Compounds and methods targeting human and mouse INSL5

Abstract

To provide compounds and methods targeting insulin-like peptide-5 (INSL5), including antibodies, to provide cells and vectors comprising DNA encoding the same, and to provide methods for producing antibodies.SOLUTION: In one aspect, provided is an antibody that binds to insulin-like peptide-5 comprising a VH and a VL, the VH comprising HCDR1, HCDR2, and HCDR3, the VL comprising LCDR1, LCDR2, and LCDR3, the HCDR1 comprising TVSGIDLTTYAMG, the HCDR2 comprising IIGGGGRTY, the HCDR3 comprising VRGGDFFDL, the LCDR1 comprising QASEDISKYLS, the LCDR2 comprising YYVSNLEF, and the LCDR3 comprising HQGYTGVNVENV.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of medicine. More specifically, the present invention relates to compounds, diagnostic methods, and methods comprising antibodies or fragments thereof against human and mouse insulin-like peptide-5 (INSL5). The compounds and methods of the present invention are expected to be useful in the fields of oncology, reproductive, and metabolic diseases, such as diabetes and obesity, including for the diagnosis thereof. [Background technology]

[0002] The gut hormone INSL5 belongs to the relaxin / insulin-like family of peptides. Like other INSL family members, INSL5 consists of a B chain and an A chain linked by two disulfide bonds. Although INSL5 is expressed in various tissues, it is primarily co-secreted with other hormones by specialized enteroendocrine cells (EECs) called L cells in the distal intestine, particularly the colon and rectum. These cells regulate metabolic and physiological processes, including intestinal motility, hormone secretion, glucose homeostasis, and appetite. INSL5 is a ligand for the receptor RXFR4 / GPCR142 / GPR100, and this receptor-ligand interaction results in the inhibition of intracellular cAMP levels. INSL5 is considered an orexigenic (appetite-stimulating) hormone, and its secretion is increased by caloric restriction; however, the biological consequences of the INSL5-RXFP4-activated signaling pathway remain largely elusive.

[0003] To study INSL5, a reliable and sensitive assay for INSL5 is needed. INSL5 has been studied in patients with metabolic dysfunction, polycystic ovary syndrome (PCOS), colorectal cancer (CRC), infertility, and in preclinical models of these diseases. However, there remains a need to provide antibodies that bind to human and mouse INSL5 to further study these effects. In particular, there remains a need for INSL5 antibodies with high affinity to both human and mouse INSL5. Currently available assays have limitations in sensitivity, reproducibility, performance, and / or quantitation. Therefore, additional methods for testing human or mouse samples are needed. Summary of the Invention

[0004] In certain embodiments of the present disclosure, an antibody that binds to INSL5 is provided, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises TASGFSLSSYDMG (SEQ ID NO: 10), HCDR2 comprises TISAGGYTY (SEQ ID NO: 11), HCDR3 comprises ARERWNYDRSGGAGAGYFDL (SEQ ID NO: 12), LCDR1 comprises QASQSITSSYLS (SEQ ID NO: 14), LCDR2 comprises YPAANLAS (SEQ ID NO: 15), and LCDR3 comprises LYGYFSSSIDFA (SEQ ID NO: 16).

[0005] According to some embodiments, an antibody of the present disclosure comprises a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 13. According to some embodiments, an antibody of the present disclosure comprises a heavy chain (HC) comprising SEQ ID NO: 5 and a light chain (LC) comprising SEQ ID NO: 7. According to some embodiments, an antibody of the present disclosure comprises a heavy chain (HC) comprising amino acids 2-446 of SEQ ID NO: 5, and a light chain (LC) comprising SEQ ID NO: 7. According to some embodiments, an antibody of the present disclosure comprises a HC consisting of SEQ ID NO: 5 and a LC consisting of SEQ ID NO: 7.

[0006] According to some embodiments, the present disclosure provides a nucleic acid comprising a sequence encoding SEQ ID NO:5 or SEQ ID NO:7, or both. According to some embodiments, one or more vectors comprise one or more nucleic acids encoding SEQ ID NO:5 or SEQ ID NO:7, or both. According to some embodiments, the present disclosure provides a composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:5 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:7. According to other embodiments, the present disclosure provides a composition comprising a vector comprising a nucleic acid encoding SEQ ID NO:5 and SEQ ID NO:7. In another embodiment, the present disclosure provides a cell comprising one or more vectors comprising one or more nucleic acids encoding SEQ ID NO:5 or SEQ ID NO:7, or both. According to some embodiments, the present disclosure provides a cell comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:5 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:7. According to other embodiments, the present disclosure provides a cell comprising a vector comprising a nucleic acid encoding SEQ ID NO:5 and a second nucleic acid sequence encoding SEQ ID NO:7. In a further embodiment, the cell is a mammalian cell.

[0007] In certain embodiments of the present disclosure, an antibody that binds to INSL5 is provided, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises TVSGIDLTTYAMG (SEQ ID NO: 22), HCDR2 comprises IIGGGGRTY (SEQ ID NO: 23), HCDR3 comprises VRGGDFFDL (SEQ ID NO: 24), LCDR1 comprises QASEDISKYLS (SEQ ID NO: 26), LCDR2 comprises YYVSNLEF (SEQ ID NO: 27), and LCDR3 comprises HQGYTGVNVENV (SEQ ID NO: 28).

[0008] According to some embodiments, an antibody of the disclosure comprises a VH comprising SEQ ID NO: 21 and a VL comprising SEQ ID NO: 25. In further embodiments, an antibody of the disclosure comprises a heavy chain (HC) comprising SEQ ID NO: 17 and a light chain (LC) comprising SEQ ID NO: 19. In some embodiments, an antibody of the disclosure comprises a heavy chain (HC) comprising amino acids 2-435 of SEQ ID NO: 17, and a light chain (LC) comprising SEQ ID NO: 19. According to some embodiments, an antibody of the disclosure comprises a HC consisting of SEQ ID NO: 17 and a LC consisting of SEQ ID NO: 19.

[0009] According to some embodiments, the present disclosure provides a nucleic acid comprising a sequence encoding SEQ ID NO: 17 or SEQ ID NO: 19, or both. According to some embodiments, one or more vectors comprise one or more nucleic acids encoding SEQ ID NO: 17 or SEQ ID NO: 19, or both. According to some embodiments, the present disclosure provides a composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 17 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 19. According to some embodiments, the present disclosure provides a composition comprising a vector comprising a nucleic acid encoding SEQ ID NO: 17 and SEQ ID NO: 19. In another embodiment, the present disclosure provides a cell comprising one or more vectors comprising one or more nucleic acids encoding SEQ ID NO: 17 or SEQ ID NO: 19, or both. In some embodiments, the present disclosure provides a cell comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 17 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 19. According to other embodiments, the present disclosure provides a cell comprising a vector comprising a nucleic acid encoding SEQ ID NO: 17 and a second nucleic acid sequence encoding SEQ ID NO: 19. In a further embodiment, the cell is a mammalian cell.

[0010] According to some embodiments, the antibodies of the present disclosure bind to human INSL5. According to some embodiments, the antibodies of the present disclosure bind to the A chain (SEQ ID NO: 1) and B chain (SEQ ID NO: 2) of human INSL5. According to some embodiments, the antibodies of the present disclosure bind to mouse INSL5. According to some embodiments, the antibodies of the present disclosure bind to the A chain (SEQ ID NO: 3) and B chain (SEQ ID NO: 4) of mouse INSL5.

[0011] According to some embodiments, the invention provides methods of producing an antibody comprising culturing cells under conditions in which the antibody is expressed and then recovering the expressed antibody from the culture medium. According to some embodiments, the invention provides methods of producing an antibody produced by culturing cells under conditions in which the antibody is expressed and then recovering the expressed antibody from the culture medium.

[0012] According to further embodiments of the present disclosure, there is provided a method for detecting human or mouse INSL5 in a patient sample, comprising contacting the sample with an antibody of the present disclosure that specifically binds to human or mouse INSL5, and detecting a signal resulting from the contacting step.

[0013] According to some embodiments, there is provided a method for quantifying human or mouse INSL5 in a sample. Such a method comprises contacting the sample with an antibody of the present disclosure that specifically binds to INSL5 and detecting a signal provided by the contacting step. In some embodiments, such a method further comprises contacting a control standard with the antibody and detecting a signal provided by the contacting of the control standard.

[0014] According to some embodiments of the methods of the present disclosure, such methods further comprise quantifying INSL5 in the sample, hi such embodiments, quantifying INSL5 comprises quantifying INSL5 in the sample relative to a reference standard.

[0015] According to some embodiments of the methods of the present disclosure, the sample is one of blood, plasma, serum, or cerebrospinal fluid (CSF).

[0016] According to some embodiments of the methods of the present disclosure, the methods further include contacting the sample with an antibody that specifically binds to human or mouse INSL5 and a second antibody, where the second antibody also binds to human or mouse INSL5. In some such methods, one of the antibody or the second antibody comprises a detectable label, and the detecting step comprises detecting a signal provided by the detectable label upon formation of a complex comprising the antibody, the second antibody, and human or mouse INSL5. According to some such embodiments, the antibody and one of the second antibody are immobilized on a substrate. In some embodiments of the methods of the present disclosure, the steps of contacting the sample with the antibody and contacting the sample with the second antibody occur simultaneously. According to some more specific embodiments, the second antibody comprises an antibody of the present disclosure that specifically binds to human or mouse INSL5 as disclosed herein.

[0017] According to some embodiments of the disclosed methods, the method further comprises contacting the sample with an antibody that specifically binds to INSL5 and a second antibody, where the second antibody also specifically binds to INSL5. In some such methods, one of the antibody or the second antibody comprises a detectable label, and the detecting step comprises detecting a signal provided by the detectable label upon formation of a complex comprising the antibody, the second antibody, and INSL5. According to some such embodiments, one of the antibody and the second antibody is immobilized on a substrate. In other embodiments, the second antibody binds to a different epitope of INSL5. In some embodiments of the disclosed methods, the steps of contacting the sample with the antibody and contacting the patient sample with the second antibody occur simultaneously. In any such embodiment, the antibody or the second antibody, or both, are antibodies that bind to INSL5 as disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0018] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Antibody embodiments include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, or chimeric antibodies. The antibody may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0019] An exemplary antibody of the present disclosure is an immunoglobulin G (IgG)-type antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 amino acids or more that is primarily responsible for antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region that is primarily responsible for effector function. Each heavy chain is composed of a heavy chain variable region (HB) and a heavy chain constant region. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. IgG isotypes can be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0020] The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). The CDRs are exposed on the surface of the protein and are critical regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Herein, 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." The CDRs contain most of the residues that form specific interactions with the antibody. The assignment of amino acid residues to CDRs is based on the work of 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)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 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).The assignment of amino acids to the CDR domains within the LCVR and HCVR regions of the antibodies of the present invention is based on North. According to some embodiments of the present disclosure, the LC is classified as kappa or lambda, each characterized by a specific constant region known in the art. According to some embodiments of the present disclosure, the HC is classified as gamma, mu, alpha, delta, or epsilon, defining the antibody's isotype as IgG, IgM, IgA, IgD, or IgE, respectively. According to some embodiments, the antibody comprises an IgG HC, which can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, and IgG4. The carboxy-terminal portion of each HC defines a constant region primarily responsible for effector function. In certain embodiments, the antibodies of the present disclosure have one or more modifications within the constant region of each HC that reduce effector function.

[0021] The antibodies of the present invention are monoclonal antibodies. A monoclonal antibody is an antibody that is derived from a single copy or clone (including, for example, any eukaryotic, prokaryotic, or phage clone), and not the method by which it is produced. Monoclonal antibodies can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic techniques such as CDR grafting, or a combination of such techniques or other techniques known in the art.

[0022] Methods for producing and purifying antibodies are well known in the art and can be found, for example, in Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 5-8 and 15, ISBN 0-87969-314-2. For example, mice or rabbits can be immunized with human or mouse INSL5, and the resulting antibodies can be recovered and purified, and the amino acid sequence can be determined using conventional methods well known in the art. Similarly, thousands of Fab fragments can be screened for interaction with human or mouse INSL5, and the resulting interacting antibodies can be recovered and purified, and the amino acid sequence can be determined using conventional methods well known in the art, to construct initial lead antibodies.

[0023] In certain embodiments of the invention, the antibody, or nucleic acid encoding the antibody, is provided in isolated form. As used herein, the term "isolated" refers to a protein, peptide, or nucleic acid that is free or substantially free from other macromolecular species that are found in a cellular environment.

[0024] The term "bind," as used herein, unless otherwise specified, refers to the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, bringing the two proteins or molecules into proximity as determined by common methods known in the art.

[0025] Anti-INSL5 antibodies of the present disclosure that bind to human or mouse INSL5 can be used to isolate and / or detect isoforms of human or mouse INSL5 by techniques such as affinity chromatography, immunoprecipitation, immunohistochemistry, or ELISA-based assays. Such assays can be used to detect and / or assess the abundance and / or pattern of INSL5, e.g., to monitor polypeptide levels in serum, plasma, blood, or CSF as part of a clinical testing procedure; to determine the effectiveness of a given therapeutic regimen; or for diagnostic, prognostic, or therapeutic purposes. As understood in the art, antibodies of the present invention can be conjugated to a detectable substance or label to facilitate their detection. Examples of detectable substances or labels include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, chemiluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichloronaphthalene fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, ruthenium, and aequorin; examples of suitable radioactive materials include 125 I, 131 I, 35 S or 3 H. The antibodies of the present invention may also be useful in pharmacogenomic analyses. Such embodiments may be used to identify individuals who may benefit from a particular or modified therapeutic approach and / or to monitor the effectiveness of current therapeutic regimens.

[0026] As used herein, "control standard" refers to a sample that can be used to compare results obtained from a sample in the methods of the present invention. A control standard can be a concentration of a known protein spiked into cells, blood, plasma, CSF, tissue, or culture medium. The concentration level in the control standard can be an absolute or relative amount, a range of amounts, or a minimum, average, and / or median amount of INSL5. A control standard can also serve as a baseline for INSL5 against which patient samples are compared. A control standard can include concentration values ​​from the same patient or a known normal reference for INSL5. Furthermore, in some embodiments, a control standard can represent INSL5 concentrations in the form of a standard curve.

[0027] As used herein, the term "capture antibody" refers to an antibody that binds to INSL5. In such embodiments, the capture antibody can bind to and capture INSL5 such that the capture antibody-INSL5 complex can be separated from the remainder of the sample, e.g., can specifically bind to INSL5 in a sample under suitable conditions. In some embodiments, the capture antibody can be an antibody that specifically binds to INSL5, and the antibody that specifically binds to INSL5 is used as a secondary (or detection) antibody. In further embodiments, the capture antibody is an antibody provided herein. In further embodiments, the capture antibody is an antibody having an HCDR corresponding to SEQ ID NOS: 10-12 and an LCDR corresponding to SEQ ID NOS: 14-16, respectively. In further embodiments, the capture antibody is an antibody having an HCDR corresponding to SEQ ID NOS: 22-24 and an LCDR corresponding to SEQ ID NOS: 26-28, respectively. In some embodiments, the capture antibody is immobilized. In some embodiments, the detection antibody is labeled with a detectable label. In some embodiments, the secondary (or detection) antibody is an antibody provided herein. In a further embodiment, the secondary (or detection) antibody is an antibody having an HCDR corresponding to SEQ ID NOs: 10 to 12 and an LCDR corresponding to SEQ ID NOs: 14 to 16, respectively. In a further embodiment, the secondary (or detection) antibody is an antibody having an HCDR corresponding to SEQ ID NOs: 22 to 24 and an LCDR corresponding to SEQ ID NOs: 26 to 28, respectively.

[0028] In some embodiments, the capture antibody is immobilized in a "sandwich" immunoassay, and the capture or primary antibody specifically binds to human or mouse INSL5. In some such embodiments, the capture antibody is an antibody of the present invention that specifically binds to human or mouse INSL5. In further embodiments, the capture antibody is an antibody having an HCDR corresponding to SEQ ID NOs: 10-12 and an LCDR corresponding to SEQ ID NOs: 14-16. In further embodiments, the capture antibody is an antibody having an HCDR corresponding to SEQ ID NOs: 22-24 and an LCDR corresponding to SEQ ID NOs: 26-28. In such sandwich immunoassays, a "detection (or secondary) antibody" is also utilized. According to some embodiments, the detection or secondary antibody is capable of specifically binding to the capture antibody and may be labeled with a detectable label. In some embodiments, the detection secondary antibody specifically binds to human or mouse INSL5 that has already been bound or captured by the capture or primary antibody. In some such embodiments, the secondary antibody is an antibody of the present invention that specifically binds to human or mouse INSL5. In some embodiments, the secondary (or detection) antibody is an antibody provided herein. In a further embodiment, the secondary (or detection) antibody is an antibody having an HCDR corresponding to SEQ ID NOs: 10 to 12 and an LCDR corresponding to SEQ ID NOs: 14 to 16. In a further embodiment, the secondary (or detection) antibody is an antibody having an HCDR corresponding to SEQ ID NOs: 22 to 24 and an LCDR corresponding to SEQ ID NOs: 26 to 28.

[0029] As used herein, a "detectable label" is a moiety, composition, or technique that can be used to detect the formation of a complex between an antibody of the invention that specifically binds to the A and B chains of human or mouse INSL5. According to some embodiments, the detectable label can be directly or indirectly conjugated to the antibody (either capture or detection, as the case may be). Exemplary embodiments of detectable labels include biotin, radioisotopes, fluorophores or other fluorescent moieties, and enzymatic moieties.

[0030] Antigen-binding fragments of such antibodies include, for example, Fab fragments, Fab' fragments, F(ab')2 fragments, and single-chain Fv fragments.

[0031] A "framework region" or "framework sequence" refers to any one of framework regions 1-4. Humanized antibodies and antigen-binding fragments thereof encompassed by the present invention include molecules in which any one or more of framework regions 1-4 are humanized, i.e., any possible combination of individual humanized regions 1-4 is present. For example, this includes molecules in which framework region 1 and framework region 2, framework region 1 and framework region 3, framework regions 1, 2, and 3, etc. are humanized. A humanized framework is one that has at least about 80% sequence identity to a known human germline framework sequence. Germline sequences of human frameworks can be obtained from ImMunoGeneTics (IMGT) or from The 20 Immunoglobulin Facts Book by Marie-Paule Lefranc and Gerard Lefranc (Academic Press, 2001, ISBN 012441351). For example, the germline light chain framework can be selected from the group consisting of A11, A17, A18, A19, A20, A27, A30, L1, L11, L12, L2, L5, L15, L6, L8, O12, O2, and O8, and the germline heavy chain framework region can be selected from the group consisting of VH2-5, VH2-26, VH2-70, VH3-20, VH3-72, VH1-46, VH3-9, VH3-66, VH3-74, VH4-31, VH1-18, VH1-69, VH1-13-7, VH3-11, VH3-15, VH3-21, VH3-23, VH3-30, VH3-48, VH4-39, VH4-59, and VH5-51.

[0032] The protein "INSL5" (also known as insulin-like peptide-5) refers to a gastrointestinal hormone encoded by the Insl5 gene. INSL5 is a peptide produced in enteroendocrine cells of the distal colon in humans and mice.

[0033] The results of the following assays demonstrate that the exemplified monoclonal antibodies of the invention and antigen-binding fragments thereof bind to and / or neutralize INSL5 and therefore can be used in diagnostic assays, including but not limited to sandwich ELISA assays, to study the role of INSL5 in metabolic diseases, including but not limited to diabetes and obesity.

[0034] Disclosed herein are antibodies for use in diagnostics, such as in diagnostic assays to detect the presence or level of INSL5. [Example]

[0035] The following non-limiting examples are offered by way of illustration and not limitation.

[0036] Example 1: Recombinant expression of Antibody I Antibody I is an antibody having a heavy chain amino acid sequence of SEQ ID NO:5 and a light chain amino acid sequence of SEQ ID NO:7.

[0037] Antibody I is produced in a mammalian cell expression system using a CHO-K1 cell derivative (Lonza Biologics Inc.). cDNA sequences encoding SEQ ID NO:5 and SEQ ID NO:7 are subcloned into a GS-containing expression plasmid backbone (pEE12.4-based plasmid, Lonza Biologics Inc.). The cDNA sequences are fused in frame with the coding sequence for the signal peptide sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO:29) to enhance secretion of the antibody into tissue culture medium. Expression of both cDNA sequences is driven by the viral CMV promoter.

[0038] For antibody production via transient transfection, CHO-K1 cells are transfected with recombinant expression plasmids at the same stoichiometric ratio using a PEI-based method. Briefly, at a density of 4 x 10 6 An appropriate volume of CHO-K1 suspension cells, in cells / mL, is transferred to a shake flask and both PEI and recombinant plasmid DNA are added to the cells. The cells are incubated in suspension culture at 32°C for 6 days. At the end of the incubation period, the cells are removed by low-speed centrifugation and the antibody is purified from the conditioned medium.

[0039] Antibodies secreted into the medium from CHO-K1 cells are purified by Protein A affinity chromatography followed by size exclusion chromatography (SEC) and / or ion exchange chromatography. Specifically, antibodies from harvested medium are captured on Mab Select Protein A resin (GE). The resin is then briefly washed with a running buffer, such as phosphate-buffered saline (PBS, pH 7.4) or a Tris-containing buffer, to remove nonspecifically bound material. The protein is eluted from the resin with a low pH solution, such as 10 mM citric acid, pH 3. Fractions containing the antibody can be pooled at low pH to inactivate potential viruses. The pH can be neutralized by adding a base, such as 0.1 M Tris, pH 8.0. The antibody is further purified by SEC by loading the concentrated Protein A pool onto a Superdex 200 (GE Healthcare) in PBS, pH 7.4, using isocratic elution. The antibody can be further purified by an ion exchange chromatography step using a resin such as Poros 50 HS (ThermoFisher). The antibody is then eluted from the column using a 0-500 mM NaCl gradient in 20 mM NaOAc at pH 5.0 over 15 column volumes. The purified antibody may be buffer exchanged into PBS at pH 7.4 and concentrated, for example, by using a centrifugal filter unit (such as an Amicon Ultra 50K centrifugal filter unit) or by tangential flow ultrafiltration on a regenerated cellulose membrane (Millipore).

[0040] Antibodies are therefore prepared in this manner, or in a similar manner readily determined by one of skill in the art.

[0041] Example 2: Recombinant expression of Antibody II Antibody II is an antibody having a heavy chain amino acid sequence of (SEQ ID NO: 17) and a light chain amino acid sequence of (SEQ ID NO: 19).

[0042] Here, SEQ ID NO:17 and SEQ ID NO:19 are produced essentially as described for Example 1, except that cDNA sequences encoding SEQ ID NO:17 and SEQ ID NO:19 are used in expression plasmids.

[0043] In vitro function Example 3: Antibody binding to human and mouse INSL5 via SPR The in vitro binding of the antibodies of Examples 1 and 2 to human and mouse INSL5 is determined by surface plasmon resonance (SPR) at 25° C. and 37° C. In particular, the affinities of the antibodies of Examples 1 and 2 are summarized in Tables 1 and 2 below.

[0044] Binding of the antibodies of Examples 1 and 2 to human and mouse INSL5 was performed using a Biacore 8K (GE Healthcare) instrument. 1x HBS-EP+ (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20) (Teknova) was used as the running buffer. Protein A (Calbiochem No. 539202-5mg) was immobilized on a Series S Sensor Chip CM5 (GE Healthcare) at 25°C according to the manufacturer's instructions (Amine Coupling Kit BR-1000-50). Briefly, the carboxyl groups on the sensor chip surface (flow cells 1 and 2) were activated by injecting 70 μL of a mixture containing 75 mg / mL EDC and 11.5 mg / mL NHS at 10 μL / min. A 50 μg / mL solution of Protein A is prepared by diluting 10 μL of a 5 mg / mL stock solution into 1 mL of 10 mM sodium acetate, pH 4.5. 70 μL of this solution is injected over the activated chip surface (flow cells 1 and 2, channels 1-8) at 10 μL / min for 7 minutes. Excess reactive groups on the surface (flow cells 1 and 2) are then deactivated by injecting 70 μL of 1 M ETA HCl-NaOH, pH 8.5, at 10 μL / min. The chip is then conditioned with six 15-second injections of 10 mM glycine-HCl, pH 1.5, at 30 μL / min.

[0045] Human INSL5 (Phoenix Pharmaceuticals catalog number 035-70A) and mouse INSL5 (Phoenix Pharmaceuticals 035-40) are reconstituted in DMSO to 0.5 (99 μM) or 1 mg / mL (195 μM), respectively. A two-fold dilution series of the peptides is prepared in 1×HBS-EP+ buffer at concentrations of 20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, and 0 nM. Antibodies from Examples 1 and 2, or negative controls, are prepared by diluting them to 1 μg / mL in 1×HBS-EP+ buffer.

[0046] The experiment was performed at 25°C and 37°C and began with five start-up cycles of 1x HBS-EP+ buffer injections. Then, in each cycle, the antibody was captured on flow cell 2 of channels 1-8 at 10 μL / min for 20 seconds. Next, 180 μL of each INSL5 peptide sample was injected individually across flow cells 1 and 2 at 60 μL / min for 180 seconds, followed by dissociation for 1200 seconds at a flow rate of 60 μL / min. The surface was regenerated by injecting two 15-second pulses of 10 mM glycine-HCl pH 1.5 (BR-1003-54) at 30 μL / min. After a 60-second stabilization period at a flow rate of 60 μL / min, the next cycle began.

[0047] The resulting sensorgrams are analyzed using Biacore 8K Evaluation Software. A 1:1 binding kinetics model fitting is used to determine the binding kinetic parameters on rate (ka), off rate (kd), and equilibrium dissociation constant (K D ) is calculated. [Table 1]

[0048] For antibody I, K at 25°C D is determined to be 41 pM for human INSL5 and 4.6 pM for mouse INSL5. For Antibody II, the K at 25°C D is determined as 21 pM for human INSL5 and 6.1 pM for mouse INSL5. [Table 2]

[0049] For antibody I, K at 37°C D is determined as 110 pM for human INSL5 and 6.7 pM for mouse INSL5. For Antibody II, the K at 37°C D is determined to be 81 pM for human INSL5 and 14 pM for mouse INSL5.

[0050] Example 4: In vitro neutralization of INSL5 activity increases cAMP production In vitro neutralization of human and mouse INSL5 activity by Antibody I and Antibody II is determined using the cAMP Kinetic II Assay (Cisbio Catalog No. 62AM4PEJ). This kit is based on HTRF technology, which measures intracellular cAMP accumulation. Cells stimulated with a fixed concentration of forskolin increase cAMP production. Introducing a fixed concentration of human and / or mouse INSL5 into cells inhibits cAMP production by binding to the RXFP4 receptor. Antibody I and Antibody II bind to and neutralize the effects of human and mouse INSL5 before binding to the RXFP4 receptor. EC values ​​derived from antibody serial dilution data for Antibody I and Antibody II are shown. 50The values ​​are summarized in Table 3 below and show that cAMP production increases with increasing antibody concentration. Cell Plate Preparation: Mouse RXFP4 CHO-K1 cells (DiscoveRx Part No. 93-0929E2) and human CHO RXFP4 cells (DiscoveRx 93-0701E2) are quickly thawed in a 37°C water bath and reconstituted with 500 μL of preheated Cell Plating Reagent 2 (DiscoveRx 93-0563R series) (in a 37°C water bath). The cells are mixed by pipetting up and down and then added to 11.5 mL of preheated Cell Plating Reagent 2 (in a 37°C water bath). The cells are plated at 8,000 cells / well in 100 μL per well in white full-area plates (Corning-Costar 3917). The plates are incubated at 37°C for 48 hours to allow the cells to attach to the plates. After 48 hours, the growth medium is aspirated, and 40 μL of Cell Assay Buffer [HBSS (Hyclone SH30028.03), 20 mM HEPES (Hyclone SH30237.01), 1% FBS (Gibco 10438-026), 1 mM IBMX (Sigma15879, diluted in DMSO)] is added to each well. 30 μL of compound assay buffer (HBSS + 20 mM HEPES + 1% FBS) containing 17 μM forskolin (Sigma Aldrich - CAS number F3917 (10 mM in DMSO)) is diluted into each well.

[0051] Preparation of sample (antibody-INSL5 peptide mixture): Human INSL5 peptide (Phoenix Pharmaceuticals, Cat. No. 035-70) or mouse INSL5 peptide (Phoenix Pharmaceuticals, Cat. No. 035-40) was diluted to the respective IC 80Then, 10 μL of each INSL5 peptide (16 times the final concentration in the assay, i.e., 144 nM for mouse INSL5 and 1280 nM for human INSL5) is mixed with 10 μL of a serial dilution (starting at 160 nM) of antibody from Example 1 or 2 (final antibody starting concentration is 10 nM). The samples are incubated in a PCR plate (Fisherbrand catalog number 14230244) with gentle shaking for 2 hours at room temperature.

[0052] Performing the cell assay: Add 10 μL of the antibody-INSL5 peptide mixture to 70 μL of the buffer mixture in the assay plate (containing the cells) and incubate at room temperature for 1 hour with moderate shaking. During the incubation, prepare the cAMP detection reagent. Dilute 500 μL of cAMP-d2 and Anti-cAMP-Cryptate stock reagent solution (cAMP Dynamic 2 100,000 Test Kit Catalog No. 62AM4PEJ) each in 9.5 mL of Conjugate / Lysis Buffer. After the 1-hour incubation, add 40 μL of the cAMP-d2 detection reagent to each well of the cell assay plate. Then, immediately add 40 μL of the Anti-cAMP-Cryptate detection reagent to the cell assay plate. Incubate the plate with an aluminum seal at room temperature for 1 hour. The plate is then read on a Perkin Elmer Envision, and the results are calculated from the instrument's 665 nm / 620 nm ratio.

[0053] Statistical analysis of data: Data are imported from the Perkin Elmer Envision reader into GraphPad Prism® software (GraphPad Software, LLC, La Jolla, CA). 50 Values ​​are generated by variable slope-four parameter dose-response curve analysis. SEM is the EC between independent experiments. 50 Calculate the standard deviation (n) by dividing by the square root of the number of independent experiments. [Table 3]

[0054] For antibody I, EC 50 is determined as 4.35 nM for human INSL5 and 0.84 nM for mouse INSL5. For antibody II, the EC 50 is determined as 1.27 nM for human INSL5 and 0.45 pM for mouse INSL5.

[0055] Example 5: Thermal stability with TDF The thermal stability of Antibody I and Antibody II is determined by thermal denaturation fluorimetry (TDF). In particular, the melting temperatures (T m ) are reported in Table 4.

[0056] Thermofluorescence assays are performed on a LightCycler 480II PCR machine using SYPRO Orange dye (5000x concentrate, Invitrogen S6651). The excitation and emission filters are set at 465 nm and 580 nm, respectively, and the temperature is increased continuously from 25°C to 95°C at a rate of 1°C / sec. Final assay conditions include 0.2 mg / mL of Example 1 or 2 and 10x SYPRO Orange dye in PBS pH 7.4 (Corning 21-040-CV).

[0057] The experiment is performed by diluting Example 1 or 2 to 0.4 mg / mL and mixing equal volumes with 20x SYPRO Orange dye in PBS pH 7.4 to reach a final volume of 30 μL per sample (BE05746-005). The mixture is dispensed in triplicate 6 μL aliquots into a 384 multiwell assay plate (Roche 04-729-749-001). The T of Examples 1 and 2 m The values ​​are determined by the first derivative method using Thermal Shift Analysis Software (Roche). This analysis software smooths the raw fluorescence data and calculates the T mis collected by determining the temperature at which the slope of the increase in fluorescence versus temperature is greatest (the inflection point). m Values ​​and SD are calculated for each set of triplicates. [Table 4]

[0058] For Antibody I, the Tm is determined as 76.1° C. for the Fc domain and 87.9° C. for the Fab domain. For Antibody II, the Tm values ​​are determined as 76.4° C. for the Fc domain and 82.5° C. for the Fab domain.

[0059] Example 6: INSL5 detection assay Antibody I and Antibody II are used to detect and quantify human and mouse INSL5 concentrations by sandwich ELISA. Notably, the detection range of this assay approximates between 30 pg / mL for the lower limit of detection and 100,000 pg / mL for the upper limit of detection, as summarized in Table 5 below.

[0060] Labeling Antibody I with MSD SulfoTag (NHS Ester 150 nmole, Catalog No. R91AN-1): Resuspend MSD Sulfo-Tag powder in 50 μL of ice-cold Milli Q water in the original vial for a concentration of 3 nmol / μμL. Using a labeling challenge ratio of 12, add 2.7 μL of the reagent to 100 μL of 1 mg / mL antibody. Mix the solution well and protect from light while shaking at room temperature for 2 hours. Toward the end of the incubation, prepare a Zeba Spin Desalting Column (Thermo Scientific Catalog No. 87766). Invert the column until all the resin is a homogenous mixture. Open the valve at the bottom of the column and drain the liquid by centrifugation at 1500 × g for 1 minute. Wash the column three times with 500 μL of 1 × PBS using a 1-minute 1500 × g spin. After 2 hours of incubation, the antibody labeling reaction solution is dropped into the column resin. The column is placed in an Eppendorf tube and spun at 1500xg for 2 minutes to collect the labeled antibody. The labeled antibody is stored in the dark at 4°C.

[0061] For sandwich ELISA assays, on day 1, antibody I is diluted to 4 μg / mL in 1x PBS (Gibco Life Technologies catalog number 14190-144). 35 μL / well of this solution is applied to a MesoScale Discovery (MSD) plate (catalog number L15XA-3). The plate is tapped to evenly coat the wells. The plate is sealed and incubated overnight at room temperature. On day 2, the plate is washed three times with 1x PBS with 0.05% Tween 20 (20x PBS Tween 20 Thermo Scientific catalog number 28352) using an automated plate washer (Biotek ELx405). The plate is tapped dry on a paper towel. 100 μL of Superblock buffer (Superblock (T20) Thermo Scientific catalog number 37536) is added to each well and gently shaken at room temperature for 2 hours. The plate is washed three times and tapped dry as described above. 25 μL of Superblock buffer is added to all wells of the plate. 25 μL of a 4-fold serial dilution (made in Superblock (T20)) starting with 500 ng / mL of either human INSL5 (Phoenix Pharmaceuticals catalog number 035-70 made in DMSO) or mouse INSL5 (Phoenix Pharmaceuticals catalog number 035-40 made in DMSO) is then added to each well in the plate. The plate is sealed and gently shaken at room temperature for 2 hours. The plate is washed three times and tapped dry as described above. The MSD Sulfotag-labeled antibody from Example 2 is diluted to 1 μg / mL (i.e., approximately 1000-fold) in 0.2× Superblock (T20) buffer in 1× PBS / Tween 0.05%. 25 μL of this solution is added to each well of the plate and incubated at room temperature for 1 hour. The plate is washed three times and tapped dry as described above. Add 150 μL of 1×MSD ​​Read Buffer (MSD Read Buffer (4×) Catalog No. R92TC-2) to each well and read on an MSD Sector plate reader.The signal is measured in electrochemiluminescence units (ECLU).

[0062] Statistical Analysis of Data: Data were imported from the Mesoscale Discovery Sector reader into Microsoft Excel and GraphPad Prism® software (GraphPad Software, LLC, La Jolla, CA) to determine the approximate linear range for accurate INSL5 detection. The detection range is approximated at 30 pg / mL to 100,000 pg / mL for both human and mouse INSL5. [Table 5]

[0063] Amino acid and nucleotide sequences SEQ ID NO: 1: Human INSL5 A chain QDLQTLCCTDGCSMTDLSALC SEQ ID NO: 2: Human INSL5 B chain KESVRLCGLEYIRTVIYICASSRW SEQ ID NO: 3: Mouse INSL5 A chain RDLQALCCREGCSMKELSTLC SEQ ID NO: 4: Mouse INSL5 B chain RQTVKLCGLDYVRTVIYICASSRW SEQ ID NO: 5: HC of antibody I QSVEESGGRLVTPGTPLTLTCTASGFSLSSYDMGWVRQTPGEGLEWVGTISAGGTYYAHWAKGRFTISKSSTTVDLKMTSLTTEDTATYFCARERWNYDRSGGAGAGYFDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTC SKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPITHQDWLRGKEFCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSSRSVSLTCMINGFYPSDISVEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK SEQ ID NO: 6: antibody I of HC DNA SEQ ID NO: 7: Antibodies I of LC ADVVMTQTASPVSAAVGGTVTINCQASQSITSSYLSWYQQKPGQPPKLLIYPAANLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCLYGYFSSSIDFAFGGGTEVVVRGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC SEQ ID NO:8: antibody I of LC DNA GCCGATGTCGTGATGACCCAGACTGCATCCCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTATTACTAGTAGCTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATCCTGCAGCCAATCTGGCATCTGGAGTCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACAGCGGCGTGCAGTGACGATGCTGCCACTTACTACTGTCTATACGGTTATTTAGTTCTAGTATTTTGCTTTCGGCGGAGGGACCGAG GTGGTGGTCAGAGGTGATCCAGTGCACCTACTGTCCTCATCTTCCCACCAGCTGTGATCAAGTCGCAACAGGTACTGTGACGATCGTGTGTGTCGCGAAAAACTTTCCCGACGTGACCGTGACGTGGGGAAGTCGACGGAACAACCCAGAGCGACCGGGATCGAAAACTCAAAGACCCCGCAAAACTCGGCCGATTGCACATACAATTTGTCCTCTACGCTTACACTCACGTCGACGCAGTACAATAGTCACAAGGATATACATGCAAAGTTACTCAAGGAACTACGAGCGTGCAGTCATTCAATAGAGGGATTGT SEQ ID NO: 9: Antibodies I of VH QSVEESGGRLVTPGTPLTLTCTASGFSLSSYDMGWVRQTPGEGLEWVGTISAGGYTYYAHWAKGRFTISKSSTTVDLKMTSLTTEDTATYFCARERWNYDRSGGAGAGYFDLWGPGTLVTVSS SEQ ID NO: 10: HCDR1 of Antibody I TASGFSLSSYDMG SEQ ID NO: 11: HCDR2 of Antibody I TISAGGYTY SEQ ID NO: 12: HCDR3 of Antibody I ARERWNYDRSGGAGAGYFDL SEQ ID NO: 13: VL of antibody I ADVVMTQTASPVSAAVGGTVTINCQASQSITSSYLSWYQQKPGQPPKLLIYPAANLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCLYGYFSSSIDFAFGGGTEVVVR SEQ ID NO: 14: LCDR1 of antibody I QASQSITSSYLS SEQ ID NO: 15: LCDR2 of antibody I YPAANLAS SEQ ID NO: 16: LCDR3 of antibody I LYGYFSSSIDFA SEQ ID NO: 17: HC of antibody II QSVEESGGGLVTPGGSLTLTCTVSGIDLTTYAMGGVRQAPGEGLEWIGIIGGGGRTYYAAWAKGRFTISKTSTTVDLRITSPATEDTATYFCVRGGDFFDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPITHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK SEQ ID NO:18: antibody II of HC DNA SEQ ID NO: 19: LC II AQVLTQTPASVSAAVGGTVTIKCQASEDISKYLSWYQQKPGQRPKLLIYYVSNLEFGVPSRFKGSGSGTEYTLTISDLECDDAATYYCHQGYTGVNVFGGGTEVVVRGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC SEQ ID NO:20: antibody II of LC DNA GCTCAAGTGCTGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGGATATTAGCAAGTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCGCCCCAAACTCCTGATCTATTATGTATCCAATCTGGAATTTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGTACACTTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCACCAGGGTTATACCGGTGTAATGTTGAAAATGTTTTCGGCGGAGGGACCGAGGTG GTGGTCAGAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACAGCTGCTGATCAAGTCGCAACAGGTACTGTGACGATCGTGTGTGTCGCGAACAAATACTTTCCCGACGTGACCGTGACGTGGGAAGTCGACGGAACAACCCAGAGCGACCGGGATCGAAAACTCAAAGACCCCGCAAAACTCGGCCGATTGCACATACAATTTGTCCTCTACGCTTACACTCACGTCGACGCAGTACAATAGTCACAAGGATTATACATGCAAAGTTACTCAAGGAACTACGAGCGTGCAGTCATTCAATAGAGGGATTGT SEQ ID NO: 21: VH QSVEESGGGLVTPGGSLTLTCTVSGIDLTTYAMGWVRQAPGEGLEWIGIIGGGGRTYYAAWAKGRFTISKTSTTVDLRITSPATEDTATYFCVRGGDFFDLWGPGTLVTVSS SEQ ID NO: 22: HCDR1 of Antibody II TVSGIDLTTYAMG SEQ ID NO: 23: HCDR2 of Antibody II IIGGGGRTY SEQ ID NO: 24: HCDR3 of Antibody II VRGGDFFDL SEQ ID NO: 25: VL of antibody II AQVLTQTPASVSAAVGGTVTIKCQASEDISKYLSWYQQKPGQRPKLLIYYVSNLEFGVPSRFKGSGSGTEYTLTISDLECDDAATYYCHQGYTGVNVENVFGGGTEVVVR SEQ ID NO: 26: LCDR1 of antibody II QASEDISKYLS SEQ ID NO: 27: LCDR2 of antibody II YYVSNLEF SEQ ID NO: 28: LCDR3 of antibody II HQGYTGVNVENV SEQ ID NO: 29: Signal peptide METDTLLLWVLLLWVPGSTG

Claims

1. An antibody that binds to INSL5, comprising a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3; the HCDR1 comprises TVSGIDLTTYAMG (SEQ ID NO: 22); the HCDR2 comprises IIGGGGRTY (SEQ ID NO: 23); the HCDR3 comprises VRGGDFFDL (SEQ ID NO: 24); the LCDR1 comprises QASEDISKYLS (SEQ ID NO: 26); the LCDR2 comprises YYVSNLEF (SEQ ID NO:27); and An antibody wherein the LCDR3 comprises HQGYTGVNVENV (sequence number 28).

2. The antibody of claim 1, wherein the VH comprises SEQ ID NO: 21 and the VL comprises SEQ ID NO:

25.

3. The antibody of claim 1 or 2, wherein the antibody comprises a heavy chain (HC) comprising SEQ ID NO: 17 and a light chain (LC) comprising SEQ ID NO:

19.

4. The antibody of claim 1 or 2, wherein the antibody comprises a heavy chain (HC) comprising amino acids 2 to 435 of SEQ ID NO: 17 and a light chain (LC) comprising SEQ ID NO:

19.

5. The antibody of claim 1 or 2, wherein the antibody comprises an HC consisting of SEQ ID NO: 17 and an LC consisting of SEQ ID NO:

19.

6. The antibody of any one of claims 1 to 5, wherein the antibody binds to human INSL5.

7. The antibody according to any one of claims 1 to 5, which binds to the A chain (SEQ ID NO: 1) and B chain (SEQ ID NO: 2) of human INSL5.

8. The antibody of any one of claims 1 to 5, wherein the antibody binds to mouse INSL5.

9. The antibody according to any one of claims 1 to 5, which binds to the A chain (SEQ ID NO: 3) and B chain (SEQ ID NO: 4) of mouse INSL5.

10. A nucleic acid comprising a sequence encoding SEQ ID NO:17 and SEQ ID NO:

19.

11. A vector comprising the nucleic acid of claim 10.

12. A composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:17 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:

19.

13. (a) a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 17 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 19; or (b) a cell comprising a vector comprising a first nucleic acid sequence encoding SEQ ID NO:17 and a second nucleic acid sequence encoding SEQ ID NO:

19.

14. The cell of claim 13 , wherein the cell is a mammalian cell.

15. 15. A method for producing an antibody, comprising culturing a cell according to claim 13 or 14 under conditions in which the antibody is expressed, and then recovering the expressed antibody from the culture medium.

16. An antibody produced by culturing the cell of claim 13 or 14 under conditions in which the antibody is expressed, and then recovering the expressed antibody from the culture medium.

17. A method for detecting INSL5 in a sample, comprising the steps of contacting the sample with an antibody according to any one of claims 1 to 9, and detecting a signal generated by the contacting step.

18. 10. A method for quantifying INSL5 in a sample, comprising contacting the sample with an antibody according to any one of claims 1 to 9 and detecting a signal generated by the contacting step.

19. 20. The method of claim 18, further comprising contacting a control standard with the antibody and detecting a signal provided by the contacting of the control standard.

20. 20. The method of any one of claims 17 to 19, further comprising contacting the sample with a second antibody, wherein one of the antibody or the second antibody comprises a detectable label, and wherein the contacting step comprises detecting a signal provided by the detectable label upon formation of a complex comprising the antibody, the second antibody, and INSL5.

Images