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WO2024094076A1 - 一种gipr结合蛋白及其应用 - Google Patents

一种gipr结合蛋白及其应用 Download PDF

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Publication number
WO2024094076A1
WO2024094076A1 PCT/CN2023/129127 CN2023129127W WO2024094076A1 WO 2024094076 A1 WO2024094076 A1 WO 2024094076A1 CN 2023129127 W CN2023129127 W CN 2023129127W WO 2024094076 A1 WO2024094076 A1 WO 2024094076A1
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Prior art keywords
gipr
seq
binding protein
fusion protein
present disclosure
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English (en)
French (fr)
Inventor
徐霆
金宇灏
朱旭国
逄敏洁
恽丽红
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Suzhou Alphamab Co Ltd
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Suzhou Alphamab Co Ltd
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Priority to JP2025526318A priority Critical patent/JP2025537215A/ja
Priority to EP23885002.8A priority patent/EP4613768A1/en
Publication of WO2024094076A1 publication Critical patent/WO2024094076A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present disclosure relates to the field of biomedicine, and discloses single domain antibodies and derived proteins thereof against glucose-dependent insulinotropic polypeptide receptor (GIPR). Specifically, the present disclosure discloses a binding protein against glucose-dependent insulinotropic polypeptide receptor and its use.
  • GIPR glucose-dependent insulinotropic polypeptide receptor
  • Glucose-dependent insulinotropic polypeptide is an intestinal incretin hormone that has the ability to enhance glucose-stimulated insulin secretion.
  • GIP is secreted by K cells located in the proximal small intestine. Dietary carbohydrates and fats are effective stimulators of GIP secretion in humans. Studies have shown that rodents fed a high-fat diet have increased GIP secretion from K cells. Similarly, acute high-fat dietary intake in humans also leads to a 42% increase in GIP concentrations, followed by a significant increase in body weight, indicating a positive correlation between exposure to a high-fat diet and GIP concentrations. Therefore, a high-fat diet and increased systemic concentrations of GIP may underlie differences in obesity in human subjects.
  • GIPR glucose-dependent insulinotropic polypeptide receptor
  • BMI body mass index
  • the present invention uses phage display technology to screen and obtain anti-GIPR single domain antibodies (VHH) and their derivative proteins.
  • the anti-GIPR single domain antibodies (VHH) and their derivative proteins disclosed in the present invention have significant high specificity and high affinity effects.
  • the present disclosure provides a glucose-dependent insulinotropic polypeptide receptor (GIPR) binding protein, wherein the GIPR binding protein comprises at least one immunoglobulin single variable domain, wherein the at least one immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:5 and/or SEQ ID NO:6.
  • GIPR glucose-dependent insulinotropic polypeptide receptor
  • the CDR1, CDR2, and CDR3 are defined according to the following definition systems: Kabat, AbM, Chothia, or IMGT.
  • the immunoglobulin single variable domain is camelid, humanized, or chimeric.
  • the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:5 are selected from any one of the following groups: SEQ ID NO:74-76, SEQ ID NO:77-79, SEQ ID NO:80-82 and SEQ ID NO:83-85.
  • the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 5 are selected from any one of the following groups: SEQ ID NO: 74-76, SEQ ID NO: 77-79, SEQ ID NO: 80-82 and SEQ ID NO: 83-85.
  • 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 NO: 8-14. In some embodiments, the at least one immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO: 5 and SEQ ID NO: 8-14.
  • the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:6 are selected from any one of the following groups: SEQ ID NO:86-88, SEQ ID NO:89-91, SEQ ID NO:92-94 and SEQ ID NO:95-97.
  • 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 NO:15-25. 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 NO:15-25.
  • 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 NO: 8-14, and one or more of the amino acid sequences shown in SEQ ID NO: 6 and SEQ ID NO: 15-25. 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 NO: 8-14, and one or more of the amino acid sequences shown in SEQ ID NO: 6 and SEQ ID NO: 15-25.
  • the GIPR binding protein comprises 1 of the immunoglobulin single variable domains. In some embodiments, the GIPR binding protein comprises a plurality, such as 2, 3, 4 or 5, of the immunoglobulin single variable domains.
  • the GIPR binding protein further comprises an immunoglobulin Fc region, preferably an Fc region of a human immunoglobulin, more preferably an Fc region of a human IgG1, IgG2, IgG3 or IgG4.
  • the Fc region of the immunoglobulin comprises an amino acid sequence as shown in SEQ ID NO: 110 or SEQ ID NO: 113.
  • the amino acid sequence of the Fc region of the immunoglobulin is as shown in SEQ ID NO: 110 or SEQ ID NO: 113.
  • the Fc region of the immunoglobulin is directly linked to the at least one immunoglobulin single variable domain or indirectly linked via a linker.
  • the GIPR binding protein has at least one of the following characteristics:
  • the present disclosure provides a fusion protein comprising the GIPR binding protein described in the first aspect of the present disclosure.
  • the present disclosure provides a multispecific antibody comprising the GIPR binding protein described in the first aspect of the present disclosure and/or the fusion protein described in the second aspect of the present disclosure, and one or more additional antigen binding regions, wherein the one or more additional antigen binding regions bind to different antigens or different epitopes of the same antigen with the GIPR binding protein and/or the fusion protein.
  • the present disclosure provides a conjugate comprising the GIPR binding protein described in the first aspect of the present disclosure and/or the fusion protein described in the second aspect of the present disclosure and/or the multispecific antibody described in the third aspect of the present disclosure, and the conjugate further comprises a molecule conjugated to the GIPR binding protein and/or the fusion protein and/or the multispecific antibody.
  • the present disclosure provides a nucleic acid molecule encoding the GIPR binding protein of the first aspect of the present disclosure and/or the fusion protein of the second aspect of the present disclosure and/or the multispecific antibody of the third aspect of the present disclosure.
  • 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 regulatory element.
  • the present disclosure provides a recombinant cell, which comprises the nucleic acid molecule described in the fifth aspect of the present disclosure and/or is transformed with the expression vector described in the sixth aspect of the present disclosure, and is capable of expressing the GIPR binding protein and/or the fusion protein and/or the multispecific antibody.
  • the present disclosure provides a pharmaceutical composition comprising the GIPR binding protein described in the first aspect of the present disclosure and/or the fusion protein described in the second aspect of the present disclosure and/or the multispecific antibody described in the third aspect of the present disclosure and/or the conjugate described in the fourth aspect of the present disclosure and/or the nucleic acid molecule described in the fifth aspect of the present disclosure and/or the expression vector described in the sixth aspect of the present disclosure and/or the recombinant cell described in the seventh aspect of the present disclosure, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a kit comprising the GIPR binding protein described in the first aspect of the present disclosure, the fusion protein described in the second aspect of the present disclosure, the multispecific antibody described in the third aspect of the present disclosure, the conjugate described in the fourth aspect of the present disclosure and/or the pharmaceutical composition described in the eighth aspect of the present disclosure.
  • the present disclosure provides a method for determining the presence and/or content of a GIPR protein, comprising providing the GIPR binding protein described in the first aspect of the present disclosure, the fusion protein described in the second aspect of the present disclosure, the multispecific antibody described in the third aspect of the present disclosure, and/or the conjugate described in the fourth aspect of the present disclosure.
  • the present disclosure provides a method for inhibiting the binding of a GIPR protein to its ligand, comprising providing the GIPR binding protein described in the first aspect of the present disclosure, the fusion protein described in the second aspect of the present disclosure, the multispecific antibody described in the third aspect of the present disclosure, and/or the conjugate described in the fourth aspect of the present disclosure.
  • the The ligand is GIP.
  • the present disclosure provides a method for treating and/or preventing metabolic diseases and/or symptoms, comprising administering to a subject in need thereof an effective amount of the GIPR binding protein of the first aspect of the present disclosure, the fusion protein of the second aspect of the present disclosure, the multispecific antibody of the third aspect of the present disclosure, the conjugate of the fourth aspect of the present disclosure, and/or the pharmaceutical composition of the eighth aspect of the present disclosure.
  • the metabolic disease and/or symptom is obesity, overweight and/or diabetes.
  • the present disclosure provides the use of the GIPR binding protein described in the first aspect of the present disclosure, the fusion protein described in the second aspect of the present disclosure, the multispecific antibody described in the third aspect of the present disclosure, the conjugate described in the fourth aspect of the present disclosure, and/or the pharmaceutical composition described in the eighth aspect of the present disclosure in the preparation of a drug for treating and/or preventing metabolic diseases and/or symptoms.
  • the metabolic diseases and/or symptoms are obesity, overweight and/or diabetes.
  • the present disclosure provides the GIPR binding protein described in the first aspect of the present disclosure, the fusion protein described in the second aspect of the present disclosure, the multispecific antibody described in the third aspect of the present disclosure, the conjugate described in the fourth aspect of the present disclosure, and/or the pharmaceutical composition described in the eighth aspect of the present disclosure for use in treating and/or preventing metabolic diseases and/or symptoms.
  • the metabolic diseases and/or symptoms are obesity, overweight and/or diabetes.
  • FIG1 shows the blocking activity of GIPR single domain antibody-Fc fusion protein on GIPR, wherein ⁇ represents iGI-72-Ld-Fc and ⁇ represents the positive control AMG-GIPR-mab2.
  • FIG2 shows the effects of multiple administrations of GIPR single domain antibody-Fc fusion protein on the body weight of DIO mice.
  • FIG3 shows the effects of multiple administrations of GIPR single domain antibody-Fc fusion protein on fasting blood glucose in DIO mice.
  • FIG4 shows the effect of multiple administrations of GIPR single domain antibody-Fc fusion protein on insulin secretion in DIO mice.
  • FIG5 shows the effect of multiple administrations of GIPR single domain antibody-Fc fusion protein on the insulin resistance index of DIO mice.
  • FIG6 shows the neutralizing activity of humanized GIPR single domain antibody-Fc fusion protein.
  • FIG. 7 shows the effect of humanized GIPR single domain antibody-Fc fusion protein on blood glucose in C57BL/6 mice stimulated by DA-GIP.
  • FIG8 shows the effect of humanized GIPR single domain antibody-Fc fusion protein on DA-GIP-stimulated insulin secretion in C57BL/6 mice.
  • FIG. 9 shows the effects of multiple administrations of humanized GIPR single domain antibody-Fc fusion protein on the body weight of ob mice.
  • FIG. 10 shows the binding ability of humanized GIPR single domain antibody-Fc fusion protein and Maridebart to GIPR.
  • FIG. 11 shows the neutralization ability of humanized GIPR single domain antibody-Fc fusion protein and Maridebart against GIPR.
  • antibody or “immunoglobulin” used interchangeably, whether referring to heavy chain antibodies or conventional 4-chain antibodies herein, are used as general terms to include full-length antibodies, their individual chains, and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments, such as VHH domains or VH/VL domains, respectively).
  • sequence used herein (e.g., in terms of “immunoglobulin sequence”, “antibody sequence”, “single variable domain sequence”, “VHH sequence” or “protein sequence”, etc.) is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.
  • domain refers to a folded protein structure that is able to maintain its tertiary structure independently of the rest of the protein.
  • a domain is responsible for a single functional property of a protein and in many cases can be added, removed or transferred to other proteins without loss of function of the rest of the protein and/or the domain.
  • immunoglobulin domain refers to a globular region of an antibody chain (e.g., a chain of a conventional 4-chain antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a globular region.
  • An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristic of an antibody molecule.
  • immunoglobulin variable domain refers to an immunoglobulin domain that is essentially composed of four "framework regions” referred to in the art and hereinafter as “framework region 1" or “FR1”, “framework region 2" or “FR2”, “framework region 3” or “FR3”, and “framework region 4" or “FR4", respectively, wherein the framework regions are separated by three “complementarity determining regions” or “CDRs” referred to in the art and hereinafter as “complementarity determining region 1" or “CDR1", “complementarity determining region 2" or “CDR2", and “complementarity determining region 3" or “CDR3".
  • an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer specificity to antibodies for antigens by having antigen binding sites.
  • immunoglobulin single variable domain refers to an immunoglobulin variable domain that is capable of specifically binding to an antigenic epitope without pairing with other immunoglobulin variable domains.
  • An example of an immunoglobulin single variable domain of the present disclosure is a "domain antibody”, such as an immunoglobulin single variable domain VH and VL (VH domain and VL domain).
  • Another example of an immunoglobulin single variable domain is a "VHH domain” (or simply "VHH") of Camelidae as defined below.
  • VHH domain also known as heavy chain single domain antibody, VHH, VHH antibody fragment and VHH antibody, is the variable domain of the antigen-binding immunoglobulin called “heavy chain antibody” (i.e. "antibody lacking light chain”) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)).
  • VHH domain is used to distinguish the variable domain from the heavy chain variable domain present in conventional 4-chain antibodies (which is referred to herein as "VH domain”) and the light chain variable domain present in conventional 4-chain antibodies (which is referred to herein as "VL domain”).
  • VHH domain specifically binds to an epitope without the need for other antigen-binding domains (this is in contrast to the VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by both the VL and VH domains).
  • the VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • VHH domain In the context of the present disclosure, the terms “heavy chain single domain antibody”, “VHH domain”, “VHH”, “VHH antibody fragment”, and “VHH antibody” are used interchangeably.
  • the amino acid residues used for the VHH domain of Camelidae can be numbered according to the general numbering method for VH domains given by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the CDR of an antibody can also be IMGT-CDR, which is a CDR definition method based on the IMGT antibody code, which is obtained by integrating the structural information of more than 5,000 sequences.
  • IMGT-CDR is a CDR definition method based on the IMGT antibody code, which is obtained by integrating the structural information of more than 5,000 sequences.
  • CDR1 27-38
  • CDR2 56-65
  • CDR3 105-117.
  • the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering).
  • the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • the CDRs may include "extended CDRs", such as: 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2) and 89-97 or 89-96 (LCDR3) in VL; 26-35 (HCDR1), 50-65 or 49-65 (HCDR2) and 93-102, 94-102 or 95-102 (HCDR3) in VH.
  • extended CDRs such as: 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2) and 89-97 or 89-96 (LCDR3) in VL; 26-35 (HCDR1), 50-65 or 49-65 (HCDR2) and 93-102, 94-102 or 95-102 (HCDR3) in VH.
  • the total number of amino acid residues in a VHH domain will usually be in the range of 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described herein.
  • VHH domains and polypeptides containing them can be summarized as follows:
  • VHH domains (which have been naturally "designed” to functionally bind to antigens in the absence of, and without interacting with, light chain variable domains) 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 4-chain antibodies.
  • VH and VL domains themselves are generally not suitable for practical use as single antigen-binding proteins or immunoglobulin single variable domains, but need to be combined in some form or another to provide a functional antigen-binding unit (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).
  • a functional antigen-binding unit 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.
  • VHH domains - alone or as part of a larger polypeptide - offers a number of significant advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (e.g. Fab- or F(ab')2-fragments): only a single domain is required to bind antigen with high affinity and selectivity, thereby eliminating the need for the presence of two separate domains and for ensuring that the two domains are present in the proper spatial conformation and configuration (e.g.
  • VHH domains can be expressed from a single gene and do not require post-translational folding or modification; VHH domains can be easily engineered into multivalent and multispecific formats (formatting); VHH domains are highly soluble and have no tendency to aggregate; VHH domains Highly stable to heat, pH, proteases and other denaturing agents or conditions, and therefore, no refrigeration equipment may be used during preparation, storage or transportation, thereby achieving cost, time and environmental savings; VHH domains are easy to prepare and relatively inexpensive, even on the scale required for production; VHH domains are relatively small compared to conventional 4-chain antibodies and antigen-binding fragments thereof (approximately 15 kDa or 1/10 the size of conventional IgG), and therefore, compared to conventional 4-chain antibodies and antigen-binding fragments thereof, they exhibit higher tissue penetration and can be administered at higher doses; VHH domains may exhibit so-called cavity binding properties (particularly due to their extended CDR3 loops compared to conventional VH domains
  • the VHH domain derived from Camelidae can be "humanized” (also referred to herein as "sequence optimization") by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at the corresponding positions in the VH domain of a conventional human 4-chain antibody.
  • sequence optimization may also encompass other modifications of the sequence by one or more mutations that provide improved properties of the VHH, such as removal of potential post-translational modification sites.
  • Humanized VHH sequences may contain one or more fully human framework region sequences. Humanization can be accomplished using a protein surface amino acid resurfacing method and/or a humanized universal framework CDR grafting method, for example, as exemplified in the Examples.
  • 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., immunoglobulin single variable domains, heavy chain single domain antibodies, or GIPR binding proteins disclosed herein) can bind.
  • the specificity of an antigen binding protein can be determined based on its affinity and/or avidity.
  • Affinity represented by the dissociation equilibrium constant (KD) of an antigen and an antigen binding protein, is a measure of the binding strength between an epitope and an antigen binding site on an antigen binding protein: the smaller the KD value, the stronger the binding strength between the epitope and the antigen binding protein (or, affinity can also be expressed as an association constant (KA), which is 1/KD).
  • affinity can be determined in a known manner, depending on the specific antigen of interest.
  • Avidity is a measure of the binding strength between an antigen binding protein (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing it) and a related antigen. Avidity is related to the following two things: the affinity between an antigen binding site on an antigen binding protein and a related antigen, and the number of related binding sites present on an antigen binding protein.
  • GIPR glucose-dependent insulinotropic polypeptide receptor
  • glucose-dependent insulinotropic polypeptide receptor (GIPR) binding protein means any protein that can specifically bind to the glucose-dependent insulinotropic polypeptide receptor (GIPR).
  • the GIPR binding protein may include a heavy chain single domain antibody as defined herein to the GIPR.
  • the GIPR binding protein also encompasses an immunoglobulin superfamily antibody (IgSF) or a CDR-grafted molecule.
  • IgSF immunoglobulin superfamily antibody
  • the "GIPR binding protein" of the present disclosure may comprise at least one immunoglobulin single variable domain, such as VHH, that binds to GIPR.
  • the "GIPR binding protein” of the present disclosure may comprise 2, 3, 4 or more immunoglobulin single variable domains, such as VHH, that bind to GIPR.
  • the GIPR binding protein of the present disclosure may also comprise a linker and/or a portion having effector function, such as a half-life extending portion (such as an immunoglobulin single variable domain that binds to serum albumin), and/or a fusion partner (such as serum albumin) and/or a conjugated polymer (such as PEG) and/or an Fc region, in addition to the immunoglobulin single variable domain that binds to GIPR.
  • the "GIPR binding protein" of the present disclosure also encompasses bispecific antibodies that contain antigen binding domains or immunoglobulin single variable domains that bind to different antigens or different regions of the same antigen (such as different epitopes).
  • the GIPR binding proteins of the present disclosure will have a dissociation constant (KD) of preferably 10-7 to 10-10 moles/liter (M), more preferably 10-8 to 10-10 moles/liter, even more preferably 10-9 to 10-10 or less, as measured in a Biacore or KinExA or Fortibio assay, and/or a KD of at least 107 M -1 , preferably at least 108 M -1 , more preferably at least 109 M -1 , more preferably at least 1010 M- 1. 1.
  • KD dissociation constant
  • Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and/or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competitive assays) as described herein.
  • SPR surface plasmon resonance
  • RIA radioimmunoassays
  • EIA enzyme immunoassays
  • sandwich competitive assays as described herein.
  • amino acid residues will be represented according to the standard three-letter or one-letter amino acid code as is known and agreed upon in the art.
  • amino acid difference refers to the insertion, deletion or substitution of a specified number of amino acid residues at a position in a reference sequence compared to another sequence.
  • substitutions will preferably be a conservative amino acid substitution, which refers to the replacement of an amino acid residue by another amino acid residue of similar chemical structure and which has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.
  • conservative amino acid substitutions are preferably substitutions of an amino acid within the following groups (i) to (v) by another amino acid residue within the same group: (i) smaller aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (iii) polar positively charged residues: His, Arg and Lys; (iv) larger aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln or Glu; Met is replaced by Leu, Tyr or Ile; Phe is replaced by Met, Leu or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; Val is replaced by Ile or Leu.
  • sequence identity between two polypeptide sequences indicates the percentage of identical amino acids between the sequences.
  • sequence similarity indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity.
  • sequence identity For the determination of sequence identity, see, for example, Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., 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.
  • isolated generally means substantially free from the substances that normally accompany or are associated with the environment in which it is found in nature.
  • the biological material of separation is optionally included in its natural environment (for example, nucleic acid or protein) other materials that the biological material is not found to have.
  • isolated when it comes to proteins, “isolated” generally refers to the separation and separation of the molecule from the whole organism in which the molecule is found to be naturally present, or the absence of other biological macromolecules of the same type.
  • nucleic acid molecules it is completely or partially separated from the sequence naturally associated with it, or the nucleic acid has a heterologous sequence associated with it, or the nucleic acid is separated from the chromosome.
  • a polypeptide or nucleic acid molecule is considered “isolated” when it has been separated from at least one other component (e.g., another protein/polypeptide, another nucleic acid, another biological component or macromolecule or at least one contaminant, impurity or trace component) with which it is normally associated in the source or medium (culture medium) compared to its natural biological source and/or the reaction medium or culture medium from which it was obtained.
  • a polypeptide or nucleic acid molecule is considered “isolated” when it has been purified at least 2 times, particularly at least 10 times, more particularly at least 100 times and up to 1000 times or more.
  • An "isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous as determined by a suitable technique (e.g., a suitable chromatographic technique, such as polyacrylamide gel electrophoresis).
  • an effective amount is meant an amount of a GIPR binding protein or pharmaceutical composition of the disclosure that results in a decrease in severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability resulting from disease affliction.
  • metabolic disease refers to a disorder that affects the energy production of human (or animal) cells, also known as metabolic disorders. Most metabolic diseases are hereditary diseases, while some are acquired due to diet, toxins, and infection. Common metabolic diseases can be divided into three major categories: disorders that affect carbohydrate metabolism, disorders that affect fat metabolism, and disorders that affect mitochondria in cells.
  • the term "subject” means a mammal, particularly a primate, especially a human.
  • the term "antigen binding protein” generally refers to a protein comprising a portion that binds an antigen, and optionally a scaffold or skeleton portion that allows the portion that binds the antigen to adopt a conformation that promotes the antigen binding protein to bind to the antigen.
  • Antigen binding proteins may typically include an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both, and functional fragments thereof.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • the term “antigen binding protein” also encompasses single domain antibodies and proteins comprising a single variable domain of an immunoglobulin. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen.
  • 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, etc., as long as they show the desired antigen binding activity.
  • polypeptide or “protein” are used interchangeably and generally refer to a polymer of amino acid residues.
  • the term also applies to amino acid polymers in which one or more amino acid residues is an analog or mimetic of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers.
  • the term may also include modified amino acid polymers, such as
  • the polypeptides and proteins may be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and may include molecules having the amino acid sequence of a native protein, or molecules having one or more amino acids deleted, added and/or substituted from the native sequence.
  • polypeptide and “protein” specifically include sequences that are deleted, added and/or substituted from one or more amino acids of the antigen binding proteins described in the present disclosure.
  • conjugate generally refers to a substance formed by linking an antigen binding protein with other active agents, which may be small molecule active agents, such as therapeutic agents, imaging probes, or spectroscopy probes.
  • nucleic acid generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogs thereof, isolated from their natural environment or artificially synthesized.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vector may include a vector mainly used to insert DNA or RNA into a cell, a vector mainly used to replicate DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA.
  • the vector also includes vectors with a variety 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.
  • the vector can produce a desired expression product by culturing a suitable host cell containing the vector.
  • the term "cell” generally refers to an individual cell, cell line or cell culture that may contain or already contain a plasmid or vector including a nucleic acid molecule described in the present disclosure, or that can express an antigen-binding protein described in the present disclosure.
  • the cell may include progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cell may not necessarily be completely identical to the original parent cell in morphology or genome, but may be able to express the antibody or antigen-binding fragment thereof described in the present disclosure.
  • the cell may be obtained by transfecting cells in vitro using the vector described in the present disclosure.
  • the cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NS0 cell or a myeloma cell).
  • a prokaryotic cell e.g., Escherichia coli
  • a eukaryotic cell e.g., a yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NS0 cell or a myeloma cell.
  • the cell may be a mammalian cell.
  • the mammalian cell may be a CHO-K1 cell.
  • the term "pharmaceutical composition” generally refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective, and contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.
  • treatment generally refers to the desire to change the natural course of the individual being treated, and can be achieved by prevention or treatment or clinical intervention during clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.
  • antigen binding proteins e.g., antibodies specific to the present disclosure
  • Antibodies to a specific antigen can be used to delay disease development or slow disease progression.
  • administering generally refers to a method of administering a dose of a compound or pharmaceutical composition to a subject (e.g., a patient).
  • Administration can be performed by any suitable means, including parenteral, intrapulmonary, and intranasal, and (if desired for local treatment) intralesional administration.
  • Parenteral infusions include, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the term “include” generally means to include, encompass, contain or encompass. In some cases, it also means “for”, “consisting of.".
  • the term "about” generally refers to variations within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.
  • the present disclosure relates to a glucose-dependent insulinotropic polypeptide receptor (GIPR) binding protein comprising at least one immunoglobulin single variable domain.
  • GIPR glucose-dependent insulinotropic polypeptide receptor
  • the at least one immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in a VHH as shown in any one of SEQ ID NOs: 1-7.
  • the CDRs may be defined according to any of the following definition systems: Kabat CDR, AbM CDR, Chothia CDR or IMGT CDR.
  • the at least one immunoglobulin single variable domain comprises one or more groups of CDR1, CDR2, and CDR3 selected from the following Table 2:
  • the at least one immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:5 and/or SEQ ID NO:6.
  • the GIPR binding protein of the present disclosure comprises at least one immunoglobulin single variable domain, wherein 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 NO: 8-14.
  • “Multiple” can be 2, 3, 4 or 5, and the multiple amino acid sequences can be the same or different.
  • the at least one immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO: 5 and SEQ ID NO: 8-14.
  • the GIPR binding protein of the present disclosure comprises the sequence contained in the following general formula (wherein [] refers to the type of amino acid that can be selected at this position):
  • the GIPR binding protein of the present invention comprises at least one immunoglobulin single variable domain, and 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 NO:15-25.
  • the at least one immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 15-25.
  • the GIPR binding protein of the present disclosure comprises the sequence contained in the following general formula (wherein [] refers to the type of amino acid that can be selected at this position):
  • the immunoglobulin single variable domain is a humanized VHH, and the humanized VHH comprises at least 80%, preferably at least 90%, more preferably at least 95%, or even More preferably, an amino acid sequence with at least 99% sequence identity.
  • 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.
  • 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.
  • the at least one immunoglobulin single variable domain may simultaneously contain one or more of the amino acid sequences shown in SEQ ID NO:5 and SEQ ID NO:8-14, and one or more of the amino acid sequences shown in SEQ ID NO:6 and SEQ ID NO:15-25.
  • the at least one immunoglobulin single variable domain may simultaneously contain the amino acid sequence shown in SEQ ID NO:5 and one of SEQ ID NO:8-14, and the amino acid sequence shown in one of SEQ ID NO:6 and SEQ ID NO:15-25.
  • the GIPR binding proteins of the present disclosure in addition to at least one immunoglobulin single variable domain, further comprise an immunoglobulin Fc region.
  • an immunoglobulin Fc region in the GIPR binding proteins of the present disclosure can allow the binding molecules to form dimers.
  • the Fc regions that can be used in the present disclosure can be from immunoglobulins of different subtypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM.
  • mutations can be introduced into the wild-type Fc sequence to change Fc-mediated related activities.
  • the mutations include but are not limited to: a). mutations that change Fc-mediated CDC activity; b). mutations that change Fc-mediated ADCC activity; or c). mutations that change FcRn-mediated in vivo half-life.
  • Such mutations are described in the following literature: 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; Paul R.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids on the CH2 region can be used to increase or remove Fc-mediated ADCC or CDC activity or to enhance or weaken the affinity of FcRn.
  • the stability of the protein can be increased by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in the hinge region.
  • mutations can be introduced into the Fc sequence, so that the mutated Fc is more likely to form homodimers or heterodimers.
  • the knob-hole model using the spatial effect of the amino acid side chain groups at the Fc contact interface mentioned in Ridgway, Presta et al.
  • the immunoglobulin Fc region is preferably a human immunoglobulin Fc region, for example, the Fc region of human IgG1, IgG2, IgG3, or IgG4.
  • the amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO: 110 or SEQ ID NO: 113.
  • the immunoglobulin Fc region (eg, the Fc region of human IgG1) is directly or indirectly linked via a linker to the C-terminus of the immunoglobulin single variable domain (eg, VHH).
  • the GIPR binding protein of the present disclosure comprises an immunoglobulin single variable domain that specifically binds to GIPR, and the immunoglobulin single variable domain that specifically binds to GIPR is directly or indirectly connected to the immunoglobulin Fc region, and the immunoglobulin Fc region allows 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.
  • the dimer is a homodimer.
  • the GIPR binding protein of the present disclosure comprises two immunoglobulin single variable domains that specifically bind to GIPR and an immunoglobulin Fc region that are directly or indirectly connected to each other via a linker, and the immunoglobulin Fc region allows the GIPR binding protein to form a dimeric molecule comprising four GIPR binding domains.
  • Such GIPR binding proteins are also referred to as tetravalent GIPR binding proteins.
  • the dimer is a homodimer.
  • the two immunoglobulin single variable domains that specifically bind to GIPR in the GIPR binding protein bind to different regions or different epitopes of GIPR, respectively.
  • the multiple immunoglobulin single variable domains disclosed herein can be directly connected to each other, and between the immunoglobulin single variable domain and the immunoglobulin Fc region, or indirectly connected through a linker.
  • the linker can be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary structure or above.
  • the linker is a flexible linker, such as one or more of (GGG) n , (GGGS) n , (GGGA) n , (GGGAA) n and (GGGGS) n , wherein 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.
  • the linker is GGGGS, GS, GAP, (GGGGS) x 3, etc.
  • the GIPR binding protein of the present disclosure has at least one of the following characteristics:
  • the present disclosure also relates to a fusion protein, wherein the fusion protein comprises the GIPR binding protein described in the present disclosure.
  • the fusion protein in addition to the GIPR binding protein, further comprises one or more other biologically active proteins, which may be any protein having biological, therapeutic, preventive or diagnostic significance or function, and when administered to a subject, mediating biological activity can prevent or alleviate a disease, disorder or condition.
  • the biologically active protein may be an agonist, antagonist, regulator, ligand, cytokine, enzyme or hormone, in particular a biologically active protein that can be used to prevent and/or metabolic diseases, especially obesity, overweight and/or diabetes.
  • the present disclosure also relates to a multispecific antibody, wherein the multispecific antibody comprises the GIPR binding protein described in the present disclosure.
  • the multispecific antibody in addition to the GIPR binding protein, 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.
  • the present disclosure relates to nucleic acid molecules 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.
  • the nucleic acid of the present disclosure is a substantially isolated nucleic acid.
  • the nucleic acid of the present disclosure may also be in the form of a vector, may be present in a vector and/or may be part of a vector, such as a plasmid, a cosmid or a YAC.
  • the vector may be, in particular, an expression vector, i.e., a vector that provides for 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).
  • the expression vector typically comprises at least one nucleic acid of the present disclosure, which is operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.).
  • regulatory elements and other elements useful or necessary for the expression of the GIPR binding protein of the present disclosure such as promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.
  • nucleic acids of the present disclosure can be prepared or obtained by known means (e.g., by automated DNA synthesis and/or recombinant DNA technology) based on the information on the amino acid sequences of the polypeptides of the present disclosure given herein, and/or can be isolated from suitable natural sources.
  • the present disclosure relates to recombinant host cells that express or are capable of expressing one or more GIPR binding proteins, fusion proteins, and/or contain nucleic acids or vectors of the present disclosure.
  • Preferred host cells of the present disclosure are bacterial cells, fungal cells, bacterial cells or mammalian cells.
  • Suitable bacterial cells include cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).
  • Gram-negative bacterial strains e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains
  • Gram-positive bacterial strains e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains.
  • Suitable fungal cells include cells of species of Trichoderma, Neurospora and Aspergillus; or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica) and Hansenula.
  • Saccharomyces e.g., Saccharomyces cerevisiae
  • Schizosaccharomyces e.g., Schizosaccharomyces pombe
  • Pichia e.g., Pichia pastoris and Pichia methanolica
  • Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, or COS cells.
  • the present disclosure may also use amphibian cells, insect cells, and any other cells known in the art for expressing heterologous proteins.
  • the GIPR binding proteins or fusion proteins of the present disclosure can be produced intracellularly in cells as described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified.
  • the present disclosure provides a composition, such as a pharmaceutical composition, containing 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.
  • pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, stabilizers, isotonic and absorption delaying agents, etc. that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound i.e., the antibody molecule
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated and the particular route of administration.
  • the amount of active ingredient that can be combined with a carrier material to prepare a single dosage form is generally the amount of the composition that produces a therapeutic effect. Typically, based on 100%, this amount ranges from about 0.01% to about 99% of the active ingredient, such as about 0.1% to about 70%, or about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.
  • the selected dosage level depends on a variety of pharmacokinetic factors, including the activity of the particular composition of the present disclosure or its ester, salt, or amide being applied, the route of administration, the time of administration, the rate of excretion of the particular compound being applied, the duration of treatment, other drugs, compounds, and/or materials being used in combination with the particular composition being applied, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.
  • compositions of the present disclosure can be administered by one or more routes of administration using one or more methods known in the art. It will be appreciated by those skilled in the art that the route and/or mode of administration will vary depending on the desired outcome.
  • routes of administration for the GIPR binding proteins of the present disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion.
  • parenteral administration refers to modes of administration other than enteral and topical administration, typically injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • the present disclosure also provides a method for preventing and/or treating a disease, particularly a method for treating and/or preventing a metabolic disease, comprising administering an effective amount of a GIPR binding protein, fusion protein, multispecific antibody, conjugate or pharmaceutical composition of the present disclosure to a subject in need thereof.
  • the metabolic disease comprises obesity, overweight and/or diabetes.
  • other therapeutic drugs may be administered to a subject in need thereof, such as insulin (including porcine or bovine insulin, human insulin, aspart insulin, lispro insulin, low-protamine zinc insulin, glargine insulin, detemir insulin, LY3209590, etc.), biguanides (such as metformin, etc.), thiazolidinediones (including pioglitazone, rosiglitazone, etc.), sulfonylureas (including glitazone, etc.), Pyrazine, gliclazide, glibenclamide, glibourea, gliclazide, glisipate, glimepiride, etc.), phenylalanine (including repaglinide, nateglinide, mitiglinide, etc.), ⁇ -
  • the GLP-1R agonist can be: GLP-1 (7-37), GLP-1 (7-36) -NH2, liraglutide, albiglutide, lixisenatide, dulaglutide, semaglutide, Exendin-4, Exendin-3, polyethylene glycol loxenatide, tilportide, GMA102, PB119, ibenatide, Mazdutide, supaglutide, cotadutide, Danuglipron, HM-15211, Efinopegdutide, etc.
  • the other therapeutic drugs and the binding protein/fusion protein/multispecific antibody/conjugate/drug composition of the present invention can be administered simultaneously or sequentially; when administered sequentially, the interval between the administration of the two shall not exceed 1 week, preferably not exceed 3 days, more preferably not exceed 2 days, and even more preferably not exceed 1 day.
  • the present disclosure also provides use of the GIPR binding protein, fusion protein, multispecific antibody, conjugate or pharmaceutical composition of the present disclosure in the preparation of a drug for treating and/or preventing metabolic diseases.
  • the present disclosure provides a GIPR binding protein, fusion protein, multispecific antibody, conjugate or pharmaceutical composition for treating and/or preventing metabolic diseases.
  • 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.
  • the method may be an ex vivo or in vitro method.
  • the method may be a method for non-therapeutic purposes.
  • 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.
  • the present disclosure provides a method for detecting the content of GIPR protein in a sample, which comprises 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.
  • the present invention also provides use of the GIPR binding protein, fusion protein, multispecific antibody or conjugate disclosed herein in preparing a kit for detecting the presence and/or content of a GIPR protein in a sample.
  • the sample can be a sample of blood, plasma, amniotic fluid, etc.
  • the present disclosure provides a kit that may include the GIPR binding protein, fusion protein, multispecific antibody, conjugate and/or pharmaceutical composition described in the present disclosure. It may include the GIPR binding protein, fusion protein, multispecific antibody, conjugate and/or pharmaceutical composition described in the present disclosure in a single common container, and may also be optionally combined with one or more therapeutic agents, optionally formulated together in a pharmaceutical composition.
  • the present disclosure provides a drug delivery device that can be used to administer the GIPR binding protein, fusion protein, multispecific antibody, conjugate and/or pharmaceutical composition described in the present disclosure.
  • the present disclosure provides the following embodiments:
  • a glucose-dependent insulinotropic polypeptide receptor GIPR binding protein which comprises at least one immunoglobulin single variable domain, and the at least one immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:5 and/or 6.
  • GIPR binding protein of embodiment 1, wherein the CDRs are defined according to the following definition systems: Kabat, AbM, Chothia or IMGT.
  • CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:5 are selected from any one of the following groups: SEQ ID NO:74-76, SEQ ID NO:77-79, SEQ ID NO:80-82 and SEQ ID NO:83-85.
  • 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 NO:8-14.
  • At least one immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:8-14.
  • CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:6 are selected from any one of the following groups: SEQ ID NO:86-88, SEQ ID NO:89-91, SEQ ID NO:92-94 and SEQ ID NO:95-97.
  • 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 NO:15-25.
  • At least one immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:6, one of SEQ ID NO:15-25.
  • At least one immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:5, one of SEQ ID NO:8-14, and/or the amino acid sequence shown in SEQ ID NO:6, one of SEQ ID NO:15-25.
  • the GIPR binding protein according to any one of embodiments 1-9, further comprising an immunoglobulin Fc region, preferably a human immunoglobulin Fc region, more preferably a human IgG1, IgG2, IgG3 or IgG4 Fc region.
  • the amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO:110 or SEQ ID NO:113.
  • the GIPR binding protein according to embodiment 10 or 11, wherein the immunoglobulin Fc region is bound to the at least one immunoglobulin The immunoglobulin single variable domains are linked directly or indirectly via a linker.
  • GIPR binding protein according to any one of embodiments 1-12, having at least one of the following features:
  • a fusion protein comprising the GIPR binding protein according to any one of embodiments 1-13.
  • a conjugate comprising the GIPR binding protein according to any one of embodiments 1 to 13 and/or the fusion protein according to embodiment 14.
  • An expression vector comprising the nucleic acid molecule of embodiment 15 operably linked to an expression regulatory element.
  • a recombinant cell comprising the nucleic acid molecule of embodiment 15 and/or transformed with the expression vector of embodiment 16, and capable of expressing the GIPR binding protein or the fusion protein comprising the GIPR binding protein.
  • a pharmaceutical composition comprising the GIPR binding protein of any one of embodiments 1-13, the fusion protein of embodiment 14 and/or the conjugate of embodiment 15, and a pharmaceutically acceptable carrier.
  • kits comprising the GIPR binding protein of any one of embodiments 1-13, the fusion protein of embodiment 14, the conjugate of embodiment 15 and/or the pharmaceutical composition of embodiment 19.
  • a method for determining the presence and/or content of a GIPR protein, and/or inhibiting the binding of a GIPR protein to its ligand comprising providing the GIPR binding protein of any one of embodiments 1-13, the fusion protein of embodiment 14, and/or the conjugate of embodiment 15.
  • a method for treating and/or preventing metabolic diseases and/or symptoms comprising administering to a subject in need thereof an effective amount of the GIPR binding protein of any one of embodiments 1-13, the fusion protein of embodiment 14, the conjugate of embodiment 15, or the pharmaceutical composition of embodiment 18.
  • alpaca arterial blood was collected in a vacuum blood collection tube, and the supernatant was collected as pre-immune serum. Healthy alpacas were selected, and antigens containing human GIPR were injected into the alpacas at multiple points in the neck muscle. The alpacas were immunized once every two weeks, for a total of six immunizations. At the end of the last immunization, 50 mL of alpaca arterial blood was collected in a vacuum blood collection tube, and the supernatant was collected as post-immune serum.
  • Lymphocytes were separated by density gradient centrifugation, and total RNA was extracted using the RNA extraction kit provided by QIAGEN. The extracted RNA was reverse transcribed into cDNA using the Super-Script III FIRST STRAND SUPERMIX kit according to the instructions, and the nucleic acid fragment encoding the variable region of the heavy chain antibody was amplified by nested PCR.
  • the nucleic acid fragment of the target heavy chain single domain antibody was recovered and cloned into the phage display vector pComb3XSS using the restriction endonuclease SfiI.
  • the product was then electrotransformed into the E. coli electrocompetent cell TG1 to construct an anti-GIPR immune single domain antibody phage display library and identify the library.
  • the library capacity was calculated to be 1.69 ⁇ 10 9 .
  • 50 clones were randomly selected for sequencing, and 50 clones had the correct exogenous fragment insertion, with a correct rate of 100%.
  • By analyzing and comparing the DNA and amino acid sequences of the sequenced clones it was confirmed that all sequences were alpaca VHH sequences, and it can be estimated that their diversity is above 95%.
  • the phage library obtained in Example 1.1 was panned, and the bound phages were screened using the proteins GIPR-ECD-muFc (a fragment of the extracellular region of human GIPR fused to mouse Fc), GIPR-ECD-chis (a fragment of the extracellular region of human GIPR fused to his tag), and GIPR-ECD-laFc (a fragment of the extracellular region of human GIPR fused to alpaca Fc).
  • negative panning was performed using the fusion protein Control-1 (C1) containing muFc and the fusion protein Control-2 (C2) containing laFc to remove non-specifically bound phages.
  • the binding-positive phages obtained after the above panning were used to infect blank Escherichia coli and plated. Subsequently, the colonies were selected and inoculated into 2TY-AG (containing 10% glycerol) and allowed to stand at room temperature overnight. The next day, 1% of the inoculation volume was transferred to 200 ⁇ L 2TY-AG, at 37°C, 250rpm, until the OD600 was about 0.5, and the helper phage M13KO7 was added for infection (infection index was 1:20), and allowed to stand at 37°C for 15 minutes, and then at 220rpm for 45 minutes.
  • 2TY-AG containing 10% glycerol
  • Primers were designed for PCR amplification of the GIPR single domain antibody VHH fragment, fused with a DNA fragment encoding human IgG1-Fc (amino acid sequence SEQ ID NO: 110), and cloned into a conventional mammalian expression vector to obtain a recombinant plasmid for expressing the GIPR single domain antibody-Fc fusion protein in mammals.
  • Universal primers were used to amplify different VHH fragments. Universal primers are as follows:
  • the plasmid vector obtained in 2.1 was transfected into HEK293 cells for transient expression of the antibody.
  • the recombinant expression plasmid was diluted with Freestyle293 medium and the PEI (Polyethylenimine) solution required for transformation was added.
  • Each group of plasmid/PEI mixtures was added to the HEK293 cell suspension and placed at 37°C, 5% CO 2 for suspension culture. After 5 to 6 days of culture, the supernatant of the transient expression culture was collected and purified by Protein A affinity chromatography to obtain the target GIPR single domain antibody-Fc fusion protein, and the protein purity was detected by SDS-PAGE.
  • the expression levels of each protein are shown in Table 4, and the SDS purity of each protein after one-step purification is greater than 95%.
  • 293T-GIPR cells (HEK293T cells expressing human GIPR) were plated in a cell culture plate, and GIPR single domain antibody-Fc fusion protein samples were added at final concentrations of 100 ⁇ g/ml and 10 ⁇ g/ml, 2 concentrations. After adding anti-human IgG-APC secondary antibody and incubating, the mean fluorescence intensity (MFI) was detected using a fluorescence activated cell sorter. The results are shown in Table 5.
  • MFI mean fluorescence intensity
  • GIP-C12H4-Fc4 human GIP-IgG4Fc fusion protein
  • 25 ⁇ L GIPR single domain antibody-Fc sample were added to a 96-well plate. Then 293T-GIPR cells were added and incubated.
  • the cAMP kit was used to detect the absorbance at 450 nm/550 nm using an ELISA reader. The results are shown in Table 6.
  • GIPR single domain antibody-Fc fusion protein The binding kinetics of GIPR single domain antibody-Fc fusion protein to GIPR-ECD-chis were detected by biolayer interferometry (BLI) technology.
  • GIPR single domain antibody-Fc fusion protein was directly immobilized on the AHC sensor, and then GIPR-ECD-chis was diluted to 7 concentrations and combined with the immobilized GIPR single domain antibody-Fc fusion protein.
  • the equilibrium dissociation constant (KD), association rate (K a ) and dissociation rate (K dis ) were calculated using Octet K2 data analysis software 9.0. The results are shown in Tables 8 and 9.
  • the positive control AMG-GIPR-mab2 was synthesized according to the sequence in the literature MAbs.2020 Jan-Dec; 12(1):1710047, and then prepared by transient expression in 293 cells according to the above method.
  • CHOK1 empty cells were resuspended in 3% BSA-PBS, and the final concentrations of GIPR single domain antibody-Fc fusion protein were selected to be 5 ⁇ g/mL and 50 ⁇ g/mL, and negative control and blank control were set at the same time.
  • the secondary antibody APC anti-human IgG Fc was added.
  • the cells were resuspended in PBS-BSA Buffer and detected by flow cytometry.
  • the results are shown in Table 10. iGI-72-Ld-Fc There was no non-specific binding to iGI-1198-Ld-Fc, but there was non-specific binding to iGI-1225-Ld-Fc and iGI-1225NA-Ld-Fc.
  • the iGI-72-Ld-Fc treatment group significantly reduced the body weight of DIO mice, while improving fasting blood glucose, fasting insulin and insulin resistance HOMA-IR.
  • the combination of iGI-72-Ld-Fc and dulaglutide can further enhance the efficacy and have a synergistic effect.
  • the humanization method uses protein surface amino acid humanization (resurfacing) and VHH humanization universal framework transplantation (CDR grafting to a universal framework) to complete.
  • VHH humanized universal framework transplantation method obtain highly homologous human antibody sequences through IMGT, and refer to the universal humanized VHH framework hNbBcII10FGLA (PDB number: 3EAK) to humanize the target sequence. Humanization. Using a highly homologous sequence framework as a framework template, the CDR is replaced with the CDR region of the target antibody strain. Then, based on the modeling, the non-surface amino acids on the framework are back-mutated to complete the humanization of the target antibody.
  • the antibody strains iGI-72 and iGI-1198 were humanized to obtain 8 and 11 humanized variants huGI of the antibody strains, respectively.
  • Example 4 The humanized sequence in Example 4 was subjected to gene synthesis, fused with a DNA fragment encoding human IgG1-Fc, and cloned into a conventional mammalian expression vector to obtain a recombinant plasmid for expressing GIPR single domain antibody Fc fusion protein in mammals.
  • the vector obtained by constructing 5.1 was transfected into HEK293 cells for transient expression of the antibody.
  • the recombinant expression plasmid was diluted with Freestyle293 medium and the PEI (Polyethylenimine) solution required for transformation was added.
  • Each group of plasmid/PEI mixtures was added to the HEK293 cell suspension and cultured at 37°C, 5% CO2, and 130rpm.
  • the transient expression culture supernatant was collected, and the target huGI single-domain antibody Fc fusion protein was purified by Protein A affinity chromatography, and the protein was examined for purity 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 is greater than 95%.
  • 293T-GIPR cells were plated in a cell culture plate, and humanized GIPR single domain antibody-Fc fusion protein or Maridebart sample was added at a final concentration of 0.00128nM-100nM.
  • Anti-human secondary antibody SULFO-TAG (MSD, Cat. No. R32AJ-1) was added and incubated, and the mean fluorescence intensity (MFI) was detected using MSD.
  • Maridebart is an anti-GIPR antibody, which was independently cloned and prepared with reference to the sequence disclosed in WHO Drug Information, Volume 36. Number 4.2022. Proposed INN: List 128. Similar sequences are also found in construct 2G10 LC1.006 in patent application WO2017112824.
  • Example 3.2 the neutralizing activity of humanized GIPR single domain antibody-Fc fusion protein was determined. The results are shown in Figure 6. The neutralizing activity of humanized GIPR single domain antibody-Fc fusion protein is better than that of the non-humanized molecule iGI-1198-Fc fusion protein.
  • GIP-C12H4-Fc4 and 25 ⁇ L humanized GIPR single domain antibody-Fc or Maridebart samples were added to the 96-well plate; then 293T-GIPR cells were added, and after incubation, the cAMP kit was used to detect it, and the absorbance at 450nm/550nm was detected by an ELISA reader, and the inhibition rate (%) was calculated.
  • the results are shown in Figure 11, and the neutralizing activity of the humanized GIPR single domain antibody-Fc fusion protein is better than that of Maridebart.
  • mice After fasting overnight, 9 C57BL/6 mice were randomly divided into 3 groups. After returning to normal diet for 24 hours, huGI-72v5-Ld-Fc 203nmol/kg was administered according to the experimental protocol. 12 hours after administration, overnight fasting began. 48 hours after administration, each mouse was ip-administered DA-GIP 50nmol/kg. After administration of DA-GIP, 2g/kg glucose load was immediately administered orally. Blood was collected before, 5, 10, 15, 30, and 60 minutes after glucose load to measure blood glucose and insulin levels. The results are shown in Figures 7 and 8.
  • DA-GIP significantly reduced OGTT blood glucose
  • huGI-72v5-Ld-Fc blocked the hypoglycemic effect of DA-GIP, indicating that huGI-72v5-Ld-Fc has significant blocking activity in mice.
  • iGI-9 (SEQ ID NO: 1):
  • iGI-27 SEQ ID NO: 2
  • iGI-44 SEQ ID NO: 3
  • iGI-51 SEQ ID NO: 4
  • iGI-72 (SEQ ID NO: 5):
  • iGI-1198 SEQ ID NO: 6
  • iGI-1225NA SEQ ID NO: 7
  • huGI-72v3 (SEQ ID NO: 10):
  • huGI-72v4 (SEQ ID NO: 11):
  • huGI-72v5 (SEQ ID NO: 12):
  • huGI-72v6 (SEQ ID NO: 13):
  • huGI-1198n1 (SEQ ID NO: 15):
  • huGI-1198n3 (SEQ ID NO: 17):
  • huGI-1198n4 (SEQ ID NO: 18):
  • huGI-1198n5 (SEQ ID NO: 19):
  • huGI-1198n6 (SEQ ID NO: 20):
  • huGI-1198n8 (SEQ ID NO: 22):
  • IgG1 Fc C220S (SEQ ID NO: 113)

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Abstract

提供了一种GIPR结合蛋白及其应用。具体提供了一种特异性结合葡萄糖依赖性促胰岛素多肽受体的单域抗体、其衍生蛋白及医药用途。

Description

一种GIPR结合蛋白及其应用 技术领域
本公开涉及生物医药领域，公开了针对葡萄糖依赖性促胰岛素多肽受体(GIPR)的单域抗体及其衍生蛋白。具体而言，本公开公开了一种针对葡萄糖依赖性促胰岛素多肽受体的结合蛋白及其用途。
背景技术
葡萄糖依赖性促胰岛素多肽(Glucose-dependent insulinotropic polypeptide，GIP)是一种肠源性肠促胰岛素激素，具有增强葡萄糖刺激的胰岛素分泌的能力。GIP由位于小肠近端的K细胞分泌，膳食中的碳水化合物和脂肪是人类GIP分泌的有效刺激物，研究表明，高脂饮食喂养的啮齿动物增加了K细胞的GIP分泌，同样，人类的急性高脂饮食摄入也会导致GIP浓度增加42％，之后体重明显增加，这表明暴露于高脂饮食和GIP浓度之间呈正相关。因此，高脂饮食和GIP的全身浓度升高可能是人类受试者肥胖差异的基础。
近来，多项人类遗传学研究展现出了葡萄糖依赖性促胰岛素多肽受体(GIPR)与体重指数(BMI)之间的联系。此外，GIPR敲除的小鼠表现出了对高脂饮食所引起肥胖的抵抗力，再加上GIPR在胰腺细胞和脂肪细胞中的既定作用，开发GIPR拮抗剂用于治疗肥胖症似乎是一种可行的策略。然而，现有技术中尚缺乏具有明确治疗作用的GIPR拮抗剂。
发明内容
本公开利用噬菌体展示技术，通过筛选得到了抗GIPR单域抗体(VHH)及其衍生蛋白。本公开的抗GIPR单域抗体(VHH)及其衍生蛋白具有显著的高特异性、高亲和力的效果。
在第一方面，本公开提供了葡萄糖依赖性促胰岛素多肽受体(GIPR)结合蛋白，所述GIPR结合蛋白包含至少一个免疫球蛋白单一可变结构域，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5和/或SEQ ID NO:6所示的VHH中的CDR1、CDR2和CDR3。
在一些实施方案中，所述CDR1、CDR2和CDR3根据以下定义系统定义：Kabat、AbM、Chothia或IMGT。
在一些实施方案中，所述免疫球蛋白单一可变结构域是骆驼科的、人源化的、或嵌合的。
在一些实施方案中，SEQ ID NO:5所示的VHH中的CDR1、CDR2和CDR3选自以下的任意一组：SEQ ID NO:74-76、SEQ ID NO:77-79、SEQ ID NO:80-82和SEQ ID NO:83-85。 在一些实施方案中，SEQ ID NO:5所示的VHH中的CDR1、CDR2和CDR3选自以下的任意一组：SEQ ID NO:74-76、SEQ ID NO:77-79、SEQ ID NO:80-82和SEQ ID NO:83-85。在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5和SEQ ID NO:8-14所示氨基酸序列中的一个或多个。在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5和SEQ ID NO:8-14之一的所示氨基酸序列。
在一些实施方案中，SEQ ID NO:6所示的VHH中的CDR1、CDR2和CDR3选自以下的任意一组：SEQ ID NO:86-88、SEQ ID NO:89-91、SEQ ID NO:92-94和SEQ ID NO:95-97。在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:6和SEQ ID NO:15-25所示氨基酸序列中的一个或多个。在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:6和SEQ ID NO:15-25之一的所示氨基酸序列。
在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5和SEQ ID NO:8-14所示氨基酸序列中的一个或多个，以及SEQ ID NO:6和SEQ ID NO:15-25所示氨基酸序列中的一个或多个。在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5和SEQ ID NO:8-14之一的所示氨基酸序列，和SEQ ID NO:6和SEQ ID NO:15-25之一的所示氨基酸序列。
在一些实施方案中，所述GIPR结合蛋白包含1个所述免疫球蛋白单一可变结构域。在一些实施方案中，所述GIPR结合蛋白包含多个，例如2个、3个、4个或5个，所述免疫球蛋白单一可变结构域。
在一些实施方案中，所述GIPR结合蛋白还包含免疫球蛋白Fc区，优选人免疫球蛋白的Fc区，更优选人IgG1、IgG2、IgG3或IgG4的Fc区。在一些实施方案中，所述免疫球蛋白的Fc区包含如SEQ ID NO:110或SEQ ID NO:113所示的氨基酸序列。在一些实施方案中，所述免疫球蛋白的Fc区的氨基酸序列如SEQ ID NO:110或SEQ ID NO:113所示。
在一些实施方案中，所述免疫球蛋白的Fc区与所述至少一个免疫球蛋白单一可变结构域直接连接或通过接头间接连接。
在一些实施方案中，所述GIPR结合蛋白具有下述特征中的至少一项：
(a)与人GIPR具有特异性的结合；
(b)与人GIPR结合的KD值小于1x10-7M，优选小于1x10-8M；
(c)阻断GIP与GIPR的相互作用；和
(d)能够降低受试者的体重和/或血糖。
在第二方面，本公开提供了一种融合蛋白，其包含本公开的第一方面所述的GIPR结合蛋白。
在第三方面，本公开提供了一种多特异性抗体，其包含本公开的第一方面所述的GIPR结合蛋白和/或本公开的第二方面所述的融合蛋白，以及另外的一个或多个抗原结合区，所述另外的一个或多个抗原结合区与所述GIPR结合蛋白和/或所述融合蛋白结合不同的抗原或相同抗原的不同表位。
在第四方面，本公开提供了一种缀合物，其包含本公开的第一方面所述的GIPR结合蛋白和/或本公开的第二方面所述的融合蛋白和/或本公开的第三方面所述的多特异性抗体，所述缀合物还包含与所述GIPR结合蛋白和/或所述融合蛋白和/或所述多特异性抗体缀合的分子。
在第五方面，本公开提供了一种核酸分子，其编码本公开的第一方面所述的GIPR结合蛋白和/或本公开的第二方面所述的融合蛋白和/或本公开的第三方面所述的多特异性抗体。
在第六方面，本公开提供了一种表达载体，其包含与表达调控元件可操作地连接的本公开的第五方面所述的核酸分子。
在第七方面，本公开提供了一种重组细胞，其包含本公开的第五方面所述的核酸分子和/或以本公开的第六方面所述的表达载体转化，并能够表达所述GIPR结合蛋白和/或所述融合蛋白和/或所述多特异性抗体。
在第八方面，本公开提供了一种药物组合物，其包含本公开的第一方面所述的GIPR结合蛋白和/或本公开的第二方面所述的融合蛋白和/或本公开的第三方面所述的多特异性抗体和/或本公开的第四方面所述的缀合物和/或本公开的第五方面所述的核酸分子和/或本公开的第六方面所述的表达载体和/或本公开的第七方面所述的重组细胞，以及药学上可接受的载剂。
在第九方面，本公开提供了一种试剂盒，其包含本公开的第一方面所述的GIPR结合蛋白、本公开的第二方面所述的融合蛋白、本公开的第三方面所述的多特异性抗体、本公开的第四方面所述的缀合物和/或本公开的第八方面所述的药物组合物。
在第十方面，本公开提供了一种确定GIPR蛋白的存在和/或含量的方法，其包含提供本公开的第一方面所述的GIPR结合蛋白、本公开的第二方面所述的融合蛋白、本公开的第三方面所述的多特异性抗体、和/或本公开的第四方面所述的缀合物。
在第十一方面，本公开提供了一种抑制GIPR蛋白与其配体结合的方法，其包含提供本公开的第一方面所述的GIPR结合蛋白、本公开的第二方面所述的融合蛋白、本公开的第三方面所述的多特异性抗体、和/或本公开的第四方面所述的缀合物。在优选的实施方案中，所 述配体是GIP。
在第十二方面，本公开提供了一种治疗和/或预防代谢性疾病和/或症状的方法，其包括向有需要的对象施用有效量的本公开的第一方面所述的GIPR结合蛋白、本公开的第二方面所述的融合蛋白、本公开的第三方面所述的多特异性抗体、本公开的第四方面所述的缀合物、和/或本公开的第八方面所述的药物组合物。在一些实施方案中，所述代谢性疾病和/或症状为肥胖、超重和/或糖尿病。
在第十三方面，本公开提供了本公开的第一方面所述的GIPR结合蛋白、本公开的第二方面所述的融合蛋白、本公开的第三方面所述的多特异性抗体、本公开的第四方面所述的缀合物、和/或本公开的第八方面所述的药物组合物在制备用于治疗和/或预防代谢性疾病和/或症状的药物中的用途。在一些实施方案中，所述代谢性疾病和/或症状为肥胖、超重和/或糖尿病。
在第十四方面，本公开提供了用于治疗和/或预防代谢性疾病和/或症状中使用的本公开的第一方面所述的GIPR结合蛋白、本公开的第二方面所述的融合蛋白、本公开的第三方面所述的多特异性抗体、本公开的第四方面所述的缀合物、和/或本公开的第八方面所述的药物组合物。在一些实施方案中，所述代谢性疾病和/或症状为肥胖、超重和/或糖尿病。
本领域技术人员能够从下文的详细描述中容易地洞察到本公开的其它方面和优势。下文的详细描述中仅显示和描述了本公开的示例性实施方式。如本领域技术人员将认识到的，本公开的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本公开所涉及发明的精神和范围。相应地，本公开的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本公开所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本公开所涉及发明的特点和优势。对附图简要说明如下：
图1显示了GIPR单域抗体-Fc融合蛋白对GIPR的阻断活性，其中△代表iGI-72-Ld-Fc，○代表阳性对照AMG-GIPR-mab2。
图2显示了GIPR单域抗体-Fc融合蛋白多次给药对DIO小鼠体重的影响。
图3显示了GIPR单域抗体-Fc融合蛋白多次给药对DIO小鼠空腹血糖的影响。
图4显示了GIPR单域抗体-Fc融合蛋白多次给药对DIO小鼠胰岛素分泌的影响。
图5显示了GIPR单域抗体-Fc融合蛋白多次给药对DIO小鼠胰岛素抵抗指数的影响。
图6显示了人源化GIPR单域抗体-Fc融合蛋白的中和活性。
图7显示了人源化GIPR单域抗体-Fc融合蛋白对DA-GIP刺激的C57BL/6小鼠血糖的影响。
图8显示了人源化GIPR单域抗体-Fc融合蛋白对DA-GIP刺激的C57BL/6小鼠胰岛素分泌的影响。
图9显示了人源化GIPR单域抗体-Fc融合蛋白多次给药对ob小鼠体重的影响。
图10显示了人源化GIPR单域抗体-Fc融合蛋白与Maridebart对GIPR的结合能力。
图11显示了人源化GIPR单域抗体-Fc融合蛋白与Maridebart对GIPR的中和能力。
具体实施方式
以下由特定的具体实施例说明本公开发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本公开发明的其他优点及效果。
术语定义
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook等人,“Molecular Cloning:A Laboratory Manual”(第2版)，第1-3卷，Cold Spring Harbor Laboratory Press(1989)；Lewin,“Genes IV”，Oxford University Press,New York,(1990)；及Roitt等人,“Immunology”(第2版),Gower Medical Publishing,London,New York(1989)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
除非另有说明，否则可互换使用的术语“抗体”或“免疫球蛋白”，在本文中无论是指重链抗体还是指常规4链抗体，均用作一般术语以包括全长抗体、其单个的链以及其所有部分、结构域或片段(包括但不限于抗原结合结构域或片段，分别例如VHH结构域或VH/VL结构域)。此外，本文所用的术语“序列”(例如在“免疫球蛋白序列”、“抗体序列”、“单一可变结构域序列”、“VHH序列”或“蛋白质序列”等的术语中)一般应理解为既包括相关氨基酸序列，又包括编码所述序列的核酸序列或核苷酸序列，除非本文需要更限定的解释。
如本文所用，术语(多肽或蛋白质的)“结构域”是指折叠蛋白质结构，其能够独立于蛋白质的其余部分维持其三级结构。一般而言，结构域负责蛋白质的单个的功能性质，且在许多情况下可添加、移除或转移至其他蛋白质而不损失蛋白质的其余部分和/或结构域的功能。
如本文所用的术语“免疫球蛋白结构域”是指抗体链(例如常规4链抗体的链或重链抗体的链)的球形区域，或是指基本上由这类球形区域组成的多肽。免疫球蛋白结构域的特征在于其维持抗体分子的免疫球蛋白折叠特征。
如本文所用的术语“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予抗体对抗原的特异性。
如本文所用的术语“免疫球蛋白单一可变结构域”是指能够在不与其他免疫球蛋白可变结构域配对的情况下特异性结合抗原表位的免疫球蛋白可变结构域。本公开的免疫球蛋白单一可变结构域的一个实例为“结构域抗体”，例如免疫球蛋白单一可变结构域VH及VL(VH结构域及VL结构域)。免疫球蛋白单一可变结构域的另一实例为如下文定义的骆驼科的“VHH结构域”(或简称为“VHH”)。
“VHH结构域”，亦称为重链单域抗体、VHH、VHH抗体片段和VHH抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域(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))。使用术语“VHH结构域”以将所述可变结构域与存在于常规4链抗体中的重链可变结构域(其在本文中称为“VH结构域”)以及存在于常规4链抗体中的轻链可变结构域(其在本文中称为“VL结构域”)进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(此与常规4链抗体中的VH或VL结构域相反，在该情况下表位由VL结构域与VH结构域一起识别)。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。
在本公开的上下文中，术语“重链单域抗体”、“VHH结构域”、“VHH”、“VHH抗体片段”、以及“VHH抗体”可互换使用。
例如Riechmann及Muyldermans,J.Immunol.Methods 231,25-38(1999)的图2中所示，对于骆驼科的VHH结构域所应用的氨基酸残基，可以根据Kabat等人给出的VH结构域的一般编号法来编号(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))。
本领域中已知对VH结构域的氨基酸残基进行编号的替代方法，所述替代方法也可以类似地应用于VHH结构域。例如，Chothia CDR指的是结构环的位置(Chothia and Lesk,J.Mol.Biol.196:901-917(1987))。AbM CDR代表的是Kabat超变区和Chothia结构环的折中，并且在Oxford Molecular's AbM抗体建模软件中使用。“接触(Contact)”CDR则基于对可获得的复合物晶体结构的分析。来自每种方法的CDR的残基描述如下表1所示：
表1.根据各种编号系统的CDR的残基
抗体的CDR还可以是IMGT-CDR，这是一种基于IMGT抗体编码的CDR定义方式，该编码通过综合超过5000个序列的结构信息后获得。在IMGT的VH CDR编码中，CDR1：27-38；CDR2：56-65；CDR3：105-117。
然而应注意，如本领域中对于VH结构域及VHH结构域所公知的，各CDR中的氨基酸残基的总数可能不同，且可能不对应于由Kabat编号指示的氨基酸残基的总数(即根据Kabat编号的一个或多个位置可能在实际序列中未被占据，或实际序列可能含有多于Kabat编号所允许数目的氨基酸残基)。这意味着一般而言，根据Kabat的编号可能对应或可能不对应于实际序列中氨基酸残基的实际编号。
例如，CDR可以包括“扩展的(extended)CDR”，例如：在VL中的24-36或24-34(LCDR1)，46-56或50-56(LCDR2)以及89-97或89-96(LCDR3)；在VH中的26-35(HCDR1)，50-65或49-65(HCDR2)以及93-102、94-102或95-102(HCDR3)。
VHH结构域中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本文所述的目的。
VHH结构域及含有其的多肽的其他结构特性及功能性质可总结如下：
VHH结构域(其已经天然“设计”以在不存在轻链可变结构域且不与轻链可变结构域相互作用的情况下与抗原功能性结合)可用作单一且相对较小的功能性抗原结合结构单元、结构域或多肽。该特性区分VHH结构域与常规4链抗体的VH及VL结构域。这些VH及VL结构域自身通常不适于作为单一抗原结合蛋白或免疫球蛋白单一可变结构域进行实际应用，但需要以某种形式或另一形式组合以提供功能性抗原结合单元(如以诸如Fab片段等常规抗体片段的形式；或以由与VL结构域共价连接的VH结构域组成的scFv的形式)。
由于这些独特性质，使用VHH结构域—单独或作为较大多肽的一部分—提供许多优于使用常规VH及VL结构域、scFv或常规抗体片段(例如Fab-或F(ab’)2-片段)的显著优势：仅需要单一结构域以高亲和力及高选择性结合抗原，从而使得既不需要存在两个单独结构域，也不需要确保该两个结构域以适当空间构象及构型存在(例如scFv一般需要使用经特别设计的接头)；VHH结构域可自单一基因表达且不需要翻译后折叠或修饰；VHH结构域可容易地改造成多价及多特异性格式(格式化)；VHH结构域高度可溶且无聚集趋势；VHH结构域对热、pH、蛋白酶及其他变性剂或条件高度稳定，且因此可在制备、储存或运输中不使用冷冻设备，从而达成节约成本、时间及环境；VHH结构域易于制备且相对廉价，甚至在生产所需的规模上亦如此；VHH结构域与常规4链抗体及其抗原结合片段相比相对较小(大约15kDa或大小为常规IgG的1/10)，因此相比于常规4链抗体及其抗原结合片段，显示较高的组织渗透性且可以较高剂量给药；VHH结构域可显示所谓腔结合性质(尤其由于与常规VH结构域相比其延长的CDR3环)，从而可到达常规4链抗体及其抗原结合片段不可到达的靶及表位。
获得结合特定抗原或表位的VHH的方法，先前已公开于以下文献中：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.2645-2652,17June,2009和WO94/04678。
源自骆驼科的VHH结构域可通过以人常规4链抗体VH结构域中相应位置处存在的一个或多个氨基酸残基置换原始VHH序列的氨基酸序列中的一个或多个氨基酸残基而经“人源化”(本文中亦称为“序列优化”。除人源化外，“序列优化”也可涵盖通过提供VHH改良性质的一个或多个突变对序列进行的其他修饰，例如移除潜在的翻译后修饰位点)。人源化VHH结 构域可含有一个或多个完全人框架区序列。人源化可以使用蛋白表面氨基酸人源化(resurfacing)的方法和/或人源化通用框架CDR移植法(CDR grafting to a universal framework)完成，例如，如实施例中所示例。
一般而言，术语“特异性”是指特定抗原结合分子或抗原结合蛋白(例如本公开的免疫球蛋白单一可变结构域、重链单域抗体、或GIPR结合蛋白)可结合的不同类型抗原或表位的数目。可基于抗原结合蛋白的亲和力和/或亲合力确定其特异性。由抗原与抗原结合蛋白的解离平衡常数(KD)所表示的亲和力，是表位与抗原结合蛋白上的抗原结合位点之间结合强度的量度：KD值越小，表位与抗原结合蛋白之间的结合强度越强(或者，亲和力也可表示为缔合常数(KA)，其为1/KD)。如本领域技术人员将了解，取决于具体感兴趣的抗原，可以以已知方式测定亲和力。亲合力为抗原结合蛋白(例如免疫球蛋白、抗体、免疫球蛋白单一可变结构域或含有其的多肽)与相关抗原之间结合强度的量度。亲合力与以下两者有关：抗原结合蛋白上的抗原结合位点与相关抗原之间的亲和力，以及存在于抗原结合蛋白上的相关结合位点的数目。
如本文所用，术语“葡萄糖依赖性促胰岛素多肽受体”，简称为GIPR，涵盖任何物种的GIPR。优选地，GIPR为人GIPR或小鼠GIPR。
如本文所用，术语“葡萄糖依赖性促胰岛素多肽受体(GIPR)结合蛋白”意指任何能够特异性结合葡萄糖依赖性促胰岛素多肽受体(GIPR)的蛋白。GIPR结合蛋白可以包括针对GIPR的如本文定义的重链单域抗体。GIPR结合蛋白还涵盖免疫球蛋白超家族抗体(IgSF)或CDR移植分子。
本公开的“GIPR结合蛋白”可以包含至少一个结合GIPR的免疫球蛋白单一可变结构域如VHH。在一些实施方案中，本公开的“GIPR结合蛋白”可以包含2、3、4或更多个结合GIPR的免疫球蛋白单一可变结构域如VHH。本公开的GIPR结合蛋白除结合GIPR的免疫球蛋白单一可变结构域外也可包含接头和/或具有效应器功能的部分，例如半衰期延长部分(如结合血清白蛋白的免疫球蛋白单一可变结构域)、和/或融合配偶体(如血清白蛋白)和/或缀合的聚合物(如PEG)和/或Fc区。在一些实施方案中，本公开的“GIPR结合蛋白”还涵盖双特异性抗体，其含有结合不同抗原或相同抗原不同区域(如不同表位)的抗原结合结构域或免疫球蛋白单一可变结构域。
通常，本公开的GIPR结合蛋白将以如于Biacore或KinExA或Fortibio测定中测量的优选10-7至10-10摩尔/升(M)、更优选10-8至10-10摩尔/升、甚至更优选10-9至10-10或更低的解离常数(KD)，和/或以至少107M-1、优选至少108M-1、更优选至少109M-1，更优选至少1010M- 1的缔合常数(KA)结合所要结合的抗原(即GIPR)。任何大于10-4M的KD值一般都视为指示非特异性结合。抗原结合蛋白对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如本文所述的表面等离子体共振术(SPR)测定、Scatchard测定和/或竞争性结合测定(例如放射免疫测定(RIA)、酶免疫测定(EIA)及夹心式竞争性测定)。
氨基酸残基将根据如本领域中公知且达成一致的标准三字母或一字母氨基酸编码加以表示。在比较两个氨基酸序列时，术语“氨基酸差异”是指与另一序列相比，在参考序列某一位置处指定数目氨基酸残基的插入、缺失或取代。在取代的情况下，所述取代将优选为保守氨基酸取代，所述保守氨基酸是指氨基酸残基被化学结构类似的另一氨基酸残基置换，且其对多肽的功能、活性或其他生物性质影响较小或基本上无影响。所述保守氨基酸取代在本领域中是公知的，例如保守氨基酸取代优选是以下组(i)-(v)内的一个氨基酸被同一组内的另一氨基酸残基所取代：(i)较小脂族非极性或弱极性残基：Ala、Ser、Thr、Pro及Gly；(ii)极性带负电残基及其(不带电)酰胺：Asp、Asn、Glu及Gln；(iii)极性带正电残基：His、Arg及Lys；(iv)较大脂族非极性残基：Met、Leu、Ile、Val及Cys；及(v)芳族残基：Phe、Tyr及Trp。特别优选的保守氨基酸取代如下：Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。
两个多肽序列之间的“序列同一性”指示序列之间相同氨基酸的百分比。“序列相似性”指示相同或代表保守氨基酸取代的氨基酸的百分比。用于评价氨基酸或核苷酸之间的序列相同性程度的方法是本领域技术人员已知的。例如，氨基酸序列相同性通常使用序列分析软件来测量。例如，可使用NCBI数据库的BLAST程序来确定相同性。对于序列相同性的确定，可以参见例如：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988；Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993；Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994；Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987和Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991。
在本公开中，术语“分离的”通常是指大体上不含其天然存在的环境中通常伴随或与之相 互作用的组分的生物材料(例如病毒、核酸或蛋白质)。所述分离的生物材料任选地包含在其天然环境(例如，核酸或蛋白质)中所述生物材料未发现具有的另外的材料。在本公开中，当涉及蛋白质时，“分离的”通常是指所述的分子从发现该分子天然存在的整个生物体中分离和分开，或基本不存在其它相同类型的生物大分子。当涉及核酸分子时，它与天然与其结合的序列完全或部分分离，或该核酸具有与其结合的异源序列，或该核酸从染色体分离。
相比于其天然生物来源和/或获得该多肽或核酸分子的反应介质或培养基，当其已与至少一种在该来源或介质(培养基)中通常与之相关的其他组分(例如另一蛋白/多肽、另一核酸、另一生物组分或大分子或至少一种污染物、杂质或微量组分)分离时，多肽或核酸分子视为“分离的”。特别地，多肽或核酸分子在其已纯化至少2倍、特别是至少10倍、更特别是至少100倍且多达1000倍或1000倍以上时被视为“分离的”。经适合的技术(例如适合色谱技术，如聚丙烯酰胺凝胶电泳)确定，“分离的”多肽或核酸分子优选基本上为均质的。
“有效量”意指本公开的GIPR结合蛋白或药物组合物的量能导致疾病症状的严重性降低，疾病无症状期的频率和持续时间增加，或者防止因疾病痛苦而引起的损伤或失能。
如本文所用，“代谢性疾病”是指一种影响人类(或动物)细胞生成能量的障碍，又被称为新陈代谢失调症。大部分代谢性疾病为遗传性疾病，而有部分则是因饮食、毒素、感染而获得。常见的代谢性疾病主要可分为三大类：影响糖类代谢的障碍、影响脂肪代谢的障碍以及影响细胞内线粒体的障碍。
如本文所用的术语“对象”意指哺乳动物，尤其灵长类动物，尤其是人。
在本公开中，术语“抗原结合蛋白”通常是指包含结合抗原部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。抗原结合蛋白可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者，及其功能性片段。在本公开中，术语“抗原结合蛋白”还涵盖单域抗体以及包含免疫球蛋白单一可变结构域的蛋白质。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗原结合蛋白的实例包括但不限于抗体、抗原结合片段、单域抗体、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本公开中，术语“多肽”或“蛋白质”可互换地使用，通常是指氨基酸残基的聚合物。该术语也适用于其中一个或多个氨基酸残基是相应的天然存在的氨基酸的类似物或模拟物的氨基酸聚合物、以及天然存在的氨基酸聚合物。该术语也可包括修饰的氨基酸聚合物，例如，通 过添加糖残基以形成糖蛋白或被磷酸化修饰。多肽和蛋白质可由天然存在的和非重组的细胞或由遗传工程改造的或重组的细胞产生，并且可包含具有天然蛋白质的氨基酸序列的分子、或具有天然序列的一个或多个氨基酸的缺失、添加和/或取代的分子。术语“多肽”和“蛋白质”特别包括本公开所述的抗原结合蛋白的一个或多个氨基酸的缺失、添加和/或取代的序列。
在本公开中，术语“缀合物”通常是指抗原结合蛋白与其它活性剂连接形成的物质，其他活性剂可以是小分子活性剂，例如治疗剂、成像探针或光谱探针。
在本公开中，术语“核酸”分子通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本公开中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本公开中，术语“细胞”通常是指可以包含或已经含有包括本公开所述的核酸分子的质粒或载体，或者能够表达本公开所述的抗原结合蛋白的个体细胞、细胞系或细胞培养物。所述细胞可以包括单个宿主细胞的子代。由于天然的、意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本公开所述的抗体或其抗原结合片段即可。所述细胞可以通过使用本公开所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在某些情形中，所述细胞可以是哺乳动物细胞。例如，所述哺乳动物细胞可以是CHO-K1细胞。
在本公开中，术语“药物组合物”通常是指这样的制剂，其以允许活性成分的生物学活性有效的形式存在，并且不包含对将施用所述组合物的对象具有不可接受的毒性的另外的成分。
在本公开中，术语“治疗”通常是指期望改变所治疗个体的天然病程，且可为实现防治或在临床病变过程中进行的临床介入。合乎需要的治疗效果包括但不限于防止疾病发生或复发性、减轻症状、减弱疾病的任何直接或间接病理学后果、防止转移、降低疾病进展速率、改善或缓解疾病状态以及缓和或改善预后。在一些情形中，抗原结合蛋白(例如，抗本公开特 定抗原的抗体)可用来延迟疾病发展或减缓疾病进展。
在本公开中，术语“施用”通常是指向受试者(例如，患者)给予一定剂量的化合物或药物组合物的方法。施用可通过任何合适的方式进行，包括肠胃外、肺内和鼻内，以及(如果局部治疗需要)损伤内施用。胃肠外输注包括例如肌肉内、静脉内、动脉内、腹膜内或皮下施用。
在本公开中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本公开中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下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％、或10％的范围内变动。
本公开的GIPR结合蛋白
一方面，本公开涉及一种葡萄糖依赖性促胰岛素多肽受体(GIPR)结合蛋白，其包含至少一个免疫球蛋白单一可变结构域。
在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域可以包含SEQ ID NO:1-7任一所示的VHH中的CDR1、CDR2和CDR3。所述CDR可以是按照以下任一种定义系统定义的：Kabat CDR、AbM CDR、Chothia CDR或IMGT CDR。
在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域包含选自以下表2的一组或多组的CDR1、CDR2和CDR3：
表2.本发明的VHH的CDR

在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5和/或SEQ ID NO:6所示的VHH中的CDR1、CDR2和CDR3。
在一些实施方案中，本公开的GIPR结合蛋白包含至少一个免疫球蛋白单一可变结构域，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5和SEQ ID NO:8-14所示氨基酸序列中的一个或多个。“多个”可以是2、3、4或5个，多个氨基酸序列可以相同，也可以不相同。
在一个具体的实施方案中，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5和SEQ ID NO:8-14之一的所示氨基酸序列。例如，本公开的GIPR结合蛋白包含以下通式所包含的序列(其中[]所指的是在该位置可以选择的氨基酸种类)：
在另一些实施方案中，本公开的GIPR结合蛋白包含至少一个免疫球蛋白单一可变结构域，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:6和SEQ ID NO:15-25所示氨基酸序列中的一个或多个。
在一个具体的实施方案中，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:6、SEQ ID NO:15-25之一的所示氨基酸序列。例如，本公开的GIPR结合蛋白包含以下通式所包含的序列(其中[]所指的是在该位置可以选择的氨基酸种类)：
在一些实施方案中的，所述免疫球蛋白单一可变结构域为人源化的VHH，所述人源化的VHH包含与SEQ ID NO:5或6具有至少80％、优选地至少90％、更优选地至少95％、甚至 更优选地至少99％序列同一性的氨基酸序列。在一些实施方案中，所述人源化的VHH的氨基酸序列与SEQ ID NO:5或6相比包含一个或多个氨基酸取代，优选保守氨基酸取代。例如，所述人源化的免疫球蛋白单一可变结构域的氨基酸序列与SEQ ID NO:5或6相比包含1、2、3、4、5、6、7、8、9或10个保守氨基酸取代。
在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域可同时包含SEQ ID NO:5和SEQ ID NO:8-14所示氨基酸序列中的一个或多个，以及SEQ ID NO:6和SEQ ID NO:15-25所示氨基酸序列中的一个或多个。
在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域可同时包含SEQ ID NO:5和SEQ ID NO:8-14之一的所示的氨基酸序列，以及SEQ ID NO:6、SEQ ID NO:15-25之一的所示的氨基酸序列。
在一些实施方案中，本公开的GIPR结合蛋白，除了至少一个免疫球蛋白单一可变结构域外，还包含免疫球蛋白Fc区。在本公开的GIPR结合蛋白中包含免疫球蛋白Fc区可以使所述结合分子形成二聚体。可用于本公开的Fc区可以来自不同亚型的免疫球蛋白，例如，IgG(例如，IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM。
在一些实施方案中，可以在野生型的Fc序列上引入突变用于改变Fc介导的相关活性。所述突变包括但不限于：a).改变Fc介导的CDC活性的突变；b).改变Fc介导的ADCC活性的突变；或c).改变FcRn介导的体内半衰期的突变。此类突变描述于下列文献中：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；Paul R.Hinton et al.,J Immunol,2006,176:346-356。例如，可以通过突变CH2区上的1、2、3、4、5、6、7、8、9或10个氨基酸用于增加或去除Fc介导的ADCC或CDC活性或是增强或减弱FcRn的亲和力。此外，可以通过突变铰链区的1、2、3、4、5、6、7、8、9或10个氨基酸增加蛋白的稳定性。
在一些实施方案中，Fc序列上可以引入突变，从而使得突变的Fc更容易形成同二聚体或者异二聚体。如Ridgway,Presta et al.1996以及Carter 2001中提到的利用Fc接触界面氨基酸侧链基团空间作用的knob-hole模型，使得不同Fc突变之间更容易形成异二聚体；再比如如CN102558355A或者CN103388013A中，通过改变Fc接触界面氨基酸所带的电荷，进而改变Fc接触界面之间的离子相互作用力，使得不同的Fc突变对之间更容易形成异二聚体(CN102558355A)，亦或是具有相同突变的Fc之间更容易形成同二聚体(CN 103388013A)。
所述免疫球蛋白Fc区优选是人免疫球蛋白Fc区，例如是人IgG1、IgG2、IgG3、或IgG4的Fc区。在一些具体实施方案中，所述免疫球蛋白Fc区的氨基酸序列示于SEQ ID NO:110或SEQ ID NO:113。
在一些具体实施方案中，本公开的GIPR结合蛋白中，所述免疫球蛋白Fc区(例如人IgG1的Fc区)直接或经由接头间接连接至所述免疫球蛋白单一可变结构域(如VHH)的C端。
在一些实施方案中，本公开的GIPR结合蛋白包含一个特异性结合GIPR的免疫球蛋白单一可变结构域，所述特异性结合GIPR的免疫球蛋白单一可变结构域直接或间接与免疫球蛋白Fc区连接，所述免疫球蛋白Fc区允许所述GIPR结合蛋白形成包含两个GIPR结合结构域的二聚体分子。这样的GIPR结合蛋白也称为二价GIPR结合蛋白。在一些实施方案中，所述二聚体是同二聚体。
在一些实施方案中，本公开的GIPR结合蛋白包含直接或经由接头间接相互连接的两个特异性结合GIPR的免疫球蛋白单一可变结构域和一个免疫球蛋白Fc区，所述免疫球蛋白Fc区允许所述GIPR结合蛋白形成包含四个GIPR结合结构域的二聚体分子。这样的GIPR结合蛋白也称为四价GIPR结合蛋白。在一些实施方案中，所述二聚体是同二聚体。在一些实施方案中，所述GIPR结合蛋白中的两个特异性结合GIPR的免疫球蛋白单一可变结构域分别结合GIPR的不同区域或不同表位。
本公开的多个免疫球蛋白单一可变结构域相互之间，以及免疫球蛋白单一可变结构域与免疫球蛋白Fc区之间可直接连接，或通过接头间接连接。所述接头可以是长1-20或更多个氨基酸、无二级以上结构的非功能性氨基酸序列。例如，所述接头是柔性接头，例如(GGG)n、(GGGS)n、(GGGA)n、(GGGAA)n和(GGGGS)n中的一个或多个，其中n为选自1至30的整数，选自1至20的整数，选自1至10的整数，选自1至9的整数，选自1至8的整数，选自1至7的整数，选自1至6的整数，选自1至5的整数，选自1至4的整数，或选自1至3的整数。在一些实施方案中，所述接头是GGGGS、GS、GAP、(GGGGS)x 3等。
在一些实施方案中，本公开的GIPR结合蛋白，其具有下述特征中的至少一项：
(a)与人GIPR具有特异性的结合；
(b)与人GIPR结合的KD值小于1x10-7M，优选小于1x10-8M；
(c)阻断GIP与GIPR的相互作用；
(d)能够降低受试者的体重和/或血糖。
融合蛋白
另一方面，本公开还涉及一种融合蛋白，所述融合蛋白包含本公开所述的GIPR结合蛋白。
在一些实施方案中，除所述GIPR结合蛋白外，所述融合蛋白还包含另一种或多种生物活性蛋白，所述生物活性蛋白可以具有有生物学、治疗、预防或诊断意义或功能的任何蛋白质，当其被施用于受试者时，介导生物活性可预防或缓解疾病、障碍或病症。具体来说，所述生物活性蛋白可以是激动剂、拮抗剂、调节剂、配体、细胞因子、酶或者激素，特别是可用于预防和/或代谢性疾病，尤其是肥胖、超重和/或糖尿病的生物活性蛋白。
多特异性抗体
另一方面，本公开涉及还涉及一种多特异性抗体，所述多特异性抗体包含本公开所述的GIPR结合蛋白。
在一些实施方案中，除所述GIPR结合蛋白外，所述多特异性抗体还包含另外的一个或多个抗原结合区，所述另外的一个或多个抗原结合区与所述GIPR结合蛋白和/或所述融合蛋白结合不同的抗原或相同抗原的不同表位。
核酸、载体、宿主细胞
在另一方面中，本公开涉及编码本公开的GIPR结合蛋白或融合蛋白的核酸分子。本公开的核酸可为RNA、DNA或cDNA。根据本公开的一个实施方案，本公开的核酸是基本上分离的核酸。
本公开的核酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒或YAC。载体可尤其为表达载体，即可提供GIPR结合蛋白体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本公开的核酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主中的表达对所述元件及其序列进行选择为本领域技术人员的常识。对本公开的GIPR结合蛋白的表达有用或必需的调控元件及其他元件的具体实例，例如启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
本公开的核酸可基于关于本文给出的本公开的多肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
在另一方面中，本公开涉及表达或能够表达一种或多种本公开的GIPR结合蛋白、融合蛋白和/或含有本公开的核酸或载体的重组宿主细胞。本公开的优选宿主细胞为细菌细胞、真 菌细胞或哺乳动物细胞。
适合的细菌细胞包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
适合的真菌细胞包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
适合的哺乳动物细胞包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、或COS细胞等。
然而，本公开也可使用两栖类细胞、昆虫细胞等已知细胞及本领域中用于表达异源蛋白的任何其他细胞。
本公开的GIPR结合蛋白或融合蛋白可在如上所述细胞中以细胞内方式(例如在细胞质中、在周质中或在包涵体中)产生，接着从宿主细胞分离且任选进一步纯化；或其可以细胞外方式(例如在培养宿主细胞的培养基中)产生，接着自培养基分离且任选进一步纯化。
用于重组产生多肽的方法及试剂，例如特定适合表达载体、转化或转染方法、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。类似地，适用于制造本公开的GIPR结合蛋白的方法中的蛋白分离及纯化技术为本领域技术人员所公知。
药物组合物
另一方面，本公开提供一种组合物，例如药物组合物，其含有与药学上可接受的载剂配制在一起的一种或组合的本公开的GIPR结合蛋白、融合蛋白、多特异性抗体、或缀合物。
本文使用的“药学上可接受的载剂”包括生理学相容的任何和所有的溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、缓冲剂、稳定剂、等渗剂和吸收延迟剂等。优选地，该载剂适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。根据施用途径，可将活性化合物即抗体分子包裹于一种材料中，以保护该化合物免受可使该化合物失活的酸和其他天然条件的作用。
可以与载剂材料组合制备单一剂量形式的活性成分的量根据所治疗的对象和特定给药方 式而不同。可以与载剂材料组合制备单一剂量形式的活性成分的量一般是产生治疗效果的组合物的量。通常，以100％计，这个量的范围是大约0.01％至大约99％的活性成分，例如大约0.1％至大约70％，或大约1％至大约30％的活性成分，与药学上可接受的载剂相组合。
本公开药物组合物中活性成分的实际剂量水平可能改变，以获得可有效实现对特定患者、组合物和给药方式的所需治疗反应，而对患者无毒性的活性成分的量。选择的剂量水平取决于多种药物代谢动力学因素，包括应用的本公开特定组合物或其酯、盐或酰胺的活性，给药途径，给药时间，应用的特定化合物的排泄速率，治疗的持续时间，与应用的特定组合物联合应用的其他药物、化合物和/或材料，接受治疗的患者的年龄、性别、体重、状况、总体健康情况和病史，以及医学领域中公知的类似因素。
本公开的组合物可以利用本领域公知的一种或多种方法通过一种或多种给药途径给药。本领域技术人员应当理解，给药途径和/或方式根据期望的结果而不同。本公开GIPR结合蛋白的优选给药途径包括静脉内、肌肉内、皮内、腹膜内、皮下、脊柱或其他肠胃外给药途径，例如注射或输注。本文使用的短语“肠胃外给药”是指除肠和局部给药以外的给药模式，通常是注射，包括但不限于静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注。
预防和/或治疗疾病
在另一方面，本公开还提供了一种预防和/或治疗疾病，特别是治疗和/或预防代谢性疾病的方法，包括向有需要的对象施用有效量的本公开的GIPR结合蛋白、融合蛋白、多特异性抗体、缀合物或药物组合物。所述代谢性疾病包括肥胖、超重和/或糖尿病。
在一些实施方案中，在预防和/或治疗代谢性疾病的过程中，除本公开的GIPR结合蛋白、融合蛋白、多特异性抗体、缀合物或药物组合物外，还可向有需要的对象施用其他治疗药物，例如胰岛素(包括猪或牛胰岛素、人胰岛素、门冬胰岛素、赖脯胰岛素、低精蛋白锌胰岛素、甘精胰岛素、地特胰岛素、LY3209590等)、双胍类(如二甲双胍等)、噻唑烷二酮类(包括吡格列酮、罗格列酮等)、硫酰基尿素类(包括格列吡嗪、格列齐特、格列本脲、格列波脲、格列喹酮、格列派特、格列美脲等)、苯丙胺酸类(包括瑞格列奈、那格列奈、米格列奈等)、α-葡萄糖苷酶抑制剂(包括阿卡波糖、伏格列波糖等)、DPP4抑制剂(包括西格列汀、利格列汀、戈格列汀、维格列汀、沙格列汀、阿格列汀、吉格列汀、替格列汀等)、SGLT2抑制剂(包括达格列净、坎格列净、恩格列净等)、奥利司他、GCGR激动剂，以及GLP-1R激动剂等。
其中，所述GLP-1R激动剂可以为：GLP-1(7-37)、GLP-1(7-36)-NH2、利拉鲁肽、阿必鲁肽、利司那肽、度拉糖肽、司美格鲁特、Exendin-4、Exendin-3、聚乙二醇洛塞那肽、替尔泊肽、GMA102、PB119、艾本那肽、Mazdutide、苏帕鲁肽、Cotadutide、Danuglipron、HM-15211、Efinopegdutide等。
所述其他治疗药物与本公开的结合蛋白/融合蛋白/多特异性抗体/缀合物/药物组合物可同时施用，也可先后施用；先后施用时，两者的施用间隔不超过1周，优选不超过3日，更优选不超过2日，甚至更优选不超过1日。
在另一些实施方案中，本公开还提供了本公开的GIPR结合蛋白、融合蛋白、多特异性抗体、缀合物或药物组合物在制备治疗和/或预防代谢性疾病药物中的用途。
而在另一些实施方案中，本公开提供了一种GIPR结合蛋白、融合蛋白、多特异性抗体、缀合物或药物组合物，其用于治疗和/或预防代谢性疾病。
在另一些实施方案中，本公开提供了一种用于检测GIPR蛋白的存在和/或含量的方法，其包括提供所述GIPR结合蛋白和/或所述融合蛋白和/或所述多特异性抗体和/或所述缀合物。例如，所述方法可以是离体或体外方法。例如，所述方法可以是非治疗目的的方法。
在一些实施方案中，本公开提供了一种用于检测样品中GIPR蛋白的存在的方法，其包括使所述样品与所述GIPR结合蛋白和/或所述融合蛋白和/或所述多特异性抗体和/或所述缀合物接触，所述GIPR结合蛋白和/或所述融合蛋白和/或所述多特异性抗体和/或所述缀合物与所述GIPR蛋白的结合指示所述样品中存在所述GIPR蛋白。
在一些实施方案中，本公开提供了一种用于检测样品中GIPR蛋白的含量的方法，其包括使所述样品与所述GIPR结合蛋白和/或所述融合蛋白和/或所述多特异性抗体和/或所述缀合物接触，所述GIPR结合蛋白和/或所述融合蛋白和/或所述多特异性抗体和/或所述缀合物与所述GIPR蛋白的结合的量指示所述样品中所述GIPR蛋白的含量。
本发明还提供了本公开的GIPR结合蛋白、融合蛋白、多特异性抗体或缀合物在制备用于检测样品中GIPR蛋白的存在和/或含量的试剂盒中的用途。
所述样品可以是血液、血浆、羊水等样品。
另一方面，本公开提供了一种试剂盒，其可以包含本公开所述的GIPR结合蛋白、融合蛋白、多特异性抗体、缀合物和/或药物组合物。其可在单一常用容器中包括本公开所述的GIPR结合蛋白、融合蛋白、多特异性抗体、缀合物和/或药物组合物，也可任选地与一种或多种治疗剂组合，任选地一起配制于药物组合物中。
另一方面，本公开提供了一种给药装置，它可以用来施用本公开所述的GIPR结合蛋白、融合蛋白、多特异性抗体、缀合物和/或药物组合物。
另一方面，本公开提供了以下实施方案：
1.一种葡萄糖依赖性促胰岛素多肽受体GIPR结合蛋白，其包含至少一个免疫球蛋白单一可变结构域，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5和/或6所示的VHH中的CDR1、CDR2和CDR3。
2.根据实施方案1所述的GIPR结合蛋白，其中所述CDR根据以下定义系统定义：Kabat、AbM、Chothia或IMGT。
3.根据实施方案1所述的GIPR结合蛋白，其中SEQ ID NO:5所示的VHH中的CDR1、CDR2和CDR3选自以下的任意一组：SEQ ID NO:74-76、SEQ ID NO:77-79、SEQ ID NO:80-82和SEQ ID NO:83-85。
4.根据实施方案3所述的GIPR结合蛋白，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5、SEQ ID NO:8-14所示氨基酸序列中的一个或多个。
5.根据实施方案4所述的GIPR结合蛋白，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5、SEQ ID NO:8-14之一的所示氨基酸序列。
6.根据实施方案1所述的GIPR结合蛋白，其中SEQ ID NO:6所示的VHH中的CDR1、CDR2和CDR3选自以下的任意一组：SEQ ID NO:86-88、SEQ ID NO:89-91、SEQ ID NO:92-94和SEQ ID NO:95-97。
7.根据实施方案6所述的GIPR结合蛋白，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:6、SEQ ID NO:15-25所示氨基酸序列中的一个或多个。
8.根据实施方案7所述的GIPR结合蛋白，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:6、SEQ ID NO:15-25之一的所示氨基酸序列。
9.根据实施方案1-8中任一所述的GIPR结合蛋白，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5、SEQ ID NO:8-14之一的所示氨基酸序列，和/或SEQ ID NO:6、SEQ ID NO:15-25之一的所示氨基酸序列。
10.根据实施方案1-9中任一所述的GIPR结合蛋白，其还包含免疫球蛋白Fc区，优选为人免疫球蛋白Fc区，更优选为人IgG1、IgG2、IgG3或IgG4的Fc区。
11.根据实施方案10所述的GIPR结合蛋白，所述免疫球蛋白Fc区的氨基酸序列如SEQ ID NO:110或SEQ ID NO:113所示。
12.根据实施方案10或11所述的GIPR结合蛋白，所述免疫球蛋白Fc区与所述至少一个免 疫球蛋白单一可变结构域直接连接或通过接头间接连接。
13.根据实施方案1-12中任一所述的GIPR结合蛋白，其具有下述特征中的至少一项：
(a)与人GIPR具有特异性的结合；
(b)与人GIPR结合的KD值小于1x10-7M，优选小于1x10-8M；
(c)阻断GIP与GIPR的相互作用；和
(d)能够降低受试者的体重和/或血糖。
14.一种融合蛋白，其包含实施方案1-13中任一所述的GIPR结合蛋白。
15.一种缀合物，其包含实施方案1-13中任一所述GIPR结合蛋白和/或实施方案14所述融合蛋白。
16.一种核酸分子，其编码实施方案1-13中任一所述GIPR结合蛋白和/或实施方案14所述融合蛋白。
17.一种表达载体，其包含与表达调控元件可操作地连接的实施方案15的核酸分子。
18.一种重组细胞，其包含实施方案15的核酸分子和/或以实施方案16的表达载体转化，并能够表达所述GIPR结合蛋白或所述包含GIPR结合蛋白的融合蛋白。
19.一种药物组合物，其包含实施方案1-13中任一所述的GIPR结合蛋白、实施方案14所述融合蛋白和/或实施方案15所述的缀合物，以及药学上可接受的载剂。
20.一种试剂盒，其包含实施方案1-13中任一所述的GIPR结合蛋白、实施方案14所述融合蛋白、实施方案15所述的缀合物和/或实施方案19所述的药物组合物。
21.一种确定GIPR蛋白存在和/或含量、和/或抑制GIPR蛋白与其配体结合的方法，包含提供实施方案1-13中任一所述的GIPR结合蛋白、实施方案14所述融合蛋白和/或实施方案15所述的缀合物。
22.一种治疗和/或预防代谢性疾病和/或症状的方法，其包括向有需要的对象施用有效量的实施方案1-13中任一所述的GIPR结合蛋白、实施方案14所述的融合蛋白、实施方案15所述的缀合物或实施方案18所述的药物组合物。
23.根据实施方案22的方法，所述代谢性疾病和/或症状为肥胖、超重和/或糖尿病。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本公开发明的各个技术方案，而不用于限制本公开发明的范围。
实施例
实施例1
针对GIPR重链单域抗体的筛选
1.1文库的构建
免疫前收集50mL羊驼动脉血于真空采血管，收集上清作为免疫前血清。选取健康羊驼，取包含人GIPR的抗原对羊驼采用颈部肌肉多点注射，每两周免疫一次，共计免疫六次。最后一次免疫结束时，收集50mL羊驼动脉血于真空采血管内，收集上清作为免疫后血清。
利用密度梯度离心法分离出淋巴细胞，使用QIAGEN公司提供的RNA提取试剂盒提取总RNA。使用Super-Script III FIRST STRANDSUPERMIX试剂盒按照说明书将提取的RNA全部反转录为cDNA，用巢式PCR扩增编码重链抗体的可变区的核酸片段。
回收目标重链单域抗体的核酸片段，并使用限制性内切酶SfiI将其克隆进入噬菌体展示用载体pComb3XSS中。产物随后电转化至大肠杆菌电转感受态细胞TG1中，构建抗GIPR的免疫单域抗体噬菌体展示文库并对文库进行鉴定。通过梯度稀释铺板，计算库容的大小为1.69×109。为检测文库的插入率，随机挑选50个克隆测序检测，具有正确的外源片段插入的克隆为50个，正确率为100％。通过对测序克隆的DNA和氨基酸序列分别分析比对，证实所有序列均为羊驼VHH序列，可估测其多样性在95％以上。
1.2针对GIPR重链单域抗体的淘选
将实施例1.1获得的噬菌体文库进行淘选，采用蛋白GIPR-ECD-muFc(小鼠Fc融合的人GIPR胞外区片段)、GIPR-ECD-chis(his标签融合的人GIPR胞外区片段),及GIPR-ECD-laFc(羊驼Fc融合的人GIPR胞外区片段)筛选出结合的噬菌体，同时，使用包含muFc的融合蛋白Control-1(C1)和包含laFc的融合蛋白Control-2(C2)进行负向淘选，去除非特异性结合的噬菌体。
将以上淘选后获得的结合阳性的噬菌体感染空白大肠杆菌并铺板。随后挑选菌落分别接种至2TY-AG(含10％甘油)，室温静置过夜。次日，按1％接种量分别转接至200μL 2TY-AG中，37℃，250rpm，至OD600为0.5左右，加入辅助噬菌体M13KO7进行感染(感染指数为1:20)，37℃静置15min，之后220rpm，45min。每孔加入800μL 2TY-AG，30℃，220rpm，过夜，次日离心，收集上清用于ELISA检测。分别用GIPR-ECD-Chis、GIPR-ECD-laFc、C1和 C2包被平板4℃过夜，将获得的上清液加入，室温下反应2小时。洗涤之后加入二抗Goat anti-HA tag HRP(购自Abcam)，室温反应2小时。洗涤之后加入TMB显色液，读取450nm和650nm波长吸收值，450nm波长的吸光度值减去650nm波长的吸光度值为最终的吸光度值。
淘选所得的297个克隆中，OD值>1.0的阳性克隆有191个，其中与GIPR-ECD-laFc具有特异性结合的部分阳性克隆如表3所示。将上述具有特异性结合的阳性克隆进行测序，得到抗体序列。
表3.与GIPR-ECD-laFc具有特异性结合的阳性克隆

实施例2
用哺乳动物细胞制备GIPR单域抗体的Fc融合蛋白
2.1制备表达GIPR单域抗体-Fc融合蛋白的质粒
设计引物进行PCR扩增GIPR单域抗体VHH片段，与编码人IgG1-Fc的DNA片段(氨基酸序列SEQ ID NO:110)融合，并克隆至常规哺乳动物表达载体，获得用于在哺乳动物中表达GIPR单域抗体-Fc融合蛋白的重组质粒。其中使用通用引物扩增不同VHH片段。通用引物如下：
上游引物cccACCGGTCAGGTGCAGCTGCAGGAGTC(SEQ ID NO:111)
下游引物cccGGATCCTGAGGAGACGGTGACCTGG(SEQ ID NO:112)
2.2制备GIPR单域抗体的Fc融合蛋白
将2.1构建获得的质粒载体转染至HEK293细胞进行抗体的瞬时表达。将重组表达质粒用Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液，将每组质粒/PEI混合物分别加入HEK293细胞悬液中，放置在37℃，5％CO2，悬浮培养。培养5～6天后，收集瞬时表达培养物的上清液，通过Protein A亲和层析法，纯化得到目标GIPR单域抗体-Fc融合蛋白，通过SDS-PAGE检测蛋白纯度。各蛋白的表达量见表4，并且一步纯化后各蛋白的SDS纯度都大于95％。
表4.GIPR单域抗体的Fc融合蛋白的表达量

实施例3
鉴定GIPR单域抗体-Fc融合蛋白的功能
3.1 GIPR单域抗体-Fc融合蛋白的GIPR的结合能力
向细胞培养板中铺293T-GIPR细胞(表达人GIPR的HEK293T细胞)，加入GIPR单域抗体-Fc融合蛋白样品，终浓度为100μg/ml和10μg/ml，2个浓度。加入抗人IgG-APC二抗后孵育，使用荧光激活细胞分选仪检测平均荧光强度(MFI)。结果如表5所示。
表5.GIPR单域抗体的Fc融合蛋白的结合GIPR的能力

3.2 GIPR单域抗体-Fc融合蛋白的中和活性
(1)96孔板中分别加入GIP-C12H4-Fc4(人GIP-IgG4Fc融合蛋白)以及25μL GIPR单域抗体-Fc样品。然后加入293T-GIPR细胞，孵育之后用cAMP试剂盒检测，酶标仪检测450nm/550nm的吸光度。结果如表6所示。
表6.GIPR单域抗体-Fc融合蛋白的中和活性
(2)96孔板中分别加入GIP-C12H4-Fc4，以及25μL GIPR单域抗体-Fc样品；然后加入细胞膜上稳定表达全长人GIPR蛋白的293T细胞293T-GIPRFL-GFP-PURO-5细胞，孵育之后用cAMP试剂盒检测，读取RLU值，计算得到中和活性(％)。结果如表7所示。
表7.GIPR单域抗体-Fc融合蛋白的中和活性
3.3检测GIPR单域抗体-Fc融合蛋白的亲和力
通过生物膜干涉(Biolayerinterferometry，BLI)技术，检测GIPR单域抗体-Fc融合蛋白对GIPR-ECD-chis的结合动力学。GIPR单域抗体-Fc融合蛋白直接固化到AHC sensor上，然后将GIPR-ECD-chis稀释至7个浓度，和固化的GIPR单域抗体-Fc融合蛋白进行结合。使用Octet K2数据分析软件9.0计算平衡解离常数(KD)、结合速率(Ka)和解离速率(Kdis)。结果如表8和表9所示。
表8.GIPR单域抗体-Fc融合蛋白的亲和力
表9.GIPR单域抗体-Fc融合蛋白的亲和力
3.4检测GIPR单域抗体-Fc融合蛋白的阻断活性
包被5μg/ml小鼠GIPR-Chis蛋白，BSA封闭之后加入梯度稀释的AMG-GIPR-mab2和iGI72-Ld-Fc充分孵育，稀释液中含有1.2μg/mL GIP-C12H4-Fc4。再加入1:2000稀释的Mouse Anti-Human IgG4 pFc’[HP6023](HRP)(货号ab99817)，TMB显色后于酶标仪上检测。结果如图1所示。
阳性对照AMG-GIPR-mab2参照文献MAbs.2020 Jan-Dec；12(1):1710047中的序列进行基因合成后，根据上述方法通过293细胞进行瞬时表达制备获得。
3.5检测GIPR单域抗体-Fc融合蛋白与空细胞的非特异结合
CHOK1空细胞重悬于3％BSA-PBS，选取GIPR单域抗体-Fc融合蛋白终浓度为5μg/mL和50μg/mL，同时设置阴性对照和空白对照。洗涤后加入二抗APC anti-human IgG Fc。洗涤后将细胞重悬于PBS-BSA Buffer中，流式细胞仪进行检测。结果如表10所示，iGI-72-Ld-Fc 与iGI-1198-Ld-Fc无非特异性结合，而iGI-1225-Ld-Fc与iGI-1225NA-Ld-Fc存在非特异性结合。
表10.GIPR单域抗体-Fc融合蛋白与空细胞的非特异结合
3.6 GIPR单域抗体-Fc融合蛋白的体内药效研究
16只DIO小鼠在16周龄禁食过夜后，测量动物的血糖和体重，随机分成4组。分组给药当天记为D0，每周2次分别给予PBS、Dulaglutide 17nmol/kg、iGI-72-Ld-Fc 375nmol/kg、Dulaglutide 17nmol/kg+iGI-72-Ld-Fc 17+375nmol/kg，共10次。给药后每周一次一般临床观察、每周两次称量体重，每周1次测空腹血糖及空腹胰岛素。结果如图2至图5所示，iGI-72-Ld-Fc处理组明显降低DIO小鼠体重，同时改善空腹血糖、空腹胰岛素和胰岛素抵抗HOMA-IR，iGI-72-Ld-Fc和dulaglutide联用组可以进一步增强药效，具有协同作用。
实施例4
GIPR单域抗体的人源化
人源化方法采用蛋白表面氨基酸人源化(resurfacing)的方法及VHH人源化通用框架移植法(CDR grafting to a universal framework)完成。
首先获取Cécile Vincke等人根据序列同源性设计完成的通用性人源化VHH框架hNbBcII10FGLA(PDB编号为：3EAK)，该框架设计基于纳米抗体NbBcII10抗体(PDB编号为：3DWT)。使用Modeller9进行建模，根据蛋白三维结构计算框架上氨基酸的相对溶剂可及性(relative solvent accessibility)。
VHH人源化通用框架移植法具体步骤如下：通过IMGT获取高度同源的人源抗体序列，并参考通用性人源化VHH框架hNbBcII10FGLA(PDB编号为：3EAK)，对目标序列进行人 源化。使用高度同源序列框架作为框架模板，将CDR替换为目标抗体株的CDR区。然后根据建模，将框架上的非表面氨基酸进行回复突变，完成目标抗体人源化。
对抗体株iGI-72和iGI-1198进行人源化，分别获得8种和11种抗体株的人源化变体huGI。
实施例5
人源化单域抗体的Fc融合蛋白的制备
5.1制备人源化单域抗体的Fc融合质粒
将实施例4中的人源化序列进行基因合成，与编码人IgG1-Fc的DNA片段融合，并克隆至常规哺乳动物表达载体，获得用于在哺乳动物中表达GIPR单域抗体Fc融合蛋白的重组质粒。
5.2制备人源化单域抗体的Fc融合蛋白
将5.1构建获得载体转染至HEK293细胞进行抗体的瞬时表达。将重组表达质粒用Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液，将每组质粒/PEI混合物分别加入HEK293细胞悬液中，放置在37℃，5％CO2，130rpm中培养。四小时后补加EXCELL293培养基，2mM谷氨酰胺，130rpm培养。24小时后加3.8mM VPA，72小时后加入4g/L葡萄糖。培养5～6天后，收集瞬时表达培养上清液，通过Protein A亲和层析法，纯化得到目标huGI单域抗体Fc融合蛋白，蛋白通过SDS-PAGE考察纯度。各蛋白的表达量见表11，并且一步纯化后各蛋白的SDS纯度都大于95％。
表11.人源化单域抗体的Fc融合蛋白的表达量

实施例6
鉴定人源化单域抗体的Fc融合蛋白的功能
6.1人源化GIPR单域抗体-Fc融合蛋白的GIPR的结合能力
向细胞培养板中铺293T-GIPR细胞，加入人源化GIPR单域抗体-Fc融合蛋白或Maridebart样品，终浓度为0.00128nM-100nM。加入抗人二抗SULFO-TAG(MSD，货号R32AJ-1)后孵育，使用MSD检测平均荧光强度(MFI)。
Maridebart为抗GIPR抗体，参照WHO Drug Information,Volume 36.Number 4.2022.Proposed INN:List 128中公开的序列自主克隆并制备，相似序列还见于专利申请WO2017112824中的构建体2G10 LC1.006。
结果如图10所示，人源化GIPR单域抗体-Fc融合蛋白对GIPR的结合能力略优于Maridebart。
6.2人源化GIPR单域抗体-Fc融合蛋白的GIPR的亲和力
参照3.3的实验步骤，测定并计算人源化单域抗体Fc融合蛋白对GIPR-ECD-chis的平衡解离常数(KD)、结合速率(Ka)和解离速率(Kdis)，结果如表12至表14所示，人源化后的融合蛋白与GIPR的亲和力与未人源化前相当。
表12.人源化GIPR单域抗体-Fc融合蛋白的GIPR的亲和力
表13.人源化GIPR单域抗体-Fc融合蛋白的GIPR的亲和力
表14.人源化GIPR单域抗体-Fc融合蛋白的GIPR的亲和力
采用相同的实验步骤，检测人源化单域抗体Fc融合蛋白对GIPR-ECD-chis的平衡解离常数(KD)、结合速率(Ka)和解离速率(Kdis)，并与Maridebart进行比较。结果如表15所示，人源化单域抗体Fc融合蛋白的亲和力优于抗体Maridebart。
表15.人源化GIPR单域抗体-Fc融合蛋白的GIPR的亲和力
6.3人源化GIPR单域抗体-Fc融合蛋白的中和活性
参照实施例3.2的实验步骤，测定人源化GIPR单域抗体-Fc融合蛋白的中和活性，结果如图6所示，人源化GIPR单域抗体-Fc融合蛋白的中和活性均优于未人源化的分子iGI-1198-Fc融合蛋白。
96孔板中分别加入GIP-C12H4-Fc4，以及25μL人源化GIPR单域抗体-Fc或Maridebart样品；然后加入293T-GIPR细胞，孵育之后用cAMP试剂盒检测，酶标仪检测450nm/550nm的吸光度，计算得到抑制率(％)。结果如图11所示，人源化GIPR单域抗体-Fc融合蛋白的中和活性优于Maridebart。
6.4人源化GIPR单域抗体-Fc融合蛋白的体内药效研究
(1)DA-GIP刺激C57BL/6小鼠实验
9只C57BL/6小鼠禁食过夜后，测量动物的血糖水平，随机分成3组，恢复正常饮食24h后，按照实验方案给药huGI-72v5-Ld-Fc 203nmol/kg，给药12h后开始禁食过夜，药后48h每只小鼠ip给药DA-GIP 50nmol/kg，给药DA-GIP后立即进行口服2g/kg葡萄糖负荷。于葡萄糖负荷前，负荷后5，10，15，30，60min采血测量血糖和胰岛素水平。结果如图7-图8所示，和PBS组相比，DA-GIP明显降低OGTT血糖，huGI-72v5-Ld-Fc具有阻断DA-GIP降血糖作用，说明huGI-72v5-Ld-Fc在小鼠体内具有明显的阻断活性。
(2)ob小鼠多次给药
24只ob/ob小鼠根据血糖和体重随机分成6组,6-8周开始给药。分组给药当天记为D0，每周一次给药，共6次。药前，药后D0、7、16、20、28、35称量体重。结果如图9所示，dulaglutide 0.5mg/kg、huGI-72v5-Ld-Fc 3mg/kg和huGI-72v5-Ld-Fc 30mg/kg处理组都明显降低ob/ob小鼠的体重，dulaglutide和huGI-72v5-Ld-Fc联用组可以进一步增强降体重作用。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本公开所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附 的权利要求和其等同方式的范围内。
序列信息
iGI-9(SEQ ID NO:1)：

iGI-27(SEQ ID NO:2):

iGI-44(SEQ ID NO:3)：

iGI-51(SEQ ID NO:4)：

iGI-72(SEQ ID NO:5)：

iGI-1198(SEQ ID NO:6)：


iGI-1225NA(SEQ ID NO:7)：

huGI-72v1(SEQ ID NO:8)：
huGI-72v2(SEQ ID NO:9)：
huGI-72v3(SEQ ID NO:10)：
huGI-72v4(SEQ ID NO:11)：
huGI-72v5(SEQ ID NO:12)：
huGI-72v6(SEQ ID NO:13)：
huGI-72v7(SEQ ID NO:14)：
huGI-1198n1(SEQ ID NO:15)：
huGI-1198n2(SEQ ID NO:16)：
huGI-1198n3(SEQ ID NO:17)：
huGI-1198n4(SEQ ID NO:18)：
huGI-1198n5(SEQ ID NO:19)：
huGI-1198n6(SEQ ID NO:20)：
huGI-1198n7(SEQ ID NO:21)：
huGI-1198n8(SEQ ID NO:22)：
huGI-1198n9(SEQ ID NO:23)：
huGI-1198n10(SEQ ID NO:24)：
huGI-1198n11(SEQ ID NO:25)：
IgG1 Fc(SEQ ID NO:110)
IgG1 Fc(C220S)(SEQ ID NO:113)

Claims (29)

  1. 一种葡萄糖依赖性促胰岛素多肽受体(GIPR)结合蛋白，其包含至少一个免疫球蛋白单一可变结构域，其中所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5和/或SEQ ID NO:6所示的VHH中的CDR1、CDR2和CDR3。
  2. 根据权利要求1所述的GIPR结合蛋白，其中所述CDR1、CDR2和CDR3根据以下定义系统定义：Kabat、AbM、Chothia或IMGT。
  3. 根据权利要求1或2所述的GIPR结合蛋白，其中所述免疫球蛋白单一可变结构域是骆驼科的、人源化的、或嵌合的。
  4. 根据权利要求1-3中任一项所述的GIPR结合蛋白，其中SEQ ID NO:5所示的VHH中的CDR1、CDR2和CDR3选自以下的任意一组：SEQ ID NO:74-76、SEQ ID NO:77-79、SEQ ID NO:80-82和SEQ ID NO:83-85。
  5. 根据权利要求4所述的GIPR结合蛋白，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5和SEQ ID NO:8-14所示的氨基酸序列中的一个或多个。
  6. 根据权利要求5所述的GIPR结合蛋白，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5和SEQ ID NO:8-14之一的所示的氨基酸序列。
  7. 根据权利要求1-3中任一项所述的GIPR结合蛋白，其中SEQ ID NO:6所示的VHH中的CDR1、CDR2和CDR3选自以下的任意一组：SEQ ID NO:86-88、SEQ ID NO:89-91、SEQ ID NO:92-94和SEQ ID NO:95-97。
  8. 根据权利要求7所述的GIPR结合蛋白，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:6和SEQ ID NO:15-25所示氨基酸序列中的一个或多个。
  9. 根据权利要求8所述的GIPR结合蛋白，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:6和SEQ ID NO:15-25之一的所示氨基酸序列。
  10. 根据权利要求1-9中任一项所述的GIPR结合蛋白，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5和SEQ ID NO:8-14所示氨基酸序列中的一个或多个，以及SEQ ID NO:6和SEQ ID NO:15-25所示氨基酸序列中的一个或多个。
  11. 根据权利要求10所述的GIPR结合蛋白，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5和SEQ ID NO:8-14之一的所示氨基酸序列，和SEQ ID NO:6和SEQ ID NO:15-25之一的所示氨基酸序列。
  12. 根据权利要求1-11中任一项所述的GIPR结合蛋白，其中所述GIPR结合蛋白包含1个所述免疫球蛋白单一可变结构域。
  13. 根据权利要求1-11中任一项所述的GIPR结合蛋白，其中所述GIPR结合蛋白包含多个，例如2个、3个、4个或5个，所述免疫球蛋白单一可变结构域。
  14. 根据权利要求1-13中任一项所述的GIPR结合蛋白，其还包含免疫球蛋白Fc区，优选人免疫球蛋白的Fc区，更优选人IgG1、IgG2、IgG3或IgG4的Fc区。
  15. 根据权利要求14所述的GIPR结合蛋白，所述免疫球蛋白的Fc区的氨基酸序列如SEQ ID NO:110或SEQ ID NO:113所示。
  16. 根据权利要求14或15所述的GIPR结合蛋白，所述免疫球蛋白的Fc区与所述至少一个免疫球蛋白单一可变结构域直接连接或通过接头间接连接。
  17. 根据权利要求1-16中任一项所述的GIPR结合蛋白，其具有下述特征中的至少一项：
    (a)与人GIPR具有特异性的结合；
    (b)与人GIPR结合的KD值小于1x10-7M，优选小于1x10-8M；
    (c)阻断GIP与GIPR的相互作用；和
    (d)能够降低受试者的体重和/或血糖。
  18. 一种融合蛋白，其包含权利要求1-17中任一项所述的GIPR结合蛋白。
  19. 一种多特异性抗体，其包含权利要求1-17中任一项所述的GIPR结合蛋白和/或权利要求18所述的融合蛋白，以及另外的一个或多个抗原结合区，所述另外的一个或多个抗原结合区与所述GIPR结合蛋白和/或所述融合蛋白结合不同的抗原或相同抗原的不同表位。
  20. 一种缀合物，其包含权利要求1-17中任一项所述的GIPR结合蛋白和/或权利要求18所述的融合蛋白和/或权利要求19所述的多特异性抗体，所述缀合物还包含与所述GIPR结合蛋白和/或所述融合蛋白和/或所述多特异性抗体缀合的分子。
  21. 一种核酸分子，其编码权利要求1-17中任一项所述的GIPR结合蛋白和/或权利要求18所述的融合蛋白和/或权利要求19所述的多特异性抗体。
  22. 一种表达载体，其包含与表达调控元件可操作地连接的权利要求20所述的核酸分子。
  23. 一种重组细胞，其包含权利要求21所述的核酸分子和/或以权利要求22所述的表达载体转化，并能够表达所述GIPR结合蛋白和/或所述融合蛋白和/或所述多特异性抗体。
  24. 一种药物组合物，其包含权利要求1-17中任一项所述的GIPR结合蛋白和/或权利要求18所述的融合蛋白和/或权利要求19所述的多特异性抗体和/或权利要求20所述的缀合物 和/或权利要求21所述的核酸分子和/或权利要求22所述的表达载体和/或权利要求23所述的重组细胞，以及药学上可接受的载剂。
  25. 一种试剂盒，其包含权利要求1-17中任一项所述的GIPR结合蛋白、权利要求18所述的融合蛋白、权利要求19所述的多特异性抗体、权利要求20所述的缀合物和/或权利要求24所述的药物组合物。
  26. 一种确定GIPR蛋白的存在和/或含量的方法，其包含提供权利要求1-17中任一项所述的GIPR结合蛋白、权利要求18所述的融合蛋白、权利要求19所述的多特异性抗体、和/或权利要求20所述的缀合物。
  27. 一种抑制GIPR蛋白与其配体结合的方法，其包含提供权利要求1-17中任一项所述的GIPR结合蛋白、权利要求18所述的融合蛋白、权利要求19所述的多特异性抗体、和/或权利要求20所述的缀合物，优选地所述配体是GIP。
  28. 一种治疗和/或预防代谢性疾病和/或症状的方法，其包括向有需要的对象施用有效量的权利要求1-17中任一项所述的GIPR结合蛋白、权利要求18所述的融合蛋白、权利要求19所述的多特异性抗体、权利要求20所述的缀合物、和/或权利要求24所述的药物组合物。
  29. 根据权利要求28的方法，其中所述代谢性疾病和/或症状为肥胖、超重和/或糖尿病。
PCT/CN2023/129127 2022-11-02 2023-11-01 一种gipr结合蛋白及其应用 Ceased WO2024094076A1 (zh)

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