Bifunctional fusion protein

Abstract

Provided is a bifunctional fusion protein. Specifically provided are a bifunctional fusion protein which specifically binds to a glucose-dependent insulinotropic polypeptide receptor and a glucagon-like peptide-1 receptor and the use thereof.

Description

Bifunctional fusion protein

[0001] This application claims priority to Chinese invention patent application number 202410237970.7, filed March 1, 2024, entitled “Dual Function Fusion Protein,” the contents of which are incorporated herein by reference. Technical Field

[0002] The present disclosure relates to the field of biomedicine and discloses a fusion protein having a therapeutic and / or preventive effect on metabolic diseases. Specifically, the present disclosure relates to a fusion protein comprising a GLP-1R antagonist and a GLP-1R agonist. Background Art

[0003] Glucose-dependent insulinotropic polypeptide (GIP) is a gut-derived incretin hormone with the ability to enhance glucose-stimulated insulin secretion. GIP is secreted by K cells located in the proximal small intestine. Dietary carbohydrates and fat are potent stimulators of GIP secretion in humans. Studies have shown that high-fat diets increase GIP secretion from K cells in rodents. Similarly, acute high-fat diet ingestion in humans leads to a 42% increase in GIP concentrations, followed by significant weight gain, indicating a positive correlation between high-fat diet exposure and GIP concentrations. Therefore, high-fat diets and elevated systemic GIP concentrations may underlie differences in obesity in human subjects.

[0004] Recently, several human genetic studies have demonstrated a link between the glucose-dependent insulinotropic polypeptide receptor (GIPR) and body mass index (BMI). Furthermore, GIPR knockout mice exhibit resistance to high-fat diet-induced obesity. Combined with the established role of GIPR in pancreatic cells and adipocytes, developing GIPR antagonists for the treatment of obesity appears to be a viable strategy.

[0005] Furthermore, glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted by the L-cells of the small intestinal epithelium. In healthy individuals, GLP-1 responds to nutrient intake, promoting insulin release and inhibiting glucagon secretion. GLP-1 specifically binds to the GLP-1 receptor (GLP-1R), activating intracellular pathways and causing an increase in cAMP. GLP-1 promotes glucose-dependent insulin secretion and inhibits glucagon synthesis. It can also act on the central nervous system, suppressing the feeding center to reduce food intake and thus leading to weight loss. It can also act on the gastrointestinal tract to delay gastric emptying and increase satiety, thereby achieving the effects of lowering blood sugar and weight loss. Summary of the Invention

[0006] The present disclosure provides a bifunctional fusion protein consisting of an anti-GIPR single-domain antibody and a GLP-1R agonist, wherein the fusion protein has a significant metabolic regulation effect.

[0007] In a first aspect, the present disclosure provides a fusion protein comprising: a) an antigen binding region that binds to a glucose-dependent insulinotropic polypeptide receptor (GIPR), for example, an immunoglobulin single variable domain that binds to a GIPR; and b) a glucagon-like peptide-1 receptor (GLP-1R) agonist.

[0008] In some embodiments, the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO: 1. In some embodiments, the CDR1, CDR2, and CDR3 are defined according to the following definition systems: Kabat, AbM, Chothia, or IMGT.

[0009] In some embodiments, the immunoglobulin single variable domain is camelid, humanized, or chimeric.

[0010] In some embodiments, the CDR1, CDR2, and CDR3 in the VHH represented by SEQ ID NO: 1 are selected from any one of the following groups: SEQ ID NOs: 9-11, SEQ ID NOs: 12-14, SEQ ID NOs: 15-17, and SEQ ID NOs: 18-20.

[0011] In some embodiments, the immunoglobulin single variable domain comprises the amino acid sequence of any one of SEQ ID NOs: 1-8. In some embodiments, the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 6.

[0012] In some embodiments, the antigen binding region comprises a plurality (e.g., 2, 3, or 4) of immunoglobulin single variable domains. In some embodiments, each of the plurality of immunoglobulin single variable domains independently comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-8. In some embodiments, at least one, e.g., each, of the plurality of immunoglobulin single variable domains comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, at least one, e.g., each, of the plurality of immunoglobulin single variable domains comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0013] In some embodiments, the GLP-1R agonist is human GLP-1 or a variant thereof, or human GLP-1 or a functional fragment thereof, or exenatide or a variant thereof, or a functional fragment of exenatide or a variant thereof.

[0014] In some embodiments, the GLP-1R agonist is human GLP-1, e.g., comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the GLP-1R agonist is a human GLP-1 variant, e.g., comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the GLP-1R agonist is a human GLP-1 variant, which further comprises an amino acid mutation at one or more of Y19, Q23, K26, and W31, compared to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the GLP-1R agonist is a human GLP-1 variant, which further comprises an amino acid mutation at one of Y19, Q23, and W31, compared to the amino acid sequence of SEQ ID NO: 22.

[0015] In some embodiments, the GLP-1R agonist is a human GLP-1 variant, which further comprises an amino acid mutation at a position selected from the group consisting of:

[0016] 1)Y19;

[0017] 2)Q23;

[0018] 3)W31;

[0019] 4) Y19 and K26:

[0020] 5) Y19 and W31;

[0021] 6) Q23 and K26;

[0022] 7) Q23 and W31;

[0023] 8) K26 and W31;

[0024] 9) Y19, K26, and W31; and

[0025] 10)Q23, K26 and W31.

[0026] In some embodiments, the amino acid mutation at position W31 is a substitution selected from the group consisting of W31Y, W31L, and W31A. In some embodiments, the amino acid mutation at position K26 is K26R. In some embodiments, the amino acid mutation at position Q23 is a substitution selected from the group consisting of Q23E, Q23T, Q23S, and Q23N. In some embodiments, the amino acid mutation at position Y19 is a substitution selected from the group consisting of Y19A, Y19L, Y19T, Y19F, Y19I, Y19V, and Y19S. In some embodiments, the GLP-1R agonist comprises the amino acid sequence of any one of SEQ ID NOs: 23-36. In some embodiments, the GLP-1R agonist comprises the amino acid sequence of any one of SEQ ID NOs: 37-59. In some embodiments, the GLP-1R agonist comprises the amino acid sequence of any one of SEQ ID NOs: 60-70.

[0027] In some embodiments, the GLP-1R agonist is located at the N-terminus or C-terminus of the antigen binding region.

[0028] In some embodiments, the fusion protein further comprises an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is derived from an IgG; preferably, the immunoglobulin Fc region is derived from an IgG1, IgG2, IgG3, or IgG4. In some embodiments, the immunoglobulin Fc region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 71-74; preferably, the amino acid sequence set forth in SEQ ID NO: 74.

[0029] In some embodiments, the immunoglobulin Fc region comprises an amino acid mutation that selectively enhances the binding affinity of the immunoglobulin Fc region to an Fc receptor compared to an unmutated immunoglobulin Fc region; preferably, the amino acid mutation selectively enhances the binding affinity of the immunoglobulin Fc region to the neonatal Fc receptor (FcRn). In some embodiments, the immunoglobulin Fc region comprises amino acid mutations at positions M252, S254, and T256 according to the EU index of Kabat. In some embodiments, the amino acid mutations comprised in the immunoglobulin Fc region are M252Y, S254T, and T256E. In some embodiments, the immunoglobulin Fc region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 75-78; preferably, it comprises an amino acid sequence as shown in SEQ ID NO: 78.

[0030] In some embodiments, the fusion protein comprises a configuration selected from any one of formulas (A)-(D):

[0031] (A)

[0032] (B)

[0033] (C) and

[0034] (D)

[0035] Wherein, GIPR BM is the antigen binding region that binds to GIPR as described above, GLP-1Ra is the GLP-1R agonist as described above, and Fc is the immunoglobulin Fc region as described above;

[0036] L1 and L2 are each independently a peptide linker, or are absent;

[0037] HR is the hinge region of an immunoglobulin, for example, HR comprises a hinge region derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the HR comprises the amino acid sequence shown in any one of SEQ ID NOs: 79-82, preferably comprising the sequence shown in SEQ ID NO: 79.

[0038] In some embodiments, the peptide linker has a length of 2-20 amino acids. In some embodiments, the peptide linker comprises GS, GAP, or an amino acid sequence as shown in any one of SEQ ID NOs: 83-87, preferably comprising the sequence shown in SEQ ID NO: 85 or SEQ ID NO: 87.

[0039] In some embodiments, the fusion protein comprises any one of the combinations of L1, L2, and HR shown in (I) to (XIV):

[0040] (I) L1 is the amino acid sequence set forth in SEQ ID NO: 85, L2 is the amino acid sequence set forth in SEQ ID NO: 85, and HR is the amino acid sequence set forth in SEQ ID NO: 82;

[0041] (II) L1 is the amino acid sequence set forth in SEQ ID NO:85, L2 is the amino acid sequence set forth in SEQ ID NO:85, and HR is the amino acid sequence set forth in SEQ ID NO:79;

[0042] (III) L1 is the amino acid sequence set forth in SEQ ID NO:85, L2 is the amino acid sequence set forth in SEQ ID NO:87, and HR is the amino acid sequence set forth in SEQ ID NO:82;

[0043] (IV) L1 is the amino acid sequence set forth in SEQ ID NO:85, L2 is the amino acid sequence set forth in SEQ ID NO:87, and HR is the amino acid sequence set forth in SEQ ID NO:79;

[0044] (V) L1 is the amino acid sequence set forth in SEQ ID NO:87, L2 is the amino acid sequence set forth in SEQ ID NO:85, and HR is the amino acid sequence set forth in SEQ ID NO:82;

[0045] (VI) L1 is the amino acid sequence set forth in SEQ ID NO:87, L2 is the amino acid sequence set forth in SEQ ID NO:85, and HR is the amino acid sequence set forth in SEQ ID NO:79;

[0046] (VII) L1 has the amino acid sequence set forth in SEQ ID NO: 85, L2 is absent, and HR has the amino acid sequence set forth in SEQ ID NO: 82;

[0047] (VIII) L1 has the amino acid sequence set forth in SEQ ID NO:85, L2 is absent, and HR has the amino acid sequence set forth in SEQ ID NO:79;

[0048] (IX) L1 has the amino acid sequence set forth in SEQ ID NO: 87, L2 is absent, and HR has the amino acid sequence set forth in SEQ ID NO: 82;

[0049] (X) L1 represents the amino acid sequence set forth in SEQ ID NO:87, L2 is absent, and HR represents the amino acid sequence set forth in SEQ ID NO:79;

[0050] (XI) L1 is absent, L2 has the amino acid sequence set forth in SEQ ID NO: 85, and HR has the amino acid sequence set forth in SEQ ID NO: 82;

[0051] (XII) L1 is absent, L2 has the amino acid sequence set forth in SEQ ID NO:85, and HR has the amino acid sequence set forth in SEQ ID NO:79;

[0052] (XIII) L1 is absent, L2 has the amino acid sequence set forth in SEQ ID NO: 87, and HR has the amino acid sequence set forth in SEQ ID NO: 82; and

[0053] (XIV) L1 is absent, L2 has the amino acid sequence shown in SEQ ID NO: 87, and HR has the amino acid sequence shown in SEQ ID NO: 79.

[0054] In some embodiments, the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 88-93. In some embodiments, the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 94-97.

[0055] In some embodiments, the fusion protein is a homodimer.

[0056] In a second aspect, the present disclosure provides a nucleic acid molecule encoding the fusion protein described in the first aspect of the present disclosure.

[0057] In a third aspect, the present disclosure provides an expression vector comprising the nucleic acid molecule according to the second aspect operably linked to an expression regulatory element.

[0058] In a fourth aspect, the present disclosure provides a recombinant cell comprising the nucleic acid molecule of the second aspect and / or the expression vector of the third aspect, and capable of expressing the fusion protein of the first aspect.

[0059] In a fifth aspect, the present disclosure further provides a pharmaceutical composition comprising the fusion protein described in the first aspect and / or the nucleic acid molecule described in the second aspect and / or the expression vector described in the third aspect and / or the recombinant cell described in the fourth aspect, and a pharmaceutically acceptable carrier.

[0060] In a sixth aspect, the present disclosure further provides a kit comprising the fusion protein described in the first aspect and / or the pharmaceutical composition described in the fifth aspect.

[0061] In a seventh aspect, the present disclosure also provides a method for treating and / or preventing metabolic diseases and / or symptoms, comprising administering an effective amount of the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, and / or the pharmaceutical composition described in the fifth aspect to a subject in need.

[0062] In some embodiments, the metabolic disease and / or condition is obesity, overweight, diabetes, hyperlipidemia, and / or non-alcoholic fatty liver disease.

[0063] Those skilled in the art will readily appreciate other aspects and advantages of the present disclosure from the detailed description below. The detailed description below only shows and describes exemplary embodiments of the present disclosure. As will be appreciated by those skilled in the art, the disclosure enables those skilled in the art to modify the disclosed embodiments without departing from the spirit and scope of the invention to which the present disclosure relates. Accordingly, the drawings and descriptions of the present disclosure are intended to be exemplary only and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The specific features of the invention disclosed herein are set forth in the appended claims. The features and advantages of the invention disclosed herein can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0065] FIG1 shows the blocking activity of GIPR single domain antibody-Fc fusion protein on GIPR, wherein △ represents iGI-72-Ld-Fc, and ○ represents the positive control AMG-GIPR-mab2.

[0066] FIG2 shows the effects of multiple administrations of GIPR single domain antibody-Fc fusion protein on the body weight of DIO mice.

[0067] FIG3 shows the effects of multiple administrations of GIPR single domain antibody-Fc fusion protein on fasting blood glucose in DIO mice.

[0068] FIG4 shows the effects of multiple administrations of GIPR single domain antibody-Fc fusion protein on insulin secretion in DIO mice.

[0069] FIG5 shows the effects of multiple administrations of GIPR single domain antibody-Fc fusion protein on the insulin resistance index of DIO mice.

[0070] FIG6 shows the binding ability of humanized GIPR single domain antibody-Fc fusion protein and Maridebart to GIPR.

[0071] FIG7 shows the neutralizing activity of humanized GIPR single domain antibody-Fc fusion protein.

[0072] FIG8 shows the neutralization ability of humanized GIPR single domain antibody-Fc fusion protein and Maridebart against GIPR.

[0073] FIG9 shows the effect of humanized GIPR single domain antibody-Fc fusion protein on blood glucose in C57BL / 6 mice stimulated by DA-GIP.

[0074] FIG10 shows the effect of humanized GIPR single domain antibody-Fc fusion protein on DA-GIP-stimulated insulin secretion in C57BL / 6 mice.

[0075] FIG11 shows the effects of multiple administrations of humanized GIPR single domain antibody-Fc fusion protein on the body weight of ob mice.

[0076] FIG. 12a to FIG. 12c show the neutralization activity of the bifunctional fusion protein of the present disclosure on GIPR.

[0077] FIG. 13 a - FIG. 13 c show the agonistic activity of the bifunctional fusion protein of the present disclosure on GLP-1R.

[0078] FIG14 shows the biological effects of the bifunctional fusion protein of the present disclosure on GIPR-GLP-1R dual receptor cells.

[0079] FIG15 shows the effects of bifunctional fusion proteins comprising different GLP-1 variants on body weight in DIO mice.

[0080] FIG16 shows the effects of bifunctional fusion proteins containing different linkers and hinge regions on the body weight of DIO mice.

[0081] Figures 17a-17g show the effects of the bifunctional fusion protein of the present disclosure on body weight, non-fasting blood glucose, OGTT, ITT, fat-to-body ratio, TG, TC, LDL-C, HDL-C and liver TG in DIO mice.

[0082] FIG18 shows the PK results of the bifunctional fusion protein of the present disclosure in FcRn humanized mice.

[0083] FIG19 shows the binding of the bifunctional fusion protein of the present disclosure to HEK293-GLP1R-CREB cells.

[0084] FIG20 shows the results of the bifunctional fusion protein of the present disclosure acting on mouse pancreatic islets to promote glucose-dependent insulin release. DETAILED DESCRIPTION

[0085] The following describes the embodiments of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0086] Definition of terms

[0087] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which will be understood by those skilled in the art. Reference is made, for example, to standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd edition), Volumes 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al., "Immunology" (2nd edition), Gower Medical Publishing, London, New York (1989), as well as the general prior art cited herein; in addition, unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can and have been carried out in a manner known per se, which will be understood by those skilled in the art. Reference is also made, for example, to standard manuals, the above-mentioned general prior art and other references cited therein.

[0088] Unless otherwise indicated, the terms "antibody" or "immunoglobulin" are used interchangeably, whether referring to heavy chain antibodies or conventional four-chain antibodies herein, and are used as general terms to include full-length antibodies, their individual chains, and all parts, domains, or fragments thereof (including but not limited to antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively). In addition, the term "sequence" used herein (e.g., in terms such as "immunoglobulin sequence," "antibody sequence," "single variable domain sequence," "VHH sequence," or "protein sequence") is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.

[0089] As used herein, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and, in many cases, can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or the domain.

[0090] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional 4-chain antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a globular region. An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristic of an antibody molecule.

[0091] As used herein, the term "immunoglobulin variable domain" refers to an immunoglobulin domain that essentially consists of four "framework regions," referred to in the art and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3," respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity to an antibody for an antigen by having an antigen-binding site.

[0092] As used herein, the term "immunoglobulin single variable domain" refers to an immunoglobulin variable domain that is capable of specifically binding to an antigenic epitope without being paired with other immunoglobulin variable domains. An example of an immunoglobulin single variable domain of the present disclosure is a "domain antibody," such as an immunoglobulin single variable domain VH and VL (VH domain and VL domain). Another example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") of Camelidae, as defined below.

[0093] "VHH domain", also known as heavy chain single domain antibody, VHH, VHH antibody fragment and VHH antibody, is the variable domain of the antigen-binding immunoglobulin called "heavy chain antibody" (i.e., "antibody lacking light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain present in conventional four-chain antibodies (which is referred to herein as "VH domain") and the light chain variable domain present in conventional four-chain antibodies (which is referred to herein as "VL domain"). The VHH domain specifically binds an epitope without the need for additional antigen-binding domains (in contrast to the VH or VL domains in conventional four-chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and efficient antigen-recognition unit formed by a single immunoglobulin domain.

[0094] In the context of the present disclosure, the terms "heavy chain single domain antibody", "single domain antibody", "VHH domain", "VHH", "VHH antibody fragment", and "VHH antibody" are used interchangeably.

[0095] For example, as shown in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999), the amino acid residues used for the VHH domain of Camelidae can be numbered according to the general numbering method for VH domains given by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0096] Alternative methods for numbering and defining the amino acid residues of VH domains are known in the art and can be similarly applied to VHH domains. For example, Chothia CDRs refer to the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM CDRs represent a compromise between the Kabat hypervariable regions and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. "Contact" CDRs are based on analysis of available complex crystal structures. The residue descriptions of the CDRs from each method are shown in Table 1 below:

[0097] Table 1. CDR residues according to various definition systems

[0098] The antibody CDRs can also be IMGT-CDRs, which is a CDR definition based on the IMGT antibody encoding, which was obtained by integrating structural information from over 5,000 sequences. In the IMGT VH CDR encoding, CDR1: 27-38; CDR2: 56-65; CDR3: 105-117.

[0099] It should be noted, however, that, as is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

[0100] For example, the CDRs may comprise "extended CDRs", e.g., 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2), and 89-97 or 89-96 (LCDR3) in VL; 26-35 (HCDR1), 50-65 or 49-65 (HCDR2), and 93-102, 94-102, or 95-102 (HCDR3) in VH.

[0101] The total number of amino acid residues in a VHH domain will generally range from 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0102] Other structural characteristics and functional properties of VHH domains and polypeptides containing them can be summarized as follows:

[0103] The VHH domain (which has been naturally "designed" to functionally bind to an antigen in the absence of, and without interacting with, a light chain variable domain) can be used as a single, relatively small, functional antigen-binding structural unit, domain, or polypeptide. This property distinguishes the VHH domain from the VH and VL domains of conventional four-chain antibodies. These VH and VL domains themselves are generally not suitable for practical application as a single antigen-binding protein or immunoglobulin single variable domain, but need to be combined in some form or another to provide a functional antigen-binding unit (e.g., in the form of a conventional antibody fragment such as a Fab fragment; or in the form of an scFv consisting of a VH domain covalently linked to a VL domain).

[0104] Due to these unique properties, the use of VHH domains—alone or as part of a larger polypeptide—offers a number of significant advantages over the use of conventional VH and VL domains, scFvs, or conventional antibody fragments (e.g., Fab- or F(ab')2-fragments): only a single domain is required to bind antigen with high affinity and selectivity, thereby eliminating the need for the presence of two separate domains and the need to ensure that the two domains are in the proper spatial conformation and configuration (e.g., scFvs generally require the use of specially designed linkers); VHH domains can be expressed from a single gene and do not require post-translational folding or modification; VHH domains can be easily engineered into multivalent and multispecific formats (formatting); VHH domains are highly soluble and have no tendency to aggregate; VHH domains They are highly stable to heat, pH, proteases, and other denaturing agents or conditions, and therefore can be prepared, stored, or transported without the use of refrigeration equipment, thereby saving cost, time, and the environment; VHH domains are easy to prepare and relatively inexpensive, even at the scale required for production; VHH domains are relatively small compared to conventional four-chain antibodies and antigen-binding fragments thereof (approximately 15 kDa, or 1 / 10 the size of conventional IgG), and therefore exhibit higher tissue penetration and can be administered at higher doses than conventional four-chain antibodies and antigen-binding fragments thereof; VHH domains can exhibit so-called cavity-binding properties (particularly due to their extended CDR3 loops compared to conventional VH domains), thereby being able to access targets and epitopes that are inaccessible to conventional four-chain antibodies and antigen-binding fragments thereof.

[0105] Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185–195; Li et al., J Biol Chem., 287 (2012) 13713–13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645-2652, 17 June, 2009 and WO94 / 04678.

[0106] A VHH domain derived from Camelidae can be "humanized" (also referred to herein as "sequence optimization") by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at the corresponding positions in a conventional human four-chain antibody VH domain. In addition to humanization, "sequence optimization" can also encompass other modifications to the sequence by one or more mutations that provide improved properties of the VHH, such as removal of potential post-translational modification sites. The humanized VHH domain may contain one or more fully human framework region sequences. Humanization can be accomplished using protein surface amino acid humanization methods and / or humanized universal framework CDR grafting, for example, as exemplified in the Examples.

[0107] In general, the term "specificity" refers to the number of different types of antigens or epitopes to which a particular antigen binding molecule or antigen binding protein (e.g., an immunoglobulin single variable domain, a heavy chain single domain antibody, or a GIPR binding protein of the present disclosure) can bind. Specificity can be determined based on the affinity and / or avidity of an antigen binding protein. Affinity, represented by the dissociation equilibrium constant (KD) of an antigen and an antigen binding protein, is a measure of the binding strength between an epitope and an antigen binding site on an antigen binding protein: the smaller the KD value, the stronger the binding strength between the epitope and the antigen binding protein (alternatively, affinity can also be expressed as an association constant (KA), which is 1 / KD). As will be appreciated by those skilled in the art, affinity can be determined in a known manner, depending on the specific antigen of interest. Avidity is a measure of the binding strength between an antigen binding protein (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing the same) and a related antigen. Avidity is related to both the affinity between an antigen binding site on an antigen binding protein and the relevant antigen, and the number of relevant binding sites present on the antigen binding protein.

[0108] As used herein, the term "glucose-dependent insulinotropic polypeptide receptor," abbreviated as GIPR, encompasses GIPRs of any species. Preferably, the GIPR is a human GIPR or a mouse GIPR.

[0109] As used herein, the term "glucose-dependent insulinotropic polypeptide receptor (GIPR) binding protein" means any protein that can specifically bind to the glucose-dependent insulinotropic polypeptide receptor (GIPR). GIPR binding proteins can include heavy chain single domain antibodies as defined herein against GIPR. GIPR binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules.

[0110] Typically, the fusion proteins of the present disclosure will be expressed as measured in a Biacore or KinExA or Fortibio assay at a concentration of preferably 10 -7 to 10 -10 Mole / liter (M), more preferably 10 -8 to 10 -10 mol / L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (KD), and / or with a dissociation constant of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , more preferably at least 10 10 M -1 The association constant (KA) of the target antigen (i.e., GIPR) is greater than 10 -4 KD values ​​of M are generally considered to indicate nonspecific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays) as described herein.

[0111] As used herein, the term "GLP-1" generally refers to glucagon-like peptide 1. Natural GLP-1 molecules are processed in vivo, with the first six amino acids cleaved off. Therefore, it is customary in the art to define the first amino acid at the N-terminus of the GLP-1 amino acid sequence as position 7, and the last amino acid at the C-terminus as position 37. The processed peptide can be further modified in vivo by removing the C-terminal glycine residue and replacing it with an amide group. GLP-1 generally has two biologically active forms, namely GLP-1(7-37)OH and GLP-1(7-36)NH2. The "GLP-1" described in this application includes natural, synthetic, or modified GLP-1 proteins, as well as complete GLP-1 proteins or functional fragments thereof, and GLP-1 proteins in different biologically active forms. For example, wild-type human GLP-1 may comprise the amino acid sequence shown in SEQ ID NO:21, with the N-terminal amino acid residue H being designated as position 7. Therefore, the term "K26" in this application generally refers to the position of the 26th amino acid, calculated from the 7th H at the N-terminus of the GLP-1 protein, in which the amino acid at position 26 in the amino acid sequence set forth in SEQ ID NO:21 is K; the term "W31" generally refers to the position of the 31st amino acid, calculated from the 7th H at the N-terminus of the GLP-1 protein, in which the amino acid at position 31 in the amino acid sequence set forth in SEQ ID NO:21 is W. In this application, when describing amino acid mutations and / or substitutions, the numbering of amino acid residues in GLP-1 polypeptide variants is distinguished from the numbering of amino acid residues in the Fc region, in which the numbering of amino acid residues in the Fc region is based on the EU numbering system.

[0112] The term "GLP-1R" generally refers to the glucagon like peptide 1 receptor. Binding of GLP-1R to GLP-1 activates a signaling cascade, leading to activation of adenylate cyclase and an increase in intracellular cAMP levels. The GLP-1R described herein may include full-length molecules, variants, fragments, and synthetic forms of native GLP-1R, as long as the activity of GLP-1R is retained. For example, the GLP-1R described herein may comprise the amino acid sequence shown in NCBI database accession number NP_002053.3.

[0113] The term "GLP-1R agonist" generally refers to substances that bind to and activate the GLP-1 receptor, including but not limited to GLP-1 and its active variants.

[0114] As used herein, the term "at least a portion of the activity of human GLP-1" generally refers to a polypeptide having one or more activities of a human GLP-1 protein, or having at least 20% (e.g., at least 25%, 30%, 35%, 40%, 45%, or 50% or more) of the activity of a human GLP-1 protein. The human GLP-1 referred to as "at least a portion of the activity of human GLP-1" may be wild-type, such as the amino acid sequence set forth in SEQ ID NO: 21. The human GLP-1 referred to as "at least a portion of the activity of human GLP-1" may be a GLP-1 variant engineered from wild-type human GLP-1, such as the amino acid sequence set forth in SEQ ID NO: 22. For example, the human GLP-1 polypeptide variant of the present application may have at least a portion of the activity of the human GLP-1 with an amino acid sequence such as SEQ ID NO: 21. In other cases, the human GLP-1 polypeptide variant of the present application may have at least a portion of the activity of the human GLP-1 with an amino acid sequence such as SEQ ID NO: 22.

[0115] The activity is not required to be the same level as that of human GLP-1 protein, and may be higher, similar or lower than that of human GLP-1 protein. In some cases, "at least part of the activity of human GLP-1" may refer to one or more selected from the following groups: activity of binding to GLP-1 receptor, activity of activating GLP-1 receptor, activity of activating adenylate cyclase, activity of promoting the increase of intracellular cyclic adenosine monophosphate (cAMP) level, activity of positively regulating intracellular Ca 2+ The activity of a human GLP-1 receptor agonist can be detected by measuring the activity of binding to the GLP-1 receptor or the expression level of cAMP. For example, the activity of a human GLP-1 receptor agonist can be detected by measuring the activation level of the cAMP / PKA signaling pathway using a luciferase assay. The "at least partial activity of human GLP-1" can be at least partial activity of a fusion protein comprising human GLP-1 (e.g., a fusion protein with an Fc region).

[0116] As used herein, the term "fusion protein" refers to a fusion polypeptide molecule comprising a glucose-dependent insulinotropic polypeptide receptor (GIPR) binding protein and a GLP-1R agonist, wherein the components of the fusion protein are linked to each other directly by peptide bonds or via a peptide linker.

[0117] As used herein, the term "Fc region" generally refers to a domain derived from the C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of an intact antibody. The Fc region can be a native sequence Fc region or a variant Fc region, wherein "the immunoglobulin Fc region without the amino acid mutation" refers to the amino acid sequence shown in any one of SEQ ID NOs: 71-74, which correspond to the Fc sequences of human IgG1, IgG2, IgG3, and IgG4, respectively. The Fc region of the immunoglobulin described herein generally comprises two constant domains (CH2 domain and CH3 domain), does not comprise a hinge region unless otherwise specified, and may optionally comprise a CH4 domain.

[0118] The term "hinge region" generally refers to a flexible amino acid sequence that allows the polypeptide portion at its N-terminus or C-terminus to move independently. The hinge region is typically derived from the region between the CH1 and CH2 functional domains of an immunoglobulin heavy chain. The hinge region is typically derived from IgG, for example, IgG1, IgG2, IgG3, or IgG4. Compared to the natural IgG-derived hinge region, the hinge region of the present application may have one or more amino acid residues added to, deleted from, or replaced at the N-terminus or C-terminus, as long as the function of the hinge region is maintained.

[0119] As used herein, the term "linker" generally refers to an amino acid sequence that connects two heterologous polypeptides or fragments thereof. In this application, a linker can be an amino acid sequence that covalently links polypeptides to form a fusion polypeptide. The length of the linker can be 2-20 amino acids in length. The linker can be flexible or rigid.

[0120] Amino acid residues will be represented according to the standard three-letter or one-letter amino acid code as is well known and agreed upon in the art. When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion, or substitution of a specified number of amino acid residues at a position in a reference sequence compared to another sequence. In the case of substitutions, the substitution will preferably be a conservative amino acid substitution, which refers to the replacement of an amino acid residue with another amino acid residue of similar chemical structure and which has little or substantially no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art. For example, conservative amino acid substitutions are preferably substitutions of an amino acid within the following groups (i) to (v) by another amino acid residue within the same group: (i) smaller aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (iii) polar positively charged residues: His, Arg and Lys; (iv) larger aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gln or His; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln or Glu; Met is substituted by Leu, Tyr or Ile; Phe is substituted by Met, Leu or Tyr; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is substituted by Trp or Phe; Val is substituted by Ile or Leu.

[0121] "Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program from the NCBI database can be used to determine identity. For the determination of sequence identity, see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0122] In this disclosure, the term "isolated" generally refers to a biological material (e.g., a virus, nucleic acid, or protein) that is substantially free of components that normally accompany or interact with the environment in which it occurs naturally. The isolated biological material optionally contains additional materials that the biological material is not found to have in its natural environment (e.g., nucleic acid or protein). In this disclosure, "isolated" when referring to proteins generally refers to the separation and separation of the molecule from the entire organism in which the molecule is found naturally, or the substantial absence of other biological macromolecules of the same type. When referring to a nucleic acid molecule, it is completely or partially separated from the sequence to which it is naturally associated, or the nucleic acid has a heterologous sequence to which it is associated, or the nucleic acid is separated from a chromosome.

[0123] A polypeptide or nucleic acid molecule is considered "isolated" when it has been separated from at least one other component with which it is normally associated in that source or medium (e.g., another protein / polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or trace component) compared to its natural biological source and / or the reaction medium or culture medium from which it was obtained. In particular, a polypeptide or nucleic acid molecule is considered "isolated" when it has been purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold and up to 1000-fold or more. An "isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous as determined by a suitable technique (e.g., a suitable chromatographic technique, such as polyacrylamide gel electrophoresis).

[0124] "Effective amount" means an amount of the fusion protein or pharmaceutical composition of the present disclosure that results in a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevents damage or disability caused by disease affliction.

[0125] As used herein, "metabolic disease" refers to a disorder that affects how human (or animal) cells produce energy, also known as a metabolic disorder. Most metabolic diseases are hereditary, while others are acquired through diet, toxins, or infection. Common metabolic diseases can be divided into three main categories: disorders that affect carbohydrate metabolism, disorders that affect fat metabolism, and disorders that affect the mitochondria within cells.

[0126] As used herein, the term "subject" means a mammal, particularly a primate, especially a human.

[0127] In the present disclosure, the term "antigen binding protein" generally refers to a protein comprising an antigen-binding portion, and optionally a scaffold or backbone portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen. Antigen binding proteins can typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both, and functional fragments thereof. In the present disclosure, the term "antigen binding protein" also encompasses single domain antibodies and proteins comprising a single variable domain of an immunoglobulin. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. Examples of antigen binding proteins include, but are not limited to, antibodies, antigen binding fragments, single domain antibodies, immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, as long as they show the desired antigen-binding activity.

[0128] In the present disclosure, the terms "polypeptide" or "protein" are used interchangeably and generally refer to polymers of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The term may also include modified amino acid polymers, for example, by the addition of sugar residues to form glycoproteins or modified by phosphorylation. Polypeptides and proteins can be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and can include molecules having the amino acid sequence of a native protein, or molecules having one or more amino acids deleted, added and / or substituted from the native sequence. The terms "polypeptide" and "protein" particularly include sequences that are deleted, added and / or substituted from one or more amino acids of the antigen binding proteins described in the present disclosure.

[0129] In this disclosure, the term "nucleic acid" molecule generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or their analogs, either isolated from their natural environment or artificially synthesized.

[0130] In the present disclosure, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into a host cell and / or between host cells. The vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression by transcription and / or translation of DNA or RNA. The vector also includes vectors with a variety of the above-mentioned functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0131] In the present disclosure, the term "cell" generally refers to an individual cell, cell line or cell culture that may contain or already contain a plasmid or vector comprising a nucleic acid molecule described in the present disclosure, or that is capable of expressing the antigen-binding proteins described in the present disclosure. The cell may include the progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cell, but may be capable of expressing the antibody or antigen-binding fragment thereof described in the present disclosure. The cell may be obtained by in vitro transfection of cells using the vectors described in the present disclosure. The cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, e.g., a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NSO cell or a myeloma cell). In some cases, the cell may be a mammalian cell. For example, the mammalian cell may be a CHO-K1 cell.

[0132] In this disclosure, the term "pharmaceutical composition" generally refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective, and contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0133] In the present disclosure, the term "treatment" generally refers to the desire to change the natural course of the disease in the individual being treated, and can be a clinical intervention to achieve prevention or treatment or during the clinical course of the disease. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some cases, antigen binding proteins (e.g., antibodies against specific antigens of the present disclosure) can be used to delay disease development or slow disease progression.

[0134] In the present disclosure, the term "administering" generally refers to a method of administering a dose of a compound or pharmaceutical composition to a subject (e.g., a patient). Administration can be performed by any suitable means, including parenteral, intrapulmonary, and intranasal, and (if desired for local treatment) intralesional administration. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0135] In this disclosure, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0136] In this disclosure, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0137] Detailed Description of the Invention

[0138] Fusion protein

[0139] In one aspect, the present disclosure relates to a fusion protein comprising:

[0140] a) an antigen binding region that binds to the glucose-dependent insulinotropic polypeptide receptor (GIPR), for example, an immunoglobulin single variable domain that binds to GIPR; and

[0141] b) Glucagon-like peptide-1 receptor (GLP-1R) agonists.

[0142] i) Immunoglobulin single variable domain that binds to GIPR

[0143] In some embodiments, the immunoglobulin single variable domain may comprise CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO: 1. The CDR1, CDR2, and CDR3 may be defined according to the following definition systems: Kabat, AbM, Chothia, or IMGT.

[0144] In some embodiments, the immunoglobulin single variable domain may be camelid, humanized, or chimeric.

[0145] In some embodiments, the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1 are selected from any one of the following groups: SEQ ID NO: 9-11 (Kabat), SEQ ID NO: 12-14 (AbM), SEQ ID NO: 15-17 (Chothia) and SEQ ID NO: 18-20 (IMGT).

[0146] In some embodiments, the immunoglobulin single variable domain may comprise a humanized VHH comprising an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the humanized VHH comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to SEQ ID NO: 1. For example, the amino acid sequence of the humanized immunoglobulin single variable domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to SEQ ID NO: 1.

[0147] In some embodiments, the immunoglobulin single variable domain may comprise the amino acid sequence of any one of SEQ ID NOs: 1-8. In some embodiments, the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 1; in other embodiments, the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 6.

[0148] In some embodiments, the antigen binding region comprises one or more immunoglobulin single variable domains, and "plurality" can be an integer of 2, 3, 4, 5 or more. The amino acid sequences of the multiple immunoglobulin single variable domains can be the same or different.

[0149] In some embodiments, each of the plurality of immunoglobulin single variable domains independently comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 8. For example, in some embodiments, at least one of the plurality of immunoglobulin single variable domains, such as each, comprises the amino acid sequence of SEQ ID NO: 1; in some embodiments, at least one of the plurality of immunoglobulin single variable domains, such as each, comprises the amino acid sequence of SEQ ID NO: 2; in some embodiments, at least one of the plurality of immunoglobulin single variable domains, such as each, comprises the amino acid sequence of SEQ ID NO: 3; in some embodiments, at least one of the plurality of immunoglobulin single variable domains, such as each, comprises the amino acid sequence of SEQ ID NO: 4; in some embodiments, at least one of the plurality of immunoglobulin single variable domains, such as each, comprises the amino acid sequence of SEQ ID NO: 5; in some embodiments, at least one of the plurality of immunoglobulin single variable domains, such as each, comprises the amino acid sequence of SEQ ID NO: 6; in some embodiments, at least one of the plurality of immunoglobulin single variable domains, such as each, comprises the amino acid sequence of SEQ ID NO: 7; in some embodiments, at least one of the plurality of immunoglobulin single variable domains, such as each, comprises the amino acid sequence of SEQ ID NO: 8.

[0150] In some specific embodiments, at least one of the plurality of immunoglobulin single variable domains, for example, each, has an amino acid sequence as shown in SEQ ID NO: 1; at least one of the plurality of immunoglobulin single variable domains, for example, each, has an amino acid sequence as shown in SEQ ID NO: 2; at least one of the plurality of immunoglobulin single variable domains, for example, each, has an amino acid sequence as shown in SEQ ID NO: 3; at least one of the plurality of immunoglobulin single variable domains, for example, each, has an amino acid sequence as shown in SEQ ID NO: 4; at least one of the plurality of immunoglobulin single variable domains, for example, each, has an amino acid sequence as shown in SEQ ID NO: 5; at least one of the plurality of immunoglobulin single variable domains, for example, each, has an amino acid sequence as shown in SEQ ID NO: 6; at least one of the plurality of immunoglobulin single variable domains, for example, each, has an amino acid sequence as shown in SEQ ID NO: 7; or at least one of the plurality of immunoglobulin single variable domains, for example, each, has an amino acid sequence as shown in SEQ ID NO: 8.

[0151] In some embodiments, the fusion protein may comprise a GIPR-binding immunoglobulin single variable domain as described above.

[0152] In some embodiments, the fusion protein may comprise an immunoglobulin single variable domain that binds to GIPR, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH set forth in SEQ ID NO: 1. Preferably, the CDR1, CDR2, and CDR3 of the VHH set forth in SEQ ID NO: 1 are selected from any one of the following groups: SEQ ID NOs: 9-11, SEQ ID NOs: 12-14, SEQ ID NOs: 15-17, and SEQ ID NOs: 18-20.

[0153] In some embodiments, the fusion protein may comprise an immunoglobulin single variable domain that binds to GIPR, wherein the immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-8, or the amino acid sequence of the immunoglobulin single variable domain is as shown in any one of SEQ ID NOs: 1-8.

[0154] In some embodiments, the fusion protein may comprise one or more GIPR-binding immunoglobulin single variable domains, wherein each of the multiple immunoglobulin single variable domains independently comprises the amino acid sequence of any one of SEQ ID NOs: 1-8; or the amino acid sequence of each of the multiple immunoglobulin single variable domains independently comprises any one of SEQ ID NOs: 1-8. For example, at least one of the multiple immunoglobulin single variable domains, such as each comprises the amino acid sequence of SEQ ID NO: 1, or at least one of the multiple immunoglobulin single variable domains, such as each comprises the amino acid sequence of SEQ ID NO: 1; or at least one of the multiple immunoglobulin single variable domains, such as each comprises the amino acid sequence of SEQ ID NO: 6, or at least one of the multiple immunoglobulin single variable domains, such as each comprises the amino acid sequence of SEQ ID NO: 6.

[0155] In some embodiments, the fusion protein comprising a GIPR-binding immunoglobulin single variable domain described in the present disclosure has at least one of the following characteristics:

[0156] (a) specifically binds to human GIPR;

[0157] (b) The KD value for binding to human GIPR is less than 1×10 -7 M, preferably less than 1×10 -8 M, 1×10 -9 M or 1×10 -10 M;

[0158] (c) blocking the interaction between GIP and GIPR; and

[0159] (d) Neutralization of human GIPR on the cell surface.

[0160] ii) GLP-1R agonists

[0161] In some embodiments, the GLP-1R agonist is human GLP-1 or a variant thereof, or human GLP-1 or a functional fragment thereof, or exenatide or a variant thereof, or a functional fragment of exenatide or a variant thereof.

[0162] The GLP-1R agonist has at least some of the activity of human GLP-1. For example, in some cases, the GLP-1R agonist may have one or more activities selected from the group consisting of: activity of binding to the GLP-1 receptor, activity of activating the GLP-1 receptor, activity of activating adenylate cyclase, activity of promoting an increase in intracellular cyclic adenosine monophosphate (cAMP) levels, activity of positively regulating intracellular Ca 2+ The activity of a GLP-1R agonist can be detected by detecting the ability to bind to the GLP-1 receptor and the expression level of cAMP. The activity of the GLP-1R agonist can be at least 1% (e.g., at least 5%, at least 10%, at least 20%, at least 25%, 30%, 35%, 40%, 45%, or 50% or more) of the activity of human GLP-1.

[0163] In some embodiments, the GLP-1R agonist is human GLP-1 (SEQ ID NO: 21).

[0164] In some embodiments, the GLP-1R agonist is a human GLP-1 variant, such as:

[0165] Arg 34 -GLP-1(7-37);

[0166] Glu 30 -GLP-1(7-37);

[0167] Lys 22 -GLP-1(7-37);

[0168] Gly 8,36 ,Glu 22 -GLP-1(7-37);

[0169] Val8 ,Glu 22 ,Gly 36 -GLP-1(7-37);

[0170] Gly 8,36 ,Glu 22 ,Lys 33 ,Asn 34 -GLP-1(7-37);

[0171] Val 8 ,Glu 22 ,Lys 33 ,Asn 34 ,Gly 36 -GLP-1(7-37);

[0172] Gly 8,36 ,Glu 22 ,Pro 37 -GLP-1(7-37);

[0173] Val 8 ,Glu 22 ,Gly 36 ,Pro 37 -GLP-1(7-37);

[0174] Gly 8,36 ,Glu 22 ,Lys 33 ,Asn 34 ,Pro 37 -GLP-1(7-37);

[0175] Val 8 ,Glu 22 ,Lys 33 ,Asn 34 ,Gly 36 ,Pro 37 -GLP-1(7-37);

[0176] Aib 8 ,Lys 37 -GLP-1(7-37);

[0177] Aib 8 ,Lys 36 -GLP-1(7-37);

[0178] Aib 8,22 ,Gly 36 -GLP-1(7-37);

[0179] Aib 8,Glu 22 ,Gly 36 -GLP-1(7-37);

[0180] Aib 8 ,Arg 34 -GLP-1(7-37);

[0181] Aib 8 ,Tyr 16 ,Glu 22 ,Gly 36 -GLP-1(7-37);

[0182] Aib 8 ,Lys 18,33 ,Glu 22,23,30 ,Val 25 ,Arg 26 ,Leu 27 ,Asn 34 ,Gly 36 -GLP-1(7-37);

[0183] Aib 8 ,Lys 18,33 ,Glu 22,23,30 ,Leu 27 ,Gly 36 -GLP-1(7-37);

[0184] Aib 8,22 ,Ile 9 ,Gly 36 -GLP-1(7-37);

[0185] Aib 8,22 ,Glu 15 ,Gly 36 -GLP-1(7-37);

[0186] Gly 8,36 ,Glu 22 -GLP-1(7-36);

[0187] Val 8 ,Glu 22 ,Gly 36 -GLP-1(7-36);

[0188] Val 8 ,Glu 22 ,Asn 34 ,Gly 36 -GLP-1(7-36);

[0189] Gly8,36 ,Glu 22 ,Asn 34 -GLP-1(7-36);

[0190] In some embodiments, the GLP-1R agonist is Exendin-4 (Ex-4) or a variant thereof, such as:

[0191] Exendin-4 (SEQ ID NO: 98);

[0192] Leu 14 -Ex-4;

[0193] Leu 14 ,Phe 25 -Ex-4;

[0194] Leu 14 ,Ala 19 ,Phe 25 -Ex-4;

[0195] Leu 14 ,Lys 17,20 ,Ala 19 ,Glu 21 ,Phe 25 ,Gln 28 -Ex-4;

[0196] Leu 14 ,Lys 17,20 ,Ala 19 ,Glu 21 ,Gln 28 -Ex-4;

[0197] Phe 4 ,Leu 14 ,Lys 17,20 ,Ala 19 ,Glu 21 ,Gln 28 -Ex-4;

[0198] Val 11 ,Ile 13 ,Leu 14 ,Ala 16 ,Lys 21 ,Phe 25 -Ex-4;

[0199] Ex-4-Lys 40 .

[0200] The "AA m", means that the original amino acid residue at position m is mutated to AA relative to wild-type human GLP-1 (SEQ ID NO: 21) or exenatide (SEQ ID NO: 98), wherein m is an integer (relative to human GLP-1, m starts from 7; relative to exenatide, m starts from 1), and AA is the abbreviation of any amino acid residue.

[0201] In some embodiments, the GLP-1R agonist is a human GLP-1 variant having, for example, the amino acid sequence shown in SEQ ID NO:22.

[0202] In some embodiments, the human GLP-1 variant is selected from the GLP-1 variant sequences disclosed in the prior patent application WO2022143516, which is incorporated herein by reference in its entirety.

[0203] Specifically, in some embodiments, the GLP-1R agonist is a human GLP-1 variant, which further comprises an amino acid mutation at one or more positions of Y19, Q23, K26 and W31 compared to the amino acid sequence shown in SEQ ID NO: 22.

[0204] In other embodiments, the GLP-1R agonist is a human GLP-1 variant, which further comprises an amino acid mutation at one of Y19, Q23, and W31 compared to the amino acid sequence shown in SEQ ID NO: 22.

[0205] In some embodiments, the GLP-1R agonist is a human GLP-1 variant, which may further comprise an amino acid mutation selected from the following positions compared to the amino acid sequence shown in SEQ ID NO: 22:

[0206] 1)Y19;

[0207] 2)Q23;

[0208] 3)W31;

[0209] 4) Y19 and K26:

[0210] 5) Y19 and W31;

[0211] 6) Q23 and K26;

[0212] 7) Q23 and W31;

[0213] 8) K26 and W31;

[0214] 9) Y19, K26, and W31; and

[0215] 10)Q23, K26 and W31.

[0216] In some embodiments, the amino acid substitution at position W31 is selected from W31Y, W31L, and W31A.

[0217] In some embodiments, the amino acid substitution at position K26 is K26R.

[0218] In some embodiments, the amino acid substitution at position Q23 is selected from Q23E, Q23T, Q23S, and Q23N.

[0219] In some embodiments, the amino acid substitution at position Y19 is selected from Y19A, Y19L, Y19T, Y19F, Y19I, Y19V, and Y19S.

[0220] In the present disclosure, the amino acid substitution "XnY" means that the residue X at position n in the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 22 is substituted with the amino acid residue Y, wherein n is a positive integer (for GLP-1, n starts at 7), X and Y are each independently an abbreviation for any amino acid residue, and X is different from Y. For example, the amino acid substitution "W31Y" means that the amino acid residue W at position 31 in the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 22 is substituted with the amino acid residue Y.

[0221] In some embodiments, the GLP-1R agonist is a human GLP-1 variant, which may further comprise an amino acid substitution at position Y19 compared to the amino acid sequence shown in SEQ ID NO: 22; for example, the amino acid substitution at position Y19 is selected from Y19A, Y19L, Y19T, Y19F, Y19I, Y19V and Y19S.

[0222] In some embodiments, the GLP-1R agonist comprises the amino acid sequence set forth in any one of SEQ ID NOs: 23-29.

[0223] In some embodiments, the GLP-1R agonist is a human GLP-1 variant, which may further comprise an amino acid substitution at position Q23 compared to the amino acid sequence shown in SEQ ID NO: 22; for example, the amino acid substitution at position Q23 is selected from Q23E, Q23T, Q23S and Q23N.

[0224] In some embodiments, the GLP-1R agonist comprises the amino acid sequence set forth in any one of SEQ ID NOs: 30-33.

[0225] In some embodiments, the GLP-1R agonist is a human GLP-1 variant, which may further comprise an amino acid substitution at position W31 compared to the amino acid sequence shown in SEQ ID NO: 22; for example, the amino acid substitution at position W31 is selected from W31Y, W31L and W31A.

[0226] In some embodiments, the GLP-1R agonist comprises the amino acid sequence set forth in any one of SEQ ID NOs: 34-36.

[0227] In other embodiments, the GLP-1R agonist is a human GLP-1 variant, which may further comprise amino acid substitutions at positions Y19 and K26 compared to the amino acid sequence set forth in SEQ ID NO: 22; for example, the amino acid substitutions at positions Y19 and K26 are selected from the following mutation combinations:

[0228] 4a) Y19A and K26R;

[0229] 4b) Y19L and K26R;

[0230] 4c) Y19T and K26R;

[0231] 4d) Y19F and K26R;

[0232] 4e) Y19I and K26R;

[0233] 4f) Y19V and K26R; and

[0234] 4g)Y19S and K26R.

[0235] In some embodiments, the GLP-1R agonist comprises the amino acid sequence set forth in any one of SEQ ID NOs: 37-43.

[0236] In some embodiments, the GLP-1R agonist is a human GLP-1 variant, which may further comprise amino acid substitutions at positions Y19 and W31 compared to the amino acid sequence set forth in SEQ ID NO: 22; for example, the amino acid substitutions at positions Y19 and W31 are selected from the following mutation combinations:

[0237] 5a) Y19A and W31Y;

[0238] 5b) Y19L and W31Y;

[0239] 5c) Y19T and W31Y;

[0240] 5d) Y19F and W31Y;

[0241] 5e) Y19I and W31Y;

[0242] 5f) Y19V and W31Y; and

[0243] 5g)Y19S and W31Y.

[0244] In some embodiments, the GLP-1R agonist comprises the amino acid sequence set forth in any one of SEQ ID NOs: 44-50.

[0245] In some embodiments, the GLP-1R agonist is a human GLP-1 variant, which may further comprise amino acid substitutions at positions Q23 and K26 compared to the amino acid sequence set forth in SEQ ID NO: 22; for example, the amino acid substitutions at positions Q23 and K26 are selected from the following mutation combinations:

[0246] 6a) Q23N and K26R;

[0247] 6b) Q23T and K26R;

[0248] 6c) Q23S and K26R; and

[0249] 6d)Q23E and K26R.

[0250] In some embodiments, the GLP-1R agonist comprises the amino acid sequence set forth in any one of SEQ ID NOs: 51-54.

[0251] In some embodiments, the GLP-1R agonist is a human GLP-1 variant, which may further comprise amino acid substitutions at positions Q23 and W31 compared to the amino acid sequence set forth in SEQ ID NO: 22; for example, the amino acid substitutions at positions Q23 and W31 are selected from the following mutation combinations:

[0252] 7a) Q23N and W31Y;

[0253] 7b) Q23T and W31Y;

[0254] 7c) Q23S and W31Y; and

[0255] 7d)Q23E and W31Y.

[0256] In some embodiments, the GLP-1R agonist comprises the amino acid sequence set forth in any one of SEQ ID NOs: 55-58.

[0257] In some embodiments, the GLP-1R agonist is a human GLP-1 variant, which may further comprise amino acid substitutions at positions K26 and W31 compared to the amino acid sequence shown in SEQ ID NO: 22; for example, the amino acid substitutions at positions K26 and W31 are K26R and W31Y.

[0258] In some embodiments, the GLP-1R agonist comprises the amino acid sequence shown in SEQ ID NO:59.

[0259] In some embodiments, the GLP-1R agonist is a human GLP-1 variant, which may further comprise amino acid substitutions at positions Y19, K26, and W31 compared to the amino acid sequence set forth in SEQ ID NO: 22; for example, the amino acid substitutions at positions Y19, K26, and W31 are selected from the following mutation combinations:

[0260] 9a) Y19A, K26R, and W31Y;

[0261] 9b) Y19F, K26R, and W31Y;

[0262] 9c) Y19L, K26R, and W31Y;

[0263] 9d) Y19T, K26R, and W31Y;

[0264] 9e) Y19I, K26R, and W31Y;

[0265] 9f) Y19V, K26R, and W31Y; and

[0266] 9g)Y19S, K26R and W31Y.

[0267] In some embodiments, the GLP-1R agonist comprises the amino acid sequence set forth in any one of SEQ ID NOs: 60-66.

[0268] In some embodiments, the GLP-1R agonist is a human GLP-1 variant, which may further comprise amino acid substitutions at positions Q23, K26, and W31 compared to the amino acid sequence set forth in SEQ ID NO: 22; for example, the amino acid substitutions at positions Q23, K26, and W31 are selected from the following mutation combinations:

[0269] 10a) Q23N, K26R, and W31Y;

[0270] 10b) Q23T, K26R, and W31Y;

[0271] 10c) Q23S, K26R, and W31Y; and

[0272] 10d)Q23E, K26R and W31Y.

[0273] In some embodiments, the GLP-1R agonist comprises the amino acid sequence set forth in any one of SEQ ID NOs: 67-70.

[0274] In some embodiments, the GLP-1R agonist is located at the N-terminus or C-terminus of the aforementioned immunoglobulin single variable domain. For example, when the GLP-1R agonist is located at the N-terminus of the aforementioned immunoglobulin single variable domain, the C-terminus of the GLP-1R agonist is fused to the N-terminus of the immunoglobulin single variable domain; when the GLP-1R agonist is located at the C-terminus of the aforementioned immunoglobulin single variable domain, the N-terminus of the GLP-1R agonist is fused to the C-terminus of the immunoglobulin single variable domain.

[0275] When the aforementioned GLP-1R agonist is fused with an immunoglobulin single variable domain, a protein and / or polypeptide fragment, such as a linker, a hinge region and / or an Fc region, may optionally be contained between the two.

[0276] iii) Immunoglobulin Fc region

[0277] In some embodiments, the fusion protein further comprises an immunoglobulin Fc region.

[0278] In some embodiments, the immunoglobulin Fc region is an Fc region derived from IgG, preferably, the immunoglobulin Fc region is an Fc region derived from IgG1, IgG2, IgG3, or IgG4. For example, the immunoglobulin Fc region comprises the amino acid sequence shown in any one of SEQ ID NOs: 71-74.

[0279] In some embodiments, the immunoglobulin Fc region comprises amino acid mutations that selectively enhance the binding affinity of the immunoglobulin Fc region to an Fc receptor compared to an unmutated immunoglobulin Fc region; in some preferred embodiments, the amino acid mutations selectively enhance the binding affinity of the immunoglobulin Fc region to the neonatal Fc receptor (FcRn). In some embodiments, the selective enhancement of binding affinity refers to an enhancement of binding affinity under acidic conditions compared to the pre-mutated state.

[0280] In some embodiments, the aforementioned immunoglobulin Fc region may comprise amino acid mutations at positions M252, S254 and / or T256 according to the EU index numbering of Kabat.

[0281] In some embodiments, the immunoglobulin Fc region may comprise amino acid mutations at positions M252, S254, and T256, numbered according to the EU index of Kabat.

[0282] In some embodiments, the amino acid mutation at M252 may be M252Y; in some embodiments, the amino acid mutation at S254 may be S254T; in some embodiments, the amino acid mutation at T256 may be T256E.

[0283] In the present disclosure, the Fc region may be an Fc region derived from IgG1, IgG2, IgG3, or IgG4, and may comprise amino acid mutations of M252Y, S254T, and T256E.

[0284] For example, the Fc region may be an Fc region derived from IgG1 and may include amino acid mutations of M252Y, S254T, and T256E. For example, the Fc region may be an Fc region derived from IgG2 and may include amino acid mutations of M252Y, S254T, and T256E. For example, the Fc region may be an Fc region derived from IgG3 and may include amino acid mutations of M252Y, S254T, and T256E. For example, the Fc region may be an Fc region derived from IgG4 and may include amino acid mutations of M252Y, S254T, and T256E.

[0285] In some embodiments, the immunoglobulin Fc region comprises the amino acid sequence shown in any one of SEQ ID NOs: 75-78.

[0286] iv) Configuration of fusion protein

[0287] The fusion proteins disclosed herein can have a variety of different formats. For example, from the N-terminus to the C-terminus, the fusion protein can comprise, in order, an immunoglobulin single variable domain that binds to GPIR, a GLP-1R agonist, and an immunoglobulin Fc region; or, from the N-terminus to the C-terminus, the fusion protein can comprise, in order, a GLP-1R agonist, an immunoglobulin single variable domain that binds to GPIR, and an immunoglobulin Fc region; or, from the N-terminus to the C-terminus, the fusion protein can comprise, in order, an immunoglobulin single variable domain that binds to GPIR, an immunoglobulin Fc region, and a GLP-1R agonist; or, from the N-terminus to the C-terminus, the fusion protein can comprise, in order, a GLP-1R agonist, an immunoglobulin Fc region, and an immunoglobulin single variable domain that binds to GPIR. The immunoglobulin single variable domain that binds to GPIR, the GLP-1R agonist, and the immunoglobulin Fc region are as defined in sections i) to iii).

[0288] In some embodiments, the three aforementioned functional domains (GPIR-binding immunoglobulin single variable domain, GLP-1R agonist, immunoglobulin Fc region) may each optionally comprise a linker, preferably a peptide linker, for example, the peptide linker having a length of 2-20 amino acids.

[0289] In some embodiments, the peptide linker may comprise GS or comprise GAP or comprise an amino acid sequence as shown in any one of SEQ ID NOs: 83-87.

[0290] In some embodiments, the N-terminus of the immunoglobulin Fc region optionally comprises a hinge region, for example, a hinge region derived from IgG1, IgG2, IgG3, or IgG4.

[0291] In some embodiments, the N-terminus of the immunoglobulin Fc region, such as the hinge region, may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 79-82.

[0292] In some specific embodiments, the fusion protein may comprise a configuration selected from any one of formulas (A)-(D):

[0293] (A)

[0294] (B)

[0295] (C) and

[0296] (D)

[0297] Wherein, GIPR BM is a GIPR-binding immunoglobulin single variable domain, GLP-1Ra is a GLP-1R agonist, and Fc is an immunoglobulin Fc region, and their definitions are as described in Sections i) to iii);

[0298] L1 and L2 are each independently a peptide linker, or are absent;

[0299] HR is the hinge region of an immunoglobulin, for example, HR comprises a hinge region derived from IgG1, IgG2, IgG3, or IgG4.

[0300] In some embodiments, the HR comprises the amino acid sequence shown in any one of SEQ ID NOs: 79-82.

[0301] In some embodiments, the peptide linker has a length of 2-20 amino acids.Preferably, the peptide linker comprises GS or GAP or an amino acid sequence as shown in any one of SEQ ID NOs: 83-87.

[0302] In some embodiments, the fusion protein comprises any one of the combinations of L1, L2, and HR shown in (I) to (XIV):

[0303] (I) L1 is the amino acid sequence set forth in SEQ ID NO: 85, L2 is the amino acid sequence set forth in SEQ ID NO: 85, and HR is the amino acid sequence set forth in SEQ ID NO: 82;

[0304] (II) L1 is the amino acid sequence set forth in SEQ ID NO:85, L2 is the amino acid sequence set forth in SEQ ID NO:85, and HR is the amino acid sequence set forth in SEQ ID NO:79;

[0305] (III) L1 is the amino acid sequence set forth in SEQ ID NO:85, L2 is the amino acid sequence set forth in SEQ ID NO:87, and HR is the amino acid sequence set forth in SEQ ID NO:82;

[0306] (IV) L1 is the amino acid sequence set forth in SEQ ID NO:85, L2 is the amino acid sequence set forth in SEQ ID NO:87, and HR is the amino acid sequence set forth in SEQ ID NO:79;

[0307] (V) L1 is the amino acid sequence set forth in SEQ ID NO:87, L2 is the amino acid sequence set forth in SEQ ID NO:85, and HR is the amino acid sequence set forth in SEQ ID NO:82;

[0308] (VI) L1 is the amino acid sequence set forth in SEQ ID NO:87, L2 is the amino acid sequence set forth in SEQ ID NO:85, and HR is the amino acid sequence set forth in SEQ ID NO:79;

[0309] (VII) L1 has the amino acid sequence set forth in SEQ ID NO: 85, L2 is absent, and HR has the amino acid sequence set forth in SEQ ID NO: 82;

[0310] (VIII) L1 has the amino acid sequence set forth in SEQ ID NO:85, L2 is absent, and HR has the amino acid sequence set forth in SEQ ID NO:79;

[0311] (IX) L1 has the amino acid sequence set forth in SEQ ID NO: 87, L2 is absent, and HR has the amino acid sequence set forth in SEQ ID NO: 82;

[0312] (X) L1 represents the amino acid sequence set forth in SEQ ID NO:87, L2 is absent, and HR represents the amino acid sequence set forth in SEQ ID NO:79;

[0313] (XI) L1 is absent, L2 has the amino acid sequence set forth in SEQ ID NO: 85, and HR has the amino acid sequence set forth in SEQ ID NO: 82;

[0314] (XII) L1 is absent, L2 has the amino acid sequence set forth in SEQ ID NO:85, and HR has the amino acid sequence set forth in SEQ ID NO:79;

[0315] (XIII) L1 is absent, L2 has the amino acid sequence set forth in SEQ ID NO: 87, and HR has the amino acid sequence set forth in SEQ ID NO: 82; and

[0316] (XIV) L1 is absent, L2 has the amino acid sequence shown in SEQ ID NO: 87, and HR has the amino acid sequence shown in SEQ ID NO: 79.

[0317] In some embodiments, the fusion protein comprises configuration (A) and comprises a combination of L1, L2 and HR shown in any one of (I) to (XIV); preferably, comprises a combination of L1, L2 and HR shown in any one of (I), (VI), (VIII) and (X).

[0318] In some embodiments, the fusion protein comprises configuration (B) and comprises a combination of L1, L2 and HR shown in any one of (I) to (XIV); preferably, comprises a combination of L1, L2 and HR shown in any one of (I), (IV), (XII) and (XIV).

[0319] In some embodiments, the fusion protein comprises the (C) configuration and comprises a combination of L1, L2 and HR shown in any one of (I) to (XIV); preferably, comprises a combination of L1, L2 and HR shown in any one of (I), (VI), (VIII) and (X).

[0320] In some embodiments, the fusion protein comprises the (D) configuration and comprises a combination of L1, L2 and HR shown in any one of (I) to (XIV); preferably, comprises a combination of L1, L2 and HR shown in any one of (I), (IV), (XII) and (XIV).

[0321] In some embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 88-93.

[0322] In other embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 94-97.

[0323] In some embodiments, the fusion protein is a dimer; preferably, the fusion protein is a homodimer.

[0324] Nucleic acids, vectors, host cells

[0325] In another aspect, the present disclosure relates to a nucleic acid molecule encoding a fusion protein of the present disclosure. The nucleic acid of the present disclosure may be RNA, DNA or cDNA. According to one embodiment of the present disclosure, the nucleic acid of the present disclosure is a substantially isolated nucleic acid.

[0326] Nucleic acid of the present disclosure can also be in the form of a vector, can be present in a vector and / or can be a part of a vector, such as a plasmid, a cosmid or a YAC. The vector can be an expression vector in particular, can provide a vector for expressing the fusion protein in vitro and / or in vivo (i.e., in a suitable host cell, host organism and / or expression system). The expression vector generally comprises at least one nucleic acid of the present disclosure, which is operably connected to one or more suitable expression control elements (such as promoters, enhancers, terminators, etc.). It is common sense for those skilled in the art to select the elements and their sequences for expression in a specific host. The specific examples of the control elements and other elements useful or necessary to the expression of the fusion protein of the present disclosure include, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0327] The nucleic acids of the present disclosure can be prepared or obtained by known means (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on the information on the amino acid sequences of the polypeptides of the present disclosure given herein, and / or can be isolated from suitable natural sources.

[0328] In another aspect, the present disclosure relates to recombinant host cells that express or are capable of expressing one or more fusion proteins of the present disclosure and / or contain nucleic acids or vectors of the present disclosure.Preferred host cells of the present disclosure are bacterial cells, fungal cells or mammalian cells.

[0329] Suitable bacterial cells include cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0330] Suitable fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0331] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, or COS cells.

[0332] However, the present disclosure may also utilize amphibian cells, insect cells, and any other cells known in the art for expressing heterologous proteins.

[0333] The fusion proteins of the present disclosure can be produced intracellularly in the cells as described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified.

[0334] Methods and reagents for recombinant production of polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc., are known in the art. Similarly, protein isolation and purification techniques suitable for use in methods for producing the fusion proteins of the present disclosure are well known to those skilled in the art.

[0335] Pharmaceutical composition

[0336] In another aspect, the present disclosure provides a composition, such as a pharmaceutical composition, containing one or a combination of fusion proteins of the present disclosure formulated together with a pharmaceutically acceptable carrier.

[0337] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, stabilizers, isotonic and absorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody molecule, may be encapsulated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0338] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular route of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of the composition that produces a therapeutic effect. Typically, based on 100%, this amount ranges from about 0.01% to about 99% of the active ingredient, for example, about 0.1% to about 70%, or about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0339] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors, including the activity of the particular composition of the present disclosure being administered, or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion of the particular compound being administered, the duration of treatment, other drugs, compounds, and / or materials being administered in combination with the particular composition being administered, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.

[0340] Compositions of the present disclosure can be administered by one or more methods well known in the art through one or more routes of administration. It will be understood by those skilled in the art that routes of administration and / or modes are different according to the desired result. Preferred routes of administration for fusion proteins of the present disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. Phrase "parenteral administration" as used herein refers to the mode of administration except enteral and topical administration, typically injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intra-epidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0341] Prevent and / or treat disease

[0342] In another aspect, the present disclosure further provides a method for preventing and / or treating a disease, particularly a method for treating and / or preventing a metabolic disease, comprising administering an effective amount of a fusion protein, nucleic acid molecule, or pharmaceutical composition of the present disclosure to a subject in need thereof. The metabolic diseases include obesity, overweight, diabetes, hyperlipidemia, and / or non-alcoholic fatty liver disease.

[0343] In some embodiments, in the process of preventing and / or treating metabolic diseases, in addition to the fusion protein or pharmaceutical composition of the present invention, other therapeutic drugs may be administered to subjects in need thereof, such as insulin (including porcine or bovine insulin, human insulin, aspart insulin, lispro insulin, protamine zinc insulin, glargine insulin, detemir insulin, LY3209590, etc.), biguanides (such as metformin, etc.), thiazolidinediones (including pioglitazone, rosiglitazone, etc.), sulfonylureas (including glipizide, glipizide, etc.), Zidane, glibenclamide, gliprexa, glimepiride, glimepiride, etc.), phenylalanines (including repaglinide, nateglinide, mitiglinide, etc.), α-glucosidase inhibitors (including acarbose, voglibose, etc.), DPP4 inhibitors (including sitagliptin, linagliptin, goagliflozin, vildagliptin, saxagliptin, alogliptin, gemagliptin, tenegliptin, etc.), SGLT2 inhibitors (including dapagliflozin, canagliflozin, empagliflozin, etc.), orlistat and GCGR agonists, etc.

[0344] The other therapeutic drugs and the fusion protein / drug composition of the present invention can be administered simultaneously or sequentially; when administered sequentially, the interval between the two administrations shall not exceed 1 week, preferably not exceed 3 days, more preferably not exceed 2 days, and even more preferably not exceed 1 day.

[0345] In other embodiments, the present disclosure also provides use of the fusion protein or pharmaceutical composition of the present disclosure in preparing a drug for treating and / or preventing metabolic diseases.

[0346] In yet other embodiments, the present disclosure provides a fusion protein or pharmaceutical composition for treating and / or preventing metabolic diseases.

[0347] In another aspect, the present disclosure provides a kit that may include the fusion protein or pharmaceutical composition described herein. The kit may include the fusion protein or pharmaceutical composition described herein in a single common container, optionally in combination with one or more therapeutic agents, optionally formulated together in a pharmaceutical composition.

[0348] In another aspect, the present disclosure provides a drug delivery device that can be used to administer the fusion protein or pharmaceutical composition described in the present disclosure.

[0349] Without intending to be bound by any theory, the following embodiments are merely intended to illustrate various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0350] Example

[0351] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. The scope of the present invention is defined by the appended claims. In conjunction with this specification and common knowledge in the art, a person of ordinary skill in the art will clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, those skilled in the art may make any modifications or changes to the technical solution of the present invention, and such modifications and changes are also included in the scope of the present invention.

[0352] Example 1 Screening of GIPR Heavy Chain Single Domain Antibodies

[0353] 1.1 Library Construction

[0354] Before immunization, 50 mL of alpaca arterial blood was collected into a vacutainer tube, and the supernatant was collected as pre-immune serum. Healthy alpacas were selected and immunized with antigens containing human GIPR via multiple injections into the neck muscle. Immunizations were repeated every two weeks for a total of six times. At the end of the final immunization, 50 mL of alpaca arterial blood was collected into a vacutainer tube, and the supernatant was collected as post-immune serum.

[0355] Lymphocytes were isolated by density gradient centrifugation, and total RNA was extracted using an RNA extraction kit provided by QIAGEN. The extracted RNA was completely reverse-transcribed into cDNA using the Super-Script III FIRST STRAND SUPERMIX kit according to the manufacturer's instructions, and the nucleic acid fragment encoding the variable region of the heavy chain antibody was amplified by nested PCR.

[0356] The nucleic acid fragment of the target heavy chain single domain antibody was recovered and cloned into the phage display vector pComb3XSS using the restriction endonuclease SfiI. The product was then electroporated into E. coli electrocompetent cells TG1 to construct and characterize an anti-GIPR immune single domain antibody phage display library. The library size was calculated to be 1.69×10 9 To test the insertion rate of the library, 50 clones were randomly selected for sequencing. 50 clones had the correct exogenous fragment inserted, a 100% accuracy rate. Analysis and alignment of the DNA and amino acid sequences of the sequenced clones confirmed that all sequences were alpaca VHH sequences, with an estimated diversity of over 95%.

[0357] 1.2 Selection of GIPR heavy chain single domain antibodies

[0358] The phage library obtained in Example 1.1 was panned using the proteins GIPR-ECD-muFc (a fragment of the extracellular region of human GIPR fused to mouse Fc), GIPR-ECD-chis (a fragment of the extracellular region of human GIPR fused to his tag), and GIPR-ECD-laFc (a fragment of the extracellular region of human GIPR fused to alpaca Fc). At the same time, negative panning was performed using the fusion protein Control-1 (C1) containing muFc and the fusion protein Control-2 (C2) containing laFc to remove non-specifically binding phage.

[0359] Phages that bind positively after panning were infected with blank E. coli and plated. Selected colonies were then inoculated into 2TY-AG (containing 10% glycerol) and allowed to stand overnight at room temperature. The next day, 1% of the inoculum was transferred to 200 μL of 2TY-AG and incubated at 37°C, 250 rpm, until the OD600 reached approximately 0.5. Helper phage M13KO7 was added for infection (infection index 1:20), and the cells were incubated at 37°C for 15 minutes, followed by 45 minutes at 220 rpm. 800 μL of 2TY-AG was added to each well and incubated at 30°C, 220 rpm, overnight. The cells were centrifuged the next day, and the supernatant was collected for ELISA analysis. Plates were coated with GIPR-ECD-Chis, GIPR-ECD-laFc, C1, and C2 overnight at 4°C. The supernatants were added and reacted at room temperature for 2 hours. After washing, the secondary antibody, Goat anti-HA tag HRP (purchased from Abcam), was added and reacted at room temperature for 2 hours. After washing, TMB colorimetric solution was added and the absorbance values ​​at 450 nm and 650 nm were read. The final absorbance value was obtained by subtracting the absorbance value at 650 nm from the absorbance value at 450 nm.

[0360] Of the 297 clones obtained by panning, 191 clones exhibited an OD value > 1.0. Table 2 lists some of the positive clones that specifically bound to GIPR-ECD-1aFc. These positive clones were sequenced to obtain antibody sequences. The amino acid sequence of the antibody corresponding to clone iGI-72 is shown in SEQ ID NO: 1.

[0361] Table 2. Positive clones that specifically bind to GIPR-ECD-1aFc

[0362] Example 2 Preparation of Fc Fusion Protein of GIPR Single Domain Antibody Using Mammalian Cells

[0363] 2.1 Preparation of plasmid expressing GIPR single domain antibody-Fc fusion protein

[0364] Primers were designed for PCR amplification of the GIPR single-domain antibody VHH fragment, which was fused to a DNA fragment encoding human IgG1-Fc (including the hinge region) and cloned into a conventional mammalian expression vector to obtain a recombinant plasmid for expressing the GIPR single-domain antibody-Fc fusion protein in mammals. Universal primers were used to amplify different VHH fragments. The universal primers are as follows:

[0365] Upstream primer cccACCGGTCAGGTGCAGCTGCAGGAGTC (SEQ ID NO: 99)

[0366] Downstream primer cccGGATCCTGAGGAGACGGTGACCTGG (SEQ ID NO: 100)

[0367] 2.2 Preparation of Fc fusion protein of GIPR single domain antibody

[0368] The plasmid vector constructed in 2.1 was transfected into HEK293 cells for transient expression of the antibody. The recombinant expression plasmid was diluted with Freestyle293 medium and the PEI (Polyethylenimine) solution required for transformation was added. Each group of plasmid / PEI mixtures was added to the HEK293 cell suspension and placed at 37°C, 5% CO2 for suspension culture. After culturing for 5 to 6 days, the supernatant of the transient expression culture was collected and purified by Protein A affinity chromatography to obtain the target GIPR single domain antibody-Fc fusion protein. The protein purity was detected by SDS-PAGE. The expression levels of each protein are shown in Table 3, and the SDS purity of each protein after one-step purification is greater than 95%.

[0369] Table 3. Expression levels of Fc fusion proteins of GIPR single domain antibodies

[0370] Example 3 Identification of the Function of GIPR Single Domain Antibody-Fc Fusion Protein

[0371] 3.1 GIPR Binding Ability of GIPR Single Domain Antibody-Fc Fusion Protein

[0372] 293T-GIPR cells (HEK293T cells expressing human GIPR) were plated onto cell culture plates and GIPR single-domain antibody-Fc fusion protein samples were added at final concentrations of 100 μg / ml and 10 μg / ml. Following incubation with an anti-human IgG-APC secondary antibody, the mean fluorescence intensity (MFI) was measured using a fluorescence-activated cell sorter. The results are shown in Table 4.

[0373] Table 4. GIPR-binding ability of Fc fusion proteins of GIPR single-domain antibodies

[0374] Neutralizing activity of GIPR single-domain antibody-Fc fusion protein

[0375] (1) GIP-C12H4-Fc4 (human GIP-IgG4 Fc fusion protein) and 25 μL of GIPR single-domain antibody-Fc sample were added to a 96-well plate. 293T-GIPR cells stably expressing full-length human GIPR protein on their cell membranes were then added. After incubation, the cAMP assay was performed using a cAMP kit and the absorbance at 450 nm / 550 nm was measured using a microplate reader. The results are shown in Table 5.

[0376] Table 5. Neutralizing activity of GIPR single domain antibody-Fc fusion proteins

[0377] (2) GIP-C12H4-Fc4 and 25 μL of GIPR single-domain antibody-Fc sample were added to a 96-well plate. 293T-GIPRFL-GFP-PURO-5 cells stably expressing full-length human GIPR protein were then added. After incubation, the cells were assayed using a cAMP assay kit, and the RLU values ​​were read to calculate the neutralization activity (%). The results are shown in Table 6.

[0378] Table 6. Neutralizing activity of GIPR single domain antibody-Fc fusion proteins

[0379] 3.3 Detection of affinity of GIPR single domain antibody-Fc fusion protein

[0380] The binding kinetics of the GIPR single-domain antibody-Fc fusion protein to the GIPR-ECD-chis were detected using biolayer interferometry (BLI) technology. The GIPR single-domain antibody-Fc fusion protein was directly immobilized on the AHC sensor, and then the GIPR-ECD-chis was diluted to 7 concentrations and bound to the immobilized GIPR single-domain antibody-Fc fusion protein. The equilibrium dissociation constant (KD), association rate (Ka), and dissociation rate (Kdis) were calculated using Octet K2 data analysis software 9.0. The results are shown in Tables 7 and 8.

[0381] Table 7. Affinity of GIPR single domain antibody-Fc fusion proteins

[0382] Table 8. Affinity of GIPR single domain antibody-Fc fusion proteins

[0383] 3.4 Detection of the blocking activity of GIPR single domain antibody-Fc fusion protein

[0384] The cells were coated with 5 μg / mL mouse GIPR-Chis protein and blocked with BSA. A serial dilution of AMG-GIPR-mab2 and iGI72-Ld-Fc was added and incubated thoroughly in the dilution buffer containing 1.2 μg / mL GIP-C12H4-Fc4. Mouse Anti-Human IgG4 pFc'[HP6023](HRP) (Cat. No. ab99817) was then added at a 1:2000 dilution. After development with TMB, the cells were detected on a microplate reader. The results are shown in Figure 1.

[0385] The positive control AMG-GIPR-mab2 was synthesized according to the sequence in the literature MAbs.2020 Jan-Dec; 12(1):1710047, and then transiently expressed in 293 cells according to the above method.

[0386] 3.5 Detection of nonspecific binding of GIPR single domain antibody-Fc fusion protein to empty cells

[0387] CHOK1 cells were resuspended in 3% BSA-PBS. Final concentrations of GIPR single-domain antibody-Fc fusion protein were selected at 5 μg / mL and 50 μg / mL, and negative and blank controls were also set up. After washing, the secondary antibody APC anti-human IgG Fc was added. After washing, the cells were resuspended in PBS-BSA buffer and analyzed by flow cytometry. The results are shown in Table 9. iGI-72-Ld-Fc showed no nonspecific binding to iGI-1198-Ld-Fc, while iGI-1225-Ld-Fc showed nonspecific binding to iGI-1225NA-Ld-Fc.

[0388] Table 9. Non-specific binding of GIPR single domain antibody-Fc fusion protein to empty cells

[0389] 3.6 In vivo efficacy study of GIPR single domain antibody-Fc fusion protein

[0390] Sixteen DIO mice were fasted overnight at 16 weeks of age, and their blood glucose and body weight were measured. The mice were then randomly divided into four groups. The day of group administration was recorded as D0, and PBS, Dulaglutide 17nmol / kg, iGI-72-Ld-Fc 375nmol / kg, and Dulaglutide 17nmol / kg + iGI-72-Ld-Fc 17 + 375nmol / kg were administered twice a week, for a total of 10 times. After administration, general clinical observations were performed once a week, body weight was measured twice a week, and fasting blood glucose and fasting insulin were measured once a week. The results are shown in Figures 2 to 5. The iGI-72-Ld-Fc treatment group significantly reduced the body weight of DIO mice, while improving fasting blood glucose, fasting insulin and insulin resistance HOMA-IR. The combination of iGI-72-Ld-Fc and dulaglutide further enhanced the efficacy of the drug, showing a synergistic effect.

[0391] Example 4 Humanization of GIPR Single Domain Antibodies

[0392] The humanization method uses the protein surface amino acid humanization (resurfacing) method and VHH humanization universal framework transplantation method (CDR grafting to a universal framework) to complete.

[0393] First, we obtained the universal humanized VHH framework hNbBcII10FGLA (PDB ID: 3EAK), designed by Cécile Vincke et al. based on sequence homology. This framework is based on the nanobody NbBcII10 antibody (PDB ID: 3DWT). We performed modeling using Modeller9, and calculated the relative solvent accessibility of the amino acids on the framework based on the protein's three-dimensional structure.

[0394] The specific steps for the universal framework transplantation method for VHH humanization are as follows: A highly homologous human antibody sequence is obtained through IMGT. The target sequence is then humanized using the universal humanized VHH framework hNbBcII10FGLA (PDB ID: 3EAK) as a reference. Using the highly homologous framework as a framework template, the CDRs are replaced with the CDR regions of the target antibody strain. Based on the modeling, non-surface amino acids on the framework are then backmutated to complete the humanization of the target antibody.

[0395] Humanization of the antibody strains iGI-72 and iGI-1198 yielded eight and eleven humanized huGI variants, respectively, designated huGI-72v1 to huGI-72v8 and huGI-1198v1 to huGI-1198v11. The amino acid sequences of huGI-72v1 to huGI-72v7 are shown in SEQ ID NOs: 2-8, respectively.

[0396] Example 5 Preparation of Fc fusion protein of humanized single domain antibody

[0397] 5.1 Preparation of Fc fusion plasmids for humanized single-domain antibodies

[0398] The humanized sequence in Example 4 was synthesized, fused with a DNA fragment encoding human IgG1-Fc (including the hinge region), and cloned into a conventional mammalian expression vector to obtain a recombinant plasmid for expressing the GIPR single-domain antibody Fc fusion protein in mammals.

[0399] 5.2 Preparation of Fc Fusion Proteins of Humanized Single Domain Antibodies

[0400] The vector obtained in 5.1 was transfected into HEK293 cells for transient expression of the antibody. The recombinant expression plasmid was diluted with Freestyle293 medium and the PEI (Polyethylenimine) solution required for transformation was added. Each group of plasmid / PEI mixtures was added to the HEK293 cell suspension and cultured at 37°C, 5% CO2, and 130rpm. Four hours later, EXCELL293 medium, 2mM glutamine, and cultured at 130rpm. After 24 hours, 3.8mM VPA was added, and 4g / L glucose was added after 72 hours. After 5-6 days of culture, the transient expression culture supernatant was collected and purified by Protein A affinity chromatography to obtain the target huGI single-domain antibody Fc fusion protein. The protein was examined for purity by SDS-PAGE. The expression levels of each protein are shown in Table 10, and the SDS purity of each protein after one-step purification is greater than 95%.

[0401] Table 10. Expression levels of Fc fusion proteins of humanized single domain antibodies

[0402] Example 6 Identification of the Function of Fc Fusion Proteins of Humanized Single Domain Antibodies

[0403] 6.1 GIPR Binding Ability of Humanized GIPR Single Domain Antibody-Fc Fusion Protein

[0404] 293T-GIPR cells were plated onto a cell culture plate and humanized GIPR single-domain antibody-Fc fusion protein or Maridebart sample was added at a final concentration of 0.00128 nM to 100 nM. Anti-human secondary antibody SULFO-TAG (MSD, Cat. No. R32AJ-1) was added and incubated, and mean fluorescence intensity (MFI) was measured using MSD.

[0405] Maridebart is an anti-GIPR antibody, which was independently cloned and prepared with reference to the sequence disclosed in WHO Drug Information, Volume 36. Number 4.2022. Proposed INN: List 128. A similar sequence is also found in construct 2G10 LC1.006 in patent application WO2017112824.

[0406] The results are shown in FIG6 , and the binding ability of the humanized GIPR single domain antibody-Fc fusion protein to GIPR is slightly better than that of Maridebart.

[0407] 6.2 GIPR Affinity of Humanized GIPR Single Domain Antibody-Fc Fusion Protein

[0408] Refer to the experimental steps in 3.3 to determine and calculate the equilibrium dissociation constant (KD) and binding rate (K a ) and dissociation rate (K dis ), the results are shown in Tables 11 to 13, and the affinity of the humanized fusion protein to GIPR is comparable to that before humanization.

[0409] Table 11. Affinity of humanized GIPR single domain antibody-Fc fusion proteins for GIPR

[0410] Table 12. Affinity of humanized GIPR single domain antibody-Fc fusion proteins for GIPR

[0411] Table 13. Affinity of humanized GIPR single domain antibody-Fc fusion proteins for GIPR

[0412] The same experimental steps were used to detect the equilibrium dissociation constant (KD) and binding rate (K a ) and dissociation rate (K dis ) and compared with Maridebart. The results are shown in Table 14. The affinity of the humanized single-domain antibody Fc fusion protein is better than that of the antibody Maridebart.

[0413] Table 14. Affinity of humanized GIPR single domain antibody-Fc fusion proteins for GIPR

[0414] 6.3 Neutralizing Activity of Humanized GIPR Single Domain Antibody-Fc Fusion Protein

[0415] Referring to the experimental steps of Example 3.2, the neutralizing activity of the humanized GIPR single domain antibody-Fc fusion protein was determined. As shown in FIG7 , the neutralizing activity of the humanized GIPR single domain antibody-Fc fusion protein was superior to that of the non-humanized molecule iGI-1198-Fc fusion protein.

[0416] GIP-C12H4-Fc4 and 25 μL of humanized GIPR single-domain antibody-Fc or Maridebart samples were added to a 96-well plate; 293T-GIPR cells were then added and incubated. After incubation, cAMP was detected using a cAMP kit and absorbance at 450 nm / 550 nm was measured on a microplate reader. The inhibition rate (%) was calculated. As shown in Figure 8, the neutralizing activity of the humanized GIPR single-domain antibody-Fc fusion protein was superior to that of Maridebart.

[0417] 6.4 In vivo efficacy studies of humanized GIPR single-domain antibody-Fc fusion proteins

[0418] (1) DA-GIP stimulation experiment in C57BL / 6 mice

[0419] Nine C57BL / 6 mice were fasted overnight, and blood glucose levels were measured. The mice were then randomly divided into three groups. After 24 hours of resuming a normal diet, they were administered 203 nmol / kg of huGI-72v5-Ld-Fc according to the experimental protocol. 12 hours after dosing, an overnight fast was resumed. 48 hours after dosing, each mouse was given 50 nmol / kg of DA-GIP per ip. Immediately following DA-GIP administration, a 2 g / kg oral glucose load was administered. Blood glucose and insulin levels were measured before, 5, 10, 15, 30, and 60 minutes after the glucose load. As shown in Figures 9 and 10, compared with the PBS group, DA-GIP significantly lowered OGTT blood glucose. HuGI-72v5-Ld-Fc blocked the hypoglycemic effect of DA-GIP, demonstrating that huGI-72v5-Ld-Fc has significant blocking activity in mice. DA-GIP can be found in Nature Communications, volume 11, article number: 4981 (2020).

[0420] (2) Multiple administration of ob mice

[0421] Twenty-four ob / ob mice were randomly divided into six groups based on blood glucose and body weight. Dosing began at 6-8 weeks. The day of group dosing was designated D0, and dosing was continued weekly for a total of six times. Body weights were measured before and after treatment on D0, 7, 16, 20, 28, and 35. As shown in Figure 11, treatment with 0.5 mg / kg dulaglutide, 3 mg / kg huGI-72v5-Ld-Fc, and 30 mg / kg huGI-72v5-Ld-Fc significantly reduced the body weight of ob / ob mice. The combination of dulaglutide and huGI-72v5-Ld-Fc further enhanced the body weight-reducing effect.

[0422] Example 7 Preparation of GIPR Antagonist-GLP1R Activator Bifunctional Fusion Protein

[0423] Fusion proteins of huGI-72v5 and the GLP-1R agonist (GLP-1Ra) were constructed using plasmids constructed according to the protocols in Examples 2 and 5. The fusion proteins were transiently expressed by transfection into HEK293 cells and purified using Protein A. The amino acid sequences of the fusion proteins and their fragments are shown in Table 15.

[0424] Table 15. Amino acid sequence of fusion protein

[0425] Example 8 Identification of the biological function of the fusion protein

[0426] 8.1 Neutralizing Activity of Fusion Protein against GIPR

[0427] (1) Prepare 1 μM concentration of fusion protein sample (working concentration 0.25 μM) in experimental culture medium and dilute 4-fold for a total of 9 concentrations. Add GIP-C12H4-Fc4 (working concentration 10 ng / ml) and 10 μL of fusion protein Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE sample to 96-well plates, then add 293T-GIPRFL-GFP-PURO cells, incubate for 30 minutes, and then use cAMP kit (cAMP-Glo TM Max Assay (Promega, Catalog No. V1681) was used to detect the RLU value. As shown in Figure 12a, the fusion protein retained the neutralizing activity of the GIPR single domain antibody against GIPR.

[0428] The fusion proteins Duyakrwy-huGI-72v5-Fc4, Duyawy-huGI-72v5-Fc4, and Duqewy-huGI-72v5-Fc4 were tested using a similar method. A standard curve was generated using the standard sample provided with the cAMP kit to determine the relative RLU values ​​for cAMP expression. The RLU values ​​for the test fusion proteins were then used to calculate cAMP expression levels using the standard curve. The results are shown in Figure 12b.

[0429] (2) 100 nM fusion protein sample (working concentration: 25 nM) was prepared in experimental culture medium and diluted 5-fold stepwise for a total of 6 concentrations. GIP-C12H4-Fc4 (working concentration 10 ng / ml) and culture medium of fusion protein Duyakrwy-huGI-72v5-Fc4, Duyakrwy-C12-huGI-72v5-H1Fc4YTE, Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE or Duyakrwy-G4S-huGI-72v5-H1Fc4YTE were added to 96-well white plates, and then HEK293-GIPR-CREB (HEK293 cells expressing human GIPR protein on the cell membrane with CREB response element protein) cells were added. After incubation for 30 minutes, the cells were stained with Bio-Glo. TM Reporter gene expression was detected using the Luciferase Assay System (Promega, G7940), and the RLU values ​​were read. The results are shown in Figure 12c. Similarly, the above results show that the fusion protein retains the neutralizing activity of the GIPR single-domain antibody against GIPR.

[0430] 8.2 Agonistic Activity of Fusion Protein on GLP-1R

[0431] The fusion protein sample was prepared at a concentration of 10 nM in the experimental culture medium (working concentration: 5 nM), and then diluted 5-fold for a total of 8 concentrations. 50 μL of protein sample was added to each well of a 96-well white plate, and then HEK293-GLP1R-CREB (HEK293 cells expressing human GLP1R protein on the cell membrane with CREB response element protein) cells were added. After incubation for 30 minutes, the cells were assayed using Bio-Glo TM Reporter gene expression was detected using the Luciferase Assay System (Promega, G7940) and the RLU values ​​were read. As shown in Figure 13a, the fusion protein Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE had no effect on its GLP-1R agonist activity.

[0432] A similar method was used to test several other fusion proteins, and the results are shown in Figures 13b and 13c. Similarly, the above results showed that the fusion proteins retained the agonist activity on GLP-1R.

[0433] 8.3 Biological Effects of Fusion Protein on GIPR-GLP-1R Dual Receptor Cells

[0434] The fusion protein sample was prepared in the experimental culture medium and then diluted 5-fold for a total of 8 concentrations. 25 μl of GIP and 25 μl of fusion protein were added to each well of a 96-well plate. CHO-CREB-GIPR-GLP1R cells (CHO cells expressing human GLP1R protein and GIPR protein on the cell membrane, with CREB response element protein) were added to each well. After being placed in the incubator for 6 hours, the cells were assayed using Bio-Glo TM Reporter gene expression was detected using the Luciferase Assay System (Promega, G7940) and the RLU values ​​were read. As shown in Figure 14, the bifunctional protein of the present disclosure had a higher RLU value than the GLP-1R agonist protein, indicating that it can simultaneously bind to two different receptors and lead to a stronger downstream activation signal. Furthermore, it can partially block the binding of GIP to GIPR.

[0435] 8.4 Affinity of Fusion Proteins for Human GIPR or Human GLP-1R

[0436] The binding kinetics of different bifunctional fusion proteins to human GLP1R-chis or GIPR-ECD-chis were detected by biolayer interferometry (BLI) technology. The fusion proteins were directly immobilized on the AHC sensor, and then GLP1R-chis or GIPR-ECD-chis was diluted to 4 concentrations and bound to the immobilized proteins. The equilibrium dissociation constant (KD), association rate (Ka), and dissociation rate (Kdis) were calculated using Data Analysis HT 12.0.2.59 software. The results are shown in Tables 16 and 17.

[0437] Table 16. Affinity of fusion proteins for human GIPR

[0438] Table 17. Affinity of fusion proteins for human GLP-1R

[0439] A similar method was used to test the affinity of fusion proteins with YTE mutations and different linkers to the two receptors. The results are shown in Tables 18 and 19.

[0440] Table 18. Affinity of fusion proteins for human GLP-1R

[0441] Table 19. Affinity of fusion proteins for human GIPR

[0442] 8.5 In vivo efficacy of fusion proteins

[0443] (1) Forty-two male DIO mice were randomly divided into seven groups of six mice each based on their non-fasting blood glucose and body weight. The groups included G1, PBS, G2, huGI-72v5-Ld-Fc 27.8 nmol / kg, G3, Duyakrwy-C12-Fc4 27.8 nmol / kg, G4, Duyakrwy-huGI72v5-Fc4 27.8 nmol / kg, G5, Duyawy-huGI72v5-Fc4 27.8 nmol / kg, G6, Duqewy-huGI72v5-Fc4 27.8 nmol / kg, and G7, huGI-72v5-Ld-Fc + Duyakrwy-C12-Fc4 27.8 nmol / kg, respectively.

[0444] The day of group administration was designated D0, and the experimental endpoint was D28 (after overnight fasting on D27). Mice were administered intraperitoneally twice weekly for a total of 10 doses (D0, 3, 7, 10, 14, 17, 21, 24, 25, and 27). Body weights were measured twice weekly. As shown in Figure 15, all bifunctional molecules exhibited significant weight loss effects. Duyakrwy-huGI72v5-Fc4 achieved the greatest weight loss, outperforming the equimolar combination group.

[0445] (2) 30 DIO mice were randomly divided into 5 groups, with 6 animals in each group. PBS, Duyakrwy-C12-huGI-72v5-H1Fc4, Duyakrwy-C12-huGI-72v5-G4S-H1Fc4, Duyakrwy-G4S-huGI-72v5-H1Fc4 and Duyakrwy-huGI72v5-Fc4 were intraperitoneally administered, with a dose of 27.8 nmol / kg for each group. The administration frequency was twice a week for 6 consecutive times (D0, D3, D7, D10, D14 and D17). Clinical observation was performed once a day after administration, and body weight was measured twice a week. As shown in Figure 16, the body weight of mice in each administration group decreased significantly after administration compared with the PBS group.

[0446] (3) Twenty-five male DIO mice were randomly divided into five groups according to their non-fasting blood glucose and body weight, namely G1 PBS, G2 huGI-72v5-Ld-Fc (55.6 nmol / kg), G3 Duyakrwy-C12-Fc4 (55.6 nmol / kg), G4 Duyakrwy-C12-huGI-72v5-G4S-H1Fc4 (55.6 nmol / kg) and G5 Duyakrwy-C12-huGI-72v5-G4S-H1Fc4 (222.4 nmol / kg), with 5 mice in each group.

[0447] The day of group administration was recorded as D0, and the experimental endpoint was D21. Subcutaneous administration was performed twice a week for a total of 6 times (D0, D3, D7, D10, D14, and D17). During the experiment, general clinical observations were performed on the day of each administration, mice were weighed twice a week, non-fasting blood glucose was measured once a week, and OGTT and ITT experiments were performed on D16 and D17, respectively.

[0448] OGTT experiment: Mice were fasted overnight on Day 15 and orally administered with 2 g / kg glucose on Day 16. Tail tip blood glucose levels were measured using a handheld glucometer (Roche, Accu-chek) before, and 5, 10, 20, 30, and 60 minutes after the glucose loading.

[0449] ITT (Insulin Tolerance Test) experiment: On the morning of D17, mice were fasted for 4 hours. Two hours after dosing on D17, mice were administered an insulin load of 0.75 U / kg (intraperitoneal injection). Tail tip blood glucose levels were measured using a handheld glucometer (Roche, Accu-chek) before and 10, 20, 40, 60, and 90 minutes after the insulin load.

[0450] In addition, mouse serum was collected on D19 to detect the levels of TG (triglyceride), TC (total cholesterol), LDL-C (low-density lipoprotein cholesterol), and HDL-C (high-density lipoprotein cholesterol) in the serum; mice were dissected on D21, the kidneys and attached fat, epididymal attached fat, and inguinal fat were weighed, and the organ-to-body ratio was calculated; and part of the mouse liver was collected on D21 to detect the TG content.

[0451] As shown in Figures 17a-17b, the bifunctional fusion protein can significantly reduce the body weight and non-fasting blood glucose of mice. The results in Figure 17c show that compared with the G1 group, the OGTT blood glucose exposure of G3 to G5 mice was significantly reduced, and the rates of change in blood glucose exposure were -23.50%, -35.10%, and -29.95%, respectively. The results in Figure 17d show that compared with the G1 group, the insulin sensitivity of mice in the bifunctional fusion protein group was significantly improved. The results in Figures 17e-17g show that compared with the G1 group, TC, LDL-C, and HDL-C in the serum of G4 and G5 mice were significantly reduced (Figure 17f), the proportion of fat body was significantly reduced (Figure 17e), and the TG in the mouse liver was significantly reduced (Figure 17g).

[0452] 8.6 Affinity of YTE Variants for FcRn

[0453] The YTE variant of Fc can improve the binding to FcRn under acidic pH conditions without affecting the binding to FcRn under neutral pH conditions, thereby obtaining a longer half-life than the parent.

[0454] The binding kinetics of Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE to human FcRn (acro, Catalog No. FCN-H52W7) at acidic pH 6.0 and neutral pH 7.4 were assessed using biotin-labeled Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE. Human FcRn (acro, Catalog No. FCN-H52W7) was then directly immobilized onto the SA sensor. Human FcRn (acro, Catalog No. FCN-H52W7) was then diluted to five different concentrations and allowed to bind to the immobilized protein. The equilibrium dissociation constant (KD), association rate (ka), and dissociation rate (kdis) were calculated using Data Analysis HT 12.0.2.59 software. The results are shown in Table 20. The YTE-containing fusion protein binds to human FcRn at pH 6.0, with similar fitted KD values ​​at different concentrations. No binding was observed at pH 7.4.

[0455] Table 20. Binding kinetics of YTE-containing fusion proteins to FcRn

[0456] PK results of 8.7 YTE variants in FcRn humanized mice

[0457] B-hFcRn mice (Biocytogen) were given a single subcutaneous (SC) injection of the bifunctional molecules Duyakrwy-C12-huGI-72v5-H1Fc4YTE at 5 mg / kg, Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE at 5 mg / kg, and Duyakrwy-G4S-huGI-72v5-H1Fc4YTE at 5 mg / kg. Mice were randomly divided into three groups based on body weight, with 10 animals in each group. Blood samples were collected at different time points, and serum was collected. Serum test compound concentrations were determined using ELISA, and pharmacokinetic parameters were calculated using a non-compartmental model using Pheonix WinNonlin software. The results are shown in Table 21 and Figure 18. The T values ​​for Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE were 0. 1 / 2 The longest one is 85.31h, which are 1.2 times and 2.3 times that of Duyakrwy-C12-huGI-72v5-H1Fc4YTE and Duyakrwy-G4S-huGI-72v5-H1Fc4YTE, respectively.

[0458] Table 21. PK results of fusion proteins in FcRn humanized mice

[0459] 8.8 Binding of the Fusion Protein to HEK293-GLP1R-CREB Cells

[0460] Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE or Duyakrwy-C12-H1Fc4m (final concentration of 500 nM, 5-fold serial dilution, 8 concentrations in total) was incubated with HEK293-GLP1R-CREB cells on ice for 1 h, washed three times with 1% BSA / PBS, and then SMLFO-TAG was added. TM The labeled Goat Anti-Human Antibody (purchased from MSD, product number R32AJ-1) was incubated on ice in the dark for 40 minutes, washed three times with 1% BSA / PBS, and then the electrochemiluminescence signal was detected using an MSD ultrasensitive multi-factor electrochemiluminescence analyzer. The binding of each drug to the cells was evaluated based on the signal strength. The results showed that with the increase of drug concentration, the signal gradually increased and was concentration-dependent. By fitting the concentration-binding signal with a 4-parameter curve, the maximum binding signal values ​​of Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE and Duyakrwy-C12-H1Fc4m were 8191 and 5273, respectively. 50The values ​​were 11.635 nM and 13.756 nM, respectively, indicating that Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE has a higher binding affinity to HEK293-GLP1R-CREB cells than Duyakrwy-C12-H1Fc4m. The relevant results are shown in FIG19 .

[0461] 8.9 Binding of Fusion Protein to 293T-GIPR Cells

[0462] 293T-GIPR cells were incubated with Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE (final concentration of 0.024-100 μg / ml) at 4°C for 1 hour, washed once with 1% BSA / PBS, and then APC anti-human IgG Fc was added. The cells were incubated in the dark for 30 minutes at 4°C, washed twice with 1% BSA / PBS, and the average fluorescence intensity of the cell surface was detected by flow cytometry to investigate the binding of Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE to the cells. The results showed that with the increase of drug concentration, the fluorescence intensity gradually increased in a concentration-dependent manner. The EC value of Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE binding to 293T-GIPR cells was 0. 50 It is 0.89μg / ml.

[0463] 8.10 Fusion protein blocks GIP binding to 293T-GIPR cells

[0464] 293T-GIPR cells were incubated with biotin-labeled GIP-C12H4-Fc4 (working concentration 20 μg / ml) and Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE (final concentration of 20 μg / ml, 5-fold serial dilution, a total of 8 concentrations) were added at the same time. They were incubated at 4°C for 1 hour, washed once with 1% BSA / PBS, and then APC-streptavidin was added. The cells were incubated in the dark at 4°C for 30 minutes. After washing twice with 1% BSA / PBS, the mean fluorescence intensity of the cell surface was detected by flow cytometry, and the inhibition rate was calculated. The results showed that with the increase of drug concentration, the mean fluorescence intensity of GIP-C12H4-Fc4 binding to cells gradually decreased in a concentration-dependent manner. Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE blocked the IC of GIP-C12H4-Fc4 binding to 293T-GIPR cells. 50 The maximum inhibition rate at the highest concentration of 20 μg / mL was 81.362%.

[0465] 8.11 fusion protein blocks the biological activity of GIP in stimulating cAMP production in 293T-GIPR cells

[0466] After GIP binds to the GIP receptor, it activates adenylate cyclase (AC) in the cell to produce cAMP. The fusion protein disclosed herein can bind to GIPR and block the biological function of GIP. Therefore, the blocking activity of the fusion protein can be evaluated by detecting the cAMP content in the cell. 293T-GIPR cells were cultured at 5×10 4 Cells / well were plated, GIP-C12H4-Fc4 was added at a working concentration of 10 ng / ml, and Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE was added at a final concentration of 0.061nM-1uM. The cells were reacted at 37°C for 30 minutes, 25μl of cell lysate was added to each well, and the cells were placed at room temperature for 30 minutes. 50μL of cell lysate was taken out from each well and the cAMP level was quantitatively detected by Elisa kit. The inhibition rate of the drug was calculated based on the cAMP level. The results showed that compared with the blank control (blank group), GIP alone significantly stimulated the production of cAMP in HEK293T-GIPR cells. Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE concentration-dependently reduced the level of cAMP produced by GIP stimulation. The IC of the inhibition rate of Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE was 0. 50 It is 32.503nM.

[0467] 8.12 fusion protein promotes glucose-dependent insulin release in mouse pancreatic islets

[0468] Both GIP and GLP1 act on mouse pancreatic islets, leading to glucose-stimulated insulin secretion (GSIS). To evaluate the effect of the fusion protein on GSIS, islets isolated from mouse pancreas were cultured in vitro and insulin release was measured. Islet tissue was isolated from the mouse pancreas and cultured in a cell culture incubator. A solution of KRBH buffer, 0.5% BSA, and 11.1 mM glucose was prepared and the islets were equilibrated for 1 hour. Three islets of uniform size were selected and placed in a 24-well cell culture plate and incubated with Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE (final concentrations of 10 nM, 1 nM, 0.1 nM, and 0 nM) for 80 minutes. Every 20 minutes, 10 μL of supernatant was removed for insulin detection, and 10 μL of the diluent was added to each well. The results showed that Duyakrwy-C12-huGI-72v5-G4S-H1Fc4YTE promoted GSIS in mouse pancreatic islets. As the drug concentration increased, the amount of insulin secretion increased in a concentration-dependent manner. The relevant results are shown in Figure 20.

[0469] Sequence information:

Claims

1. A fusion protein comprising: a) an antigen binding region that binds to the glucose-dependent insulinotropic polypeptide receptor (GIPR), said antigen binding region comprising an immunoglobulin single variable domain, and b) Glucagon-like peptide-1 receptor (GLP-1R) agonists. 2 . The fusion protein according to claim 1 , wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:

1.

3. The fusion protein of claim 2, wherein the CDR1, CDR2 and CDR3 are defined according to the following definition systems: Kabat, AbM, Chothia or IMGT.

4. The fusion protein of claim 2 or 3, wherein the immunoglobulin single variable domain is camelid, humanized, or chimeric.

5. The fusion protein according to any one of claims 2 to 4, wherein the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1 are selected from any one of the following groups: SEQ ID NOs: 9 to 11, SEQ ID NOs: 12 to 14, SEQ ID NOs: 15 to 17 and SEQ ID NOs: 18 to 20.

6. The fusion protein according to any one of claims 1 to 5, wherein the immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 1 to 8. The fusion protein according to claim 6 , wherein the immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:

1. The fusion protein according to claim 6 , wherein the immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:

6.

9. The fusion protein of any one of claims 1 to 6, wherein the antigen binding region comprises multiple (eg, 2, 3, or 4) immunoglobulin single variable domains.

10. The fusion protein according to claim 9, wherein the plurality of immunoglobulin single variable domains each independently comprise the amino acid sequence shown in any one of SEQ ID NOs: 1-8.

11. The fusion protein of claim 10, wherein at least one, for example each, of the plurality of immunoglobulin single variable domains comprises the amino acid sequence shown in SEQ ID NO:

1.

12. The fusion protein of claim 10, wherein at least one, for example each, of the plurality of immunoglobulin single variable domains comprises the amino acid sequence shown in SEQ ID NO:

6.

13. The fusion protein according to any one of claims 1 to 12, wherein the GLP-1R agonist is human GLP-1 or a variant thereof, or human GLP-1 or a functional fragment thereof, or exenatide or a variant thereof, or a functional fragment of exenatide or a variant thereof. The fusion protein according to claim 13 , wherein the GLP-1R agonist is human GLP-1, for example comprising the amino acid sequence shown in SEQ ID NO:

21. 15 . The fusion protein according to claim 13 , wherein the GLP-1R agonist is a human GLP-1 variant, for example comprising the amino acid sequence shown in SEQ ID NO:

22.

16. The fusion protein according to claim 13, wherein the GLP-1R agonist is a human GLP-1 variant, which further comprises an amino acid mutation at one or more of Y19, Q23, K26 and W31 compared to the amino acid sequence shown in SEQ ID NO:

22.

17. The fusion protein according to claim 13, wherein the GLP-1R agonist is a human GLP-1 variant, which further comprises an amino acid mutation at one of Y19, Q23 and W31 compared to the amino acid sequence shown in SEQ ID NO:

22.

18. The fusion protein according to claim 13, wherein the GLP-1R agonist is a human GLP-1 variant, and the variant further comprises an amino acid mutation selected from the following positions compared to the amino acid sequence shown in SEQ ID NO: 22: 1)Y19； 2)Q23； 3)W31； 4) Y19 and K26: 5) Y19 and W31; 6) Q23 and K26; 7) Q23 and W31; 8) K26 and W31; 9) Y19, K26, and W31; and 10)Q23, K26 and W31. The fusion protein according to any one of claims 16 to 18, wherein the amino acid mutation at position W31 is a substitution selected from the group consisting of W31Y, W31L and W31A.

20. The fusion protein according to any one of claims 16 to 19, wherein the amino acid at position K26 is substituted with K26R.

21. The fusion protein according to any one of claims 16 to 20, wherein the amino acid mutation at position Q23 is a substitution selected from the group consisting of Q23E, Q23T, Q23S and Q23N.

22. The fusion protein according to any one of claims 16 to 21, wherein the amino acid mutation at the Y19 position is a substitution selected from the group consisting of Y19A, Y19L, Y19T, Y19F, Y19I, Y19V and Y19S. 23 . The fusion protein according to claim 13 , wherein the GLP-1R agonist comprises the amino acid sequence shown in any one of SEQ ID NOs: 23-36. The fusion protein according to claim 13 , wherein the GLP-1R agonist comprises the amino acid sequence shown in any one of SEQ ID NOs: 37-59. 25 . The fusion protein according to claim 13 , wherein the GLP-1R agonist comprises the amino acid sequence shown in any one of SEQ ID NOs: 60-70. The fusion protein according to any one of claims 1 to 25 , wherein the GLP-1R agonist is located at the N-terminus or C-terminus of the antigen binding region.

27. The fusion protein of any one of claims 1-26, further comprising an immunoglobulin Fc region.

28. The fusion protein according to claim 27, wherein the immunoglobulin Fc region is an Fc region derived from IgG; preferably, the immunoglobulin Fc region is an Fc region derived from IgG1, IgG2, IgG3 or IgG4.

29. The fusion protein according to claim 27 or 28, wherein the immunoglobulin Fc region comprises the amino acid sequence shown in any one of SEQ ID NOs: 71-74; preferably comprises the amino acid sequence shown in SEQ ID NO:

74.

30. The fusion protein according to any one of claims 27 to 29, wherein the immunoglobulin Fc region comprises an amino acid mutation, and the amino acid mutation selectively enhances the binding affinity of the immunoglobulin Fc region to an Fc receptor compared to an unmutated immunoglobulin Fc region; preferably, the amino acid mutation selectively enhances the binding affinity of the immunoglobulin Fc region to a neonatal Fc receptor (FcRn).

31. The fusion protein according to any one of claims 27 to 30, wherein the immunoglobulin Fc region comprises amino acid mutations at positions M252, S254 and T256, as numbered by the EU index of Kabat. The fusion protein according to claim 31 , wherein the amino acid mutations contained in the immunoglobulin Fc region are M252Y, S254T and T256E.

33. The fusion protein according to any one of claims 27 to 32, wherein the immunoglobulin Fc region comprises the amino acid sequence shown in any one of SEQ ID NOs: 75 to 78, preferably comprises the amino acid sequence shown in SEQ ID NO:

78.

34. The fusion protein according to any one of claims 1 to 33, wherein the fusion protein comprises a configuration selected from any one of formulas (A) to (D): (A) (B) (C) and (D) in, GIPR BM is an antigen binding region as defined in any one of claims 1 to 12 that binds to GIPR, GLP-1Ra is a GLP-1R agonist as defined in any one of claims 13 to 26, and Fc is an immunoglobulin Fc region as defined in any one of claims 27 to 33; L1 and L2 are each independently a peptide linker, or are absent; HR is the hinge region of an immunoglobulin, for example, HR comprises a hinge region derived from IgG1, IgG2, IgG3, or IgG4.

35. The fusion protein according to claim 34, wherein the HR comprises the amino acid sequence shown in any one of SEQ ID NOs: 79-82; preferably comprises the sequence shown in SEQ ID NO:

79.

36. The fusion protein according to claim 34 or 35, wherein the peptide linker has a length of 2-20 amino acids.

37. The fusion protein according to any one of claims 34 to 36, wherein the peptide linker comprises GS or GAP or an amino acid sequence as shown in any one of SEQ ID NOs: 83 to 87; preferably comprises a sequence as shown in SEQ ID NO: 85 or SEQ ID NO:

87.

38. The fusion protein according to any one of claims 34-37, comprising a combination of L1, L2 and HR as shown in any one of (I) to (XIV): (I) L1 is the amino acid sequence set forth in SEQ ID NO: 85, L2 is the amino acid sequence set forth in SEQ ID NO: 85, and HR is the amino acid sequence set forth in SEQ ID NO: 82; (II) L1 is the amino acid sequence set forth in SEQ ID NO:85, L2 is the amino acid sequence set forth in SEQ ID NO:85, and HR is the amino acid sequence set forth in SEQ ID NO:79; (III) L1 is the amino acid sequence set forth in SEQ ID NO:85, L2 is the amino acid sequence set forth in SEQ ID NO:87, and HR is the amino acid sequence set forth in SEQ ID NO:82; (IV) L1 is the amino acid sequence set forth in SEQ ID NO:85, L2 is the amino acid sequence set forth in SEQ ID NO:87, and HR is the amino acid sequence set forth in SEQ ID NO:79; (V) L1 is the amino acid sequence set forth in SEQ ID NO:87, L2 is the amino acid sequence set forth in SEQ ID NO:85, and HR is the amino acid sequence set forth in SEQ ID NO:82; (VI) L1 is the amino acid sequence set forth in SEQ ID NO:87, L2 is the amino acid sequence set forth in SEQ ID NO:85, and HR is the amino acid sequence set forth in SEQ ID NO:79; (VII) L1 has the amino acid sequence set forth in SEQ ID NO: 85, L2 is absent, and HR has the amino acid sequence set forth in SEQ ID NO: 82; (VIII) L1 has the amino acid sequence set forth in SEQ ID NO:85, L2 is absent, and HR has the amino acid sequence set forth in SEQ ID NO:79; (IX) L1 has the amino acid sequence set forth in SEQ ID NO: 87, L2 is absent, and HR has the amino acid sequence set forth in SEQ ID NO: 82; (X) L1 represents the amino acid sequence set forth in SEQ ID NO:87, L2 is absent, and HR represents the amino acid sequence set forth in SEQ ID NO:79; (XI) L1 is absent, L2 has the amino acid sequence set forth in SEQ ID NO: 85, and HR has the amino acid sequence set forth in SEQ ID NO: 82; (XII) L1 is absent, L2 has the amino acid sequence set forth in SEQ ID NO:85, and HR has the amino acid sequence set forth in SEQ ID NO:79; (XIII) L1 is absent, L2 has the amino acid sequence set forth in SEQ ID NO: 87, and HR has the amino acid sequence set forth in SEQ ID NO: 82; and (XIV) L1 is absent, L2 has the amino acid sequence shown in SEQ ID NO: 87, and HR has the amino acid sequence shown in SEQ ID NO:

79.

39. The fusion protein according to any one of claims 1-38, comprising the amino acid sequence shown in any one of SEQ ID NOs: 88-93.

40. The fusion protein according to any one of claims 1-38, comprising the amino acid sequence shown in any one of SEQ ID NOs: 94-97.

41. The fusion protein of any one of claims 1-40, which is a homodimer.

42. A nucleic acid molecule encoding the fusion protein of any one of claims 1-41.

43. An expression vector comprising the nucleic acid molecule of claim 42 operably linked to an expression control element.

44. A recombinant cell comprising the nucleic acid molecule of claim 42 and / or transformed with the expression vector of claim 43, and capable of expressing the fusion protein.

45. A pharmaceutical composition comprising the fusion protein of any one of claims 1-41 and / or the nucleic acid molecule of claim 42 and / or the expression vector of claim 43 and / or the recombinant cell of claim 44, and a pharmaceutically acceptable carrier. A kit comprising the fusion protein of any one of claims 1 to 41 and / or the pharmaceutical composition of claim 45.

47. A method for treating and / or preventing a metabolic disease and / or symptom, comprising administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1-41, the nucleic acid molecule of claim 42, and / or the pharmaceutical composition of claim 45.

48. The method according to claim 47, wherein the metabolic disease and / or condition is obesity, overweight, diabetes, hyperlipidemia and / or non-alcoholic fatty liver disease.

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