GHR agonist antibodies and their applications

Abstract

The invention provides an agonist-type single-domain antibody or antigen-binding fragment thereof that specifically binds to the growth hormone receptor, a polypeptide construct containing said single-domain antibody or antigen-binding fragment thereof, and further provides the use of said single-domain antibody or antigen-binding fragment thereof and polypeptide construct in the prevention and / or treatment of diseases.

Description

GHR agonistic antibodies and their applications

[0001] This application is based on the application with CN application number 202410020660.X and application date January 5, 2024, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of biomedicine, and more specifically, to an agonist single-domain antibody or an antigen-binding fragment thereof that specifically binds to a growth hormone receptor, a polypeptide construct containing the single-domain antibody or the antigen-binding fragment thereof, and related uses. Background Art

[0003] Human growth hormone (hGH) is a protein hormone secreted by eosinophils in the anterior pituitary gland. Composed of 191 amino acids, its primary physiological function is to promote metabolism and growth. hGH acts on cartilage tissue through growth factors or insulin-like growth factors, increasing bone length and thereby promoting linear growth. In recent years, additional functions of hGH have been discovered, including promoting skeletal and cardiac muscle cell growth, promoting protein synthesis, regulating immune system function, and enhancing immune defenses. The large-scale clinical application of hGH has expanded from its initial use in the prevention and treatment of dwarfism in children and adult growth hormone deficiency to include burns, acute pancreatitis, and anti-aging in the elderly, and its clinical indications continue to expand.

[0004] Currently marketed and under development GHR (GH receptor) agonists are daily or weekly formulations, and all utilize GH or GH modifications. However, GH replacement therapy may be less effective in patients with GHR receptor point mutations or those with GH neutralizing antibodies. Furthermore, due to receptor-mediated clearance, GH and related modifications require longer dosing intervals and higher doses, which can lead to excessively elevated IGF-1 levels and raise safety concerns. Summary of the Invention

[0005] After extensive research, the inventors of the present application have obtained agonist single-domain antibodies that can specifically bind to GHR and polypeptide constructs containing the same. The single-domain antibodies or polypeptide constructs can mimic the in vitro and in vivo functions of GH, that is, activate the GHR downstream signaling pathway to achieve effects such as promoting animal weight and bone growth.

[0006] Therefore, in a first aspect, the present application provides a single domain antibody or an antigen-binding fragment thereof that specifically binds to growth hormone receptor (GHR).

[0007] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises:

[0008] CDR1 or a variant thereof, CDR2 or a variant thereof, and CDR3 or a variant thereof contained in the heavy chain variable region (VHH) as shown in any one of SEQ ID NOs: 61, 67-75, 90-92;

[0009] The variant has one or more amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived.

[0010] In certain embodiments, the substitutions are conservative substitutions.

[0011] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises: three CDRs contained in the heavy chain variable region (VHH) as shown in any one of SEQ ID NOs: 61, 67-75, 90-92.

[0012] In certain embodiments, the CDRs are defined by the Kabat, IMGT, or Chothia numbering systems.

[0013] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises:

[0014] (a)(i) CDR1 having: the sequence shown in SEQ ID NO: 7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 7;

[0015] (ii) CDR2 having: a sequence as shown in any one of SEQ ID NOs: 22-24, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 22-24; and,

[0016] (iii) CDR3 having: a sequence as shown in SEQ ID NO: 48, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 48;

[0017] wherein the CDRs are defined by the Kabat numbering system;

[0018] (b)(i) CDR1 having: the sequence shown in SEQ ID NO: 9, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 9;

[0019] (ii) CDR2 having: a sequence as shown in SEQ ID NO: 27 or 28, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence as shown in SEQ ID NO: 27 or 28; and,

[0020] (iii) CDR3 having: a sequence as shown in SEQ ID NO: 49, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 49;

[0021] wherein the CDRs are defined by the IMGT numbering system;

[0022] or,

[0023] (c)(i) CDR1 having: the sequence shown in SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 8;

[0024] (ii) CDR2 having: a sequence as shown in SEQ ID NO: 25 or 26, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence as shown in SEQ ID NO: 25 or 26; and,

[0025] (iii) CDR3 having: a sequence as shown in SEQ ID NO: 48, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 48;

[0026] The CDRs are defined by the Chothia numbering system.

[0027] In certain embodiments, the substitutions are conservative substitutions.

[0028] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises:

[0029] (a) a CDR1 as set forth in SEQ ID NO:7, a CDR2 as set forth in any one of SEQ ID NOs:22-24; and a CDR3 as set forth in SEQ ID NO:48; wherein the CDRs are defined by the Kabat numbering system;

[0030] (b) a CDR1 as set forth in SEQ ID NO:9, a CDR2 as set forth in SEQ ID NO:27 or 28, and a CDR3 as set forth in SEQ ID NO:49; wherein the CDRs are defined by the IMGT numbering system;

[0031] or,

[0032] (c) CDR1 as shown in SEQ ID NO: 8, CDR2 as shown in SEQ ID NO: 25 or 26, and CDR3 as shown in SEQ ID NO: 48; wherein the CDRs are defined by the Chothia numbering system.

[0033] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a CDR1 as set forth in SEQ ID NO:7, a CDR2 as set forth in SEQ ID NO:22; and a CDR3 as set forth in SEQ ID NO:48; wherein the CDRs are defined by the Kabat numbering system.

[0034] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a CDR1 as set forth in SEQ ID NO:7, a CDR2 as set forth in SEQ ID NO:23; and a CDR3 as set forth in SEQ ID NO:48; wherein the CDRs are defined by the Kabat numbering system.

[0035] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a CDR1 as set forth in SEQ ID NO:7, a CDR2 as set forth in SEQ ID NO:24; and a CDR3 as set forth in SEQ ID NO:48; wherein the CDRs are defined by the Kabat numbering system.

[0036] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a CDR1 as set forth in SEQ ID NO:9, a CDR2 as set forth in SEQ ID NO:27; and a CDR3 as set forth in SEQ ID NO:49; wherein the CDRs are defined by the IMGT numbering system.

[0037] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a CDR1 as set forth in SEQ ID NO:9, a CDR2 as set forth in SEQ ID NO:28; and a CDR3 as set forth in SEQ ID NO:49; wherein the CDRs are defined by the IMGT numbering system.

[0038] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a CDR1 as set forth in SEQ ID NO:8, a CDR2 as set forth in SEQ ID NO:25; and a CDR3 as set forth in SEQ ID NO:48; wherein the CDRs are defined by the Chothia numbering system.

[0039] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a CDR1 as set forth in SEQ ID NO:8, a CDR2 as set forth in SEQ ID NO:26; and a CDR3 as set forth in SEQ ID NO:48; wherein the CDRs are defined by the Chothia numbering system.

[0040] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of:

[0041] (i) the sequence shown in SEQ ID NO: 61;

[0042] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 61; or

[0043] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:61.

[0044] In certain embodiments, the substitutions are conservative substitutions.

[0045] In certain embodiments, the single domain antibody or antigen-binding fragment thereof is humanized.

[0046] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises a heavy chain framework region of a human immunoglobulin.

[0047] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises a heavy chain framework region in the amino acid sequence encoded by a human heavy chain germline antibody gene.

[0048] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a heavy chain framework region of an amino acid sequence encoded by a human IGHV gene selected from the group consisting of IGHV3-23, IGHV3-30, IGHV3-34, IGHV3-7, and IGHV3-74. In certain embodiments, the heavy chain framework region is defined by the IMGT, Kabat, or Chothia numbering systems. In certain embodiments, the heavy chain framework region is defined by the IMGT numbering system.

[0049] In certain embodiments, the heavy chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) backmutations from a human residue to a camel residue.

[0050] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises:

[0051] (a) FR1 as set forth in SEQ ID NO: 2 or 6, FR2 as set forth in any one of SEQ ID NO: 10-13, FR3 as set forth in any one of SEQ ID NO: 30-33, and / or FR4 as set forth in SEQ ID NO: 51; wherein the FRs are defined by the Kabat numbering system;

[0052] (b) FR1 as set forth in SEQ ID NO: 4 or 5, FR2 as set forth in any one of SEQ ID NOs: 18-21, FR3 as set forth in any one of SEQ ID NOs: 42-47, and / or FR4 as set forth in SEQ ID NO: 51; wherein the FRs are defined by the IMGT numbering system;

[0053] or,

[0054] (c) FR1 as set forth in SEQ ID NO: 4 or 5, FR2 as set forth in any one of SEQ ID NOs: 14-17, FR3 as set forth in any one of SEQ ID NOs: 35-40, and / or FR4 as set forth in SEQ ID NO: 51; wherein the FRs are defined by the Chothia numbering system.

[0055] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises:

[0056] (i) a sequence as shown in any one of SEQ ID NOs: 67-75, 90-92;

[0057] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 67-75, 90-92; or

[0058] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 67-75, 90-92.

[0059] In certain embodiments, the substitutions are conservative substitutions.

[0060] In certain embodiments, the single domain antibody is a GHR agonistic single domain antibody.

[0061] In certain embodiments, the single domain antibody or antigen-binding fragment thereof has cross-binding activity to human GHR and murine (eg, rat) GHR.

[0062] In certain embodiments, the GHR is human GHR or murine (eg, rat) GHR.

[0063] In a second aspect, the present application provides a polypeptide construct comprising the single domain antibody or antigen-binding fragment thereof according to the first aspect, an Fc peptide derived from an immunoglobulin, and optionally a hinge peptide.

[0064] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof is covalently linked to the Fc peptide and the optional hinge peptide (e.g., covalently linked via covalent bonds comprising peptide bonds, isopeptide bonds, and / or disulfide bonds).

[0065] In certain embodiments, the polypeptide construct comprises the single domain antibody or antigen-binding fragment thereof of the first aspect, and the Fc peptide and the hinge peptide, and the single domain antibody or antigen-binding fragment thereof is covalently linked to the Fc peptide and the hinge peptide via a covalent bond comprising a peptide bond.

[0066] In certain embodiments, the polypeptide construct comprises a first peptide chain, the first peptide chain comprising: a first single-domain antibody or an antigen-binding fragment thereof, a first hinge peptide, and a first Fc peptide, wherein the first single-domain antibody or an antigen-binding fragment thereof is selected from the single-domain antibody or antigen-binding fragment thereof of the first aspect. In certain embodiments, the first peptide chain comprises, from N-terminus to C-terminus, the first single-domain antibody or an antigen-binding fragment thereof, the first hinge peptide, and the first Fc peptide.

[0067] In certain embodiments, the polypeptide construct further comprises a second peptide chain comprising a second hinge peptide and a second Fc peptide. In certain embodiments, the second peptide chain further comprises a second single domain antibody or an antigen-binding fragment thereof. In certain embodiments, the second single domain antibody is selected from a single domain antibody that specifically binds to GHR. In certain embodiments, the second single domain antibody or its antigen-binding fragment is selected from the single domain antibody or its antigen-binding fragment of the first aspect.

[0068] In certain embodiments, the second peptide chain comprises, from N-terminus to C-terminus, the second single-domain antibody or antigen-binding fragment thereof, the second hinge peptide, and the second Fc peptide.

[0069] In certain embodiments, the polypeptide construct is a heavy chain antibody.

[0070] In certain embodiments, there are no additional amino acid residues between the first single-domain antibody or antigen-binding fragment thereof and the first hinge peptide, and / or there are no additional amino acid residues between the second single-domain antibody or antigen-binding fragment thereof and the second hinge peptide.

[0071] In certain embodiments, the first hinge peptide and the first Fc peptide are optionally connected via a peptide linker, and / or the second hinge peptide and the second Fc peptide are optionally connected via a peptide linker.

[0072] In certain embodiments, there are no additional amino acid residues between the first hinge peptide and the first Fc peptide, and / or there are no additional amino acid residues between the second hinge peptide and the second Fc peptide.

[0073] In certain embodiments, the polypeptide construct has one or more of the following features:

[0074] (i) the first single domain antibody or antigen-binding fragment thereof is the same as or different from the second single domain antibody or antigen-binding fragment thereof;

[0075] (ii) the first hinge peptide and the second hinge peptide are derived from the same or different immunoglobulins;

[0076] (iii) the first Fc peptide and the second Fc peptide are derived from the same or different immunoglobulins;

[0077] (iv) the first hinge peptide and the second hinge peptide are the same or different;

[0078] (v) the first Fc peptide and the second Fc peptide are the same or different;

[0079] (vi) the first peptide chain and the second peptide chain are the same or different;

[0080] (vii) The first peptide chain and the second peptide chain are connected via a disulfide bond.

[0081] In certain embodiments, the hinge peptide (eg, the first hinge peptide and / or the second hinge peptide) is a hinge peptide derived from an immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).

[0082] In certain embodiments, the hinge peptide (eg, the first hinge peptide and / or the second hinge peptide) is a hinge peptide derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).

[0083] It is easy for those skilled in the art to understand that the hinge peptide derived from immunoglobulin (e.g., human immunoglobulin) includes both hinge peptides directly derived from immunoglobulin (e.g., human immunoglobulin) and hinge peptides obtained by transforming or modifying hinge peptides derived from immunoglobulin (e.g., human immunoglobulin) (e.g., artificially designed hinge peptides).

[0084] In certain embodiments, the hinge peptide (eg, the first hinge peptide and / or the second hinge peptide) is selected from the hinge peptides of wild-type human immunoglobulins IgG1, IgG2, and IgG4 and artificially designed hinge peptides.

[0085] In certain embodiments, the artificially designed hinge peptide has enhanced FcRn binding activity compared to the hinge peptides of wild-type human immunoglobulins IgG1, IgG2, and IgG4 (eg, the hinge peptide of wild-type human immunoglobulin IgG4).

[0086] In certain embodiments, the artificially designed hinge peptide has a structure as shown in Formula I from N-terminus to C-terminus: Z-CPPCP (Formula I)

[0087] Wherein, Z is absent or selected from a short peptide consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues.

[0088] In certain embodiments, Z is linked to the short peptide CPPCP via a peptide bond.

[0089] In certain embodiments, Z does not comprise a cysteine ​​residue.

[0090] In certain embodiments, the hinge peptide (eg, the first hinge peptide and / or the second hinge peptide) is selected from the hinge peptide of wild-type human immunoglobulin IgG1 and an artificially designed hinge peptide.

[0091] In certain embodiments, the artificially designed hinge peptide has a structure as shown in Formula II: X1X2X3X4X5X6X7X8X9X 10 CPPCP (Formula II);

[0092] wherein X1 is absent or selected from (i) amino acid residue E and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., D);

[0093] X2 is absent or selected from (i) amino acid residue P and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., A, V, L, I, M);

[0094] X3 is absent or selected from (i) amino acid residue K and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., R, H);

[0095] X4 is absent or selected from (i) amino acid residue S and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., N, Q, G, T, C, Y, W);

[0096] X5 is absent or selected from (i) amino acid residue S and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., N, Q, G, T, C, Y, W);

[0097] X6 is absent or selected from (i) amino acid residue D and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., E);

[0098] X7 is absent or selected from (i) amino acid residue K and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., R, H);

[0099] X8 is absent or selected from (i) amino acid residue T and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., N, Q, G, S, C, Y, W);

[0100] X9 is absent or selected from (i) amino acid residue H and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., R, K);

[0101] X 10 It is absent or selected from (i) amino acid residue T and (ii) amino acid residues that are conservative substitutions relative to (i) (e.g., N, Q, G, S, C, Y, W).

[0102] In certain embodiments, the artificially designed hinge peptide has one or more features selected from the following: (1) X1 is absent or is an amino acid residue E, (2) X2 is absent or is an amino acid residue P, (3) X3 is absent or is an amino acid residue K, (4) X4 is absent or is an amino acid residue S, (5) X5 is absent or is an amino acid residue S, (6) X6 is absent or is an amino acid residue D, (7) X7 is absent or is an amino acid residue K, (8) X8 is absent or is an amino acid residue T, (9) X9 is absent or is an amino acid residue H, (10) X 10 Absent or represented by amino acid residue T.

[0103] In certain embodiments, X1 is absent or is an amino acid residue E, X2 is absent or is an amino acid residue P, X3 is absent or is an amino acid residue K, X4 is absent or is an amino acid residue S, X5 is absent or is an amino acid residue S, X6 is absent or is an amino acid residue D, X7 is absent or is an amino acid residue K, X8 is absent or is an amino acid residue T, X9 is absent or is an amino acid residue H, X 10 Absent or represented by amino acid residue T.

[0104] In certain embodiments, X1 to X 10 Optional 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 amino acid residues are absent.

[0105] In certain embodiments, X1 is absent, X1 to X2 are absent, X1 to X3 are absent, X1 to X4 are absent, X1 to X5 are absent, X1 to X6 are absent, X1 to X7 are absent, X1 to X8 are absent, X1 to X9 are absent, or X1 to X 10 Does not exist.

[0106] In certain embodiments, wherein X1 to X5 are absent, X1 to X6 are absent, X1 to X7 are absent, X1 to X8 are absent, X1 to X9 are absent, or X1 to X 10 In certain embodiments, X1 to X7 are absent, or X1 to X8 are absent.

[0107] In certain embodiments, the hinge peptide (eg, the first hinge peptide and / or the second hinge peptide) is selected from the hinge peptide of wild-type human immunoglobulin IgG2 and an artificially designed hinge peptide.

[0108] In certain embodiments, the artificially designed hinge peptide has a structure as shown in Formula III: i X ii X iii X ivX v X vi X vii CPPCP (Formula III);

[0109] Among them, X i Absent or selected from (i) amino acid residue E and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., D);

[0110] X ii Absent or selected from (i) amino acid residue R and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., K or H);

[0111] X iii Absent or selected from (i) amino acid residue K and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., R or H);

[0112] X iv is absent or selected from non-cysteine ​​residues; preferably, X iv Absent or selected from (i) amino acid residue S and (ii) amino acid residues that are conservative substitutions relative to (i) (e.g., N, Q, G, T, C, Y, W);

[0113] X v is absent or selected from non-cysteine ​​residues; preferably, X v Absent or selected from (i) amino acid residue S and (ii) amino acid residues that are conservative substitutions relative to (i) (e.g., N, Q, G, T, C, Y, W);

[0114] X vi Absent or selected from (i) amino acid residue V and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., A, P, L, I, M);

[0115] X vii It is absent or selected from (i) amino acid residue E and (ii) an amino acid residue that is a conservative substitution relative to (i) (eg, D).

[0116] In certain embodiments, the artificially designed hinge peptide has one or more features selected from the following: (1) X i Absent or amino acid residue E, (2) X ii Absent or amino acid residue R, (3)X iii Absent or amino acid residue K, (4)X iv Absent or amino acid residue S, (5)X v Absent or amino acid residue S, (6)X vi Absent or amino acid residue V, (7) Xvii Absent or amino acid residue E.

[0117] In certain embodiments, X i Not present or amino acid residue E, X ii Absent or amino acid residue R, X iii Not present or amino acid residue K, X iv Not present or amino acid residue S, X v Not present or amino acid residue S, X vi Absent or amino acid residue V, X vii Absent or amino acid residue E.

[0118] In certain embodiments, X i To X vii In certain embodiments, X iv To X vii Does not exist.

[0119] In certain embodiments, the hinge peptide (eg, the first hinge peptide and / or the second hinge peptide) has an amino acid sequence as shown in any one of SEQ ID NOs: 62-64, 80-89.

[0120] In certain embodiments, the Fc peptide (eg, the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from an immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).

[0121] In certain embodiments, the Fc peptide (eg, the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).

[0122] It is easy for those skilled in the art to understand that the Fc peptide derived from an immunoglobulin (e.g., human immunoglobulin) includes both an Fc peptide directly derived from an immunoglobulin (e.g., human immunoglobulin) and an Fc peptide obtained by transforming or modifying an Fc peptide derived from an immunoglobulin (e.g., human immunoglobulin) (e.g., Fc peptide variant).

[0123] In certain embodiments, the Fc peptide (eg, the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from human immunoglobulin IgG1 or IgG4.

[0124] In certain embodiments, the Fc peptide (eg, the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from human immunoglobulin IgG1.

[0125] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is selected from the Fc peptide of wild-type human immunoglobulin IgG1 and variants thereof; wherein, compared to the Fc peptide of wild-type human immunoglobulin IgG1, the Fc peptide variant has reduced Fc effector function (e.g., has reduced ADCC, CDC and / or ADCP activity) and / or prolonged half-life.

[0126] In certain embodiments, the Fc peptide variant has reduced FcγR binding activity, reduced serum complement molecule (C1q) binding activity, and / or enhanced FcRn binding activity compared to the Fc peptide of wild-type human immunoglobulin IgG1.

[0127] In certain embodiments, the Fc peptide variant comprises a mutation selected from the group consisting of L234A, L235A, M252Y, S254T, T256E, and any combination thereof, compared to the Fc peptide of wild-type human immunoglobulin IgG1 (e.g., selected from the group consisting of mutations: (i) L234A and L235A, (ii) M252Y, S254T, and T256E, and (iii): a combination of (i) and (ii)).

[0128] In certain embodiments, the Fc peptide variant comprises mutations L234A and L235A compared to the Fc peptide of wild-type human immunoglobulin IgG1. In certain embodiments, the Fc peptide variant further comprises mutations M252Y, S254T, and T256E.

[0129] In certain embodiments, the mutation site of the mutation is defined by the EU numbering system.

[0130] In certain embodiments, the Fc peptide (eg, the first Fc peptide and / or the second Fc peptide) comprises a sequence as shown in any one of SEQ ID NOs: 52, 65, 76, and 93.

[0131] In certain embodiments, the Fc peptide (eg, the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from human immunoglobulin IgG4.

[0132] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is selected from the Fc peptide of wild-type human immunoglobulin IgG4 and variants thereof; wherein, compared to the Fc peptide of wild-type human immunoglobulin IgG4, the Fc peptide variant has reduced Fc effector function (e.g., reduced ADCC, CDC and / or ADCP activity) and / or prolonged half-life.

[0133] In certain embodiments, the Fc peptide variant has reduced FcγR binding activity, reduced serum complement molecule (C1q) binding activity, and / or enhanced FcRn binding activity compared to the Fc peptide of wild-type human immunoglobulin IgG4.

[0134] In certain embodiments, the Fc peptide variant comprises the mutation L235E compared to the Fc peptide of wild-type human immunoglobulin IgG4.

[0135] In certain embodiments, the mutation site of the mutation is defined by the EU numbering system.

[0136] In certain embodiments, the Fc peptide (eg, the first Fc peptide and / or the second Fc peptide) comprises the sequence shown in SEQ ID NO: 79 or 99.

[0137] In certain embodiments, the polypeptide construct comprises a sequence as shown in any one of SEQ ID NOs: 94-97, 103-126.

[0138] In certain embodiments, the polypeptide construct comprises the first peptide chain and, optionally, the second peptide chain; wherein the first peptide chain comprises a sequence as shown in any one of SEQ ID NOs: 94-97, 103-126, and / or the second peptide chain comprises a sequence as shown in any one of SEQ ID NOs: 94-97, 103-126.

[0139] In certain embodiments, the polypeptide construct comprises the first peptide chain and optionally the second peptide chain; wherein:

[0140] (1) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 94;

[0141] (2) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 95;

[0142] (3) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 96;

[0143] (4) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 97;

[0144] (5) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 103;

[0145] (6) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 104;

[0146] (7) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 105;

[0147] (8) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 106;

[0148] (9) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 107;

[0149] (10) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 108;

[0150] (11) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 109;

[0151] (12) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 110;

[0152] (13) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 111;

[0153] (14) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 112;

[0154] (15) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 113;

[0155] (16) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 114;

[0156] (17) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 115;

[0157] (18) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 116;

[0158] (19) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 117;

[0159] (20) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 118;

[0160] (21) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 119;

[0161] (22) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 120;

[0162] (23) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 121;

[0163] (24) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 122;

[0164] (25) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 123;

[0165] (26) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 124;

[0166] (27) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 125; or,

[0167] (28) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 126.

[0168] In certain embodiments, the polypeptide construct is a GHR agonist construct (eg, the polypeptide construct can act as a GHR agonist).

[0169] In certain embodiments, the polypeptide construct has cross-binding activity to both human GHR and murine (eg, rat) GHR.

[0170] In certain embodiments, the GHR is human GHR or murine (eg, rat) GHR.

[0171] In a third aspect, the present application provides a polypeptide construct comprising a single domain antibody or an antigen-binding fragment thereof, a hinge peptide and an Fc peptide derived from an immunoglobulin;

[0172] The hinge peptide has a structure from N-terminus to C-terminus as shown in Formula I: Z-CPPCP (Formula I)

[0173] Wherein, Z is absent or selected from a short peptide consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-cysteine ​​amino acid residues.

[0174] In certain embodiments, Z is linked to the short peptide CPPCP via a peptide bond.

[0175] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof is covalently linked to the Fc peptide and the hinge peptide (e.g., covalently linked via covalent bonds comprising peptide bonds, isopeptide bonds, and / or disulfide bonds).

[0176] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof is covalently linked to the Fc peptide and the hinge peptide via a covalent bond comprising a peptide bond.

[0177] In certain embodiments, the polypeptide construct comprises a first peptide chain, the first peptide chain comprising: a first single-domain antibody or an antigen-binding fragment thereof, a first hinge peptide, and a first Fc peptide. In certain embodiments, the first hinge peptide has the same meaning as the hinge peptide described above in this aspect (third aspect). In certain embodiments, the first peptide chain comprises, from N-terminus to C-terminus, the first single-domain antibody or an antigen-binding fragment thereof, the first hinge peptide, and the first Fc peptide.

[0178] In certain embodiments, the polypeptide construct further comprises a second peptide chain comprising a second hinge peptide and a second Fc peptide. In certain embodiments, the second peptide chain further comprises a second single domain antibody or an antigen-binding fragment thereof.

[0179] In certain embodiments, the second peptide chain comprises, from N-terminus to C-terminus, the second single-domain antibody or antigen-binding fragment thereof, the second hinge peptide, and the second Fc peptide.

[0180] In certain embodiments, the polypeptide construct is a heavy chain antibody.

[0181] In certain embodiments, there are no additional amino acid residues between the first single-domain antibody or antigen-binding fragment thereof and the first hinge peptide, and / or there are no additional amino acid residues between the second single-domain antibody or antigen-binding fragment thereof and the second hinge peptide.

[0182] In certain embodiments, the first hinge peptide and the first Fc peptide are optionally connected via a peptide linker, and / or the second hinge peptide and the second Fc peptide are optionally connected via a peptide linker.

[0183] In certain embodiments, there are no additional amino acid residues between the first hinge peptide and the first Fc peptide, and / or there are no additional amino acid residues between the second hinge peptide and the second Fc peptide.

[0184] In certain embodiments, the polypeptide construct has one or more of the following features:

[0185] (i) the first single domain antibody or antigen-binding fragment thereof is the same as or different from the second single domain antibody or antigen-binding fragment thereof;

[0186] (ii) the first hinge peptide and the second hinge peptide are derived from the same or different immunoglobulins;

[0187] (iii) the first Fc peptide and the second Fc peptide are derived from the same or different immunoglobulins;

[0188] (iv) the first hinge peptide and the second hinge peptide are the same or different;

[0189] (v) the first Fc peptide and the second Fc peptide are the same or different;

[0190] (vi) the first peptide chain and the second peptide chain are the same or different;

[0191] (vii) The first peptide chain and the second peptide chain are connected via a disulfide bond.

[0192] In certain embodiments, the first single domain antibody is selected from a single domain antibody that specifically binds to growth hormone receptor (GHR).

[0193] In certain embodiments, the first single domain antibody is selected from a GHR agonistic antibody.

[0194] In certain embodiments, the first single domain antibody or antigen-binding fragment thereof is selected from the single domain antibody or antigen-binding fragment thereof of the first aspect.

[0195] In certain embodiments, the second single domain antibody is selected from a single domain antibody that specifically binds to growth hormone receptor (GHR).

[0196] In certain embodiments, the second single domain antibody is selected from a GHR agonistic antibody.

[0197] In certain embodiments, the second single domain antibody or antigen-binding fragment thereof is selected from the single domain antibody or antigen-binding fragment thereof of the first aspect.

[0198] In certain embodiments, the hinge peptide (eg, the first hinge peptide and / or the second hinge peptide) has a structure as shown in Formula II: X1X2X3X4X5X6X7X8X9X10 CPPCP (Formula II);

[0199] wherein X1 is absent or selected from (i) amino acid residue E and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., D);

[0200] X2 is absent or selected from (i) amino acid residue P and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., A, V, L, I, M);

[0201] X3 is absent or selected from (i) amino acid residue K and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., R, H);

[0202] X4 is absent or selected from (i) amino acid residue S and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., N, Q, G, T, C, Y, W);

[0203] X5 is absent or selected from (i) amino acid residue S and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., N, Q, G, T, C, Y, W);

[0204] X6 is absent or selected from (i) amino acid residue D and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., E);

[0205] X7 is absent or selected from (i) amino acid residue K and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., R, H);

[0206] X8 is absent or selected from (i) amino acid residue T and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., N, Q, G, S, C, Y, W);

[0207] X9 is absent or selected from (i) amino acid residue H and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., R, K);

[0208] X 10 It is absent or selected from (i) amino acid residue T and (ii) amino acid residues that are conservative substitutions relative to (i) (e.g., N, Q, G, S, C, Y, W).

[0209] In certain embodiments, the hinge peptide has one or more of the following features: (1) X1 is absent or is an amino acid residue E, (2) X2 is absent or is an amino acid residue P, (3) X3 is absent or is an amino acid residue K, (4) X4 is absent or is an amino acid residue S, (5) X5 is absent or is an amino acid residue S, (6) X6 is absent or is an amino acid residue D, (7) X7 is absent or is an amino acid residue K, (8) X8 is absent or is an amino acid residue T, (9) X9 is absent or is an amino acid residue H, (10) X 10 Absent or represented by amino acid residue T.

[0210] In certain embodiments, X1 is absent or is an amino acid residue E, X2 is absent or is an amino acid residue P, X3 is absent or is an amino acid residue K, X4 is absent or is an amino acid residue S, X5 is absent or is an amino acid residue S, X6 is absent or is an amino acid residue D, X7 is absent or is an amino acid residue K, X8 is absent or is an amino acid residue T, X9 is absent or is an amino acid residue H, X 10 Absent or represented by amino acid residue T.

[0211] In certain embodiments, X1 to X 10 Optional 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 amino acid residues are absent.

[0212] In certain embodiments, X1 is absent, X1 to X2 are absent, X1 to X3 are absent, X1 to X4 are absent, X1 to X5 are absent, X1 to X6 are absent, X1 to X7 are absent, X1 to X8 are absent, X1 to X9 are absent, or X1 to X 10 Does not exist.

[0213] In certain embodiments, wherein X1 to X5 are absent, X1 to X6 are absent, X1 to X7 are absent, X1 to X8 are absent, X1 to X9 are absent, or X1 to X 10 In certain embodiments, X1 to X7 are absent, or X1 to X8 are absent.

[0214] In certain embodiments, X1 to X 10 It does not contain cysteine ​​residues.

[0215] In certain embodiments, the hinge peptide (eg, the first hinge peptide and / or the second hinge peptide) has a structure as shown in Formula III: X i X ii X iii X iv X v Xvi X vii CPPCP (Formula III);

[0216] Among them, X i Absent or selected from (i) amino acid residue E and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., D);

[0217] X ii Absent or selected from (i) amino acid residue R and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., K or H);

[0218] X iii Absent or selected from (i) amino acid residue K and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., R or H);

[0219] X iv is absent or selected from non-cysteine ​​residues; preferably, X iv Absent or selected from (i) amino acid residue S and (ii) amino acid residues that are conservative substitutions relative to (i) (e.g., N, Q, G, T, C, Y, W);

[0220] X v is absent or selected from non-cysteine ​​residues; preferably, X v Absent or selected from (i) amino acid residue S and (ii) amino acid residues that are conservative substitutions relative to (i) (e.g., N, Q, G, T, C, Y, W);

[0221] X vi Absent or selected from (i) amino acid residue V and (ii) an amino acid residue that is a conservative substitution relative to (i) (e.g., A, P, L, I, M);

[0222] X vii It is absent or selected from (i) amino acid residue E and (ii) an amino acid residue that is a conservative substitution relative to (i) (eg, D).

[0223] In certain embodiments, the hinge peptide has one or more of the following characteristics: (1) X i Absent or amino acid residue E, (2) X ii Absent or amino acid residue R, (3)X iii Absent or amino acid residue K, (4)X iv Absent or amino acid residue S, (5)X v Absent or amino acid residue S, (6)X vi Absent or amino acid residue V, (7) X vii Absent or amino acid residue E.

[0224] In certain embodiments, X i Not present or amino acid residue E, X ii Absent or amino acid residue R, X iii Not present or amino acid residue K, X iv Not present or amino acid residue S, X v Not present or amino acid residue S, X vi Absent or amino acid residue V, X vii Absent or amino acid residue E.

[0225] In certain embodiments, X i To X vii In certain embodiments, X iv To X vii Does not exist.

[0226] In certain embodiments, X i To X vii It does not contain cysteine ​​residues.

[0227] In certain embodiments, the hinge peptide (eg, the first hinge peptide and / or the second hinge peptide) has an amino acid sequence as shown in any one of SEQ ID NOs: 80-89.

[0228] In certain embodiments, the Fc peptide (eg, the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from an immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).

[0229] In certain embodiments, the Fc peptide (eg, the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).

[0230] It is easy for those skilled in the art to understand that the Fc peptide derived from an immunoglobulin (e.g., human immunoglobulin) includes both an Fc peptide directly derived from an immunoglobulin (e.g., human immunoglobulin) and an Fc peptide obtained by transforming or modifying an Fc peptide derived from an immunoglobulin (e.g., human immunoglobulin) (e.g., Fc peptide variant).

[0231] In certain embodiments, the Fc peptide (eg, the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from human immunoglobulin IgG1 or IgG4.

[0232] In certain embodiments, the immunoglobulin Fc peptide (eg, the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from human immunoglobulin IgG1.

[0233] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is selected from the Fc peptide of wild-type human immunoglobulin IgG1 and variants thereof; wherein, compared to the Fc peptide of wild-type human immunoglobulin IgG1, the Fc peptide variant has reduced Fc effector function (e.g., has reduced ADCC, CDC and / or ADCP activity) and / or prolonged half-life.

[0234] In certain embodiments, the Fc peptide variant has reduced FcγR binding activity, reduced serum complement molecule (C1q) binding activity, and / or enhanced FcRn binding activity compared to the Fc peptide of wild-type human immunoglobulin IgG1.

[0235] In certain embodiments, the Fc peptide variant comprises a mutation selected from the group consisting of L234A, L235A, M252Y, S254T, T256E, and any combination thereof, compared to the Fc peptide of wild-type human immunoglobulin IgG1 (e.g., selected from mutations (i): L234A and L235A, (ii) M252Y, S254T, and T256E, and (iii): a combination of (i) and (ii)).

[0236] In certain embodiments, the Fc peptide variant comprises mutations L234A and L235A compared to the Fc peptide of wild-type human immunoglobulin IgG1. In certain embodiments, the Fc peptide variant further comprises mutations M252Y, S254T, and T256E.

[0237] In certain embodiments, the mutation site of the mutation is defined by the EU numbering system.

[0238] In certain embodiments, the Fc peptide (eg, the first Fc peptide and / or the second Fc peptide) comprises a sequence as shown in any one of SEQ ID NOs: 52, 65, 76, and 93.

[0239] In certain embodiments, the Fc peptide (eg, the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from human immunoglobulin IgG4.

[0240] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is selected from the Fc peptide of wild-type human immunoglobulin IgG4 and variants thereof; wherein, compared to the Fc peptide of wild-type human immunoglobulin IgG4, the Fc peptide variant has reduced Fc effector function (e.g., reduced ADCC, CDC and / or ADCP activity) and / or prolonged half-life.

[0241] In certain embodiments, the Fc peptide variant has reduced FcγR binding activity, reduced serum complement molecule (C1q) binding activity, and / or enhanced FcRn binding activity compared to the Fc peptide of wild-type human immunoglobulin IgG4.

[0242] In certain embodiments, the Fc peptide variant comprises the mutation L235E compared to the Fc peptide of wild-type human immunoglobulin IgG4.

[0243] In certain embodiments, the mutation site of the mutation is defined by the EU numbering system.

[0244] In certain embodiments, the Fc peptide (eg, the first Fc peptide and / or the second Fc peptide) comprises the sequence shown in SEQ ID NO: 79 or 99.

[0245] In certain embodiments, the polypeptide construct comprises a sequence as shown in any one of SEQ ID NOs: 94-97, 113-126.

[0246] In certain embodiments, the polypeptide construct comprises the first peptide chain and, optionally, the second peptide chain; wherein the first peptide chain comprises a sequence as shown in any one of SEQ ID NOs: 94-97, 113-126, and / or the second peptide chain comprises a sequence as shown in any one of SEQ ID NOs: 94-97, 113-126.

[0247] In certain embodiments, the polypeptide construct comprises the first peptide chain and optionally the second peptide chain; wherein:

[0248] (1) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 94;

[0249] (2) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 95;

[0250] (3) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 96;

[0251] (4) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 97;

[0252] (5) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 113;

[0253] (6) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 114;

[0254] (7) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 115;

[0255] (8) the first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 116;

[0256] (9) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 117;

[0257] (10) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 118;

[0258] (11) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 119;

[0259] (12) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 120;

[0260] (13) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 121;

[0261] (14) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 122;

[0262] (15) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 123;

[0263] (16) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 124;

[0264] (17) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 125; or,

[0265] (18) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 126.

[0266] In certain embodiments, the polypeptide construct is a GHR agonist construct (eg, the polypeptide construct can act as a GHR agonist).

[0267] In certain embodiments, the polypeptide construct has cross-binding activity to both human GHR and murine (eg, rat) GHR.

[0268] In certain embodiments, the GHR is human GHR or murine (eg, rat) GHR.

[0269] In a fourth aspect, the present application provides an isolated nucleic acid molecule encoding the single domain antibody or antigen-binding fragment thereof of the first aspect, or the polypeptide construct of the second aspect or the third aspect.

[0270] In certain embodiments, the isolated nucleic acid molecule encodes the single domain antibody or antigen-binding fragment thereof of the first aspect.

[0271] In certain embodiments, the isolated nucleic acid molecule encodes the polypeptide construct of the second aspect or the third aspect.

[0272] In certain embodiments, the polypeptide construct comprises a first peptide chain and a second peptide chain, and the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the first peptide chain and a second nucleotide sequence encoding the second peptide chain, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule described in the present application comprises a first nucleic acid molecule containing the first nucleotide sequence and a second nucleic acid molecule containing the second nucleotide sequence.

[0273] In a fifth aspect, the present application provides a vector comprising the isolated nucleic acid molecule of the fourth aspect. In certain embodiments, the vector is a cloning vector or an expression vector.

[0274] In certain embodiments, the vector comprises a nucleotide sequence encoding the single domain antibody or antigen-binding fragment thereof of the first aspect.

[0275] In certain embodiments, the vector comprises a nucleotide sequence encoding the polypeptide construct of the second aspect or the third aspect.

[0276] In certain embodiments, the polypeptide construct comprises a first peptide chain and a second peptide chain, and the vector comprises a first nucleotide sequence encoding the first peptide chain and a second nucleotide sequence encoding the second peptide chain, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector described in the present application comprises a first vector containing the first nucleotide sequence and a second vector containing the second nucleotide sequence.

[0277] In a sixth aspect, the present application provides a host cell comprising the isolated nucleic acid molecule of the fourth aspect or the vector of the fifth aspect.

[0278] Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as E. coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell is a microorganism.

[0279] The single-domain antibodies or antigen-binding fragments thereof or polypeptide constructs of the present invention can be prepared by various methods known in the art, for example, by genetic engineering recombinant technology. For example, a DNA molecule encoding the single-domain antibody or antigen-binding fragment thereof or polypeptide construct of the present invention is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. The transfected host cell is then cultured under specific conditions and expresses the single-domain antibody or antigen-binding fragment thereof or polypeptide construct of the present invention. The antigen-binding fragment of the present invention can be obtained by hydrolyzing the intact antibody molecule.

[0280] In the seventh aspect, the present application provides a method for preparing the single domain antibody or antigen binding fragment thereof of the first aspect or the polypeptide construct of the second aspect or the third aspect, comprising culturing the host cell of the sixth aspect under conditions allowing expression of the single domain antibody or antigen binding fragment thereof or the polypeptide construct, and recovering the single domain antibody or antigen binding fragment thereof or the polypeptide construct from the cultured host cell culture.

[0281] In an eighth aspect, the present application provides a bispecific or multispecific molecule comprising the single domain antibody or antigen-binding fragment thereof of the first aspect or the polypeptide construct of the second aspect or the third aspect.

[0282] In certain embodiments, the bispecific or multispecific molecule specifically binds GHR and additionally specifically binds one or more other targets.

[0283] In certain embodiments, the bispecific or multispecific molecule further comprises at least one molecule with a second binding specificity for a second target (eg, a second antibody or antigen-binding fragment thereof).

[0284] In the ninth aspect, the present application provides an immunoconjugate comprising the single domain antibody or antigen-binding fragment thereof of the first aspect or the polypeptide construct of the second aspect or the third aspect, and a therapeutic agent linked to the single domain antibody or antigen-binding fragment thereof or the polypeptide construct.

[0285] In certain embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0286] In the tenth aspect, the present application provides a pharmaceutical composition comprising the single domain antibody or antigen-binding fragment thereof of the first aspect, the polypeptide construct of the second aspect or the third aspect, the isolated nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, the host cell of the sixth aspect, the bispecific or multispecific molecule of the eighth aspect or the immunoconjugate of the ninth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0287] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0288] In certain embodiments, the additional pharmaceutically active agent is selected from drugs used to treat diseases associated with GH, IGF-1 and / or GHRH deficiency.

[0289] In the eleventh aspect, the present application provides use of the single domain antibody or antigen-binding fragment thereof of the first aspect, the polypeptide construct of the second aspect or the third aspect, the isolated nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, the host cell of the sixth aspect, the bispecific or multispecific molecule of the eighth aspect, the immunoconjugate of the ninth aspect, or the pharmaceutical composition of the tenth aspect for preparing a medicament for preventing and / or treating a disease associated with GHR signaling in a subject.

[0290] In certain embodiments, prevention and / or treatment of diseases associated with GHR signaling will benefit from agonism of GHR signaling.

[0291] In certain embodiments, the disease associated with GHR signaling is selected from diseases associated with GH (growth hormone), IGF-1 (insulin-like growth factor-1) and / or GHRH (growth hormone-releasing hormone) deficiency.

[0292] In certain embodiments, the disease associated with GHR signaling is selected from the group consisting of growth hormone deficiency (GHD) (e.g., growth hormone deficiency caused by hypothalamic-pituitary disease), renal insufficiency (CKD), Turner syndrome, Prader-Will syndrome, small for gestational age infant (SGA), idiopathic short stature (ISS), SHOX gene defect, Noonan syndrome, childhood short stature (e.g., short stature caused by achondroplasia (ACH), etc.), severe burns, adult growth hormone deficiency, adult short bowel syndrome, HIV wasting syndrome, aging, and reproductive disorders (e.g., reproductive disorders related to the growth hormone-growth hormone receptor signaling pathway, such as reproductive disorders caused by gonadal dysgenesis due to growth disorders caused by Turner syndrome).

[0293] In certain embodiments, the subject is a mammal, such as a human or a mouse.

[0294] In certain embodiments, the single domain antibody or antigen-binding fragment thereof, polypeptide construct, isolated nucleic acid molecule, vector, host cell or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (e.g., simultaneously or sequentially).

[0295] In certain embodiments, the additional pharmaceutically active agent is selected from drugs used to treat diseases associated with GH, IGF-1 and / or GHRH deficiency.

[0296] In the twelfth aspect, the present application provides a method for preventing and / or treating a disease associated with GHR signaling in a subject, comprising: administering to a subject in need thereof an effective amount of the single domain antibody or antigen-binding fragment thereof of the first aspect, the polypeptide construct of the second aspect or the third aspect, the isolated nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, the host cell of the sixth aspect, the bispecific or multispecific molecule of the eighth aspect, the immunoconjugate of the ninth aspect, or the pharmaceutical composition of the tenth aspect.

[0297] In certain embodiments, prevention and / or treatment of diseases associated with GHR signaling will benefit from agonism of GHR signaling.

[0298] In certain embodiments, the disease associated with GHR signaling is selected from diseases associated with GH (growth hormone), IGF-1 (insulin-like growth factor-1) and / or GHRH (growth hormone-releasing hormone) deficiency.

[0299] In certain embodiments, the disease associated with GHR signaling is selected from the group consisting of growth hormone deficiency (GHD) (e.g., growth hormone deficiency caused by hypothalamic-pituitary disease), renal insufficiency (CKD), Turner syndrome, Prader-Will syndrome, small for gestational age infant (SGA), idiopathic short stature (ISS), SHOX gene defect, Noonan syndrome, childhood short stature (e.g., short stature caused by achondroplasia (ACH), etc.), severe burns, adult growth hormone deficiency, adult short bowel syndrome, HIV wasting syndrome, aging, and reproductive disorders (e.g., reproductive disorders related to the growth hormone-growth hormone receptor signaling pathway, such as reproductive disorders caused by gonadal dysgenesis due to growth disorders caused by Turner syndrome).

[0300] In certain embodiments, the subject is a mammal, such as a human or mouse.

[0301] In certain embodiments, the single domain antibody or antigen-binding fragment thereof, polypeptide construct, isolated nucleic acid molecule, vector, host cell or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (e.g., simultaneously or sequentially).

[0302] In certain embodiments, the additional pharmaceutically active agent is selected from drugs used to treat diseases associated with GH, IGF-1 and / or GHRH deficiency.

[0303] The single-domain antibody or its antigen-binding fragment or polypeptide construct or pharmaceutical composition of the present application can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The single-domain antibody or its antigen-binding fragment or polypeptide construct or pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: incorporating the required dose of the single-domain antibody or its antigen-binding fragment or polypeptide construct or pharmaceutical composition of the present invention into an appropriate solvent, and optionally, incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives, stabilizers, or any combination thereof), followed by filtration and sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0304] The single domain antibody or its antigen-binding fragment or polypeptide construct or pharmaceutical composition of the present application can be applied by any suitable method known in the art, including but not limited to oral, oral, sublingual, ophthalmic, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (such as, powder, ointment or drops), or nasal route. However, for many therapeutic uses, the preferred route of administration / mode is parenteral administration (such as intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). It should be understood by the technician that the route of administration and / or mode will change according to the intended purpose. In certain embodiments, the single domain antibody or its antigen-binding fragment or polypeptide construct or pharmaceutical composition of the present invention is administered by intravenous injection or push injection.

[0305] In a thirteenth aspect, the present application also provides a conjugate comprising the single domain antibody or antigen-binding fragment thereof of the first aspect or the polypeptide construct of the second aspect or the third aspect, and a detectable label connected to the single domain antibody or antigen-binding fragment thereof or the polypeptide construct.

[0306] In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0307] In a fourteenth aspect, the present application further provides a kit comprising the single domain antibody or antigen-binding fragment thereof of the first aspect, or the polypeptide construct of the second aspect or the third aspect, or the conjugate of the thirteenth aspect.

[0308] In certain embodiments, the kit comprises a detection buffer.

[0309] In certain embodiments, the kit comprises the conjugate of the thirteenth aspect.

[0310] In certain embodiments, the kit comprises the single domain antibody or antigen-binding fragment thereof of the first aspect or the polypeptide construct of the second aspect or the third aspect, and a second antibody that specifically recognizes the single domain antibody or antigen-binding fragment thereof or the polypeptide construct; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0311] In a fifteenth aspect, the present application also provides a method for detecting the presence or level of GHR in a sample, which comprises using the single domain antibody or antigen-binding fragment thereof of the first aspect, or the polypeptide construct of the second or third aspect, or the conjugate of the thirteenth aspect.

[0312] In certain embodiments, the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic, non-diagnostic purposes.

[0313] In certain embodiments, the method is an immunological assay, such as immunoblotting, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0314] In certain embodiments, the method comprises use of the conjugate of the thirteenth aspect.

[0315] In certain embodiments, the method comprises using the single domain antibody or antigen-binding fragment thereof of the first aspect or the polypeptide construct of the second aspect or the third aspect, and the method further comprises using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin) to detect the single domain antibody or antigen-binding fragment thereof or the polypeptide construct.

[0316] In certain embodiments, the method comprises: (1) contacting the sample with a single domain antibody or antigen-binding fragment thereof, polypeptide construct, or conjugate of the present invention; and (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of GHR or cells expressing GHR.

[0317] The present application also provides a method for diagnosing a disease associated with GHR (e.g., a disease associated with abnormal levels or structure of GHR), comprising detecting the presence or level of GHR in a sample from a subject using the method described in Aspect 15. In certain embodiments, when GHR is present or the level of GHR is increased compared to a reference level (e.g., compared to a healthy control), it indicates that the subject suffers from a disease associated with GHR (e.g., a disease associated with abnormal levels or structure of GHR).

[0318] In certain embodiments, the GHR-related disease is selected from Laron syndrome.

[0319] In certain embodiments, the GHR is human GHR or murine (eg, rat) GHR.

[0320] In certain embodiments, the subject is a mammal (preferably a human or mouse).

[0321] In certain embodiments, the sample is a tissue sample or a cell sample from a subject (eg, a mammal, preferably a human or mouse).

[0322] In a sixteenth aspect, the present application also provides use of the single-domain antibody or antigen-binding fragment thereof of the first aspect, the polypeptide construct of the second aspect or the third aspect, or the conjugate of the thirteenth aspect in the preparation of a detection reagent, wherein the detection reagent is used to detect the presence or level of GHR in a sample and / or diagnose a disease associated with GHR (e.g., a disease associated with abnormal levels or structure of GHR).

[0323] In certain embodiments, the detection reagent detects the presence or level of GHR in a sample by the method of aspect 15.

[0324] In certain embodiments, the detection reagent detects the presence or level of GHR in a sample by the method described in aspect 15 to diagnose a disease associated with GHR (e.g., a disease associated with abnormal levels or structure of GHR). In certain embodiments, the presence of GHR or an increase in the level of GHR compared to a reference level (e.g., compared to a healthy control) indicates that the subject suffers from a disease associated with GHR (e.g., a disease associated with abnormal levels or structure of GHR).

[0325] In certain embodiments, the GHR-related disease is selected from Laron syndrome.

[0326] In certain embodiments, the GHR is human GHR or murine (eg, rat) GHR.

[0327] In certain embodiments, the subject is a mammal (preferably a human or mouse).

[0328] In certain embodiments, the sample is a tissue sample or a cell sample from a subject (eg, a mammal, preferably a human or mouse).

[0329] Definition of terms

[0330] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0331] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0332] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0333] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, which refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region, VHH), usually derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody).

[0334] Single-domain antibodies are also called nanobodies, and the two are used interchangeably. Typically, nanobodies are composed of four framework regions and three complementary determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N-terminus or C-terminus to include only part of FR1 and / or FR4, or lack one or two of those framework regions, as long as they substantially maintain antigen binding and specificity.

[0335] As used herein, the term "antigen-binding fragment" of a single-domain antibody refers to a polypeptide comprising a fragment of a single-domain antibody that retains the ability to specifically bind to the same antigen to which the single-domain antibody binds, and / or competes with the single-domain antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of the Nanobodies of the invention can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the Nanobodies of the invention. In some embodiments, the "antigen-binding fragment" of the single-domain antibody may be truncated at the N-terminus or C-terminus compared to the full-length single-domain antibody so that it comprises only part of FR1 and / or FR4, or lacks one or both of those framework regions, as long as it substantially retains antigen binding and specificity.

[0336] Antigen-binding fragments of single-domain antibodies can be obtained from a given single-domain antibody (such as the Nanobodies provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and antigen-binding fragments of single-domain antibodies can be screened for specificity in the same manner as for intact Nanobodies.

[0337] Herein, unless the context clearly indicates otherwise, when referring to the term "single domain antibody", it includes not only intact single domain antibodies, but also antigen-binding fragments of single domain antibodies.

[0338] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. In nanobodies, there are three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given nanobody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). In the present invention, the CDRs contained in the single-domain antibodies or antigen-binding fragments thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the single-domain antibodies or antigen-binding fragments thereof of the present invention are preferably determined by the Kabat, Chothia or IMGT numbering systems.

[0339] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0340] In this article, the term "polypeptide construct" is used in its broadest sense. Generally speaking, the polypeptide construct is generally used to refer to a construct comprising one or more polypeptides or protein components, wherein the one or more polypeptides or protein components can independently have different sources or different biological activities or functions, and are connected by covalent and / or non-covalent means (for example, by forming a covalent bond comprising a peptide bond, an isopeptide bond and / or a disulfide bond, and / or forming a non-covalent bond by a hydrogen bond). The polypeptide construct of the present invention is not limited in the number of its molecular chains (for example, peptide chains). For example, the polypeptide construct of the present invention can comprise only one molecular chain (for example, a peptide chain), or comprise two or more molecular chains (for example, peptide chains), and the two or more molecular chains (for example, peptide chains) are covalently and / or non-covalently connected (for example, by forming a covalent bond comprising a peptide bond, an isopeptide bond and / or a disulfide bond, and / or forming a non-covalent bond by a hydrogen bond). Similarly, it is readily understood by those skilled in the art that, in embodiments comprising multiple polypeptide or protein components, the multiple polypeptide or protein components comprised in the polypeptide construct of the present invention may be located entirely or partially in the same molecular chain (e.g., peptide chain), or may be located in different molecular chains (e.g., peptide chains).

[0341] As used herein, the terms "Fc peptide", "Fc", "Fc fragment" or "Fc region" refer to an antibody fragment formed by the second constant region (CH2) and the third constant region (CH3) of the immunoglobulin heavy chain. The Fc fragment of an antibody has a variety of different functions but does not participate in the binding of an antigen. For example, the Fc peptide can mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system. In certain embodiments, the Fc peptide comprises a hinge region located between the CH1 region and the CH2 region. In certain embodiments, the Fc peptide does not comprise a hinge region located between the CH1 region and the CH2 region.

[0342] As used herein, the term "hinge peptide" or "hinge region" refers to the hinge peptide of an immunoglobulin heavy chain, which is located at the C-terminus of the immunoglobulin heavy chain CH1 region and is used to connect the immunoglobulin heavy chain CH2 region. The hinge peptide generally contains cysteine ​​and can be used to form a disulfide bond between two heavy chains of an immunoglobulin containing the hinge peptide, thereby mediating dimerization between the two Fc peptides contained in the two heavy chains.

[0343] As used herein, the term "heavy chain antibody" (HCAb), or "heavy chain antibody only" refers to an antibody that lacks a conventional antibody light chain. In certain embodiments, the heavy chain antibody includes, but is not limited to, a dimeric antibody comprising a VHH antigen-binding domain and a CH2 and / or CH3 constant domain, and optionally a hinge region, in the absence of a CH1 domain. In certain embodiments, the heavy chain antibody consists of a VHH antigen-binding domain, at least a portion of a hinge region, and a CH2 domain and / or a CH3 domain.

[0344] As used herein, the term "bispecific antibody" or "bispecific molecule" refers to an antibody that has binding specificity for two different antigens (or epitopes). The term "multispecific antibody" or "multispecific molecule" refers to an antibody that has binding specificity for at least two or more (e.g., three or four) different antigens (or epitopes). A bispecific antibody or multispecific antibody comprises multiple antigen-binding domains that have binding specificity for different antigens (or epitopes), thereby being able to bind to at least two different binding sites and / or target molecules.

[0345] As used herein, the term "antibody" refers to an immunoglobulin-derived molecule that is capable of specifically binding to a target antigen through at least one antigen-binding site located in its variable region. A "complete antibody" typically consists of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with the heavy chain also containing a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of a single domain, CL. The constant domain is not directly involved in antibody-antigen binding but exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly variable regions, known as complementarity-determining regions (CDRs), interspersed with more conserved regions known as framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding site. The distribution of amino acids among regions or domains can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0346] As used herein, the term "humanization" refers to the modification of a non-human antibody by genetic engineering, wherein the amino acid sequence of the non-human antibody is modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). In certain embodiments, the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, etc. In the present application, the donor antibody can be a camel-derived antibody with expected properties (e.g., antigen specificity, affinity, reactivity, etc.). To prepare a humanized antibody, the CDR region of the donor antibody can be inserted into a human framework sequence using methods known in the art. In some cases, the humanized framework sequence may include amino acid mutations replaced by corresponding non-human residues. In addition, the humanized antibody may also include residues that are not found in the initial donor antibody variable region (e.g., light chain variable region or heavy chain variable region) or human framework sequence, to further improve or optimize the performance of the humanized antibody. In certain embodiments, when humanizing a single-domain antibody, all or part of the CDR region of the humanized antibody is derived from a non-human single-domain antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) is derived from the heavy chain of a human immunoglobulin (receptor antibody). In certain embodiments, the CDR region of the humanized antibody is derived from a non-human single-domain antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) is derived from the heavy chain of a human immunoglobulin (receptor antibody).

[0347] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0348] The determination of percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403.

[0349] As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived.

[0350] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) indicates. In the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0351] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant K D(See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D , kon and kdis values. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0352] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0353] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0354] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0355] The twenty conventional amino acids referred to herein are compiled according to conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0356] Unless otherwise indicated herein or clearly contradicted by the context, "A, B and / or C" or similar expressions should be understood to mean "A, B, C or any combination thereof", for example, it can be understood to mean any one selected from A, B, C, A and B, A and C, B and C, A and B and C.

[0357] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives, and stabilizers. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0358] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to extending survival compared to expected survival (e.g., survival without treatment).

[0359] As used herein, the term "subject" refers to a mammal, such as a human or mouse (e.g., rat). In certain embodiments, the subject (e.g., human or mouse) suffers from a disease associated with GHR signaling (e.g., a disease associated with GH (growth hormone), IGF-1 (insulin-like growth factor-1) and / or GHRH (growth hormone-releasing hormone) deficiency), or is at risk of suffering from the above-mentioned disease.

[0360] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing a disease (e.g., a disease associated with GH (growth hormone), IGF-1 (insulin-like growth factor-1) and / or GHRH (growth hormone-releasing hormone) deficiency) refers to an amount sufficient to prevent, prevent, or delay the onset of the disease; an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0361] Advantageous Effects of the Invention

[0362] Compared to existing GHR agonists (e.g., GH or its modifications), the agonist single-domain antibodies specifically binding to GHR or polypeptide constructs comprising the same provided in the present application have at least one or more of the following beneficial effects:

[0363] 1) Existing GHR agonists derived from GH itself (e.g., GH-Fc fusion protein GHR agonists) are not ideal for GH replacement therapy in patients with GHR mutations (e.g., point mutations) or the presence of GH neutralizing antibodies. The agonistic antibodies provided in this application are theoretically effective for these two types of patients.

[0364] 2) There is no interaction between the agonist antibodies provided in this application and the prolactin receptor, and no adverse reactions related to the mammary gland;

[0365] 3) The polypeptide construct provided in this application has a long half-life similar to that of general antibodies in vivo, which can greatly improve patient compliance compared to existing GHR agonist formulations;

[0366] 4) The agonistic single-domain antibodies or polypeptide constructs provided herein stimulate IGF-1 levels more gradually than GH, thus avoiding potential safety risks associated with high-dose use;

[0367] 5) The polypeptide construct provided in this application is a heavy chain antibody and has high stability and drugability.

[0368] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0369] Figure 1: ELISA binding activity test results of P15164-1 molecule and hGHBP-mFc.

[0370] Figure 2: Activity detection results of P15164-1 molecule in promoting the proliferation of Baf3-hGHR cells.

[0371] Figure 3: Activity detection results of SZ0022~SZ0030 in promoting the proliferation of Baf3-hGHR cells.

[0372] FIG4 is a schematic diagram showing the structure of an exemplary heavy chain antibody molecule of the present invention.

[0373] Figure 5: Activity detection results of SZ0022 and SZ0035~SZ0044 in promoting the proliferation of Baf3-hGHR cells.

[0374] FIG6A : Binding activity test results of SZ20022 and SZ0051 to SZ0053 to HEK293-hGHR cells.

[0375] Figure 6B: Binding activity test results of SZ20022 and SZ0054~SZ0056 to HEK293-hGHR cells.

[0376] Figure 7: Activity detection results of SZ0022 and SZ0051~SZ0056 in promoting the proliferation of Baf3-hGHR cells.

[0377] Figure 8: Activity detection results of SZ0055~SZ0056 in promoting the proliferation of Baf3-rGHR cells.

[0378] FIG9A : The results of the detection of the binding activity of SZ0055 to rat PRLR.

[0379] Figure 9B: Detection results of the binding activity of SZ0055 to human PRLR.

[0380] Figure 10: GX-H9 binding activity test results with human PRLR and rat PRLR.

[0381] Figure 11: Body weight changes of animals in each group after drug administration.

[0382] Figure 12: Changes in serum IGF-1 levels in each group of animals after drug administration.

[0383] Figure 13: Body length of animals in each group after drug administration.

[0384] Figure 14: Tibia length of animals in each group after drug administration.

[0385] Figure 15: Results of PK experiment in cynomolgus monkeys.

[0386] Sequence information

[0387] A description of the sequences involved in this application is provided in the table below.

[0388] Table 1.1: Sequence information DETAILED DESCRIPTION

[0389] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0390] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Molecular Biology: A Compendium of Laboratory Manuals, 3rd edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0391] Reagent preparation and experimental methods

[0392] 1. Protein Sample Preparation

[0393] The desired protein amino acid sequence (SEQ ID NOs: 53-60) was reverse-translated into a nucleotide sequence, and a eukaryotic expression vector was constructed for transient transfection into CHO or 293 cells. The target protein was purified using affinity chromatography (protein A, protein G, or a nickel column). The purified protein passed SDS-PAGE, SEC-HPLC, and endotoxin testing before use in subsequent experiments. hGHBP is the extracellular domain of human GHR.

[0394] 2. Cell Line Construction

[0395] The genes encoding human GHR protein (Uniprot accession number: P10912), cynomolgus macaque GHR protein (Uniprot accession number: A0A7N9D090), and rat GHR protein (Uniprot accession number: P16310) were ligated into plasmids to construct lentiviral plasmids overexpressing hGHR, cGHR, and rGHR proteins, respectively. Lentiviral packaging and concentration were performed to obtain lentiviral solutions. The lentiviral solutions were used to infect Baf3 (CTCC) and HEK293 cells, respectively. Following infection, the infected cells were pressure-selected with puromycin (Puro) to obtain resistant cell pools. GHR protein expression was then assessed by flow cytometry. The identified positive pool cells were monocloned using the limiting dilution method. After expansion culture and identification, Baf3 and HEK293 cell lines overexpressing GHR protein were obtained and named Baf3-hGHR, Baf3-cGHR, Baf3-rGHR, HEK293-hGHR, HEK293-cGHR, and HEK293-rGHR cell lines.

[0396] 3. Cell Proliferation Activity Detection

[0397] Baf3-hGHR cell proliferation activity was assayed using a cell proliferation-toxicity assay kit (Dongren Chemical). Baf3-hGHR cells were washed twice with experimental medium (RPMI-1640 + 3% fetal bovine serum + 50 μM mercaptoethanol). After starvation for 4 hours, cells were counted and plated at a density of 40,000 cells / 50 μL / well. GH was diluted to 25 nM in experimental medium, with the starting concentration of the test sample ranging from 75 to 300 nM. Four-fold dilutions were performed in 12 steps at 50 μL / well in triplicate. 50 μL of cells were plated, and 50 μL of the test sample was added to a total volume of 100 μL. The cells were incubated for 18 hours. CCK8 (5-fold dilution) was added at a density of 50 μL / well, and OD450 values ​​were measured 3 hours later using a microplate reader.

[0398] The Baf3-rGHR cell proliferation activity detection method is similar to the above-mentioned Baf3-hGHR cell proliferation activity detection method, except that the detection cells are Baf3-rGHR and are incubated for 72 hours; 100 μL / well of CellCountingLite2.0 working solution is added, and the luminescence value is detected by the microplate detection system.

[0399] 4. Affinity Determination

[0400] The experiment used a Biacore molecular interaction analyzer (cytiva, Biacore 8K+). Anti-human antibodies were coupled to a CM-5 chip (cytiva, 29149603) according to the instructions of the anti-human capture kit, type 2 (cytiva, 29234600). The chip was then used to capture the sample. The analyte was passed over the chip, with binding for 180 seconds and dissociation for 300 seconds. Kinetic analysis was performed based on the acquired data using Biacore Insight Evaluation Software 3.0.12. hIgG1 was used as a negative control.

[0401] 5. Rat Growth Experiment

[0402] The animals were rGHR (rat GHR) knockout rats with simultaneous hGHR (human GHR) knock-in. In these rats, rGH only partially activates hGHR, resulting in GH deficiency. The animals were smaller and weighed less than wild-type Sprague-Dawley rats of the same age. hGHR transgenic rats aged 7 to 9 weeks were randomly divided into groups based on body weight, with 6 to 8 rats per group. Administration was via subcutaneous injection in the neck; an equal volume of vehicle was administered subcutaneously in the negative control group. PEG-GH was prepared according to the method described in CN101385858B, diluted with vehicle according to the dose, and injected subcutaneously in an equal volume.

[0403] For single-dose studies, rats were weighed daily for 14-18 days after administration. Blood was collected at appropriate time points to measure IGF-1 levels. Body length and tibia length were measured at the end of the experiment. Body length refers to the distance from the tip of the rat's nose to its anus.

[0404] According to the blood collection plan, use a syringe to collect blood from the jugular vein. Collect 100-150 μL of whole blood from each animal. Place the collected whole blood in a coagulant tube and let it rest at 2-8°C for 2 hours. Then centrifuge it twice at 4°C at 1500g for 10 minutes. After centrifugation, transfer the collected serum to a new, labeled centrifuge tube and store it below -70°C.

[0405] Serum IGF-1 levels were measured using a commercially available ELISA kit (R&D Systems; Cat. No. SMG100). The assay is a sandwich ELISA using a highly IGF-1-specific polyclonal antibody as a capture agent and a horseradish peroxidase-labeled high-affinity polyclonal antibody as a detector.

[0406] Example 1: Discovery of GHR agonistic antibody molecules

[0407] 1. Immunization, library construction and screening

[0408] Alpaca were immunized with hGH / hGHBP-His complex and hGHBP-His. The first seven immunizations were performed with hGH / hGHBP-His complex and the eighth immunization was performed with hGHBP-His as a booster immunization. Peripheral blood was collected after the eighth immunization to construct an alpaca immune library.

[0409] 50 ml of fresh peripheral blood was drawn from a recipient of immunoprecipitated cells (PBMCs), and total RNA was extracted and reverse transcribed to synthesize first-strand cDNA. Two rounds of PCR were performed using the reverse transcription product as a template. The first round of PCR used primers designed for the signal peptide and heavy chain constant regions, amplifying two bands of approximately 1000 bp and 750 bp. The 750 bp band was recovered by gel extraction and used as the template for the second round of PCR. The second round of PCR used primers designed for the FR1 and FR4 regions, and introduced restriction sites for the phagemid vector. After restriction digestion and agarose gel recovery, the second round of PCR products were ligated to the restriction-digested vector and electroporated into Escherichia coli TG1 to complete the phage display library construction.

[0410] After library construction and packaging into phage, liquid-phase panning was performed using biotinylated hGHBP-His as the antigen. Bacterial cultures from rounds 1 to 4 of panning were plated with limiting dilutions and incubated overnight. Single clones were selected for soluble expression. Soluble expression supernatants were analyzed by ELISA using hGHBP-mFc as the antigen. Finally, 1406 positive clones were selected for sequencing. Finally, based on sequence characteristics, 11 clones were selected from 226 different sequences for further soluble expression. A series of in vitro activity assays were performed, and the preferred clone, P15164, was selected for further research. The VHH and CDR sequences of clone P15164 are shown in Tables 1.1 and 1.2.

[0411] 2. Construction of full-length vector

[0412] 2.1 Molecular construction

[0413] The P15164 single-domain antibody (SDO) was selected for fusion protein design. From N-terminus to C-terminus, the design sequence was: P15164 SDO VHH (SEQ ID NO: 61), hIgG2 hinge region (SEQ ID NO: 62), and hIgG1 Fc region (T366Y, Δ477K, i.e., amino acid T at position 366 is mutated to Y and amino acid K at position 447 is deleted) (SEQ ID NO: 65). The resulting fusion protein was named P15164-1.

[0414] 2.2 ELISA binding activity detection

[0415] The P15164-1 molecule was purified and its binding activity to hGHBP was detected by ELISA.

[0416] ELISA plates were coated with hGHBP-mFc protein, with 50 μl per well incubated overnight at 4°C. After washing the plates three times with PBST, they were blocked with 1% BSA, with 250 μl per well incubated at room temperature for 2 hours. After washing the plates three times with PBST, 50 μl of P15164-1 sample (starting at 10 μg / ml and diluted three-fold) was added to each well and incubated for 1 hour at room temperature. After washing the plates six times with PBST, 50 μl of a 1:5000 dilution of goat anti-human Fc secondary antibody was added to each well and incubated for 1 hour at room temperature. After washing the plates six times with PBST, 50 μl of TMB colorimetric solution was added to each well and color was developed at room temperature in the dark for 3-5 minutes. The reaction was terminated by adding 50 μl of stop solution per well. Immediately after termination, the plate was placed in a microplate reader and the OD was measured at 450 nm. The raw data were saved and analyzed. The data were analyzed using software, as shown in Figure 1.

[0417] The results showed that P15164-1 had good hGHBP protein binding activity with an EC50 of 0.2135 nM.

[0418] 2.3 Cell proliferation activity detection

[0419] The cells tested were Baf3-hGHR, and the starting concentration of the sample was 75 nM. The results are shown in Figure 2 and Table 2.

[0420] Table 2 Cell proliferation activity detection results

[0421] The results showed that P15164-1 had a certain degree of Baf3-hGHR cell proliferation activity.

[0422] Example 2: Humanization

[0423] 1. Humanized design

[0424] Humanization involved transplanting the CDRs (defined by IMGT) from P15164-1 into the human germline gene IgHV3-23, testing the importance of different amino acids within the CDR and FR regions, and combining the corresponding amino acids. The humanized variable region sequences are shown in SEQ ID NOs: 67-75.

[0425] Heavy chain antibodies were constructed by fusion of the humanized variable region with the human IgG2 hinge region and human IgG1 Fc region. The human IgG1 Fc region employed the L234A / L235A (LALA) mutation to minimize Fc effector function. The sequences of the human IgG2 hinge region and human IgG1 Fc (LALA) region are shown in SEQ ID NOs: 62 and 76, respectively. The resulting heavy chain antibody molecules were designated SZ0022 to SZ0030.

[0426] 2. Affinity Determination

[0427] The experiment used a Biacore molecular interaction analyzer (cytiva, Biacore 8K+), the test samples were SZ0022-SZ0030 series molecules (concentration 0.5 μg / ml), and the analyte was hGHBP-His (concentration 50 nM). The results are shown in Table 3.

[0428] Table 3 Affinity test results of SZ0022~SZ0030

[0429] The results showed that the humanized molecules SZ0022~SZ0030 all had good affinity.

[0430] 3. Cell Proliferation Activity Detection

[0431] The cells tested were Baf3-hGHR cells, and the starting concentration of the sample was 300 nM. The results are shown in Figure 3 and Table 4.

[0432] Table 4 Cell proliferation activity detection results

[0433] The results showed that the humanized molecules SZ0022~SZ0030 all had good Baf3-hGHR cell proliferation activity.

[0434] Example 3: Hinge region sequence optimization

[0435] 1. Sequence Design

[0436] The hinge region of SZ0022 uses the human IgG2 hinge region, which contains four cysteines that form interchain disulfide bonds with the other heavy chain within the molecule. However, these disulfide bonds are unstable and are prone to the presence of free sulfhydryl groups, which may form disulfide bonds with free sulfhydryl groups of other molecules, leading to the formation of aggregates. This example optimizes the hinge region based on SZ0022. The optimization strategy includes one or more of the following:

[0437] (1) Mutate the two upstream cysteines of the IgG2 hinge region to serine; (2) Use the hinge regions of IgG1 and IgG4; (3) Mutate one upstream cysteine ​​of the IgG1 hinge region to serine and / or gradually shorten the IgG1 hinge region to find the optimal hinge region length; (4) Optimize the amino acid composition upstream of the disulfide bond in the hinge region; (5) Mutate the residue S at position 228 defined by the EU numbering system in the IgG4 hinge region to P.

[0438] A series of hinge regions were designed, and the heavy chain antibody molecules constructed were named SZ0035 to SZ0044. The molecular information is shown in Table 5, and the exemplary structure of the construct is shown in Figure 4.

[0439] Table 5 Information of constructed heavy chain antibody molecules

[0440] 2. Protein Sample Preparation

[0441] SZ0035-SZ0044 were constructed using vectors and expressed in the same batch as the original molecule SZ0022. Following a one-step protein A affinity chromatography step, the proteins were identified by SEC-HPLC. The overall expression, purification, and identification results are shown in Table 6.

[0442] Table 6 Protein expression, purification and identification results

[0443] The results showed that the purity of SZ0022 monomer was above 60%, while the SEC-HPLC purity of SZ0035 to SZ0044 was above 99% after the hinge region sequence was optimized.

[0444] 3. Affinity Determination

[0445] The experiment used a Biacore molecular interaction analyzer (cytiva, Biacore 8K+), the test samples were SZ0035-SZ0044 series molecules (concentration 0.5 μg / ml), and the analyte was hGHBP-His (concentration 50 nM). The results are shown in Table 7.

[0446] Table 7 Protein affinity test results

[0447] The results showed that when only the constant region was changed without changing the variable region, the hinge region mutation did not affect the affinity of SZ0035~SZ0044.

[0448] 4. Cell Proliferation Activity Assay

[0449] The cells tested were Baf3-hGHR, and the starting concentration of the sample was 300 nM. The results are shown in Figure 5 and Table 8.

[0450] Table 8 Cell proliferation activity detection results

[0451] The results showed that SZ0035 to SZ0044 all had good Baf3-hGHR cell proliferation activity. As the hinge region upstream of the disulfide bond shortened, the cell proliferation activity gradually increased. The activity (highest value) was strongest when it was shortened to only two amino acids (SZ0041, i.e., hinge region HTCPPCP), and the activity decreased as it continued to shorten.

[0452] Example 4: Optimization of variable region and hinge region combinations

[0453] This example further combines and optimizes the variable region and hinge region based on Examples 2 and 3, with the goal of combining the most humanized variable region sequence and the most optimized hinge region sequence.

[0454] 1. Molecular design

[0455] Based on the results of previous humanization, amino acids with minimal impact on activity were selected for combination to improve the degree of humanization. Three new humanized variable region sequences, P15164-1-12 to P15164-1-15, were designed (see SEQ ID NOs: 90-92). The hinge region used the optimized hinge region sequences HTCPPCP (SEQ ID NO: 86) and TCPPCP (SEQ ID NO: 87). The Fc region used IgG1 Fc (LALA) (SEQ ID NO: 76).

[0456] The constructed heavy chain antibody molecules were named SZ0051 to SZ0056, and the molecular information is shown in Table 9.

[0457] Table 9 Constructed heavy chain antibody molecule information

[0458] 2. Affinity Determination

[0459] The experiment used a Biacore molecular interaction analyzer (cytiva, Biacore 8K+), the test samples were SZ0051-SZ0056 series molecules (concentration 0.5 μg / ml), and the analyte was hGHBP-His (concentration 50 nM). The results are shown in Table 10.

[0460] Table 10 Affinity determination results

[0461] The results showed that SZ0051~SZ0056 had better affinity.

[0462] 3. Detection of cross-species activity

[0463] The experiment used a Biacore molecular interaction analyzer (cytiva, Biacore 8K+), the test samples were SZ0051-SZ0056 series molecules (concentration was 2 μg / ml), and the analyte was cyno / rat GHBP-His (concentration was 200 nM). The results are shown in Table 11.

[0464] Table 11 Results of species cross-activity detection

[0465] The results showed that SZ0051~SZ0056 had certain cross-reactivity with rat GHBP (rGHBP-His), but almost no cross-reactivity with cynomolgus monkey GHBP (cGHBP-His).

[0466] 4. Cell Binding Activity Assay

[0467] The binding activity of the test antibodies to HEK293-hGHR was detected using the iQue3 system (Sartorius): 10 μg / mL, 3-fold serial dilutions (10 steps), incubation at 4°C for 1 hour. The secondary antibody used was R-Phycoerythrin AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (Jackson). The results are shown in Figures 6A-6B.

[0468] The results showed that SZ0051~SZ0056 had good binding activity with HEK293-hGHR cells.

[0469] 5. Cell Proliferation Activity Detection

[0470] 5.1 Baf3-hGHR cell proliferation activity

[0471] The cells tested were Baf3-hGHR, and the starting concentration of the test sample was 300 nM. The results are shown in Figure 7 and Table 12.

[0472] Table 12 Cell proliferation activity detection results

[0473] The results showed that SZ0051~SZ0056 all had good Baf3-hGHR cell proliferation activity.

[0474] 5.2 Baf3-rGHR cell proliferation activity

[0475] The cells tested were Baf3-rGHR, and the starting concentration of the sample was 300 nM. The results are shown in Figure 8 and Table 13.

[0476] Table 13 Cell proliferation activity detection results

[0477] The results showed that both SZ0055 and SZ0056 had Baf3-rGHR cell proliferation activity.

[0478] 6. PRLR (prolactin receptor) binding activity verification

[0479] The experiment used a Biacore molecular interaction analyzer (cytiva, Biacore 8K+). The test samples were SZ0055 or GX-H9 molecules (concentration was 2 μg / ml), and the analytes were human PRLR (hum PRLR) and rat PRLR (rat PRLR) (Sino Biological) (concentrations were 12.5, 25, 50, 100, 200, and 400 nM). The results are shown in Figures 9A-9B, Figure 10, and Table 14.

[0480] Table 14PRLR binding activity detection results

[0481] The results showed that SZ0055 did not bind to either human or rat PRLR, indicating that SZ0055 did not activate the prolactin receptor.

[0482] 7. Rat Growth Experiment

[0483] hGHR transgenic male rats aged 7 to 9 weeks were randomly divided into groups based on body weight. The dosing information for each group is as follows:

[0484] Table 15 Dosage information for each group

[0485] Among them, SZ0055-YTE (also known as SZ0112, SEQ ID NO: 96) is a construct obtained by replacing the Fc region of SZ0055 with IgG1Fc (AA, YTE), and SZ0056-YTE (also known as SZ0113, SEQ ID NO: 97) is a construct obtained by replacing the Fc region of SZ0056 with IgG1Fc (AA, YTE); wherein, IgG1Fc (AA, YTE) contains L234A / L235A and M252Y / S254T / T256E mutations, and the amino acid sequence is shown in SEQ ID NO: 93.

[0486] Body weight, body length, tibia length and IGF-1 level were tested respectively, and the results are shown in Figures 11-14.

[0487] The results showed that each drug-treated group could significantly increase the weight, body length and tibia length of the animals compared with the vehicle control group; serum IGF-1 detection showed that compared with the vehicle control group, the test group significantly increased the IGF-1 level, which lasted until it returned to the vehicle control group level after 7 days. Compared with PEG-GH, which returned to the vehicle control group after 3 days, the duration of the increased IGF-1 level was significantly prolonged, and the IGF-1 peak was lower than that of PEG-GH.

[0488] 8. Crab-eating monkey PK (select YTE)

[0489] After a single subcutaneous injection of SZ0055, SZ0056, SZ0055-YTE and SZ0056-YTE in male cynomolgus monkeys, the sera of the cynomolgus monkeys at different time points were collected to investigate the changes in the exposure of SZ0055, SZ0056, SZ0055-YTE and SZ0056-YTE molecules in male cynomolgus monkeys after administration. The pharmacokinetic characteristics of the four molecules, SZ0055, SZ0056, SZ0055-YTE and SZ0056-YTE, were evaluated and compared.

[0490] 8.1 Preparation

[0491] The test samples were SZ0055, SZ0056, SZ0055-YTE, and SZ0056-YTE subcutaneous injection (SC) liquid formulations, provided by Changchun Jinsai Pharmaceutical Co., Ltd. According to Table 17, the test samples were diluted with the formulation vehicle (vehicle (no drug)) to the corresponding concentration (5 mg / mL) before administration.

[0492] 8.2 Experimental Animals

[0493] The experimental animals were housed in the animal room of Suzhou Xihua New Drug Development Co., Ltd. (Animal Use License No.: SYXK(Su)2021-0018). The sources and numbers of animals used in this experiment are shown in Table 16.

[0494] Table 16 Source and number of experimental animals

[0495] 8.3 Experimental Design

[0496] 8.3.1 Experimental Animal Grouping

[0497] The dosage regimen design is shown in Table 17.

[0498] Table 17 Animal grouping

[0499] 8.3.2 Sample collection and processing

[0500] The collection time was 0h before administration (within 10 minutes before administration); 1h, 2h, 4h, 8h, 12h, 18h, 24h (D1), 36h, 48h (D2), 72h (D3), 96h (D4), 120h (D5), 144h (D6), 168h (D7), 192h (D8), 216h (D9), 240h (D10), 264h (D11), 288h (D12), 312h (D13), 336h (D14), 384h (D16), 432h (D18), 480h (D20), 576h (D24), and 672h (D28) after administration.

[0501] 8.4 Experimental Data

[0502] The experimental results are shown in Figure 15. The results showed that the Fc segment YTE mutation significantly prolonged the PK of SZ0055 and SZ0056 molecules.

[0503] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A single-domain antibody or an antigen-binding fragment thereof that specifically binds to the growth hormone receptor (GHR).

2. The single-domain antibody or an antigen-binding fragment thereof according to claim 1, comprising: CDR1 or a variant thereof, CDR2 or a variant thereof, and CDR3 or a variant thereof contained in the heavy-chain variable region (VHH) as shown in any one of SEQ ID NOs: 61, 67-75, 90-92; Among them, The variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; Preferably, the substitution is a conservative substitution; Preferably, the single-domain antibody or an antigen-binding fragment thereof comprises 3 CDRs contained in the heavy-chain variable region (VHH) as shown in any one of SEQ ID NOs: 61, 67-75, 90-92.

3. The single-domain antibody or an antigen-binding fragment thereof according to claim 1 or 2, comprising: (a)(i) CDR1, which has a sequence as shown in SEQ ID NO: 7, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 7; (ii) CDR2, which has a sequence as shown in any one of SEQ ID NOs: 22-24, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 22-24; and, (iii) CDR3, which has a sequence as shown in SEQ ID NO: 48, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 48; Among them, The CDRs are defined by the Kabat numbering system; (b)(i) CDR1, which has a sequence as shown in SEQ ID NO: 9, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 9; (ii) CDR2, which has a sequence as shown in SEQ ID NO: 27 or 28, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 27 or 28; and, (iii) CDR3, which has a sequence as shown in SEQ ID NO: 49, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 49; wherein, the CDRs are defined by the IMGT numbering system; Or, (c)(i) A CDR1 having: the sequence as shown in SEQ ID NO:8, or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence shown in SEQ ID NO:8; (ii) A CDR2 having: the sequence as shown in SEQ ID NO:25 or 26, or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence shown in SEQ ID NO:25 or 26; and, (iii) A CDR3 having: the sequence as shown in SEQ ID NO:48, or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence shown in SEQ ID NO:48; wherein, the CDRs are defined by the Chothia numbering system; Preferably, the substitution is a conservative substitution.

4. A single-domain antibody or an antigen-binding fragment thereof according to any one of claims 1-3, comprising: (a) CDR1 as shown in SEQ ID NO:7, CDR2 as shown in any one of SEQ ID NO:22 - 24; and, CDR3 as shown in SEQ ID NO:48; wherein, the CDRs are defined by the Kabat numbering system; (b) CDR1 as shown in SEQ ID NO:9, CDR2 as shown in SEQ ID NO:27 or 28, and CDR3 as shown in SEQ ID NO:49; wherein, the CDRs are defined by the IMGT numbering system; Or, (c) CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:25 or 26, and CDR3 as shown in SEQ ID NO:48; wherein, the CDRs are defined by the Chothia numbering system; Preferably, the single-domain antibody or an antigen-binding fragment thereof comprises: (i) CDR1 as shown in SEQ ID NO:7, CDR2 as shown in SEQ ID NO:22; and CDR3 as shown in SEQ ID NO:48; wherein, the CDRs are defined by the Kabat numbering system; (ii) CDR1 as shown in SEQ ID NO:7, CDR2 as shown in SEQ ID NO:23; and CDR3 as shown in SEQ ID NO:48; wherein, the CDRs are defined by the Kabat numbering system; (iii) CDR1 as shown in SEQ ID NO:7, CDR2 as shown in SEQ ID NO:24; and CDR3 as shown in SEQ ID NO:48; wherein, the CDRs are defined by the Kabat numbering system; (iv) CDR1 as shown in SEQ ID NO:9, CDR2 as shown in SEQ ID NO:27; and CDR3 as shown in SEQ ID NO:49; wherein, the CDRs are defined by the IMGT numbering system; (v) CDR1 as shown in SEQ ID NO:9, CDR2 as shown in SEQ ID NO:28; and, CDR3 as shown in SEQ ID NO:49; wherein, said CDRs are defined by the IMGT numbering system; (vi) CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:25; and, CDR3 as shown in SEQ ID NO:48; wherein, said CDRs are defined by the Chothia numbering system; or, (vii) CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:26; and, CDR3 as shown in SEQ ID NO:48; wherein, said CDRs are defined by the Chothia numbering system.

5. The single-domain antibody or antigen-binding fragment thereof according to any one of claims 1-4, which comprises an amino acid sequence selected from the following: (i) The sequence as shown in SEQ ID NO:61; (ii) A sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) as compared with the sequence as shown in SEQ ID NO:61; or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence as shown in SEQ ID NO:61; Preferably, said substitution is a conservative substitution.

6. The single-domain antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein, The single-domain antibody or antigen-binding fragment thereof is humanized; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises a heavy chain framework region of a human immunoglobulin; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises a heavy chain framework region in the amino acid sequence encoded by a human heavy chain germline antibody gene; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises a heavy chain framework region in the amino acid sequence encoded by a human IGHV gene, and said human IGHV gene is selected from IGHV3-23, IGHV3-30, IGHV3-34, IGHV3-7 and IGHV3-74; Preferably, the heavy chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to camelid residues; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises: (a) FR1 as shown in SEQ ID NO:2 or 6, FR2 as shown in any one of SEQ ID NOs:10-13, FR3 as shown in any one of SEQ ID NOs:30-33, and / or, FR4 as shown in SEQ ID NO:51; wherein, said FRs are defined by the Kabat numbering system; (b) FR1 as shown in SEQ ID NO:4 or 5, FR2 as shown in any one of SEQ ID NO:18-21, FR3 as shown in any one of SEQ ID NO:42-47, and / or FR4 as shown in SEQ ID NO:51; wherein, the FRs are defined by the IMGT numbering system; Or (c) FR1 as shown in SEQ ID NO:4 or 5, FR2 as shown in any one of SEQ ID NO:14-17, FR3 as shown in any one of SEQ ID NO:35-40, and / or FR4 as shown in SEQ ID NO:51; wherein, the FRs are defined by the Chothia numbering system; Preferably, the single-domain antibody or its antigen-binding fragment comprises: (i) a sequence as shown in any one of SEQ ID NO:67-75, 90-92; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence as shown in any one of SEQ ID NO:67-75, 90-92; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in any one of SEQ ID NO:67-75, 90-92; Preferably, the substitution is a conservative substitution.

7. A polypeptide construct, which comprises the single-domain antibody or its antigen-binding fragment according to any one of claims 1-6, and an Fc peptide derived from an immunoglobulin and an optional hinge peptide; Preferably, the single-domain antibody or its antigen-binding fragment is covalently linked to the Fc peptide and the optional hinge peptide (e.g., a covalent bond is formed through a covalent bond including a peptide bond, an isopeptide bond and / or a disulfide bond); Preferably, the polypeptide construct comprises the single-domain antibody or its antigen-binding fragment according to any one of claims 1-6, and the Fc peptide and the hinge peptide, and the single-domain antibody or its antigen-binding fragment forms a covalent bond with the Fc peptide and the hinge peptide through a covalent bond including a peptide bond; Preferably, the polypeptide construct comprises a first peptide chain, the first peptide chain comprising: a first single domain antibody or an antigen-binding fragment thereof, a first hinge peptide, and a first Fc peptide, wherein, The first single-domain antibody or its antigen-binding fragment is selected from the single-domain antibody or its antigen-binding fragment according to any one of claims 1-6; preferably, the first peptide chain sequentially comprises from the N-terminus to the C-terminus: the first single-domain antibody or its antigen-binding fragment, the first hinge peptide, and the first Fc peptide; Preferably, the polypeptide construct further comprises a second peptide chain, the second peptide chain comprises a second hinge peptide and a second Fc peptide; preferably, the second peptide chain further comprises a second single-domain antibody or its antigen-binding fragment; preferably, the second single-domain antibody is selected from single-domain antibodies that specifically bind to GHR; preferably, the second single-domain antibody or its antigen-binding fragment is selected from the single-domain antibody or its antigen-binding fragment according to any one of claims 1-6; Preferably, the second peptide chain sequentially comprises, from the N-terminus to the C-terminus: the second single-domain antibody or its antigen-binding fragment, the second hinge peptide, and the second Fc peptide; Preferably, the polypeptide construct has one or more of the following characteristics: (i) The first single-domain antibody or its antigen-binding fragment is the same as or different from the second single-domain antibody or its antigen-binding fragment; (ii) The first hinge peptide and the second hinge peptide are derived from the same or different immunoglobulins; (iii) The first Fc peptide and the second Fc peptide are derived from the same or different immunoglobulins; (iv) The first hinge peptide and the second hinge peptide are the same or different; (v) The first Fc peptide and the second Fc peptide are the same or different; (vi) The first peptide chain is the same as or different from the second peptide chain; (vii) A disulfide bond is formed between the first peptide chain and the second peptide chain for connection.

8. The polypeptide construct of claim 7, wherein, The hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) is a hinge peptide derived from an immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4); Preferably, the hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) is a hinge peptide derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4); Preferably, the hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) is selected from the hinge peptides of wild-type human immunoglobulins IgG1, IgG2, and IgG4 and artificially designed hinge peptides; Preferably, the artificially designed hinge peptide has a structure shown in Formula I from the N-terminus to the C-terminus: Z-CPPCP (Formula I) wherein Z is absent or is selected from a short peptide composed of 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 amino acid residues; Preferably, Z is connected to the short peptide CPPCP by a peptide bond; Preferably, Z does not contain cysteine residues.

9. The polypeptide construct of claim 7 or 8, wherein, The hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) is selected from the hinge peptide of wild-type human immunoglobulin IgG1 and artificially designed hinge peptides; Preferably, the artificially designed hinge peptide has a structure shown in Formula II: X1X2X3X4X5X6X7X8X9X 10 CPPCP (Formula II); wherein X1 is absent or is selected from (i) the amino acid residue E and (ii) amino acid residues that are conservative substitutions relative to (i); X2 is absent or is selected from (i) the amino acid residue P and (ii) amino acid residues that are conservative substitutions relative to (i); X3 is absent or is selected from (i) the amino acid residue K and (ii) amino acid residues that are conservative substitutions relative to (i); X4 is absent or is selected from (i) the amino acid residue S and (ii) amino acid residues that are conservative substitutions relative to (i); X5 is absent or is selected from (i) the amino acid residue S and (ii) amino acid residues that are conservative substitutions relative to (i); X6 is absent or is selected from (i) the amino acid residue D and (ii) amino acid residues that are conservative substitutions relative to (i); X7 is absent or selected from (i) the amino acid residue K and (ii) an amino acid residue that is a conservative substitution relative to (i); X8 is absent or selected from (i) the amino acid residue T and (ii) an amino acid residue that is a conservative substitution relative to (i); X9 is absent or selected from (i) the amino acid residue H and (ii) an amino acid residue that is a conservative substitution relative to (i); X 10 is absent or is selected from (i) amino acid residue T and (ii) an amino acid residue that is a conservative substitution relative to (i); Preferably, the artificially designed hinge peptide has one or more of the following characteristics: (1) X1 is absent or is the amino acid residue E, (2) X2 is absent or is the amino acid residue P, (3) X3 is absent or is the amino acid residue K, (4) X4 is absent or is the amino acid residue S, (5) X5 is absent or is the amino acid residue S, (6) X6 is absent or is the amino acid residue D, (7) X7 is absent or is the amino acid residue K, (8) X8 is absent or is the amino acid residue T, (9) X9 is absent or is the amino acid residue H, (10) X 10 is absent or is the amino acid residue T; Preferably, X1 is absent or is the amino acid residue E, X2 is absent or is the amino acid residue P, X3 is absent or is the amino acid residue K, X4 is absent or is the amino acid residue S, X5 is absent or is the amino acid residue S, X6 is absent or is the amino acid residue D, X7 is absent or is the amino acid residue K, X8 is absent or is the amino acid residue T, X9 is absent or is the amino acid residue H, X 10 is absent or is the amino acid residue T; Preferably, one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten amino acid residues selected from X1 to X 10 are absent; Preferably, X1 is absent, X1 to X2 are absent, X1 to X3 are absent, X1 to X4 are absent, X1 to X5 are absent, X1 to X6 are absent, X1 to X7 are absent, X1 to X8 are absent, X1 to X9 are absent, or X1 to X 10 is absent; Preferably, X1 to X5 are absent, X1 to X6 are absent, X1 to X7 are absent, X1 to X8 are absent, X1 to X9 are absent, or X1 to X 10 are absent; preferably, X1 to X7 are absent, or X1 to X8 are absent.

10. The polypeptide construct of claim 7 or 8, wherein, The hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) is selected from the hinge peptide of wild-type human immunoglobulin IgG2 and an artificially designed hinge peptide; Preferably, the artificially designed hinge peptide has the structure shown in Formula III; X i X ii X iii X iv X v X vi X vii CPPCP (Formula III); wherein, X i is absent or selected from (i) the amino acid residue E and (ii) an amino acid residue that is a conservative substitution relative to (i); X ii is absent or selected from (i) an amino acid residue R and (ii) an amino acid residue that is a conservative substitution relative to (i); X iii is absent or is selected from (i) amino acid residue K and (ii) amino acid residues that are conservative substitutions relative to (i); X iv is absent or selected from non-cysteine residues; preferably, X iv is absent or selected from (i) the amino acid residue S and (ii) amino acid residues that are conservative substitutions relative to (i); X v is absent or selected from non-cysteine residues; preferably, X v is absent or selected from (i) the amino acid residue S and (ii) an amino acid residue that is a conservative substitution relative to (i); X vi is absent or is selected from (i) amino acid residue V and (ii) amino acid residues that are conservative substitutions relative to (i); X vii is absent or selected from (i) amino acid residue E and (ii) an amino acid residue that is a conservative substitution relative to (i); Preferably, the artificially designed hinge peptide has one or more of the following characteristics: (1) X i is absent or is the amino acid residue E, (2) X ii is absent or is the amino acid residue R, (3) X iii is absent or is the amino acid residue K, (4) X iv is absent or is the amino acid residue S, (5) X v is absent or is the amino acid residue S, (6) X vi is absent or is the amino acid residue V, (7) X vii is absent or is the amino acid residue E; Preferably, X i is absent or is the amino acid residue E, X ii is absent or is the amino acid residue R, X iii is absent or is the amino acid residue K, X iv is absent or is the amino acid residue S, X v is absent or is the amino acid residue S, X vi is absent or is the amino acid residue V, X vii is absent or is the amino acid residue E; Preferably, X i to X vii One, or two, or three, or four, or five, or six, or seven amino acid residues selected therefrom are absent; preferably, X iv to X vii is absent.

11. The polypeptide construct according to any one of claims 7-10, wherein, The hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) has the amino acid sequence shown in any one of SEQ ID NO: 62-64, 80-89; 12. The polypeptide construct according to any one of claims 7-11, wherein, The Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from an immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4); Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4); Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from human immunoglobulin IgG1 or IgG4; 13. The polypeptide construct of claim 12, wherein, The Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from human immunoglobulin IgG1; Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is selected from the Fc peptide of wild-type human immunoglobulin IgG1 and its variants; wherein, compared with the Fc peptide of wild-type human immunoglobulin IgG1, the Fc peptide variant has reduced Fc effector function (e.g., has reduced ADCC, CDC and / or ADCP activities) and / or an extended half-life; Preferably, compared with the Fc peptide of wild-type human immunoglobulin IgG1, the Fc peptide variant has reduced FcγR binding activity, reduced serum complement molecule (C1q) binding activity, and / or enhanced FcRn binding activity; Preferably, compared with the Fc peptide of wild-type human immunoglobulin IgG1, the Fc peptide variant contains mutations selected from the following: L234A, L235A, M252Y, S254T, T256E and any combination thereof (e.g., selected from mutation (i): L234A and L235A, (ii): M252Y, S254T and T256E, and (iii): the combination of (i) and (ii)); Preferably, compared with the Fc peptide of wild-type human immunoglobulin IgG1, the Fc peptide variant contains the mutations L234A and L235A; preferably, the Fc peptide variant further contains the mutations M252Y, S254T and T256E; Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) comprises a sequence shown in any one of SEQ ID NO: 52, 65, 76, 93.

14. The polypeptide construct of claim 12, wherein, The Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from human immunoglobulin IgG4; Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is selected from the Fc peptide of wild-type human immunoglobulin IgG4 and its variants; wherein, compared with the Fc peptide of wild-type human immunoglobulin IgG4, the Fc peptide variant has reduced Fc effector function (e.g., has reduced ADCC, CDC and / or ADCP activity) and / or an extended half-life; Preferably, compared with the Fc peptide of wild-type human immunoglobulin IgG4, the Fc peptide variant has reduced FcγR binding activity, reduced serum complement molecule (C1q) binding activity, and / or enhanced FcRn binding activity; Preferably, compared with the Fc peptide of wild-type human immunoglobulin IgG4, the Fc peptide variant comprises the mutation L235E; Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) comprises a sequence shown in SEQ ID NO: 79 or 99.

15. A polypeptide construct, which comprises a single-domain antibody or an antigen-binding fragment thereof, a hinge peptide, and an Fc peptide derived from an immunoglobulin; Among them, The hinge peptide has a structure shown in Formula I from the N-terminus to the C-terminus: Z-CPPCP (Formula I) wherein, Z is absent or is selected from a short peptide composed of 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 non-cysteine amino acid residues; Preferably, Z is linked to the short peptide CPPCP by a peptide bond; Preferably, the single-domain antibody or an antigen-binding fragment thereof is covalently linked to the Fc peptide and the hinge peptide (e.g., forms a covalent link through a covalent bond comprising a peptide bond, an isopeptide bond, and / or a disulfide bond); Preferably, the single-domain antibody or an antigen-binding fragment thereof forms a covalent link with the Fc peptide and the hinge peptide through a covalent bond comprising a peptide bond; Preferably, the polypeptide construct comprises a first peptide chain, and the first peptide chain comprises: a first single-domain antibody or an antigen-binding fragment thereof, a first hinge peptide, and a first Fc peptide; preferably, the first peptide chain sequentially comprises from the N-terminus to the C-terminus: the first single-domain antibody or an antigen-binding fragment thereof, the first hinge peptide, and the first Fc peptide; Preferably, the polypeptide construct further comprises a second peptide chain, and the second peptide chain comprises a second hinge peptide and a second Fc peptide; preferably, the second peptide chain further comprises a second single-domain antibody or an antigen-binding fragment thereof; Preferably, the second peptide chain sequentially comprises from the N-terminus to the C-terminus: the second single-domain antibody or an antigen-binding fragment thereof, the second hinge peptide, and the second Fc peptide; Preferably, the polypeptide construct has one or more of the following characteristics: (i) The first single-domain antibody or its antigen-binding fragment is the same as or different from the second single-domain antibody or its antigen-binding fragment; (ii) The first hinge peptide and the second hinge peptide are derived from the same or different immunoglobulins; (iii) The first Fc peptide and the second Fc peptide are derived from the same or different immunoglobulins; (iv) The first hinge peptide and the second hinge peptide are the same as or different from each other; (v) The first Fc peptide and the second Fc peptide are the same as or different from each other; (vi) The first peptide chain and the second peptide chain are the same as or different from each other; (vii) A disulfide bond is formed between the first peptide chain and the second peptide chain to form a connection.

16. The polypeptide construct of claim 15, wherein, The first single-domain antibody is selected from single-domain antibodies that specifically bind to the growth hormone receptor (GHR); preferably, the first single-domain antibody is selected from GHR agonist antibodies; preferably, the first single-domain antibody or its antigen-binding fragment is selected from the single-domain antibodies or their antigen-binding fragments of any one of claims 1-6; Preferably, the second single-domain antibody is selected from single-domain antibodies that specifically bind to the growth hormone receptor (GHR); preferably, the second single-domain antibody is selected from GHR agonist antibodies; preferably, the second single-domain antibody or its antigen-binding fragment is selected from the single-domain antibodies or their antigen-binding fragments of any one of claims 1-6.

17. The polypeptide construct of claim 15 or 16, wherein, The hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) has a structure shown in Formula II: X1X2X3X4X5X6X7X8X9X 10 CPPCP (Formula II); Wherein, X1 is absent or selected from (i) the amino acid residue E and (ii) amino acid residues that are conservative substitutions relative to (i); X2 is absent or selected from (i) the amino acid residue P and (ii) amino acid residues that are conservative substitutions relative to (i); X3 is absent or selected from (i) the amino acid residue K and (ii) amino acid residues that are conservative substitutions relative to (i); X4 is absent or selected from (i) the amino acid residue S and (ii) amino acid residues that are conservative substitutions relative to (i); X5 is absent or selected from (i) the amino acid residue S and (ii) amino acid residues that are conservative substitutions relative to (i); X6 is absent or selected from (i) the amino acid residue D and (ii) amino acid residues that are conservative substitutions relative to (i); X7 is absent or selected from (i) the amino acid residue K and (ii) amino acid residues that are conservative substitutions relative to (i); X8 is absent or selected from (i) the amino acid residue T and (ii) amino acid residues that are conservative substitutions relative to (i); X9 is absent or selected from (i) the amino acid residue H and (ii) amino acid residues that are conservative substitutions relative to (i); X 10 is absent or is selected from (i) the amino acid residue T and (ii) an amino acid residue that is a conservative substitution relative to (i); Preferably, the hinge peptide has one or more of the following characteristics: (1) X1 is absent or is the amino acid residue E, (2) X2 is absent or is the amino acid residue P, (3) X3 is absent or is the amino acid residue K, (4) X4 is absent or is the amino acid residue S, (5) X5 is absent or is the amino acid residue S, (6) X6 is absent or is the amino acid residue D, (7) X7 is absent or is the amino acid residue K, (8) X8 is absent or is the amino acid residue T, (9) X9 is absent or is the amino acid residue H, (10) X 10 is absent or is the amino acid residue T; Preferably, X1 is absent or is the amino acid residue E, X2 is absent or is the amino acid residue P, X3 is absent or is the amino acid residue K, X4 is absent or is the amino acid residue S, X5 is absent or is the amino acid residue S, X6 is absent or is the amino acid residue D, X7 is absent or is the amino acid residue K, X8 is absent or is the amino acid residue T, X9 is absent or is the amino acid residue H, X 10 is absent or is the amino acid residue T; Preferably, one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten amino acid residues selected from X1 to X 10 are absent; Preferably, X1 is absent, X1 to X2 are absent, X1 to X3 are absent, X1 to X4 are absent, X1 to X5 are absent, X1 to X6 are absent, X1 to X7 are absent, X1 to X8 are absent, X1 to X9 are absent, or X1 to X 10 is absent; Preferably, X1 to X5 are absent, X1 to X6 are absent, X1 to X7 are absent, X1 to X8 are absent, X1 to X9 are absent, or X1 to X 10 are absent; preferably, X1 to X7 are absent, or X1 to X8 are absent.

18. The polypeptide construct of claim 15 or 16, wherein, The hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) has a structure shown in Formula III: X i X ii X iii X iv X v X vi X vii CPPCP (Formula III); wherein, X i is absent or selected from (i) amino acid residue E and (ii) an amino acid residue that is a conservative substitution relative to (i); X ii is absent or is selected from (i) the amino acid residue R and (ii) an amino acid residue that is a conservative substitution relative to (i); X iii is absent or selected from (i) the amino acid residue K and (ii) an amino acid residue that is a conservative substitution relative to (i); X iv is absent or selected from non-cysteine residues; preferably, X iv is absent or selected from (i) the amino acid residue S and (ii) an amino acid residue that is a conservative substitution relative to (i); X v is absent or selected from non-cysteine residues; preferably, X v is absent or selected from (i) the amino acid residue S and (ii) amino acid residues that are conservative substitutions relative to (i); X vi is absent or is selected from (i) amino acid residue V and (ii) an amino acid residue that is a conservative substitution relative to (i); X vii is absent or is selected from (i) the amino acid residue E and (ii) an amino acid residue that is a conservative substitution relative to (i); Preferably, the hinge peptide has one or more of the following characteristics: (1) X i is absent or is the amino acid residue E, (2) X ii is absent or is the amino acid residue R, (3) X iii is absent or is the amino acid residue K, (4) X iv is absent or is the amino acid residue S, (5) X v is absent or is the amino acid residue S, (6) X vi is absent or is the amino acid residue V, (7) X vii is absent or is the amino acid residue E; Preferably, X i is absent or is the amino acid residue E, X ii is absent or is the amino acid residue R, X iii is absent or is the amino acid residue K, X iv is absent or is the amino acid residue S, X v is absent or is the amino acid residue S, X vi is absent or is the amino acid residue V, X vii is absent or is the amino acid residue E; Preferably, X i to X vii any one, or two, or three, or four, or five, or six, or seven amino acid residues are absent; preferably, X iv to X vii is absent.

19. The polypeptide construct according to any one of claims 15-18, wherein, The hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) has an amino acid sequence shown in any one of SEQ ID NO: 80-89.

20. The polypeptide construct according to any one of claims 15-19, wherein, The Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from an immunoglobulin (such as IgG1, IgG2, IgG3, or IgG4); Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from a human immunoglobulin (such as IgG1, IgG2, IgG3, or IgG4); Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from human immunoglobulin IgG1 or IgG4.

21. The polypeptide construct of claim 20, wherein, The Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from human immunoglobulin IgG1; Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is selected from the Fc peptide of wild-type human immunoglobulin IgG1 and its variants; wherein, compared with the Fc peptide of wild-type human immunoglobulin IgG1, the Fc peptide variant has reduced Fc effector function (e.g., has reduced ADCC, CDC, and / or ADCP activities) and / or an extended half-life; Preferably, compared with the Fc peptide of wild-type human immunoglobulin IgG1, the Fc peptide variant has reduced FcγR binding activity, reduced serum complement molecule (C1q) binding activity, and / or enhanced FcRn binding activity; Preferably, compared with the Fc peptide of wild-type human immunoglobulin IgG1, the Fc peptide variant contains mutations selected from the following: L234A, L235A, M252Y, S254T, T256E, and any combination thereof (e.g., selected from mutation (i): L234A and L235A, (ii): M252Y, S254T, and T256E, and (iii): the combination of (i) and (ii)); Preferably, compared with the Fc peptide of wild-type human immunoglobulin IgG1, the Fc peptide variant contains the mutations L234A and L235A; preferably, the Fc peptide variant further contains the mutations M252Y, S254T, and T256E; Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) contains a sequence shown in any one of SEQ ID NO: 52, 65, 76, 93.

22. The polypeptide construct of claim 20, wherein, The Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from human immunoglobulin IgG4; Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is selected from the Fc peptide of wild-type human immunoglobulin IgG4 and its variants; wherein, compared with the Fc peptide of wild-type human immunoglobulin IgG4, the Fc peptide variant has reduced Fc effector function (e.g., has reduced ADCC, CDC, and / or ADCP activities) and / or an extended half-life; Preferably, compared with the Fc peptide of wild-type human immunoglobulin IgG4, the Fc peptide variant has reduced FcγR binding activity, reduced serum complement molecule (C1q) binding activity, and / or enhanced FcRn binding activity; Preferably, compared with the Fc peptide of wild-type human immunoglobulin IgG4, the Fc peptide variant contains the mutation L235E; Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) contains a sequence shown in SEQ ID NO: 79 or 99.

23. The polypeptide construct according to any one of claims 7-22, which comprises a sequence as shown in any one of SEQ ID NOs: 94-97, 103-126; Preferably, the polypeptide construct comprises the first peptide chain and optionally the second peptide chain; wherein, The first peptide chain comprises a sequence as shown in any one of SEQ ID NOs: 94-97, 103-126, and / or, the second peptide chain comprises a sequence as shown in any one of SEQ ID NOs: 94-97, 103-126; Preferably, the polypeptide construct comprises the first peptide chain and optionally the second peptide chain; wherein: (1) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 94; (2) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 95; (3) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 96; (4) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 97; (5) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 103; (6) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 104; (7) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 105; (8) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 106; (9) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 107; (10) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 108; (11) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 109; (12) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 110; (13) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 111; (14) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 112; (15) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 113; (16) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 114; (17) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 115; (18) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 116; (19) The first peptide chain and the second peptide chain each independently comprise the sequence as shown in SEQ ID NO: 117; (20) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 118; (21) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 119; (22) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 120; (23) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 121; (24) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 122; (25) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 123; (26) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 124; (27) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO: 125; or, (28) The first peptide chain and the second peptide chain each independently comprise the sequence shown in SEQ ID NO:

126.

24. An isolated nucleic acid molecule encoding a single-domain antibody or an antigen-binding fragment thereof according to any one of claims 1-6, or a polypeptide construct according to any one of claims 7-23.

25. A vector comprising the isolated nucleic acid molecule of claim 24; preferably, the vector is a cloning vector or an expression vector.

26. A host cell comprising the isolated nucleic acid molecule of claim 24 or the vector of claim 25.

27. A method for preparing a single-domain antibody or an antigen-binding fragment thereof according to any one of claims 1-6, or a polypeptide construct according to any one of claims 7-23, comprising culturing the host cell of claim 26 under conditions that permit the expression of the single-domain antibody or an antigen-binding fragment thereof or the polypeptide construct, and recovering the single-domain antibody or an antigen-binding fragment thereof or the polypeptide construct from the cultured host cell culture.

28. A bispecific or multispecific molecule comprising a single-domain antibody or an antigen-binding fragment thereof according to any one of claims 1-6, or a polypeptide construct according to any one of claims 7-23; preferably, the bispecific or multispecific molecule specifically binds to GHR and additionally specifically binds to one or more other targets; preferably, the bispecific or multispecific molecule further comprises at least one molecule having a second binding specificity for a second target (such as a second antibody or an antigen-binding fragment thereof).

29. An immunoconjugate comprising a single-domain antibody or an antigen-binding fragment thereof according to any one of claims 1-6, or a polypeptide construct according to any one of claims 7-23, and a therapeutic agent linked to the single-domain antibody or an antigen-binding fragment thereof or the polypeptide construct; preferably, the immunoconjugate is an antibody-drug conjugate (ADC).

30. A pharmaceutical composition comprising a single-domain antibody or an antigen-binding fragment thereof according to any one of claims 1-6, a polypeptide construct according to any one of claims 7-23, an isolated nucleic acid molecule according to claim 24, a vector according to claim 25, a host cell according to claim 26, a bispecific or multispecific molecule according to claim 28, or an immunoconjugate according to claim 29, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

31. Use of a single-domain antibody or an antigen-binding fragment thereof according to any one of claims 1-6, a polypeptide construct according to any one of claims 7-23, an isolated nucleic acid molecule according to claim 24, a vector according to claim 25, a host cell according to claim 26, a bispecific or multispecific molecule according to claim 28, an immunoconjugate according to claim 29, or a pharmaceutical composition according to claim 30 for the preparation of a medicament for preventing and / or treating a disease associated with GHR signaling in a subject; Preferably, the prevention and / or treatment of the disease associated with GHR signaling will benefit from agonism of GHR signaling; Preferably, the disease associated with GHR signaling is selected from diseases associated with deficiencies in GH (growth hormone), IGF-1 (insulin-like growth factor-1), and / or GHRH (growth hormone releasing hormone); Preferably, the disease associated with GHR signaling is selected from: growth hormone deficiency (GHD), chronic kidney disease (CKD), Turner syndrome, Prader-Willi syndrome, small for gestational age (SGA), idiopathic short stature (ISS), SHOX gene deficiency, Noonan syndrome, pediatric short stature, severe burns, adult growth hormone deficiency, adult short bowel syndrome, HIV wasting syndrome, aging, reproductive disorders; Preferably, the growth hormone deficiency (GHD) is growth hormone deficiency caused by hypothalamic-pituitary diseases, the pediatric short stature is pediatric short stature caused by achondroplasia (ACH), and / or the reproductive disorder is a reproductive disorder associated with the growth hormone-growth hormone receptor signaling pathway (such as gonadal dysgenesis caused by growth disorders in Turner syndrome); Preferably, the subject is a mammal, such as a human or a mouse; Preferably, the single-domain antibody or an antigen-binding fragment thereof, polypeptide construct, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition is used alone or in combination with an additional pharmaceutically active agent (e.g., administered simultaneously or sequentially).

32. A method for preventing and / or treating a disease associated with GHR signaling in a subject, the method comprising: Administering an effective amount of a single-domain antibody or an antigen-binding fragment thereof according to any one of claims 1-6, a polypeptide construct according to any one of claims 7-23, an isolated nucleic acid molecule according to claim 24, a vector according to claim 25, a host cell according to claim 26, a bispecific or multispecific molecule according to claim 28, an immunoconjugate according to claim 29, or a pharmaceutical composition according to claim 30 to a subject in need thereof; Preferably, the prevention and / or treatment of the diseases related to GHR signaling will benefit from agonistic effects on GHR signaling; Preferably, the diseases related to GHR signaling are selected from the diseases related to the deficiency of GH (growth hormone), IGF-1 (insulin-like growth factor-1) and / or GHRH (growth hormone releasing hormone); Preferably, the diseases related to GHR signaling are selected from: growth hormone deficiency (GHD), chronic kidney disease (CKD), Turner syndrome, Prader-Willi syndrome, small for gestational age (SGA), idiopathic short stature (ISS), SHOX gene defect, Noonan syndrome, pediatric short stature, severe burns, adult growth hormone deficiency, adult short bowel syndrome, HIV wasting syndrome, aging, reproductive disorders; Preferably, the growth hormone deficiency (GHD) is growth hormone deficiency caused by hypothalamic-pituitary diseases, the pediatric short stature is pediatric short stature caused by achondroplasia (ACH), and / or the reproductive disorder is a reproductive disorder related to the growth hormone-growth hormone receptor signaling pathway (such as gonadal dysgenesis caused by growth disorders in Turner syndrome); Preferably, the subject is a mammal, such as a human or a mouse; Preferably, the single-domain antibody or its antigen-binding fragment, polypeptide construct, isolated nucleic acid molecule, vector, host cell or pharmaceutical composition is used alone or administered in combination with another pharmaceutically active agent (for example, administered simultaneously or sequentially).

33. A conjugate comprising the single-domain antibody or its antigen-binding fragment according to any one of claims 1-6 or the polypeptide construct according to any one of claims 7-23, and a detectable label linked to the single-domain antibody or its antigen-binding fragment or the polypeptide construct; Preferably, the detectable label is selected from enzymes (such as horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent proteins), radionuclides or biotin.

34. A kit comprising the single-domain antibody or its antigen-binding fragment according to any one of claims 1-6, the polypeptide construct according to any one of claims 7-23, or the conjugate according to claim 33; Preferably, the kit comprises the conjugate according to claim 33; Preferably, the kit comprises the single-domain antibody or its antigen-binding fragment according to any one of claims 1-6 or the polypeptide construct according to any one of claims 7-23, and a second antibody that specifically recognizes the single-domain antibody or its antigen-binding fragment or the polypeptide construct; optionally, the second antibody further comprises a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent proteins), a radionuclide or biotin.

35. A method for detecting the presence or level of GHR in a sample, which comprises using a single-domain antibody or an antigen-binding fragment thereof according to any one of claims 1-6, a polypeptide construct according to any one of claims 7-23, or a conjugate according to claim 33; Preferably, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay; Preferably, the method comprises using the conjugate according to claim 33; Preferably, the method comprises using a single-domain antibody or an antigen-binding fragment thereof according to any one of claims 1-6 or a polypeptide construct according to any one of claims 7-23, and the method further comprises using a second antibody carrying a detectable label (such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide, or biotin) to detect the single-domain antibody or an antigen-binding fragment thereof or the polypeptide construct; Preferably, the method includes: (1) contacting the sample with the single-domain antibody or an antigen-binding fragment thereof or the polypeptide construct or the conjugate; (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex.

36. Use of a single-domain antibody or an antigen-binding fragment thereof according to any one of claims 1-6, a polypeptide construct according to any one of claims 7-23, or a conjugate according to claim 33 in the preparation of a detection reagent for detecting the presence or level of GHR in a sample and / or diagnosing a disease related to GHR.