Tirzepatide binding molecules, and uses thereof

Abstract

Provided herein are molecules that bind to the N-terminal and C-terminal regions of tirzepatide and uses thereof, including methods for detecting tirzepatide in a sample.

Description

TIRZEPATIDE BINDING MOLECULES, AND USES THEREOFREFERENCE TO A SEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled “31258_US_PRI” created September 10, 2024, and is 40 kilobytes in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety.FIELD

[0002] This disclosure is related to tirzepatide binding molecules that specifically bind to the N- terminal and C-terminal regions of tirzepatide and uses thereof, including methods for detecting tirzepatide.BACKGROUND

[0003] Diabetes mellitus is a chronic disorder characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. In type 2 diabetes mellitus (“T2D”), the combined effects of impaired insulin secretion and insulin resistance are associated with elevated blood glucose levels. The GIP / GLP1 dual agonist tirzepatide is described and claimed in United States patent 9474780 (“780 Patent”). Tirzepatide is approved by various regulatory authorities around the world, such as the US Food and Drug Administration, for use in the treatment of T2D and obesity. Tirzepatide is the active ingredient in the products Mounjaro® and Zepbound®.

[0004] There is a need to be able to test samples for the presence of tirzepatide. Various test kits are being developed. However, available kits are relatively slow requiring an operating time frame of 4-6 hours.

[0005] There remains a need for an assay method to test samples for the presence of tirzepatide in a quick and efficient manner.SUMMARY

[0006] Tirzepatide binding molecules that bind to the N-terminus and C-terminus of tirzepatide (SEQ ID NO: 25) are disclosed. A tirzepatide binding molecule that binds the N-terminal region of tirzepatide, can have the complementarity determining regions (CDRs) set forth in SEQ ID NOS: 1 -6; a heavy chain variable region (VH) comprising SEQ ID NO: 7 and a light chain variable region (VL) comprising SEQ ID NO: 8; or a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10. A tirzepatide binding molecule that binds the C-terminal region of tirzepatide, can have the complementarity determining regions (CDRs) set forth in SEQ ID NOS: 13-18; a VH comprising SEQ ID NO: 19 and a VL comprising SEQ ID NO: 20; or a heavy chain comprising SEQ ID NO: 21 and a light chain comprising SEQ ID NO: 22. The tirzepatide binding molecules can be an antibody, a scFv, a Fab, or fragments thereof.

[0007] Further provided herein is a method for determining the presence of tirzepatide (SEQ ID NO: 25) in a sample, comprising the steps of:(a) contacting a sample suspected of containing tirzepatide with a first tirzepatide binding molecule that binds the C-terminal region in each of tirzepatide, wherein the first tirzepatide binding molecule comprises the CDRs of SEQ ID NOS: 13- 18, thereby forming a first tirzepatide binding molecule-tirzepatide complex if tirzepatide is present;(b) contacting the results of step (a) with a second tirzepatide binding molecule that binds the N-terminal region of each of tirzepatide, wherein the second tirzepatide binding molecule comprises the CDRs of SEQ ID NOS: 1-6, thereby forming a first tirzepatide binding molecule-tirzepatide-second tirzepatide binding molecule complex if tirzepatide is present; and(c) detecting the first tirzepatide binding molecule-tirzepatide-second tirzepatide binding molecule complex if tirzepatide is present in the sample.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Several of these Figures contain color aspects that are useful in understanding the invention.

[0009] Figure 1 is an illustration of a sandwich immunoassay for detecting tirzepatide as described in Example 2.

[0010] Figure 2 is a flow diagram of the sandwich immunoassay for detecting tirzepatide as used in Example 2.

[0011] Figure 3 is an image of tirzepatide detection results seen for the microplate of Table 5 in Example 2.

[0012] Figure 4 is an illustration of a nitrocellulose membrane used in the Dot Blot Assay process for detecting tirzepatide as described in Example 3.

[0013] Figure 5 is representation of the Dot Blot Assay process for detecting tirzepatide as described in Example 3.

[0014] Figure 6 shows images of the results of Dot Blot Assays where (A) shows an image of the results of a Dot Blot Assay using 1% BSA blocking buffer, (B) shows an image of the results of a Dot Blot Assay using Pierce-protein free blocking buffer, and (C) shows an image of the results of a second Dot Blot Assay using Pierce protein free blocking buffer.

[0015] Figure 7 shows an image of tirzepatide speed of detection results from Example 4.DETAILED DESCRIPTION

[0016] Disclosed herein is a sandwich immunoassay method for detecting the presence of tirzepatide in a sample. A sandwich immunoassay is an assay that utilizes two antibodies that bind to different sites on an antigen of interest. The present immunoassay for detecting tirzepatide provides antibodies and methods that facilitate a sandwich assay method for detecting the presence of tirzepatide. The present immunoassay for detecting tirzepatide provides for the sensitive and specific detection of tirzepatide. Some embodiments of the assay provide a visual indication of the presence of tirzepatide with minimal specialized equipment needed. Some embodiments of the assay provide for a rapid result. Other embodiments of the assay provide for a qualitative indication of the presence of tirzepatide. Further embodiments of the assay provide for a quantitative indication of the concentration of tirzepatide present in a sample.

[0017] The present disclosure provides a tirzepatide binding molecule that binds the N-terminal region of tirzepatide (SEQ ID NO: 25), wherein the tirzepatide binding molecule comprises the complementarity determining regions (CDRs) set forth in SEQ ID NOS: 1-6. In one embodiment, the tirzepatide binding molecule is an antibody. In another embodiment, the tirzepatide binding molecule is an antibody fragment, wherein the antibody fragment binds the N-terminal region of each of tirzepatide (SEQ ID NO: 25) and comprises SEQ ID NOS: 1-6. In another embodiment, the antibody fragment is an scFv. In another embodiment, the antibody fragment is a Fab.

[0018] In another embodiment, the tirzepatide binding molecule is an antibody comprising a heavy chain variable region (HCVR or VH) comprising SEQ ID NO: 7 and a light chain variable region (LCVR or VL) comprising SEQ ID NO: 8. In another embodiment, the tirzepatide binding 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 tirzepatide binding molecule is an antibody comprising a heavy chain consisting of a SEQ ID NO: 9 and a light chain consisting of SEQ ID NO: 10. The present disclosure also provides a composition comprising the tirzepatide binding molecule which is an antibody comprising a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10.

[0019] The present disclosure 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 disclosure also provides a vector 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. The present disclosure also provides a composition comprising the vector. The present disclosure also provides a cell comprising the vector. In one embodiment, the cell is a mammalian cell. The present disclosure also provides a process of producing a tirzepatide binding molecule, comprising culturing the cell under conditions such that the tirzepatide binding molecule is expressed, and recovering the expressed tirzepatide binding molecule from the culture medium. The present disclosure also provides a tirzepatide binding molecule produced by the process.

[0020] The present disclosure also provides a tirzepatide binding molecule that binds the C-terminal region of tirzepatide (SEQ ID NO: 25), wherein the tirzepatide binding molecule comprises the complementarity determining regions set forth in SEQ ID NOS: 13-18. In one embodiment, the tirzepatide binding molecule is an antibody. In another embodiment, the tirzepatide binding molecule is a fragment of an antibody, wherein the fragment binds the C-terminal region of tirzepatide (SEQ ID NO: 25). In another embodiment, the fragment of the tirzepatide binding molecule is an scFv. In another embodiment, the fragment of the tirzepatide binding molecule is a Fab.

[0021] In another embodiment, the tirzepatide binding molecule is an antibody comprising a VH comprising SEQ ID NO: 19 and a VL comprising SEQ ID NO: 20. In another embodiment,the tirzepatide binding molecule is an antibody comprising a heavy chain comprising SEQ ID NO: 21 and a light chain comprising SEQ ID NO: 22. In another embodiment, the tirzepatide binding molecule is an antibody comprising a heavy chain consisting of a SEQ ID NO:21 and a light chain consisting of SEQ ID NO: 22. The present disclosure also provides a composition comprising the tirzepatide binding molecule which is an antibody comprising a heavy chain consisting of a SEQ ID NO:21 and a light chain consisting of SEQ ID NO: 22.

[0022] The present disclosure 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 disclosure also provides a vector 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. The present disclosure also provides a composition comprising the vector. The present disclosure also provides a cell comprising the vector. In one embodiment, the cell is a mammalian cell. The present disclosure also provides a process of producing a tirzepatide binding molecule, comprising culturing the cell under conditions such that the tirzepatide binding molecule is expressed, and recovering the expressed tirzepatide binding molecule from the culture medium. The present disclosure also provides a tirzepatide binding molecule produced by the process.

[0023] The present disclosure also provides a composition comprising (a) a first tirzepatide binding molecule that binds the N-terminal region of tirzepatide (SEQ ID NO: 25), wherein the first tirzepatide binding molecule comprises SEQ ID NOS: 1-6, and (b) a second tirzepatide binding molecule that binds the C-terminal region of tirzepatide (SEQ ID NO: 25), wherein second the tirzepatide binding molecule comprises SEQ ID NOS: 13-18.

[0024] The present disclosure also provides a sandwich assay method for determining the presence of tirzepatide (SEQ ID NO: 25) in a sample, comprising: (a) contacting a sample desired to be tested for the presence of tirzepatide with a first tirzepatide binding molecule that binds the C-terminal region of tirzepatide (SEQ ID NO: 25), wherein the first tirzepatide binding molecule comprises SEQ ID NOS: 13-18, thereby forming a first tirzepatide binding molecule -tirzepatide complex; (b) contacting a second tirzepatide binding molecule that binds the N-terminal region of each of tirzepatide (SEQ ID NO: 25), wherein the second tirzepatide binding molecule comprises SEQ ID NOS: 1-6, thereby forming a first tirzepatide binding molecule-tirzepatide-second tirzepatide binding molecule complex; and (c) detecting the presence of tirzepatide in the first tirzepatide binding molecule-tirzepatide-second tirzepatide binding molecule complex, for example by biotinylating the second tirzepatide binding moleculeprior to the assay and adding streptavidin-conjugated horseradish peroxidase, which produces a soluble colored product after a substrate is added.

[0025] In another embodiment of the methods of the disclosure, the first tirzepatide binding molecule is an antibody and the second tirzepatide binding molecule is an antibody. In another embodiment, the first tirzepatide binding molecule is an antibody comprising a VH comprising SEQ ID NO: 19 and a VL comprising SEQ ID NO: 20, and the second tirzepatide binding molecule is an antibody comprising a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 8. In another embodiment, the first tirzepatide binding molecule is an antibody comprising a heavy chain comprising SEQ ID NO: 21 and comprising a light chain comprising SEQ ID NO: 22, and the second tirzepatide binding molecule is an antibody comprising a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10.

[0026] In one embodiment of the methods described herein, the sample is solid. In another embodiment, the sample is liquid. In a further embodiment, the sample is a formulated liquid.

[0027] In the methods described herein, the first tirzepatide binding molecule can be attached to a solid support. In one embodiment, the solid surface is a plate. In another embodiment, the solid surface is a membrane such as a nitrocellulose membrane. In a further embodiment, the solid surface is a plurality of beads. Such solid supports include, e.g., without limitation, glass, cellulose, plastic, poly-acrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0028] In another embodiment, the first tirzepatide binding molecule is attached directly to the solid surface. In another embodiment, the first tirzepatide binding molecule is indirectly attached to the solid surface with an attachment agent. In one embodiment, the attachment agent is streptavidin, neutravidin or avidin that is coated on the solid surface, and the tirzepatide binding molecule is biotinylated.

[0029] hi another embodiment, prior to contacting the sample with the first tirzepatide binding molecule, the solid surface is contacted with a blocking solution. In another embodiment, after contacting the sample with the first tirzepatide binding molecule, the first tirzepatide binding molecule-tirzepatide complex is contacted with a wash solution, thereby removing uncomplexed tirzepatide.

[0030] In another embodiment, the first tirzepatide binding molecule-tirzepatide-second tirzepatide binding molecule complex is contacted with a wash solution, thereby removing uncomplexed second tirzepatide binding molecule.

[0031] In another embodiment of the method of determining the presence of tirzepatide (SEQ ID NO: 25) in a sample, 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 attached, with a blocking solution;(b) contacting a liquid sample comprising or suspected of comprising tirzepatide with a first antibody comprising a heavy chain comprising SEQ ID NO: 21 and a light chain comprising SEQ ID NO: 22 attached to a solid surface, thereby forming a first antibody-tirzepatide complex;(c) contacting the solid surface with a wash solution, thereby removing uncomplexed tirzepatide (if present);(d) contacting the solid surface with a second antibody comprising a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10, thereby forming a first antibody-tirzepatide-second antibody complex;(e) contacting the solid surface with a wash solution, thereby removing uncomplexed second antibody; and(f) detecting tirzepatide in the first antibody-tirzepatide-second antibody complex if tirzepatide is present in the sample.

[0032] In another embodiment of the method of determining the presence of tirzepatide (SEQ ID NO: 25) in a sample, the method comprises:(a) contacting a solid surface, to which an antibody comprising a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10 is attached, with a blocking solution;(b) contacting a liquid sample comprising or suspected of comprising tirzepatide with a first antibody comprising a heavy chain comprising SEQ ID NO: 9 and a lightchain comprising SEQ ID NO: 10 attached to a solid surface, thereby forming a first antibody-tirzepatide complex;(c) contacting the solid surface with a wash solution, thereby removing uncomplexed tirzepatide (if present);(d) contacting the solid surface with a second antibody comprising a heavy chain comprising SEQ ID NO: 21 and a light chain comprising SEQ ID NO: 22, thereby forming a first antibody-tirzepatide-second antibody complex;(e) contacting the solid surface with a wash solution, thereby removing uncomplexed second antibody; and(f) detecting tirzepatide in the first antibody-tirzepatide-second antibody complex if tirzepatide is present in the sample.

[0033] hi another embodiment of the methods described herein, the second tirzepatide binding molecule is labeled. In another embodiment, the label is a radiolabel. In another embodiment, the radiolabel is ruthenium.

[0034] In another embodiment of the methods described herein, the first tirzepatide binding molecule can be exchanged with an anti-GLP-lR antibody that will bind to tirzepatide. In this embodiment, the second tirzepatide binding molecule is as described herein. In a further embodiment of the methods described herein, the second tirzepatide binding molecule is an anti-GLP-1R antibody that will bind to tirzepatide. In this embodiment, the first tirzepatide binding molecule is as described herein. In these embodiments, either the first or the second tirzepatide binding molecule is one of the first or second tirzepatide binding molecules as described herein.

[0035] In many of the embodiments described herein, the first tirzepatide binding molecule is the C-Terminal Antibody and the second tirzepatide binding molecule is the N-Terminal binding molecule. However, it is also contemplated that the first tirzepatide binding molecule is the N-Terminal Antibody and the second tirzepatide binding molecule is the C-Terminal binding molecule. Additionally, other molecules that bind tirzepatide could be used in the assay, the other molecules that bind tirzepatide could be used in place of either the C-Terminal binding molecule or N-Terminal binding molecule as described herein.

[0036] In another embodiment of the methods described herein, the method is set up as a lateral flow immunochromatographic assay (a.k.a., a lateral flow assay or a rapid lateral flow assay). In some cases, a lateral flow assay uses a sample pad, a conjugate pad, a nitrocellulosestrip or membrane that contains test and control lines and wicking / absorbent pad as components aligned on a backing card (overlapping of the components on the backing card enables smooth capillary flow of the sample). Once a sample is applied to the sample pad, the sample starts to migrate to the conjugate pad which contains first antibodies specific to tirzepatide (could be either antibodies that bind to the N-terminal region of tirzepatide or C-terminal region of tirzepatide) and conjugated to fluorescent or colored particles (such as, for example, latex microspheres or colloidal gold) which form a complex with tirzepatide if present. The sample mixture which now contains the first antibody complexed to the tirzepatide (if present) migrates along the strop into the detection zone that contains a second antibody (e.g., the opposite antibody to the first antibody, i.e., antibodies that bind to the N-terminal region of tirzepatide if the first antibodies are antibodies that bind to the C-terminal region of tirzepatide and vice versa) immobilized on a test line and further control antibodies that binds the first antibodies in a control line. When tirzepatide is present, the second antibodies on the test line complex with the tirzepatide bound to the first antibodies and hold the first antibodies at the test line. First antibodies that flow past the test line are complexed to the control antibodies on the control line to show the sample has moved through the test strip (control antibodies to the first antibodies can be readily developed by one of skill in the art). After the control line, the sample flows into the wicking pad which maintains the capillary force for the sample liquid to flow. Further discussion of lateral flow assays can be found in Koczula KM, Gallotta A., Lateral flow assays.. Essays Biochem., 2016 Jun 30; 60(1): 111-20.

[0037] The tirzepatide binding molecules, methods and uses described herein facilitate the detection of tirzepatide.

[0038] Sequences referred to herein are set forth in Table 1. For example, the term “tirzepatide” refers to SEQ ID NO: 25."

[0039] The term “N-terminal region of tirzepatide” as used herein refers to amino acid residues 1 to 20 of the tirzepatide peptide as shown by SEQ ID NO: 26.

[0040] The term “binds the N-terminal region of tirzepatide” as used herein refers to (a) binding to one or more amino acid residue(s) located at the N-terminal region of tirzepatide (SEQ ID NO: 26), (b) not binding to the C-terminal region of tirzepatide (SEQ ID NO: 27), and (c) not binding to any amino acid portion of tirzepatide (SEQ ID NO: 25) not located in the N-terminal region of tirzepatide (SEQ ID NO: 26).

[0041] The term “C-terminal region of tirzepatide” as used herein refers to amino acid residues 22-39 of the tirzepatide peptide as shown by SEQ ID NO: 27.

[0042] The term “binds the C-terminal region of tirzepatide” as used herein refers to (a) binding to one or more of amino acid residue(s) located in the C-terminal region of tirzepatide (SEQ ID NO: 27), (b) not binding to the N-terminal region of tirzepatide (SEQ ID NO: 26), and (c) not binding to any amino acid portion of tirzepatide (SEQ ID NO: 25) not located in the C-terminal region of tirzepatide (SEQ ID NO: 27).

[0043] The term “N-Terminal Antibody” as used herein refers to the antibody that binds to the N-terminal region of tirzepatide (SEQ ID NO: 26) having a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10.

[0044] The term “C-Terminal Antibody” as used herein refers to the antibody that binds to the C-terminal region of tirzepatide (SEQ ID NO: 27) having a heavy chain amino acid sequence of SEQ ID NO: 21 and a light chain amino acid sequence of SEQ ID NO: 22.

[0045] The term “tirzepatide binding molecule” as used herein refers to a molecule such as an antibody that comprises a polymer of amino acid residues that binds to tirzepatide. In one embodiment, the tirzepatide binding molecule consists of a polymer of amino acid residues.

[0046] The term “contacting” as used herein refers to the exposing a first substance to another substance. For example, a sample can be exposed to a tirzepatide binding molecule of the disclosure, for a time and under conditions that permit the tirzepatide binding molecule to bind to tirzepatide present in the sample. Such time and conditions are known to one of skill in the art, and / or can be routinely determined by methods known in the art according to references cited herein.

[0047] The term “complex” as used herein refers to the protein-protein interaction between, e.g., a tirzepatide binding molecule and tirzepatide (SEQ ID NO: 25). “First tirzepatide binding molecule-tirzepatide complex” as used herein refers to the protein-protein interaction between a tirzepatide binding molecule as disclosed herein and a molecule of tirzepatide (SEQ ID NO: 25). “First tirzepatide binding molecule-tirzepatide-second tirzepatide binding molecule complex” as used herein refers to the concomitant protein-protein interaction between (a) a molecule of a first tirzepatide binding molecule as described herein and a molecule of tirzepatide (SEQ ID NO: 25), and (b) a molecule of a second tirzepatide binding molecule as disclosed herein and the same molecule of tirzepatide (SEQ ID NO: 25).

[0048] A tirzepatide binding molecule as described herein can be conjugated to an enzyme and used in an enzyme-linked immunosorbent assay (ELISA). Such assays are described in detail in, for example, Butler (1994) “ELISA” (Chapter 29), In: van Oss, C. J. et al., eds., Immunochemistry, Marcel Dekker, Inc., New York, pp. 759-803.

[0049] A molecule such as tirzepatide can be measured by a variety of immunoassay methods including, e.g., without limitation, competitive and non-competitive assay systems using techniques such as, e.g., without limitation, Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Many of these methods are expensive and require expensive laboratory equipment. For a review of immunological and immunoassay procedures in general, see for example Stites and Terr (eds.), Basic and Clinical Immunology (7th ed.) (1991). Moreover, 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); Diamandis & Christopoulus, Immunoassay, Academic Press (San Diego, CA) (1996).

[0050] The term “antibody,” as used herein, refers to an immunoglobulin molecule that binds an antigen. Embodiments of an antibody include a monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, chimeric antibody, bispecific or multispecific antibody, or conjugated antibody. The antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA), and any subclass (e.g., IgGl, IgG2, IgG3, IgG4).

[0051] An exemplary antibody of the present disclosure is an immunoglobulin G (IgG) type antibody comprised of four polypeptide chains: two heavy chains (HC) and two light chains (LC) that are cross-linked via inter-chain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains includes a variable region of about 100-125 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each of the four polypeptide chains contains a constant region primarily responsible for effector function. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The IgG isotype may be further divided into subclasses (e.g., IgGl, IgG2, IgG3, and IgG4).

[0052] The VH and VL regions can be further subdivided into regions of hyper-variability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). The CDRs are exposed on the surface of the protein and are important regions of the antibody for antigen binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, 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 residuesthat form specific interactions with the antigen. Assignment of amino acid residues to the CDRs may be done according to the well-known schemes, including those described in 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, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database available on at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212).

[0053] An “antibody fragment” or “antigen-binding fragment” that, as used herein, comprise at least a portion of an antibody retaining the ability to specifically interact with an antigen or an epitope of the antigen, such as Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, scFab, disulfide-linked Fvs (sdFv), a Fd fragment.

[0054] The terms “bind” and “binds” as used herein are intended to mean, unless indicated otherwise, the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, which results in proximity of the two proteins or molecules as determined by common methods known in the art, for example, the molecular interaction between two molecules, e.g., a tirzepatide binding molecule as described herein and, e.g., tirzepatide (SEQ ID NO: 25). In one embodiment, a tirzepatide binding molecule as described herein binds specifically to tirzepatide. In another embodiment, "specifically binds" means that a tirzepatide binding molecule as described herein interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to tirzepatide. In another preferred embodiment, "specifically binds" means that a tirzepatide binding molecule as described herein binds to tirzepatide with a KD of about 0.1 mM or less. In another preferred embodiment, "specifically binds" means that a tirzepatide binding molecule as described herein binds to tirzepatide with a KD of about 0.01 mM or less. In another preferred embodiment, "specifically binds" means that a tirzepatide binding molecule as described herein binds to tirzepatide with a KD of about 0.001 mM or less. In another preferred embodiment, "specifically binds" means that a tirzepatide binding molecule as described herein binds to tirzepatide with a KD of about 0.0001 mM or less.

[0055] An isolated polynucleotide molecule encoding a HCVR region may be converted to a full-length heavy chain gene by operably linking the HCVR-encoding polynucleotide to another polynucleotide molecule encoding heavy chain constant regions. The sequences of human, as well as other mammalian, heavy chain constant region genes are known in the art. Polynucleotide fragments encompassing these regions may be obtained, e.g., by standard PCR amplification.

[0056] An isolated polynucleotide molecule encoding a LCVR region may be converted to a full-length light chain gene by operably linking the LCVR-encoding polynucleotide to another polynucleotide molecule encoding a light chain constant region. The sequences of human, as well as other mammalian, light chain constant region genes are known in the art. Polynucleotide fragments encompassing these regions may be obtained by standard PCR amplification.

[0057] The term “sensitivity” as used herein refers to the lowest level of an analyte (in this case, tirzepatide) that can be measured with acceptable accuracy and precision. Sensitivity is reflected for example by the limit of quantitation (LOQ) which is determined, according to the present disclosure, by taking a sample of tirzepatide and diluting it serially until the %CV (coefficient of variation) goes above 20%.

[0058] The term “detectably labeled” means that a tirzepatide binding molecule as described herein, or a complex of tirzepatide and tirzepatide binding molecule, has attached to it, either covalently or non-covalently, a useful detectable label. In direct conjugate-labeled methods, many different useful labels can be employed including, for example, prosthetic group complexes, chromophores, chromogens (color-producing substrates), dyes, fluorescent compounds, Anorogenic compounds, radioactive isotopes, paramagnetic isotopes, and compounds that can be imaged by positron emission tomography (PET) and magnetic resonance imaging (MRI).

[0059] The nucleic acid molecules as described herein may be expressed in a host cell after the sequences are operably linked to an expression control sequence. The expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to permit detection of those cells transformed with the desired polynucleotide sequences.

[0060] An expression vector containing the nucleic acid sequences of interest (e.g., the nucleic acid sequences encoding one or more of the tirzepatide binding molecules as described hereinand expression control sequences) can be transferred into a host cell by known methods, which vary depending on the type of host cells.

[0061] A tirzepatide binding molecule as described herein may be produced in mammalian host cells, non-limiting examples of which include CHO, NSO, HEK293 or COS cells. The host cells may be cultured using techniques known in the art. Tirzepatide binding molecules as described herein may be expressed and purified essentially as follows. An appropriate host cell, such as HEK293 or CHO, may be either transiently or stably transfected with an expression system for secreting tirzepatide binding, e.g., an antibody, using an optimal predetermined heavy chai light chain vector ratio or a single vector system encoding both heavy chain and light chain. Nucleic acid encoding a tirzepatide binding molecule as described herein may be either transiently or stably transfected with an expression system for secreting tirzepatide binding moleculeusing one or more DNA molecules encoding for, e.g., an antibody heavy chain and light chain.

[0062] Murine-derived anti-tirzepatide antibodies of the present disclosure are generated employing hybridoma methodology (e.g., as first described by Kohler et al., Nature, 256:495 (1975)). Briefly, the mouse is immunized with a mix of two peptides conjugated to KLH:N-term peptide: Y-(Aib)-EGTFTSDYSI-(Aib)-LDKIAQ-Cys (SEQ ID NO: 26)C-term peptide: Cys-VQWLIAGGPSSGAPPPS-NH2(SEQ ID NO: 27)and lymphocytes capable of producing antibodies that bind tirzepatide are isolated and fused with a myeloma cell line using a suitable fusing agent for forming a hybridoma cell (Goding, Monoclonal Antibodies : Principles and Practice, pp.59-103 (Academic Press, 1986)).Hybridomas are seeded and grown in a suitable culture medium (preferably containing one or more substances inhibiting survival of unfused myeloma cells). Binding specificity of monoclonal antibodies produced by hybridomas is then determined by an in vitro binding assay (e.g., immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA)). Preferred hybridomas may be subcloned by limiting dilution procedures and grown by standard methods including in vivo as ascites tumors in an animal (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Monoclonal antibodies secreted by the hybridomas (and or subclones) are purified according to conventional procedures such as, for example, affinity chromatography (e.g., protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, or the like.

[0063] cDNA encoding antibodies of the present disclosure is sequenced using conventional procedures. cDNA sequences encoding the heavy and light chains may be cloned and engineered into a GS (glutamine synthetase) expression vector. The engineered immunoglobulin expression vector may then be stably transfected into CHO cells. As one of skill in the art will appreciate, mammalian expression of antibodies will result in glycosylation, typically at highly conserved N-glycosylation sites in the Fc region. Stable clones may be verified for expression of an antibody specifically binding to tirzepatide. Positive clones may be expanded into serum- free culture medium for antibody production in bioreactors. Media, into which an antibody has been secreted, may be purified by conventional techniques. For example, the medium may be conveniently applied to a Protein A or G Sepharose FF column that has been equilibrated with a compatible buffer, such as phosphate buffered saline. The column is washed to remove nonspecific binding components. The bound antibody is eluted, for example, by pH gradient and antibody fractions are detected, such as by SDS-PAGE, and then pooled. The antibody may be concentrated and / or sterile filtered using common techniques. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product may be immediately frozen, for example at -70°C, or may be lyophilized.

[0064] Various methods of protein purification may be employed to purify a tirzepatide binding molecule as described herein and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).100651 For example, the medium may be conveniently applied to a MabSelect column (GE Healthcare Life Sciences), or KappaSelect column (GE Healthcare Life Sciences), that has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH 7.4). The column may be washed to remove nonspecific binding components. The bound tirzepatide binding molecule may be eluted, for example, by 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). Antibody fractions may be detected, such as by UV absorbance or SDS-PAGE, and then may be pooled. Further purification is optional, depending on the intended use. The purified tirzepatide binding molecules may be concentrated and / or sterile filtered using commontechniques. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, multimodal, or hydroxyapatite chromatography. The purified tirzepatide binding molecules may be immediately frozen at -70°C or may be lyophilized.

[0066] The tirzepatide binding molecules disclosed herein are useful for detecting the presence of tirzepatide in a sample.

[0067] Useful radiolabels, which are detected simply by gamma counter, scintillation counter or autoradiography, include3H,124I,125I,131I,35S, and14C. Radionuclides can be bound to a tirzepatide binding molecule as described herein, either directly or indirectly, using a chelating agent, such as DTPA and EDTA. Examples of such radionuclides include "Tc,123I,12SI,131I,niIn,97Ru,67CU,67Ga,68Ga,72As,89Zr,90Y and2O1T1.

[0068] Other suitable labels are art-known or can be determined by routine experimentation.

[0069] Colorimetric detection can be used, employing chromogenic compounds that have, or result in, chromophores with high extinction coefficients, and which are therefore easily detectable. When later exposed to its substrate under appropriate reaction conditions, the enzyme will react with the substrate to produce a chemical label that can be detected, for example, by spectrophotometric, fluorometric, or visual means.

[0070] Colorimetric detection and the color produced can also be used to determine the concentration level of tirzepatide present in the sample. For example, a comparison chart can be created using a range of concentration levels for tirzepatide in know samples and that chart can be used to compare to the color level developed in the method. A colorimeter instrument could also be programmed with this information to provide concentration level based on the degree of color development.

[0071] 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, [3-galactosidase, ribonuclease, urease, catalase, glucoamylase, and acetylcholinesterase.

[0072] Non-limiting examples of suitable prosthetic group complexes include, e.g., streptavidin / biotin, avidin / biotin, and neutravidin / biotin. Use of chromogens is preferred because assays employing them can be easily performed in clinical diagnostic laboratories and reviewed by a pathologist with equipment commonly available in these laboratories. Commonly usedchromogens include diaminobenzidine (DAB); DAB with enhancement; 3-amino-9- ethyl carbazole (AEC); 4-chloro-l -naphthol (4-CN); Hanker-Yates reagent; alpha-naphthol pyronin; 3,3’,5,5’-tetramethylbenzidine (TMB); Fast Blue BB; Fast Red TR; new fuchsin; BCIP-NBT; tetrazolium; tetranitoblue tetrazolium (TNBT); and immunogold with silver enhancement.

[0073] Useful fluorescent labels include umbelliferone, fluorescein, fluorescein isothiocyanate, dichlorotriazinylamine fluorescein, rhodamine, a dansyl group, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, and Cy5 (Haugland ((1996) Handbook of Fluorescent Probes and Research Chemicals, Sixth Ed., Molecular Probes, Eugene, OR).

[0074] Tirzepatide binding molecules, or tirzepatide binding molecule-tirzepatide complexes can also be delectably labeled using fluorescence-emitting metals, such as152Eu+, or other members of the lanthanide series, by attaching them using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediamine-tetraacetic acid (EDTA).

[0075] Tirzepatide binding molecules can also be detectably labeled by coupling them to a phosphorescent or chemiluminescent compound that can then be detected by the phosphorescence or luminescence that arises during the course of a chemical reaction. Examples of useful chemiluminescent compounds include luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester. Likewise, a bioluminescent compound such as luciferin, luciferase, or aequorin can be used to label the antibody peptides. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.

[0076] The present disclosure also provides articles of manufacture and kits containing compositions useful for detecting tirzepatide. The article of manufacture may comprise a container with a written label. The container may hold a composition comprising tirzepatide binding molecules as described herein, which is either detectably labeled, or unlabeled.

[0077] The kit as described herein can also comprise a container comprising a first and a second tirzepatide binding molecule of the disclosure. The second tirzepatide binding molecule can be conjugated with an enzyme or other label. A chromogenic substrate of the enzyme can 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 package inserts with instructions for use in vivo, in vitro, or both.

[0078] The embodiments of the methods described herein can be performed quickly. For example, the tirzepatide incubation step can be performed in less than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 minutes. For further example,the tirzepatide incubation step of the methods can be performed in less than 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes. The rapidity of the tirzepatide incubation step allows for the overall tirzepatide detection method to be performed quickly. For example, the tirzepatide detection method can detect tirzepatide in less than 15 minutes, 20, minutes, 25 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 135 minutes, or 150 minutes. For further example, the overall tirzapatide detection method can be performed in less than 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 minutes.

[0079] The following examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention.

[0080] Example 1: Antibody Binding Studies

[0081] Kinetic analysis and binding specificity of the anti-tirzepatide N-terminal antibody and anti-tirzepatide C-terminal antibody are determined by surface plasmon resonance using Biacore T100 (GE Healthcare Life Sciences). Antibodies were loaded on to anti-mouse IgG (AMC) Octet tips. Biolayer interferometry was used to determine the antibody binding specificity.

[0082] Using Biacore analysis of the anti-tirzepatide N-terminal antibody, the equilibrium dissociation constant is 1.3x1 O’8mol / L (Table 2).

[0083] Using Biacore analysis of the anti-tirzepatide C-terminal antibody, the dissociation constant is 3.42xl0'12mol / L (Table 2).

[0084] Table 2. Binding Kinetics of the Anti-tirzepatide N-terminal antibody and Anti-tirzepatide C-terminal antibody

[0085] Example 2: A kit for detecting tirzepatide

[0086] Example 2 describes a kit for detecting tirzepatide. The provided components of the kit are included in Table 3:

[0087]

[0088] Table 3: Kit Components| Component | Details| Quantity Source

[0089] Materials Not Provided:a. Distilled, deionized or USP waterb. Test tubes, pipettes and any other standard laboratory items

[0090] STORAGE

[0091] Store the detection antibody and HRP-streptavidin at -20°C for up to one year from the kit's assembly date. The remaining components should be stored in a refrigerator (2-8°C). Long term storage, improper storage conditions and large temperature fluctuation cycles may result in the accumulation of precipitates in the TMB solution and in the ELISA buffer concentrate. These precipitates should not affect the assay noticeably. Bring these reagents to room temperature and mix gently to dissolve the precipitate.

[0092] THE ELISA ASSAY

[0093] The ELISA kit is a sandwich immunoassay (see Figure 1). Capture reagent anti-tirzepatide C-Term mAh is firstly coated on a microtiter plate. The analyte (tirzepatide) is captured by antibodies coated on a 96- well plate. A constant concentration of biotinylated detection antibody (N-term mAb) is added that will bind specifically to the bound tirzepatide. Captured biotinylated antibody is subsequently bound by streptavidin-conjugated horseradish peroxidase, which produces a soluble colored product after a substrate is added.

[0094] Quality control (high and low) should be included in each assay. Unknown sample identity is confirmed by visually comparing the blue color generated after substrate is added.

[0095] PROTOCOL

[0096] REAGENT AND SAMPLE PREPARATION

[0097] Wash Buffer (10X DPBS with 0.5% Tween 20); Supplied as 10X solution. Add 50 mL 10X PBS-Tween solution to 450 mL Distilled, deionized or USP water and mix. A 1:10 dilution is required so volumes can be modified as long as the proportions are maintained.

[0098] DPBS buffer.

[0099] The quality controls, samples, detection antibodies and Streptavidin-HRP are prepared in DPBS buffer.

[0100] Reagents, samples, and the plate should be brought to room temperature before use.

[0101] PLATE LAYOUT

[0102] Table 5 shows an Example Plate Layout. A blank, high-quality control, and low-quality control sample are included in each assay.

[0103] PREPARE SAMPLES AND STANDARD DILUTIONS

[0104] 1. Sample concentration: Kit can detect Tirzepatide at a concentration equal or more than 15 ng / mL. Since the concentration of the test sample may not be known, take a scoop full of sample and add it to 10 mL DPBS. Mix well.

[0105] 2. Quality Controls: See Table 4.

[0106] Table 4: Quality Control Preparation (QC’s)

[0107] The SO (Stock) tirzepatide concentration can change which will change the required dilution method to achieve the noted test concentrations. For example, the stock concentration could be 1.004 mg / mL.

[0108] Final test concentrations of tirzepatide: 1200, 160, 80, 40, 20, 10, and 2.5 ng / ml.

[0109] Final test concentrations: semaglutide 33 pg / ml; dulaglutide 6.3 pg / ml; cetuximab 33 g / ml; retatrutide 53 g / ml and 113 pg / ml.

[0110] Unknown Sample Preparation

[0111] For sample stocks in liquid: Dilute sample stock by adding 20 pL of sample stocks to 380 pL of IX DPBS. Mix well.

[0112] For sample stocks in powder: Use static-free micro scoop (provided in the kit) to measure one scoop full of powder and dissolve it in 10 mL IX DPBS. Mix well to ensure the powder is fully dissolved. Dilute the solution further 10-fold by adding 20 pL of solution to 380 pLof IX DPBS. Mix well.

[0113] REAGENT PREPARATION

[0114] Kit components should not be diluted until they are needed. Please read the complete protocol before proceeding.

[0115] 1) Equilibrate unopened kit components to room temperature for a minimum of 30 minutes. To avoid accumulation of moisture, do not open reagents and immunoplate while they are cold.

[0116] 2) 96- well immunoplate (strip wells) with acetate plate sealer - ELISA is supplied precoated and blocked.

[0117] 3) ELISA Wash Buffer: Mix 50 mL ELISA buffer concentrate with 950 mL with distilled, deionized or USE pure water and mix well.

[0118] 4) Biotinylated-detection antibody: Prepare approximately 6 mL for each full 96- well plate. Add 12 pL biotinylated-detection antibody in 6 mL DPBS and mix by inverting.

[0119] 5) Streptavidin-Horseradish Peroxidase: Prepare approximately 6 mL for each full 96-well plate. Add 60 p L in 6 mL DPBS and mix by inverting.

[0120] 6) TMB chromogenic solution: Prepare approximately 6 mL for each full 96- well plate.

[0121] Prepare two-component TMB chromogenic solution immediately before use by mixing 3 mL TMB Peroxidase Substrate with 3 mL Peroxidase Substrate Solution B. Mix well. Prepare immediately before use.

[0122] PROCEDURE FOR ELISA

[0123] Figure 2 shows the ELISA process.

[0124] A 96 well microplate plate was prepared as follows:

[0125] Table 5: Microplate LayoutSI: test sample#!S2: test sample #2Semaglutide (SEQ ID NO:28), 5 mg / mlDulaglutide (SEQ ID NO:29), 0.94 mg / mlCetuximab (SEQ ID NO:30 (Heavy Chain), 31 (Light Chain)), 5 mg / ml Retatrutide (SEQ ID NO:32), 8 mg / ml (low cone.), 17 mg / / l (high cone.)Each sample was diluted 150 fold for test.Final test concentrations: semaglutide 33 pg / ml; dulaglutide 6.3 pg / ml; cetuximab 33 pg / ml; retatrutide 53 pg / ml and 113 pg / ml.

[0126] The absorbance at 650nm was read with a microplate reader.

[0127] Table 6: Absorbance 650nmSee Figure 3 for a picture of the plate.

[0128] Table 7: The Average Absorbance at 650nm0129] CV: coefficient of variance

[0130] HOW TO INTERPRET DATA

[0131] The Plate can be visually analyzed for blue color development. Blank wells will either be clear or a very faint blue color. Each replicate of both high-quality control (HQC) and low-quality control (LQC) should show blue color (see Figure 3 where darker gray grayscale corresponds to higher intensity blue color). NOTE: In the absence of blue color development inQC control sample, assay will be considered invalid and should be repeated. Assay may be repeated one more time. If it fails again, use a new kit. Blue color with the intensity similar or higher to the (LQC) confirms presence of tirzepatide in a sample and the results will be reported as positive. Color similar to blank demonstrates absence of Tirzepatide in the sample and hence negative. Take a photograph to document results. The intensity of the blue color that develops could be coded to a comparison chart of known sample concentrations to provide an indication of concentration level (or a colorimeter instrument could be used for this purpose).

[0132] The results show a highly specific ELISA assay to detect tirzepatide.

[0133] Example 3: A Dot Blot Assay for detecting tirzepatide

[0134] The Dot Blot assay is performed using a nitrocellulose membrane (see Figures 4 and 5). Reagents

[0135] Preparations

[0136] Procedure

[0137] The Dot Blot Assay procedure was run three times using two different blocking buffers. The first procedure used 1% BSA blocking buffer and the second and third procedures used Pierce protein free blocking buffer. The results of the three procedures are shown in Figure 6.

[0138] Results

[0139] Figure 6A shows a picture of the results using 1% BSA blocking buffer. Figure 6B shows a picture of the results using Pierce-protein free blocking buffer. Figure 6C shows a picture of the results using Pierce protein free blocking buffer. In all three procedures shown in Figure 6, the BSA negative control shows very little color (6A), the tirzepatide positive control shows a blueish color (6B), and the sample shows a blueish color (6C) — bluish color shown in grayscale in Figure 6.

[0140] Example 4: Assay speed of binding

[0141] Following the procedure of Example 2 using a 2x titration of TZP (starting from 0.04 pg / mL), the time the sample was allowed to incubate with the C-Terminal Antibody as described herein coated on the plate (C-terminal Antibody started at lOug / mL with a 2x dilution) was varied at 5, 10, 20, and 30 minutes. As shown in Figure 7, even 5 minutes incubation was able to detect tirzepatide.

[0142] Sequences

[0143] SEQ ID NO: 1

[0144] GFTFSSY

[0145] SEQ ID NO: 2

[0146] SGGGSY

[0147] SEQ ID NO: 3

[0148] QLVYGMDY

[0149] SEQ ID NO: 4

[0150] RSSKSLLHSNGITYLY

[0151] SEQ ID NO: 5

[0152] QMSNLAS|0153| SEQ ID NO: 6

[0154] AHNLELPYT

[0155] SEQ ID NO: 7

[0156] EVQLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISG GGSYANYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCASQLVYGMDYWGPG TSVTVSS

[0157] SEQ ID NO: 8

[0158] DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQ MSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAHNLELPYTFGGGTKLEIK

[0159] SEQ ID NO: 9

[0160] EVQLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISG GGSYANYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCASQLVYGMDYWGPGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHT FPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPE VSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQF NSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQ MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKS NWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0161] SEQ ID NO: 10

[0162] DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQ MSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAHNLELPYTFGGGTKLEIKRAD AAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0163] SEQ ID NO: 11

[0164] atggaaactgacacactgctgctctgggtccttctgctttgggtgcctggttccaccggtgaagtgcagctcgtggagtctgga ggggatctggtcaagcccggaggttcgctgaagctgtcatgcgccgcaagcggttttaccttctcctcctacggaatgagctgggtcagaca gaccccagacaagcggcttgaatgggtggccactatttccggcggaggcagctacgccaactatccggattccgtgaaagggaggttcac catctcgcgcgacaacgccaagaacaccctctacttgcaaatgtcatcgctgaagtccgaggacactgctatgtactactgtgcgagccagc tggtgtacggaatggactactggggccctggcacttccgtgaccgtgtcctccgccaaaacgacacccccatctgtctatccgctagcccct ggatctgccgcccagaccaacagcatggtgaccctgggctgtctggtgaagggctacttccctgagcctgtgacagtgacctggaacagc ggctctctgtctagcggcgtgcacacattccctgccgtgctgcagagcgacctgtacaccctgagcagcagcgtgaccgtgcctagcagca catggcctagcgagacagtgacatgcaacgtggcccaccctgcctcttctaccaaggtggacaagaagatcgtgcccagagactgcggct gcaagccttgcatctgcaccgtgcctgaggtgagcagcgtgttcatcttcccacccaagcccaaggacgtgctcaccatcaccctcaccccc aaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctca gacgcaaccccgggaggagcagttcaacagcactttccgctcagtcagtgaacttcccatcatgcaccaggactggctcaatggcaagga gttcaaatgcagggtcaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggt gtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacagacttcttccctgaagacattactgt ggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaa gctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactgagaag agcctctcccactctcctggtaaatag

[0165] SEQ ID NO: 12

[0166] atggaaactgacacactgctgctctgggtccttctgctttgggtgcctggttccaccggtgatatcgtgatgacccaggccgcc ttctctaaccctgtgactctgggtacttcagcttccattagctgccggtcctcgaagtcgctgttgcattccaatgggatcacctacctctattggt accttcaaaagccgggacagagcccacagctgctgatctaccaaatgagcaacctggcatccggagtgccggacagattctcctcatcgg gctccgggactgacttcaccctccggatttcccgcgtggaagcggaggacgtgggagtctactactgtgcccacaacctcgaactgcccta cacctttggaggcggcaccaagctggagatcaaaagggctgatgcggcgcccactgtatccatcttcccaccatccagtgagcagttaaca tctggaggtgctagcgtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaat ggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatga acgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgttag

[0167] SEQ ID NO: 13

[0168] GFTFSRY

[0169] SEQ ID NO: 14

[0170] SPGGHY

[0171] SEQ ID NO: 15

[0172] DNGPDGLDY

[0173] SEQ 1D NO: 16

[0174] KSSRSLLHRDGKTYLN

[0175] SEQ ID NO: 17

[0176] LVSKRAP

[0177] SEQ ID NO: 18

[0178] WQGTHFRT

[0179] SEQ ID NO: 19

[0180] DVKMVESGGGLVKPGGSLKLSCAASGFTFSRYAMSWVRQTPEKRLEWVAYIS PGGHYKYYGDTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCTRDNGPDGLDYWG QGTSVTVSS

[0181] SEQ ID NO: 20

[0182] DFVMTQTPLTLSVTLGQPASISCKSSRSLLHRDGKTYLNWLLQRPGQSPKRLIY LVSKRAPGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFRTFGGGTKLEIK

[0183] SEQ ID NO: 21

[0184] DVKMVESGGGLVKPGGSLKLSCAASGFTFSRYAMSWVRQTPEKRLEWVAYIS PGGHYKYYGDTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCTRDNGPDGLDYWG QGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGV HTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICT VPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREE QFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPK EQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQ KSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0185] SEQ ID NO: 22

[0186] DFVMTQTPLTLSVTLGQPASISCKSSRSLLHRDGKTYLNWLLQRPGQSPKRLIY LVSKRAPGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFRTFGGGTKLEIKRA DAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDS KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSP1VKSFNRNEC

[0187] SEQ ID NO: 23

[0188] atggaaactgacacactgctgctctgggtccttctgctttgggtgcctggttccaccggtgatgtgaagatggtggagtccggt ggaggactggtcaagccgggtggaagccttaagctctcgtgtgccgcatccggcttcaccttttcgcgctatgccatgtcctgggtccggca gactcccgaaaagcgcctggaatgggtggcctacatctcaccgggagggcactacaagtactacggcgacaccgtgaaaggccggttca ccatttcgagggacaacgcgcggaacacgctgtacctccaaatgagcagcctgaagtccgaggacaccgctatgtactactgcactagag acaacggacctgacgggttggattactggggacagggcacttcagtgaccgtgtcctccgccaaaacgacacccccatctgtctatccgct agcccctggatctgccgcccagaccaacagcatggtgaccctgggctgtctggtgaagggctacttccctgagcctgtgacagtgacctgg aacagcggctctctgtctagcggcgtgcacacattccctgccgtgctgcagagcgacctgtacaccctgagcagcagcgtgaccgtgccta gcagcacatggcctagcgagacagtgacatgcaacgtggcccaccctgcctcttctaccaaggtggacaagaagatcgtgcccagagact gcggctgcaagccttgcatctgcaccgtgcctgaggtgagcagcgtgttcatcttcccacccaagcccaaggacgtgctcaccatcaccctc acccccaaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacgcaaccccgggaggagcagttcaacagcactttccgctcagtcagtgaacttcccatcatgcaccaggactggctcaatggc aaggagttcaaatgcagggtcaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctcca caggtgtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacagacttcttccctgaagacatt actgtggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctaca gcaagctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactga gaagagcctctcccactctcctggtaaatag

[0189] SEQ ID NO: 24

[0190] atggaaactgacacactgctgctctgggtccttctgctttgggtgcctggttccaccggtgatttcgtgatgacccagactcccc tcactctgagcgtgaccctgggtcagccagcctccatttcctgcaaaagctcccggtccctgctgcatcgggacggaaagacctacctgaac tggttgctgcaacggccgggacagtcacccaagcgcctcatctacctggtgtccaagagggctcctggagtgccggacagattcactgggt cgggttcgggcaccgatttcacgctgaagatcagccgcgtcgaagccgaggaccttggcgtgtattactgttggcaggggacccactttag aaccttcggaggcggcaccaagctcgaaatcaagcgcgctgatgcggcgcccactgtatccatcttcccaccatccagtgagcagttaaca tctggaggtgctagcgtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaat ggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatga acgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgttag

[0191] SEQ ID NO: 25 (Tirzepatide) YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS

[0192] wherein Xi is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with (2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(yGlu)i-CO-(CH2) 18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide

[0193] SEQ ID NO: 26Y-(Aib)-EGTFTSDYSI-(Aib)-LDKIAQ-Cys

[0194] SEQ ID NO: 27Cys-VQWLIAGGPSSGAPPPS-NFE

[0195] SEQ ID NO:28 (Semaglutide)H-(Aib)-EGTFTSDVSSYLEGQAAK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(yGlu)- CO-(CH2)16-CO2H)EFIAWLVRGRG

[0196] SEQ ID NO:29 (Dulaglutide) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPP CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLG

[0197] SEQ ID NO:30 (Cetuximab HC) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSG GNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAY WGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

[0198] SEQ ID NO 31 (Cetuximab LC) DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIP SRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0199] SEQ ID NO: 32 (Retatrutide)Y-(Aib)-QGTFTSDYSI-(aMeL)-LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)-(yGlu)- CO-(CH2)i8-CO2H)AQ-(Aib)-AFIEYLLEGGPSSGAPPPS-NH2

Claims

CLAIMSWe claim:

1. A tirzepatide binding molecule that binds the N-terminal region of tirzepatide (SEQ ID NO: 25), wherein the tirzepatide binding molecule comprises the complementarity determining regions (CDRs) set forth in SEQ ID NOS: 1-6.

2. The tirzepatide binding molecule of claim 1, wherein the tirzepatide binding molecule is an antibody.

3. The tirzepatide binding molecule of claim 1, wherein the tirzepatide binding molecule is an scFv or a Fab.

4. The tirzepatide binding molecule of claim 1 or claim 2, wherein the tirzepatide binding molecule is an antibody comprising a heavy chain variable region (VH) comprising SEQ ID NO: 7 and a light chain variable region (VL) comprising SEQ ID NO: 8.

5. The tirzepatide binding molecule of any one of claims 1, 2 or 4, wherein the tirzepatide binding molecule is an antibody comprising a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10.

6. 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.

7. The nucleic acid molecule of claim 6, wherein the first nucleic acid sequence comprises SEQ ID NO: 11, and wherein the second nucleic acid sequence comprises SEQ ID NO:

12.

8. A vector 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.

9. A composition comprising the vector of claim 8.

10. A cell comprising the vector of Claim 8.

11. The cell of claim 10, wherein the cell is a mammalian cell.

12. A process of producing a tirzepatide binding molecule, comprising culturing a cell 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 under conditions such that the tirzepatidebinding molecule is expressed, and recovering the expressed tirzepatide binding molecule from the culture medium.

13. A tirzepatide binding molecule produced by the process of claim 12.

14. A composition comprising the tirzepatide binding molecule of any one of claims 1-6 and 13.

15. A tirzepatide binding molecule that binds the C-terminal region of tirzepatide (SEQ ID NO: 25), wherein the tirzepatide binding molecule comprises the complementarity determining regions (CDRs) set forth in SEQ ID NOS: 13-18.

16. The tirzepatide binding molecule of claim 15, wherein the tirzepatide binding molecule is an antibody.

17. The tirzepatide binding molecule of claim 15, wherein the tirzepatide binding molecule is an scFv or a Fab.

18. The tirzepatide binding molecule of claim 15 or claim 16, wherein the tirzepatide binding molecule is an antibody comprising a VH comprising SEQ ID NO: 19 and a VL comprising SEQ ID NO: 20.

19. The tirzepatide binding molecule of any one of claims 15, 16 or 18, wherein the tirzepatide binding molecule is an antibody comprising a heavy chain comprising SEQ ID NO: 21 and a light chain comprising SEQ ID NO: 22.

20. The tirzepatide binding molecule of any one of claims 15, 16, 18 or 19, wherein the tirzepatide binding molecule is an antibody comprising a heavy chain consisting of a SEQ ID NO:21 and a light chain consisting of SEQ ID NO: 22.

21. 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.

22. The nucleic acid molecule of claim 21 , wherein the first nucleic acid sequence comprises SEQ ID NO: 23, and wherein the second nucleic acid sequence comprises SEQ ID NO:

24.

23. A vector comprising one or both of a first nucleic acid sequence encoding SEQ ID NO: 23 and a second nucleic acid sequence encoding SEQ ID NO: 24.

24. A composition comprising the vector of claim 23.

25. A cell comprising the vector of Claim 23.

26. The cell of claim 25, wherein the cell is a mammalian cell.

27. A process of producing a tirzepatide binding molecule, comprising culturing a cell 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 under conditions such that the tirzepatide binding molecule is expressed, and recovering the expressed tirzepatide binding molecule from the culture medium.

28. A tirzepatide binding molecule produced by the process of claim 27.

29. A composition comprising the tirzepatide binding molecule of any one of claims 15-20 or 28.

30. A composition comprising (a) a first tirzepatide binding molecule that binds the N- terminal region in each of tirzepatide (SEQ ID NO: 25), wherein the first tirzepatide binding molecule comprises the CDRs set forth in SEQ ID NOS: 1-6, and (b) a second tirzepatide binding molecule that binds the C-terminal region in each of tirzepatide (SEQ ID NO: 25), wherein second the tirzepatide binding molecule comprises the CDRs set forth in SEQ ID NOS: 13-18.

31. A method for determining the presence of tirzepatide (SEQ ID NO: 25) in a sample, comprising:(a) contacting a sample suspected of containing tirzepatide with a first tirzepatide binding molecule that binds the C-terminal region in each of tirzepatide (SEQ ID NO: 25), wherein the first tirzepatide binding molecule comprises the CDRs of SEQ ID NOS: 13-18, thereby forming a first tirzepatide binding molecule- tirzepatide complex if tirzepatide is present;(b) contacting the results of step (a) with a second tirzepatide binding molecule that binds the N-terminal region of each of tirzepatide (SEQ ID NO: 25), wherein the second tirzepatide binding molecule comprises the CDRs of SEQ ID NOS: 1-6, thereby forming a first tirzepatide binding molecule-tirzepatide-second tirzepatide binding molecule complex if tirzepatide is present; and(c) detecting the first tirzepatide binding molecule-tirzepatide-second tirzepatide binding molecule complex if tirzepatide is present in the sample.

32. The method of claim 31 , wherein one of the first tirzepatide binding molecule or second tirzepatide binding molecule is biotinylated.

33. The method of claim 32, wherein the detecting step further comprises adding streptavidin-conjugated horseradish peroxidase to produce a visual color change if tirzepatide is present in the sample.

34. The method of any one of claims 31-33, wherein the sample is a solid or a liquid.

35. The method of any one of claims 31-34, wherein the first tirzepatide binding molecule is an antibody and the second tirzepatide binding molecule is an antibody.

36. The method of any one of claims 31-35, wherein the first tirzepatide binding molecule is an tirzepatide binding comprising a VH comprising SEQ ID NO: 19 and a VL comprising SEQ ID NO: 20, and the second tirzepatide binding molecule is an antibody comprising a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 8.

37. The method of any one of claims 31-35, wherein the first tirzepatide binding molecule is an antibody comprising a heavy chain comprising SEQ ID NO: 21 and comprising a light chain comprising SEQ ID NO: 22, and the second tirzepatide binding molecule is an antibody comprising a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10.

38. The method of any one of claims 31-35, wherein the first tirzepatide binding molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 21 and comprising a light chain consisting of SEQ ID NO: 22, and the second tirzepatide binding molecule is an antibody comprising a heavy chain consisting of SEQ ID NO: 9 and a light chain consisting of SEQ ID NO: 10.

39. The method of any one of claims 31-38, wherein tirzepatide is detected in less than 60 minutes.

40. The method of any one of claims 31-38, wherein tirzepatide is detected in less than 30 minutes.

41. The method of any one of claims 31-38, wherein tirzepatide is detected in less than 20 minutes.

42. Use of a first tirzepatide binding molecule that binds the C-terminal region of tirzepatide, and a second tirzepatide binding molecule that binds the N-terminal region of tirzepatide, in detecting the presence of tirzepatide in a sample.

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