Compositions and methods for IL-17 targeted engagement assays with small molecule modulators

Abstract

The present invention provides a method for measuring free IL-17 in a sample containing IL-17 and a small molecule modulator of IL-17.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 077,978, filed on September 14, 2020; U.S. Provisional Application No. 62 / 960,031, filed on January 12, 2020; and U.S. Provisional Application No. 62 / 908,195, filed on September 30, 2019, the entire disclosures of which are hereby incorporated by reference into this document.

[0003] Reference sequence listing submitted electronically

[0004] This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with the file reference "004852.11713 / 142WO1 (JBI6152WOPCT1)" and a creation date of September 23, 2020, and is 44 kb in size. This sequence listing, submitted via EFS-Web, is part of this specification and is incorporated herein by reference in its entirety. Background Art

[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 interleukin-23 (IL-23) and transforming growth factor beta (TGF-β). IL-17 regulates the production of regulatory agents, such as antimicrobial peptides, proinflammatory cytokines and chemokines from various cell types, including epithelial cells, fibroblasts and synoviocytes involved in the neutrophil and pathological recruitment of tissue damage in inflammation or host defense. IL-17 also acts synergistically with other cytokines (such as TNF-α and IL-1 β) to enhance proinflammatory environment.

[0006] Due to its involvement in immune regulation, IL-17 inhibitors (or IL-17 modulators) are being studied as possible treatments for various autoimmune diseases. Anti-IL-17 mAbs have been clinically validated for the treatment of psoriasis, psoriatic arthritis, and ankylosing spondylitis, and have been conceptually proven to be useful for the treatment of multiple sclerosis. Although oral small molecule modulators of IL-17 have not yet entered clinical trials, they remain an attractive area of discovery because their development may broaden treatment options for many patients who cannot access biologics. (e.g., S. Liu et al., Scientific Reports, 6, 30859, https: / / doi.org / 10.1038 / srep30859).

[0007] Although the macromolecular regulators of IL-17 have clinical utility, and the small molecule regulators of IL-17 have appeared in the pharmaceutical industry, there is no report yet on the targeted engagement assay for measuring the binding activity between IL-17 and its small molecule regulators. In other words, currently available immunoassay kits can only measure the level of total IL-17 in samples with small regulators of IL-17 and IL-17 (including both IL-17 bound by regulators and free IL-17). However, there is no assay for specifically detecting only free IL-17 in the presence of small molecule regulators of IL-17 to determine targeted engagement. Therefore, there is a lack of information on the IL-17 targeted engagement level of small molecule regulators.

[0008] Furthermore, although disease-associated biomarkers have been used to monitor the effects of anti-IL-17 therapy in patients, no biomarkers have been developed to monitor the effects of anti-IL-17 in healthy subjects, who are typically included in the early stages of clinical evaluation. Measurement of the targeted engagement of IL-17 small molecule modulators with IL-17 in subjects (e.g., humans) may prove important for progressing SM modulators through clinical studies.

[0009] Therefore, methods are needed to assess IL-17 engagement of SM modulators in samples and to investigate clinically relevant parameters, such as pharmacokinetic (PK) and pharmacodynamic (PD) information, which in turn can help determine the optimal dose for safe and effective treatment. Summary of the Invention

[0010] In a general aspect, the present application relates to a method of determining the amount of free IL-17 (ie, IL-17 that is not bound to a SM modulator) in a sample comprising IL-17 and a SM modulator.

[0011] Provided herein is a method for determining the amount of free IL-17 in a sample comprising IL-17 and a small molecule (SM) modulator of IL-17, the method comprising:

[0012] i. contacting the sample with a first capture antibody to form a mixture comprising a first complex comprising the first capture antibody and IL-17 that is not bound to the SM modulator, and a second complex comprising the first capture antibody and IL-17 that is bound to the SM modulator;

[0013] ii. contacting the mixture from step i) with a first detection agent, thereby forming a third complex comprising the first capture antibody, IL-17 not bound to the SM modulator, and the first detection agent, but not forming a fourth complex comprising the first capture antibody, IL-17 bound to the SM modulator, and the first detection agent; and

[0014] iii. Determine the amount of free IL-17 by measuring the amount of the first detection agent in the third complex, preferably, the first detection agent is a first detection antibody.

[0015] In one embodiment of the method, no washing step is performed before contacting the mixture from step i) with the first detection agent.

[0016] Also provided herein is a method for determining targeted engagement of a SM modulator of IL-17 in a subject, the method comprising: a) determining the amount of free IL-17 in a sample from the subject using the method of claim 1 before administering the SM modulator; b) determining the amount of free IL-17 in a sample from the subject using the method of claim 1 after administering the SM modulator; and c) comparing the amounts of free IL-17 determined from steps a) and b).

[0017] Also provided herein is a method for determining targeted engagement of a SM modulator of IL-17 in a subject, the method comprising: a) determining the amount of free IL-17 in a sample from the subject using the method of claim 1 at two or more time points after administration of the SM modulator; and b) comparing the amount of free IL-17 at the two or more time points determined from step a).

[0018] Also provided herein is a method of determining the amount of modulator-bound IL-17 in a sample comprising IL-17 and a SM modulator of IL-17, the method comprising:

[0019] i. contacting a first portion of the sample with a first capture antibody to form a first mixture comprising a first complex comprising the first capture antibody and IL-17 that is not bound to the SM modulator and a second complex comprising the first capture antibody and IL-17 that is bound to the SM modulator;

[0020] ii. contacting the first mixture from step i) with a first detection agent, thereby forming a third complex comprising the first capture antibody, IL-17 not bound to the SM modulator, and the first detection agent, but not forming a fourth complex comprising the first capture antibody, IL-17 bound to the SM modulator, and the first detection agent;

[0021] iii. contacting a second portion of the sample with a second capture antibody to form a second mixture comprising a fifth complex comprising the second capture antibody and IL-17 that is not bound to the SM modulator and a sixth complex comprising the second capture antibody and IL-17 that is bound to the SM modulator;

[0022] iv. contacting the second mixture with a second detection agent, thereby forming a seventh complex comprising the second capture antibody, IL-17 not bound to the SM modulator, and the second detection agent, and an eighth complex comprising the second capture antibody, IL-17 bound to the SM modulator, and the second detection agent;

[0023] v. determining the amount of free IL-17 by measuring the amount of the first detection agent in the third complex;

[0024] vi. determining the total amount of IL-17 by measuring the total amount of the second detection agent in the seventh complex and the eighth complex; and

[0025] vii. determining the amount of modulator-bound IL-17 in the sample by subtracting the amount of free IL-17 determined in step (v) from the total amount of IL-17 in step (vi),

[0026] Preferably, the first detection agent is a first detection antibody and the second detection agent is a second detection antibody.

[0027] Also provided herein is a method of determining targeted engagement of a SM modulator of IL-17 in a subject, the method comprising determining the amount of modulator-bound IL-17 in a sample from the subject using the above method after administration of the SM modulator.

[0028] In one embodiment of any of the above methods, the SM modifier has a molecular weight of about 100 g / mol to 1500 g / mol.

[0029] In another embodiment of any of the above methods, the SM modulator has a molecular weight of about 400 g / mol-1050 g / mol, or about 500 g / mol-1000 g / mol, or about 600 g / mol-900 g / mol.

[0030] In yet another embodiment of any of the above methods, the SM modulator has a molecular weight of about 300 g / mol-750 g / mol, or about 500 g / mol-800 g / mol, or about 600 g / mol-900 g / mol.

[0031] In yet another embodiment of any of the above methods, the SM modifier has a molecular weight of about 300 g / mol to 750 g / mol. In yet another embodiment of any of the above methods, the SM modifier has a molecular weight of about 500 g / mol to 800 g / mol. In yet another embodiment of any of the above methods, the SM modifier has a molecular weight of about 600 g / mol to 900 g / mol.

[0032] In yet another embodiment of any of the above methods, the SM modulator is selected from the group consisting of Compound #1-6 having the following structure:

[0033]

[0034]

[0035]

[0036] In yet another embodiment of any of the above methods, the SM modulator is selected from the group consisting of Compound #2-6 having the following structure:

[0037]

[0038]

[0039] In yet another embodiment of any of the above methods, the first detection agent and / or the second detection agent is labeled with a detectable label, more preferably, the label is an enzyme or biotin.

[0040] In yet another embodiment of any of the above methods, the first capture antibody and the first detection agent are selected from the pairs in the following table:

[0041]

[0042]

[0043] In yet another embodiment of any of the above methods, the first capture antibody is the capture antibody of DuoSet ELISA kit DY317 (from R&D Systems, Inc.), and the first detection agent is human mAb4538 antibody (from Janssen Biotech, Inc.).

[0044] In yet another embodiment of any of the above methods, the second capture antibody and the second detection agent are selected from the pairs in the following table:

[0045]

[0046] In yet another embodiment of any of the above methods, the second capture antibody and the second detection agent are the capture antibody and detection antibody of DuoSet ELISA kit DY317 (from R&D Systems, Inc.), respectively.

[0047] In another embodiment of any of the above methods, the sample is a biological sample obtained from a subject treated ex vivo or in vivo with a SM modulating agent. The subject can be a mammal, preferably a human. The biological sample can be selected from the group consisting of a sample prepared from cells, tissue, or serum, plasma, or another biological fluid sample.

[0048] In a further embodiment of any of the above methods, the method has a detection sensitivity of a lower limit of quantification (LLOQ) of about 0.01 pg / ml, or about 0.1 pg / ml, or about 0.25 pg / ml, or about 0.5 pg / ml, or about 0.75 pg / ml, or about 1 pg / ml of IL-17 in the sample.

[0049] Also provided herein is a kit for determining the amount of free IL-17 in a sample comprising IL-17 and a SM modulator of IL-17, the kit comprising:

[0050] i. a first capture antibody, wherein upon contact with the sample, the first capture antibody forms a first complex comprising the first capture antibody and IL-17 that is not bound to the SM modulator and a second complex comprising the first capture antibody and IL-17 that is bound to the SM modulator; and

[0051] ii. a first detection agent, wherein upon contact with the first complex and the second complex, the first detection agent forms a third complex comprising the first capture antibody, IL-17 not bound to the SM modulator, and the first detection agent, but does not form a fourth complex comprising the first capture antibody, IL-17 bound to the SM modulator, and the first detection agent,

[0052] Preferably, the first detection agent is a first detection antibody.

[0053] Also provided herein is a kit for determining the amount of modulator-bound IL-17 in a sample comprising IL-17 and a SM modulator of IL-17, the kit comprising:

[0054] i. a first capture antibody, wherein upon contact with the sample, the first capture antibody forms a first complex comprising the first capture antibody and IL-17 that is not bound to the SM modulator and a second complex comprising the first capture antibody and IL-17 that is bound to the SM modulator;

[0055] ii. a first detection agent, wherein upon contact with the first complex and the second complex, the first detection agent forms a third complex comprising the first capture antibody, IL-17 not bound to the SM modulator, and the first detection agent, but does not form a fourth complex comprising the first capture antibody, IL-17 bound to the SM modulator, and the first detection agent; and

[0056] iii. a second capture antibody, wherein upon contact with the sample, the second capture antibody forms a fifth complex comprising the second capture antibody and IL-17 that is not bound to the SM modulator and a sixth complex comprising the second capture antibody and IL-17 that is bound to the SM modulator;

[0057] iv. a second detection agent, wherein upon contact with the fifth complex and the sixth complex, the second detection agent forms a seventh complex comprising the second capture antibody, IL-17 not bound to the SM modulator, and the second detection agent, and an eighth complex comprising the second capture antibody, IL-17 bound to the SM modulator, and the second detection agent, respectively,

[0058] Preferably, the first detection agent is a first detection antibody and the second detection agent is a second detection antibody.

[0059] In one embodiment of any of the above kits, the first detection agent and / or the second detection agent is labeled with a detectable label, more preferably, the label is an enzyme or biotin.

[0060] In another embodiment of any of the above kits, the first capture antibody and the first detection agent are selected from the pairs in the following table:

[0061]

[0062]

[0063] In yet another embodiment of any of the above kits, the first capture antibody is the capture antibody of DuoSet ELISA kit DY317 (from R&D Systems, Inc.), and the first detection agent is human mAb4538 antibody (from Janssen Biotech, Inc.).

[0064] In yet another embodiment of any of the above kits, the second capture antibody and the second detection agent are selected from the pairs in the following table:

[0065]

[0066]

[0067] In yet another embodiment of any one of the above kits, the second capture antibody and the second detection agent are the capture antibody and detection antibody of DuoSet ELISA kit DY317 (from R&D Systems, Inc.), respectively.

[0068] Other aspects, features, and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments, and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The foregoing summary and the following detailed description of preferred embodiments of the present patent application will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the present patent application is not limited to the precise embodiments shown in the drawings.

[0070] Figure 1A-Figure 1C Schematic diagram showing the IL-17 targeted engagement assay: Figure 1A - Immunoassays for detecting IL-17 using a capture antibody / detector pair; Figure 1B - an immunoassay that detects only free IL-17 (but not modulator-bound IL-17) in the presence of a small molecule (SM) modulator using a capture antibody / detector pair of the invention; and Figure 1C - Immunoassays that detect total IL-17, including both free IL-17 and modulator-bound IL-17 (ie, IL-17 bound to a SM modulator), using currently available capture antibody / detector pairs.

[0071] Figure 2A-2F Shown are the detection of free IL-17 but not modulator-bound IL-17 using the capture antibody of the DuoSet ELISA kit DY317 (R&D Systems, Inc.) paired with mAb4538 (antibody to IL-17 from Janssen Biotech, Inc.) as a detector in samples containing small molecule (SM) modulators Compound #1, Compound #2, Compound #3, Compound #4, Compound #5, and Compound #6, respectively.

[0072] Figure 3A-3F Shown are the detection of total IL-17 using DuoSet ELISA Kit DY317 in samples containing SM modulators Compound #1, Compound #2, Compound #3, Compound #4, Compound #5, and Compound #6, respectively.

[0073] Figures 4A-4F Detection of total IL-17 using the capture antibody of DuoSet ELISA kit DY317 paired with antibody ABIN1724556 (from Covalab Antibodies Online) as a detector is shown in samples containing SM modulators Compound #1, Compound #2, Compound #3, Compound #4, Compound #5, and Compound #6, respectively.

[0074] Figure 5A-5BShown is the detection of free IL-17 using the capture antibody of DuoSet ELISA kit DY317 paired with mAb4538 as a detector in samples containing a range of SM modulator concentrations. DETAILED DESCRIPTION

[0075] Various publications, articles, and patents are cited or described in the Background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, and the like included in this specification is intended to provide a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0076] 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 this invention belongs. Otherwise, certain terms used herein have the meanings set forth in this specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein.

[0077] 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.

[0078] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0079] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "include" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term "comprise" may be replaced with the term "contains" or "includes," or sometimes as used herein, with the term "having."

[0080] As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim elements. As 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.

[0081] As used herein, the connection term "and / or" between multiple listed elements is understood to include both individual 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".

[0082] Unless otherwise indicated, any numerical value, such as a concentration or concentration range described herein, is understood to be modified by the term "about" in all cases. Therefore, numerical values generally include the 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. Unless otherwise clearly indicated by the context, as used herein, the numerical range used explicitly includes all possible subranges, all individual values within the range, including integers within such ranges and fractions within the range.

[0083] The phrases "percent (%) sequence identity" or "% identity" or "% identity to" when used with reference to amino acid sequences describe the number of matches ("hits") of identical amino acids in two or more aligned amino acid sequences compared to the number of amino acid residues making up the total length of these amino acid sequences. In other words, when two or more sequences are compared and aligned for maximum correspondence as measured using a sequence comparison algorithm known in the art, or when aligned manually and visually, the alignment of these sequences can be used to determine the percentage of amino acid residues that are identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical over the full length of the amino acid sequences). Thus, sequences compared for determining sequence identity may differ by amino acid substitutions, additions or deletions. Suitable programs for aligning protein sequences are known to the skilled artisan. The percent sequence identity of protein sequences can be determined, for example, using programs such as CLUSTALW, Clustal Omega, FASTA, or BLAST, eg, using the NCBI BLAST algorithm (Altschul SF et al., (1997), Nucleic Acids Res. 25:3389-3402).

[0084] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human, that is to 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., and more preferably include humans.

[0085] In order to help the reader of the application, the description is divided into each paragraph or chapter, or relates to each embodiment of the application. These divisions should not be considered as the essence of paragraphs or chapters or embodiments being separated from the essence of another paragraph or chapter or embodiment. On the contrary, it will be understood by those skilled in the art that this description has a wide range of applications and contains all combinations of conceivable chapters, paragraphs and sentences. The discussion of any embodiment is only intended to be exemplary, and is not intended to show that the scope of the disclosure (including claims) is limited to these examples.

[0086] As used herein, "IL-17" or "IL-17A" refers to interleukin 17A. It is also known as IL17, CTLA8 or CTLA-8. Interleukin 17A is a proinflammatory cytokine. This cytokine is produced by a type of T helper cell called a T helper 17 cell in response to its stimulation by IL-23. The protein encoded by IL 17A is a founding member of the IL-17 family, which includes 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 activity of NF-κB and mitogen-activated protein kinase. It can stimulate the expression of IL-6 and cyclooxygenase-2 (PTGS2 / COX-2) and enhance the production of nitric oxide (NO). High levels of IL-17 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 set forth in GenBank Accession No. NP_002181.1, which can be encoded by a nucleic acid sequence, such as the nucleic acid sequence in GenBank Accession No. NM_002190.3.

[0087] As used herein, the term "modulator" refers to any agent or molecule that can bind to IL-17, including small molecule compounds and macromolecules, such as antibodies.

[0088] 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 that can bind to IL-17 and has a molecular weight within the range of about 100 g / mol to about 1500 g / mol, or 400 g / mol to about 1050 g / mol, or 500 g / mol to about 1000 g / mol, or about 600 g / mol to about 900 g / mol, or about 300 g / mol to about 750 g / mol, or about 500 g / mol to about 800 g / mol. Alternatively, the SM modulator may have a molecular weight ranging from a lower limit of about 100 g / mol, or 200 g / mol, or about 300 g / mol, or about 400 g / mol, or about 500 g / mol, or about 600 g / mol to an upper limit of about 400 g / mol, or about 500 g / mol, or about 600 g / mol, or about 700 g / mol, or about 800 g / mol, or about 900 g / mol, or about 1000 g / mol, or about 1100 g / mol, or about 1200 g / mol, or about 1300 g / mol, or about 1400 g / mol, or about 1500 g / mol.

[0089] 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 can bind to IL-17 and has a molecular weight greater than about 1500 Daltons, or preferably has a molecular weight in the range of about 2000 Daltons to about 250,000 Daltons. Dalton (symbol Da) is used herein as a unit of molar mass, defined as 1 Da = 1 g / mol.

[0090] For small molecules, absolute stereochemistry is assigned according to the Cahn-Ingold-Prelog RS system. Wherein the absolute configuration is a known chiral center depicted or labeled by the prefixes R and S designated using standard sequential rules methods and, where necessary, by appropriate positional precedence (Pure & Appl. Chem. 45, 1976, 11–30). Certain examples contain chemical structures depicted or labeled as (R*) or (S*). When (R*) or (S*) is used in the name of a compound or in the chemical representation of a compound, it is intended to indicate that the compound is a pure single isomer at that stereocenter; however, the absolute configuration of that stereocenter has not been determined. Thus, a compound designated as (R*) refers to a compound that is a pure single isomer at that stereocenter with an absolute configuration of (R) or (S), and a compound designated as (S*) refers to a compound that is a pure single isomer at that stereocenter with an absolute configuration of (R) or (S). For example, refers to a compound which is:

[0091] As used herein, the term "free IL-17" refers to IL-17 molecules that are not bound to small molecule modulators or macromolecular modulators in a biological sample.

[0092] The terms "sample" and "biological sample" are used interchangeably herein and encompass various sample types obtained or derived from an organism and useful for diagnostic or monitoring assays. The term encompasses blood and other liquid samples, solid tissue samples of biological origin, such as biopsy specimens or tissue cultures, or cells and progeny thereof. The term encompasses samples that have been manipulated in any way after purchase, such as by treatment with reagents, solubilization, or enrichment for 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.

[0093] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulins or antibody molecules, including human antibodies, humanized antibodies, composite 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 specific antigen. The antibody can be derived from any species and can be, for example, IgM, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgD, IgA, or IgE.

[0094] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, such as, for example, a diabody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), a single domain antibody (sdab), an scFv dimer (divalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise the structure of a complete antibody.

[0095] As used herein, the term "monoclonal antibody" refers to a preparation of antibodies of single molecular composition.

[0096] As used herein, the term "polyclonal antibody" refers to a composition of different antibody molecules capable of binding or reacting with several different specific antigenic determinants on the same or different antigens. The variability in the antigenic specificity of polyclonal antibodies is located in the variable regions of the individual antibodies that make up the polyclonal antibody, particularly in the complementarity determining regions CDR1, CDR2, and CDR3.

[0097] As used herein, the term "capture agent" refers to an agent that specifically binds to IL-17, regardless of whether IL-17 is bound to a modulator. "Capture agent" is intended to encompass agents that specifically bind to both IL-17 that is not bound to a modulator and IL-17 that is bound to a modulator, such as antibodies or soluble receptors. Such capture agents (such as capture antibodies) can be commercially available or research reagents. Capture agents (such as capture antibodies) can be provided in solution or pre-applied to a surface.

[0098] 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 to IL-17 that is bound to a modulator, or both. In a preferred embodiment, the term "detection agent" refers to an agent that specifically binds to IL-17 that is not bound to a modulator but does not specifically bind to IL-17 that is bound to a modulator. "Detection agents" (such as detection antibodies) or detection of soluble receptors can be used to detect IL-17 that is not bound to a modulator or IL-17 that is bound to a modulator. Detection agents (such as detection antibodies) can be commercially available or research-based. Detection agents can be detected by any method known in the art, such as using radioactive or fluorescent labels, or by being directly or indirectly bound to an enzyme or biotin. Preferably, detection agents (such as detection antibodies) are labeled with a detectable label (such as an enzyme or biotin).

[0099] An enzyme converts a substrate into a detectable product. For example, an enzyme could be one of the following enzymes commonly used in enzyme-linked immunosorbent assays (ELISAs):

[0100] Horseradish peroxide (HRP), commonly used for protein conjugation, converts o-phenylenediamine dihydrochloride (OPD) into an amber product;

[0101] HRP converts 3,3',5,5'-tetramethylbenzidine (TMB) into a blue product, which turns yellow in the presence of sulfuric acid or phosphoric acid;

[0102] HRP converts 2,2'-azobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) into a green product; and

[0103] • Alkaline phosphatase converts p-nitrophenyl phosphate (PNPP) into a yellow product.

[0104] 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 above-mentioned detectable enzymes to generate a signal.

[0105] The term "specific binding" refers to a 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 the other agent (e.g., an antibody) even in the presence of many other agents, i.e., showing preferential binding of one agent over the other in a heterogeneous mixture of agents. The term "KD" refers to the dissociation constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). As used herein, an antibody that "specifically binds to" an antigen refers to an antibody that binds to the antigen at a concentration of 1×10 -7 M or less, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller, or 1×10 -10 An antibody or antigen-binding fragment thereof that binds to an antigen with a KD of M or less. In view of the present disclosure, the KD value of a binding agent can be determined using methods in the art. For example, the KD of an antibody can be determined using surface plasmon resonance, such as by using a biosensor system (e.g., The smaller the KD value of the antibody, the higher the affinity of the antibody for binding to the target antigen.

[0106] As used herein, the term "test kit" refers to a combination of reagents and other materials. It is expected that the test kit may include reagents, such as buffers, protein stabilizers, signal generating systems (e.g., fluorescent signal generating systems), antibodies, control proteins, and test containers (e.g., microtiter plates, etc.). The term "test kit" is not intended to be limited to a specific combination of reagents and / or other materials. In one embodiment, the test kit also includes instructions for use of the reagents. The test kit can be packaged in any suitable manner, typically packaged as needed with the elements in a single container or various containers together with a description for testing. In some embodiments, the test kit also preferably includes a positive control sample. Test kits can be produced in various ways known in the art.

[0107] IL-17 targeted engagement assay

[0108] The present application generally relates to IL-17 target engagement assays. Using the present application to measure the amount of free IL-17 (i.e., IL-17 not bound to a SM modulator) in a sample containing IL-17 and a SM modulator, together with a total IL-17 detection assay, the IL-17 target engagement level of a SM modulator can be determined.

[0109] In studies involving SM modulators, currently available assays can only measure the total amount of IL-17 in a biological sample because the detection antibodies in the assays cannot distinguish between free IL-17 (i.e., IL-17 not bound to the SM modulator) and IL-17 bound to the SM modulator. Therefore, it is not possible to monitor the binding between IL-17 and SM modulators in such biological samples. However, the present application provides a novel method for determining the amount of free IL-17 in a sample in the presence of a small modulator of IL-17. By subtracting the amount of free IL-17 from the total amount of IL-17, the level of IL-17 binding of the SM modulator in the biological sample can be determined. Therefore, this innovative method can be used to study clinically relevant parameters such as pharmacokinetic (PK) and pharmacodynamic (PD) information, which in turn can help determine the optimal dose to maximize efficacy and safety. In addition, the methods described herein can be applied to healthy subjects or patients because the innovative method can measure free IL-17 in the presence of SM modulators without relying on disease readouts.

[0110] Therefore, the present application provides a method for determining the amount of free IL-17 in a sample containing IL-17 and a SM modulator of IL-17, the method comprising (see Figure 1B ):

[0111] i. contacting the sample with a first capture antibody to form a mixture comprising a first complex comprising the first capture antibody and IL-17 that is not bound to the SM modulator, and a second complex comprising the first capture antibody and IL-17 that is bound to the SM modulator;

[0112] ii. contacting the mixture from step i) with a first detection agent (preferably a first detection antibody) to form a third complex comprising the first capture antibody, IL-17 not bound to the SM modulator, and the first detection agent, but not forming a fourth complex comprising the first capture antibody, IL-17 bound to the SM modulator, and the first detection agent; and

[0113] iii. Determine the amount of free IL-17 by measuring the amount of the first detection agent in the third complex.

[0114] After contacting the sample with the first capture antibody, the first capture antibody specifically binds to free IL-17 and IL-17 bound to the SM modulator in the sample to form a first complex and a second complex, respectively, in the mixture. In certain embodiments, the first 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, as well as the remainder of the sample.

[0115] As used herein, the term "first detection agent" refers to an agent, such as an antibody or receptor, that specifically binds to free IL-17 captured by the first capture antibody but does not specifically bind to IL-17 bound to the modulator captured by the first capture antibody. In particular, when the first detection agent is contacted with the first complex and the second complex, it forms a third complex comprising the first capture antibody, free IL-17, and the first detection agent, but does not form a fourth complex comprising the first capture antibody, modulator-bound IL-17, and the first detection agent.

[0116] Thus, a first capture antibody and a first detection agent are used in pair to measure the amount of free IL-17 in a sample comprising IL-17 and a SM modulator of IL-17.

[0117] Exemplary first detection agents and pairs of primary antibodies can be selected from the pairs listed in Table 1 below.

[0118] Table 1. "First Detector" / "First Capture Antibody" Pairs for Measuring Free IL-17

[0119]

[0120]

[0121] Among the first detection agents listed in Table 1, IL-17 antibodies mAb7024, mAb3584, mAb3077, mAb732, mAb4538, and mAb5548 from Janssen Biotech, Inc. are disclosed in U.S. Patent 8,519,107, which is hereby incorporated by reference in its entirety. Their detailed sequence information is listed in Table 2.

[0122] Table 2. Antibody sequences

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] In certain embodiments, the first detection agent (such as a first detection antibody) is labeled with a detectable label, preferably an enzyme or biotin. In other embodiments, the first 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 for determining the amount of the first detection agent.

[0130] As used herein, the term "wash," "washing step," or "washing" refers to a process for separating the total IL-17 (including both free IL-17 and modulator-bound IL-17) that is not bound to the capture antibody from the mixture in step (i). The solution used for washing is typically a buffer ("washing buffer"). In some embodiments, the wash buffer contains a low concentration of detergent. In some embodiments, the wash buffer is 10 mM phosphate buffer at a pH of about 7.4, comprising 150 mM NaCl and 0.05% Tween 20. The pH of the wash buffer is preferably in the range of about 6 to about 9. In some embodiments, the pH 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 some embodiments, the washing step is performed three to six times.

[0131] In certain embodiments, a washing step is performed after step (i) and before step (ii) of contacting the mixture with a second detection agent.

[0132] In some embodiments, no washing step is performed. For example, in some embodiments, no washing step is performed after step (i) and before step (ii) of contacting the mixture with the second detection agent.

[0133] The present application also provides a method for determining targeted engagement of a SM modulator of IL-17 in a subject. The method comprises: a) determining the amount of free IL-17 in a sample from the subject before administering the SM modulator as described above; b) determining the amount of free IL-17 in a sample from the subject after administering the SM modulator as described above; and c) comparing the amounts of free IL-17 determined from steps a) and b). Alternatively, the method may comprise: (aa) determining the amount of free IL-17 in a sample from the subject at two or more time points after administering the SM modulator as described above; and (bb) assessing changes in the amount of free IL-17 at various time points after administering the SM modulator. Thus, the targeted engagement activity of a SM modulator of IL-17 can be assessed and used to aid in the development of therapeutic regimens that modulate the SM modulator of IL-17.

[0134] The present application also provides a method for determining the amount of total IL-17 (free IL-17 and modulator-bound IL-17) in a sample containing IL-17 and a SM modulator of IL-17. The method comprises (see, e.g., Figure 1C ):

[0135] i. contacting a portion of the sample with a second capture antibody to form a second mixture comprising a fifth complex comprising the second capture antibody and IL-17 that is not bound to the SM modulator and a sixth complex comprising the second capture antibody and IL-17 that is bound to the SM modulator;

[0136] ii. contacting the second mixture with a second detection agent (preferably a second detection antibody), thereby forming a seventh complex comprising the second capture antibody, IL-17 not bound to the SM modulator, and the second detection agent, and an eighth complex comprising the second capture antibody, IL-17 bound to the SM modulator, and the second detection agent;

[0137] iii. Determine the total amount of IL-17 by measuring the total amount of the second detection agent in the seventh complex and the eighth complex.

[0138] As used herein, a "second capture antibody" and a "second detection agent" (preferably a "second detection antibody") are used in pairs to measure the total amount of IL-17 (i.e., free IL-17 and modulator-bound IL-17) in a sample. In particular, the second detection agent binds to both free IL-17 captured by the second capture antibody and modulator-bound IL-17 captured by the second capture antibody. Thus, when the second detection agent is contacted with the second complex, it forms a seventh complex comprising the second capture antibody, IL-17 not bound to the SM modulator, and the second detection agent, and an eighth complex comprising the second capture antibody, IL-17 bound to the SM modulator, and the second detection agent.

[0139] Therefore, a second capture antibody and a second detection agent are used in pairs to measure the amount of total IL-17 in a sample.

[0140] Exemplary secondary detection agents and secondary antibody pairs can be selected from the pairs listed in Table 3 below.

[0141] Table 3. "Secondary Detection Agent" / "Secondary Capture Antibody" Pairs for Measuring Total IL-17

[0142]

[0143]

[0144] In certain embodiments, the second detection agent is labeled with a detectable label, preferably an enzyme or biotin. In other embodiments, the second 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 for determining the amount of the second detection agent (such as a second detection antibody).

[0145] In certain embodiments, a washing step is performed after step (i) and before step (ii) of contacting the second mixture with the second detection agent.

[0146] In some embodiments, no washing step is performed. For example, in some embodiments, no washing step is performed after step (i) and before step (ii) of contacting the second mixture with the second detection agent.

[0147] The present application also provides a method for determining the amount of modulator-bound IL-17 in a sample containing IL-17 and a SM modulator of IL-17. The method comprises: a) determining the amount of free IL-17 in the sample using a first capture antibody and a first detection agent as described above; b) determining the amount of total IL-17 in the sample using a second capture antibody and a second detection agent as described above; and c) subtracting the amount of free IL-17 determined in step (a) from the amount of total IL-17 in step (b) to obtain the amount of modulator-bound IL-17 in the sample.

[0148] By measuring the amount of modulator-bound IL-17 in a sample, the specificity and target engagement of a SM modulator can be assessed.

[0149] As previously stated, the terms "small molecule modulator" and / or "SM modulator" and / or "SM modulator of IL-17" refer to any small molecule compound that can bind to IL-17 and has a molecular weight within the range of about 100 g / mol to about 1500 g / mol, or 400 g / mol to about 1050 g / mol, or 500 g / mol to about 1000 g / mol, or about 600 g / mol to about 900 g / mol, or about 300 g / mol to about 750 g / mol, or about 500 g / mol to about 800 g / mol. Alternatively, the SM modulator may have a molecular weight ranging from a lower limit of about 100 g / mol, or 200 g / mol, or about 300 g / mol, or about 400 g / mol, or about 500 g / mol, or about 600 g / mol to an upper limit of about 400 g / mol, or about 500 g / mol, or about 600 g / mol, or about 700 g / mol, or about 800 g / mol, or about 900 g / mol, or about 1000 g / mol, or about 1100 g / mol, or about 1200 g / mol, or about 1300 g / mol, or about 1400 g / mol, or about 1500 g / mol.

[0150] Exemplary SM modulators of IL-17 used herein include, but are not limited to, compounds #1-6 listed in Table 4 below and pharmaceutically acceptable salts thereof:

[0151] Table 4. Exemplary small molecule modulators of IL-17

[0152]

[0153]

[0154]

[0155] The following compounds:

[0156] Disclosed in Liu, S. et al., “Binding site elucidation and structure-guided design of macrocyclic IL-17A antagonists,” Scientific Reports, 6 30859, DOI: 10.1038 / srep30859 (2016). This compound was prepared as a mixture of diastereomers and separated by supercritical fluid chromatography (SFC) (column: DAICEL CHIRALPAK AD (250 mm*30 mm, 10 μm); mobile phase: [0.1% NH3H2O IPA]; B%: 40%-40%, 5.3 minutes; 420 minutes). Using the methods disclosed herein, the more potent diastereomer, designated herein as Compound #1, was evaluated:

[0157] Compounds #2-5 are disclosed in PCT Patent Application Publication No. WO 2013 / 116682, entitled: “Macrocyclic Compounds for Modulating IL-17,” which is incorporated herein by reference (see, e.g., FIG. 12 , compounds 479, 529, 487, and 159, respectively).

[0158] Compound #6 is disclosed in PCT Patent Application Publication No. WO 2014 / 066726, entitled: “Compounds for Modulating IL-17,” which is incorporated herein by reference (see, eg, Figure 6, Compound 286).

[0159] In certain embodiments, the methods disclosed herein achieve a detection sensitivity of a lower limit of quantification (LLOQ) of about 0.01 pg / ml, or about 0.1 pg / ml, or about 0.25 pg / ml, or about 0.5 pg / ml, or about 0.75 pg / ml, or 1 pg / ml of IL-17 in a sample.

[0160] In certain embodiments, the IL-17 is human IL-17.

[0161] In certain embodiments, the sample is a biological sample obtained from a human subject treated ex vivo or in vivo with a SM modulator of human IL-17.

[0162] In certain embodiments, the sample can be a biological sample obtained from an animal (eg, a mouse) into which human IL-17 has been injected.

[0163] In certain embodiments, the biological sample is selected from the group consisting of a sample prepared from cells, tissue, or serum, plasma, or another biological fluid sample.

[0164] According to an embodiment of the present application, the method further comprises adjusting the subject's treatment regimen based on the amount of free IL-17 in a biological sample from the subject determined before and after administration of the SM modulator to the subject. For example, if the binding of the currently used SM modulator to IL-17 is found to be below a threshold level, the adjustment may be to increase or decrease the dose or frequency of treatment with the SM modulator, or to switch to a different modulator. The treatment regimen may also be adjusted based on the amount of free IL-17 in a biological sample from the subject determined at two or more time points after administration of the SM modulator to the subject.

[0165] In some embodiments, the subject suffers from an IL-17-mediated inflammatory disease. The IL-17-mediated inflammatory disease can be selected from psoriasis, psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, hidradenitis suppurativa, bullous pemphigoid, atopic dermatitis, vitiligo, multiple sclerosis, asthma, uveitis, chronic obstructive pulmonary disease, multiple myeloma, and systemic lupus erythematosus. In some embodiments, the IL-17-mediated inflammatory disease is selected from psoriasis, psoriatic arthritis, and ankylosing spondylitis. In some embodiments, the IL-17-mediated inflammatory disease is psoriasis. In some embodiments, the IL-17-mediated inflammatory disease is psoriatic arthritis. In some embodiments, the IL-17-mediated inflammatory disease is ankylosing spondylitis.

[0166] In some embodiments, the IL-17 target engagement assays of the invention disclosed herein can be used in preclinical models, to simulate doses for human trials, in human Phase 1 trials, to determine Phase 2 doses in patients, and to determine whether alternative molecules or trial redesigns require improved target engagement. In Phase 2 trials in patients, the correlation of target engagement assays with efficacy can be used to determine whether improved target engagement is needed to achieve better efficacy.

[0167] The assays of the present invention can be used to measure free IL-17 in the presence of small molecule IL-17 modulators, independent of any disease readout. The assays can be applied to healthy subjects or patients with IL-17-mediated inflammatory diseases. Targeted engagement readouts and test molecule PK can be used for modeling to determine the optimal dose to achieve efficacy and minimize signal in safety studies.

[0168] The present application also provides a kit for determining the amount of free IL-17 in a sample containing IL-17 and a SM modulator, the kit comprising:

[0169] i. a first capture antibody, wherein upon contact with the sample, the first capture antibody forms a first complex comprising the first capture antibody and IL-17 not bound to the SM modulator and a second complex comprising the first capture antibody and IL-17 bound to the SM modulator, respectively; and

[0170] ii. a first detection agent (such as a first detection antibody), wherein upon contact with the first complex and the second complex, the first detection agent forms a third complex comprising the first capture antibody, IL-17 not bound to the SM modulator, and the first detection agent, but does not form a fourth complex comprising the first capture antibody, IL-17 bound to the SM modulator, and the first detection agent.

[0171] In some embodiments, the kit further comprises one or more reagents for detecting the first detection agent.

[0172] The present application also provides a kit for determining the amount of IL-17 bound by a modulator in a sample containing IL-17 and a SM modulator, the kit comprising:

[0173] i. a first capture antibody, wherein upon contact with the sample, the first capture antibody forms a first complex comprising the first capture antibody and IL-17 that is not bound to the SM modulator and a second complex comprising the first capture antibody and IL-17 that is bound to the SM modulator;

[0174] ii. a first detection agent (such as a first detection antibody), wherein upon contact with the first complex and the second complex, the first detection agent forms a third complex comprising the first capture antibody, IL-17 not bound to the SM modulator, and the first detection agent, but does not form a fourth complex comprising the first capture antibody, IL-17 bound to the SM modulator, and the first detection agent;

[0175] as well as

[0176] iii. a second capture antibody, wherein upon contact with the sample, the second capture antibody forms a fifth complex comprising the second capture antibody and IL-17 that is not bound to the SM modulator and a sixth complex comprising the second capture antibody and IL-17 that is bound to the SM modulator;

[0177] iv. a second detection agent (such as a second detection antibody), wherein upon contact with the fifth complex and the sixth complex, the second detection agent forms a seventh complex comprising the second capture antibody, IL-17 not bound to the SM modulator, and the second detection agent, and an eighth complex comprising the second capture antibody, IL-17 bound to the SM modulator, and the second detection agent, respectively.

[0178] In some embodiments, the kit further comprises one or more reagents for detecting the first detection agent and / or the second detection agent.

[0179] Implementation Plan

[0180] The present invention also provides the following non-limiting embodiments.

[0181] Embodiment 1 is a method for determining the amount of free IL-17 in a sample comprising IL-17 and a small molecule (SM) modulator of IL-17, the method comprising:

[0182] i. contacting the sample with a first capture antibody to form a mixture comprising a first complex comprising the first capture antibody and IL-17 that is not bound to a SM modulator, and a second complex comprising the first capture antibody and IL-17 that is bound to a SM modulator;

[0183] ii. contacting the mixture from step i) with a first detection agent, thereby forming a third complex comprising the first capture antibody, the IL-17 not bound to the SM modulator, and the first detection agent, but not forming a fourth complex comprising the first capture antibody, the IL-17 bound to the SM modulator, and the first detection agent; and

[0184] iii. determining the amount of free IL-17 by measuring the amount of the first detection agent in the third complex.

[0185] Embodiment 2 is a method according to embodiment 1, wherein the first detection agent is a first antibody.

[0186] Embodiment 3 is a method according to embodiment 1 or 2, wherein the first capture antibody and the first detection agent are selected from the pairs in the following table:

[0187]

[0188]

[0189]

[0190] Embodiment 4 is a method according to embodiment 3, wherein the first capture antibody is the capture antibody of DuoSet ELISA kit DY317 (R&D Systems, Inc.), and the first detection agent is human mAb4538 antibody (from Janssen Biotech, Inc.).

[0191] Embodiment 5 is the method according to any one of embodiments 1 to 4, wherein the SM modifier has a molecular weight of about 100 g / mol to 1500 g / mol.

[0192] Embodiment 5a is the method according to embodiment 5, wherein the SM modifier has a molecular weight of about 400 g / mol to 1050 g / mol.

[0193] Embodiment 5b is the method according to embodiment 5, wherein the SM modifier has a molecular weight of about 500 g / mol to 1000 g / mol.

[0194] Embodiment 5c is the method according to embodiment 5, wherein the SM modifier has a molecular weight of about 500 g / mol to 800 g / mol.

[0195] Embodiment 5d is a method according to embodiment 5, wherein the SM modifier has a molecular weight of about 600 g / mol to 900 g / mol.

[0196] Embodiment 5e is a method according to embodiment 5, wherein the SM modifier has a molecular weight of about 300 g / mol to 750 g / mol.

[0197] Embodiment 5f is a method according to embodiment 5, wherein the SM modulator is selected from the group consisting of compound #1-6 having the following structure and pharmaceutically acceptable salts thereof:

[0198]

[0199]

[0200]

[0201] Embodiment 5g is a method according to embodiment 5, wherein the SM modulator is selected from the group consisting of compound #2-6 having the following structure:

[0202]

[0203]

[0204] Embodiment 6 is a method according to any one of embodiments 1 to 5, wherein no washing step is performed before contacting the first mixture from step i) with the first detection agent in step (ii).

[0205] Embodiment 7 is a method for determining targeted engagement of a SM modulator of IL-17 in a subject, the method comprising: a) determining the amount of free IL-17 in a sample from the subject using a method according to any one of embodiment claims 1 to 6 before administering the SM modulator; b) determining the amount of free IL-17 in a sample from the subject using a method according to any one of embodiment 1 to 6 after administering the SM modulator; and c) comparing the amounts of free IL-17 determined from steps a) and b).

[0206] Embodiment 8 is a method of determining targeted engagement of a SM modulator of IL-17 in a subject, the method comprising: a) determining the amount of free IL-17 in a sample from the subject at two or more time points after administration of the SM modulator using the method according to any one of embodiments 1 to 6; and b) comparing the amount of free IL-17 determined from step a).

[0207] Embodiment 9 is a method according to any one of embodiments 1 to 8, wherein the sample is a biological sample obtained from a human subject treated ex vivo or in vivo with the SM modulator.

[0208] Embodiment 10 is a method according to any one of embodiments 1 to 9, wherein the biological sample is selected from the group consisting of samples prepared or extracted from cells, or tissues, or serum, plasma or another biological fluid sample.

[0209] Embodiment 11 is a method according to any one of embodiments 1 to 10, wherein the method has a detection sensitivity of a lower limit of quantification (LLOQ) of about 0.01 pg / ml, or about 0.1 pg / ml, or about 0.25 pg / ml, or about 0.5 pg / ml, or about 0.75 pg / ml, or 1 pg / ml of said IL-17 in said sample.

[0210] Embodiment 12 is a method of determining the amount of modulator-bound IL-17 in a sample comprising IL-17 and a SM modulator of IL-17, the method comprising:

[0211] i. contacting a first portion of the sample with a first capture antibody to form a first mixture comprising a first complex comprising the first capture antibody and IL-17 that is not bound to a SM modulator, and a second complex comprising the first capture antibody and IL-17 that is bound to a SM modulator;

[0212] ii. contacting the first mixture from step i) with a first detection agent, thereby forming a third complex comprising the first capture antibody, the IL-17 not bound to the SM modulator, and the first detection agent, but not forming a fourth complex comprising the first capture antibody, the IL-17 bound to the SM modulator, and the first detection agent;

[0213] iii. contacting a second portion of the sample with a second capture antibody to form a second mixture comprising a fifth complex comprising the second capture antibody and the IL-17 that is not bound to the SM modulator, and a sixth complex comprising the second capture antibody and the IL-17 that is bound to the SM modulator;

[0214] iv. contacting the second mixture with a second detection agent, thereby forming a seventh complex comprising the second capture antibody, the IL-17 not bound to the SM modulator, and the second detection agent, and an eighth complex comprising the second capture antibody, the IL-17 bound to the SM modulator, and the second detection agent;

[0215] v. determining the amount of free IL-17 by measuring the amount of the first detection agent in the third complex;

[0216] vi. determining the total amount of the IL-17 by measuring the total amount of the second detection agent in the seventh complex and the eighth complex; and

[0217] vii. Determine the amount of modulator-bound IL-17 in the sample by subtracting the amount of free IL-17 determined in step (v) from the total amount of IL-17 in step (vi).

[0218] Embodiment 13 is a method according to embodiment 12, wherein the first detection agent is a first detection antibody.

[0219] Embodiment 14 is a method according to embodiment 12 or 13, wherein the second detection agent is a second detection antibody.

[0220] Embodiment 15 is a method according to any one of embodiments 12 to 14, wherein the first capture antibody and the first detection agent are selected from the pairs in the following table:

[0221]

[0222]

[0223] Embodiment 16 is a method according to embodiment 15, wherein the first capture antibody is the capture antibody of DuoSet ELISA kit DY317 (R&D Systems, Inc.), and the first detection agent is human mAb4538 antibody (from Janssen Biotech, Inc.).

[0224] Embodiment 17 is a method according to any one of embodiments 12 to 14, wherein the second capture antibody and the second detection agent are selected from the pairs in the following table:

[0225]

[0226]

[0227] Embodiment 18 is a method according to embodiment 17, wherein the second capture antibody and the second detection agent are the capture antibody and detection agent of DuoSet ELISA kit DY317 (R&D Systems, Inc.), respectively.

[0228] Embodiment 19 is a method according to any one of embodiments 12 to 18, wherein the SM modulator is selected from the group consisting of compound #1-6 having the following structure:

[0229]

[0230]

[0231]

[0232] Embodiment 19a is a method according to any one of embodiments 12 to 18, wherein the SM modulator is selected from the group consisting of compound #2-6 having the following structure:

[0233]

[0234]

[0235] Embodiment 20 is a method according to any one of embodiments 12 to 19, wherein the sample is a biological sample obtained from a human subject treated ex vivo or in vivo with the SM modulator.

[0236] Embodiment 21 is a method according to any one of embodiments 12 to 20, wherein the biological sample is selected from the group consisting of a sample prepared or extracted from a cell, or a tissue, or a serum, plasma or another biological fluid sample.

[0237] Embodiment 22 is a method according to any one of embodiments 12 to 21, wherein the method has a detection sensitivity of a lower limit of quantification (LLOQ) of about 0.01 pg / ml, or about 0.1 pg / ml, or about 0.25 pg / ml, or about 0.5 pg / ml, or about 0.75 pg / ml, or 1 pg / ml of said IL-17 in said sample.

[0238] Embodiment 23 is a kit for determining the amount of free IL-17 in a sample containing IL-17 and a SM modulator of IL-17, the kit comprising:

[0239] iii. a first capture antibody, wherein upon contact with the sample, the first capture antibody forms a first complex comprising the first capture antibody and IL-17 that is not bound to the SM modulator and a second complex comprising the first capture antibody and IL-17 that is bound to the SM modulator; and

[0240] iv. a first detection agent, wherein upon contact with the first complex and the second complex, the first detection agent forms a third complex comprising the first capture antibody, the IL-17 not bound to the SM modulator, and the first detection agent, but does not form a fourth complex comprising the first capture antibody, the IL-17 bound to the SM modulator, and the first detection agent,

[0241] Preferably, the first detection agent is a first detection antibody.

[0242] Embodiment 24 is a kit according to embodiment 23, wherein the first detection agent is a first detection antibody.

[0243] Embodiment 25 is a kit according to embodiment 23 or 24, wherein the first capture antibody and the first detection agent are selected from the pairs in the following table:

[0244]

[0245]

[0246] Embodiment 26 is a kit according to embodiment 25, wherein the first capture antibody is the capture antibody of DuoSet ELISA kit DY317 (R&D Systems, Inc.), and the first detection agent is human mAb4538 antibody (from Janssen Biotech, Inc.).

[0247] Embodiment 27 is a kit for determining the amount of modulator-bound IL-17 in a sample comprising IL-17 and a SM modulator of IL-17, the kit comprising:

[0248] i. a first capture antibody, wherein upon contact with the sample, the first capture antibody forms a first complex comprising the first capture antibody and IL-17 that is not bound to the SM modulator and a second complex comprising the first capture antibody and IL-17 that is bound to the SM modulator;

[0249] ii. a first detection agent, wherein upon contact with the first complex and the second complex, the first detection agent forms a third complex comprising the first capture antibody, the IL-17 not bound to the SM modulator, and the first detection agent, but does not form a fourth complex comprising the first capture antibody, the IL-17 bound to the SM modulator, and the first detection agent; and

[0250] iii. a second capture antibody, wherein upon contact with the sample, the second capture antibody forms a fifth complex comprising the second capture antibody and the IL-17 not bound to the SM modulator and a sixth complex comprising the second capture antibody and the IL-17 bound to the SM modulator;

[0251] iv. a second detection agent, wherein upon contact with the fifth complex and the sixth complex, the second detection agent forms a seventh complex comprising the second capture antibody, the IL-17 not bound to the SM modulator, and the second detection agent, and an eighth complex comprising the second capture antibody, the IL-17 bound to the SM modulator, and the second detection agent, respectively. Preferably, the first detection agent is a first detection antibody, and the second detection agent is a second detection antibody.

[0252] Embodiment 28 is a kit according to embodiment 27, wherein the first detection agent is a first detection antibody.

[0253] Embodiment 29 is a kit according to embodiment 27 or 28, wherein the second detection agent is a second detection antibody.

[0254] Embodiment 29 is a kit according to any one of embodiments 27 to 29, wherein the first capture antibody and the first detection agent are selected from the pairs in the following table:

[0255]

[0256]

[0257] Embodiment 31 is a kit according to embodiment 30, wherein the first capture antibody is the capture antibody of DuoSet ELISA kit DY317 (R&D Systems, Inc.), and the first detection agent is human mAb4538 antibody (from Janssen Biotech, Inc.).

[0258] Embodiment 32 is a kit according to any one of embodiments 27 to 31, wherein the second capture antibody and the second detection agent are selected from the pairs in the following table:

[0259]

[0260] Embodiment 33 is a kit according to embodiment 32, wherein the second capture antibody and the second detection agent are the capture antibody and detection agent of DuoSet ELISA kit DY317 (R&D Systems, Inc.), respectively.

[0261] Example

[0262] It will be appreciated by those skilled in the art that modifications may be made to the above-described embodiments without departing from the broad inventive concept of the present invention. It will be understood, therefore, that the present invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present application, as defined herein.

[0263] Example 1

[0264] Material

[0265] Three (3) capture antibody / detector pairs (listed in Table 5 below) were tested using Compounds #1-6 (described above in Table 5) as SM modulators.

[0266] Table 5

[0267]

[0268] method

[0269] Prepare two-fold and eight-point serial dilutions of recombinant IL-17 in diluent, starting at 1 ng / ml, in duplicate. To test for differential activity on SM modulators, prepare IL-17 calibrators in the presence of 10 μM SM modulator or vehicle control containing 0.1% DMSO. Incubate IL-17 samples at 37°C for 1 hour.

[0270] High binding 96-well plates were coated overnight at room temperature with 100 μL / well of 4 μg / ml capture antibody in PBS. The coated plates were washed 6×400 μL / well on a plate gasket (Zoom HT Microplate Washer) and blocked with 300 μL / well blocking agent (1% BSA in PBS) for 2.5 hours at room temperature, then washed 6×400 μL / well on a plate gasket.

[0271] The sample is prepared by incubating the titrated IL-17 with a final concentration of 10 μM SM regulator. IL-17 levels are measured in an ELISA assay by adding 95 μL / well samples to a plate coated with the capture antibody. The plate is kept at room temperature for 30 minutes, and then 5 μL / well detection agents are added to a final 500 ng / mL, kept at room temperature for another 30 minutes, and then washed with 6 × 400 μL / well on a plate gasket. Streptavidin-conjugated horseradish peroxidase is added with 100 μL / well and incubated in the dark for 20 minutes, then streptavidin-conjugated horseradish peroxidase is added with 6x400 μL / well on a plate gasket, and then 100 μL / well substrate solution is added, incubated in the dark for 20 minutes at room temperature to measure color development. The reaction is then terminated by adding 50 μL / well stop solution, and the plate is measured at 450nm-540nm in a plate reader.

[0272] Optical density (OD) readings from a plate reader (Molecular Devices Spectra Max 340 fpc) were analyzed using Softmax Pro 6.3 and GraphPad Prism and plotted as a dose-response curve of OD values versus IL-17 concentration.

[0273] result

[0274] like Figure 2A-2F As shown, using the capture antibody of DuoSet ELISA kit DY317 paired with mAb4538 (500 ng / ml, as disclosed in U.S. Patent 8,519,107) as a detection agent, only free IL-17 (not SM modulator-bound) but not SM modulator-bound IL-17 can be detected with a signal corresponding to the concentration of IL-17.

[0275] However, total IL-17 (free and modulator-bound forms) was detected in a dose-dependent manner using the DuoSet ELISA kit DY317 or using the capture antibody of the DuoSet ELISA kit DY317 paired with the antibody ABIN1724556 (CovalAb R&D (Antibodies Online)) ( Figure 3A-3F and Figures 4A-4F ).

[0276] Example 2

[0277] Various capture antibody / detector pairs (having a capture antibody selected from Table 6 and a detector selected from Table 7) were tested using Compound #1 as a SM modulator using an ELISA assay similar to that described in Example 1. Table 8 below lists the capture antibody / detector pairs identified by this experiment that can distinguish between free human IL-17 and human IL-17 bound to a SM modulator. Table 9 lists the capture antibody / detector pairs that cannot distinguish between free human IL-17 and human IL-17 bound to a SM modulator.

[0278] Table 6. Human IL-17 capture antibodies

[0279]

[0280] Table 7. Screening of antibodies useful as human IL-17 detection agents

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289] Example 3

[0290] In this example, the capture antibody of DuoSet ELISA kit DY317 (from R&D Systems, Inc.) was paired with mAb4538 (from Janssen Biotech, Inc.) as a detector, and samples containing 1000 pg / ml IL-17 and titrated SM modulators were assayed. Figure 5A-5B As shown, with increasing compound concentrations, decreasing levels of free IL-17 were detected, demonstrating dose-dependent target engagement of the IL-17SM modulator.

Claims

1. A method for determining the amount of free IL-17 in a sample comprising IL-17 and a small molecule modulator of IL-17, the method comprising: i. contacting the sample with a first capture antibody to form a mixture comprising a first complex comprising the first capture antibody and IL-17 that is not bound to a SM modulator, and a second complex comprising the first capture antibody and IL-17 that is bound to a SM modulator; ii. contacting the mixture from step i) with a first detection agent, thereby forming a third complex comprising the first capture antibody, the IL-17 not bound to the SM modulator, and the first detection agent, but not forming a fourth complex comprising the first capture antibody, the IL-17 bound to the SM modulator, and the first detection agent; and iii. determining the amount of free IL-17 by measuring the amount of the first detection agent in the third complex, wherein the SM modulator is selected from the group consisting of compounds #1-6 having the following structures and pharmaceutically acceptable salts thereof: The first capture antibody is the capture antibody of DuoSet ELISA kit DY317, and the first detection agent is mAb4538, and the first detection agent cannot form a complex comprising the first capture antibody, IL-17 bound to any one of compounds #1-6, and the first detection agent.

2. The method of claim 1, wherein no washing step is performed before contacting the mixture from step i) with the first detection agent.

3. A method of determining targeted engagement of a SM modulator of IL-17 in a subject, the method comprising: a) determining the amount of free IL-17 in a sample from the subject using the method of claim 1 before administering the SM modulator; b) determining the amount of free IL-17 in a sample from the subject using the method of claim 1 after administering the SM modulator; and c) comparing the amounts of free IL-17 determined from steps a) and b).

4. A method of determining targeted engagement of a SM modulator of IL-17 in a subject, the method comprising: a) determining the amount of free IL-17 in a sample from the subject using the method of claim 1 at two or more time points after administration of the SM modulator; and b) comparing the amount of free IL-17 at the two or more time points determined from step a).

5. A method for determining the amount of modulator-bound IL-17 in a sample comprising IL-17 and a SM modulator of IL-17, the method comprising: A. determining the amount of free IL-17 using the method of claim 1; B. contacting the second portion of the sample with a second capture antibody to form a second mixture comprising a fifth complex comprising the second capture antibody and the IL-17 that is not bound to the SM modulator, and a sixth complex comprising the second capture antibody and the IL-17 that is bound to the SM modulator; C. contacting the second mixture with a second detection agent, thereby forming a seventh complex comprising the second capture antibody, the IL-17 not bound to the SM modulator, and the second detection agent, and an eighth complex comprising the second capture antibody, the IL-17 bound to the SM modulator, and the second detection agent; D. determining the total amount of the IL-17 by measuring the total amount of the second detection agent in the seventh complex and the eighth complex; and E. Determine the amount of modulator-bound IL-17 in the sample by subtracting the amount of free IL-17 determined in step (A) from the total amount of IL-17 in step (D).

6. A method of determining targeted engagement of a SM modulator of IL-17 in a subject, the method comprising determining the amount of IL-17 bound by the modulator in a sample from the subject using the method of claim 5 after administration of the SM modulator.

7. The method according to any one of claims 1 to 6, wherein the SM modulator is selected from the group consisting of compound #2-6 having the following structure:

8. The method of claim 5 or 6, wherein the first detection agent and / or the second detection agent is labeled with a detectable label.

9. The method according to claim 5 or 6, wherein the first detection agent and / or the second detection agent is labeled with a detectable enzyme or biotin.

10. The method of claim 5 or 6, wherein the second capture antibody and the second detection agent are selected from the pairs in the following table: The method according to claim 10 , wherein the second capture antibody and the second detection agent are respectively the capture antibody and the detection antibody of DuoSet ELISA kit DY317.

12. The method of any one of claims 1 to 11, wherein the sample is a biological sample obtained from a subject treated ex vivo or in vivo with the SM modulating agent.

13. The method of claim 12, wherein the subject is a mammal.

14. The method of claim 12, wherein the subject is a human.

15. The method of claim 12, wherein the biological sample is selected from the group consisting of a sample prepared from cells, tissue, or serum, plasma, or another biological fluid sample.

16. The method of any one of claims 1 to 15, wherein the method has a detection sensitivity of a lower limit of quantification (LLOQ) of the IL-17 in the sample of about 0.01 pg / ml, or about 0.1 pg / ml, or about 0.25 pg / ml, or about 0.5 pg / ml, or about 0.75 pg / ml, or about 1 pg / ml.

17. A kit for determining the amount of free IL-17 in a sample comprising IL-17 and a SM modulator of IL-17, the kit comprising: i. a first capture antibody, wherein upon contact with the sample, the first capture antibody forms a first complex comprising the first capture antibody and IL-17 that is not bound to the SM modulator and a second complex comprising the first capture antibody and IL-17 that is bound to the SM modulator; and ii. a first detection agent, wherein upon contact with the first complex and the second complex, the first detection agent forms a third complex comprising the first capture antibody, the IL-17 not bound to the SM modulator, and the first detection agent, but does not form a fourth complex comprising the first capture antibody, the IL-17 bound to the SM modulator, and the first detection agent, wherein the SM modulator is selected from the group consisting of compounds #1-6 having the following structures and pharmaceutically acceptable salts thereof: The first capture antibody is the capture antibody of DuoSet ELISA kit DY317, and the first detection agent is mAb4538, and the first detection agent cannot form a complex comprising the first capture antibody, IL-17 bound to any one of compounds #1-6, and the first detection agent.

18. A kit for determining the amount of modulator-bound IL-17 in a sample comprising IL-17 and a SM modulator of IL-17, the kit comprising: A. the kit according to claim 17; B. a second capture antibody, wherein upon contact with the sample, the second capture antibody forms a fifth complex comprising the second capture antibody and the IL-17 not bound to the SM modulator and a sixth complex comprising the second capture antibody and the IL-17 bound to the SM modulator; and C. a second detection agent, wherein upon contact with the fifth complex and the sixth complex, the second detection agent forms a seventh complex comprising the second capture antibody, the IL-17 not bound to the SM modulator, and the second detection agent, and an eighth complex comprising the second capture antibody, the IL-17 bound to the SM modulator, and the second detection agent, respectively.

19. The kit of claim 18, wherein the first detection agent and / or the second detection agent is labeled with a detectable label.

20. The kit according to claim 18, wherein the first detection agent and / or the second detection agent is labeled with a detectable enzyme or biotin.

21. The kit according to any one of claims 18 to 20, wherein the second capture antibody and the second detection agent are selected from the pairs in the following table:

22. The kit according to claim 21, wherein the second capture antibody and the second detection agent are the capture antibody and the detection antibody of DuoSet ELISA kit DY317, respectively.

Images