Compositions and methods for IL-17 target engagement assays using macromolecular modulators

Abstract

The present invention provides a method for measuring total IL-17 in a biological sample containing IL-17 and a macromolecular modulator of IL-17.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 077,992, filed Sep. 14, 2020, U.S. Provisional Application No. 62 / 960,031, filed Jan. 12, 2020, and U.S. Provisional Application No. 62 / 908,195, filed Sep. 30, 2019, under 35 U.S.C. § 119(e), the entire disclosures of which are hereby incorporated by reference herein.

[0003] Reference Sequence Listing Submitted Electronically

[0004] This application contains a Sequence Listing that has been submitted electronically via EFS-Web as an ASCII formatted sequence listing, named “004852.11712 / 142W02(JBI6359WOPCT1)”, created on Sep. 23, 2020, and is 24 kb in size. The sequence listing submitted via EFS-Web is part of this specification and is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0005] Interleukin-17A (IL-17 or IL-17A) is a cytokine secreted by activated Th17 cells, CD8+ T cells, γδ T cells, and NK cells in response to cytokines such as IL-23 and TGF-β. IL-17 regulates mediators produced by a variety of cell types including epithelial cells, fibroblasts, and synoviocytes, such as antimicrobial peptides, pro-inflammatory cytokines, and chemokines, which are involved in neutrophil recruitment and the pathology of tissue damage in inflammation or host defense. IL-17 also acts in concert with other cytokines such as TNF-α and IL-1β to enhance the pro-inflammatory environment.

[0006] Due to its involvement in immune regulatory functions, inhibitors of IL-17 are being investigated as possible treatments for various autoimmune diseases. Anti-IL-17 monoclonal antibodies (mAbs) have been clinically validated for the treatment of psoriasis, psoriatic arthritis, and ankylosing spondylitis, and proof-of-concept has been demonstrated for the treatment of multiple sclerosis.

[0007] Although anti-IL-17 monoclonal antibodies (hereinafter referred to as macromolecular (LM) modulators of IL-17) have clinical utility, there is still a lack of target engagement assays for LM modulators of IL-17. Specifically, in studies involving LM modulators, commercially available assays in the art typically can only measure the amount of free IL-17 in a biological sample containing IL-17 and an LM modulator, but not the amount of LM-bound IL-17. In addition, although Peng et al. developed an assay for measuring target engagement specific to one anti-IL-17 antibody, namely MCAF5352A, this method demonstrated the utility with a single antibody. See, e.g., Peng et al., Measurement of IL-17AA and IL-17FF as Pharmacodynamic Biomarkers to Demonstrate Target Engagement in the Phase I Study of MCAF5352A, The AAPS Journal (2019) 21:9). Thus, information on the IL-17 target engagement levels of multiple LM modulators is lacking.

[0008] In addition, although disease-related biomarkers have been used to monitor the therapeutic effect of anti-IL-17 in patients, biomarkers have not been developed to monitor the anti-IL-17 effect in healthy subjects, which are typically included in the early stages of clinical evaluation. Thus, measurement of the target engagement of IL-17 LM modulators with IL-17 in a subject (e.g., a human) can be shown to be important for advancing LM modulators through clinical studies. There is also a need for one or more methods to evaluate the IL-17 engagement of an LM modulator in a sample to study clinically relevant parameters such as pharmacokinetic (PK) and pharmacodynamic (PD) information, which in turn can help determine the optimal dose for a safe and effective therapy. Thus, there is still a need for a robust immunoassay to evaluate the amount of total IL-17 (e.g., IL-17 bound and unbound to an LM modulator) and further provide target engagement information in samples involving a larger number of LM modulators. Summary of the Invention

[0009] In general aspects, the present application relates to an immunoassay that can detect total IL-17 (i.e., both free IL-17 and modulator-bound IL-17) in a sample containing IL-17 and an LM modulator, such that the target engagement of the LM modulator can be evaluated by subtracting the level of free IL-17 from the level of total IL-17.

[0010] The present invention provides a method for determining the total amount of IL-17 (free IL-17 and IL-17 bound to a large molecule (LM) modulator) in a sample comprising an IL-17 and an LM modulator of IL-17, the method comprising:

[0011] i. contacting the sample with a capture antibody to form a mixture comprising a first complex and a second complex, the first complex comprising the capture antibody and IL-17 not bound to the LM modulator, and the second complex comprising the capture antibody and IL-17 bound to the LM modulator;

[0012] ii. contacting the mixture from step i) with a detection agent, thereby forming a third complex comprising the capture antibody, IL-17 not bound to the LM modulator, and the detection agent, and a fourth complex comprising the capture antibody, IL-17 bound to the LM modulator, and the detection agent; and

[0013] iii. determining the amount of total IL-17 by measuring the amount of the detection agent in the third complex and the fourth complex,

[0014] wherein the capture antibody is anti-IL-17 antibody AF-317, and the detection agent is mAb4538.

[0015] In another embodiment of the method, a washing step is performed before contacting the first mixture from step i) with the detection agent in step (ii).

[0016] In another embodiment of the method, the LM modulator has a molecular weight in the range of about 2,000 Daltons - 250,000 Daltons, or about 5,000 Daltons - 160,000 Daltons.

[0017] In another embodiment of the method, the LM modulator is selected from the group consisting of secukinumab, mAb7024, and mAb732, preferably the LM modulator is secukinumab or mAb7024.

[0018] In another embodiment of the method, the detection agent is labeled with a detectable label.

[0019] In another embodiment of the method, the detectable label is an enzyme or biotin.

[0020] In another embodiment of the method, the sample is a biological sample obtained from a subject treated ex vivo or in vivo with an LM modulator.

[0021] In another embodiment of the method, the subject is a mammal, preferably a human.

[0022] In another embodiment of the method, the biological sample is selected from the group consisting of samples prepared from cells, tissues, or serum, plasma, or another biological fluid sample.

[0023] In another embodiment of the method, the immunoassay is an enzyme-linked immunosorbent assay (ELISA).

[0024] Also provided herein is a kit for determining the amount of total IL-17 in a sample comprising IL-17 and an LM modulator of IL-17, the kit comprising:

[0025] i. a capture antibody which, upon contact with the sample, forms a first complex comprising the capture antibody and IL-17 not bound to the LM modulator, and a second complex comprising the capture antibody and IL-17 bound to the LM modulator; and

[0026] ii. a detection agent which, upon contact with the first and second complexes, forms a third complex comprising the capture antibody, IL-17 not bound to the LM modulator, and the detection agent, and a fourth complex comprising the capture antibody, IL-17 bound to the LM modulator, and the detection agent,

[0027] wherein the capture antibody is anti-IL-17 antibody AF-317 and the detection agent is mAb4538.

[0028] In another embodiment of the kit, the detection agent is labeled with a detectable label, and more preferably, the label is an enzyme or biotin.

[0029] In another embodiment, the kit is for performing an ELISA.

[0030] Based on the following disclosure, including the detailed description of the invention, its preferred embodiments, and the appended claims, other aspects, features, and advantages of the invention will be apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The foregoing invention content and the following detailed description of the preferred embodiments of this patent application can be better understood when read in conjunction with the accompanying drawings. However, it should be understood that this patent application is not limited to the exact embodiments shown in the drawings.

[0032] Figures 1A - 1C A schematic diagram of an IL-17 detection assay is shown: Figure 1A - A conventional immunoassay for detecting a target antigen (e.g., IL-17) using a capture antibody / detection agent pair; Figure 1B - An immunoassay for detecting free IL-17 (i.e., IL-17 not bound to the modulator) using a commercially available capture antibody / detection agent pair in the presence of an LM modulator; andFigure 1C - An immunoassay for detecting total IL-17 (including free IL-17 and IL-17 bound to the modulator) using the capture antibody / detection agent of the present invention in the presence of an LM modulator.

[0033] Figure 2 Detection of free IL-17 using DuoSet ELISA DY317 (R&D Systems, Inc.) in the presence of an IL-17 LM modulator (secukinumab, 10 μg / ml, 1 μg / ml, 0.1 μg / ml, or 0.01 μg / ml) or in the absence of an LM modulator is shown.

[0034] Figure 3 Detection of total IL-17 using anti-IL-17 antibody AF-317 (Catalog No. AF-317-NA, from R&D systems) as the capture antibody and mAb4538 as the detection agent in samples with an IL-17 LM modulator (secukinumab, 10 pg / ml, 1 pg / ml, 0.1 pg / ml, 0.01 pg / ml, or 0.001 pg / ml) or in the absence of an LM modulator is shown.

[0035] Figure 4A Detection of free IL-17 using DuoSet ELISA kit DY317 in samples with an IL-17 LM modulator (secukinumab, mAb7024, or mAb732) or in the absence of an LM modulator is shown.

[0036] Figure 4B Detection of total IL-17 using anti-IL-17 antibody AF-317 (Catalog No. AF-317-NA, R&D systems) as the capture antibody and mAb4538 as the detection agent in samples with an IL-17 LM modulator (secukinumab, mAb7024, or mAb732) or in the absence of an LM modulator is shown. Detailed Description

[0037] Various publications, articles, and patents are cited or described throughout the background and the specification; each of these references is hereby incorporated by reference in its entirety. The discussions of documents, acts, materials, devices, articles, etc. included in this specification are intended to provide context for the present invention. Such discussions are not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed herein.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Otherwise, certain terms used herein have the meanings described in this specification. All patents, published patent applications, and publications cited herein are incorporated herein by reference as if fully set forth herein.

[0039] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0040] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or will be able to ascertain using only routine experimentation, a variety of equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0041] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "include" and "contain" will be understood to imply the inclusion of the stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps. When used herein, the term "comprise" may be replaced with the term "contains" or "includes", or sometimes when used herein, with the term "having".

[0042] When used herein, "consisting of excludes any element, step, or ingredient not specified in the claim elements. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Whenever used herein in the context of one aspect or embodiment of the present application, any of the foregoing terms "comprising," "containing," "including," and "having" may be replaced with the terms "consisting of" or "consisting essentially of" to alter the scope of the present disclosure.

[0043] As used herein, the connection term "and / or" between multiple listed elements is understood to include both single options and combined options. For example, where two elements are connected by "and / or", the first option refers to the application of the first element without the second element. The second option refers to the application of the second element without the first element. The third option refers to the application of the first element and the second element together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and / or" as used herein. The parallel applicability of more than one option is also understood to fall within the meaning and therefore meets the requirements of the term "and / or".

[0044] Unless otherwise indicated, any numerical values, such as concentrations or concentration ranges described herein, are understood to be modified in all cases by the term "about". Thus, the numerical values generally include the stated value ±10%. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1 mg / mL to 10 mg / mL includes 0.9 mg / mL to 11 mg / mL. As used herein, unless the context clearly indicates otherwise, numerical ranges used expressly include all possible sub-ranges, all individual numerical values within the range, including integers and fractions within such range.

[0045] When used in reference to amino acid sequences, the phrase "percent sequence identity (%)" or "identity %" or "identical to...%" describes the number of matching ( "hits") of identical amino acids of two or more aligned amino acid sequences compared to the number of amino acid residues that make up the total length of the amino acid sequence. In other words, when the maximum correspondence of the compared and aligned sequences is measured using sequence comparison algorithms known in the art, or when manually aligned and visually inspected, using an alignment, for two or more sequences, the percentage of identical amino acid residues can be determined (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity over the full length of the amino acid sequence). Thus, the sequences being compared to determine sequence identity may differ by amino acid substitutions, additions or deletions. Suitable programs for aligning protein sequences are known to those skilled in the art. The percent sequence identity of protein sequences can be determined, for example, using programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, e.g., using the NCBI BLAST algorithm (Altschul SF et al. (1997), Nucleic Acids Res. 25:3389 - 3402).

[0046] As used herein, a "subject" refers to any animal, preferably a mammal, most preferably a human, that will be or has been treated by a method according to an embodiment of the present application. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) (such as monkeys or apes), humans, etc., more preferably including humans.

[0047] To assist the reader of this application, the specification has been divided into paragraphs or sections, or the reader has been directed to different embodiments of this application. These divisions should not be construed as separating the substance of a paragraph or section or embodiment from the substance of another paragraph or section or embodiment. On the contrary, those skilled in the art will understand that this specification has broad application and encompasses all combinations of the various sections, paragraphs, and sentences that can be conceived. The discussion of any embodiment is intended to be exemplary only and is not intended to indicate that the scope of the disclosure (including the claims) is limited to these examples.

[0048] As used herein, "IL-17" or "IL-17A" refers to interleukin 17A. It is also named IL17, CTLA8, CTLA-8. Interleukin 17A is a pro-inflammatory cytokine. This cytokine is produced by a subset of T helper cells called T helper 17 cells in response to stimulation with IL-23. The protein encoded by IL17A is the founding member of the IL-17 family, including IL17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. IL-17E is also known as IL-25. All members of the IL-17 family have a similar protein structure. IL-17 regulates the activities of NF-κb and mitogen-activated protein kinases. It can stimulate the expression of IL6 and cyclooxygenase-2 (PTGS2 / COX-2), as well as enhance the production of nitric oxide (NO). High levels of IL17 are associated with several chronic inflammatory diseases, including rheumatoid arthritis, psoriasis, and multiple sclerosis. "IL-17" includes IL-17A from any animal species, as well as recombinant products of IL-17A. IL-17 can be a homodimer of IL-17A or a heterodimer of IL-17A and IL-17F. An exemplary amino acid sequence of human IL-17 is represented in GenBank accession number NP 002181.1, which can be encoded by a nucleic acid sequence such as GenBank accession number NM_002190.3.

[0049] As used herein, the term "modulator" refers to any agent or molecule capable of binding to IL-17, including small molecule compounds and macromolecules such as antibodies.

[0050] The terms "small molecule modulator" and / or "SM modulator" and / or "SM modulator of IL-17" are used interchangeably herein and refer to any small molecule compound capable of binding to IL-17 and having a molecular weight in the range of about 100 g / mol to about 1500 g / mol.

[0051] The terms "macromolecular modulator" and / or "LM modulator" and / or "LM modulator of IL-17" are used interchangeably herein and refer to any macromolecular compound (e.g., antibody, protein, etc.) that is capable of binding to IL-17 and has a molecular weight greater than about 1500 daltons or preferably has a molecular weight in the range of about 2,000 daltons to about 250,000 daltons, or about 5,000 daltons to about 160,000 daltons, or about 150,000 daltons to about 250,000 daltons. Dalton (symbol Da) is used herein as a unit of molar mass and is defined as 1 Da = 1 g / mol.

[0052] As used herein, the term "free IL-17" or "unbound IL-17" refers to IL-17 that is not bound to any modulator in a biological sample.

[0053] The terms "sample" and "biological sample" are used interchangeably herein and encompass various sample types obtained or derived from an organism and can be used in diagnostic or monitoring assays. The term encompasses blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny. The term encompasses samples that have been manipulated in any way after procurement, such as treatment with reagents, solubilization or enrichment of certain components. The term encompasses clinical samples and also includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids and tissue samples.

[0054] As used herein, the term "antibody" is used broadly and includes immunoglobulin or antibody molecules, including human antibodies, humanized antibodies, bispecific antibodies and chimeric antibodies as well as monoclonal or polyclonal antibody fragments. Generally speaking, an antibody is a protein or peptide chain that exhibits binding specificity for a particular antigen. Antibodies can be derived from any species and can be, for example, IgM, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), IgD, IgA or IgE.

[0055] As used herein, the term "antigen-binding fragment" refers to antibody fragments such as, for example, diabodies, Fab, Fab', F(ab’)2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv 1 )), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), single-domain antibodies (sdab), scFv dimers (bivalent diabodies), multispecific antibodies formed from a part of an antibody containing one or more CDRs, camelized single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies or any other antibody fragment that binds to an antigen but does not contain the complete antibody structure, or any peptide containing an antigen-binding fragment.

[0056] As used herein, the term "monoclonal antibody" refers to a preparation of an antibody of a single molecular composition.

[0057] As used herein, the term "polyclonal antibody" refers to a composition of different antibody molecules that are capable of binding to or reacting with several different antigenic determinants on the same or different antigens. The variability in the antigen specificity of polyclonal antibodies resides in the variable regions of the individual antibodies that make up the polyclonal antibody, particularly in the complementarity-determining regions CDR1, CDR2, and CDR3 regions.

[0058] As used herein, the term "capture antibody" refers to an antibody that specifically binds to IL-17, regardless of whether IL-17 is bound to a modulator. Such capture antibodies can be commercially available or investigational agents. The capture antibody can be provided in solution or pre-coated onto a surface.

[0059] As used herein, the term "detection agent" refers to an agent that specifically binds to IL-17 that is not bound to a modulator or IL-17 that is bound to a modulator or both. A "detection agent" (such as a detection antibody) or a soluble receptor can be used to detect IL-17 that is not bound to a modulator or IL-17 that is bound to a modulator. The detection agent (such as a detection antibody) can be commercially available or investigational. The detection agent can be detected by any method known in the art, such as using a radioactive label or a fluorescent label, or by direct or indirect binding to an enzyme or biotin. Preferably, the detection agent (such as a detection antibody) is labeled with a detectable label (such as an enzyme or biotin).

[0060] An enzyme can convert certain substrates into detectable products. For example, the enzyme can be one of the following enzymes commonly used in enzyme-linked immunosorbent assays (ELISAs):

[0061] · Horseradish peroxidase (HRP), commonly used for conjugation with proteins, converts o-phenylenediamine dihydrochloride (OPD) into an amber product;

[0062] · HRP converts 3,3',5,5'-tetramethylbenzidine (TMB) into a blue product that turns yellow in the presence of sulfuric acid or phosphoric acid;

[0063] · HRP converts 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) into a green product; and

[0064] · Alkaline phosphatase converts p-nitrophenyl phosphate (PNPP) into a yellow product.

[0065] Biotin is a water-soluble B vitamin, also known as vitamin B7. Biotin can bind to avidin, streptavidin, and neutravidin with considerable affinity. These proteins (avidin, streptavidin, and neutravidin) can be further conjugated with the detectable enzymes described above to generate a signal.

[0066] The term "specific binding" refers to the binding between two agents (e.g., an antigen and an antibody), characterized by the ability of one agent (e.g., an antigen) to associate with another agent (e.g., an antibody), even in the presence of many other different agents, i.e., showing a preference of one agent for another agent in a mixture of multiple agents. For example, an antibody that "specifically binds" to an antigen refers to an antibody or its antigen-binding fragment that binds to the antigen with a KD of 1×10 -7 M or less, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1×10 -10 M or less. KD is the equilibrium dissociation constant between an antibody and its antigen, i.e., the calculated ratio of K off / K on . For example, the KD of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system (e.g., system), or by using biolayer interferometry techniques (such as the Octet RED96 system). The smaller the value of the antibody KD, the higher the affinity of the antibody for the target antigen.

[0067] As used herein, the term "kit" refers to a combination of reagents and other materials. It is contemplated that the kit may include reagents such as buffers, protein stabilizing reagents, signal generation systems (e.g., fluorescence signal generation systems), antibodies, control proteins, and test containers (e.g., microtiter plates, etc.). The term "kit" is not intended to be limited to a specific combination of reagents and / or other materials. In one embodiment, the kit further includes instructions for using the reagents. The test kit can be packaged in any suitable manner, typically packaged in a single container or in components in various containers as needed, and packaged with a sheet of instructions for performing the test. In some embodiments, the kit also preferably includes a positive control sample. The kit can be produced in a variety of ways known in the art.

[0068] This application as a whole relates to an IL-17 target engagement assay for measuring the amount of total IL-17 (i.e., IL-17 that is bound or unbound to an LM modulator) in a sample comprising IL-17 and an LM modulator.

[0069] In studies involving LM modulators, commercially available assays in the art typically can only measure the amount of free IL-17 in a biological sample containing IL-17 and an LM modulator. As previously discussed, although Peng et al. developed an assay specific for measuring target engagement of an anti-IL-17 antibody, MCAF5352A, the present application provides methods for determining the amount of total IL-17 (e.g., bound and unbound IL-17) for a greater number of LM modulators. The method comprises:

[0070] i. contacting the sample with a capture antibody to form a mixture comprising a first complex and a second complex, the first complex comprising the capture antibody and IL-17 not bound to an LM modulator, and the second complex comprising the capture antibody and IL-17 bound to an LM modulator;

[0071] ii. contacting the mixture from step i) with a detection agent (preferably a detection antibody), thereby forming a third complex comprising the capture antibody, IL-17 not bound to an LM modulator, and the detection agent, and a fourth complex comprising the capture antibody, IL-17 bound to an LM modulator, and the detection agent; and

[0072] iii. determining the amount of total IL-17 by measuring the amount of the detection agent in the third and fourth complexes.

[0073] After contacting the sample with the capture antibody, the capture antibody specifically binds IL-17 in the sample (both free IL-17 and those bound to an LM modulator), forming the first and second complexes, respectively, in the mixture. In certain embodiments, the capture antibody is attached to a solid support, and the mixture contains the first IL-17 complex and the second IL-17 complex attached to the solid support and the remainder of the sample.

[0074] As used herein, the term "detection agent" refers to an agent, such as an antibody or a receptor, that specifically binds free IL-17 captured by the capture antibody and IL-17 bound to a modulator captured by the capture antibody. Specifically, when the detection agent is contacted with the first and second complexes, it forms a third complex comprising the capture antibody, free IL-17, and the detection agent, and a fourth complex comprising the capture antibody, modulator-bound IL-17, and the detection agent.

[0075] Thus, the capture antibody and the detection agent are used in pairs to measure the amount of total IL-17 in a sample.

[0076] In one embodiment, the capture antibody is anti-IL-17 antibody AF-317 (Catalog No. AF-317-NA, from R&D Systems, Inc.), and the detection agent is mAb4538 (an anti-IL-17 antibody, from Janssen Biotech, Inc.). mAb4538 is disclosed in U.S. Patent 8,519,107, which is incorporated herein by reference in its entirety. Its detailed sequence information is listed in Table 1.

[0077] Table 1. Antibody Sequences

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] In certain embodiments, the detection agent (such as a detection antibody) is labeled with a detectable label. Preferably, the label is an enzyme or biotin. In another embodiment, the detection agent is labeled with a marker (such as biotin) for interaction with a labeled enzyme (such as streptavidin-conjugated horseradish peroxidase), which acts on tetramethylbenzidine in the presence of hydrogen peroxide to generate a color signal to determine the amount of the first detection agent.

[0084] As used herein, the terms "wash", "washing step", or "washing" refer to the process for separating total IL-17 (including both free IL-17 and IL-17 bound to a modulator) that is not bound to the capture antibody from the mixture of step (i). The solution used for washing is typically a buffer ("washing buffer"). In some embodiments, the washing buffer contains a low concentration of detergent. In some embodiments, the washing buffer is a 10 mM phosphate buffer at a pH of about 7.4 containing 150 mM NaCl and 0.05% Tween 20. The pH of the washing buffer is preferably in the range of about 6 to about 9. In some embodiments, the pH value is about 7.0. The washing step can be performed one or more times (e.g., once, twice, three times, four times, five times, or six times). In a preferred embodiment, the washing step is performed three to six times.

[0085] In certain embodiments, the washing step is performed before contacting the first mixture from step (i) with the first detection agent in step (ii).

[0086] In certain embodiments, no washing step is performed before contacting the first mixture from step (i) with the first detection agent in step (ii).

[0087] As used herein, the term "LM modulator" or "LM modulator of IL-17" refers to any macromolecule, including an antibody or an antigen-binding fragment thereof, or a protein comprising an antigen-binding fragment thereof, that is capable of binding IL-17 and has a molecular weight greater than about 1500 daltons, or preferably has a molecular weight in the range of about 2,000 daltons to about 250,000 daltons, or about 5000 daltons to about 160,000 daltons, or about 15,000 daltons to about 250,000 daltons, or about 100,000 daltons to about 200,000 daltons, or about 125,000 daltons to about 175,000 daltons.

[0088] In certain embodiments, the LM modulator used herein is an antibody that specifically binds IL-17. The methods according to the embodiments of the present application can be used to measure total IL-17 in the presence of any LM modulator. Examples of LM modulators include, but are not limited to, antibodies or antigen-binding fragments thereof, or proteins comprising antigen-binding fragments thereof. Exemplary LM modulators used herein include, but are not limited to, secukinumab, mAb7024, mAb732, or binding fragments thereof.

[0089] Secukinumab (Caligor Coghlan, Secaucus, NJ), also disclosed in WO 2006 / 013107 (also published as US20090280131, which is incorporated herein by reference in its entirety), is a recombinant high-affinity fully human monoclonal anti-human interleukin-17A antibody of the IgG1 / k class. Secukinumab has a molecular weight of approximately 151 kDa; both heavy chains of secukinumab contain oligosaccharide chains. Secukinumab binds to human IL-17A and neutralizes the biological activity of the cytokine. Secukinumab has a very high affinity for IL-17, i.e., KD is approximately 100 pM - 200 pM, and IC50 is approximately 0.4 nM for in vitro neutralization of the biological activity of 0.67 nM human IL-17A.

[0090] mAb7024 and mAb732 are antibodies to IL-17 from Janssen Biotech, Inc. and are disclosed in U.S. Patent 8,519,107, which is incorporated herein by reference in its entirety. The detailed sequence information of mAb7024 and mAb732 is listed in Table 1. mAb7024 has a molecular weight of approximately 146 kDa. mAb732 has a molecular weight of approximately 150 kDa.

[0091] In certain embodiments, IL-17 is human IL-17.

[0092] In certain embodiments, the sample is a biological sample obtained from a human subject treated ex vivo or in vivo with a small molecule modulator of human IL-17.

[0093] In certain embodiments, the biological sample is selected from the group consisting of samples prepared from cells, tissues, or serum, plasma, or another biological fluid sample.

[0094] Typically, commercially available kits (e.g., ELISA kits) only detect free IL-17, which, due to its low levels, is rarely detected in healthy subjects when administered together with an anti-IL-17 mAb. However, the methods described herein detect total IL-17 (including free and LM-bound IL-17), which provides a measurable amount of IL-17 above the lower limit of detection. Thus, the claimed methods provide quantitative evidence of target engagement by LM modulators. In addition, analysis of clinical serum samples has shown that the increase in IL-17 is dose- and exposure-dependent (Peng K. et al.) due to an increased half-life of the IL-17 / LM complex, which supports the concept of measuring total IL-17 for target engagement assessment. Moreover, the inventive assays described herein measure total IL-17 in the presence of LM modulators, independent of the presence of disease, and can be applied to healthy subjects or patients.

[0095] In addition, as described above, even though Peng et al. developed an assay for measuring target engagement specific to an anti-IL-17 antibody, i.e., MCAF5352A, the method demonstrated the utility of using only a single antibody. However, the inventive methods described herein can be used to measure target engagement of a larger number of LM modulators. This target engagement readout and pharmacokinetics (PK) of test molecules can be used to model to determine the optimal dose to achieve efficacy and minimize signals in safety studies.

[0096] In addition, the inventive methods described herein can be used in combination with commercially available assays that measure free IL-17 in the presence of LM modulators to determine target engagement of LM modulators. Specifically, in a sample containing IL-17 and an LM modulator, the degree of target engagement of the LM modulator can be evaluated by subtracting the free IL-17 level (determined using a commercially available assay) from the total IL-17 level (determined using the inventive capture antibody and detection agent pair disclosed herein).

[0097] This application also provides a kit for determining the amount of total IL-17 in a sample containing IL-17 and an LM modulator, the kit comprising:

[0098] i. A capture antibody that, upon contact with a sample, forms a first complex comprising the capture antibody and IL-17 not bound to an LM modulator and a second complex comprising the capture antibody and IL-17 bound to an LM modulator; and

[0099] ii. A detection agent (such as a first detection antibody) that, upon contact with the first and second complexes, forms a third complex comprising the capture antibody, IL-17 not bound to an LM modulator, and the detection agent, and a fourth complex comprising the capture antibody, IL-17 bound to an LM modulator, and the detection agent.

[0100] In some embodiments, the kit further comprises one or more reagents for detecting the detection agent.

[0101] In one embodiment, the capture antibody is anti-IL-17 antibody AF-317 and the detection agent is mAb4538.

[0102] Embodiments

[0103] The present invention also provides the following non-limiting embodiments.

[0104] Embodiment 1 is a method for determining the amount of total IL-17 in a sample comprising IL-17 and a macromolecular (LM) modulator of IL-17, the method comprising:

[0105] i. Contacting the sample with a capture antibody to form a mixture comprising a first complex and a second complex, the first complex comprising the capture antibody and IL-17 not bound to the LM modulator, and the second complex comprising the capture antibody and IL-17 bound to the LM modulator;

[0106] ii. Contacting the mixture from step (i) with a detection agent, thereby forming a third complex comprising the capture antibody, IL-17 not bound to the LM modulator, and the detection agent, and a fourth complex comprising the capture antibody, IL-17 bound to the LM modulator, and the detection agent; and

[0107] iii. Determining the amount of total IL-17 by measuring the amount of the detection agent in the third and fourth complexes,

[0108] wherein the capture antibody is anti-IL-17 antibody AF-317 and the detection agent is mAb4538.

[0109] Embodiment 2 is the method according to Embodiment 1, wherein a washing step is performed before contacting the first mixture from step (i) with the detection agent in step (ii).

[0110] Embodiment 3 is the method according to any one of Embodiments 1 or 2, wherein the LM modulator is selected from the group consisting of secukinumab, mAb7024, and mAb732.

[0111] Embodiment 3a is the method according to Embodiment 3, wherein the LM modulator is secukinumab or mAb7024

[0112] Embodiment 3b is the method according to Embodiment 3, wherein the LM modulator is secukinumab.

[0113] Embodiment 4 is the method according to any one of Embodiments 1-3, wherein the detection agent is labeled with a detectable label.

[0114] Embodiment 5 is the method according to Embodiment 4, wherein the detectable label is an enzyme or biotin.

[0115] Embodiment 6 is the method according to any one of Embodiments 1-5, wherein the sample is a biological sample obtained from a subject treated ex vivo or in vivo with the LM modulator.

[0116] Embodiment 6a is the method according to Embodiment 6, wherein the subject is a mammal, preferably a human.

[0117] Embodiment 6b is the method according to Embodiment 6, wherein the biological sample is selected from the group consisting of samples prepared from cells, tissues, or serum, plasma, or another biological fluid sample.

[0118] Embodiment 7 is the method according to any one of Embodiments 1-6, wherein the method is an enzyme-linked immunosorbent assay (ELISA).

[0119] Embodiment 8 is a kit for determining the amount of total IL-17 in a sample comprising an LM modulator of IL-17 and IL-17, the kit comprising:

[0120] i. a capture antibody, wherein upon contact with the sample, the capture antibody forms a first complex comprising the capture antibody and IL-17 not bound to the LM modulator, and a second complex comprising the capture antibody and IL-17 bound to the LM modulator; and

[0121] ii. A detection agent, which forms, upon contact with the first complex and the second complex, a third complex comprising the capture antibody, IL-17 not bound to the LM modulator, and the detection agent, and a fourth complex comprising the capture antibody, IL-17 bound to the LM modulator, and the detection agent.

[0122] Wherein the capture antibody is anti-IL-17 antibody AF-317, and the detection agent is mAb4538.

[0123] Embodiment 9 is the kit according to Embodiment 8, wherein the detection agent is labeled with a detectable label, and more preferably, the label is an enzyme or biotin.

[0124] Embodiment 10 is the kit according to Embodiment 8 or 9, and the kit is used for performing ELISA.

[0125] Examples

[0126] Those skilled in the art should understand that modifications can be made to the above embodiments without departing from the broad inventive concept of the present invention. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but the present invention is intended to cover modifications within the essence and scope of the present invention, as defined in this specification.

[0127] Example 1

[0128] Materials

[0129] The capture antibody / detection agent pairs used in the examples are listed in Table 2.

[0130] Table 2

[0131]

[0132]

[0133] Methods

[0134] Screening of capture antibody / detection agent pairs capable of detecting total IL-17 (free and LM modulator-bound IL-17) by ELISA: Prepare two-fold and 8-point serial dilutions of recombinant IL-17 at a final concentration of 1 ng / ml in diluent, in duplicate. Prepare secukinumab (150 mg / ml injection solution; NDC: 00078-0639-98; Caligor OPCO, LLC) at final concentrations of 10 μg / ml, 1 pg / ml, 0.1 μg / ml, 0.01 μg / ml, and 0.001 μg / ml.

[0135] Coat a high-binding 96-well plate with capture antibody in 4 pg / ml PBS at 100 μL / well overnight at room temperature. Wash the coated plate on a plate washer (Zoom HT microplate washer) with 6 × 400 μL / well, and block it with 300 μL / well blocking agent (PBS containing 1% BSA) for 2.5 hours at room temperature, then wash it on the plate washer with 6 × 400 μL / well.

[0136] Measure IL-17 in the sample after incubation with the test molecule in an ELISA assay by adding 95 μL / well sample to the capture antibody-coated plate. Keep the plate at room temperature for 30 minutes, then wash it on the plate washer with 6 × 400 μL / well. Add 5 μL / well detection agent to a final concentration of 500 ng / mL, keep it at room temperature for an additional 30 minutes, then wash it on the plate washer with 6 × 400 μL / well. Perform color development for measurement by adding 100 μL / well streptavidin-conjugated horseradish peroxidase and incubating for 20 minutes at room temperature in the dark, then wash it on the plate washer with 6 × 400 μL / well, then add 100 μL / well substrate solution and incubate for 20 minutes at room temperature in the dark. Then terminate the reaction by adding 50 μL / well stop solution and measure the plate at 450 nm - 540 nm in a plate reader.

[0137] Analyze the optical density (OD) readings from the plate reader (Molecular Devices Spectra Max 340fPC) using Softmax Pro 6.3 and GraphPad Prism, and plot a dose-response curve of OD values against IL-17 concentration.

[0138] Results

[0139] Detection of free or total human IL-17 ( Figures 2 - 3 ): Figure 2 A - Figure 2 D show the IL-17 levels in samples containing 10 μg / ml, 1 μg / ml, 0.1 μg / ml, or 0.01 μg / ml secukinumab compared to the levels in samples without added IL-17LM modulator. As shown herein, using the DuoSet DY317 ELISA kit, only free IL-17 can be detected, and IL-17 bound to the modulator cannot be detected. The level of free IL-17 is secukinumab concentration-dependent, and the free IL-17 level increases as the secukinumab concentration decreases. However, as Figure 3As shown, when AF-317 / mAb4538 is used as the capture antibody / detection agent pair, total IL-17 is detected in the presence of secukinumab, and the IL-17 level is independent of the secukinumab concentration.

[0140] Example 2

[0141] In this example, using a similar ELISA assay as described in Example 1, two (2) capture antibody / detection agent pairs (listed in Table 2) were tested using three (3) different LM modulators of IL-17, namely secukinumab (10 μg / ml), mAb7024 (10 μg / ml), and mAb732 (10 μg / ml).

[0142] As Figure 4A shown, the DuoSet DY317 ELISA kit detects free IL-17. In the presence of high concentrations of LM modulators, IL-17 binds to the LM, so only zero to minimal levels of free IL-17 are detected, and no modulator-bound IL-17 is detected. When AF-317 / mAb4538 is used as the capture antibody / detection agent pair, total IL-17 is detected in a concentration-dependent manner in the presence of LM modulators (secukinumab, mAb7024, or mAb732).

Claims

1. A method for determining the total amount of IL-17, including free IL-17 and IL-17 bound to a macromolecular (LM) modulator, in a sample comprising IL-17 and a macromolecular (LM) modulator of IL-17, the method comprising: i. contacting the sample with a capture antibody to form a mixture comprising a first complex and a second complex, the first complex comprising the capture antibody and IL-17 not bound to the LM modulator, and the second complex comprising the capture antibody and IL-17 bound to the LM modulator; ii. contacting the mixture from step i) with a detection agent, thereby forming a third complex comprising the capture antibody, IL-17 not bound to the LM modulator, and the detection agent, and a fourth complex comprising the capture antibody, IL-17 bound to the LM modulator, and the detection agent; and iii. determining the amount of total IL-17 by measuring the amount of the detection agent in the third complex and the fourth complex, wherein the capture antibody is anti-IL-17 antibody AF-317, and the detection agent is mAb4538, and the macromolecular (LM) modulator is selected from the group consisting of secukinumab, mAb7024, and mAb732, and the capture antibody is capable of forming a complex comprising the capture antibody and IL-17 bound to any one of secukinumab, mAb7024, and mAb732.

2. The method according to claim 1, wherein A washing step is performed before contacting the first complex from step i) with the detection agent in step (ii).

3. The method according to claim 1 or 2, wherein The detection agent is labeled with a detectable label.

4. The method according to claim 3, wherein, The detectable label is an enzyme or biotin.

5. The method according to claim 1 or 2, wherein, The sample is a biological sample obtained from a subject treated ex vivo or in vivo with the LM modulator.

6. The method according to claim 5, wherein, The subject is a mammal.

7. The method according to claim 5, wherein The subject is a human.

8. The method according to claim 5, wherein the biological sample is selected from the group consisting of samples prepared from cells, tissues, or serum, plasma, or another biological fluid sample.

9. The method according to claim 1 or 2, wherein the method is an enzyme-linked immunosorbent assay (ELISA).

10. A kit for determining the amount of total IL-17 in a sample comprising IL-17 and a macromolecular (LM) modulator of IL-17, the kit comprising: i. a capture antibody, wherein upon contact with the sample, the capture antibody forms a first complex comprising the capture antibody and IL-17 not bound to the LM modulator, and a second complex comprising the capture antibody and IL-17 bound to the LM modulator; and ii. a detection agent, wherein upon contact with the first complex and the second complex, the detection agent forms a third complex comprising the capture antibody, IL-17 not bound to the LM modulator, and the detection agent, and a fourth complex comprising the capture antibody, IL-17 bound to the LM modulator, and the detection agent, Wherein, the capture antibody is anti-IL-17 antibody AF-317, and the detection agent is mAb4538, and the macromolecule (LM) modulator is selected from the group consisting of: secukinumab, mAb7024, and mAb732, and the capture antibody is capable of forming a complex comprising the capture antibody and IL-17 that binds to any one of secukinumab, mAb7024, and mAb732.

11. The kit according to claim 10, wherein the detection agent is labeled with a detectable label.

12. The kit according to claim 11, wherein the detectable label is an enzyme or biotin.

13. The kit according to claim 10 or 11, the kit is for performing ELISA.

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