI > drug and a labeled TNFa complex;

(c) The level of free labeled TNF [alpha] was measured after size exclusion chromatography (for example, by measuring the area under the curve of the free labeled TNF [alpha] peak after size exclusion chromatography (SEC) (AUC)); And

(d) The level of free labeled TNF [alpha] measured in step (c) is compared to the level of free labeled TNF [alpha] in the control sample (for example, by measuring the AUC of free labeled TNFa after SEC of the reference sample) This measures the percentage or level of neutralizing autoantibodies.

In some embodiments, the under-curve area (AUC) of the free labeled TNF [alpha] peak is calculated for a sample (e.g., from a patient) and a control sample. In certain embodiments, the labeled anti-TNF [alpha] drug binds to the labeled TNF [alpha] and decreases the assay signal (e.g., the AUC of the free labeled TNF [alpha] peak of the sample as compared to the AUC of the free labeled TNF [ . In another embodiment, the neutralizing form of the autoantibody (NAb) neutralizes the labeled anti-TNFα drug and restores the assay signal (eg, the AUC of the free labeled TNFα peak of the sample is compared to the free labeled TNFα Restoring or recovering to a level comparable to the AUC of the peak). In some embodiments, the NAb activity is directly proportional to the measured assay signal. In certain embodiments, the percentage of NAb in the sample is equivalent to the recovery percentage of the black signal. In a particular example, the percentage of NAb is calculated as the ratio of the AUC of the free labeled TNF [alpha] peak of the sample to the AUC of the free labeled TNF [alpha] peak of the control sample. As a non-limiting example, the percentage of NAb is calculated according to the following equation:% Recovery = (Free labeled TNFα _AUC -BKGD of sample) / (Free labeled TNFα _AUC -BKGD of control sample) * 100.

In some instances, the free labeled TNFa consists of a labeled TNFa substantially free of bound anti-TNF [alpha] drug (e.g., a label and / or unlabeled anti-TNFa drug).

In certain embodiments, the level of the free labeled TNF [alpha] is measured as a function of elution time from size exclusion chromatography (e.g., SEC-HPLC) . In other embodiments, the level of complex formed between the labeled anti-TNF [alpha] drug and the labeled TNF [alpha] is determined from a plot of signal intensity as a function of elution time from size exclusion chromatography (e.g., SEC- RTI ID = 0.0 > TNFa < / RTI >

In certain embodiments, the partial population of autoantibodies (e.g., ADA) to an anti-TNF [alpha] drug is an autoantibody in neutralization form (e.g., NAb). In some embodiments, the total level of autoantibody to the anti-TNF [alpha] drug in the sample is increased by adding both the level of autoantibody in neutralized form and the level of autoantibody in non-neutralized form, measured according to the methods of the invention Can be calculated. In certain embodiments, autoantibodies in neutralized form are present in a population of autoantibodies in which both neutralizing autoantibodies and non-neutralizing autoantibodies are included.

In some embodiments, the level of free labeled TNF [alpha] measured in step (c) is further compared with a negative control, a positive control, or a combination thereof. Non-limiting examples of negative controls include mouse monoclonal anti-human IgG ₁ Fc samples and / or rabbit monoclonal anti-human IgG ₁ Fc samples. Non-limiting examples of positive controls include samples of rabbit polyclonal antibody to the F (ab ') ₂ fragment of the anti-TNFa drug and / or serum samples of pooled ADA-positive patients.

In a further embodiment, the percentage of neutralizing forms of autoantibodies (e.g., NAb) determined in step (d) is compared to a cutoff value or reference range established, for example, from a sample having a low positive for autoantibodies . In certain embodiments, the cutoff value or reference range is expressed as a percentage threshold of NAb that the sample must have to be considered positive for NAb. In this embodiment, the sample is positive for NAb if the percentage of NAb measured in step (d) is greater than or equal to the cutoff value established in a healthy control. In another embodiment, the sample is negative with respect to NAb if the percentage of NAb measured in step (d) is less than or equal to the established cutoff value. Non-limiting examples of cutoff values or reference ranges include, for example, at least about 0.10%, 0.20%, 0.30%, 0.40%, 0.50%, 0.60%, 0.70%, 0.80%, 0.90%, 1.00% 1.30%, 1.20%, 1.21%, 1.22%, 1.23%, 1.24%, 1.25%, 1.26%, 1.27%, 1.28%, 1.29%, 1.30%, 1.40%, 1.50%, 2.00%, 2.50% , 2.70%, 2.80%, 2.90%, 3.00%, 3.01%, 3.02%, 3.03%, 3.04%, 3.05%, 3.06%, 3.07%, 3.08%, 3.09%, 3.10%, 3.20%, 3.30% %, 3.50%, 4.00%, 4.50%, 5.00%, 5.50%, 6.00%, 6.50%, 7.00%, 7.50%, 8.00%, 8.50%, 9.00%, 9.50%, 10.00% ≪ / RTI > In some examples, the cutoff value or reference range is between about 1.28% NAb or about 1.25% -1.30% NAb.

In some embodiments, all autoantibodies to the anti-TNF [alpha] drug are neutralizing antibodies, and the sample comprises 100% neutralizing anti-drug antibody (NAb) and / or 0% non-neutralizing anti- drug antibody . In these embodiments, the level of free labeled TNF [alpha] measured in step (c) is generally equal to the level of the free labeled TNF [alpha] in the control sample, and the autoantibody completely blocks or inhibits binding between the anti- TNF [alpha] It is predicted to interfere.

In certain other embodiments, none of the autoantibodies to the anti-TNFa drug are neutralizing antibodies, and the sample is defined as having 100% non-NAb and / or 0% NAb. In these embodiments, the level of free labeled TNF [alpha] measured in step (c) is generally non-existent (e.g., undetectable) compared to the level of free labeled TNF [alpha] in the control sample, It is not expected to completely block or interfere with the binding between the TNF [alpha] drug and TNF [alpha].

In a further embodiment, if both the neutralizing and non-neutralizing forms of the autoantibody are present in the sample, the percentages of each species may be used alone (e.g., 50% NAb or 50% non- NAb and non-NAb) or as a ratio. In a particular example, the ratio is calculated by dividing the percentage of NAb by the percentage of non-NAb or vice versa. In other examples, the ratio is calculated by dividing the level of NAb by the level of non-NAb or vice versa.

In some embodiments, the anti-TNF [alpha] drug is selected from the group consisting of REMICADE ™ (Infliximab), ENBREL ™ (etanercept), HUMIRA ™ (adalimumab), CIMZIA® (sertoli marbella), SIMPONI Muramov; CNTO 148), and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasma sample of a subject receiving a whole blood, serum, or plasma sample, for example, an anti-TNFa drug therapy. In a preferred embodiment, the sample is serum. In certain embodiments, the subject is a mammal such as a human, a human, a human, a human, a human, a human, a human, a human, a human, a human, Mediated diseases or disorders.

In certain embodiments, the sample is suspected to have or have an autoantibody to the anti-TNF [alpha] drug. In other embodiments, anti-TNF alpha drug autoantibodies include but are not limited to human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti-mouse antibodies .

In another particular aspect, the invention provides a method of determining whether an autoantibody of neutralization form to a first anti-TNF alpha drug comprises cross-reactivity with a second (i.e., different) anti-TNF alpha drug, Lt; / RTI >

(a) Detecting or measuring the presence, level or percentage of autoantibody in neutralization form in the sample according to the assays described herein to determine whether the sample is positive or negative for autoantibodies in neutralized form; And

If the sample is positive for neutralizing autoantibodies, then:

(b) A sample is contacted with a labeled second anti-TNF alpha drug to form a labeled second anti-TNF alpha drug and a labeled conjugate of an autoantibody in neutralized form (i.e., Not);

(c) The labeled complex is applied to size exclusion chromatography to separate the labeled complexes (e.g., from a second labeled anti-TNFα drug); And

(d) Labeled conjugate, thereby determining whether the neutralizing form of the autoantibody to the first anti-TNF [alpha] drug is cross-reactive with the second anti-TNF [alpha] drug.

In certain embodiments, the presence of the labeled complex indicates that the neutralizing autoantibody against the first anti-TNF alpha drug is cross-reactive with the second anti-TNF alpha drug, that is, the neutralizing autoantibody is the first Will inhibit the activity of both the anti-TNF [alpha] drug and the second anti-TNF [alpha] drug.

In certain other embodiments, the absence of the labeled complex indicates that the neutralizing autoantibody against the first anti-TNF alpha drug is not cross-reactive with the second anti-TNF alpha drug, i.e., 2 < / RTI > anti-TNFa drug.

In certain embodiments, the first and second anti-TNFa drugs are independently selected from the group consisting of REMICADE ™ (infliximab), ENBREL ™ (etanercept), HUMIRA ™ (adalimumab), CIMZIA® ≪ / RTI > Marbella), SIMPONI® (colloidal mab; CNTO 148), and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasma sample of a subject receiving a whole blood, serum, or plasma sample, for example, an anti-TNFa drug therapy. In a preferred embodiment, the sample is serum. In certain embodiments, the subject is a mammal such as a human, a human, a human, a human, a human, a human, a human, a human, a human, a human, Mediated diseases or disorders.

In certain embodiments, the sample is suspected to have or have an autoantibody to the anti-TNF [alpha] drug. In other embodiments, anti-TNF alpha drug autoantibodies include but are not limited to human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti-mouse antibodies .

In certain aspects, the assay of the present invention further comprises an acid dissociation step comprising contacting the sample with an acid before, during and / or after contacting the labeled anti-TNF [alpha] drug and labeled TNF [alpha] .

Methods for the detection of anti-drug antibodies using acid dissociation are described in PCT Application No. PCT / US2012 / 025437, filed February 16, 2012, which is hereby incorporated by reference in its entirety and for any purpose herein.

In certain other aspects, the assays of the invention include detecting the presence or level of one or more subtypes of autoantibodies in the neutralized and / or non-neutralized form of the anti-TNF [alpha] drug in the sample. As a non-limiting example, the assays of the present invention may be used to determine the presence of different neutralization and / or non-toxicity in samples from ADA-positive patients receiving anti-TNFa drugs such as REMICADE (infliximab) or HUMIRA - Can be used to measure neutralizing ADA subtypes. In certain embodiments, the at least one subtype comprises a plurality of at least 2, 3, 4, 5 or more subtypes. In other embodiments, the at least one subtype is selected from the group consisting of IgA, IgD, IgE, IgG, and IgM subtypes, subgroups thereof, and combinations thereof. In certain embodiments, each autoantibody subtype is analyzed, identified and / or detected by its residence time. In other embodiments, each autoantibody subtype is analyzed, identified and / or detected as a signal by proximate binding of a detector moiety, such as a labeled anti-Ig antibody specific for a different antibody subtype and a labeled anti-TNFa drug do. In a particular example, the signal comprises a fluorescent signal that can be detected by fluorescence resonance energy transfer (FRET).

Methods for detecting anti-drug antibody (ADA) subtypes are further described in PCT Publication No. WO 2012/054532, the disclosure of which is incorporated herein by reference in its entirety for any purpose.

Biologic agents (e. G., Anti-TNFa drugs) or biological binding moieties (e. G., TNFa) may be labeled as any of the various detectable group (s). In a preferred embodiment, the biological agent (e. G., Anti-TNFa drug) and the biological binding moiety (e. G., TNFa) comprise different labels. In certain embodiments, a biological agent (e. G., An anti-TNFa drug) or a biological binding moiety (e. G., TNFa) is labeled with a fluorophore or fluorescent dye. Non-limiting examples of fluorophores or fluorescent dyes, include those listed in the Molecular Probes Catalogue incorporated by reference herein (R. Haugland, The Handbook-A Guide to Fluorescent Probes and Labeling Technologies, 10 ^th Edition, Molecular probes , ≪ / RTI > Inc. (2005)). Fluorescent dyes or fluorescent dyes of the above examples include, but are not limited to, Alexa Fluor® dyes such as Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 514, Alexa Fluor ? 532, Alexa Fluor 占 546, Alexa Fluor 占 555, Alexa Fluor 占 568, Alexa Fluor 占 594, Alexa Fluor 占 610, Alexa Fluor 占 633, Alexa Fluor 占 635, Alexa Fluor 占 647, Alexa Fluor 占 660, Alexa Fluor ? 680, Alexa Fluor® 700, Alexa Fluor® 750, and / or Alexa Fluor® 790, and other fluorescent moieties including, but not limited to:

Dansyl chloride (DNS-Cl), 5- (iodoacetamid) fluorocaine (5-IAF), fluorocaine 5-isothiocyanate (FITC), tetramethylrhodamine 5- Nitrobenzo-2-oxa-1,3, -diazol-4-yl chloride (NBD-Cl), 6-acryloyl-2-dimethylaminonaphthalene (acryloyl) , Ethylidene bromide, Lucifer Yellow, 5-carboxydodamine 6G hydrochloride, Lissamine rhodamine B sulfonyl chloride, Texas Red (TM) sulfonyl chloride, BODIPY (TM), naphthalaminesulfonic acid For example, 1-anilino naphthalene-8-sulfonic acid (ANS), 6- (p-toluidinyl) Phosphatidylcholine, fluorescein-phosphatidylethanolamine, Texas Red-phosphatidylethanolamine, pyrenyl-papyridylcholine, fluorenyl-phosphatidylcholine, Vinylpyridinium betaine (naphthylstyryl), 3,3 ', 3', 4'- dipropyl thiazol not when dicarbonate _{(di-SC 3 - (5)),} 4- (p- dipentyl-amino-styryl) -l- methyl-pyridinium (di -5-ASP), Cy-3-iodo-O theta Mead , Cy-5-N-hydroxysuccinimide, Cy-7-isothiocyanate, Rhodamine 800, IR-125, Thiazole Orange, Azure B, Nile Blue, Al phthalocyanine, (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 complex, IRDye ^® 700DX, IRDye ^® 700, IRDye ^® 800RS, IRDye ^® 800CW, IRDye ^® 800 , Cy5, Cy5.5, Cy7, DY676, DY680, DY682, DY780, and mixtures thereof. Additional suitable fluorescent moieties include enzyme-cofactors; Lanthanide, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, or mutants and derivatives thereof. In one embodiment of the invention, the second member of the specific binding pair has a detectable group attached thereto.

Typically, the fluorophore is a fluorophore selected from the category of dyes including polymethine, phthalocyanine, cyanine, xanthene, fluorene, rhodamine, coumarin, fluororesin and BODIPY (TM).

In one embodiment, the fluorophore is a near-infrared (NIR) fluorophore emitting from about 650 to about 900 nm. The use of near-infrared fluorescence technology has advantages in biological assays because it substantially eliminates or alleviates the background from autobodies in living bodies. Another advantage of near-IR fluorescence techniques is that the scattered light from the excitation source is greatly reduced because the scattering intensity is proportional to the inverse 4 of the scattered light from the excitation source of the wavelength. Low background fluorescence and low scattering provide a high signal-to-noise ratio, essential for high sensitivity detection. Furthermore, visually transparent windows in the near-IR region (650 nm to 900 nm) in biological tissues are useful for applying in vivo imaging and cell culture detection of NIR fluorescence requiring the passage of light through biological components. In the example surface of the embodiment, the fluorescent group is preferably ^{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, is selected from the group consisting of Cy5, Cy5.5, Cy7, DY 676 , DY680, DY682, DY780, and. In certain embodiments, the near infrared ray generators are IRDye ^® 800CW, IRDye ^® 800, IRDye ^® 700DX, IRDye ^® 700, or Dynomic DY676.

Fluorescent labels are achieved using chemically reactive derivatives of the fluorophore. Typical reactors include isothiocyanate derivatives such as FITC and TRITC (derivatives of fluorescein and rhodamine), amine-reactive succinimidyl esters such as NHS-fluorescein, and sulfone-5-maleimide These materials are commercially available, including phyryl-reactive maleimide-activated fluorine. The reaction of a biologic agent (e. G., An anti-TNFa drug) or a biological binding moiety (e. G., TNFa) with any of these reactive dyes can be achieved using a fluorescent moiety and a biologic agent (e. G., An anti- TNFa drug) (E. G., TNFa). ≪ / RTI >

In certain instances, after the fluorescent labeling reaction, it is often desirable to remove any unreacted fluorophore from labeled target molecules. This is often achieved by size exclusion chromatography, which has the advantage of a size difference between the fluorophore and the labeled protein.

Reactive fluorescent pigments are available from many sources. They can be obtained in a number of reactors for attachment to various functional groups within the target molecule. They are also available in a label kit containing all components to perform the label reaction. In one preferred aspect, Alexa Fluor 647 C2 maleimide from Invitrogen is used (Cat. No. A-20347).

(E. G., Anti-TNFa drugs) and / or biological binding moieties (e. G., TNFa) of neutralizing and / or non-neutralizing anti-drug antibodies (e. G., NAb and / or non- Can be detected either directly or indirectly. Direct labeling includes fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, which attach to the antibody. In certain instances, a biological agent (e.g., an anti-TNFa drug) or a biological binding moiety (e.g., TNFa) labeled with a different radionuclide is used to determine the presence or level of NAb and / or non- Can be used. In another example, chemiluminescent assays using chemiluminescent biologics (e. G., Anti-TNFa drugs) and biological binding moieties (e. G., TNFa) can be used to determine the presence or level of NAb and / Sensitive, non-radioactive detection. In certain instances, biologic agents (e. G., Anti-TNFa drugs) and biological binding moieties (e. G., TNFa) labeled with different fluorescent color are suitable for detection of the presence or level of NAb and / or non- Do. Examples of fluorescent dyes include, without limitation, to: Alexa Fluor ^® dye, DAPI, fluorescein, Hoechst 33258, R- Pico when not, B- phycoerythrin, R- phycoerythrin, rhodamine, Texas red, And lysamin. Secondary antibodies linked to fluorescent dyes are commercially available, for example, chlorine F (ab ') ₂ anti-human IgG-FITC is available from Tago Immunologicals (Burlingame, Calif.).

Indirect labeling includes various enzymes well known in the art such as horseradish peroxidase (HRP), alkaline phosphatase (AP), beta -galactosidase, urease, and the like. The horseradish peroxidase detection system can be, for example, a chromogenic substrate tetramethylbenzidine (TMB) which produces a soluble product in the presence of hydrogen peroxide detectable at 450 nm. The alkaline phosphatase detection system can be, for example, a chromogenic substrate p-nitrophenyl phosphate which produces a soluble product readily detectable at 405 nm. Similarly, a chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside (ONPG), which produces a soluble product detectable at 410 nm, can be used for the? -Galactosidase detection system. A urease detection system may be a substrate such as urea-bromocresol purple (Sigma Immunochemicals; St. Louis, Mo.). Useful secondary antibodies linked to enzymes can be obtained from a number of commercial sources, for example, chlorine F (ab ') ₂ anti-human IgG-alkaline phosphatase can be obtained from Jackson ImmunoResearch (West Grove, Pa. .

Signals from direct or indirect markers include, for example, a spectrophotometer that detects color from a chromogenic substrate; A radiation counter for detecting radiation, such as a ¹²⁵ I gamma counter for detection; Or may be analyzed using a fluorometer to detect fluorescence in the presence of light of a particular wavelength. Quantitative analysis of NAb and / or non-NAb levels was performed using a spectrophotometer, such as an EMAX microplate reader (Molecular Devices, Menlo Park, Calif.) According to the manufacturer's instructions for the detection of enzyme-linked antibodies . If desired, the assays of the present invention can be automated or robotic, and signals from multiple samples can be detected simultaneously.

In certain embodiments, size exclusion chromatography is used. The basic principle of SEC is that particles of different sizes can be eluted (filtered) through the stationary phase at different rates. This separates the solution of particles by size. When all the particles are loaded simultaneously or nearly simultaneously, particles of the same size are eluted together. Each size exclusion column has a range of molecular weights that can be separated. The exclusion limit limits the molecular weight at the upper limit of the range which is too great for the molecule to be trapped on the immobilized phase. The permeability limit limits the molecular weight at the lower end of the separation range where molecules of sufficiently small size can be completely permeated into the pores of the fixed bed, and all molecules below this molecular weight are too small to be eluted into a single band.

In certain aspects, the eluent is collected in a certain volume, or fraction. The more similar the particles are, the more likely they are in the same fraction and not individually detected. Preferably, the collected fractions are evaluated by spectroscopic techniques to determine the concentration of eluted particles. Typically, spectroscopic detection techniques useful in the present invention include, but are not limited to, fluorescence analysis, refractive index (RI), and ultraviolet (UV). In certain instances, the elution volume decreases approximately linearly with the logarithm of the hydrodynamic volume of the molecule (i. E., The heavy portion emerges first).

In a further aspect, the present invention provides a method for monitoring and / or optimizing a therapy for a biological agent in a subject undergoing a therapeutic treatment with a biological agent, said method comprising:

(a) detecting or measuring the presence, level or percentage of neutralizing forms of autoantibodies to the biologics according to the assays described herein at multiple time points throughout the course of the therapy;

(b) detecting the change in the presence, level or percentage of autoantibodies in neutralization form over time; And

(c) determining whether a subsequent dose of the therapeutic regimen or a different therapeutic regimen for the subject should be administered to the subject, based on the change in the presence, level or percentage of the neutralizing form of the autoantibody over time.

In certain embodiments, the plurality of time points are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more time points.

In one particular aspect, the invention provides a method of monitoring and / or optimizing a therapy for a biological agent in a subject undergoing a therapeutic treatment with a biological agent, said method comprising:

(a) measuring, at a time point t ₀ , the level or percentage of autoantibody in neutralized form to a biological agent in the first sample from a subject as described herein;

(b) measuring, at time point t ₁ , the level or percentage of autoantibody in neutral form to the biological agent in the second sample from a subject as described herein;

(c) optionally repeating step (b) with n additional samples from the subject at time point t _{n + 1} , where n is from 1 to about 25 (e.g., n is 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, );

(d) detecting a change in the level or percentage of autoantibodies in neutralization form at time points from t ₀ to t ₁ or from time t ₀ to time t _{n + 1} ; And

(e) determining whether a subsequent dose of the therapeutic regimen or a different therapeutic regimen for the subject should be administered to the subject, based on the change in the presence, level or percentage of neutralizing autoantibodies over time.

In another specific embodiment, the level or percentage of autoantibodies (e.g., NAb) in the neutralized form is measured in one or more (e.g., plurality) of the biologic drug therapy regimens in the following week: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 80,

In some embodiments, determining the subsequent dose of the therapeutic regimen for the subject comprises maintaining, increasing or decreasing the subsequent dose of the therapeutic regimen for the subject. In another embodiment, the determination of the different therapeutic regimens for the subject comprises treatment with different biologic drugs. In another embodiment, the determination of the different therapeutic regimens for the subject comprises treatment with a current therapeutic regimen that is in conjunction with another therapeutic agent. In a further embodiment, the determination of the therapeutic regimen of the different processes for the subject comprises altering the current therapeutic regimen (e.g., altering into a different biologic or drug targeting a different mechanism).

In certain embodiments, autoantibodies in the form of a neutralization over time, such as, for example, NAb) levels or percentages indicate that treatment modalities should be recommended for the subject. In another particular embodiment, the autoantibody in neutralization form over time (e.g., Changes in the presence of NAb from its presence indicate that treatment adjustment should be recommended for the subject. In such embodiments, the subject may be treated with a current therapeutic regimen (e. G., Selecting an existing biological agent) with one or more other therapeutic agents. In certain alternative embodiments, the subject can be changed to a different biologic agent. In another specific alternative embodiment, the subject can be changed to a drug (e. G., A biological and / or non-biological agent) targeting a different mechanism.

In a further aspect, the present invention provides a method of optimizing treatment and / or reducing toxicity in a subject undergoing a therapeutic treatment with a first biocide, the method comprising:

(a) detecting or measuring the presence, level or percentage of autoantibody in neutralization form in the sample from a subject according to the assays described herein to detect the presence or absence of a second (ie, different) autoantibody of neutralizing form to the first biological agent, Determine whether it is cross-reactive with the biological agent; And

(b) if the neutralizing form of the autologous antibody is cross-reactive with the second biological agent, determining whether a different therapeutic regimen should be administered to the subject.

In certain embodiments, the determination of whether the different course of treatment regimen should be administered includes altering to a drug (e.g., a biological agent and / or a non-biological agent) targeting a different mechanism.

In some embodiments, if the neutralizing form of the autoantibody can not cross-react with the second biological agent, the method further comprises determining whether the subsequent dose of the current therapeutic regimen should be increased or decreased, And determining if the therapeutic regimen should be administered. In certain instances, the different therapeutic regimens include treatment with a second biological agent. In another specific example, the different therapeutic regimens include treatment with a first or second biological agent together with one or more other therapeutic agents.

In one particular aspect, the invention provides a method of monitoring and / or optimizing a therapeutic regimen for an anti-TNF alpha drug in a subject undergoing a therapeutic regimen with an anti-TNF alpha drug, the method comprising:

(a) detecting or measuring the presence, level or percentage of autoantibodies in the form of neutralization to the anti-TNFa drug according to the assays described herein at multiple time points throughout the course of the treatment;

(b) detecting the change in the presence, level or percentage of autoantibodies in neutralization form over time; And

(c) determining whether a subsequent dose of the therapeutic regimen or a different therapeutic regimen for the subject should be administered to the subject, based on the change in the presence, level or percentage of the neutralizing form of the autoantibody over time.

In certain embodiments, the plurality of time points are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more time points.

In another particular aspect, the invention provides a method of monitoring and / or optimizing a therapeutic regimen for an anti-TNF [alpha] drug in a subject undergoing a therapeutic regimen with an anti-TNF [alpha] drug, the method comprising:

(a) measuring the level or percentage of autoantibody in neutralized form to the anti-TNFα drug in the first sample from a subject as described herein, at time point t ₀ ;

(b) measuring, at time point t ₁ , the level or percentage of autoantibody in neutralized form in the second sample from a subject as described herein;

(c) optionally repeating step (b) with n additional samples from the subject at time point t _{n + 1} , where n is from 1 to about 25 (e.g., n is 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, );

(d) detecting a change in the level or percentage of autoantibodies in neutralization form at time points from t ₀ to t ₁ or from time t ₀ to time t _{n + 1} ; And

(e) determining whether a subsequent dose of the therapeutic regimen or a different therapeutic regimen for the subject should be administered to the subject, based on the change in the presence, level or percentage of neutralizing autoantibodies over time.

In another specific embodiment, the level or percentage of autoantibodies (e.g., NAb) of the neutralized form is measured in one or more (e.g., multiple) of the following courses of anti-TNFa drug therapy: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 80, 90, 100, etc.

In some embodiments, determining the subsequent dose of the therapeutic regimen for the subject comprises maintaining, increasing or decreasing the subsequent dose of the therapeutic regimen for the subject. In another embodiment, the determination of the different therapeutic regimens for the subject comprises treatment with different anti-TNF alpha drugs. In other embodiments, the determination of the different therapeutic regimens for such a subject includes, but is not limited to, anti-TNF therapy, immunosuppressants, corticosteroids, drugs targeting different mechanisms, nutritional therapies and other combination therapies As well as treatment with current therapies. In a further embodiment, the determination of the different therapeutic regimens for the subject can be accomplished by altering the current therapeutic regimen (e. G., By administering a different anti-TNFa drug or a different mechanism targeting drug such as IL-6 receptor- (E. G., A particular carbohydrate diet), a monoclonal antibody, an anti-integrin molecule (e. G. Tysabri, Vedaluzamab), a JAK-2 inhibitor and a tyrosine kinase inhibitor, ). ≪ / RTI >

In certain embodiments, autoantibodies in the form of neutralization over time (e. G., ≪ RTI ID = 0.0 & NAb) levels or percentages indicate that treatment modalities should be recommended for the subject. In another particular embodiment, the autoantibody in neutralization form over time (e.g., Changes in the presence of NAb from its presence indicate that treatment adjustment should be recommended for the subject. In such embodiments, the subject may be treated with a current therapeutic regimen (e.g., selecting an existing anti-TNFa drug) with one or more immunosuppressants such as methotrexate (MTX) or azathioprine (AZA) ). ≪ / RTI > In certain alternative embodiments, the subject can be changed to a different anti-TNF [alpha] drug. In another specific alternative embodiment, the subject can be changed to a drug (e.g., a non-anti-TNF alpha drug) that targets a different mechanism.

In another particular aspect, the invention provides a method of reducing the optimization and / or toxicity of a therapeutic regimen in a subject undergoing a therapeutic regimen with a first anti-TNF alpha drug, the method comprising:

(a) detecting or measuring the presence, level or percentage of an autoantibody in neutralized form in a sample from a subject according to the assays described herein to detect or measure the neutralization form of the autoantibody to the first anti-TNFa drug, Determining whether the compound is cross-reactive with an anti-TNF [alpha] drug; And

(b) if the neutralizing form of the autoantibody is cross-reactive with the second anti-TNFα drug, a different therapeutic regimen is to be administered to the subject.

In certain embodiments, the determination of whether the different therapeutic regimens should be administered involves altering to a drug targeting a different mechanism (e. G., A non-anti-TNF alpha drug). Non-limiting examples of such drugs include, but are not limited to, IL-6 receptor-inhibiting monoclonal antibodies, anti-integrin molecules (e.g., Tysabri, Vedaluzamab), JAK-2 inhibitors, tyrosine kinase Inhibitors, nutritional therapies (e. G., Specific carbohydrate diets), and mixtures thereof.

In some embodiments, if the neutralizing form of autoantibodies can not cross-react with a second anti-TNF alpha drug, the method further includes determining whether the subsequent dose of the current therapeutic regimen should be increased or decreased, Lt; RTI ID = 0.0 > a < / RTI > different therapy regimen. In certain instances, the therapeutic regimens of the different processes include treatment with a second anti-TNF alpha drug. In another specific example, the different therapeutic regimens include treatment with a first or second anti-TNF alpha drug in combination with one or more immunosuppressive agents such as MTX or AZA.

Methods for the detection of anti-TNFa drugs and anti-drug antibodies are further described in PCT Publication No. WO 2011/056590, the disclosure of which is incorporated herein by reference in its entirety for any purpose.

In certain instances, the invention further provides a method of treating a disorder or disorder (e. G., A non-hematopoietic agent) that targets a different mechanism useful for treating one or more symptoms associated with a TNFa mediated disease or disorder (e. G., IBD such as CD or UC) -TNF [alpha] drug) or an anti-TNF [alpha] drug, to a subject. For therapeutic applications, the therapeutic regimens may be administered alone or co-administered in combination with one or more additional agents as described herein. As described above, the present invention advantageously provides clinicians with information on the selection of therapeutic regimens and optimization of anti-TNF alpha drugs, as well as guidance of treatment decisions, in order to ensure that the correct drug is provided to the right patient at the right time. Personalized medicine ". < / RTI >

IV. San Harry

In a particular aspect, the assay method of the invention further comprises the step of administering to the subject a therapeutically effective amount of a neutralizing autoantibody (NAb), a non-neutralizing autoantibody (non-NAb) and a neutralizing autoantibody / RTI > and / or < / RTI > an immune complex for measuring the presence or level of its subtype. As a result, the presence or level of NAb and / or non-NAb relative to a biological agent (e. G., An anti-TNFa drug) administered to a subject in need thereof can be determined from the presence Can be measured without significant intervention. In particular, a sample of a subject is a sufficient amount to provide for the presence or level of NAb and / or non-NAb in the presence of a biological agent (e.g., anti-TNFa drug) without significant intervention from a high biological agent drug level Can be incubated with a positive acid.

In some embodiments, step (a) of the assay method of the invention may comprise:

contacting the sample with an acid to dissociate the preformed complex of (a ') autoantibodies (including, for example, neutralization and / or its non-neutralized form) and biologics (e. g., anti-TNFa drug);

(b ') after dissociation of the preformed complex, contacting the sample with a labeled biologic agent (eg, an anti-TNFα drug) and a labeled biologic agent binding moiety (eg, TNFα); And

(c ') neutralizing the acid in the sample to form:

(i) a first labeled conjugate of a labeled biologic agent (e. g., an anti-TNFa drug) and an autoantibody; And / or

(ii) a second labeled conjugate of a labeled biologic agent (eg, an anti-TNFα drug), a labeled biologic agent binding moiety (eg, TNFα), and an autoantibody.

In some alternative embodiments, steps (a ') and (b') are performed concurrently, for example when the sample is contacted with an acid, a labeled biologic agent (eg , an anti- TNFα drug) (E. G., TNFa). ≪ / RTI > In another alternative embodiment, step (b ') is performed prior to step (a'), for example the sample is first incubated with a labeled biological agent (eg, an anti- TNFα drug) (E. G., TNFa) and then contacted with the acid. In a further embodiment, steps (b ') and (c') are performed concurrently, for example the sample is labeled with a labeled biologic (e. G., An anti- TNFa drug) and a labeled biologic agent binding moiety (E. G., Contacting the sample with one or more neutralizing agents). ≪ / RTI >

In certain embodiments, the labeled biologic agent binding moiety (e. G., TNFa), labeled biologic agent (e. G., Anti-TNFa drug), unlabeled biologic agent (e. G., Anti- TNFa drug) (E. G., An anti-TNFa drug) such that the autoantibodies against the biological agent (e. G., Anti-TNFa drug) are in equilibrium and form a complex between them. Contacting the preformed composite with an amount of acid sufficient to dissociate. In certain embodiments, the sample can be contacted with a sufficient amount of acid to enable detection and / or measurement of the autoantibody in the presence of a high level of biological agent (e. G., Anti-TNFa drug).

In some embodiments, the term "high level biologic agent ", such as a high level of anti-TNFa drug, comprises about 10 to about 100 ug / mL, about 20 to about 80 ug / mL, about 30 to about 70 ug / Or from about 40 to about 80 < RTI ID = 0.0 > pg / mL. ≪ / RTI > In other embodiments, the term "high level of biological agent ", such as a high level of anti-TNFa drug, comprises a drug level of greater than about 10,20,30,40,50,60,70,80,90 or 100 占 퐂 / .

In some embodiments, the acid comprises an organic acid. In another embodiment, the acid comprises an inorganic acid. In a further embodiment, the acid comprises a mixture of an organic acid and an inorganic acid. Non-limiting examples of the organic acid include citric acid, isocitric acid, glutamic acid, acetic acid, lactic acid, formic acid, oxalic acid, uric acid, trifluoroacetic acid, benzenesulfonic acid, aminomethanesulfonic acid, camphor- Propionic 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, phosphoric acid, sulfuric acid, boric acid, hydrofluoric acid, hydrobromic acid, and combinations thereof.

In certain embodiments, the amount of acid is from about 0.01M to about 10M, from about 0.1M to about 5M, from about 0.1M to about 2M, from about 0.2M to about 1M, or from about 0.25M to about 0.75M, M < / RTI > In other embodiments, the amount of acid is 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 or more. The pH of the acid may 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 the acid for a time sufficient to dissociate the preformed complex of the autoantibody and the biological agent (e. G., Anti-TNFa drug). In certain instances, the sample is heated to a temperature ranging from about 0.1 hours to about 24 hours, from about 0.2 hours to about 16 hours, from about 0.5 hours to about 10 hours, from about 0.5 hours to about 5 hours, or from about 0.5 hours to about 2 hours (E. G., Incubated) for a period of time. In another example, the sample may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2.5, 3.5, 4.5, (E. G., Incubated) for a period of time of 9 or 10 hours or more. The sample can be contacted with acid at 4 占 폚, room temperature (RT) or 37 占 폚.

In certain embodiments, the neutralization step of the acid comprises increasing the pH of the sample to enable formation of the first and / or second labeled complexes described herein. In some embodiments, the acid is neutralized by the addition of one or more neutralizing agents such as strong bases, weak bases, buffer solutions and combinations thereof. It will be appreciated by those skilled in the art that the neutralization reaction does not necessarily mean that the pH obtained is 7. In some instances, acid neutralization results in a sample that is basic. In another example, acid neutralization results in a sample that is acidic (but higher than the pH of the sample prior to addition of the neutralizer). In certain embodiments, the neutralizing agent comprises a buffer at a pH of about 7.3, such as phosphate buffered saline (e.g., 10x PBS).

In some embodiments, step (b ') further comprises contacting the internal control with a labeled biologic agent (e.g., an anti-TNFa drug) and a labeled biologic agent binding moiety (e. G., TNFa) Forming complex, prior to, during, or after dissociation of the complex. In certain examples, the internal control comprises a labeled internal control, e. G., Biocytin-Alexa 488. In another specific example, the amount of the labeled internal control is from about 1 ng to about 25 ng, from about 5 ng to about 25 ng, from about 5 ng to about 20 ng, from about 1 ng to about 20 ng per 100 μL of the analyzed sample , From about 1 ng to about 10 ng, or from about 1 ng to about 5 ng. In a further example, the amount of the labeled internal control is about 1 ng, 5 ng, 10 ng, 15 ng, 20 ng or 25 ng or more per 100 μL of the analyzed sample.

As a non-limiting example of the method of the invention, a sample, such as a serum sample (e.g., serum from a subject receiving treatment with an anti-TNF.alpha. Drug such as Remicade (IFX) And incubated at room temperature for 1 hour. After dissociation of the preformed complex between the (unlabeled) anti-TNFα drug and the anti-TNFα drug, an autoantibody (eg, anti-drug antibody such as anti-IFX antibody (ATI) (E.g., IFX-Alexa 488), a labeled TNF alpha (e.g., TNF alpha Alexa 532) and optionally an internal control are added and the reaction mixture (e. G., Immediate) pH < / RTI > 7.3. After neutralization, the reaction mixture is incubated for another hour at room temperature (e.g., on a plate shaker) to equilibrate and the labeled TNFa, labeled anti-TNFa drug, unlabeled anti-TNFa drug, And / or to re-establish the immune complexes between autoantibodies to the anti-TNF [alpha] drug. The sample can then be filtered and analyzed by SEC-HPLC as described herein.

In certain embodiments, the methods of the invention (e.g., Including dissociation kinetic kinetics after acid dissociation, significantly increase IFX drug resistance, so that NAb and / or non -NAb ATI can be measured in the presence of IFX at about 60 / / mL or less. In other words, the method of the present invention can be used for the treatment or prevention of an anti-TNFa drug such as ATI, in the presence or absence of NAb and / or non-NAb as well as a high level of anti-TNFa drug (e.g., IFX) The presence or level of an autoantibody to another anti-TNF [alpha] drug can be detected without significant intervention of the anti-TNF [alpha] drug.

Methods for the detection of anti-drug antibodies using acid dissociation are further described in PCT Application No. PCT / US2012 / 025437 filed February 16, 2012, the disclosure of which is incorporated herein by reference for any purpose.

V. BIOLOGICAL TREATMENT THERAPY

The assays of the present invention may be used to determine the presence or absence (e.g., positive or negative) of neutralizing and / or non-neutralizing autoantibodies to any biological agent in a sample from an object (e.g., a subject undergoing biotherapeutic treatment) ), Levels or percentages. Non-limiting examples of biological agents include antibodies, antibody fragments, proteins, polypeptides, peptides, fusion proteins (e.g., Ig fusion proteins or Fc fusion proteins), polyvalent binding proteins (e.g. DVD Ig) Conjugates, vaccines, nucleic acids, sugars, recombinant forms thereof, processed forms thereof, and combinations thereof.

Examples of antibody-based biologics include, but are not limited to, therapeutic monoclonal antibodies and antigen-binding fragments thereof. In certain embodiments, the antibody is anti--TNFα drugs such as REMICADE ™ (an in-flight rigs during MAB), HUMIRA ™ (Ah otherwise free MAB (adalimumab)), CIMZIA ^® (Sergio toll riju MAB pegol (certolizumab pegol)), SIMPONI and; (CNTO 148 goaltender free MAB (golimumab)) or including a combination thereof ^®. Additional examples of antibody-based biological agents include antibody-drug conjugates such as Adcetris (TM) (bortuximab vedotin). Table 1 provides a representative list of therapeutic monoclonal antibodies that have been approved or are currently under development. An extensive list of monoclonal antibody therapeutics in clinical development and in approved products is provided in the 2006 PhRMA report, " 418 Biotechnology Medicines in Testing Promise to Bolster the Arsenal Against Disease ", the disclosure of which is incorporated herein by reference Quoted.

Non-limiting examples of protein-based or polypeptide-based biological agents include, but are not limited to, cytokines (such as interleukins), chemokines, growth factors, blood-forming stimulating proteins (e.g., erythropoietin), hormones (Eg, Elonva®, growth hormone), enzymes (eg, Pulmozyme® (Dornaya Alpha)), clotting factors, insulin, albumin, fragments thereof, conservative modifications thereof, analogues thereof, and combinations thereof .

Examples of cytokines include, but are not limited to, TNFa, TNF-related weak inducers of TNF-related weak inducers (TWEAK), osteoprotegerin (OPG), IFN- ?, IFN- ?, IFN-? Interleukins IL-1 receptor antagonist (IL-lra), IL-2, IL-4, IL-5, IL-6, soluble IL-6 receptor (sIL- IL-17, IL-23, and IL-27), adipocytokines (e.g., leptin, adiponectin, lecithin , Active or total plasminogen activator inhibitor-1 (PAI-1), non-spontaneous and retinol binding protein 4 (RBP4), and combinations thereof. In certain embodiments, the interleukin comprises IL-2, such as Proleukin® (Aldethsu leukin; recombinant IL-2).

Examples of chemokines include, but are not limited to, CXCL1 / GRO1 / GROa, CXCL2 / GRO2, CXCL3 / GRO3, CXCL4 / PF4, CXCL5 / ENA-78, CXCL6 / GCP-2, CXCL7 / NAP- CXCL10 / IP-10, CXCL11 / I-TAC, CXCL12 / SDF-1, CXCL13 / BCA-1, CXCL14 / BRAK, CXCL15, CXCL16, CXCL17 / DMC, CCL1, CCL2 / MCP- CCL4 / MCP-4, CCL4 / MCP-2, CCL9 / MIP-1 ?, CCL5 / RANTES, CCL6 / C10, CCL7 / MCP-3, CCL8 / MCP-2, CCL9 / CCL10, CCL11 / 1, CCL15 / MIP-5, CCL16 / LEC, CCL17 / TARC, CCL18 / MIP-4, CCL19 / MIP-3 ?, CCL20 / MIP-3 ?, CCL21 / SLC, CCL22 / MDC, CCL23 / MPIF1, CCL24 / Thaksin -2,2 include CCL25 / TECK, CCL26 / eotaxin -3, CCL27 / CTACK, CCL28 / MEC, CL1, CL2, CX ₃ CL1, and combinations thereof.

Non-limiting examples of growth factors include epithelial growth factor (EGF), heparin-binding epithelial growth factor (HB-EGF), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF; also known as SERPINF1) (AGF), regulatory (AREG), also known as schwannoma-derived growth factor (SDGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), transforming growth factor- (ETF-1), keratinocyte growth factor (KGF; also known as FGF7), osteogenic proteins (e.g., TGF-beta1, TGF- (Eg, BMP1-BMP15), platelet-derived growth factor (PDGF), nerve growth factor (NGF), β-nerve growth factor BDNF), neurotrophin 3 (NT3), neurotrophin 4 (NT4), etc.), growth differentiation factor-9 (GDF-9), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor ( GM-CSF), myostatin (GDF-8), erythropoietin (EPO), thrombopoietin (TPO), and combinations thereof.

Examples of receptor construct-based or fusion protein-based biological agents include, but are not limited to, naturally occurring receptors linked to immunoglobulin frames such as Orencia® (abatacept; immunoglobulin CTLA-4 fusion protein), Amevive® (Alefacept; an IgG1 fusion protein), ENBREL ^™ (etanercept; a recombinant human TNF-receptor fusion protein), an engineered protein combining two different polypeptide classes (for example, Ontak® Denileukin diftitox; an engineered protein comprising an interleukin-2 and a diphtheria toxin), and combinations thereof.

Therefore, the present invention relates to a method for neutralizing and / or treating a biological agent, such as an anti-TNFa drug treatment, in a sample from a subject receiving a biotherapeutic treatment for one or more of the diseases or disorders listed herein and in Table 1, Can be used in methods of detecting and measuring the presence or level of non-neutralizing autoantibodies:

Inflammatory diseases such as inflammatory bowel disease (IBD) (e.g., Crohn's disease (CD) and ulcerative colitis (UC)), uveitis, sarcoidosis, Wegener's granulomatosis and other diseases with inflammation as a central feature;

The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for the treatment of autoimmune diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), ankylosing spondylitis (Bechterew's disease), lupus, Erythema;

Cancer, such as gastrointestinal cancer and gastrointestinal cancer (e.g., rectal cancer, small intestine cancer, gastrointestinal stromal tumor, gastrointestinal carcinoma, colon cancer, rectal cancer, gastric (stomach) cancer, Etc); Gall bladder cancer; Liver cancer; Pancreatic cancer; Breast cancer; Lung cancer (e.g., non-small cell lung cancer); Prostate cancer; Ovarian cancer; Kidney cancer (e.g., renal cell carcinoma); Central nervous system cancer; cutaneous cancer; Choriocarcinoma; Head and neck cancer; Hematological malignancies (e.g., leukemia, lymphoma such as B-cell non-Hodgkin's lymphoma); Osteosarcoma (e. G., Ewing sarcoma); Soft tissue sarcoma (e.g., dermal fibrosarcoma (DFSP), rhabdomyosarcoma); Other soft tissue malignancies and papillary thyroid cancer;

Infectious diseases, such as C. difficile silane (C. difficile) disorders, respiratory syncytial virus (RSV), HIV, anthrax, candida, Staphylococcus infection and hepatitis C;

Blood disorders such as sepsis, septic shock, seizure haemagglutination and hemolytic uremic syndrome;

Cardiovascular diseases such as atherosclerosis, acute myocardial infarction, cardiopulmonary bypass and angina pectoris;

Metabolic disorders such as diabetes, for example, type I diabetes;

Genetic disorders such as seizure night hemochioroencephalopathy (PNH);

Neurological disorders such as osteoarthritis pain and Alzheimer's disease;

Respiratory disorders such as asthma, chronic obstructive pulmonary disease (COPD), nasal polyposis and pediatric asthma;

Psoriasis including psoriasis, such as chronic moderate to severe psoriasis;

Transplant rejection, such as acute renal transplant rejection, reversal of heart and liver transplant rejection, prevention of renal transplant rejection, prevention of acute renal transplant rejection, and kidney transplant rejection; And / or

Other disorders such as kidney inflammation, postmenopausal osteoporosis (bone disorder), eosinophilic syndrome, eosinophilic esophagitis and peanut allergy.

In certain embodiments, the subject is treated with a TNFa-mediated disease or disorder, e. G., An autoimmune disorder (e. G., Rheumatoid arthritis) or an inflammatory disorder (e. G., Inflammatory bowel disease Ulcerative colitis (UC)).

VI. Example

The invention will now be described in more detail by means of specific examples. The following examples are provided for illustrative purposes and are not intended to limit the invention in any way. Those skilled in the art will readily recognize a variety of non-critical parameters that may be varied or modified to provide essentially the same result.

PCT Application No. PCT / US2012 / 025437, filed February 16, 2012, is incorporated herein by reference in its entirety for all purposes.

Example 1. Development of a novel assay for monitoring neutralizing anti-drug antibody formation in IBD patients

This example illustrates the use of neutralizing and / or non-neutralizing agents in patient samples (e.g., sera) using size exclusion chromatography in the presence of labeled (e.g., fluorescently labeled) anti- TNF [alpha] Describes a novel homogeneity assay for detecting or measuring the presence or level of neutralizing anti-drug autoantibodies (ADA). In certain embodiments, the assay is advantageous in that a separate label (e. G., Fluorophore), which eliminates the step of rinsing to remove the low affinity ADA and allows detection in the visible and / or IR spectrum To increase the ability to detect neutralized and / or non-neutralized ADA in patients with low reverse owing to the high sensitivity of fluorescent label detection, and to occur as a liquid phase reaction, thereby resulting in an ELISA Because it reduces any chance of change in the epitope by attachment to a solid surface such as a plate.

Infliximab (IFX) and adalimumab (ADL) are anti-TNF monoclonal antibodies that are prescribed for the treatment of inflammatory bowel disease (IBD). Anti-drug antibodies (ADA) are often developed during the course of therapy. Portions of these ADA are neutralizing antibodies (NAb). While ADA negatively affects drug pharmacokinetics, the presence of NAb will additionally cause a loss of drug efficacy through the occlusion of the binding site of the drug. This example describes an assay that monitors the development of NAb in patients with IBD who are receiving IFX therapy based on a homogenous mobility shift assay (HMSA) platform, and tests for antibody-to-infliximab ATI) maturation and NAb formation.

Methods: Serum concentrations of IFX and ATI are measured, for example, in PCT Application No. PCT / US2012 / 025437 and PCT Publication No. WO 2011/056590, filed Feb. 16, 2012 (the entire disclosures of which are incorporated herein by reference in their entirety for all purposes) ≪ / RTI > as determined by HMSA. For NAb assays, patient serum containing ATI was first acid-dissociated and then neutralized after addition of two labeled proteins (e.g., IFX-Alexa488 and TNFalpha-Alexa532). The solution was diluted with 2% serum, injected by HPLC on a size exclusion column, and the complexes were monitored by fluorescence. The area under the curve (AUC) (e.g., plot or chromatogram) of the free TNF-Alexa532 peak in each spectrum was calculated for the control and patient samples, and the NAb percentage was calculated. ATI completely blocking antigen binding is defined as 100% NAb, 50% means that the same proportion of ATI is non-NAb in the sample, and 0% means that not all ATIs are non-NAb. Serum from 75 healthy volunteers was used to establish baseline ranges. ATI positive serum samples (> 3.13 U / mL) from 132 remaining IBD patient sera screened for IFX and ATI levels were analyzed for NAb. A positive control group was prepared using pooled ATI positive patient serum.

For data analysis, a peak detection algorithm is used to find all the peaks and troughs in each spectrum per experiment. A cubic smoothing spline is fitted to each spectrum and the peaks and troughs are defined as changes in the first derivative of the signal. The peak is a change in the sign of the slope of the spectrum from positive to negative. Conversely, a trough is defined as a sine change from negative to positive. The peak in the window at the predicted site of the free TNF-Alexa532 peak (for example, 11.5 to 13 minutes) is chosen to be the free peak itself. The troughs just above and below the detected free peak define the upper and lower limits of the peak itself. The bound, free (TNF and IFX) and negative control peak areas are found by integrating the peak regions within the limits described above using trapezoidal rules. The% of the TNF-Alexa532 peak region is then calculated for each sample using the following equation:

% = [(a-b) / c] * 100

(In IFN-Alexa488 + TNF-Alexa532 in normal human serum), where a = the AUC of the TNF-Alexa532 peak in the untreated sample and b = the AUC of the TNF- Alexa532 peak from the negative NAb negative control , c = the AUC of free TNF-Alexa532 in normal human serum. For calculation, "c" is set to 100% and "b" is as close as possible to 0%, although it can be variable based on reaction conditions. The range between "b" and "c" defines the maximum window of sensitivity.

Results: The NAb assay of the present invention demonstrates high reproducibility, accuracy and precision. The inter-and inter-interlaced precision is less than 20% of the CV and the accuracy of the test is less than 25%. The precision and accuracy obtained with the NAb assay of the present invention is substantially better than cell-based assays or ELISAs. IFX drug resistance is ~ 6 μg / mL while TNFα interferes above 1.0 ng / mL. Positive controls from pooled ATI positive patient sera are linearly diluted from 40-5% NAb. Analysis of a healthy control indicates that the sample returning to a value of ≥3% (eg, 3.06%) is considered NAb positive. Twenty-six ATI positive patient serum samples (3.12-199.43 U / mL) were screened against NAb and 26 (19.7%) of the 132 ATI positive patient sera samples were NAb positive (mean 22.47%, range 3.29- 51.63%). ATI levels above 60 U / mL correspond to highly neutralized Ab. Further analysis of NAb-positive samples reveals a linear correlation between ATI titers and NAb positivity. In particular, Figure 1 illustrates that there was a clear relationship between NAb% (y-axis) and ATI levels (Spearman Rank Correlation, rho = 0.564, p << 0.0001). FIG. 2 illustrates that the NAb positive rate (NAb +) is predicted with an ATI concentration of 60 U / ml or more. Sensitivity = 77.8%; Specificity = 98.1%; Oz ratio = 63.6, p << 0.0001, Fisher's Exact Test. Figure 3 illustrates ATI cutoff analysis and demonstrates that ATI predicts NAb with ROC AUC of 0.931. True positive rate (TPR) = sensitivity; False positive rate (FPR) = 1 - specificity.

Conclusion: In addition to drug and ADA levels, monitoring of NAb provides essential information about ADA responsiveness and helps guide early intervention interventions. This method can be applied to the analysis of ADA for any biological therapy.

Example 2. A patient case study to monitor the formation of neutralizing anti-drug antibodies over time

This example illustrates the use of size exclusion chromatography in the presence of labeled (e. G., Fluorescently labeled) anti-TNFa drug and labeled TNFa to neutralize and / or non-neutralize (e.g., Lt; RTI ID = 0.0 &gt; (ADA) &lt; / RTI &gt; This example also illustrates the use of anti-TNF alpha drugs to monitor changes from neutralizing and / or non-neutralizing anti-drug antibodies formation and / or non-neutralization to neutralizing anti-drug antibodies when the patient is ongoing therapy therapies Demonstrate time-lapse case studies of IBD patients for therapeutic therapy.

1. Drug and anti-drug antibody assay

Figure 4 shows the detection of ATI (i.e., antibody to IFX; "HACA") by the fluid phase mobility assay described herein. For example, 444 ng of Alexa488-labeled IFX (18.8 μg / ml in 100% serum) is spiked into the sample to outcompete free IFX. In certain embodiments, patient serum samples containing complexes of IFX and ATI may be applied to acid dissociation, followed by equilibration by acid dissociation and neutralization after addition of the label.

Figure 5 illustrates an exemplary ATI / IFX fluid phase mobility variability assay of the present invention. For example, samples containing various concentrations of ATI (standard or unknown) equilibrated with fluorescently labeled infliximab (IFX-488) were injected onto size exclusion columns in 2% serum. Figure 5 shows that the larger IFX-488 / ATI complex first eluted and then the smaller complex and then the unbound IFX-488 and Alexa-488 loading controls eluted. The unknown concentration was determined by interpolation from a standard curve. Detection of IFX followed a similar methodology.

2. Neutralizing and non-neutralizing anti-drug antibody test

Figures 6 and 7 illustrate the use of size exclusion chromatography to detect binding of these autoantibodies to fluorescently labeled anti-TNFa drugs in the presence of fluorescently labeled &lt; RTI ID = 0.0 &gt; TNFa &lt; / RTI &gt; &Lt; / RTI &gt; is an autoantibody. In one exemplary embodiment, the anti-TNF [alpha] drug, e.g., IFX, is labeled with fluorophore "F1 ", wherein the fluorophore may be detected in either or both of the visible and IR spectra. Similarly, TNFa is labeled with a fluorophore "F2 &quot;, wherein the fluorophores can be detected in either or both of the visible and IR spectra, where" F1 &quot; and "F2" The labeled anti-TNF [alpha] drug and the labeled TNF [alpha] are incubated with human serum in a liquid phase reaction to detect the presence of labeled anti-TNF [alpha] drugs (e.g., IFX), labeled TNFa, and / (E. G., ATI) to form a complex (e. G., An immune complex).

After incubation, the sample is loaded directly onto the size exclusion column and applied to the HPLC mobility assay. Figure 6 is a graphical representation of an anti-drug antibody (e. G., ATI) and labeled TNFa (e. G., Alexa532 labeled TNFa ; "TNF-532") results in a decrease in free TNF-532 levels. Figure 7 shows the binding of an anti-drug antibody (e. G., ATI) to a labeled anti-TNFa drug (e. G., IFX-488) without binding of the labeled TNF? 532 &lt; / RTI &gt; control and a substantially equivalent amount of free TNF-532 level is obtained.

3. Time-course study for monitoring neutralizing and non-neutralizing anti-drug antibodies

Figures 8-11 show data from a UC patient case study to determine whether an anti-drug antibody, e. G., ATI, is neutralizing or non-neutralizing autoantibodies using the mobility shift assay of the present invention. For example, Figure 8 shows the levels of IFX and ATI over time for five samples taken at 1, 2 or 3 months. Figure 9 shows a peak analysis for determining the percentage of free TNF [alpha] over time. Specifically, the peak area of the TNF-532 / IFX-488 complex was subtracted from the free labeled TNF alpha area of all samples, and then the% of free TNFα was calculated. In particular, Figure 9 shows an increase in the level of free TNF [alpha] over time for the five samples taken at 1, 2 or 3 months, suggesting an increase in neutralizing antibody levels. Figure 10 shows the variation from the presence of non-neutralizing autoantibodies to neutralizing autoantigens over time as illustrated in the three samples spiked with IFX taken at 2 or 3 months. In the "First Year of November" sample, the non-neutralizing antibody binds to spiked IFX and shows a decrease in TNF-532 peak. For the "second year of January" sample, the mixture of neutralizing antibody (NAb) / non-neutralizing antibody (Ab) shows a slight decrease in the TNF-532 peak relative to the level of the initial complex. As ATI is almost completely neutralized (the "April 2" sample), high IFX levels can not overcome the ATI binding to IFX and block all TNFα binding. Thus, Figure 10 shows that the ATI profile during the course of the IFX therapy regimen is a profile containing a mixture of neutralized ATI and non-neutralized ATI from a non-neutralized ATI profile, followed by a neutralized ATI profile, . Figure 11 shows a peak analysis for determining the percentage of free TNF [alpha] over time in IFX-spiked samples. Specifically, the percentage of free TNFα was calculated after subtracting the peak area of the TNF-532 / IFX-488 complex from the free TNFα area of all samples. In particular, Figure 11 shows an increase in the level of free TNF [alpha] over time for samples taken from patients with UC, indicating that the neutralizer increases antibody levels from non-neutralized ATI to neutralized ATI when the patient is undergoing IFX therapy Suggests a change.

Figures 12-14 show various control groups performed using the mobility variability assay of the present invention. In particular, Figure 12 shows the use of rabbit anti-human IgGl Fc as a non-neutralizing antibody (Ab) control. Figure 13 shows the use of ATI positive sera as a mixed neutralizing antibody (NAb) / non-neutralizing antibody (Ab) control. Figure 14 shows the loss of weak affinity NAb as a result of ATI purification from ATI positive sera. Figure 15 shows a peak analysis from a UC patient case study to determine the percentage of free TNFa in these various control groups. Specifically, the peak area of the TNF-532 / IFX-488 complex was subtracted from the free TNF alpha area of all samples and then the percentage of free TNF alpha was calculated.

Figures 16-18 show data from a CD patient case study to determine whether an anti-drug antibody, e. G. ATI, is neutralizing or non-neutralizing autoantibodies using the mobility shift assay of the present invention. For example, Figure 16 shows a peak analysis from a CD patient case study to determine the percentage of free TNF [alpha] over time for four samples taken at 7 or 8 weeks for a 30 week period. Figure 17 also shows a peak analysis from another CD patient case study to determine the percentage of free TNFa over time for the three samples taken over a 50 week period. Figure 18 also shows peak analysis from four additional CD patient case studies to determine the percentage of free TNF [alpha] in a sample during induction or maintenance of therapeutic regimens, or after certain weeks.

Example 3: HPLC mobility variation Detection of neutralizing antibody (NAb) activity through competitive ligand-binding assays

This example describes a novel homogeneous assay for detecting or determining the presence or level of neutralizing and / or non-neutralizing anti-drug autoantibodies (ADA) in a patient sample (e.g., serum) using HPLC size exclusion chromatography assays Another additional implementation of the test is shown. This example also illustrates a method for predicting and / or determining the cross-reactivity of NAb with alternative biological drugs such as other anti-TNF drugs.

In some embodiments, a multi-tiered approach to immunogenicity testing involves first screening both drug and anti-drug antibodies by rapid and sensitive screening assays. This approach is recommended by both FDA and EMEA and is a useful management tool for large-scale clinical trials and multiple views per patient. After confirming the presence of ADA, such as ATI, the patient sample is further investigated for the presence of neutralizing antibodies which may have significant negative clinical results. Neutralizing antibodies interfere with biological activity by binding to or near the active site or by inducing structural changes, resulting in loss of efficacy. Samples containing ATI can also be screened for subtype and epitope specificity. Comparing patients' clinical responses to those before and after ADA development can provide information on the interrelationships between ADA development (and antibody characteristics) and clinical response.

NAb assays using HPLC mobility variability assays were developed as disclosed herein. In certain embodiments, a multi-step approach or test comprises or consists of any one, two, or all of the following steps: (1) the sample is incubated with a NAb positive (cut from an assay of normal human serum Screening to qualitatively determine whether the data is yes or no based on a cutpoint; (2) identifying whether the sample is NAb positive using, for example, immune competition and / or immunodeficiency; And / or (3) predicting and / or determining the cross-reactivity of NAb with an alternative biological drug.

I. Screening steps

The patient sample can be screened against NAb after being identified as positive for ADA. In certain embodiments, the subset of ADA is NAb. In certain embodiments, patient serum containing ADA (e.g., an antibody to IFX, also known as "ATI" or "HACA") is first incubated in HPLC water for 1 hour at room temperature (RT) with 0.5 M citric acid Dissociate. Samples were prepared in 96 well plates and incubated in the dark on a plate shaker. Next, two labeled proteins (e. G., Alexa488 (e. G., IFX-Alexa488) and TNFa-Alexa532 in HPLC water containing 0.1% BSA) are added. The sample is neutralized by adding 10X PBS, pH 7.3, and incubating in a dark place on a plate shaker for 1 hour at RT. Samples are diluted with 2% serum with additional 10X buffer and HPLC water. The sample is then injected by HPLC on a size exclusion column. (TNF532), free IFX-Alexa488 (IFX488), TNF532 / IFX488 complex, TNF532 / IFX488 / ATI complex (non-neutralized Ab) , And ATI / IFX488 complex (NAb). Results are compared to negative (see, e.g., Figures 12 and 19) and positive (see, e.g., Figure 13) control groups according to cutoffs established from normal human serum (e.g., a baseline range of 3.06% NAb) , The sample can be designated as positive or negative for NAb, and potency can be determined.

Figure 19 shows the detection of non-neutralizing antibody activity through mobility variation assays. Upon combination of TNF532 and IFX488, there is variation in retention time of approximately 8 minutes, suggesting the formation of high molecular weight complexes. The free IFX-488 peak (approximately 10.5 min) completely disappeared and the free TNF-532 peak (approximately 12 min) also almost completely disappeared (suggesting the formation of the ATI / IFX / TNF ternary complex). Non-neutralizing Ab bound from the active site of IFX follows a similar pattern. Mouse monoclonal antibodies (e.g., approximately 7 minutes) are optionally performed.

Figure 13 shows the detection of neutralizing antibody activity through mobility variation assays. A fully neutralized Ab prevents the binding ability of IFX to TNF (e.g., due to blocking of the active site). This is confirmed by the disappearance of the IFX-488 peak along with the formation of high molecular weight species in the chromatogram. The TNF-532 peak will not change. Indeed, most patients experience a combination of non-neutralizing / neutralizing Ab as shown in the patient serum pool in Figure 13 (Pos. Serum, black solid line). Rabbit polyclonal antibodies against IFN / Humira F (ab ') 2 fragments as an improved NAb positive control are also useful.

Figure 8 shows the development of NAb responsiveness over time in patients during IFX treatment. They are positive for ATI at all times, but not until the second January (light gray arrow, third to 12 minutes above) when the NAb develops. The ATI / IFX-488 complexes vary in slightly different residence times (~ 7.8 minutes), suggesting complexes of different sizes compared to the complexes of TNF532 / IFX488 / ATI (~ 8.2 and 8.8 minutes). Confirmation of neutralizing activity in the presence of additional IFX as opposed to irrelevant protein (immune competition) can also be done. Such a patient may be an ideal candidate for treatment adjustment.

Figure 9 plots the data as a bar graph of the AUC of the percent survival of the free TNF peak, demonstrating clearly that the patient develops NAb over time. Even low-level NAb development observed at an early point can predict disease recurrence; Treatment adjustment is required for patients exhibiting this activity. For example, a patient should be treated with one or more immunosuppressive agents, such as methotrexate (MTX) or azathioprine (AZA), and / or converted to a different anti-TNF drug, while ingesting existing anti-TNF drugs.

II. Check step

In the identification step, the drug (e. G., Anti-TNFa antibody) is spiked to samples of various concentrations (e. G. 1-50 pg / mL) to determine the ability of the sample to neutralize. In parallel, non-specific IgG is spiked to similar levels. The sample spiked with the drug should show the dose response for the drug and the EC50 of NAb can be calculated. Non-specific IgG should have no effect. If necessary, immunodeficiency may also be performed to exclude the effect of the matrix.

Figure 10 shows the variation in the presence of neutralizing autoantibodies from non-neutralizing autoantibodies over time as illustrated in the three samples taken at 2 or 3 months spiked with IFX. The patient serum from each time point responds to spiked IFX, indicating the specificity of the reaction. Over time, the NAb becomes more neutral and can finally neutralize> 20 μg / mL IFX (the second April sample does not deteriorate if the IFX is not spiked). A full titration can be performed to determine the EC50 of NAb at each time point.

III. Cross-reactive step

The cross-reactive step is particularly useful in predicting whether a patient will respond to a drug or therapeutic regimen, such as an anti-TNFa drug or therapeutic regimen.

In some embodiments, the invention provides a method of treating a patient (e.g., Crohn's disease, ulcerative colitis, or a rheumatoid arthritis patient) based on the ability to cross-react with a series of different anti-TNF therapeutic agents of an anti- Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt; As a non-limiting example, one or more of the following drugs may be administered: Remicade (infliximab); Enbrel (etanercept); Humira (Adalmumab); Cimzia (Sartorius marbella); And patients with NAb for Simponi (collimate) (e.g., patients with Crohn's disease, ulcerative colitis, or rheumatoid arthritis). After a positive test for NAb by a particular drug (e. G., IFX), NAb assays may be performed by a series of other drugs (e. G., Fluorescence-labeled drug) using the initial NAb test method described above have.

The predictive test of the present invention is useful in the management of patient care by preventing the use of drugs (e.g., anti-TNF alpha drugs) to be neutralized by the antibody of the patient. Without being bound by any particular theory, the sequence of the binding sites of neutralizing ADA would probably have been developed in a manner similar to TNFa (see FIG. 20). If the NAb neutralizes any other anti-TNF drug, other than the other anti-TNF drug will probably be a poor alternative to the drug already administered since the patient will have immune response. In some embodiments, the cutoff established from normal human serum can be used to determine whether the test sample from the patient is positive or negative. The test can be conducted in a fast and cost-effective manner with laboratory settings.

The following non-limiting case study included patients 1 and 2 who were treated with Remicade (infliximab) but subsequently lost responsiveness to Remicade. Patient 1 was a UC patient and patient 2 was a CD patient. The mobility variability assay described herein clearly demonstrated that patients 1 and 2 lost responsiveness to Remicade as they developed anti-Remicade antibodies (e.g., ATI). Thereafter, these anti-Remicade antibodies were confirmed to be neutralizing antibodies (e.g., NAb).

Figure 21 illustrates that patients 1 and 2 developed a neutralizing antibody (NAb). These NAbs compete with TNF [alpha] for the Remicade binding site. Importantly, these NAbs can cross-react with other anti-TNF therapeutic agents. If NAb cross-reacts with other anti-TNF therapeutic agents, changing to another anti-TNF therapeutic will not be beneficial to these patients. Thus, the predictive assays of the present invention provide an advantage over current methods of managing patients with poor response to Remicade, where positive HACA (detectable antibodies) is being managed by altering to another anti-TNF agent (See, for example, Afif et al., Am. J. Gastroenterol ., 105 (5): 1133-9 (2010)).

In order to determine the cross-reactivity of NAb produced in response to one anti-TNF drug with other anti-TNF drugs, the developed NAb when the patient was being treated with Remicade (IFX) Lt; / RTI &gt; The data presented in Figure 21 clearly demonstrate that the NAb generated against IFX cross-reacts with Humira. Figure 21 shows that when the patient's serum containing NAb was added (~ 12 minutes, Patient Study # 1; ~ 12 minutes, Patient Study # 2; (Lower panel of each patient study). These results suggest that, to some extent, TNFa binds Humira in a way that NAb blocks access to Humira's antigen-binding site. Figure 22 schematically shows this for both NAb and non-NAb measurements.

In certain embodiments, the assays of the invention predict that these patients will not respond to Humira or any other anti-TNF therapeutic. Patients should not be treated with anti-TNF therapy, and should be treated with Actemra, Kineret, Orencia, Rituxan, and / or Arzerra in the case of rheumatoid arthritis (RA) ), Or in the case of Crohn's disease (CD), Tysabri and / or steroids.

Thus, the method of the present invention is particularly useful for predicting whether or not to respond to anti-TNFα therapy by determining the presence and / or concentration level of neutralizing antibodies (NAb) and / or non-NAbs in a sample from a patient It is advantageous. In one embodiment, when the sample contains NAb for one anti-TNF drug, these NAbs will be neutralized by cross-reacting with other anti-TNFa drugs, so that the recommended treatment adjustments for the patient are those that have a different mechanism of action To a drug (e. G., A non-anti-TNF agent). In another embodiment, where the sample contains a non-neutralizing ADA for one anti-TNF [alpha] drug, the recommended treatment adjustment for the patient would be to switch to another anti-TNF [alpha] drug.

Example 4: Assay for the presence and cross-reactivity of neutralizing anti-drug antibody (NAb)

This example demonstrates the ability of the present invention to predict and / or measure the cross-reactivity of an alternative biological drug with screening to determine whether the sample is NAb positive and NAb (see, e.g., Example 3) &Lt; / RTI &gt; In certain embodiments, the assays described herein determine whether a subject receiving the first anti-TNF alpha drug responds to an alternative anti-TNF alpha treatment regimen by determining whether the sample obtained from the subject is positive or negative for NAb . &Lt; / RTI &gt; If the sample is positive for NAb, the method comprises determining whether the NAb cross-reacts with the second anti-TNFa drug, and determining if the NAb cross-reacts with the second TNFa drug, RTI ID = 0.0 &gt; TNFa &lt; / RTI &gt; drug. If the sample is negative for NAb, the method includes recommending that the subject be converted to a second anti-TNF alpha drug.

23 shows the fabrication and use of an exemplary NAb standard curve of the present invention. A sample containing various concentrations of rabbit (Rb) anti-IFX antibody (ATI) serum (i. E., Standard or unknown) equilibrated with fluorescently labeled TNF-532 / IFX-488 was diluted in 2% Lt; / RTI &gt; After the large immune complexes are first eluted, the smaller complexes and subsequently unbound IFX-488 and TNF-532 are eluted. The unknown concentration can be determined from the standard curve by interpolation. A rabbit serum containing different mixtures of NAb and non-NAb can be combined to create a control. The NAb assay described herein has an improved cut-off of 2.72% compared to the past cut-off of 11.63% (N = 50 normal sample). Table 2 provides a summary of NAb clinical studies by patients.

NAb Clinical Summary - By Patient Research 1
n = 154
(290 samples) Research 2
n = 328
(952 samples) Research 3
n = 64
(812 samples) Research 4 ATI +
(all %) 43
(28%) 73
(22%) 58
(91%) 30 NAB +
(ATI + exam%) 12
(28%) 9
(64%) 3 (23 samples)
(60%) 5
(17%) High Nab activity (20 μg / mL)
(all %)
(NAB +%) 4
(2.6%)
(33%) 4
(1.2%)
(44%) 2
(3.1%)
(66%) 4

(80%)

The cross-reactivity assay method of the present invention is particularly useful in predicting whether it would be beneficial to switch to another biological treatment. After finding that the patient is NAb positive for one drug, fluorescence-labeled alternative drugs can be used for the assay. If the patient's serum is still capable of neutralizing, the new drug will not be successful. This method is advantageous because it will be useful for screening the panel of drugs in a cost-effective and timely manner that allows for the presentation or suggestion of the best treatment option.

24 and 25 provide additional case studies for the patient study described in Example 3 and shown in FIG. In particular, patients 3 and 4 who were treated with Remicade (infliximab, IFX) but subsequently lost responsiveness to IFX were determined to have developed NAb was cross-reactive with ADL when the patient was on IFX Was identified as a patient who would not respond to Humira (Adil Mammab, ADL).

Figure 26 shows a non-limiting example of a patient study demonstrating the development of ATI-affinity maturation and cross-reactive ATI.

Example 5: Development of a novel assay for monitoring neutralizing anti-drug antibody formation in IBD patients

Background and Purpose

Anti-TNF monoclonal antibodies such as infliximab (IFX), adalimumab (ADL), and others are prescribed for the treatment of inflammatory bowel disease (IBD). Certain patients will produce anti-drug antibodies (ADA) that can cause loss of drug efficacy and reverse reactions. Neutralized ADA (NAb) binds to the antigen binding site, blocking access to TNF and directly reducing or preventing drug efficacy. Binding ADA interacts with structural components outside the antigen binding site and does not directly affect drug efficacy, although it may alter pharmacokinetics. Applicants have developed a test to monitor the development of NAb in patients with IBD who are receiving IFX or ADL therapy based on the homogeneous mobility shift assay (HMSA) platform, Respectively.

Way

I. Competitive ligand binding to ATI Principle of NAb assay

ㆍ The ATI-positive patient serum is acid-dissociated.

ㆍ IFX-Alexa488 and TNF-Alexa532 are added and the solution is neutralized.

ㆍ The solution is diluted with 2% serum, injected by HPLC on a size exclusion column, and the complexes are monitored by fluorescence.

ㆍ The area under the curve (AUC) of the free TNF-Alexa532 peak is calculated for the control and patient samples.

ㆍ IFX-Alexa488 binds to TNF-Alexa532 and reduces the test signal.

ㆍ NAb neutralizes IFX-Alexa 488 and restores the assay signal.

ㆍ The NAb activity is directly proportional to the measured assay signal.

(TNFα _{AUC -} BKGD) / (TNF _{AUC -} free label - BKGD) * 100, for example , free labeled TNFα _{AUC -} Free labeled TNFα _AUC- BKGD) * 100.

ㆍ Figure 27 provides an illustration of a competitive ligand binding assay of the present invention.

II. Measure drug and ADA levels

Serum concentrations of infliximab, adalimumab and associated ADA were measured by HMSA as previously described (Wang et al. , J. Immunol. Methods , 2012, 382: 177).

III. sample

ㆍ Serum was from IRB-approved studies of residual samples or IBD patients, and healthy controls were obtained from Golden West Biologics, Inc. Purchased from the company.

ㆍ Isolated serum from immunized rats with infliximab or adalimumab was used to generate polyclonal antibodies against infliximab and adalimumab. The immunoglobulin fractions were purified by affinity chromatography and the concentration was determined by UV / VIS spectroscopy. For sensitivity and specificity experiments, purified ATI and ATA were spiked into normal human serum.

result

Figure 28 illustrates the clinical utility of neutralization assays. (Left) A total of 130 ATI positive residual serum samples from patients undergoing routine testing were analyzed for neutralizing capacity (0.0-430 U / mL, mean value = 49.22 U / mL, median = 19.10 U / mL). NAb levels were measured in a subset of 33 patients with low ATI positivity to determine the black cutoff. The cut-off with 99% one-side confidence limit was calculated to be 1.28% by the equation of mean + 2.33 x SD. Of the remaining patients, 37/97 (38.1%) were NAb positive (mean value = 7.43% recovery). (right) Eleven ATA positive serum samples from cross-sectional studies of patients suffering a response loss to Adalimumab therapy were tested for neutralizing capacity (7.93-147.9 U / ml, mean value = 36.97 U / mL, median = 16.39 U / mL). The black cutoff was set at 1.28% as described above. 5/11 (45.5%) of the serum samples were NAb positive (mean value = 8.19% recovery). A higher percentage of NAb in this subpopulation may reflect the response loss seen in these patients.

Figure 29 illustrates the performance characteristics of the neutralization test. ATI and ATA were purified from rabbit serum as described in the Methods section.

(A) Sensitivity and specificity of NAb assay. The purified ATI (19.3 μg / mL, left), purified ATA (49.1 μg / mL, right) and monoclonal mouse anti-human Fc (36.8 μg / mL) Spiked to normal human serum and tested in neutralization assay. The sensitivity of the test is defined as the lowest concentration of NAb in the neat serum producing a positive result in the NAb assay (i.e., greater than 1.28%, horizontal dashed line). The sensitivity of the ATI and ATA neutralization assays was measured to be 0.15 and 0.914 μg / mL, respectively (pure serum, vertical dotted line). Both tests are specific for ATI (left) or ATA (right).

(B) Interference of Infliximab with NAb black. In these experiments, a high control was tested in the presence of a two-fold serial dilution of infliximab (starting at 50 μg / mL). Data were normalized to the control maximal signal and plotted as% inhibition of the signal (inhibition% = 100 - Recovery _Norm %). The leftmost vertical dotted line represents the concentration of infliximab (4 μg / mL), which increases CV by more than 25%. The rightmost vertical dotted line represents an IC ₅₀ of 9.9 μg / mL.

(C) Precision of NAb positive control. A high, medium, and low positive control was generated by two-fold dilution of pooled ATI positive patient serum (200 U / mL ATI as measured by HMSA) with normal human serum. Each control was tested over a number of different days (n = 20). The mean values (CV%) for each control were 51.35 (10.4%), 28.50 (15.03%) and 10.38 (24.22%) for the high, medium and low control, respectively.

conclusion

A homogeneous, fluid phase NAb assay can detect both ATI and ATA with neutralization capability. At least 0.15 and 0.914 μg / mL of neutralized ATI or ATA, respectively, can be detected with high specificity. The assay can detect NAb in the presence of a higher level of drug (less than 4.0 μg / mL) than most cell-based or other competitive ligand binding assays. The NAb test has high accuracy and precision (CV <25%) over the extended dynamic range. The presence of neutralizing antibodies can predict the loss of response in patients undergoing infliximab or adalimumab therapy. Monitoring of NAb in addition to drug and ADA levels will provide the necessary information on the ADA response and help induce initial therapeutic intervention.

Example 6: Analysis of neutralizing anti-drug antibody responses in patients with a response loss to anti-TNF therapy

Anti-TNF monoclonal antibodies such as infliximab (IFX) and adalimumab (ADL) are prescribed for the treatment of inflammatory bowel disease. Certain patients will produce anti-drug antibodies (ADA) that can cause loss of drug efficacy and reverse reactions. Neutral ADA (NAb) binds to the antigen binding site, blocking access to TNF and directly preventing drug efficacy. Binding ADA interacts with structural components outside the antigen binding site and does not directly affect drug efficacy, although it may alter pharmacokinetics. Applicants have developed a test to monitor the development of NAb in patients with IBD who are receiving IFX or ADL therapy based on a uniform mobility shift assay (HMSA) platform and have shown a correlation between ATI / ATA maturation and NAb formation .

METHODS: 141 ATI or ATA-positive patients were recruited from IRB-approved studies in IBD patients. Serum concentrations of IFX, ADL and associated ADA were measured by HMSA as previously described (Wang et al. , J. Immunol. Methods , 2012, 382: 177). A healthy control was obtained from Golden West Biologics, Inc. Purchased from the company. Polyclonal antibodies against IFX and ADL were generated using serum isolated from rabbits immunized with IFX or ADL. For sensitivity and specificity experiments, purified rabbit ATI and ATA were spiked into normal human serum. For the NAb assay, ATI positive patient serum is first acid dissociated. IFX-Alexa488 and TNF-Alexa532 are added and the solution is neutralized. The solution is diluted with 2% serum, injected by HPLC on a size exclusion column, and the complexes are monitored by fluorescence. The area under the curve (AUC) of the free TNF-Alexa532 peak is calculated for the control and patient samples. IFX-Alexa488 binds to TNF-Alexa532 and reduces the test signal. NAb neutralizes IFX-Alexa 488 and restores the assay signal. The NAb activity is directly proportional to the measured assay signal.

Results: Verification of the NAB test proved high accuracy and accuracy (CV <25%) over extended dynamic range. A total of 130 ATI patients were analyzed for neutralizing capacity (0.0-430 U / mL, mean value = 49.22 U / mL, median = 19.10 U / mL). NAb levels were measured in a subset of 33 patients with low ATI positivity to determine the black cutoff. The cutoff with a 99% one-sided confidence limit was calculated to be 1.28% by the equation of mean + 2.33 x SD. Of the remaining patients, 37/97 (38.1%) were NAb positive (mean value = 7.43% recovery). A total of 11 ATA-positive patients who underwent a response loss to treatment with adalimumab were tested for neutralization ability (7.93-147.9 U / ml, mean value = 36.97 U / mL, median = 16.39 U / mL). The black cutoff was set at 1.28% as described above. 5/11 (45.5%) of the serum samples were NAb positive (mean value = 8.19% recovery).

Conclusion: The presence of neutralizing antibodies can predict response loss in patients undergoing infliximab or adalimumab therapy. Monitoring of NAb in addition to drug and ADA levels will provide the necessary information on the ADA response and help induce initial therapeutic intervention.

Example 7: Improved Homogeneous Mobility Assay (HMSA) for the Detection of Neutralizing Antibodies (Nab) in IBD Patients Treated with Infliximab or Adalimumab

Background: When treated with infliximab (IFX) and adalimumab (ADA), certain patients may develop antibody-versus-infliximab or adalimumab (ATI / ATA). ATI and ATA may be a neutralizing antibody (NAb) that binds to the antigen binding site of the drug to prevent access to TNF and directly prevents drug efficacy. This example describes the NAb HMSA of the present invention optimized for improved sensitivity and specificity and shows the correlation of CRP with drug levels in patient samples.

Methods: Serum of IFX, ADA and associated anti-drug antibodies Concentrations were determined by HMSA in healthy controls and remaining patient samples from routine testing (see, for example , Wang et al., J. Immunol. Methods , 382: 177-88 (2012)). Polyclonal antibodies against IFX and ADA were generated using sera isolated from rabbits immunized with IFX or ADA, purified and spiked into normal human serum to generate positive controls. For the NAb assay, ATI-positive patient serum is first acid-dissociated, IFX-Alexa488 is added, the solution is neutralized and TNF-Alexa488 is added. The solution is diluted with 4% serum, injected by HPLC on a size exclusion column, and the complexes monitored by fluorescence. The area under the curve of the free TNF-Alexa 488 peak is calculated for the control and patient samples. IFX-Alexa488 binds to TNF-Alexa488 and reduces the test signal. NAb neutralizes IFX-Alexa488 and restores the assay signal. The NAb activity is directly proportional to the measured assay signal.

Results: The NAb assay described in Example 6 demonstrated a high accuracy and precision (CV < 25%) over an extended dynamic range with a positive cutoff of 1.28% recovery (29.8 ng / mL). The optimization of the assay allowed detection of NAb <10 ng / mL in patients' sera and improved accuracy (CV <15%). The assay optimization involved the following changes to the NAb assay described in Example 6: (1) conversion to TNF-Alexa488 (IFX-Alexa488 maintenance); (2) Fluorescence detector wavelength change from Ex510_Em540 to Ex494_Em525 (Ex = excitation, Em = emission); (3) PMT (i.e., photomultiplier tube) change in gain value from 14 to 18; (4) adjustment of TNF: IFX molar ratio and reduction of IFX / TNF amount in assay; And (5) adjustment of incubation conditions, i.e. IFX addition, neutralization for 30 minutes, followed by addition of TNF for 30 minutes. Compared to the assays described in Example 6, ATI positive patients were analyzed for neutralizing capacity (3.1-200 U / mL) using the optimization assays described herein. The two assay formats are well correlated; Optimization assays detected a broader range of neutralizing antibodies (R ² = 0.998). The presence of detectable neutralizing antibodies was associated with both CRP elevation and drug level reduction (p &lt; 0.001).

Conclusion: The presence of neutralizing antibody (NAb) predicts loss of response, elevated CRP and reduced drug levels in patients undergoing infliximab or adalimumab therapy. Monitoring of NAb in addition to drug and anti-drug antibody levels provides the necessary information on the immune response and assists in inducing initial therapeutic intervention.

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

Claims (33)

A method for determining the percentage or level of autoantibody in neutralized form to an anti-TNF [alpha] drug in a sample, comprising:
(a) contacting the sample with a labeled anti-TNF [alpha] drug and labeled TNF [alpha] to form:
(i) a first labeled conjugate of a labeled anti-TNF [alpha] drug and an autoantibody; And / or
(ii) a second labeled complex of labeled anti-TNF [alpha] drug, labeled TNF [alpha] and autoantibody;
(b) applying a first labeled complex and / or a second labeled conjugate to size exclusion chromatography and comparing them to free labeled TNF ?, free labeled anti-TNF? drug and / or labeled anti-TNF? drug and Gt; TNFa &lt; / RTI &gt;complex;
(c) the level of free labeled TNF [alpha] was measured after size exclusion chromatography; And
(d) comparing the level of the free labeled TNF [alpha] measured in step (c) with the level of free labeled TNF [alpha] in the control sample, thereby determining the percentage or level of autoantibody in neutralized form. The pharmaceutical composition according to claim 1, wherein the anti-TNFa drug is selected from the group consisting of REMICADE (TM) (Infliximab), ENBREL (TM) (Etanercept), HUMIRA (TM) (Adalimumab), CIMZIA Gorimumab; CNTO 148), and combinations thereof. The method of claim 1 or 2 wherein the autoantibody to the anti-TNFa drug is selected from the group consisting of human anti-chimeric antibody (HACA), human anti-humanized antibody (HAHA), human anti-mouse antibody (HAMA) &Lt; / RTI &gt; 4. The method according to any one of claims 1 to 3, wherein step (c) comprises measuring the area under the curve (AUC) of the free labeled TNF [alpha] peak of the sample after size exclusion chromatography. 5. The method of claim 4 wherein the level of free labeled TNF [alpha] in the control sample is calculated by measuring the AUC of the free labeled TNF [alpha] peak in the control sample after size exclusion chromatography. 6. The method of claim 5, wherein step (d) comprises calculating the ratio of the AUC of the free labeled TNF [alpha] peak of the sample to the AUC of the free labeled TNF [alpha] peak of the control sample. 7. The method according to any one of claims 1 to 6, wherein the sample is positive for an autoantibody in neutralized form if the sample has a value exceeding the threshold of establishment of neutralizing autoantibodies. 8. The method of claim 7, wherein the threshold value is from about 1.25% to about 1.30% of the neutralizing form of the autoantibody. 9. The method according to any one of claims 1 to 8, wherein the control sample is a reference sample containing only TNF? 10. The method according to any one of claims 1 to 9, wherein the sample is serum. 11. The method according to any one of claims 1 to 10, wherein the sample is obtained in a subject undergoing treatment with an anti-TNFa drug. 12. The method according to any one of claims 1 to 11, wherein the labeled anti-TNF [alpha] drug and labeled TNF [alpha] comprise different fluorescent dyes or fluorescent dyes. 13. The method according to any one of claims 1 to 12, wherein the autoantibody in neutralized form is present in a population of autoantibodies comprising neutralizing autoantibodies and non-neutralizing autoantibodies. A method for determining whether an autoantibody in neutralization form to a first anti-TNF [alpha] drug is cross-reactive with a second anti-TNF [alpha] drug,
(a) determining the percentage or level of autoantibody in neutralization form in the sample according to the method of any one of claims 1 to 13 to determine whether the sample is positive or negative for the autoantibody in neutralization form; And
If the sample is positive for neutralizing autoantibodies, then:
(b) contacting the sample with a labeled second anti-TNF [alpha] drug to form a labeled second anti-TNF [alpha] drug and a labeled conjugate of an autoantibody in neutralized form;
(c) applying labeled conjugates to size exclusion chromatography to separate the labeled complexes; And
(d) detecting the labeled complex, thereby determining whether the neutralizing form of the autoantibody to the first anti-TNF alpha drug is cross-reactive with the second anti-TNF alpha drug. 15. The method of claim 14 wherein the first and second anti-TNFa drugs are selected from the group consisting of REMICADE (TM) (Infliximab), ENBREL (TM) (etanercept), HUMIRA (TM) Perl), SIMPONI (R), and combinations thereof. 16. The method of claim 14 or 15 wherein the autoantibody for the first anti-TNFa drug is selected from the group consisting of human anti-chimeric antibody (HACA), human anti-humanized antibody (HAHA), human anti- And combinations thereof. 17. The method of any one of claims 14 to 16, wherein the presence of the labeled complex indicates that the neutralizing autoantibody against the first anti-TNF [alpha] drug is cross-reactive with the second anti-TNF [alpha] drug / RTI &gt; 17. A method according to any one of claims 14 to 16, wherein the absence of the labeled complex indicates that the neutralizing autoantibody against the first anti-TNF [alpha] drug is not cross-reactive with the second anti-TNF [alpha] drug How it is. 19. The method according to any one of claims 14 to 18, wherein the sample is serum. 20. The method according to any one of claims 14 to 19, wherein the sample is obtained from a subject undergoing treatment with an anti-TNFa drug. 21. The method according to any one of claims 14 to 20, wherein the labeled second anti-TNF [alpha] drug comprises a fluorophore or a fluorescent dye. A method of monitoring or optimizing a therapeutic regimen for an anti-TNFα drug in a subject undergoing a therapeutic regimen with an anti-TNFα drug, comprising:
(a) determining the percentage or level of neutralizing forms of autoantibodies to the anti-TNFa drug according to the method of any one of claims 1 to 13 at multiple time points throughout the course of the treatment regimen;
(b) detecting a change in the percentage or level of neutralizing autoantibodies over time; And
(c) determine whether subsequent doses of the therapeutic regimen for the subject or different therapeutic regimens should be administered to the subject, based on a change in the percentage or level of neutralizing autoantibodies over time. 23. The method of claim 22, wherein the anti-TNFa drug is selected from the group consisting of REMICADE (TM) (Infliximab), ENBREL (TM) (etanercept), HUMIRA (TM) (Adalimumab), CIMZIA Gorimumab; CNTO 148), and combinations thereof. 24. The method of claim 22 or 23 wherein the autoantibody to the anti-TNFa drug is selected from the group consisting of human anti-chimeric antibody (HACA), human anti-humanized antibody (HAHA), human anti-mouse antibody (HAMA) &Lt; / RTI &gt; water. 25. The method according to any one of claims 22 to 24, wherein the subsequent dose of the therapeutic regimen is increased, decreased or maintained, based on a change in the percentage or level of neutralizing autoantibodies over time. 24. A method according to any one of claims 22-24, wherein the different therapeutic regimens involve the conversion of the different anti-TNFa drugs, current therapeutic regimens with immunosuppressive agents, or therapeutic regimens that are not anti-TNF alpha drugs How to. 27. The method of claim 26, wherein a different therapeutic regimen is administered when the percentage or level of autoantibody in neutralized form increases over time. A method of optimizing therapy and / or reducing toxicity in a subject undergoing a therapeutic treatment with a first anti-TNFα drug, comprising:
(a) measuring the percentage or level of neutralizing autoantibodies in the sample from a subject according to the method of any one of claims 1 to 13 to determine the neutralizing form of the autoantibody to the first anti-TNFa drug 2 &lt; / RTI &gt; anti-TNFa drug; And
(b) if the neutralizing form of the autologous antibody is cross-reactive with the second anti-TNFα drug, a different therapeutic regimen is to be administered to the subject. 29. The method of claim 28, wherein the first and second anti-TNFa drugs are selected from the group consisting of REMICADE (TM) (infliximab), ENBREL (TM) (etanercept), HUMIRA (TM) Perl), SIMPONI (R), and combinations thereof. 29. The method of claim 28 or 29 wherein the autoantibody for the first anti-TNFa drug is selected from the group consisting of human anti-chimeric antibody (HACA), human anti-humanized antibody (HAHA), human anti- And combinations thereof. 31. The method according to any one of claims 28 to 30, wherein the different therapeutic regimens comprise a conversion to a therapeutic regimen that is not an anti-TNFa drug. 32. The method of claim 31, wherein the non-anti-TNF [alpha] drug is selected from the group consisting of an IL-6 receptor-inhibiting monoclonal antibody, an anti-integrin molecule, a JAK-2 inhibitor, a tyrosine kinase inhibitor, Way. 33. The method according to any one of claims 28 to 32, wherein if the neutralizing form of the autoantibody is not cross-reactive with the second anti-TNF alpha drug then the subsequent dose of the current therapeutic regimen should be increased or decreased, Further comprising determining whether a different therapeutic regimen should be administered to the subject.

Images