GIPR-binding proteins and uses thereof

Abstract

The present invention provides a GIPR-binding protein and uses thereof. Specifically, the present invention provides a single domain antibody that specifically binds to the glucose-dependent insulinotropic polypeptide receptor, a derivative protein thereof, and pharmaceutical uses thereof.

Description

[Technical Field]

[0001] The present disclosure relates to the biopharmaceutical field and discloses single domain antibodies against glucose-dependent insulinotropic polypeptide receptor (GIPR) and their derivative proteins. Specifically, the present disclosure discloses binding proteins against glucose-dependent insulinotropic polypeptide receptor and uses thereof. [Background technology]

[0002] Glucose-dependent insulinotropic polypeptide (GIP) is an enterogenic incretin hormone that potentiates glucose-stimulated insulin secretion. GIP is secreted by K cells located in the proximal small intestine. Dietary carbohydrates and fats are potent stimulators of human GIP secretion. Studies have shown that K cell GIP secretion increases in rodents fed a high-fat diet. Similarly, acute high-fat diet intake in humans also increases GIP levels by 42%, resulting in significant weight gain. This suggests a positive correlation between exposure to a high-fat diet and GIP levels. Therefore, a high-fat diet and increased systemic GIP levels may underlie obesity differences in human subjects.

[0003] In recent years, many human genetic studies have shown a relationship between glucose-dependent insulinotropic polypeptide receptor (GIPR) and body mass index (BMI). Furthermore, GIPR knockout mice show resistance to obesity induced by high-fat diet. In addition to the established action of GIPR in pancreatic cells and adipocytes, the development of GIPR antagonists for treating obesity is considered a possible strategy. However, in the prior art, there are no GIPR antagonists with clear therapeutic effects. Summary of the Invention

[0004] The present disclosure utilizes phage display technology to obtain anti-GIPR single domain antibodies (VHH) and their derivative proteins by screening. The anti-GIPR single domain antibodies (VHH) and their derivative proteins of the present disclosure have remarkable high specificity and high affinity effects.

[0005] In a first aspect, the present disclosure provides a glucose-dependent insulinotropic polypeptide receptor (GIPR) binding protein comprising at least one immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 within a VHH as set forth in SEQ ID NO:5 and / or SEQ ID NO:6.

[0006] In some embodiments, the CDR1, CDR2, and CDR3 are defined by a definition system such as Kabat, AbM, Chothia, or IMGT.

[0007] In some embodiments, the immunoglobulin single variable domain is camelid, humanized, or chimeric.

[0008] In some embodiments, the CDR1, CDR2, and CDR3 in the VHH shown in SEQ ID NO: 5 are any one set selected from SEQ ID NOs: 74 to 76, 77 to 79, 80 to 82, and 83 to 85. In some embodiments, the CDR1, CDR2, and CDR3 in the VHH shown in SEQ ID NO: 5 are any one set selected from SEQ ID NOs: 74 to 76, 77 to 79, 80 to 82, and 83 to 85. In some embodiments, the at least one immunoglobulin single variable domain comprises one or more of the amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NOs: 8 to 14. In some embodiments, the at least one immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 5 and one of SEQ ID NOs: 8 to 14.

[0009] In some embodiments, the CDR1, CDR2, and CDR3 in the VHH shown in SEQ ID NO: 6 are any one set selected from SEQ ID NOs: 86 to 88, 89 to 91, 92 to 94, and 95 to 97. In some embodiments, the at least one immunoglobulin single variable domain comprises one or more of the amino acid sequences shown in SEQ ID NO: 6 and SEQ ID NOs: 15 to 25. In some embodiments, the at least one immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 6 and one of SEQ ID NOs: 15 to 25.

[0010] In some embodiments, the at least one immunoglobulin single variable domain comprises one or more of the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NOs:8-14, and one or more of the amino acid sequences set forth in SEQ ID NO:6 and SEQ ID NOs:15-25. In some embodiments, the at least one immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO:5 and one of SEQ ID NOs:8-14, and one of SEQ ID NO:6 and SEQ ID NOs:15-25.

[0011] In some embodiments, the GIPR-binding protein comprises one of the immunoglobulin single variable domains, hi some embodiments, the GIPR-binding protein comprises a plurality of the immunoglobulin single variable domains, e.g., two, three, four, or five of the immunoglobulin single variable domains.

[0012] In some embodiments, the GIPR binding protein further comprises an immunoglobulin Fc region, preferably a human immunoglobulin Fc region, more preferably a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the immunoglobulin Fc region comprises the amino acid sequence set forth in SEQ ID NO: 110 or SEQ ID NO: 113. In some embodiments, the immunoglobulin Fc region comprises the amino acid sequence set forth in SEQ ID NO: 110 or SEQ ID NO: 113.

[0013] In some embodiments, the Fc region of said immunoglobulin is connected to said at least one immunoglobulin single variable domain either directly or indirectly via a linker.

[0014] In some embodiments, the GIPR binding protein is (a) specifically binds to human GIPR; (b) KD value for binding to human GIPR is 1 × 10 -7 Less than M, preferably 1 x 10 -8 Less than M, (c) blocking the interaction of GIP with GIPR; and (d) capable of reducing the body weight and / or blood glucose of a subject; It has at least one of the following characteristics.

[0015] In a second aspect, the present disclosure provides a fusion protein comprising a GIPR binding protein according to the first aspect of the disclosure.

[0016] In a third aspect, the present disclosure provides a multispecific antibody comprising a GIPR-binding protein according to the first aspect of the disclosure and / or a fusion protein according to the second aspect of the disclosure and another antigen-binding region or regions that bind to a different antigen than the GIPR-binding protein and / or the fusion protein or to a different epitope of the same antigen.

[0017] In a fourth aspect, the present disclosure provides a conjugate comprising a GIPR-binding protein according to the first aspect of the disclosure and / or a fusion protein according to the second aspect of the disclosure and / or a multispecific antibody according to the third aspect of the disclosure, wherein said conjugate further comprises a molecule conjugated to said GIPR-binding protein and / or said fusion protein and / or said multispecific antibody.

[0018] In a fifth aspect, the present disclosure provides a nucleic acid molecule encoding a GIPR binding protein according to the first aspect of the disclosure and / or a fusion protein according to the second aspect of the disclosure and / or a multispecific antibody according to the third aspect of the disclosure.

[0019] In a sixth aspect, the present disclosure provides an expression vector comprising the nucleic acid molecule according to the fifth aspect of the present disclosure operably linked to an expression control element.

[0020] In a seventh aspect, the present disclosure provides a recombinant cell comprising the nucleic acid molecule according to the fifth aspect of the disclosure and / or transformed with the expression vector according to the sixth aspect of the disclosure and capable of expressing said GIPR-binding protein and / or said fusion protein and / or said multispecific antibody.

[0021] In an eighth aspect, the present disclosure provides a pharmaceutical composition comprising a GIPR-binding protein according to the first aspect of the disclosure and / or a fusion protein according to the second aspect of the disclosure and / or a multispecific antibody according to the third aspect of the disclosure and / or a conjugate according to the fourth aspect of the disclosure and / or a nucleic acid molecule according to the fifth aspect of the disclosure and / or an expression vector according to the sixth aspect of the disclosure and / or a recombinant cell according to the seventh aspect of the disclosure, and a pharmaceutically acceptable carrier.

[0022] In a ninth aspect, the present disclosure provides a reagent kit comprising a GIPR binding protein according to the first aspect of the disclosure, a fusion protein according to the second aspect of the disclosure, a multispecific antibody according to the third aspect of the disclosure, a conjugate according to the fourth aspect of the disclosure and / or a pharmaceutical composition according to the eighth aspect of the disclosure.

[0023] In a tenth aspect, the present disclosure provides a method for determining the presence and / or amount of a GIPR protein, the method comprising providing a GIPR binding protein according to the first aspect of the disclosure, a fusion protein according to the second aspect of the disclosure, a multispecific antibody according to the third aspect of the disclosure, and / or a conjugate according to the fourth aspect of the disclosure.

[0024] In an eleventh aspect, the present disclosure provides a method of inhibiting binding of a GIPR protein to its ligand, the method comprising providing a GIPR binding protein according to the first aspect of the disclosure, a fusion protein according to the second aspect of the disclosure, a multispecific antibody according to the third aspect of the disclosure, and / or a conjugate according to the fourth aspect of the disclosure. In a preferred embodiment, the ligand is GIP.

[0025] In a twelfth aspect, the present disclosure provides a method of treating and / or preventing a metabolic disease and / or condition, the method comprising administering to a subject in need thereof an effective amount of a GIPR binding protein described in the first aspect of the disclosure, a fusion protein described in the second aspect of the disclosure, a multispecific antibody described in the third aspect of the disclosure, a conjugate described in the fourth aspect of the disclosure, and / or a pharmaceutical composition described in the eighth aspect of the disclosure. In some embodiments, the metabolic disease and / or condition is obesity, overweight, and / or diabetes.

[0026] In a thirteenth aspect, the present disclosure provides use of a GIPR binding protein according to the first aspect of the disclosure, a fusion protein according to the second aspect of the disclosure, a multispecific antibody according to the third aspect of the disclosure, a conjugate according to the fourth aspect of the disclosure, and / or a pharmaceutical composition according to the eighth aspect of the disclosure in the manufacture of a medicament for treating and / or preventing a metabolic disease and / or condition. In some embodiments, the metabolic disease and / or condition is obesity, overweight, and / or diabetes.

[0027] In a fourteenth aspect, the present disclosure provides a GIPR binding protein according to the first aspect of the disclosure, a fusion protein according to the second aspect of the disclosure, a multispecific antibody according to the third aspect of the disclosure, a conjugate according to the fourth aspect of the disclosure, and / or a pharmaceutical composition according to the eighth aspect of the disclosure, for use in treating and / or preventing a metabolic disease and / or condition. In some embodiments, the metabolic disease and / or condition is obesity, overweight, and / or diabetes.

[0028] Those skilled in the art will readily appreciate other aspects and advantages of the present disclosure from the following detailed description. The following detailed description shows and describes only exemplary embodiments of the present disclosure. As will be apparent to those skilled in the art, the present disclosure may enable those skilled in the art to modify the specific embodiments disclosed without departing from the spirit and scope of the invention according to the present disclosure. Accordingly, the drawings and description of the present disclosure are merely illustrative, rather than limiting.

[0029] Specific features of the presently disclosed invention are set forth in the appended claims. A better understanding of the features and advantages of the presently disclosed invention can be obtained by reference to the exemplary embodiments and drawings described in detail below, the brief description of which follows. [Brief explanation of the drawings]

[0030] [Figure 1] The blocking activity of GIPR single domain antibody-Fc fusion proteins against GIPR is shown, where △ represents iGI-72-Ld-Fc and ○ represents the positive control AMG-GIPR-mab2. [Figure 2] 1 shows the effect of multiple administrations of GIPR single domain antibody-Fc fusion protein on the body weight of DIO mice. [Figure 3] 1 shows the effect of multiple administrations of GIPR single domain antibody-Fc fusion protein on fasting blood glucose in DIO mice. [Figure 4]1 shows the effect of multiple administrations of GIPR single domain antibody-Fc fusion protein on insulin secretion in DIO mice. [Figure 5] 1 shows the effect of multiple administrations of GIPR single domain antibody-Fc fusion protein on the insulin resistance index in DIO mice. [Figure 6] 1 shows the neutralizing activity of humanized GIPR single domain antibody-Fc fusion proteins. [Figure 7] 1 shows the effect of humanized GIPR single domain antibody-Fc fusion protein on blood glucose in DA-GIP-stimulated C57BL / 6 mice. [Figure 8] 1 shows the effect of humanized GIPR single domain antibody-Fc fusion protein on DA-GIP-stimulated insulin secretion in C57BL / 6 mice. [Figure 9] 1 shows the effect of multiple administrations of humanized GIPR single domain antibody-Fc fusion protein on ob mouse body weight. [Figure 10] 1 shows the binding ability of humanized GIPR single domain antibody-Fc fusion protein and Maridebart to GIPR. [Figure 11] 1 shows the neutralizing ability of humanized GIPR single domain antibody-Fc fusion protein and Maridebart to GIPR. DETAILED DESCRIPTION OF THE INVENTION

[0031] Hereinafter, embodiments of the present disclosure will be described with reference to specific examples, and those skilled in the art will easily understand other advantages and effects of the present disclosure from the contents disclosed herein.

[0032] Definition of Terms Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art as known to those skilled in the art. For example, see Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990); Roitt et al., "Immunology" (2nd Ed.), Gower Medical Publishing, London, New York (1989), and the general prior art cited therein as manuals. Furthermore, unless otherwise specified, all methods, steps, techniques, and procedures not specifically detailed can be and have been performed in a known manner, and such manners are known to those skilled in the art. Also, for example, see the manuals, the general prior art mentioned above, and other references cited therein.

[0033] Unless otherwise specified, the terms "antibody" and "immunoglobulin," which can be used interchangeably, are used herein as a general term to include full-length antibodies, single chains thereof, 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), regardless of whether they refer to heavy-chain antibodies or conventional four-chain antibodies. Furthermore, as used herein, the term "sequence" (e.g., in terms such as "immunoglobulin sequence," "antibody sequence," "single variable domain sequence," "VHH sequence," or "protein sequence") should generally be understood to include not only the related amino acid sequence, but also the nucleic acid or nucleotide sequence encoding said sequence, unless a more restrictive interpretation is required herein.

[0034] As used herein, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of the protein and can often be added, removed, or transferred to other proteins without loss of function of the other parts and / or domains of the protein.

[0035] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional four-chain antibody or a chain of a heavy-chain antibody), or a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized by maintaining the immunoglobulin fold characteristic of antibody molecules.

[0036] The term "immunoglobulin variable domain," as used herein, refers to an immunoglobulin domain consisting essentially of four "framework regions," referred to in the art and below 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 spaced apart by three "complementarity-determining regions" or "CDRs," referred to in the art and below as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively. The general structure or sequence of an immunoglobulin variable domain may therefore be depicted as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain contains the antigen-binding site and thus confers antigen specificity to the antibody.

[0037] The term "immunoglobulin single variable domain" as used herein refers to a variable domain of an immunoglobulin that is capable of specifically binding to an epitope of an antigen without pairing with a variable domain of another immunoglobulin. An example of an immunoglobulin single variable domain of the present disclosure is a "domain antibody", such as the immunoglobulin single variable domains VH and VL (VH domain and VL domain). Another example of an immunoglobulin single variable domain is a camelid "VHH domain" (or abbreviated "VHH"), as defined below.

[0038] A "VHH domain" is also called a heavy-chain single-domain antibody, VHH, VHH antibody fragment, or VHH antibody, and is a variable domain of an antigen-binding immunoglobulin called a "heavy-chain antibody" (i.e., a "light-chain-deleted antibody") (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 above variable domain from the heavy-chain variable domain (herein referred to as "VH domain") present in a conventional four-chain antibody and the light-chain variable domain (herein referred to as "VL domain") present in a conventional four-chain antibody. VHH domains specifically bind to epitopes without the need for other antigen-binding domains (this is the opposite of the VH or VL domains in conventional four-chain antibodies, where the epitope is recognized by both the VL and VH domains). VHH domains are small, stable, and efficient antigen-recognition units formed by a single immunoglobulin domain.

[0039] In the context of the present disclosure, the terms "heavy chain single domain antibody", "VHH domain", "VHH", "VHH antibody fragment", and "VHH antibody" can be used interchangeably.

[0040] For example, as shown in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999), the amino acid residues used in Camelidae VHH domains may be numbered according to the general numbering scheme for VH domains proposed 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)).

[0041] Alternative methods for numbering amino acid residues in VH domains are known in the art and are applicable to VHH domains as well. For example, Chothia CDRs refer to the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM CDRs represent intermediate states between Kabat hypervariable regions and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. "Contact" CDRs are based on analysis of crystal structures of available complexes. A description of the CDR residues from each method is provided in Table 1 below. [Table 1]

[0042] The CDRs of an antibody may be IMGT-CDRs, which are a CDR definition system based on the IMGT antibody code, and the code is obtained by integrating structural information from over 5,000 sequences. The IMGT VH CDR code is CDR1: 27-38, CDR2: 56-65, and CDR3: 105-117.

[0043] It should also be noted that, as is 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 based on the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering), which generally means that the Kabat numbering may or may not correspond to the actual number of amino acid residues in the actual sequence.

[0044] For example, the CDRs may include "extended CDRs", such as 24 to 36 or 24 to 34 (LCDR1), 46 to 56 or 50 to 56 (LCDR2), and 89 to 97 or 89 to 96 (LCDR3) in VL, and 26 to 35 (HCDR1), 50 to 65 or 49 to 65 (HCDR2), and 93 to 102, 94 to 102, or 95 to 102 (HCDR3) in VH.

[0045] The total number of amino acid residues in a VHH domain is usually in the range of 110 to 120, and often between 112 and 115. It should be noted that both relatively short and relatively long sequences are also applicable for the purposes described herein.

[0046] Other structural and functional properties of VHH domains and polypeptides comprising them can be summarized as follows.

[0047] VHH domains (which are naturally "designed" to functionally bind antigen without the presence or interaction of a light chain variable domain) can be used as single and relatively small functional antigen-binding structural units, domains, or polypeptides. This property distinguishes VHH domains from the VH and VL domains of conventional four-chain antibodies. These VH and VL domains by themselves are usually not suitable for practical use as single antigen-binding proteins or immunoglobulin single variable domains, but must be combined in one form or another (e.g., in the form of conventional antibody fragments such as Fab fragments, or in the form of scFvs consisting of a VH domain covalently linked to a VL domain) to provide a functional antigen-binding unit.

[0048] Due to their unique properties, the use of VHH domains, either alone or as part of a larger polypeptide, offers many significant advantages over the use of conventional VH and VL domains, scFvs, or conventional antibody fragments (e.g., Fab or F(ab')2 fragments). By binding antigen with high affinity and specificity using only a single domain, the presence of two separate domains is eliminated, as is the need to ensure the proper spatial conformation and orientation of the two domains (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. Multivalent and multispecific formats can be easily engineered from VHH domains. VHH domains have excellent solubility and are not prone to aggregation. VHH domains are highly stable against heat, pH, proteases, and other denaturing agents or conditions, eliminating the need for refrigeration during production, storage, or transportation, resulting in cost, time, and environmental savings. VHH domains are easy to produce and relatively inexpensive, even on a production scale. VHH domains are relatively small (approximately 15 kDa, or 1 / 10 the size of a normal IgG) compared to conventional four-chain antibodies and their antigen-binding fragments, and therefore exhibit relatively high tissue penetration and can be administered at relatively high doses compared to conventional four-chain antibodies and their antigen-binding fragments. VHH domains can exhibit so-called cavity-binding properties (especially their CDR3 loops, which are elongated compared to conventional VH domains), enabling them to reach targets and epitopes that are inaccessible to conventional four-chain antibodies and their antigen-binding fragments.

[0049] Methods for obtaining VHHs that bind to specific antigens or epitopes have previously been disclosed in the following publications: 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.

[0050] A VHH domain derived from Camelidae may be "humanized" by substituting 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 human conventional four-chain antibody VH domain (also referred to herein as "sequence optimization." In addition to humanization, "sequence optimization" may also cover other modifications of the sequence by one or more mutations that provide improved properties of the VHH, such as potential removal of post-translational modification sites). A humanized VHH domain may comprise one or more fully human framework region sequences. Humanization can be performed, for example, by resurfacing amino acids on the protein surface and / or by CDR grafting to a universal framework, as shown in the Examples.

[0051] Generally, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein (e.g., an immunoglobulin single variable domain, heavy chain single domain antibody, or GIPR-binding protein of the present disclosure) can bind. The specificity can be determined based on the affinity and / or affinity of the antigen-binding protein. The affinity, as indicated by the dissociation equilibrium constant (KD) of the antigen and the antigen-binding protein, is a measure of the binding strength between an epitope and an antigen-binding site on the antigen-binding protein; i.e., the smaller the KD value, the stronger the binding strength between the epitope and the antigen-binding protein (alternatively, affinity may be indicated by the association constant (KA), which is 1 / KD). As known to those skilled in the art, affinity may be measured in known ways depending on the specific antigen of interest. Affinity is a measure of the binding strength between an antigen-binding protein (e.g., an immunoglobulin, antibody, immunoglobulin single variable domain, or polypeptide containing it) and the relevant antigen. Affinity is related to the affinity between the antigen-binding site on the antigen-binding protein and the relevant antigen, and the number of relevant binding sites present on the antigen-binding protein.

[0052] As used herein, the term "glucose-dependent insulinotropic polypeptide receptor" is abbreviated as GIPR and covers the GIPR of any species. Preferably, the GIPR is human GIPR or mouse GIPR.

[0053] As used herein, the term "glucose-dependent insulinotropic polypeptide receptor (GIPR) binding protein" refers to any protein capable of specifically binding to the glucose-dependent insulinotropic polypeptide receptor (GIPR). GIPR binding proteins may include heavy chain single domain antibodies against GIPR as defined herein. GIPR binding proteins further cover immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules.

[0054] A "GIPR-binding protein" of the present disclosure may comprise at least one immunoglobulin single variable domain, e.g., a VHH, that binds to GIPR. In some embodiments, a "GIPR-binding protein" of the present disclosure may comprise two, three, four, or more immunoglobulin single variable domains, e.g., a VHH, that bind to GIPR. A GIPR-binding protein of the present disclosure may comprise, in addition to an immunoglobulin single variable domain that binds to GIPR, a linker and / or a moiety with effector function, e.g., a half-life extending moiety (e.g., an immunoglobulin single variable domain that binds to serum albumin), and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region. In some embodiments, a "GIPR-binding protein" of the present disclosure also covers bispecific antibodies comprising antigen-binding domains or immunoglobulin single variable domains that bind different antigens or different regions of the same antigen (e.g., different epitopes).

[0055] Typically, the GIPR binding proteins of the present disclosure have a GIPR activity of 1000 kJ / kg, preferably 100 kJ / kg, as measured in a Biacore or KinExA or Fortibio assay. -7 ~10 -10 mol / L (M), more preferably 10 -8 ~10 -10 mol / L, more preferably 10 -9 ~10 -10 or lower dissociation constant (KD), and / or 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 It binds to the antigen (i.e., GIPR) with an association constant (KA) of 10. -4Any KD value greater than M is generally considered to indicate non-specific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be measured by any suitable known method, such as, for example, a surface plasmon resonance (SPR) assay, a Scatchard assay, and / or a competitive binding assay (e.g., radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assay) as described herein.

[0056] Amino acid residues are designated by standard three-letter or one-letter amino acid codes that are consistent with those known in the art. When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion, or substitution of a given number of amino acid residues at a particular position in a reference sequence compared to another sequence. In the case of substitution, the substitution is preferably a conservative amino acid substitution, where the amino acid residue is replaced with another amino acid residue that has a similar chemical structure and has a relatively small or essentially no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are known in the art. For example, a conservative amino acid substitution is preferably a substitution of one amino acid residue in the following groups (i) to (v) with another amino acid residue in the same group: (i) relatively small 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) relatively large 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 substitution of Ala with Gly or Ser, substitution of Arg with Lys, substitution of Asn with Gln or His, substitution of Asp with Glu, substitution of Cys with Ser, substitution of Gln with Asn, substitution of Glu with Asp, substitution of Gly with Ala or Pro, substitution of His with Asn or Gln, substitution of Ile with Leu or Val, substitution of Leu with Ile or Val, substitution of Lys with Arg, Gln or Glu, substitution of Met with Leu, Tyr or Ile, substitution of Phe with Met, Leu or Tyr, substitution of Ser with Thr, substitution of Thr with Ser, substitution of Trp with Tyr, substitution of Tyr with Trp or Phe, and substitution of Val with Ile or Leu.

[0057] "Sequence identity" between two polypeptide sequences refers to the percentage of identical amino acids between the sequences. "Sequence similarity" refers to the percentage of amino acids that represent the same amino acids or conservative amino acid substitutions. Methods for assessing 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 usually measured using sequence analysis software. For example, identity can be determined using the BLAST program in the NCBI database. For determining sequence identity, reference may be made, for example, to 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.

[0058] In this disclosure, the term "isolated" generally refers to a biological material (e.g., a virus, nucleic acid, or protein) that is substantially free from components that are normally associated with or interact with it in its naturally occurring environment. Such isolated biological material optionally includes other materials that the biological material does not have in its natural environment (e.g., a nucleic acid or protein). In this disclosure, when a protein is involved, "isolated" generally refers to the molecule being isolated and separated from the whole organism in which it is naturally found, or being essentially free of other biological macromolecules of the same type. When a nucleic acid molecule is involved, it may be completely or partially isolated from sequences that are naturally associated with it, or the nucleic acid may have heterologous sequences associated with it, or the nucleic acid may be isolated from a chromosome.

[0059] A polypeptide or nucleic acid molecule is considered "isolated" when it is separated from at least one other component typically associated with it (e.g., another protein / polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or minor component) of its native biological origin and / or the reaction medium or culture medium from which the polypeptide or nucleic acid molecule was obtained. In particular, a polypeptide or nucleic acid molecule is considered "isolated" when it is purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold, and even 1000-fold or more. Preferably, an "isolated" polypeptide or nucleic acid molecule is essentially homogeneous, as determined by a suitable technique (e.g., a suitable chromatographic technique, e.g., polyacrylamide gel electrophoresis).

[0060] By "effective amount" is meant the amount of a GIPR binding protein or pharmaceutical composition of the disclosure that results in a decrease in the severity of symptoms of the disease, an increase in the frequency and duration of symptom-free periods of the disease, or prevention of damage or disability due to pain of the disease.

[0061] As used herein, "metabolic disease" refers to a disorder that affects the energy production of human (or animal) cells, also known as a metabolic disorder. The majority of metabolic diseases are genetic, and some are acquired through diet, toxins, or infection. Common metabolic diseases may be classified into three main categories: disorders that affect carbohydrate metabolism, disorders that affect fat metabolism, and disorders that affect intracellular mitochondria.

[0062] As used herein, the term "subject" means a mammal, particularly a primate, especially a human.

[0063] In the present disclosure, the term "antigen-binding protein" generally refers to a protein comprising an antigen-binding portion and, optionally, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding protein to an antigen. An antigen-binding protein may 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" further covers single-domain antibodies and proteins comprising immunoglobulin single variable domains. The variable regions of the heavy and light chains comprise a binding domain that interacts with an 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 exhibit the desired antigen-binding activity.

[0064] In this disclosure, the terms "polypeptide" and "protein" are used interchangeably and generally refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are analogs or mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms also include modified amino acid polymers that have been modified, for example, by the addition of sugar residues to form glycoproteins or by phosphorylation. Polypeptides and proteins may be produced from naturally occurring and non-recombinant cells, or from genetically engineered or recombinant cells, and may include molecules having the amino acid sequence of a native protein or molecules with one or more amino acid deletions, additions, and / or substitutions of the native sequence. The terms "polypeptide" and "protein" specifically include sequences with one or more amino acid deletions, additions, and / or substitutions of the antigen binding proteins described in this disclosure.

[0065] In this disclosure, the term "conjugate" generally refers to a substance formed when an antigen-binding protein is linked to another active agent, which may be a small molecule active agent such as a therapeutic agent, an imaging probe, or a spectroscopic probe.

[0066] In this disclosure, the term "nucleic acid molecule" generally refers to nucleotides, deoxyribonucleotides or ribonucleotides, or analogs thereof, of any length in isolated form, either isolated from their natural environment or artificially synthesized.

[0067] In the present disclosure, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating in a suitable host that transfers an inserted nucleic acid molecule into and / or between host cells. Such vectors may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and vectors primarily for expressing DNA or RNA by transcription and / or translation. The vectors further include vectors having multiple of the above 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.

[0068] In the present disclosure, the term "cell" generally refers to an individual cell, cell line, or cell culture that may contain or already contains a plasmid or vector containing a nucleic acid molecule described in the present disclosure, or that is capable of expressing an antigen-binding protein described in the present disclosure. The cell may include the progeny of a single host cell. Due to natural, unexpected, or deliberate mutations, the progeny cell may not necessarily be completely identical in morphology or genome to the original parent cell, but may still express an antibody or antigen-binding fragment thereof described in the present disclosure. The cell may be obtained by transfecting the cell in vitro with a vector described in the present disclosure. The cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, an NS0 cell, or a myeloma cell). In certain cases, the cell may be a mammalian cell. For example, the mammalian cell may be a CHO-K1 cell.

[0069] In this disclosure, the term "pharmaceutical composition" generally refers to a formulation in which the active ingredient is present in a form that effectively activates its biological activity and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0070] In the present disclosure, the term "treatment" generally refers to the desire to alter the natural course of the individual being treated and can be a process that achieves prophylaxis, treatment, or clinical intervention in the process of clinical disease. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some cases, antigen binding proteins (e.g., antibodies to specific antigens of the present disclosure) can be used to slow or delay disease progression.

[0071] In this disclosure, the term "administration" generally refers to a method of administering a compound or pharmaceutical composition to a subject (e.g., a patient) in a dose. Administration can be by any suitable method, including parenteral, intrapulmonary, intranasal, and (when local treatment is required) intralesional administration. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0072] In this disclosure, the term "comprise" generally refers to the meaning of including, summarizing, containing or including. In some cases, it also refers to the meaning of "is" or "consisting of."

[0073] In this disclosure, the term "about" generally refers to a variation within 0.5% to 10% above or below the specified value, for example, a variation within 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 the specified value.

[0074] GIPR-binding proteins of the present disclosure In one aspect, the present disclosure relates to glucose-dependent insulinotropic polypeptide receptor (GIPR) binding proteins comprising at least one immunoglobulin single variable domain.

[0075] In some embodiments, the at least one immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in a VHH as set forth in any one of SEQ ID NOs: 1 to 7. The CDRs may be defined according to any one of the following definition systems: Kabat CDR, AbM CDR, Chothia CDR or IMGT CDR.

[0076] In some embodiments, the at least one immunoglobulin single variable domain comprises one or more sets of CDR1, CDR2 and CDR3 selected from Table 2 below. [Table 2]

[0077] In some embodiments, the at least one immunoglobulin single variable domain comprises the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:5 and / or SEQ ID NO:6.

[0078] In some embodiments, a GIPR-binding protein of the present disclosure comprises at least one immunoglobulin single variable domain comprising one or more of the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NOs: 8-14. "Multiple" may be 2, 3, 4, or 5, and the multiple amino acid sequences may be the same or different.

[0079] In one specific embodiment, the at least one immunoglobulin single variable domain comprises an amino acid sequence set forth in SEQ ID NO: 5 and one of SEQ ID NOs: 8 to 14. For example, a GIPR-binding protein of the present disclosure comprises a sequence encompassed by the following general formula (where [ ] indicates the type of amino acid that can be selected at that position):

[0080] [EQ]VQLVESGGGLVQPGGSLRLSC[AS]ASRYTLDYYAIGWFRQAPGK[EG][LR]EGVSCINSKDGSTYYADSVKGRFTIS[KR]DNSKNTVYLQMNSLRAEDTAVYYCAAGYDPWSGIPPVQAMCVMGYDYWGQGTLVTVSS

[0081] In some other embodiments, the GIPR binding proteins of the present disclosure comprise at least one immunoglobulin single variable domain comprising one or more of the amino acid sequences set forth in SEQ ID NO:6 and SEQ ID NOs:15-25.

[0082] In a specific embodiment, the at least one immunoglobulin single variable domain comprises an amino acid sequence set forth in one of SEQ ID NOs: 6, 15 to 25. For example, a GIPR-binding protein of the present disclosure comprises a sequence encompassed by the following general formula (where [ ] indicates the type of amino acid that can be selected at that position): QVQL[QV]ESGGGLVQPGGSLRLSCSASGSGFSIVAMGW[VY]RQ[AT]PGK[GQ][LR]ELVAAITSGGNTNYADSVKGRFTISRDN[AS]KNTIYLQMNSL[KR][AP]EDTAVYYCNADPGVVAGPYAGMDFWGKGTTVTVSS

[0083] In some embodiments, the immunoglobulin single variable domain is 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: 5 or 6. 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: 5 or 6. 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: 5 or 6.

[0084] In some embodiments, the at least one immunoglobulin single variable domain may simultaneously comprise one or more of the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NOs: 8-14, and one or more of the amino acid sequences set forth in SEQ ID NO: 6 and SEQ ID NOs: 15-25.

[0085] In some embodiments, the at least one immunoglobulin single variable domain may simultaneously comprise the amino acid sequence set forth in SEQ ID NO: 5 and one of SEQ ID NOs: 8 to 14, as well as the amino acid sequence set forth in SEQ ID NO: 6 and one of SEQ ID NOs: 15 to 25.

[0086] In some embodiments, the GIPR-binding proteins of the present disclosure further comprise an immunoglobulin Fc region in addition to at least one immunoglobulin single variable domain. The inclusion of an immunoglobulin Fc region in the GIPR-binding proteins of the present disclosure allows the binding molecules to form dimers. The Fc regions that can be used in the present disclosure may be derived from different subtypes of immunoglobulins, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM.

[0087] In some embodiments, the wild-type Fc sequence may be mutated to alter Fc-mediated related activities. These mutations include, but are not limited to, mutations that alter Fc-mediated CDC activity, Fc-mediated ADCC activity, or FcRn-mediated in vivo half-life. These mutations are described in Leonard G. Presta, Current Opinion in Immunology 2008, 20:460-470; Esohe E. Idusogie et al., J. Immunol. 2000, 164: 4178-4184; Raphael A. Clynes et al., Nature Medicine, 2000, Volume 6, Number 4:443-446; and Paul R. Hinton et al., J. Immunol. 2006, 176:346-356. For example, mutating 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in the CH2 region can increase or eliminate Fc-mediated ADCC or CDC activity, or enhance or decrease affinity for FcRn. Furthermore, mutating 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in the hinge region can improve protein stability.

[0088] In some embodiments, introducing mutations into the Fc sequence makes the mutant Fc more susceptible to homodimer or heterodimer formation. As described in Ridgway, Presta et al. (1996) and Carter (2001), heterodimer formation between different Fc mutants is facilitated by the knob-hole model, which utilizes the spatial interaction of amino acid side chains at the Fc contact interface. Furthermore, by altering the amino acids at the Fc contact interface, for example, in CN102558355A or CN103388013A, the ionic interaction forces at the Fc contact interface can be altered to facilitate heterodimer formation between different pairs of Fc mutants (CN102558355A) or homodimer formation between Fc mutants with the same mutations (CN103388013A).

[0089] The immunoglobulin Fc region is preferably a human immunoglobulin Fc region, for example, a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some specific embodiments, the amino acid sequence of the immunoglobulin Fc region is set forth in SEQ ID NO: 110 or SEQ ID NO: 113.

[0090] In some specific embodiments, in the GIPR binding proteins of the present disclosure, the immunoglobulin Fc region (e.g., the Fc region of a human IgG1) is joined, directly or indirectly via a linker, to the C-terminus of the immunoglobulin single variable domain (e.g., a VHH).

[0091] In some embodiments, the GIPR-binding protein of the present disclosure comprises an immunoglobulin single variable domain that specifically binds to GIPR, which is directly or indirectly connected to an immunoglobulin Fc region, allowing the GIPR-binding protein to form a dimeric molecule comprising two GIPR-binding domains. Such GIPR-binding proteins are also referred to as bivalent GIPR-binding proteins. In some embodiments, the dimer is a homodimer.

[0092] In some embodiments, the GIPR-binding protein of the present disclosure comprises two immunoglobulin single variable domains that specifically bind to GIPR and an Fc region of an immunoglobulin, interconnected directly or indirectly via a linker, wherein the immunoglobulin Fc region enables the GIPR-binding protein to form a dimeric molecule comprising four GIPR-binding domains. Such a GIPR-binding protein is also referred to as a tetravalent GIPR-binding protein. In some embodiments, the dimer is a homodimer. In some embodiments, the two immunoglobulin single variable domains that specifically bind to GIPR in the GIPR-binding protein each bind to a different region or epitope of GIPR.

[0093] The immunoglobulin single variable domains of the present disclosure may be directly connected to each other, and between the immunoglobulin single variable domains and the immunoglobulin Fc region, or may be indirectly connected via a linker. The linker may be a non-functional amino acid sequence with a length of 1 to 20 or more amino acids and no secondary or higher structure. For example, the linker may be a flexible linker, such as (GGG) n , (GGGS) n , (GGGA) n , (GGGAA) n and (GGGGS) n where n is an integer selected from 1 to 30, an integer selected from 1 to 20, an integer selected from 1 to 10, an integer selected from 1 to 9, an integer selected from 1 to 8, an integer selected from 1 to 7, an integer selected from 1 to 6, an integer selected from 1 to 5, an integer selected from 1 to 4, or an integer selected from 1 to 3. In some embodiments, the linker is GGGGS, GS, GAP, (GGGGS)x3, or the like.

[0094] In some embodiments, the GIPR binding protein of the disclosure is (a) specifically binds to human GIPR; (b) KD value for binding to human GIPR is 1 × 10 -7 Less than M, preferably 1 x 10 -8 Less than M, (c) blocking the interaction of GIP with GIPR; (d) capable of reducing the body weight and / or blood glucose of a subject; It has at least one of the following characteristics.

[0095] fusion proteins In another aspect, the present disclosure further relates to a fusion protein comprising a GIPR binding protein described in this disclosure.

[0096] In some embodiments, in addition to the GIPR-binding protein, the fusion protein further comprises one or more additional biologically active proteins, which may be any protein with biological, therapeutic, prophylactic, or diagnostic significance or function, and which, when administered to a subject, mediate a biological activity that can prevent or alleviate a disease, disorder, or condition. Specifically, the biologically active protein may be an agonist, antagonist, modulator, ligand, cytokine, enzyme, or hormone, and is particularly a biologically active protein that can be used to prevent and / or treat metabolic diseases, particularly obesity, overweight, and / or diabetes.

[0097] multispecific antibodies In another aspect, the present disclosure further relates to a multispecific antibody comprising a GIPR binding protein described in this disclosure.

[0098] In some embodiments, in addition to the GIPR binding protein, the multispecific antibody further comprises one or more additional antigen-binding regions that bind to a different antigen or a different epitope of the same antigen as the GIPR binding protein and / or the fusion protein.

[0099] Nucleic acids, vectors, and host cells In another aspect, the present disclosure relates to a nucleic acid molecule encoding the GIPR-binding protein or 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 an essentially isolated nucleic acid.

[0100] The nucleic acid of the present disclosure may be in the form of a vector, may be present within a vector, and / or may be part of a vector, such as a plasmid, cosmid, or YAC. The vector may in particular be an expression vector, i.e., a vector capable of providing expression of the GIPR-binding protein in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). Such an expression vector typically comprises at least one nucleic acid of the present disclosure operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the skill of the art. Specific examples of control elements and other elements useful or essential for expression of the GIPR-binding protein of the present disclosure include, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.

[0101] Nucleic acids of the present disclosure can be produced or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA techniques) based on the amino acid sequence information of the polypeptides of the present disclosure described herein, and / or can be isolated from suitable natural sources.

[0102] In another aspect, the present disclosure relates to recombinant host cells that express or are capable of expressing one or more of the GIPR-binding proteins, fusion proteins, and / or nucleic acids or vectors of the present disclosure. Preferred host cells of the present disclosure are bacterial, fungal, or mammalian cells.

[0103] Suitable bacterial cells include cells of Gram-negative strains (e.g., Escherichia coli, Proteus, and Pseudomonas) and Gram-positive strains (e.g., Bacillus, Streptomyces, Staphylococcus, and Lactococcus).

[0104] Suitable fungal cells include cells of Trichoderma, Neurospora, and Aspergillus species, or cells of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris, Pichia methanolica), and Hansenula species.

[0105] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, or COS cells.

[0106] However, the present disclosure may also be used with any other cells known in the art for expressing heterologous proteins, such as amphibian cells, insect cells, etc.

[0107] The GIPR-binding proteins or fusion proteins of the present disclosure may be produced in an intracellular manner in cells such as those described above (e.g., in the cytoplasm, periplasm, or in inclusion bodies), and then isolated from the host cells and optionally further purified, or may be produced in an extracellular manner (e.g., in the medium in which the host cells are cultured), and then isolated from the medium and optionally further purified.

[0108] Methods and reagents for recombinantly producing polypeptides are known in the art, such as particular appropriate expression vectors, transformation or transfection methods, selectable markers, methods for inducing protein expression, culture conditions, etc. Similarly, protein isolation and purification techniques applicable to the methods for producing GIPR-binding proteins of the present disclosure are known to those of skill in the art.

[0109] Pharmaceutical Composition In another aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, comprising one or a combination of GIPR binding proteins, fusion proteins, multispecific antibodies, or conjugates of the present disclosure formulated together with a pharmaceutically acceptable carrier.

[0110] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, stabilizers, isotonic and absorption delaying agents, and the like. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, intraspinal, or epidermal administration (e.g., via injection or infusion). Depending on the route of administration, the active compound, i.e., antibody molecule, can be encapsulated in a material to protect the compound from the action of acids and other natural conditions that inactivate the compound.

[0111] 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 mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the composition that achieves a therapeutic effect. Typically, on a 100% basis, this amount ranges from about 0.01% to about 99% of the active ingredient, e.g., from about 0.1% to about 70%, or from about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0112] While the pharmaceutical compositions of the present disclosure can effectively achieve the therapeutic response required for a particular patient, composition, and mode of administration, the actual dosage level of the active ingredient may be varied to obtain an amount of the active ingredient that is not toxic to the patient. The selection of the dosage level is determined by various pharmacokinetic factors, including the activity of the particular composition of the present disclosure or its ester, salt, or amide used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors known in the medical arts.

[0113] The compositions of the present disclosure may be administered via one or more routes of administration using one or more methods known in the art. It should be understood by those skilled in the art that the route and / or mode of administration will vary depending on the desired results. Preferred routes of administration for the GIPR binding proteins of the present disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intraspinal, or other parenteral routes of administration, such as injection or infusion. As used herein, the term "parenteral administration" refers to a route of administration other than enteral and topical administration, typically injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0114] Prevention and / or treatment of diseases In another aspect, the present disclosure further provides a method for preventing and / or treating a disease, in particular treating and / or preventing a metabolic disease, comprising administering to a subject in need thereof an effective amount of a GIPR-binding protein, fusion protein, multispecific antibody, conjugate, or pharmaceutical composition of the present disclosure. The metabolic disease includes obesity, overweight, and / or diabetes.

[0115] In some embodiments, in the process of preventing and / or treating a metabolic disorder, in addition to the GIPR binding protein, fusion protein, multispecific antibody, conjugate, or pharmaceutical composition of the present disclosure, a subject in need thereof is administered other therapeutic agents, such as insulin (porcine or bovine insulin, human insulin, aspart insulin, lispro insulin, low protamine zinc insulin, glargine insulin, detemir insulin, LY3209590, etc.), biguanides (such as metformin), thiazolidinediones (including pioglitazone, rosiglitazone, etc.), sulfonylureas (glipizide, gliclazide, glipizide, gliclaz ... may be administered), 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, GCGR agonists, GLP-1R agonists, etc.

[0116] Here, the GLP-1R agonist may be GLP-1(7-37), GLP-1(7-36)-NH2, liraglutide, albiglutide, lixisenatide, dulaglutide, semaglutide, exendin-4, exendin-3, polyethylene glycol loxenatide, tirzepatide, GMA102, PB119, arbenatide, mazdutide, spaglutide, cotadutide, danuglipron, HM-15211, efinopegidutide, etc.

[0117] The other therapeutic agent may be administered simultaneously with the binding protein / fusion protein / multispecific antibody / conjugate / pharmaceutical composition of the present disclosure, or may be administered sequentially, with the interval between administrations being one week or less, preferably three days or less, more preferably two days or less, and even more preferably one day or less.

[0118] In some other embodiments, the present disclosure further provides for the use of a GIPR binding protein, fusion protein, multispecific antibody, conjugate, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating and / or preventing a metabolic disease.

[0119] In some other embodiments, the present disclosure provides a GIPR binding protein, a fusion protein, a multispecific antibody, a conjugate, or a pharmaceutical composition for treating and / or preventing a metabolic disease.

[0120] In some other embodiments, the present disclosure provides a method for detecting the presence and / or content of a GIPR protein, comprising providing the GIPR binding protein and / or the fusion protein and / or the multispecific antibody and / or the conjugate. For example, the method may be an ex vivo or in vitro method. For example, the method may be a method for non-therapeutic purposes.

[0121] In some embodiments, the present disclosure provides a method for detecting the presence of a GIPR protein in a sample, comprising contacting the sample with the GIPR binding protein and / or the fusion protein and / or the multispecific antibody and / or the conjugate, wherein binding of the GIPR binding protein and / or the fusion protein and / or the multispecific antibody and / or the conjugate to the GIPR protein indicates the presence of the GIPR protein in the sample.

[0122] In some embodiments, the present disclosure provides a method for detecting the content of GIPR protein in a sample, comprising contacting the sample with the GIPR binding protein and / or the fusion protein and / or the multispecific antibody and / or the conjugate, wherein the amount of binding of the GIPR binding protein and / or the fusion protein and / or the multispecific antibody and / or the conjugate to the GIPR protein indicates the content of the GIPR protein in the sample.

[0123] The present invention further provides the use of a GIPR binding protein, fusion protein, multispecific antibody or conjugate of the present disclosure in the manufacture of a reagent kit for detecting the presence and / or content of a GIPR protein in a sample.

[0124] The sample may be a sample of blood, plasma, amniotic fluid, etc.

[0125] In another aspect, the present disclosure provides a reagent kit that may include the GIPR-binding proteins, fusion proteins, multispecific antibodies, conjugates, and / or pharmaceutical compositions described herein, which may be contained in a single common container, optionally in combination with one or more therapeutic agents, and optionally formulated together into a pharmaceutical composition.

[0126] In another aspect, the present disclosure provides an administration device that can be used to administer the GIPR binding proteins, fusion proteins, multispecific antibodies, conjugates and / or pharmaceutical compositions described in this disclosure.

[0127] In another aspect, the present disclosure provides the following embodiments: 1. A glucose-dependent insulinotropic polypeptide receptor GIPR binding protein comprising at least one immunoglobulin single variable domain, wherein said at least one immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 within a VHH as set forth in SEQ ID NOs: 5 and / or 6. 2. The GIPR binding protein of embodiment 1, wherein the CDRs are defined by a definition system such as Kabat, AbM, Chothia, or IMGT. 3. A GIPR-binding protein according to embodiment 1, in which the CDR1, CDR2, and CDR3 in the VHH shown in SEQ ID NO: 5 are any one set selected from SEQ ID NOs: 74 to 76, 77 to 79, 80 to 82, and 83-85. 4. The GIPR-binding protein of embodiment 3, wherein the at least one immunoglobulin single variable domain comprises one or more of the amino acid sequences set forth in SEQ ID NO: 5, SEQ ID NOs: 8-14. 5. The GIPR-binding protein of embodiment 4, wherein the at least one immunoglobulin single variable domain comprises an amino acid sequence as set forth in one of SEQ ID NO: 5, SEQ ID NOs: 8-14. 6. A GIPR-binding protein according to embodiment 1, wherein the CDR1, CDR2, and CDR3 in the VHH shown in SEQ ID NO: 6 are any one set selected from SEQ ID NOs: 86 to 88, 89 to 91, 92 to 94, and 95 to 97. 7. The GIPR-binding protein of embodiment 6, wherein the at least one immunoglobulin single variable domain comprises one or more of the amino acid sequences set forth in SEQ ID NO: 6, SEQ ID NOs: 15-25. 8. The GIPR-binding protein of embodiment 7, wherein the at least one immunoglobulin single variable domain comprises an amino acid sequence as set forth in one of SEQ ID NO: 6, SEQ ID NOs: 15-25. 9. The GIPR-binding protein according to any one of embodiments 1 to 8, wherein the at least one immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 5, one of SEQ ID NOs: 8 to 14, and / or the amino acid sequence set forth in SEQ ID NO: 6, one of SEQ ID NOs: 15 to 25. 10. The GIPR-binding protein of any one of embodiments 1 to 9, further comprising an immunoglobulin Fc region, preferably a human immunoglobulin Fc region, more preferably the Fc region of human IgG1, IgG2, IgG3, or IgG4. 11. A GIPR binding protein described in embodiment 10, wherein the amino acid sequence of the immunoglobulin Fc region is set forth in SEQ ID NO: 110 or SEQ ID NO: 113. 12. The GIPR binding protein of embodiment 10 or 11, wherein the immunoglobulin Fc region is connected to the at least one immunoglobulin single variable domain directly or indirectly via a linker. 13. (a) specifically binds to human GIPR; (b) K binding to human GIPR D The value is 1 x 10 -7 Less than M, preferably 1 x 10 -8 Less than M, (c) blocking the interaction of GIP with GIPR; and (d) capable of reducing the body weight and / or blood glucose of a subject; 13. A GIPR binding protein according to any one of embodiments 1 to 12, having at least one of the following characteristics: 14. A fusion protein comprising a GIPR binding protein according to any one of embodiments 1 to 13. 15. A conjugate comprising a GIPR binding protein according to any one of embodiments 1 to 13 and / or a fusion protein according to embodiment 14. 16. A nucleic acid molecule encoding a GIPR-binding protein according to any one of embodiments 1 to 13 and / or a fusion protein according to embodiment 14. 17. An expression vector comprising the nucleic acid molecule of embodiment 15 operably linked to an expression control element. 18. A recombinant cell comprising the nucleic acid molecule of embodiment 16 and / or transformed with the expression vector of embodiment 17, and capable of expressing the GIPR-binding protein or a fusion protein comprising the GIPR-binding protein. 19. A pharmaceutical composition comprising a GIPR binding protein according to any one of embodiments 1 to 13, a fusion protein according to embodiment 14, and / or a conjugate according to embodiment 15, and a pharmaceutically acceptable carrier. 20. A reagent kit comprising a GIPR binding protein according to any one of embodiments 1 to 13, a fusion protein according to embodiment 14, a conjugate according to embodiment 15, and / or a pharmaceutical composition according to embodiment 19. 21. A method for determining the presence and / or content of a GIPR protein and / or inhibiting binding of a GIPR protein to its ligand, the method comprising providing a GIPR binding protein according to any one of embodiments 1 to 13, a fusion protein according to embodiment 14 and / or a conjugate according to embodiment 15. 22. A method for treating and / or preventing a metabolic disease and / or condition, comprising administering to a subject in need thereof an effective amount of a GIPR binding protein described in any one of embodiments 1 to 13, a fusion protein described in embodiment 14, a conjugate described in embodiment 15, or a pharmaceutical composition described in embodiment 18. 23. The method of embodiment 22, wherein the metabolic disease and / or condition is obesity, overweight and / or diabetes.

[0128] Without intending to be limited by any theory, the following examples are merely intended to illustrate the technical solutions of the presently disclosed invention, and are not intended to limit the scope of the presently disclosed invention. [Example]

[0129] Example 1 Screening for GIPR heavy chain single domain antibodies 1.1 Building the library Prior to immunization, 50 mL of alpaca arterial blood was collected using a vacuum blood collection tube, and the supernatant was collected as pre-immune serum. Healthy alpacas were selected, and an antigen containing human GIPR was collected and injected into multiple sites in the alpaca's neck muscle. The animals were immunized once every two weeks for a total of six times. At the end of the final immunization, 50 mL of alpaca arterial blood was collected using a vacuum blood collection tube, and the supernatant was collected as post-immune serum.

[0130] Lymphocytes were isolated using density gradient centrifugation, and total RNA was extracted using a QIAGEN RNA extraction reagent kit. All extracted RNA was reverse transcribed into cDNA using the Super-Script III FIRST STRAND SUPERMIX reagent kit according to the manufacturer's instructions, and nucleic acid fragments encoding the variable regions of the heavy chain antibodies were amplified by nested PCR.

[0131] The nucleic acid fragments of the target heavy chain single domain antibody were recovered and cloned into the phage display vector pComb3XSS using the restriction enzyme SfiI. The resulting fragments were then electrotransformed into E. coli electrocompetent cells TG1 to construct a phage display library of anti-GIPR immune single domain antibodies, which were then identified. The library volume size was 1.69 x 10 . 9 To determine the insertion rate of the library, 50 clones were randomly selected and sequenced. 50 clones contained the correct foreign fragment, resulting in an accuracy rate of 100%. Analysis and comparison of the DNA and amino acid sequences of the sequenced clones confirmed that all sequences were alpaca VHH sequences, and the diversity was estimated to be over 95%.

[0132] 1.2 Panning against GIPR heavy chain single domain antibodies The phage library obtained in Example 1.1 was panned, and bound phages were screened using the proteins GIPR-ECD-muFc (mouse Fc-fused human GIPR extracellular domain fragment), GIPR-ECD-chis (his-tag-fused human GIPR extracellular domain fragment), and GIPR-ECD-laFc (alpaca Fc-fused human GIPR extracellular domain fragment). At the same time, negative panning was performed using the muFc-containing fusion protein Control-1 (C1) and the laFc-containing fusion protein Control-2 (C2) to remove nonspecifically bound phages.

[0133] The positively binding phage obtained from the panning was infected with blank E. coli and plated. Colonies were then selected and inoculated into 2TY-AG (containing 10% glycerol) and incubated overnight at room temperature. The next day, 200 μL of 2TY-AG was added at a 1% inoculum volume and infected with helper phage M13KO7 (infectivity index 1:20) at 37°C and 250 rpm until an OD600 of approximately 0.5 was reached. The plates were incubated at 37°C for 15 min, followed by 45 min at 220 rpm. 800 μL of 2TY-AG was added to each well and incubated overnight at 30°C and 220 rpm. The plates were then centrifuged the next day, and the supernatants were collected and used for ELISA detection. Plates were coated with GIPR-ECD-Chis, GIPR-ECD-laFc, C1, and C2 overnight at 4°C. The resulting supernatants were added and incubated at room temperature for 2 hours. After washing, the secondary antibody Goat anti-HA tag HRP (purchased from Abcam) was added and allowed to react for 2 hours at room temperature. After washing, TMB color development solution was added and the absorbance values ​​at 450 nm and 650 nm were read. The final absorbance value was determined by subtracting the absorbance value at 650 nm from the absorbance value at 450 nm.

[0134] Of the 297 clones obtained by panning, 191 were positive clones with an OD value > 1.0, and some of these positive clones that specifically bound to GIPR-ECD-laFc are shown in Table 3. The positive clones with the above specific binding were sequenced to obtain the antibody sequences. [Table 3]

[0135] Example 2 Production of Fc fusion proteins of GIPR single domain antibodies using mammalian cells 2.1 Preparation of plasmids expressing GIPR single domain antibody-Fc fusion proteins Primers were designed to PCR amplify the GIPR single domain antibody VHH fragments, which were then fused to a DNA fragment encoding human IgG1-Fc (amino acid sequence of SEQ ID NO: 110), and cloned into a standard mammalian expression vector to obtain a recombinant plasmid for expressing the GIPR single domain antibody-Fc fusion protein in mammals. Different VHH fragments were amplified using universal primers. The universal primers were as follows: Upstream primer cccACCGGTCAGGTGCAGCTGCAGGAGTC (SEQ ID NO: 111) Downstream primer cccGGATCCTGAGGAGACGGTGACCTGG (SEQ ID NO: 112)

[0136] 2.2 Preparation of GIPR single domain antibody Fc fusion protein The plasmid vectors constructed in Section 2.1 were transfected into HEK293 cells for transient antibody expression. The recombinant expression plasmids were diluted in Freestyle 293 medium, and the PEI (Polyethylenimine) solution required for transformation was added. Each plasmid / PEI mixture was then added to a suspension of HEK293 cells and cultured at 37°C and 5% CO2. After 5-6 days of culture, the transiently expressed culture supernatants were collected and purified by Protein A affinity chromatography to obtain the target GIPR single domain antibody-Fc fusion proteins. Protein purity was then determined by SDS-PAGE. The expression levels of each protein are shown in Table 4, and after one-step purification, the SDS purity of each protein was greater than 95%. [Table 4]

[0137] Example 3 Identification of the function of GIPR single domain antibody-Fc fusion proteins 3.1 GIPR-binding ability of GIPR single-domain antibody-Fc fusion proteins 293T-GIPR cells (HEK293T cells expressing human GIPR) were plated on a cell culture plate, and GIPR single domain antibody-Fc fusion protein samples were added at two final concentrations: 100 μg / mL and 10 μg / mL. After incubation with an anti-human IgG-APC secondary antibody, the mean fluorescence intensity (MFI) was detected using a fluorescence-activated cell sorter. The results are shown in Table 5. [Table 5]

[0138] 3.2 Neutralizing activity of GIPR single domain antibody-Fc fusion proteins (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 were then added. After incubation, the cAMP was detected using a cAMP reagent kit and the absorbance at 450 nm / 550 nm was measured using a microplate reader. The results are shown in Table 6. [Table 6]

[0139] (2) GIP-C12H4-Fc4 and 25 μL of the GIPR single domain antibody-Fc sample were added to a 96-well plate, followed by 293T cells (293T-GIPRFL-GFP-PURO-5) stably expressing full-length human GIPR protein in the cell membrane. After incubation, cAMP was detected using a cAMP reagent kit, and the RLU values ​​were read and the neutralization activity (%) was calculated. The results are shown in Table 7. [Table 7]

[0140] 3.3 Affinity detection of GIPR single domain antibody-Fc fusion proteins Biolayer interferometry (BLI) was used to detect the binding kinetics of the GIPR single domain antibody-Fc fusion protein to GIPR-ECD-chis. The GIPR single domain antibody-Fc fusion protein was directly immobilized on an AHC sensor. GIPR-ECD-chis was then diluted to seven concentrations and allowed to bind to the immobilized GIPR single domain antibody-Fc fusion protein. The equilibrium dissociation constant (KD), binding rate (K), and binding rate (K) were calculated using Octet K2 data analysis software 9.0. a ) and dissociation rate (K dis ) was calculated. The results are shown in Figure 8 and Table 9. [Table 8] [Table 9]

[0141] 3.4 Detection of blocking activity of GIPR single domain antibody-Fc fusion proteins After coating with 5 μg / mL mouse GIPR-Chis protein and blocking with BSA, gradient dilutions of AMG-GIPR-mab2 and iGI72-Ld-Fc were added and incubated thoroughly. The dilution contained 1.2 μg / mL GIP-C12H4-Fc4. Mouse Anti-Human IgG4 pFc' [HP6023] (HRP) (catalog no. ab99817) diluted 1:2000 was then added, followed by TMB color development and detection using a microplate reader. The results are shown in Figure 1.

[0142] The positive control AMG-GIPR-mab2 was synthesized by gene synthesis using the sequence in the literature (MAbs. 2020 Jan-Dec; 12(1): 1710047), and then produced by transient expression in 293 cells according to the method described above.

[0143] 3.5 Detection of non-specific binding of GIPR single domain antibody-Fc fusion proteins to null cells CHOK1 null cells were resuspended in 3% BSA-PBS and treated with GIPR single domain antibody-Fc fusion proteins at final concentrations of 5 μg / mL and 50 μg / mL, along with negative and blank controls. After washing, the secondary antibody APC anti-human IgG Fc was added. After washing, the cells were resuspended in PBS-BSA buffer and detected by flow cytometry. The results, as shown in Table 10, show that iGI-72-Ld-Fc does not nonspecifically bind to iGI-1198-Ld-Fc, while iGI-1225-Ld-Fc nonspecifically binds to iGI-1225NA-Ld-Fc. [Table 10]

[0144] 3.6 In vivo efficacy studies of GIPR single domain antibody-Fc fusion proteins Sixteen DIO mice, aged 16 weeks, were fasted overnight, blood glucose and body weight were measured, and the mice were randomly divided into four groups. The day of group administration was designated D0. They received PBS, 17 nmol / kg Dulaglutide, 375 nmol / kg iGI-72-Ld-Fc, or 17 nmol / kg Dulaglutide + iGI-72-Ld-Fc 17 + 375 nmol / kg twice weekly for a total of 10 doses. After administration, general clinical observations were performed once weekly, and animals were weighed twice weekly. Fasting blood glucose and fasting insulin levels were measured once weekly. As shown in Figures 2 to 5, the results showed that the iGI-72-Ld-Fc treatment group significantly reduced the body weight of DIO mice and simultaneously improved 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, demonstrating a synergistic effect.

[0145] Example 4 Humanization of GIPR single domain antibodies The humanization methods used were protein surface amino acid resurfacing and VHH humanization CDR grafting to a universal framework.

[0146] First, we obtained the universal humanized VHH framework hNbBcII10FGLA (PDB number 3EAK), designed by Cecile Vincke et al. based on sequence homology. The design of this framework was based on the nanoantibody NbBcII10 antibody (PDB number 3DWT). Modeller9 was used to model the framework and calculate the relative solvent accessibility of amino acids in the framework based on the protein's three-dimensional structure.

[0147] The specific steps of the VHH humanization universal framework grafting method are to obtain a highly homologous human antibody sequence using IMGT and then humanize the target sequence using the universal humanized VHH framework hNbBcII10FGLA (PDB number 3EAK). Using the highly homologous sequence framework as a framework template, the CDRs were replaced with the CDR regions of the target antibody strain. Next, based on modeling, non-surface amino acids in the framework were backmutated to complete the humanization of the target antibody.

[0148] The antibody strains iGI-72 and iGI-1198 were humanized to obtain humanized variants huGI of 8 and 11 antibody strains, respectively.

[0149] Example 5 Production of humanized single-domain antibody Fc fusion proteins 5.1 Preparation of humanized single domain antibody Fc fusion plasmids The humanized sequence of Example 4 was gene synthesized, fused with a DNA fragment encoding human IgG1-Fc, 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.

[0150] 5.2 Preparation of Humanized Single Domain Antibody Fc Fusion Proteins The vectors constructed in Section 5.1 were transfected into HEK293 cells for transient antibody expression. The recombinant expression plasmids were diluted in Freestyle 293 medium, and the PEI (Polyethylenimine) solution required for transformation was added. Each plasmid / PEI mixture was then added to the HEK293 cell suspension and cultured at 37°C, 5% CO2, and 130 rpm. After 4 hours, EXCELL 293 medium supplemented with 2 mM glutamine was added and cultured at 130 rpm. After 24 hours, 3.8 mM VPA was added, and after 72 hours, 4 g / L glucose was added. After 5–6 days of culture, the transient expression culture supernatants were collected and purified by Protein A affinity chromatography to obtain the target huGI single-domain antibody Fc fusion proteins. The purity of the proteins was examined by SDS-PAGE. The expression levels of each protein are shown in Table 11, and the SDS purity of each protein after one-step purification was greater than 95%. [Table 11]

[0151] Example 6 Identification of the functions of humanized single-domain antibody Fc fusion proteins 6.1 GIPR Binding Ability of Humanized GIPR Single Domain Antibody-Fc Fusion Proteins 293T-GIPR cells were plated on cell culture plates and either humanized GIPR single domain antibody-Fc fusion proteins or Maridebart samples were added at final concentrations of 0.00128 nM to 100 nM. After incubation, the anti-human secondary antibody SULFO-TAG (MSD, catalog number R32AJ-1) was added and the mean fluorescence intensity (MFI) was measured using MSD.

[0152] Maridebart is an anti-GIPR antibody, produced by autocloning with reference to the sequence disclosed in WHO Drug Information, Volume 36. Number 4. 2022. Proposed INN: List 128; a similar sequence was also found in construct 2G10 LC1.006 of patent application WO2017112824.

[0153] The results, as shown in Figure 10, showed that the binding ability of the humanized GIPR single domain antibody-Fc fusion protein to GIPR was slightly better than that of Maridebart.

[0154] 6.2 GIPR Affinity of Humanized GIPR Single Domain Antibody-Fc Fusion Proteins Referring to the experimental step 3.3, the equilibrium dissociation constant (KD) and binding rate (K a ) and dissociation rate (K dis ) was measured and calculated, and the results, as shown in Tables 12 to 14, showed that the affinity of the humanized fusion protein for GIPR was equivalent to that before humanization. [Table 12] [Table 13] [Table 14]

[0155] Using the same experimental steps, we determined the equilibrium dissociation constant (KD), binding rate (K a ) and dissociation rate (K dis ) were detected and compared with Maridebart. The results, as shown in Table 15, show that the affinity of the humanized single domain antibody Fc fusion protein was superior to that of the antibody Maridebart. [Table 15]

[0156] 6.3 Neutralizing Activity of Humanized GIPR Single Domain Antibody-Fc Fusion Proteins Referring to the experimental steps in Example 3.2, the neutralizing activity of the humanized GIPR single domain antibody-Fc fusion proteins was measured. As shown in Figure 6, the neutralizing activity of all humanized GIPR single domain antibody-Fc fusion proteins was superior to that of the non-humanized molecule iGI-1198-Fc fusion protein. GIP-C12H4-Fc4 and 25 μL of humanized GIPR single domain antibody-Fc or Maridebart sample were added to a 96-well plate, followed by 293T-GIPR cells. After incubation, cAMP was detected using a cAMP reagent kit and absorbance at 450 nm / 550 nm was measured using a microplate reader. The inhibition rate (%) was calculated. As shown in Figure 11, the neutralizing activity of the humanized GIPR single domain antibody-Fc fusion protein was superior to that of Maridebart.

[0157] 6.4 In vivo efficacy studies of humanized GIPR single domain antibody-Fc fusion proteins (1) DA-GIP-stimulated C57BL / 6 mice experiment Nine C57BL / 6 mice were fasted overnight, and their blood glucose levels were measured. They were then randomly divided into three groups. After 24 h of normal feeding, they were administered huGI-72v5-Ld-Fc (203 nmol / kg) according to the experimental design. 12 h after administration, the mice were fasted overnight. 48 h after administration, each mouse received DA-GIP (50 nmol / kg) intravenously. Immediately after DA-GIP administration, a 2 g / kg oral glucose load was administered. Blood glucose and insulin levels were measured before and 5, 10, 15, 30, and 60 min after the glucose load. As shown in Figures 7 and 8, DA-GIP significantly reduced OGTT blood glucose compared to the PBS group. huGI-72v5-Ld-Fc blocked the DA-GIP-induced blood glucose reduction, demonstrating that huGI-72v5-Ld-Fc exhibited significant blocking activity in vivo in mice.

[0158] (2) Multiple administration of ob mice Twenty-four ob / ob mice were randomly divided into six groups based on blood glucose and body weight, and treatment began between weeks 6 and 8. The day of group administration was designated D0, and treatment was administered once a week for a total of six doses. Body weights were measured before and after administration on D0, 7, 16, 20, 28, and 35. As shown in Figure 9, 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, and the combined use of dulaglutide and huGI-72v5-Ld-Fc further enhanced the weight loss effect.

[0159] The foregoing detailed description has been provided for purposes of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications to the exemplary embodiments of the present disclosure will now be apparent to those skilled in the art and are intended to remain within the scope of the appended claims and their equivalents.

[0160] Sequence information iGI-9 (SEQ ID NO: 1): QVQLVESGGGLVQPGGSLRLSCETSKILFSSAYTMGWYRQAPGKQRELVATITTDYITNYVDSVKGRFTISRDNAKNTVYLQMNTLKLEDTAVYYCNARWGRDYWGQGTQVTVSS [Table 16]

[0161] iGI-27 (SEQ ID NO: 2): QVQLVESGGALVQTGGSLRLFCAASGDTICITNMNWYRQAPGKGREFVAAITRSGRTLYADYVKGRFTISRDNARNTMSLQMSMTSEDTAVYYCNADQNQTICAAEPSAWGRGTQVTVSS [Table 17]

[0162] iGI-44 (SEQ ID NO: 3): QVQLVESGGGLVQAGGSLKLSCAASGRTFSSKAMGWFRQAPGKEREFVAAINWSGDRTYHVNSIKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTADRRDRATIPFQWHYWGQGTQVTVSS [Table 18]

[0163] iGI-51 (SEQ ID NO: 4): QVQLVESGGKLVQAGGSLRLDCVASGRTFSYYAIGWYRQAPGKEREFVAAIRASGGSTYYADSVKGRFTASRDNAKNTGYLQMNSLKPEDTAVYFCYAATIVPITPSTHGYWGQGTQVTVSS [Table 19]

[0164] iGI-72 (SEQ ID NO: 5): QVQLVESGGGLVQPGGSLRLSCAASRYTLDYYAIGWFRQAPGKEREGVSCINSKDGSTYYADSVKGRFTISKDNAKNTVYLQMNSLKPEDTAVYYCAAGYDPWSGIPPVQAMCVMGYDYWGQGTQVTVSS [Table 20]

[0165] iGI-1198 (SEQ ID NO: 6): QVQLQESGGGLVQPGGSLRLSCVASGSGFSIVAMGWYRQTPGKQRELVAAITSGGNTNYADSVKGRFTISRDNAKNTIYLQMNSLKPEDTAVYYCNADPGVVAGPYAGMDFWGKGTQVTVSS [Table 21]

[0166] iGI-1225NA (SEQ ID NO: 7): QVQLQESGGSLRLSCAASGPIFSFTTMAWYRQVPGKQRELVASITTGGSTAYTDSVKGRFTISRDNAKSTLYLQMNNLKPEDTAVYYCNTGPRNVWAAAWGQGTQVTVSS [Table 22]

[0167] huGI-72v1 (SEQ ID NO: 8): EVQLVESGGGLVQPGGSLRLSCAASRYTLDYYAIGWFRQAPGKEREGVSCINSKDGSTYYADSVKGRFTISKDNSKNTVYLQMNSLRAEDTAVYYCAAGYDPWSGIPPVQAMCVMGYDYWGQGTLVTVSS

[0168] huGI-72v2 (SEQ ID NO: 9): EVQLVESGGGLVQPGGSLRLSCAASRYTLDYYAIGWFRQAPGKEREGVSCINSKDGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGYDPWSGIPPVQAMCVMGYDYWGQGTLVTVSS

[0169] huGI-72v3 (SEQ ID NO: 10): EVQLVESGGGLVQPGGSLRLSCAASRYTLDYYAIGWFRQAPGKGLEGVSCINSKDGSTYYADSVKGRFTISKDNSKNTVYLQMNSLRAEDTAVYYCAAGYDPWSGIPPVQAMCVMGYDYWGQGTLVTVSS

[0170] huGI-72v4 (SEQ ID NO: 11): EVQLVESGGGLVQPGGSLRLSCAASRYTLDYYAIGWFRQAPGKGLEGVSCINSKDGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGYDPWSGIPPVQAMCVMGYDYWGQGTLVTVSS

[0171] huGI-72v5 (SEQ ID NO: 12): QVQLVESGGGLVQPGGSLRLSCSASRYTLDYYAIGWFRQAPGKEREGVSCINSKDGSTYYADSVKGRFTISKDNSKNTVYLQMNSLRAEDTAVYYCAAGYDPWSGIPPVQAMCVMGYDYWGQGTLVTVSS

[0172] huGI-72v6 (SEQ ID NO: 13): QVQLVESGGGLVQPGGSLRLSCSASRYTLDYYAIGWFRQAPGKEREGVSCINSKDGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGYDPWSGIPPVQAMCVMGYDYWGQGTLVTVSS

[0173] huGI-72v7 (SEQ ID NO: 14): QVQLVESGGGLVQPGGSLRLSCSASRYTLDYYAIGWFRQAPGKGLEGVSCINSKDGSTYYADSVKGRFTISKDNSKNTVYLQMNSLRAEDTAVYYCAAGYDPWSGIPPVQAMCVMGYDYWGQGTLVTVSS

[0174] huGI-1198n1 (SEQ ID NO: 15): QVQLVESGGGLVQPGGSLRLSCSASGSGFSIVAMGWVRQAPGKGLELVAAITSGGNTNYADSVKGRFTISRDNSKNTIYLQMNSLRAEDTAVYYCNADPGVVAGPYAGMDFWGKGTTVTVSS

[0175] huGI-1198n2 (SEQ ID NO: 16): QVQLQESGGGLVQPGGSLRLSCSASGSGFSIVAMGWVRQTPGKGLELVAAITSGGNTNYADSVKGRFTISRDNSKNTIYLQMNSLRAEDTAVYYCNADPGVVAGPYAGMDFWGKGTTVTVSS

[0176] huGI-1198n3 (SEQ ID NO: 17): QVQLQESGGGLVQPGGSLRLSCSASGSGFSIVAMGWVRQTPGKGLELVAAITSGGNTNYADSVKGRFTISRDNAKNTIYLQMNSLRAEDTAVYYCNADPGVVAGPYAGMDFWGKGTTVTVSS

[0177] huGI-1198n4 (SEQ ID NO: 18): QVQLVESGGGLVQPGGSLRLSCSASGSGFSIVAMGWVRQAPGKQRELVAAITSGGNTNYADSVKGRFTISRDNSKNTIYLQMNSLRAEDTAVYYCNADPGVVAGPYAGMDFWGKGTTVTVSS

[0178] huGI-1198n5 (SEQ ID NO: 19): QVQLQESGGGLVQPGGSLRLSCSASGSGFSIVAMGWVRQTPGKQRELVAAITSGGNTNYADSVKGRFTISRDNAKNTIYLQMNSLRAEDTAVYYCNADPGVVAGPYAGMDFWGKGTTVTVSS

[0179] huGI-1198n6 (SEQ ID NO: 20): QVQLQESGGGLVQPGGSLRLSCSASGSGFSIVAMGWVRQTPGKQRELVAAITSGGNTNYADSVKGRFTISRDNAKNTIYLQMNSLKPEDTAVYYCNADPGVVAGPYAGMDFWGKGTTVTVSS

[0180] huGI-1198n7 (SEQ ID NO: 21): QVQLVESGGGLVQPGGSLRLSCSASGSGFSIVAMGWYRQTPGKGLELVAAITSGGNTNYADSVKGRFTISRDNSKNTIYLQMNSLRAEDTAVYYCNADPGVVAGPYAGMDFWGKGTTVTVSS

[0181] huGI-1198n8 (SEQ ID NO: 22): QVQLQESGGGLVQPGGSLRLSCSASGSGFSIVAMGWYRQTPGKGLELVAAITSGGNTNYADSVKGRFTISRDNSKNTIYLQMNSLRAEDTAVYYCNADPGVVAGPYAGMDFWGKGTTVTVSS

[0182] huGI-1198n9 (SEQ ID NO: 23): QVQLVESGGGLVQPGGSLRLSCSASGSGFSIVAMGWYRQTPGKQRELVAAITSGGNTNYADSVKGRFTISRDNSKNTIYLQMNSLRAEDTAVYYCNADPGVVAGPYAGMDFWGKGTTVTVSS

[0183] huGI-1198n10 (SEQ ID NO: 24): QVQLQESGGGLVQPGGSLRLSCSASGSGFSIVAMGWYRQTPGKQRELVAAITSGGNTNYADSVKGRFTISRDNAKNTIYLQMNSLRAEDTAVYYCNADPGVVAGPYAGMDFWGKGTTVTVSS

[0184] huGI-1198n11 (SEQ ID NO: 25): QVQLQESGGGLVQPGGSLRLSCSASGSGFSIVAMGWYRQTPGKQRELVAAITSGGNTNYADSVKGRFTISRDNAKNTIYLQMNSLKPEDTAVYYCNADPGVVAGPYAGMDFWGKGTTVTVSS

[0185] IgG1 Fc (SEQ ID NO: 110) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0186] IgG1 Fc(C220S) (SEQ ID NO: 113) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. A glucose-dependent insulinotropic polypeptide receptor (GIPR) binding protein comprising at least one immunoglobulin single variable domain, said at least one immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 within a VHH as set forth in SEQ ID NO: 5 and / or SEQ ID NO: 6, Glucose-dependent insulinotropic polypeptide receptor (GIPR) binding protein.

2. The CDR1, CDR2 and CDR3 are defined by a definition system such as Kabat, AbM, Chothia or IMGT.

2. The GIPR binding protein of claim 1.

3. the immunoglobulin single variable domain is camelid, humanized, or chimeric; 3. The GIPR binding protein of claim 1 or 2.

4. CDR1, CDR2, and CDR3 in the VHH shown in SEQ ID NO: 5 are any one set selected from SEQ ID NOs: 74 to 76, 77 to 79, 80 to 82, and 83 to 85. A GIPR binding protein described in any one of claims 1 to 3.

5. the at least one immunoglobulin single variable domain comprises one or more of the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NOs: 8 to 14; The GIPR binding protein of claim 4.

6. the at least one immunoglobulin single variable domain comprises an amino acid sequence set forth in one of SEQ ID NO: 5 and SEQ ID NOs: 8 to 14; The GIPR binding protein of claim 5.

7. CDR1, CDR2, and CDR3 in the VHH shown in SEQ ID NO: 6 are any one set selected from SEQ ID NOs: 86 to 88, 89 to 91, 92 to 94, and 95 to 97. A GIPR binding protein described in any one of claims 1 to 3.

8. the at least one immunoglobulin single variable domain comprises one or more of the amino acid sequences set forth in SEQ ID NO: 6 and SEQ ID NOs: 15-25; The GIPR binding protein of claim 7.

9. the at least one immunoglobulin single variable domain comprises an amino acid sequence set forth in one of SEQ ID NO: 6 and SEQ ID NOs: 15 to 25; 9. The GIPR binding protein of claim 8.

10. the at least one immunoglobulin single variable domain comprises one or more of the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NOs:8-14, and one or more of the amino acid sequences set forth in SEQ ID NO:6 and SEQ ID NOs:15-25; 10. A GIPR binding protein according to any one of claims 1 to 9.

11. the at least one immunoglobulin single variable domain comprises an amino acid sequence set forth in one of SEQ ID NO: 5 and SEQ ID NO: 8 to 14, and an amino acid sequence set forth in one of SEQ ID NO: 6 and SEQ ID NO: 15 to 25; 11. The GIPR binding protein of claim 10.

12. the GIPR-binding protein comprises one of the immunoglobulin single variable domains, A GIPR binding protein described in any one of claims 1 to 11.

13. the GIPR-binding protein comprises a plurality, e.g., two, three, four, or five, of said immunoglobulin single variable domains; A GIPR binding protein described in any one of claims 1 to 11.

14. further comprising an immunoglobulin Fc region, preferably a human immunoglobulin Fc region, more preferably a human IgG1, IgG2, IgG3 or IgG4 Fc region; A GIPR binding protein described in any one of claims 1 to 13.

15. The amino acid sequence of the Fc region of the immunoglobulin is set forth in SEQ ID NO: 110 or SEQ ID NO:

113.

15. The GIPR binding protein of claim 14.

16. the Fc region of said immunoglobulin is connected to said at least one immunoglobulin single variable domain either directly or indirectly via a linker; 16. The GIPR binding protein of claim 14 or 15.

17. (a) specifically binds to human GIPR; (b) KD value of binding to human GIPR is 1 × 10 -7 Less than M, preferably 1 x 10 -8 Less than M, (c) blocking the interaction of GIP with GIPR; and (d) capable of reducing the body weight and / or blood glucose of a subject; having at least one of the following characteristics:

17. A GIPR binding protein according to any one of claims 1 to 16.

18. The GIPR binding protein according to any one of claims 1 to 17, Fusion proteins.

19. A GIPR-binding protein according to any one of claims 1 to 17 and / or a fusion protein according to claim 18, and one or more additional antigen-binding regions that bind to a different antigen or a different epitope of the same antigen than the GIPR-binding protein and / or the fusion protein. Multispecific antibodies.

20. A conjugate comprising a GIPR-binding protein according to any one of claims 1 to 17 and / or a fusion protein according to claim 18 and / or a multispecific antibody according to claim 19, wherein the conjugate further comprises a molecule that conjugates to the GIPR-binding protein and / or the fusion protein and / or the multispecific antibody. Conjugates.

21. Encoding a GIPR-binding protein according to any one of claims 1 to 17 and / or a fusion protein according to claim 18 and / or a multispecific antibody according to claim 19. Nucleic acid molecule.

22. 21. The nucleic acid molecule of claim 20 operably linked to an expression control element. Expression vector.

23. 23. A method for the production of a GIPR-binding protein comprising the steps of: (a) transforming a vector comprising the nucleic acid molecule of claim 21 and / or the expression vector of claim 22, and capable of expressing said GIPR-binding protein and / or said fusion protein and / or said multispecific antibody; Recombinant cells.

24. 20. A method for the preparation of a GIPR-binding protein according to claim 1, comprising administering to a subject a therapeutically effective amount of a GIPR-binding protein comprising administering to a subject a therapeutically effective amount of a GIPR-binding protein according to claim 1, or a fusion protein according to claim 18, or a multispecific antibody according to claim 19, or a conjugate according to claim 20, or a nucleic acid molecule according to claim 21, or an expression vector according to claim 22, or a recombinant cell according to claim 23, and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

25. 24. A pharmaceutical composition comprising a GIPR-binding protein according to any one of claims 1 to 17, a fusion protein according to claim 18, a multispecific antibody according to claim 19, a conjugate according to claim 20 and / or a pharmaceutical composition according to claim 24. Reagent kit.

26. 20. A method for determining the presence and / or content of a GIPR protein, comprising providing a GIPR binding protein according to any one of claims 1 to 17, a fusion protein according to claim 18, a multispecific antibody according to claim 19 and / or a conjugate according to claim 20. method.

27. 20. A method for inhibiting binding of a GIPR protein to its ligand, comprising providing a GIPR binding protein according to any one of claims 1 to 17, a fusion protein according to claim 18, a multispecific antibody according to claim 19, and / or a conjugate according to claim 20, wherein preferably said ligand is GIP. method.

28. 20. A method for treating and / or preventing metabolic diseases and / or conditions, comprising administering to a subject in need thereof an effective amount of a GIPR binding protein according to any one of claims 1 to 17, a fusion protein according to claim 18, a multispecific antibody according to claim 19, a conjugate according to claim 20, and / or a pharmaceutical composition according to claim 24. method.

29. the metabolic disease and / or condition is obesity, overweight and / or diabetes; 29. The method of claim 28.

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