Oxin-modulin binding molecules and their uses

Abstract

Provided herein are novel N-terminal and C-terminal oxyntomodulin polypeptide binding molecules and their uses, including methods for quantifying oxyntomodulin.

Description

[Background technology]

[0001] Oxintomodulin is a proglucagon-derived peptide agonist of both glucagon-like peptide-1 (GLP-1) and the glucagon receptor, and is an important regulator of gastric acid secretion and energy expenditure. Oxintomodulin is a potential target for therapeutic intervention (Pocai A, Mol. Metab. 2014;3:241-51) and has been proposed as a potential biomarker to predict whether subjects are at risk of developing type 2 diabetes after acute pancreatitis (Bharmal, SH, et al., Clin. Transl. Gastroenterology 2020;11:page e00132, doi:10.14309 / ctg.00000000 00000132).

[0002] Human proglucagon is cleaved into numerous different peptides in a tissue-specific manner (Holst JJ, et al., Peptides 2018;100:48-53), and the sequence similarities between proglucagon cleaved peptides make it difficult to quantify endogenous levels of oxytomodulin. Traditionally, polyclonal antibodies against specific regions of glucagon (33-61) or C-terminal octapeptides (62-69) have been used to quantify oxytomodulin-like immunoreactivity (OLI) components in various tissues and plasmas. Initially, a two-step subtractive radioimmunoassay was used to estimate OLI after specific subtraction of pancreatic glucagon levels (Kervran A, et al., Endocrinology 1987;121:704-1). Subsequent assays directly measure the OLI component using polyclonal antibodies against the C-terminal octapeptide (Blache P, et al., Anal Biochem 1988;173:151-9, Collie NL, et al., Proc Natl Acad Sci USA 1994;91:9362-6, Le Quellec A, et al., J. Clin. Endocrinol. Metab 1992;74:1405), and assays based on this approach are now commercially available.

[0003] Nevertheless, estimates of endogenous OLI in human plasma vary widely from 60 to 5000 ng / L in a fasted state (Laferrere B, et al., J. Clin. Endocrinol. Metab. 2010; 95: 4072-6; Le Quellec A, et al., J. Clin. Endocrinol. Metab 1992; 74: 1405-9). Furthermore, the proportion of OLI expressed by glycentin confuses accurate oxytomodulin quantification and obscures the physiological role of oxytomodulin (Bak MJ, et al., Eur. J. Endocrinol. 2014; 170: 529-3). Oxintomodulin can be distinguished from glycentin using HPLC or LC-MS, but these approaches lack sufficient analytical sensitivity to quantify endogenous levels of oxintomodulin in plasma (Lee AY, et al., Clin. Chem. 2015;62:227-235). A sandwich assay has been reported (Albrechtsen, NJ, et al., EBioMedicine 2016;7:112-120), but it lacks specificity as it is characterized by 10% cross-reactivity with glycentin (Holst JJ, et al., Peptides 2018;100:48-53).

[0004] Therefore, decades after the discovery of oxyntomodulin, the research community has been unable to selectively and reliably measure endogenous levels of oxyntomodulin in human plasma samples, leaving a need for an assay that is not only selective and highly sensitive but also modifiable for high-throughput applications.

[0005] A sandwich immunoassay is an assay that utilizes two antibodies that bind to different sites on a target antigen. However, performing an oxytomodulin sandwich immunoassay requires antibodies that (a) bind to different epitopes of each proglucagon fragment and (b) detect oxytomodulin only when used together in the assay. The present invention provides antibodies and methods that facilitate a sandwich assay method for quantifying oxytomodulin and a predictive method for determining whether a subject is at risk of developing diabetes.

[0006] The present invention provides polypeptide molecules that bind to the N-terminal regions of human oxyntmodulin (SEQ ID NO: 25) and human glucagon (SEQ ID NO: 30), respectively, and include complementarity-determining regions (CDRs) shown in SEQ ID NOs: 1 to 6. In one embodiment, the polypeptide molecule is an antibody. In another embodiment, the polypeptide molecule is an antibody fragment, which binds to the N-terminal regions of human oxyntmodulin (SEQ ID NO: 25) and human glucagon (SEQ ID NO: 30), respectively, and includes SEQ ID NOs: 1 to 6. In another embodiment, the antibody fragment is scFv. In yet another embodiment, the antibody fragment is Fab.

[0007] In another embodiment, the polypeptide molecule is an antibody comprising a heavy chain variable region (HCVR or VH) containing SEQ ID NO: 7 and a light chain variable region (LCVR or VL) containing SEQ ID NO: 8. In yet another embodiment, the polypeptide molecule is an antibody comprising a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO: 10. In yet another embodiment, the polypeptide molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 9 and a light chain consisting of SEQ ID NO: 10. The present invention also provides compositions comprising polypeptide molecules.

[0008] The present invention also provides a nucleic acid molecule comprising one or both of a first nucleic acid sequence encoding SEQ ID NO: 9 and a second nucleic acid sequence encoding SEQ ID NO: 10. In another embodiment, the first nucleic acid sequence encoding SEQ ID NO: 9 comprises the nucleic acid of SEQ ID NO: 11, and the second nucleic acid sequence encoding SEQ ID NO: 10 comprises the nucleic acid of SEQ ID NO: 12. The present invention also provides a vector comprising one or both of the first nucleic acid sequence encoding SEQ ID NO: 9 and the second nucleic acid sequence encoding SEQ ID NO: 10. The present invention also provides a composition comprising the vector. The present invention also provides a cell comprising the vector. In one embodiment, the cell is a mammalian cell. The present invention also provides a process for producing a polypeptide molecule, comprising culturing the cell under conditions such that the polypeptide molecule is expressed, and recovering the expressed polypeptide molecule from the culture medium. The present invention also provides the polypeptide molecule produced by the process.

[0009] The present invention also provides polypeptide molecules that bind to the C-terminal regions of human oxyntomodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28), respectively, and that include complementarity-determining regions as shown in SEQ ID NOs: 13-18. In one embodiment, the polypeptide molecule is an antibody. In another embodiment, the polypeptide molecule is a fragment of an antibody, and the fragment binds to the C-terminal regions of human oxyntomodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28). In another embodiment, the fragment of the polypeptide molecule is scFv. In yet another embodiment, the fragment of the polypeptide molecule is Fab.

[0010] In another embodiment, the polypeptide molecule is an antibody comprising VH containing SEQ ID NO: 19 and VL containing SEQ ID NO: 20. In yet another embodiment, the polypeptide molecule is an antibody comprising a heavy chain containing SEQ ID NO: 21 and a light chain containing SEQ ID NO: 22. In yet another embodiment, the polypeptide molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 21 and a light chain consisting of SEQ ID NO: 22. The present invention also provides compositions comprising polypeptide molecules.

[0011] The present invention also provides a nucleic acid molecule comprising one or both of a first nucleic acid sequence encoding SEQ ID NO: 21 and a second nucleic acid sequence encoding SEQ ID NO: 22. In one embodiment, the first nucleic acid sequence encoding SEQ ID NO: 21 comprises the nucleic acid of SEQ ID NO: 23, and the second nucleic acid sequence encoding SEQ ID NO: 22 comprises the nucleic acid of SEQ ID NO: 24. The present invention also provides a vector comprising one or both of the first nucleic acid sequence encoding SEQ ID NO: 23 and the second nucleic acid sequence encoding SEQ ID NO: 24. The present invention also provides a composition comprising the vector. The present invention also provides a cell comprising the vector. In one embodiment, the cell is a mammalian cell. The present invention also provides a process for producing a polypeptide molecule, comprising culturing the cell under conditions such that the polypeptide molecule is expressed, and recovering the expressed polypeptide molecule from the culture medium. The present invention also provides the polypeptide molecule produced by the process.

[0012] The present invention also provides a composition comprising: (a) a first polypeptide molecule that binds to the N-terminal region of human oxyntmodulin (SEQ ID NO: 25) and human glucagon (SEQ ID NO: 30), comprising SEQ ID NOs: 1 to 6; and (b) a second polypeptide molecule that binds to the C-terminal region of human oxyntmodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28), comprising SEQ ID NOs: 13 to 18.

[0013] The present invention also provides a sandwich assay method for determining the amount of human oxyntmodulin (SEQ ID NO: 25) in a liquid sample, comprising: (a) contacting a liquid sample containing human oxyntmodulin with a first polypeptide molecule bound to the C-terminal region of human oxyntmodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28), wherein the first polypeptide molecule comprises SEQ ID NOs: 13-18, thereby forming a first polypeptide molecule-human oxyntmodulin complex; (b) contacting a second polypeptide molecule bound to the N-terminal region of human oxyntmodulin (SEQ ID NO: 25) and human glucagon (SEQ ID NO: 30), wherein the second polypeptide molecule comprises SEQ ID NOs: 1-6, thereby forming a first polypeptide molecule-human oxyntmodulin-second polypeptide molecule complex; and (c) quantifying the amount of oxyntmodulin in the first polypeptide molecule-human oxyntmodulin-second polypeptide molecule complex by comparison with a standard curve of a known amount of human oxyntmodulin (SEQ ID NO: 25).

[0014] In another embodiment of the method of the present invention, the first polypeptide molecule is an antibody, and the second polypeptide molecule is an antibody. In another embodiment, the first polypeptide molecule is an antibody comprising VH containing SEQ ID NO: 19 and VL containing SEQ ID NO: 20, and the second polypeptide molecule is an antibody comprising VH containing SEQ ID NO: 7 and VL containing SEQ ID NO: 8. In another embodiment, the first polypeptide molecule is an antibody comprising a heavy chain comprising SEQ ID NO: 21 and a light chain comprising SEQ ID NO: 22, and the second polypeptide molecule is an antibody comprising a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10. In another embodiment, the first polypeptide molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 21 and a light chain consisting of SEQ ID NO: 22, and the second polypeptide molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 9 and a light chain consisting of SEQ ID NO: 10.

[0015] The present invention also relates to a method for predicting whether a subject is at risk of developing type 2 diabetes, comprising determining the concentration of oxyntmodulin in a serum or plasma sample from the subject, the method comprising: (a) contacting a serum or plasma sample containing human oxyntmodulin with a first polypeptide molecule bound to the C-terminal region of human oxyntmodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28), wherein the first polypeptide molecule includes SEQ ID NOs: 13-18, thereby forming a first polypeptide molecule-human oxyntmodulin complex; and (b) human oxy The present invention provides a method comprising (c) contacting a second polypeptide molecule bound to the N-terminal region of oxintmodulin (SEQ ID NO: 25) and human glucagon (SEQ ID NO: 30), wherein the second polypeptide molecule comprises SEQ ID NOs: 1-6, thereby forming a first polypeptide molecule-human oxintmodulin-second polypeptide molecule complex, and (c) quantifying the amount of oxintmodulin in the first polypeptide molecule-human oxintmodulin-second polypeptide molecule complex by comparison with a standard curve of a known amount of human oxintmodulin (SEQ ID NO: 25). In one embodiment of the method, the subject is diagnosed with acute pancreatitis.

[0016] The present invention also provides the use of a first polypeptide molecule bound to the C-terminal octapeptide present in human oxyntmodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28), respectively, and a second polypeptide molecule bound to the N-terminal region of human oxyntmodulin (SEQ ID NO: 25) and human glucagon (SEQ ID NO: 30), respectively, in measuring the concentration of oxyntmodulin in a liquid sample.

[0017] In one embodiment of the method of the present invention, the liquid sample is serum. In another embodiment, the liquid sample is plasma.

[0018] In the method of the present invention, the first polypeptide molecule can bind to a solid support. In one embodiment, the solid surface is a plate. In another embodiment, the solid surface is a plurality of beads. Such solid supports include, for example, without limitation, glass, cellulose, plastic, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0019] In another embodiment of the method of the present invention, the first polypeptide molecule is directly bound to the solid surface. In another embodiment, the first polypeptide molecule is indirectly bound to the solid surface by a binder. In one embodiment, the binder is streptavidin, neutravidin, or avidin coated on the solid surface, and the polypeptide molecule is biotinylated.

[0020] In another embodiment of the method of the present invention, before contacting the liquid sample with the first polypeptide molecule, the solid surface is contacted with a blocking solution. In another embodiment, after contacting the liquid sample with the first polypeptide molecule, the first polypeptide molecule - human oxyntomodulin complex is contacted with a washing solution to thereby remove uncomplexed human oxyntomodulin.

[0021] In another embodiment of the method of the present invention, the first polypeptide molecule - human oxyntomodulin - second polypeptide molecule complex is contacted with a washing solution to thereby remove uncomplexed second polypeptide molecules.

[0022] In another embodiment of a method for determining the amount of human oxyntomodulin (SEQ ID NO: 25) in a liquid sample, e.g., plasma, the method comprises (a) contacting a solid surface to which an antibody comprising a heavy chain comprising SEQ ID NO: 21 and a light chain comprising SEQ ID NO: 22 is bound with a blocking solution; (b) A liquid sample containing human oxyntomodulin is contacted with a first antibody comprising a heavy chain containing SEQ ID NO: 21 and a light chain containing SEQ ID NO: 22, which are bound to a solid surface, thereby forming a first antibody - human oxyntomodulin complex. (c) The solid surface is contacted with a washing solution, thereby removing uncomplexed human oxyntomodulin. (d) The solid surface is contacted with a second antibody comprising a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO: 10, thereby forming a first antibody - human oxyntomodulin - second antibody complex. (e) The solid surface is contacted with a washing solution, thereby removing uncomplexed second antibody. (f) Quantifying the amount of oxyntomodulin in the first antibody - human oxyntomodulin - second antibody complex by comparison to a standard curve of a known amount of human oxyntomodulin (SEQ ID NO: 25).

[0023] In another embodiment of the prediction method of the present invention, the method comprises: (a) Contacting a solid surface to which an antibody comprising a heavy chain containing SEQ ID NO: 21 and a light chain containing SEQ ID NO: 22 is bound with a blocking solution. (b) A liquid sample containing human oxyntomodulin is contacted with a first antibody comprising a heavy chain containing SEQ ID NO: 21 and a light chain containing SEQ ID NO: 22, which are bound to a solid surface, thereby forming a first antibody - human oxyntomodulin complex. (c) The solid surface is contacted with a washing solution, thereby removing uncomplexed human oxyntomodulin. (d) The solid surface is contacted with a second antibody comprising a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO: 10, thereby forming a first antibody - human oxyntomodulin - second antibody complex. (e) The solid surface is contacted with a washing solution, thereby removing uncomplexed second antibody. (f) The method includes quantifying the amount of oxytomodulin in the first antibody-human oxytomodulin-second antibody complex by comparing it to a standard curve of known amounts of human oxytomodulin (SEQ ID NO: 25).

[0024] In another embodiment of the method of the present invention, a second polypeptide molecule is labeled. In another embodiment, the amount of the second polypeptide is quantified by quantifying the label. In another embodiment, the label is a radioactive label. In another embodiment, the radioactive label is ruthenium.

[0025] The polypeptide molecule, method, and use of the present invention facilitate the selective and highly sensitive quantification of oxyntomodulin. In one embodiment, the method yields one or more of the following: an oxyntomodulin lower limit of quantification (LLOQ) of 0.4 ng / L, no detection of glucagon, and / or cross-reactivity with glycentin less than 0.5%. An additional advantage of the method of the present invention is that it facilitates the quantification of pre- and post-meal levels of oxyntomodulin, with results that correlate highly with those obtained using conventional orthogonal IA-LC-MS assays.

[0026] Human proglucagon (SEQ ID NO: 36) contains 180 amino acid residues and is encoded by the GCG gene (GCG-proglucagon precursor-Homo sapiens (human)-GCG gene and protein, unprot.org / uniprot / P01275#sequences(2007)). This polypeptide precursor is selectively cleaved in a tissue-specific manner into numerous different peptides, generating a panel of peptides with different biological activities: signal peptide (amino acid residues 1-20), human glycentin (amino acid residues 21-89), human glycentin-related pancreatic polypeptide (amino acid residues 21-50), human oxytomodulin (amino acid residues 53-89), human glucagon (amino acid residues 53-81), human glucagon-like peptide 1 (GLP-1) (amino acid residues 92-128), human GLP-1 (7-37) (amino acid residues 98-128), human GLP-1 (7-36) (amino acid residues 98-127), and human glucagon-like peptide 2 (GLP-2) (amino acid residues 146-178). One consequence of the tissue-specific cleavage pattern from the common polypeptide precursor (SEQ ID NO: 36) is the generation of biologically active peptides with some sequence duplication. For example, human oxyntomodulin (amino acid residues 53-89) contains all the sequences found in human glucagon (amino acid residues 53-81), in addition to the 8 amino acids that extend beyond amino acid residue 81. As another example, the sequences present in human oxyntomodulin (amino acid residues 53-89) and human glucagon (amino acid residues 53-81) are also present in human glycentin (amino acid residues 21-89), but human glycentin (amino acid residues 21-89) contains the additional 32 amino acids that are present before residue 53 in both human oxyntomodulin (amino acid residues 53-89) and human glucagon (amino acid residues 53-81).

[0027] The sequences referred to herein are shown in Table 1. In Table 1, the numbering in parentheses in specific sequence names refers to the respective amino acid residues in human proglucagon, including the signal peptide sequence (residues 1-20). For example, in the term "human oxyntmodulin (53-89)," the term "(53-89)" refers to amino acid residues 53-89 in human proglucagon. Deleted versions of human sequences in Table 1 are provided without the term "human." For example, the term "oxyntmodulin (55-89)" refers to a human oxyntmodulin fragment that corresponds to amino acid residues 55-59 of human proglucagon and does not contain the first two N-terminal amino acid residues found in human oxyntmodulin. [Table 1]

[0028] As used herein, the terms “the N-terminal region of human glucagon and human oxytomodulin” refer to the first three amino acid residues in each of those peptides where the N-terminal histidine is located.

[0029] As used herein, the term "binding to the N-terminal region of human glucagon and human oxyntmodulin" means (a) binding to human oxyntmodulin (53-59) (SEQ ID NO: 25) and human glucagon (SEQ ID NO: 30), each of which has an N-terminal histidine residue; (b) oxyntmodulin (55-89) and oxyntmodulin (56-89) do not bind to oxyntmodulin (55-89) or oxyntmodulin (56-89) because they do not contain the N-terminal histidine residue found in human oxyntmodulin (53-59) and human glucagon (SEQ ID NO: 30); and (c) glycentinspane (29-69) does not bind to glycentinspane (49-89) because, although it contains histidine, it is not present at the free amine terminus.

[0030] As used herein, the terms "the C-terminal regions of human oxyntmodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28)" refer to amino acid residues 30-37 in human oxyntmodulin and amino acid residues 61-69 in human glycentin (SEQ ID NO: 28).

[0031] As used herein, the term "binding to the C-terminal region of human oxyntmodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28)" refers to binding to one or more amino acid residues 30-37 in human oxyntmodulin and one or more amino acid residues 61-69 in human glycentin (SEQ ID NO: 28).

[0032] As used herein, the term "N-terminal antibody" refers to an antibody having the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10.

[0033] As used herein, the term "C-terminal antibody" refers to an antibody having the heavy chain amino acid sequence of SEQ ID NO: 21 and the light chain amino acid sequence of SEQ ID NO: 22.

[0034] As used herein, the term "polypeptide molecule" refers to a molecule containing a polymer of amino acid residues. In one embodiment, the polypeptide molecule consists of a polymer of amino acid residues.

[0035] As used herein, the term “contact” refers to the exposure of one substance to another. For example, a sample can be exposed to the polypeptide molecule of the present invention for a time and under conditions that allow the polypeptide molecule to bind to human oxytomodulin present in the sample. Such time and conditions are known to those skilled in the art and / or can be routinely determined by methods known in the art in accordance with the references cited herein.

[0036] As used herein, the term “complex” refers to, for example, a protein-protein interaction between a polypeptide molecule and human oxyntmodulin (SEQ ID NO: 25). As used herein, the “first polypeptide molecule-human oxyntmodulin complex” refers to a protein-protein interaction between the polypeptide molecule of the present invention and the human oxyntmodulin molecule (SEQ ID NO: 25). As used herein, the “first polypeptide molecule-human oxyntmodulin-second polypeptide molecule complex” refers to a simultaneous protein-protein interaction between (a) the molecule of the first polypeptide molecule of the present invention and the human oxyntmodulin molecule (SEQ ID NO: 25), and (b) the molecule of the second polypeptide molecule of the present invention and the human oxyntmodulin molecule (SEQ ID NO: 25).

[0037] As used herein, the term "quantifying the amount of oxytomodulin" refers to measuring the amount of oxytomodulin in a sample, for example, a liquid sample. Suitable assays, such as ELISA, are known to those skilled in the art.

[0038] The polypeptide molecule of the present invention can be bound to enzymes and used in enzyme-linked immunosorbent assays (ELISA). Such assays are described in detail, for example, Butler (1994) “ELISA” (Chapter 29), In: van Oss, C.Jet al., eds., Immunochemistry, Marcel Dekker, Inc., New York, pp. 759-803. The polypeptide molecule can also be used in radioimmunoassays and fluorescence-activated cell sorting (FACS) analyses of oxytomodulin expression.

[0039] Certain proteins, such as human oxytomodulin, can be measured by a variety of immunoassay methods, including, without limitation, competitive and non-competitive assay systems, using techniques such as, for example, Western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitater reaction, gel diffusion precipitater reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescence immunoassay, and protein A immunoassay. For an overview of general immunological and immunoassay procedures, see, for example, Stites and Terr (eds.), Basic and Clinical Immunology (7th ed.) (1991). Furthermore, immunoassays can be performed in many configurations, as is known in the art (see, for example, Maggio (ed.), Enzyme Immunoassay CRC Press, Boca Raton, Florida (1980), Gosling JP, Immunoassays: A Practical Approach (Practical Approach Series), Oxford Univ Press (2000), and Diamandis & Christopoulus, Immunoassay, Academic Press (San Diego, CA) (1996)).

[0040] As used herein, the term “antibody” refers to an immunoglobulin molecule that binds to an antigen. Embodiments of an antibody include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. The antibody may be any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0041] The exemplary antibodies of this disclosure are immunoglobulin G (IgG) type antibodies comprising four polypeptide chains: two heavy chains (HC) and two light chains (LC) crosslinked via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of approximately 100 to 125 or more amino acids, primarily involved in antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region, primarily involved in effector function. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (VL) and a light chain constant region. IgG isotypes may be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0042] The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which contain more conserved regions called framework regions (FRs). CDRs are exposed on the surface of the protein and are important regions of the antibody for antigen-binding specificity. Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Hereinafter, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." CDRs contain the majority of the residues that form specific interactions with the antigen. The assignment of amino acid residues to CDRs is described by Kabat (Kabat et al., “Sequences of Proteins of Immunological Interest”, National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., “Canonical structures for the hypervariable regions of immunoglobulins”, Journal of Molecular Biology, 196, 901-917 (1987), Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), and North (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology) This may be carried out according to a well-known scheme, including those described in Biology, 406, 228-256 (2011), or IMGT (the international ImMunoGeneTics database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27: 209-212).

[0043] The “antibody fragments” or “antigen-binding fragments” used herein, such as Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, scFab, disulfide-bonded Fv(sdFv), and Fd fragment, comprise at least a portion of an antibody that retains the ability to specifically interact with an antigen or an antigenic epitope.

[0044] As used herein, the terms “bind” and “binds” are intended to mean the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, unless otherwise specified, resulting in proximity of two proteins or molecules as determined by common methods known in the art, e.g., molecular interactions between two molecules, e.g., the polypeptide molecule of the present invention and e.g., human oxyntmodulin (SEQ ID NO: 25). In one embodiment, the polypeptide molecule of the present invention specifically binds to human oxyntmodulin. In another embodiment, “specifically binds” means that the polypeptide molecule of the present invention interacts more frequently, more quickly, for a longer duration, with greater affinity, or with any combination of the above-mentioned human oxyntmodulin. In another preferred embodiment, “specifically binds” means that the polypeptide molecule of the present invention interacts with a K2 of about 0.1 mM or less. D This means that it binds to human oxytomodulin. In another preferred embodiment, "specifically binds" means that the polypeptide molecule of the present invention binds to K at a concentration of about 0.01 mM or less. D This means that it binds to human oxytomodulin. In another preferred embodiment, "specifically binds" means that the polypeptide molecule of the present invention binds to K at a concentration of about 0.001 mM or less. D This means that it binds to human oxytomodulin. In another preferred embodiment, "specifically binds" means that the polypeptide molecule of the present invention binds to K at a concentration of about 0.0001 mM or less. D This means that it binds to human oxytomodulin.

[0045] Isolated polynucleotides encoding the HCVR region can be converted into full-length heavy-chain genes by operably ligating the HCVR-encoding polynucleotide to another polynucleotide molecule encoding the heavy-chain constant region. The sequences of heavy-chain constant region genes in humans and other mammals are known in the art. Polynucleotide fragments containing these regions can be obtained, for example, by standard PCR amplification.

[0046] An isolated polynucleotide molecule encoding the LCVR region can be converted into a full-length light chain gene by operably ligating the LCVR-encoding polynucleotide to another polynucleotide molecule encoding the light chain constant region. The sequences of human and other mammalian light chain constant region genes are known in the art. Polynucleotide fragments containing these regions can be obtained by standard PCR amplification.

[0047] As used herein, the term “sensitivity” refers to the lowest level of analyte (in this case, human oxyntomodulin) that can be measured with acceptable accuracy and precision. Sensitivity is reflected in the limit of quantification (LLOQ), which is determined, for example, according to the present invention, by taking a sample of oxyntomodulin and progressively diluting it until the coefficient of variation (CV)% exceeds 20%.

[0048] The term "detectably labeled" means that the polypeptide molecule of the present invention, or the complex of oxyntomodulin and the polypeptide molecule, is bound to a useful detectable label by either covalent or non-covalent bonds. Direct binding labeling methods can utilize many different useful labels, including, for example, prosthetic group complexes, chromophores, chromogenic substrates, dyes, fluorescent compounds, fluorescence-generating compounds, radioisotopes, paramagnetic isotopes, and compounds that can be imaged by positron emission tomography (PET) and magnetic resonance imaging (MRI).

[0049] The nucleic acid molecules of the present invention can be expressed in host cells after their sequences have been operably bound to an expression control sequence. Expression vectors are typically replicable in the host organism as either episomes or integrated portions of host chromosomal DNA. Generally, expression vectors contain selection markers, such as tetracycline, neomycin, and dihydrofolate reductase, to enable the detection of those cells transformed with the desired polynucleotide sequence.

[0050] An expression vector containing the target nucleic acid sequence (for example, a nucleic acid sequence encoding one or more polypeptide molecules of the present invention and an expression control sequence) can be introduced into host cells by known methods that vary depending on the type of host cell.

[0051] The polypeptide molecules of the present invention can be produced in mammalian host cells, non-limiting examples of which include CHO, NS0, HEK293, or COS cells. Host cells can be cultured using techniques known in the art. The polypeptide molecules of the present invention can be expressed and purified essentially as follows: A suitable host cell, such as HEK293 or CHO, can be transfected transiently or stably in an expression system for secreting polypeptides, e.g., antibodies, using an optimal predetermined heavy-chain:light-chain vector ratio or a single-vector system encoding both heavy and light chains. Nucleic acids encoding the polypeptide molecules of the present invention can be transfected transiently or stably in an expression system for secreting polypeptides, e.g., using one or more DNA molecules encoding antibody heavy and light chains.

[0052] Various methods of protein purification can be used to purify the polypeptide molecules of the present invention, and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0053] For example, the medium can be conveniently applied to a MabSelect column (GE Healthcare Life Sciences), or a KappaSelect column (GE Healthcare Life Sciences), equilibrated with a compatible buffer such as phosphate-buffered saline (pH 7.4). The column can be washed to remove non-specific binding components. The bound polypeptide molecules can be eluted, for example, by a pH gradient (such as 20 mM Tris buffer pH 7.0 to 10 mM sodium citrate buffer pH 3.0, or phosphate-buffered saline pH 7.4 to 100 mM glycine buffer pH 3.0, etc.). The antibody fraction can be detected by ultraviolet absorbance or SDS-PAGE, etc., and then pooled if desired. Further purification is optional depending on the intended use. The purified polypeptide molecules can be concentrated and / or sterile filtered using common techniques. Soluble aggregates and multimers can be effectively removed by common techniques including size exclusion, hydrophobic interaction, ion exchange, multimodal, or hydroxyapatite chromatography. The purified polypeptide can be immediately frozen at -70 °C or lyophilized.

[0054] The polypeptide molecules disclosed herein are useful for diagnostic, prognostic, and / or patient monitoring procedures by detecting the level of oxyntomodulin present in serum or plasma.

[0055] Useful radiolabels that are simply detected by a gamma counter, scintillation counter, or autoradiography include 3 H, 124 I, 125 I, 131 I, 35 S, and 14 C. The radionuclides can be bound to the polypeptide molecules described herein either directly or indirectly using chelating agents such as DTPA and EDTA. Examples of such radionuclides include 99 Tc,123 I, 125 I, 131 I, 111 In, 97 Ru, 67 Cu, 67 Ga, 68 Ga, 72 As, 89 Zr, 90 Y, and 201 Tl is included.

[0056] Other suitable labels are known in the art or can be determined by routine experiments. For example, an antibody, such as a T-terminal antibody, can be bound to an enzyme. The binding of another antibody to human oxytomodulin, which is itself bound to a primary antibody, such as a C-terminal antibody, can then be detected by reaction of the enzyme with a chromogenic substrate under appropriate conditions that produce a detectable signal.

[0057] By using a chromophore with a high absorption coefficient, or a resulting chromogenic compound, easily detectable colorimetric detection can be employed. When subsequently exposed to the substrate under appropriate reaction conditions, the enzyme reacts with the substrate to produce a chemical label that can be detected, for example, by spectrophotometry, fluorescence spectroscopy, or visual means.

[0058] Enzymes commonly used for this purpose include horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucoamylase, and acetylcholinesterase.

[0059] Non-limiting examples of suitable prosthetic group complexes include, for example, streptavidin / biotin, avidin / biotin, and neutravidin / biotin. The use of chromogens is preferred because assays using them can be easily performed in clinical diagnostic laboratories and reviewed by pathologists using equipment commonly available in these laboratories. Commonly used chromogens include diaminobenzidine (DAB), enhanced DAB, 3-amino-9-ethylcarbazole (AEC), 4-chloro-1-naphthol (4-CN), Hanker-Yates reagent, alpha-naphtholpyronin, 3,3',5,5'-tetramethylbenzidine (TMB), Fast Blue BB, Fast Red TR, New Fuchsin, BCIP-NBT, tetrazolium, tetranitrite blue tetrazolium (TNBT), and silver-enhanced Immunogold.

[0060] Useful fluorescent labels include umbelliferone, fluorescein, fluorescein isothiocyanate, dichlorotriazinylamine fluorescein, rhodamine, dansyl group, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde (phthaldehyde), fluorescein, and Cy5 (Haugland ((1996) Handbook of Fluorescent Probes and Research Chemicals, Sixth Ed., Molecular Probes, Eugene, OR).

[0061] Polypeptide molecules, or polypeptide molecule-oxynmodulin complexes, 152 EU + They can be detected by using a fluorescent metal, such as another member of the lanthanide series, or by using a metal chelating group such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) to bind them.

[0062] Polypeptide molecules can also be detected by coupling them with phosphorescent or chemiluminescent compounds, which can then be detected by phosphorescence or luminescence produced during the course of a chemical reaction. Examples of useful chemiluminescent compounds include luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters. Similarly, antibody peptides can be labeled using bioluminescent compounds such as luciferin, luciferase, or aequorin. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.

[0063] The present invention also provides manufactured articles and kits containing compositions useful for quantifying human oxytomodulin. The manufactured articles may include labeled containers. The containers may hold compositions comprising polypeptide molecules of the present invention, which are either detectably labeled or unlabeled.

[0064] The kit of the present invention may also include a container containing the first and second polypeptide molecules of the present invention. The second polypeptide molecule can be bound to an enzyme or other label. A chromogenic substrate for the enzyme may also be included in the kit. The kit may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and accompanying documentation containing instructions for use in vivo, in vitro, or both.

[0065] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0066] Example 1 Antibody binding research The kinetics and binding specificity of the N-terminal and C-terminal antibodies are determined by surface plasmon resonance using Biacore T100 (GE Healthcare Life Sciences). Goat anti-mouse IgG antibody (Southern Biotech) is immobilized on a Series S CM5 sensor chip and used to capture approximately 1500 response units of anti-oxyntomodulin IgG. Binding to two injections of various concentrations of human oxyntomodulin (SEQ ID NO: 25) (1.1–90 nmol / L) diluted in running buffer (HEPES-buffered saline containing 3 mmol / L EDTA and 0.05% Tween) and either 22.5 nmol / L human glycentinuspan (49–89) (SEQ ID NO: 29) or human glucagon (53–81) (SEQ ID NO: 30) peptides is measured at a flow rate of 30 μL / min. Dynamical constants are determined using a 1:1 binding model in the T100 Evaluation software.

[0067] Using Biacore analysis of the N-terminal antibody, the equilibrium dissociation constant is 1.5 × 10⁻⁶. -10 The concentration is mol / L (Table 2). The N-terminal antibody shows minimal binding to the glycentinuspan (48-89) peptide (SEQ ID NO: 29). [Table 2]

[0068] Using Biacore analysis of the C-terminal antibody, the C-terminal antibody dissociation constant was determined to be 8.3 × 10⁻⁶. -11 The concentration is mol / L (Table 2). The C-terminal antibody shows minimal binding to human glucagon (53-81) (SEQ ID NO: 30).

[0069] Example 2 standard curve The optimal antibody pairing for the assay utilizes the C-terminal antibody as the capture antibody and the N-terminal antibody as the detection antibody. A standard curve is prepared with synthetic human oxyntomodulin (SEQ ID NO: 25) serially diluted in assay buffer from a starting concentration of 50,000 ng / L. The LLOQ is determined to be 0.4 ng / L based on a 3-SD assessment from the zero calibrator. The assay exhibits an excellent dynamic range with an upper limit of quantification (ULOQ) of 2500 ng / L (Table 3). Since oxyntomodulin is a substrate of DDP-4, and this cleavage removes the N-terminal neoepitope recognized by the reported reagent antibody, six sets of matched human plasma collected in either P800 or K2 EDTA collection tubes are compared to evaluate the effect of ex vivo proteolysis after sample collection. A reduction of approximately 35–40% in the amount of oxyntomodulin measured in blood collected in K2 EDTA compared to P800 tubes is observed.

[0070] Additional important attributes of the immunoassay are outlined in Table 3. [Table 3]

[0071] Intra-batch and inter-batch variability is less than 10% when using high (2500 ng / L), medium (500 ng / L), or low (100 ng / L) oxyntomodulin concentrations. The assay exhibits excellent dilution linearity with <15% CV after 16-fold dilutions of both the assay buffer and the human plasma pool spiked with 2500 ng / L oxyntomodulin standard. Assay parallelism is observed in dilutions of two different human plasma samples, resulting in a CV of less than 20% in the calculated analyte values. The robustness of the immunoassay using pooled P800 human plasma spiked with 2500, 500, 100, or 0 ng / L oxyntomodulin is demonstrated for many parameters. Recovery ranges from 90–113% for all runs, and variability of less than 11% in calculated oxyntomodulin values ​​is observed after 6 freeze-thaw cycles. Finally, the stability of the analytes in P800-collected plasma is demonstrated at <25% CV under three different temperature conditions outlined in Table 3.

[0072] The selectivity of oxyntomodulin in the immunoassay is demonstrated by testing the binding of proglucagon or other biologically relevant incretin peptides to a panel. Human glycentin spane (49-89) (SEQ ID NO: 29), human glucagon (53-81) (SEQ ID NO: 30), glucose-dependent insulinotropic polypeptide (GIP) (SEQ ID NO: 33), GLP-1 (98-128) (SEQ ID NO: 34), GLP-1 (100-128) (SEQ ID NO: 35), oxyntomodulin (55-89) (SEQ ID NO: 26), and oxyntomodulin (56-89) (SEQ ID NO: 27) are measured at three hyperphysiological concentrations ranging from 2500 to 100 ng / L. Only glycentin spane (49-89) (SEQ ID NO: 29) and GIP (SEQ ID NO: 33) peptides are measured above background levels at the highest concentrations. Glycentenspane (49-89) (SEQ ID NO: 29) at a concentration of 500 ng / L also provides a signal slightly higher than that seen for the buffer blank control. For comparison, in stark contrast to the glycentenspane results, human oxytomodulin (53-89) (SEQ ID NO: 25) at 2500, 500, and 100 ng / L yielded over 170,000, over 30,000, and over 8,000 ECL units, respectively.

[0073] Cross-reactivity in the oxyntmodulin immunoassay is minimal, less than 0.5% for glycentin-span (49-89) peptide (SEQ ID NO: 29) (a surrogate for glycentin cross-reactivity) and 0.12% for GIP peptide (SEQ ID NO: 33). No signal is observed for oxyntmodulin (55-89) peptide (SEQ ID NO: 26) or oxyntmodulin (56-89) peptide (SEQ ID NO: 27), which are N-terminal cleavages of 2 or 3 residues of human oxyntmodulin, respectively. No signal is observed when sampling human plasma with isotype-matched, unrelated antibodies substituted for either the capture or detection reagent.

[0074] Example 3 Sandwich assay Meso Scale Discovery (MSD) Streptavidin Gold Multi-array 96-well plates (Meso Scale Diagnostics) are washed three times with 1x Tris-buffered saline (TBS) containing 10 mmol / L Tris pH 7.4, 150 mmol / L NaCl, and 1 mL / L Tween 20, and then blocked with 200 μL of TBS containing 1% (w / v) BSA (Sigma). After 1 hour, 50 μL of biotin-labeled C-terminal antibody at a concentration of 1 mg / L is conjugated to the plate at room temperature (RT) for an additional 1 hour. Oxintomodulin peptide standards, diluted in an assay buffer consisting of 50 mmol / L HEPES, pH 7.4, 150 mmol / L NaCl, 10 mL / L Triton X-100, 5 mmol / L each of EDTA and EGTA, 1% (w / v) BSA, both protease (Roche) and dipeptidyl peptidase-4 (DPP-4) (Millipore) inhibitors, and 100 mg / L Heterophilic Blocking Reagent 1 (Scantibodies), are added to the wells to generate a standard calibration curve. Plasma samples are diluted 1:2 in the same assay buffer, and both samples and standards are incubated overnight at 4°C. The plates are washed, and 50 μL of 1 ng / mL ruthenium-labeled N-terminal antibody is added to the wells and incubated at room temperature for 1 hour. Following the final washing step, 150 μL of 2x MSD read buffer is added, and the ruthenium electrochemiluminescence units (ECL) are measured using an MSD SECTOR Imager 600 (Meso Scale Diagnostics) reader.

[0075] Human plasma samples. Blood samples from healthy men and women aged 18–65 years are collected in P800 EDTA BD® Vacutainer (BD Biosciences) collection tubes with informed consent. Samples are stored on ice and spun down at 4°C using a benchtop centrifuge at 2000g for 20 minutes within 1 hour of collection. The resulting plasma samples are stored at -70°C until analysis of oxytomodulin levels. K2 and P800 EDTA plasma are also obtained from an additional 19 healthy volunteers under conditions of 10 hours fasting, 5–10 minutes postprandial, and 90–120 minutes postprandial. Postprandial collection follows a mixed diet challenge consisting of approximately 262 fat calories, 274 carbohydrate calories, and 100 protein calories.

[0076] Oxintmodulin levels in normal human plasma. A sandwich immunoassay is used to determine the effect of a nutritional challenge on oxintmodulin plasma levels in healthy individuals. Blood is collected in both P800 and K2 EDTA tubes from 19 volunteers after overnight fasting. Samples are also collected both within minutes and within 2 hours after ingestion of a standardized mixed diet. Oxintmodulin levels from samples collected in K2 EDTA tubes are lower than those from samples collected in P800 collection tubes for all three time points analyzed. Oxintmodulin levels increase immediately after ingestion. The mean baseline, early, and late time points for P800 samples are 11±9 ng / L, 21±9 ng / L, and 33±14 ng / L, respectively. The mean baseline, early, and late time points for K2 EDTA samples are 8±7 ng / L, 14±6 ng / L, and 23±11 ng / L, respectively.

[0077] All data are expressed as mean ± SEM. MSD Workbench software is used for each of the four PL-fit calibration curves and for interpolating unknown values. Data are plotted with SigmaPlot version 11.0, and Microsoft Office Excel 2010 or GraphPad Prism 6 is used for data analysis. In each example, a p-value of 0.05 or less is considered statistically significant. Cross-reactivity percentage is determined as the ratio of ECL counts with the peptide of interest to ECL counts with the reference human oxytomodulin (53-89) (SEQ ID NO: 25) peptide, after subtracting the buffer blank ECL counts for each concentration tested.

[0078] Oxintmodulin antibody affinity increased by 6 to over 200-fold. Intra-assay and inter-assay CVs were 7–10% and 3–10%, respectively. Spike recovery ranged from 90–113%, linearity was evident up to 16-fold dilution, and glycentin cross-reactivity was 0.5%. Oxintmodulin levels were lower in K2 EDTA compared to P800 plasma. Postprandial increases in oxintmodulin occurred within minutes, and levels correlated significantly with those obtained using IA-LC-MS.

[0079] The oxytomodulin sandwich immunoassay is appropriately sensitive and selective, and can be modified for high-throughput applications for reliable determination of endogenous levels of intact oxytomodulin from human samples.

[0080] The combination of antibody pairs that bind to specific human oxytomodulin N-terminal and C-terminal epitopes present only on each proglucagon fragment facilitates the creation of immunoassays with sufficient selectivity and sensitivity to determine the endogenous level of human oxytomodulin.

[0081] The selectivity of the immunoassay is first evaluated with other proglucagon-derived and incretin peptides of interest. A small signal is observed at the highest spike of the glycentin span (49-89) peptide (SEQ ID NO: 29) in the oxyntmodulin sandwich immunoassay, but this signal is two orders of magnitude lower than that obtained with the same concentration of human oxyntmodulin (SEQ ID NO: 25). Endogenous glycentin has been reported to increase postprandially, peaking at 130 pmol / L (Naito H, et al., Regul. Pept. 1999; 79: 55-61), and its level is still lower than the high-spike concentration (2500 ng / L [560 pmol / L]) used in the studies herein, indicating that endogenous glycentin levels do not affect the human oxyntmodulin sandwich immunoassay herein.

[0082] Human oxyntomodulin has a short half-life (minutes) in circulation (Schjoldager BT, et al., Eur.J.Clin.Invest.1988;18:499-5) and has been shown to be a substrate for proteolysis by DPP-4 (Yi J, et al., PLoS One 2015;10:e0134427, Zhu L, et al., J.Biol.Chem.2003;278:22418-23), which removes the first two amino-terminal residues. The selectivity of the novel N-terminal neoepitope antibodies disclosed herein (SEQ ID NOs. 9 and 10) is confirmed by the complete lack of reactivity to synthetic oxyntomodulin peptides lacking the first two or three residues (SEQ ID NOs. 26 and 27, respectively). A small but reproducible reduction in the amount of human oxyntmodulin (SEQ ID NO: 25) measured using the sandwich immunoassay described herein was observed in serum collected with K2 EDTA against P800 tubes, confirming that endogenous human oxyntmodulin is processed by DDP-4 or some other relevant protease that removes the N-terminal amino acid, and that the sandwich immunoassay disclosed herein selectively measures only the intact human oxyntmodulin (SEQ ID NO: 25) component.

[0083] Human K2 EDTA and P800 plasma (500 μL) were spiked with stable isotope-labeled internal standard peptides (CPC Scientific) of human oxytomodulin (53-89) SEQ ID NO: 25, oxytomodulin (55-89) SEQ ID NO: 26, and oxytomodulin (56-89) SEQ ID NO: 27, and diluted with I buffer (25 mmol / L Tris-HCl, 25 mmol / L HEPES, 300 mmol / L NaCl, 0.1% (v / v) octyl β-D-glucopyranoside, pH 7.5). Immunoaffinity enrichment was carried out overnight at 4°C after the addition of 2 μg of biotinylated anti-glucagon antibody (Sloan JH, et al., Clin. Biochem. 2012;45:1640-4). After incubation, 50 μL of Dynabeads® MyOne® streptavidin T1 magnetic beads (ThermoFisher Scientific) are added to the mixture and incubated at room temperature for 30 minutes. The beads are then washed sequentially once with 1 mL of the following three buffers: radioimmunoprecipitation assay buffer (ThermoFisher Scientific), 25 mmol / L Tris-HCl, 25 mmol / L HEPES, 500 mmol / L NaCl, and a buffer consisting of 0.1% (v / v) octyl β-D-glucopyranoside pH 7.5, and deionized water. The bound analytes are eluted with 50 μL of 0.2% (v / v) formic acid / 1x Invitrosol® (ThermoFisher Scientific) / 10% (v / v) acetonitrile. Protein quantification is achieved via high-resolution, precise mass LC-MS using a Thermo Scientific Q Exactive mass spectrometer.

[0084] In healthy human volunteers, fasting oxytomodulin levels were found to range from 7 to 11 ng / L, depending on the collection method used, rising within 10 minutes of feeding and reaching levels in the range of 23 to 33 ng / L at the most recent time of collection. These results are consistent with those obtained using the IA-LC-MS assay described later and by the LC-MS assay of Lee et al. (Lee AY, et al., Clin. Chem. 2015;62:227-235).

[0085] Example 4 Correlation between sandwich assay results and immunoassay-LC-MS assay results A reference standard for oxytomodulin analysis to measure the accuracy of immunoassays is not currently available. However, immunoassay-LC-MS has a clear advantage in identifying the molecular form of the target analyte present in a given sample (Bouillon R, et al., Clin. Chem. 2016;62:6-8).

[0086] The correlation of oxytomodulin levels obtained by the sandwich immunoassay was investigated using the same set of plasma samples used in the sandwich assay, obtained using the immunoassay-LC-MS method described by Cox et al. (Cox JM, et al., Bioanalysis 2016;8:1579-95). The correlation between the sandwich assay and the immunoassay-LC-MS method was high (Spearman coefficient 0.9236, P<0.0001), confirming the selectivity of the sandwich immunoassay of the present invention.

[0087] Furthermore, the sandwich immunoassay of the present invention enables scalability and high-throughput applications, reduces sample volume and assay time, and avoids the need for extensive investment in equipment and expertise associated with the use of immunoassay-LC-MS assays. The use of the highly sensitive and selective sandwich immunoassay disclosed herein for measuring human oxytomodulin in human samples can also facilitate an improved understanding of oxytomodulin biology. array Sequence ID 1 GYTFTDYAFS Sequence ID 2 WITTNTGEATYADDFKG Sequence ID 3 ETEYGDSSWFGH Sequence ID 4 RASESVDGWGNSFMH Sequence ID 5 LATYRVA Sequence ID 6 MQSSEDPYT Sequence ID 7 QIQLVQSGPELKKPGETVKISCKASGYTFTDYAFSWVKQAPGKGLKWMGWITTNTGEATYADDFKGRFAFSLETSASTAYLQISNLKNEDTATYFCARETEYGDSSWFGHWGQGTLVTVSA Sequence ID 8 DIVLTQSPASLAVSLGQRATISCRASESVDGWGNSFMHWYQQKPGQPPKLLIYLATYRVAGIPARFSGSGSRTDFTLTINPVEADDVATYYCMQSSEDPYTFGGGTKLEIK Sequence ID 9 QIQLVQSGPELKKPGETVKISCKASGYTFTDYAFSWVKQAPGKGLKWMGWITTNTGEATYADDFKGRFAFSLETSASTAYLQISNLKNEDTATYFCARETEYGDSSWFGHWG QGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTI KPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIER TISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK Sequence ID 10 DIVLTQSPASLAVSLGQRATISCRASESVDGWGNSFMHWYQQKPGQPPKLLIYLATYRVAGIPARFSGSGSRTDFTLTINPVEADDVATYYCMQSSEDPYTFGGGTKLE IKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Sequence ID 11 Sequence ID 12 gacattgtgctgacccaatctccagcttctttggccgtgtctctagggcagaggggccaccatatcctgcagagccagtgaaagtgttgatggatggggcaatagtttcatgcactggtaccagcagaaaccaggacagccacccaaactcctcatctatctcg ctacctatcgcgtagctgggatccctgccaggttcagtggcagtgggtctaggacagacttcaccctcaccattaatcctgtggaggctgatgatgttgcaacctattattgtatgcagagtagtgaagatccgtacacgttcggaggggggaccaagctggaa ataaaacgggctgatgcggcgcccactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgctagcgtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagatgatggcagtgaacgacaaaatggcgtcc tgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt Sequence ID 13 GYNFTNYWLH Sequence ID 14 ELDPEYGFANYNQKFKG Sequence ID 15 GFMDY Sequence ID 16 RSSRSLLDPDGKTYLN Sequence ID 17 LVSKLDS Sequence ID 18 WQGTHLPVT Sequence ID 19 QVQVQQSGAELVMPGTSVKLSCKASGYNFTNYWLHWVKQRPGQGLEWIGELDPEYGFANYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCSAGFMDYWGQGTSVTVSS Sequence ID 20 DVVMTQTPLTLSVNIGQPASISCRSSRSLLDPDGKTYLNWLLQRPGQSPKRLIYLVSKLDSRVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHLPVTFGGGTKLEIK Sequence ID 21 QVQVQQSGAELVMPGTSVKLSCKASGYNFTNYWLHWVKQRPGQGLEWIGELDPEYGFANYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCSAGFMDYWGQGTSVT VSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCP PCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTI SKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK Sequence ID 22 DVVMTQTPLTLSVNIGQPASISCRSSRSLLDPDGKTYLNWLLQRPGQSPKRLIYLVSKLDSRVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHLPVTFGGGTKL EIKRADAAPTVSIFPPSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Sequence ID 23 sequence number 24 gatgttgtgatgacccagactccactcactttgtcggttaacattggacaaccagcctccatctcttgcaggtcaagtcggagcctcttagatccagatggaaagacatatttgaattggttgttacagaggccaggccagtctccaaagcgcctaatctatct ggtgtctaaactggactctagatccctgacaggttcactggcagtggatcaggcacagatttcacactgaaaatcagcagagtgggaggctgaggatttgggagtttattattgctggcaaggtacacatcttcctgtaacgttcggtggaggcaccaagctgg aaatcaaacgggctgatgcggcgccccactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgctagcgtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggagaattgatggcagtgaacgacaaaatggcgtc ctgaacagttggactgatcaggacagcaaagacagcaccctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt sequence no. 25 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA sequence number 26 QGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA sequence number 27 GTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA sequence no. 28 RSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA Sequence ID 29 EDKRHSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA Sequence ID 30 HSQGTFTSDYSKYLDSRRAQDFVQWLMNT Sequence ID 31 QGTFTSDYSKYLDSRRAQDFVQWLMNT Sequence ID 32 GTFTSDYSKYLDSRRAQDFVQWLMNT Sequence ID 33 YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ Sequence ID 34 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG Sequence ID 35 EGTFTSDVSSYLEGQAAKEFIAWLVKGRG Sequence ID 36 MKSIYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNED KRHSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAKRHDEFERHAE GTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAIVEELGRRHADGS FSDEMNTILDNLAARDFINWLIQTKITDRK This application also relates to the following aspects. (1) A polypeptide molecule that binds to the N-terminal regions of human oxytomodulin (SEQ ID NO: 25) and human glucagon (SEQ ID NO: 30), and comprises a complementarity-determining region (CDR) as shown in SEQ ID NOs: 1 to 6. (2) The polypeptide molecule according to (1) above, wherein the polypeptide molecule is an antibody. (3) The polypeptide molecule according to (1), wherein the polypeptide molecule is scFv or Fab. (4) The polypeptide molecule according to (1) or (2), wherein the polypeptide molecule is an antibody comprising a heavy chain variable region (VH) containing SEQ ID NO: 7 and a light chain variable region (VL) containing SEQ ID NO: 8. (5) The polypeptide molecule according to any one of (1), (2), or (4), wherein the polypeptide molecule is an antibody comprising a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO: 10. (6) The polypeptide molecule according to any one of (1), (2), (4), or (5), wherein the polypeptide molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 9 and a light chain consisting of SEQ ID NO: 10. (7) A nucleic acid molecule comprising one or both of the first nucleic acid sequence encoding sequence number 9 and the second nucleic acid sequence encoding sequence number 10. (8) The nucleic acid molecule according to (7), wherein the first nucleic acid sequence includes sequence number 11 and the second nucleic acid sequence includes sequence number 12. (9) A vector comprising one or both of the following: a first nucleic acid sequence encoding sequence number 9 and a second nucleic acid sequence encoding sequence number 10. (10) A composition comprising the vector described in (9) above. (11) A cell containing the vector described in (9) above. (12) The cell according to (11) above, wherein the cell is a mammalian cell. (13) A process for producing a polypeptide molecule, comprising: culturing a cell containing one or both of a first nucleic acid sequence encoding SEQ ID NO: 9 and a second nucleic acid sequence encoding SEQ ID NO: 10 under conditions such that the polypeptide molecule is expressed; and recovering the expressed polypeptide molecule from the culture medium. (14) A polypeptide molecule produced by the process described in (13) above. (15) A composition comprising a polypeptide molecule as described in any one of the above items (1) to (6) and (14). (16) A polypeptide molecule that binds to the C-terminal region of human oxytomodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28), and comprises a complementarity-determining region (CDR) as shown in SEQ ID NOs: 13-18. (17) The polypeptide molecule according to (16), wherein the polypeptide molecule is an antibody. (18) The polypeptide molecule according to (16), wherein the polypeptide molecule is scFv or Fab. (19) The polypeptide molecule according to (16) or (17), wherein the polypeptide molecule is an antibody comprising VH containing SEQ ID NO: 19 and VL containing SEQ ID NO: 20. (20) The polypeptide molecule according to any one of claims (16), (17), or (19), wherein the polypeptide molecule is an antibody comprising a heavy chain containing SEQ ID NO: 21 and a light chain containing SEQ ID NO: 22. (21) The polypeptide molecule according to any one of (16), (17), (19), or (20), wherein the polypeptide molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 21 and a light chain consisting of SEQ ID NO: 22. (22) A nucleic acid molecule comprising one or both of the first nucleic acid sequence encoding SEQ ID NO: 21 and the second nucleic acid sequence encoding SEQ ID NO: 22. (23) The nucleic acid molecule according to (22), wherein the first nucleic acid sequence includes sequence number 23 and the second nucleic acid sequence includes sequence number 24. (24) A vector comprising one or both of the following: a first nucleic acid sequence encoding sequence number 23 and a second nucleic acid sequence encoding sequence number 24. (25) A composition comprising the vector described in (24) above. (26) A cell containing the vector described in (24) above. (27) The cell according to (26) above, wherein the cell is a mammalian cell. (28) A process for producing a polypeptide molecule, comprising: culturing a cell containing one or both of a first nucleic acid sequence encoding SEQ ID NO: 21 and a second nucleic acid sequence encoding SEQ ID NO: 22 under conditions such that the polypeptide molecule is expressed; and recovering the expressed polypeptide molecule from the culture medium. (29) A polypeptide molecule produced by the process described in (28) above. (30) A composition comprising a polypeptide molecule as described in any one of the preceding items (16) to (21) or (29). (31) A composition comprising: (a) a first polypeptide molecule that binds to the N-terminal region of human oxyntmodulin (SEQ ID NO: 25) and human glucagon (SEQ ID NO: 30), the first polypeptide molecule comprising the CDR shown in SEQ ID NOs: 1 to 6; and (b) a second polypeptide molecule that binds to the C-terminal region of human oxyntmodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28), the second polypeptide molecule comprising the CDR shown in SEQ ID NOs: 13 to 18. (32) A method for determining the concentration of human oxytomodulin (SEQ ID NO: 25) in a liquid sample, (a) Contacting a liquid sample containing human oxyntmodulin with a first polypeptide molecule bound to the C-terminal region of human oxyntmodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28), wherein the first polypeptide molecule contains the CDRs of SEQ ID NOs: 13-18, thereby forming a first polypeptide molecule-human oxyntmodulin complex. (b) Contacting the first polypeptide molecule-human oxyntmodulin complex with a second polypeptide molecule bound to the N-terminal region of human oxyntmodulin (SEQ ID NO: 25) and human glucagon (SEQ ID NO: 30), wherein the second polypeptide molecule contains the CDRs of SEQ ID NOs: 1-6, thereby forming the first polypeptide molecule-human oxyntmodulin-second polypeptide molecule complex. (c) A method comprising quantifying the amount of oxynmodulin in the first polypeptide molecule-human oxynmodulin-second polypeptide molecule complex by comparison with a standard curve of a known amount of human oxynmodulin (SEQ ID NO: 25). (33) The method according to (32), wherein the liquid sample is serum or plasma. (34) The method according to any one of (31) to (33), wherein the first polypeptide molecule is an antibody and the second polypeptide molecule is an antibody. (35) The method according to any one of claims (31) to (34), wherein the first polypeptide molecule is an antibody comprising VH containing SEQ ID NO: 19 and VL containing SEQ ID NO: 20, and the second polypeptide molecule is an antibody comprising VH containing SEQ ID NO: 7 and VL containing SEQ ID NO: 8. (36) The method according to any one of claims (31) to (34), wherein the first polypeptide molecule is an antibody comprising a heavy chain containing SEQ ID NO: 21 and a light chain containing SEQ ID NO: 22, and the second polypeptide molecule is an antibody comprising a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO: 10. (37) The method according to any one of (30) to (34), wherein the first polypeptide molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 21 and a light chain consisting of SEQ ID NO: 22, and the second polypeptide molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 9 and a light chain consisting of SEQ ID NO: 10. (38) In determining the amount of oxyntomodulin in a liquid sample, a first polypeptide molecule that binds to the C-terminal region of human oxyntomodulin and human glycentene, respectively, and The use of a second polypeptide molecule that binds to the N-terminal region of human oxytomodulin and human glucagon, respectively. (39) A method for predicting whether a subject is at risk of developing type 2 diabetes, comprising determining the amount of oxymodulin in a serum or plasma sample from the subject by: (a) Contacting a serum or plasma sample containing human oxyntmodulin with a first polypeptide molecule bound to the C-terminal region of human oxyntmodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28), wherein the first polypeptide molecule contains SEQ ID NOs: 13-18, thereby forming a first polypeptide molecule-human oxyntmodulin complex. (b) Contacting the first polypeptide molecule-human oxyntmodulin complex with the N-terminal region of the second polypeptide molecules of human oxyntmodulin (SEQ ID NO: 25) and human glucagon (SEQ ID NO: 30), wherein the second polypeptide molecules include SEQ ID NOs: 1 to 6, thereby forming the first polypeptide molecule-human oxyntmodulin-second polypeptide molecule complex, and (c) Quantify the amount of oxynmodulin in the first polypeptide molecule-human oxynmodulin-second polypeptide molecule complex by comparing it to a standard curve of a known amount of human oxynmodulin (SEQ ID NO: 25). (40) The method according to (37) above, wherein the subject has been diagnosed with acute pancreatitis. (41) The method according to (39) or (40), wherein the first polypeptide molecule is an antibody and the second polypeptide molecule is an antibody. (42) The method according to any one of claims (39) to (41), wherein the first polypeptide molecule is an antibody comprising a variable weight region comprising SEQ ID NO: 19 and a variable light region comprising SEQ ID NO: 20, and the second polypeptide molecule is an antibody comprising a variable weight region comprising SEQ ID NO: 7 and a variable light region comprising SEQ ID NO: 8. (43) The method according to any one of claims (39) to (42), wherein the first polypeptide molecule is an antibody comprising a heavy chain containing SEQ ID NO: 21 and a light chain containing SEQ ID NO: 22, and the second polypeptide molecule is an antibody comprising a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO: 10. (44) The method according to any one of claims (39) to (43), wherein the first polypeptide molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 21 and a light chain consisting of SEQ ID NO: 22, and the second polypeptide molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 9 and a light chain consisting of SEQ ID NO: 10.

Claims

1. (a) A first polypeptide molecule that binds to the N-terminal region of human oxytomodulin (SEQ ID NO: 25) and human glucagon (SEQ ID NO: 30), Heavy chains including CDR1, CDR2, and CDR3, as indicated by SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, The first polypeptide molecule comprises light chains including CDR1, CDR2, and CDR3, as indicated by SEQ ID NOs: 4, 5, and 6, respectively, (b) A second polypeptide molecule that binds to the C-terminal region of human oxynmodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28), comprising a heavy chain containing CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, The second polypeptide molecule comprises a light chain containing CDR1, CDR2, and CDR3, as indicated by SEQ ID NOs: 16, 17, and 18, respectively. The aforementioned N-terminal region refers to the first three amino acid residues in a polypeptide molecule where the N-terminal histidine is located. The C-terminal region refers to amino acid residues 30-37 in human oxyntomodulin or amino acid residues 61-69 in human glycentin (SEQ ID NO: 28), A composition in which the first polypeptide molecule and the second polypeptide molecule are antibodies or antibody fragments.

2. A method for determining the concentration of human oxytomodulin (SEQ ID NO: 25) in a liquid sample, (a) A step of contacting a liquid sample containing human oxyntmodulin with a second polypeptide molecule bound to the C-terminal region of human oxyntmodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28), wherein the second polypeptide molecule comprises a heavy chain including CDR1, CDR2, and CDR3, as indicated by SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, It contains light chains comprising CDR1, CDR2, and CDR3, as indicated by SEQ ID NOs: 16, 17, and 18, respectively, thereby forming a second polypeptide molecule-human oxyntomodulin complex. The second polypeptide molecule is an antibody or antibody fragment, (b) A step of contacting the second polypeptide molecule-human oxyntmodulin complex with a first polypeptide molecule bound to the N-terminal region of human oxyntmodulin (SEQ ID NO: 25) and human glucagon (SEQ ID NO: 30), wherein the first polypeptide molecule comprises a heavy chain including CDR1, CDR2, and CDR3, as indicated by SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, It contains light chains comprising CDR1, CDR2, and CDR3, as indicated by SEQ ID NOs: 4, 5, and 6, respectively, thereby forming a first polypeptide molecule-human oxynmodulin-second polypeptide molecule complex. The second polypeptide molecule is an antibody or antibody fragment, step, (c) The step of quantifying the amount of oxynmodulin in the first polypeptide molecule-human oxynmodulin-second polypeptide molecule complex by comparison with a standard curve of a known amount of human oxynmodulin (SEQ ID NO: 25), The C-terminal region refers to amino acid residues 30-37 in human oxyntomodulin or amino acid residues 61-69 in human glycentin (SEQ ID NO: 28), The N-terminal region refers to the first three amino acid residues in a polypeptide molecule where the N-terminal histidine is located.

3. The method according to claim 2, wherein the liquid sample is serum or plasma.

4. The method according to any one of claims 2 or 3, wherein the second polypeptide molecule is an antibody, and the first polypeptide molecule is an antibody.

5. The method according to any one of claims 2 to 4, wherein the second polypeptide molecule is an antibody comprising VH containing SEQ ID NO: 19 and VL containing SEQ ID NO: 20, and the first polypeptide molecule is an antibody comprising VH containing SEQ ID NO: 7 and VL containing SEQ ID NO:

8.

6. The method according to any one of claims 2 to 4, wherein the second polypeptide molecule is an antibody comprising a heavy chain containing SEQ ID NO: 21 and a light chain containing SEQ ID NO: 22, and the first polypeptide molecule is an antibody comprising a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO:

10.

7. The method according to any one of claims 2 to 4, wherein the second polypeptide molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 21 and a light chain consisting of SEQ ID NO: 22, and the first polypeptide molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 9 and a light chain consisting of SEQ ID NO:

10.

8. Use of the composition according to claim 1 in determining the amount of oxytomodulin in a liquid sample.

9. A method for predicting whether a subject is at risk of developing type 2 diabetes, comprising determining the amount of oxymodulin in a serum or plasma sample from the subject by: (a) A serum or plasma sample containing human oxyntomodulin is brought into contact with a second polypeptide molecule bound to the C-terminal region of human oxyntomodulin (SEQ ID NO: 25) and human glycentin (SEQ ID NO: 28), The second polypeptide molecule comprises a heavy chain containing CDR1, CDR2, and CDR3, as indicated by SEQ ID NOs: 13, 14, and 15, respectively, It contains light chains comprising CDR1, CDR2, and CDR3, as indicated by SEQ ID NOs: 16, 17, and 18, respectively, thereby forming a second polypeptide molecule-human oxyntomodulin complex. The C-terminal region refers to amino acid residues 30-37 in human oxyntomodulin or amino acid residues 61-69 in human glycentin (SEQ ID NO: 28), The second polypeptide molecule is an antibody or antibody fragment. (b) The step of contacting the second polypeptide molecule-human oxyntmodulin complex with the N-terminal region of the first polypeptide molecule of human oxyntmodulin (SEQ ID NO: 25) and human glucagon (SEQ ID NO: 30), wherein the first polypeptide molecule Heavy chains including CDR1, CDR2, and CDR3, as indicated by SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, It contains light chains comprising CDR1, CDR2, and CDR3, as indicated by SEQ ID NOs: 4, 5, and 6, respectively, thereby forming a second polypeptide molecule-human oxynmodulin-first polypeptide molecule complex. The N-terminal region of the first polypeptide molecule refers to the first three amino acid residues in the polypeptide molecule where the N-terminal histidine is located. The first polypeptide molecule is an antibody or antibody fragment, and (c) A step of quantifying the amount of oxynmodulin in the second polypeptide molecule-human oxynmodulin-first polypeptide molecule complex by comparison with a standard curve of a known amount of human oxynmodulin (SEQ ID NO: 25).

10. The method according to claim 7, wherein the subject has been diagnosed with acute pancreatitis.

11. The method according to claim 9 or 10, wherein the second polypeptide molecule is an antibody, and the first polypeptide molecule is an antibody.

12. The method according to any one of claims 9 to 11, wherein the second polypeptide molecule is an antibody comprising a variable weight region comprising SEQ ID NO: 19 and a variable light region comprising SEQ ID NO: 20, and the first polypeptide molecule is an antibody comprising a variable weight region comprising SEQ ID NO: 7 and a variable light region comprising SEQ ID NO:

8.

13. The method according to any one of claims 9 to 12, wherein the second polypeptide molecule is an antibody comprising a heavy chain containing SEQ ID NO: 21 and a light chain containing SEQ ID NO: 22, and the first polypeptide molecule is an antibody comprising a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO:

10.

14. The method according to any one of claims 9 to 13, wherein the second polypeptide molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 21 and a light chain consisting of SEQ ID NO: 22, and the first polypeptide molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 9 and a light chain consisting of SEQ ID NO: 10.

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