Ghr agonistic antibody and use thereof

Abstract

Provided in the present invention are an agonistic single-domain antibody specifically binding to a growth hormone receptor or an antigen-binding fragment thereof, and a polypeptide construct containing the single-domain antibody or the antigen-binding fragment thereof. Further provided in the present invention are the use of the single-domain antibody or the antigen-binding fragment thereof and the polypeptide construct in the prevention and / or treatment of diseases.

Description

GHR agonistic antibodies and uses thereof

[0001] This application is based on and claims priority to the application with Chinese Patent Application No. 202410543523.4, filed on April 30, 2024, the disclosure of which is incorporated herein in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine, and more particularly, to an agonistic 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

[0003] Human growth hormone (hGH) is a protein hormone secreted by the acidophilic cells of the anterior pituitary, which is composed of 191 amino acids. Its physiological function is mainly to promote material metabolism and growth and development. hGH acts on cartilage tissue through growth factors or insulin-like growth factors, increasing bone length, thereby promoting the linear growth of the human body. In recent years, more functions of hGH have been discovered, including promoting the growth of skeletal muscle and myocardial cells, promoting protein synthesis, regulating immune system function, and enhancing immune defense ability. The large-scale clinical application of hGH has also expanded from the initial prevention and treatment of growth hormone deficiency in children with short stature and adult growth hormone deficiency to burn injuries, acute pancreatitis, and aging in the elderly. Its clinical indications are also expanding.

[0004] Currently marketed and in development GHR (GH receptor) agonists are all daily or weekly preparations, all of which are GH or GH modifications. However, for patients with point mutations in the GHR receptor or the presence of GH neutralizing antibodies, GH supplementation therapy may not be ideal. In addition, due to receptor-mediated clearance, GH and related modifications require higher doses to achieve longer dosing intervals, which can lead to excessively high levels of stimulated IGF-1, raising safety concerns. SUMMARY

[0005] The present inventors have obtained an agonistic single-domain antibody that can specifically bind to GHR and a polypeptide construct containing the same through extensive research. The single-domain antibody and the polypeptide construct can mimic the in vitro and in vivo functions of GH, i.e., activate the downstream signaling pathway of GHR to achieve the effect of promoting animal weight and bone growth, etc.

[0006] Accordingly, in a first aspect, the present application provides a single domain antibody or antigen binding fragment thereof that specifically binds to a growth hormone receptor (GHR), wherein one or more of the amino acid residues in the single domain antibody located at the following positions is a histidine residue:

[0007] position 27, position 28, position 29, position 35, position 36, position 37, position 58, position 62, position 65, position 106, position 108, position 111, position 111.1, position 112.2, position 112.1, position 112, position 113, position 114, position 115, position 116, position 117;

[0008] wherein the positions are defined by the IMGT numbering system.

[0009] The definition of the positions of the amino acid residues in the variable region of the light chain (VL) and the variable region of the heavy chain (VH) according to the IMGT numbering system is well known to the person skilled in the art. For example, the CDRs of an antibody VH (or single domain antibody VHH) domain can generally be defined as comprising the amino acids corresponding to amino acid residues in the heavy chain variable domain at positions 27 to 38 (CDR1), positions 56 to 65 (CDR2) and positions 105 to 117 (CDR3), see for example:

[0010] https: / / www.imgt.org / IMGTScientificChart / Numbering / IMGTIGVLsuperfamily.html; Lefranc, M.-P., Immunology Today, 18, 509 (1997) PMID: 9386342; Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); or Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) PMID: 12477501, the entire contents of which are incorporated herein by reference.

[0011] In certain embodiments, the GHR is a human or murine GHR.

[0012] In certain embodiments, one or more of the amino acid residues in the single domain antibody located at the following positions is a histidine residue:

[0013] position 28, position 112.2, position 112, position 114, position 115, position 117;

[0014] wherein the positions are defined by the IMGT numbering system.

[0015] In certain embodiments, one or more of the amino acid residues in the single domain antibody located at:

[0016] position 112.2, position 112, position 114, position 115;

[0017] wherein the positions are defined by the IMGT numbering system.

[0018] In certain embodiments, the single domain antibody or antigen binding fragment thereof possesses one or more of the features selected from the group consisting of:

[0019] (1) the amino acid residue located at position 30 is amino acid residue F;

[0020] (2) the amino acid residue located at position 38 is amino acid residue A;

[0021] (3) the amino acid residue located at position 56 is amino acid residue I;

[0022] (4) the amino acid residue located at position 57 is amino acid residue T;

[0023] (5) the amino acid residue located at position 59 is amino acid residue L;

[0024] (6) the amino acid residue located at position 63 is amino acid residue G;

[0025] (7) the amino acid residue located at position 64 is amino acid residue H;

[0026] (8) the amino acid residue located at position 105 is amino acid residue H;

[0027] (9) the amino acid residue located at position 107 is amino acid residue A;

[0028] (10) the amino acid residue located at position 109 is amino acid residue Y;

[0029] (11) the amino acid residue located at position 110 is amino acid residue Y.

[0030] In certain embodiments, the single domain antibody or antigen binding fragment thereof comprises:

[0031] (i) a CDR1 having the structure as set forth in X1X2X3FX4X5X6A (Formula I, SEQ ID NO: 18);

[0032] (ii) a CDR2 having the structure as set forth in ITX7LX8GHX9 (Formula II, SEQ ID NO: 53); and

[0033] (iii) a CDR3 having a structure as set forth in HX 10 AX 11 YYX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 (Structure III, SEQ ID NO: 72);

[0034] wherein the CDRs are defined by the IMGT numbering system; and,

[0035] X1is an amino acid residue H or G; X2is an amino acid residue H or F; X3is an amino acid residue H or T; X4is an amino acid residue H or I; X5is an amino acid residue H or N; X6is an amino acid residue H or Y; X7is an amino acid residue H or S; X8is an amino acid residue H or G; X9is an amino acid residue H or T; X 10 is an amino acid residue H or A; X 11 is an amino acid residue H or Q; X 12 is an amino acid residue H or R; X 13 is an amino acid residue H or E; X 14 is an amino acid residue H or V; X 15 is an amino acid residue H or L; X 16 is an amino acid residue H or G; X 17 is an amino acid residue H or G; X 18 is an amino acid residue H or Q; X 19 is an amino acid residue H or Y; X 20 is an amino acid residue H or D; X 21 is an amino acid residue H or Y.

[0036] In certain embodiments, at least one of X1to X 21 is H.

[0037] In certain embodiments, X1is an amino acid residue G; X2is an amino acid residue H or F; X3is an amino acid residue T; X4is an amino acid residue I; X5is an amino acid residue N; X6is an amino acid residue Y; X7is an amino acid residue S; X8is an amino acid residue G; X9is an amino acid residue T; X 10 is an amino acid residue A; X 11 is an amino acid residue Q; X 12 is an amino acid residue R; X 13 is an amino acid residue E; X 14 is an amino acid residue H; X 15is an amino acid residue L; X 16 is an amino acid residue H or G; X 17 is an amino acid residue G; X 18 is an amino acid residue H or Q; X 19 is an amino acid residue H or Y; X 20 is an amino acid residue D; X 21 is an amino acid residue H or Y.

[0038] In certain embodiments, X1is an amino acid residue G; X2is an amino acid residue F; X3is an amino acid residue T; X4is an amino acid residue I; X5is an amino acid residue N; X6is an amino acid residue Y; X7is an amino acid residue S; X8is an amino acid residue G; X9is an amino acid residue T; X 10 is an amino acid residue A; X 11 is an amino acid residue Q; X 12 is an amino acid residue R; X 13 is an amino acid residue E; X 14 is an amino acid residue H; X 15 is an amino acid residue L; X 16 is an amino acid residue H or G; X 17 is an amino acid residue G; X 18 is an amino acid residue H or Q; X 19 is an amino acid residue H or Y; X 20 is an amino acid residue D; X 21 is an amino acid residue Y.

[0039] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises:

[0040] a CDR1 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a CDR2 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, and a CDR3 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136.

[0041] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises:

[0042] a CDR1 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a CDR2 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, and a CDR3 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136.

[0043] In certain embodiments, the CDRs are defined by the IMGT, Kabat or Chothia numbering system.

[0044] In a second aspect, the present application provides a single-domain antibody or an antigen binding fragment thereof that specifically binds to growth hormone receptor (GHR), comprising:

[0045] (i) a CDR1 having a structure as set forth in X1X2X3FX4X5X6A (Formula I, SEQ ID NO: 18);

[0046] (ii) a CDR2 having a structure as set forth in ITX7LX8GHX9 (Formula II, SEQ ID NO: 53); and

[0047] (iii) a CDR3 having a structure as set forth in HX 10 AX 11 YYX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 (Formula III, SEQ ID NO: 72);

[0048] wherein the CDRs are defined by the IMGT numbering system; and,

[0049] X1is an amino acid residue H or G; X2is an amino acid residue H or F; X3is an amino acid residue H or T; X4is an amino acid residue H or I; X5is an amino acid residue H or N; X6is an amino acid residue H or Y; X7is an amino acid residue H or S; X8is an amino acid residue H or G; X9is an amino acid residue H or T; X 10 is an amino acid residue H or A; X 11 is an amino acid residue H or Q; X 12 is an amino acid residue H or R; X 13 is an amino acid residue H or E; X 14 is an amino acid residue H or V; X 15 is an amino acid residue H or L; X 16 is an amino acid residue H or G; X 17 is an amino acid residue H or G; X 18 is an amino acid residue H or Q; X 19 is an amino acid residue H or Y; X 20 is an amino acid residue H or D; X 21 is an amino acid residue H or Y;

[0050] and at least one of the amino acid residues in X 21 is H.

[0051] In certain embodiments, X1is an amino acid residue G; X2is an amino acid residue H or F; X3is an amino acid residue T; X4is an amino acid residue I; X5is an amino acid residue N; X6is an amino acid residue Y; X7is an amino acid residue S; X8is an amino acid residue G; X9is an amino acid residue T; X 10 is an amino acid residue A; X 11 is an amino acid residue Q; X 12 is an amino acid residue R; X 13 is an amino acid residue E; X 14 is an amino acid residue H; X 15 is an amino acid residue L; X 16 is an amino acid residue H or G; X 17 is an amino acid residue G; X 18 is an amino acid residue H or Q; X 19 is an amino acid residue H or Y; X 20 is an amino acid residue D; X 21 is an amino acid residue H or Y.

[0052] In certain embodiments, X1is an amino acid residue G; X2is an amino acid residue F; X3is an amino acid residue T; X4is an amino acid residue I; X5is an amino acid residue N; X6is an amino acid residue Y; X7is an amino acid residue S; X8is an amino acid residue G; X9is an amino acid residue T; X 10 is an amino acid residue A; X 11 is an amino acid residue Q; X 12 is an amino acid residue R; X 13 is an amino acid residue E; X 14 is an amino acid residue H; X 15 is an amino acid residue L; X 16 is an amino acid residue H or G; X 17 is an amino acid residue G; X 18 is an amino acid residue H or Q; X 19 is an amino acid residue H or Y; X 20 is an amino acid residue D; X 21 is an amino acid residue Y.

[0053] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises:

[0054] a CDR1 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a CDR2 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, and a CDR3 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136.

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

[0056] a CDR1 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a CDR2 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, and a CDR3 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136.

[0057] In certain embodiments, the CDRs are defined by the IMGT, Kabat, or Chothia numbering system.

[0058] In a third aspect, the present application provides a single-domain antibody or antigen-binding fragment thereof that specifically binds to growth hormone receptor (GHR), comprising:

[0059] a CDR1 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a CDR2 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, and a CDR3 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136.

[0060] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises:

[0061] a CDR1 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a CDR2 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, and a CDR3 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136.

[0062] In certain embodiments, the CDRs are defined by the IMGT, Kabat, or Chothia numbering system.

[0063] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof of any one of the first to third aspects comprises:

[0064] a CDR1 as set forth in any one of SEQ ID NOs: 2-12; a CDR2 as set forth in any one of SEQ ID NOs: 14, 15, 17, 19; and a CDR3 as set forth in any one of SEQ ID NOs: 16, 21-52, 54-60;

[0065] and the single-domain antibody or antigen-binding fragment thereof does not simultaneously comprise:

[0066] a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and a CDR3 as set forth in SEQ ID NO: 23;

[0067] wherein the CDRs are defined by the IMGT numbering system.

[0068] In certain embodiments, the GHR is a human or murine GHR.

[0069] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof of any one of the first through third aspects comprises:

[0070] (1) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and a CDR3 as set forth in SEQ ID NO: 21;

[0071] (2) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and a CDR3 as set forth in SEQ ID NO: 22;

[0072] (3) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 15; and a CDR3 as set forth in SEQ ID NO: 23;

[0073] (4) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and a CDR3 as set forth in SEQ ID NO: 24;

[0074] (5) a CDR1 as set forth in SEQ ID NO: 3; a CDR2 as set forth in SEQ ID NO: 14; and a CDR3 as set forth in SEQ ID NO: 23;

[0075] (6) a CDR1 as set forth in SEQ ID NO: 4; a CDR2 as set forth in SEQ ID NO: 14; and a CDR3 as set forth in SEQ ID NO: 23;

[0076] (7) a CDR1 as set forth in SEQ ID NO: 5; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 23;

[0077] (8) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 25;

[0078] (9) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 26;

[0079] (10) a CDR1 as set forth in SEQ ID NO: 6; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 27;

[0080] (11) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 28;

[0081] (12) a CDR1 as set forth in SEQ ID NO: 7; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 29;

[0082] (13) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 29;

[0083] (14) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 30;

[0084] (15) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 31;

[0085] (16) a CDR1 as set forth in SEQ ID NO: 4; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 31;

[0086] (17) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 15; and, a CDR3 as set forth in SEQ ID NO: 32;

[0087] (18) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 27;

[0088] (19) a CDR1 as set forth in SEQ ID NO: 8; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 33;

[0089] (20) a CDR1 as set forth in SEQ ID NO: 7; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 34;

[0090] (21) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 34;

[0091] (22) a CDR1 as set forth in SEQ ID NO: 9; a CDR2 as set forth in SEQ ID NO: 17; and, a CDR3 as set forth in SEQ ID NO: 35;

[0092] (23) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 33;

[0093] (24) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 36;

[0094] (25) a CDR1 as set forth in SEQ ID NO: 9; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 37;

[0095] (26) a CDR1 as set forth in SEQ ID NO: 10; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 23;

[0096] (27) a CDR1 as shown in SEQ ID NO: 2; a CDR2 as shown in SEQ ID NO: 14; and, a CDR3 as shown in SEQ ID NO: 38;

[0097] (28) a CDR1 as shown in SEQ ID NO: 2; a CDR2 as shown in SEQ ID NO: 14; and, a CDR3 as shown in SEQ ID NO: 39;

[0098] (29) a CDR1 as shown in SEQ ID NO: 2; a CDR2 as shown in SEQ ID NO: 14; and, a CDR3 as shown in SEQ ID NO: 40;

[0099] (30) a CDR1 as shown in SEQ ID NO: 6; a CDR2 as shown in SEQ ID NO: 14; and, a CDR3 as shown in SEQ ID NO: 35;

[0100] (31) a CDR1 as shown in SEQ ID NO: 4; a CDR2 as shown in SEQ ID NO: 14; and, a CDR3 as shown in SEQ ID NO: 34;

[0101] (32) a CDR1 as shown in SEQ ID NO: 2; a CDR2 as shown in SEQ ID NO: 14; and, a CDR3 as shown in SEQ ID NO: 41;

[0102] (33) a CDR1 as shown in SEQ ID NO: 6; a CDR2 as shown in SEQ ID NO: 14; and, a CDR3 as shown in SEQ ID NO: 42;

[0103] (34) a CDR1 as shown in SEQ ID NO: 9; a CDR2 as shown in SEQ ID NO: 19; and, a CDR3 as shown in SEQ ID NO: 34;

[0104] (35) a CDR1 as shown in SEQ ID NO: 6; a CDR2 as shown in SEQ ID NO: 14; and, a CDR3 as shown in SEQ ID NO: 43;

[0105] (36) a CDR1 as shown in SEQ ID NO: 6; a CDR2 as shown in SEQ ID NO: 14; and, a CDR3 as shown in SEQ ID NO: 44;

[0106] (37) CDR1 as shown in SEQ ID NO: 9; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 42;

[0107] (38) CDR1 as shown in SEQ ID NO: 2; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 45;

[0108] (39) CDR1 as shown in SEQ ID NO: 9; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 46;

[0109] (40) CDR1 as shown in SEQ ID NO: 6; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 23;

[0110] (41) CDR1 as shown in SEQ ID NO: 2; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 47;

[0111] (42) CDR1 as shown in SEQ ID NO: 4; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 43;

[0112] (43) CDR1 as shown in SEQ ID NO: 6; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 36;

[0113] (44) CDR1 as shown in SEQ ID NO: 11; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 48;

[0114] (45) CDR1 as shown in SEQ ID NO: 6; CDR2 as shown in SEQ ID NO: 19; and, CDR3 as shown in SEQ ID NO: 34;

[0115] (46) CDR1 as shown in SEQ ID NO: 2; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 49;

[0116] (47) CDR1 as shown in SEQ ID NO: 12; CDR2 as shown in SEQ ID NO: 17; and, CDR3 as shown in SEQ ID NO: 50;

[0117] (48) CDR1 as shown in SEQ ID NO: 6; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 51;

[0118] (49) CDR1 as shown in SEQ ID NO: 4; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 52;

[0119] (50) CDR1 as shown in SEQ ID NO: 6; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 48;

[0120] (51) CDR1 as shown in SEQ ID NO: 2; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 54;

[0121] (52) CDR1 as shown in SEQ ID NO: 2; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 55;

[0122] (53) CDR1 as shown in SEQ ID NO: 2; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 56;

[0123] (54) CDR1 as shown in SEQ ID NO: 2; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 57;

[0124] (55) CDR1 as shown in SEQ ID NO: 2; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 58;

[0125] (56) CDR1 as shown in SEQ ID NO: 2; CDR2 as shown in SEQ ID NO: 14; and, CDR3 as shown in SEQ ID NO: 59;

[0126] (57) CDR1 as set forth in SEQ ID NO: 2; CDR2 as set forth in SEQ ID NO: 14; and, CDR3 as set forth in SEQ ID NO: 60;

[0127] (58) CDR1 as set forth in SEQ ID NO: 2; CDR2 as set forth in SEQ ID NO: 14; and, CDR3 as set forth in SEQ ID NO: 16;

[0128] (59) CDR1 as set forth in SEQ ID NO: 9; CDR2 as set forth in SEQ ID NO: 14; and, CDR3 as set forth in SEQ ID NO: 60;

[0129] (60) CDR1 as set forth in SEQ ID NO: 6; CDR2 as set forth in SEQ ID NO: 14; and, CDR3 as set forth in SEQ ID NO: 60; or,

[0130] (61) CDR1 as set forth in SEQ ID NO: 4; CDR2 as set forth in SEQ ID NO: 14; and, CDR3 as set forth in SEQ ID NO: 60;

[0131] wherein the CDRs are defined by the IMGT numbering system.

[0132] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof of any one of the first aspect to the third aspect comprises a heavy chain framework region derived from an immunoglobulin.

[0133] As will be readily understood by those skilled in the art, the heavy chain framework region derived from an immunoglobulin includes both a heavy chain framework region directly derived from an immunoglobulin and a heavy chain framework region obtained by modification or modification of a heavy chain framework region derived from an immunoglobulin. Without being limited by theory, the immunoglobulin can be derived from any species, for example, from a mammal (e.g., a human or a camelid).

[0134] In certain embodiments, the heavy chain framework region does not comprise a histidine residue, or the heavy chain framework region optionally comprises one or more histidine residues.

[0135] In certain embodiments, the amino acid residue at position 118 in the heavy chain framework region is an amino acid residue H or R; wherein the position is defined by the IMGT numbering system.

[0136] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof of any one of the first aspect to the third aspect comprises a heavy chain framework region derived from a human immunoglobulin.

[0137] As will be readily understood by those skilled in the art, the heavy chain framework regions derived from human immunoglobulin include both heavy chain framework regions directly derived from human immunoglobulin, and heavy chain framework regions obtained by engineering or modifying heavy chain framework regions derived from human immunoglobulin.

[0138] In certain embodiments, the single domain antibody or antigen binding fragment thereof of any one of the first aspect to the third aspect comprises a heavy chain framework region in an amino acid sequence encoded by a human heavy chain germline antibody gene.

[0139] In certain embodiments, the single domain antibody or antigen binding fragment thereof of any one of the first aspect to the third aspect comprises a heavy chain framework region in an amino acid sequence encoded by a human IGHV gene selected from IGHV3-23, IGHV3-30, IGHV3-34, IGHV3-7 and IGHV3-74.

[0140] In certain embodiments, the heavy chain framework region is defined by the IMGT, Kabat or Chothia numbering system. In certain embodiments, the heavy chain framework region is defined by the IMGT numbering system.

[0141] 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) back mutations from a human-derived residue to a camelid-derived residue.

[0142] In certain embodiments, the single domain antibody or antigen binding fragment thereof of any one of the first aspect to the third aspect comprises: FR1 as set forth in SEQ ID NO: 1, FR2 as set forth in SEQ ID NO: 13, FR3 as set forth in SEQ ID NO: 20, and / or, FR4 as set forth in SEQ ID NO: 61 or 62; wherein the FRs are defined by the IMGT numbering system.

[0143] In certain embodiments, the single domain antibody or antigen binding fragment thereof of any one of the first aspect to the third aspect comprises:

[0144] (i) a sequence as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136;

[0145] (ii) a sequence having one or several (e.g., 1, 2, 3, 4 or 5) amino acid substitutions, deletions or additions compared to the sequence as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136; or

[0146] (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 of any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136.

[0147] In certain embodiments, the substitution is a conservative substitution.

[0148] In certain embodiments, the single-domain antibody of any one of the first aspect to the third aspect is a GHR agonistic single-domain antibody.

[0149] In a fourth aspect, the present application provides a polypeptide construct comprising a single-domain antibody or antigen-binding fragment thereof of any one of the first aspect to the third aspect, and an Fc peptide and an optional hinge peptide derived from an immunoglobulin.

[0150] 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 by a covalent bond comprising a peptide bond, an isopeptide bond, and / or a disulfide bond).

[0151] In certain embodiments, the polypeptide construct comprises a single-domain antibody or antigen-binding fragment thereof of any one of the first aspect to the third 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 by a covalent bond comprising a peptide bond.

[0152] In certain embodiments, the polypeptide construct comprises a first peptide chain comprising: a first single-domain antibody or antigen-binding fragment thereof, a first hinge peptide, and a first Fc peptide, wherein the first single-domain antibody or antigen-binding fragment thereof is selected from a single-domain antibody or antigen-binding fragment thereof of any one of the first aspect to the third aspect. In certain embodiments, the first peptide chain comprises, in order from N-terminus to C-terminus: the first single-domain antibody or antigen-binding fragment thereof, the first hinge peptide, and the first Fc peptide.

[0153] 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 antigen-binding fragment thereof. In certain embodiments, the second single-domain antibody or antigen-binding fragment thereof is selected from a single-domain antibody or antigen-binding fragment thereof that specifically binds to GHR. In certain embodiments, the second single-domain antibody or antigen-binding fragment thereof is selected from a single-domain antibody or antigen-binding fragment thereof of any one of the first aspect to the third aspect.

[0154] In certain embodiments, the second peptide chain comprises, in order 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.

[0155] In certain embodiments, the polypeptide construct is a heavy chain antibody.

[0156] 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.

[0157] In certain embodiments, the first hinge peptide and the first Fc peptide are optionally connected by or without a peptide linker, and / or, the second hinge peptide and the second Fc peptide are optionally connected by or without a peptide linker.

[0158] 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.

[0159] In certain embodiments, the polypeptide construct possesses one or more features selected from the group consisting of:

[0160] (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;

[0161] (ii) the first hinge peptide and the second hinge peptide are derived from the same or different immunoglobulin;

[0162] (iii) the first Fc peptide and the second Fc peptide are derived from the same or different immunoglobulin;

[0163] (iv) the first hinge peptide and the second hinge peptide are the same or different;

[0164] (v) the first Fc peptide and the second Fc peptide are the same or different;

[0165] (vi) the first peptide chain is the same as or different from the second peptide chain;

[0166] (vii) the first peptide chain and the second peptide chain are connected by disulfide bond formation.

[0167] In certain embodiments, 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., IgGl, IgG2, IgG3, or IgG4).

[0168] In certain embodiments, 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., IgGl, IgG2, IgG3, or IgG4).

[0169] As will be readily understood by those skilled in the art, the hinge peptide derived from an immunoglobulin (e.g., a human immunoglobulin) includes both a hinge peptide directly derived from an immunoglobulin (e.g., a human immunoglobulin) and a hinge peptide obtained by engineering or modifying a hinge peptide derived from an immunoglobulin (e.g., a human immunoglobulin) (e.g., an artificially designed hinge peptide).

[0170] In certain embodiments, the hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) is selected from the group consisting of hinge peptides of wild-type human immunoglobulins IgGl, IgG2, and IgG4, and artificially designed hinge peptides.

[0171] In certain embodiments, the hinge peptide comprises a sequence as set forth in SEQ ID NO: 137.

[0172] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from an immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0173] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0174] As will be readily understood by those skilled in the art, the Fc peptide derived from an immunoglobulin (e.g., a human immunoglobulin) includes both a Fc peptide directly derived from an immunoglobulin (e.g., a human immunoglobulin) and a Fc peptide obtained by engineering or modifying a Fc peptide derived from an immunoglobulin (e.g., a human immunoglobulin) (e.g., a Fc peptide variant).

[0175] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from a human immunoglobulin IgGl or IgG4.

[0176] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from a human immunoglobulin IgGl.

[0177] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is selected from the group consisting of a Fc peptide of wild-type human immunoglobulin IgG1 and variants thereof; wherein the Fc peptide variant has reduced Fc effector function (e.g., has reduced ADCC, CDC and / or ADCP activity) and / or prolonged half-life as compared to the Fc peptide of wild-type human immunoglobulin IgG1.

[0178] In certain embodiments, the Fc peptide variant has reduced FcyR binding activity, reduced serum complement molecule (C1q) binding activity, and / or enhanced FcRn binding activity as compared to the Fc peptide of wild-type human immunoglobulin IgG1.

[0179] In certain embodiments, the Fc peptide variant comprises mutations selected from the group consisting of L234A, L235A, M252Y, S254T, T256E, and any combination thereof (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)) as compared to the Fc peptide of wild-type human immunoglobulin IgG1.

[0180] In certain embodiments, the Fc peptide variant comprises mutations L234A and L235A; in certain embodiments, the Fc peptide variant further comprises mutations M252Y, S254T, and T256E as compared to the Fc peptide of wild-type human immunoglobulin IgG1.

[0181] In certain embodiments, the mutation site of the mutation is defined by the EU numbering system.

[0182] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) comprises a sequence as set forth in any one of SEQ ID NOs: 138-140.

[0183] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from human immunoglobulin IgG4.

[0184] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is selected from the group consisting of a Fc peptide of wild-type human immunoglobulin IgG4 and variants thereof; wherein the Fc peptide variant has reduced Fc effector function (e.g., has reduced ADCC, CDC and / or ADCP activity) and / or prolonged half-life as compared to the Fc peptide of wild-type human immunoglobulin IgG4.

[0185] In certain embodiments, the Fc peptide variant has reduced FcyR binding activity, reduced serum complement molecule (Clq) binding activity, and / or, enhanced FcRn binding activity compared to the Fc peptide of wild-type human immunoglobulin IgG4.

[0186] In certain embodiments, the Fc peptide variant comprises mutation L235E compared to the Fc peptide of wild-type human immunoglobulin IgG4.

[0187] In certain embodiments, the mutation site of the mutation is defined by the EU numbering system.

[0188] In certain embodiments, the polypeptide construct comprises a single-domain antibody or an antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in any one of SEQ ID NOs: 138-140.

[0189] In certain embodiments, the polypeptide construct comprises a first peptide chain and optionally a second peptide chain; wherein the first peptide chain comprises, in order from N-terminus to C-terminus: a single-domain antibody or an antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in any one of SEQ ID NOs: 138-140; and / or, the second peptide chain comprises, in order from N-terminus to C-terminus: a single-domain antibody or an antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in any one of SEQ ID NOs: 138-140.

[0190] In certain embodiments, the first peptide chain comprises, in order from N-terminus to C-terminus: a single-domain antibody or an antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in SEQ ID NO: 138; and / or, the second peptide chain comprises, in order from N-terminus to C-terminus: a single-domain antibody or an antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in SEQ ID NO: 138. In certain embodiments, the Fc peptide variant has reduced FcyR binding activity, reduced serum complement molecule (Clq) binding activity, and / or, enhanced FcRn binding activity compared to the Fc peptide of wild-type human immunoglobulin IgG4.

[0186] In certain embodiments, the Fc peptide variant comprises mutation L235E compared to the Fc peptide of wild-type human immunoglobulin IgG4.

[0187] In certain embodiments, the mutation site of the mutation is defined by the EU numbering system.

[0188] In certain embodiments, the polypeptide construct comprises a single-domain antibody or an antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in any one of SEQ ID NOs: 138-140.

[0189] In certain embodiments, the polypeptide construct comprises a first peptide chain and optionally a second peptide chain; wherein the first peptide chain comprises, in order from N-terminus to C-terminus: a single-domain antibody or an antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in any one of SEQ ID NOs: 138-140; and / or, the second peptide chain comprises, in order from N-terminus to C-terminus: a single-domain antibody or an antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in any one of SEQ ID NOs: 138-140.

[0190] In certain embodiments, the first peptide chain comprises, in order from N-terminus to C-terminus: a single-domain antibody or an antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in SEQ ID NO: 138; and / or, the second peptide chain comprises, in order from N-terminus to C-terminus: a single-domain antibody or an antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in SEQ ID NO: 138.

[0191] In certain embodiments, the first peptide chain comprises, in order from N-terminus to C-terminus: a single-domain antibody or antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in SEQ ID NO: 139; and / or, the second peptide chain comprises, in order from N-terminus to C-terminus: a single-domain antibody or antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in SEQ ID NO: 139.

[0192] In certain embodiments, the first peptide chain comprises, in order from N-terminus to C-terminus: a single-domain antibody or antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in SEQ ID NO: 140; and / or, the second peptide chain comprises, in order from N-terminus to C-terminus: a single-domain antibody or antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in SEQ ID NO: 140.

[0193] In certain embodiments, (i) the first peptide chain and the second peptide chain comprise the same or different single-domain antibody or antigen-binding fragment thereof, and / or, (ii) the first peptide chain and the second peptide chain comprise the same or different Fc peptide.

[0194] In certain embodiments, the polypeptide construct comprises a sequence as set forth in any one of SEQ ID NOs: 141-146.

[0195] 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 set forth in any one of SEQ ID NOs: 141-146, and / or the second peptide chain comprises a sequence as set forth in any one of SEQ ID NOs: 141-146.

[0196] In certain embodiments, the polypeptide construct comprises the first peptide chain and, optionally, the second peptide chain; wherein:

[0197] (1) the first peptide chain and the second peptide chain each independently comprises a sequence as set forth in SEQ ID NO: 141;

[0198] (2) the first peptide chain and the second peptide chain each independently comprises a sequence as set forth in SEQ ID NO: 142;

[0199] (3) the first peptide chain and the second peptide chain each independently comprises a sequence as set forth in SEQ ID NO: 143;

[0200] (4) the first peptide chain and the second peptide chain each independently comprises a sequence as set forth in SEQ ID NO: 144;

[0201] (5) the first peptide chain and the second peptide chain each independently comprises a sequence as set forth in SEQ ID NO: 145; or,

[0202] (6) the first peptide chain and the second peptide chain each independently comprises a sequence as set forth in SEQ ID NO: 146.

[0203] In certain embodiments, the polypeptide construct is a GHR agonistic construct (e.g., the polypeptide construct can act as a GHR agonist).

[0204] In certain embodiments, the GHR is a human GHR or a murine GHR.

[0205] In a fifth aspect, the present application provides an isolated nucleic acid molecule encoding the single domain antibody or antigen binding fragment thereof of any one of the first aspect to the third aspect, or the polypeptide construct of the fourth aspect.

[0206] In certain embodiments, the isolated nucleic acid molecule encodes the single domain antibody or antigen binding fragment thereof of any one of the first aspect to the third aspect.

[0207] In certain embodiments, the isolated nucleic acid molecule encodes the polypeptide construct of the fourth aspect.

[0208] In certain embodiments, the polypeptide construct comprises a first peptide chain and a second peptide chain, 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 herein comprises a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

[0209] In a sixth aspect, the present application provides a vector comprising the isolated nucleic acid molecule of the fifth aspect. In certain embodiments, the vector is a cloning vector or an expression vector.

[0210] In certain embodiments, the vector comprises a nucleotide sequence encoding the single domain antibody or antigen binding fragment thereof of any one of the first aspect to the third aspect.

[0211] In certain embodiments, the vector comprises a nucleotide sequence encoding the polypeptide construct of the fourth aspect.

[0212] In certain embodiments, the polypeptide construct comprises a first peptide chain and a second peptide chain, 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 of the present application comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0213] In a seventh aspect, the present application provides a host cell comprising the isolated nucleic acid molecule of the fifth aspect or the vector of the sixth aspect.

[0214] Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell is a microorganism.

[0215] The single domain antibody or antigen binding fragment thereof or polypeptide construct of the present application can be prepared by various methods known in the art, for example, by genetic engineering recombination techniques. For example, a DNA molecule encoding the single domain antibody or antigen binding fragment thereof or polypeptide construct of the present application is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector, which is then transfected into a host cell. The transfected host cell is then cultured under specific conditions, and the single domain antibody or antigen binding fragment thereof or polypeptide construct of the present application is expressed. The antigen binding fragment of the present application can be obtained by hydrolyzing an intact antibody molecule.

[0216] In an eighth aspect, the present application provides a method of preparing the single domain antibody or antigen binding fragment thereof of any one of the first aspect to the third aspect or the polypeptide construct of the fourth aspect, comprising culturing the host cell of the seventh aspect under conditions permitting 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 culture of the host cell.

[0217] In a ninth aspect, the present application provides a bispecific or multispecific molecule comprising the single domain antibody or antigen binding fragment thereof of any one of the first through third aspects or the polypeptide construct of the fourth aspect.

[0218] In certain embodiments, the bispecific or multispecific molecule specifically binds to GHR and additionally specifically binds to one or more target of interest.

[0219] In certain embodiments, the bispecific or multispecific molecule further comprises at least one molecule (e.g., a second antibody or antigen binding fragment thereof) having a second binding specificity for a second target.

[0220] In a tenth aspect, the present application provides an immunoconjugate comprising the single domain antibody or antigen binding fragment thereof of any one of the first through third aspects or the polypeptide construct of the fourth aspect, and a therapeutic agent linked to the single domain antibody or antigen binding fragment thereof or the polypeptide construct.

[0221] In certain embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0222] In an eleventh aspect, the present application provides a pharmaceutical composition comprising the single domain antibody or antigen binding fragment thereof of any one of the first through third aspects, the polypeptide construct of the fourth aspect, the isolated nucleic acid molecule of the fifth aspect, the vector of the sixth aspect, the host cell of the seventh aspect, the bispecific or multispecific molecule of the ninth aspect, or the immunoconjugate of the tenth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0223] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0224] In certain embodiments, the additional pharmaceutically active agent is selected from an agent for treating a disease associated with deficiency of GH (growth hormone), IGF-1 (insulin-like growth factor-1), and / or GHRH (growth hormone releasing hormone).

[0225] In a twelfth aspect, the present application provides use of the single domain antibody or antigen binding fragment thereof of any one of the first through third aspects, the polypeptide construct of the fourth aspect, the isolated nucleic acid molecule of the fifth aspect, the vector of the sixth aspect, the host cell of the seventh aspect, the bispecific or multispecific molecule of the ninth aspect, the immunoconjugate of the tenth aspect, or the pharmaceutical composition of the eleventh aspect for the manufacture of a medicament for preventing and / or treating a disease associated with GHR signaling in a subject.

[0226] In certain embodiments, the prevention and / or treatment of the disease related to GHR signaling would benefit from an agonism of GHR signaling.

[0227] In certain embodiments, the disease related to GHR signaling is selected from a disease associated with GH (growth hormone), IGF-1 (insulin-like growth factor-1) and / or GHRH (growth hormone releasing hormone) deficiency.

[0228] In certain embodiments, the disease related to GHR signaling is selected from: growth hormone deficiency (GHD), renal insufficiency (CKD), Turner syndrome, Prader-Will syndrome, small for gestational age infant (SGA), idiopathic short stature (ISS), SHOX gene deficiency, Noonan syndrome, pediatric short stature, severe burn, adult growth hormone deficiency, adult short bowel syndrome, HIV wasting syndrome, aging, reproductive disorders, Laron syndrome.

[0229] In certain embodiments, the growth hormone deficiency (GHD) is growth hormone deficiency due to hypothalamic-pituitary disease, the pediatric short stature is pediatric short stature due to achondroplasia (ACH), and / or the reproductive disorder is a reproductive disorder associated with the growth hormone-growth hormone receptor signaling pathway (e.g. gonadal dysgenesis due to growth impairment in Turner syndrome).

[0230] In certain embodiments, the subject is a mammal, for example a human or a murine.

[0231] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof, polypeptide construct, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule, immunoconjugate or pharmaceutical composition is used alone, or is administered in combination with any of them, or is administered in combination with (e.g. simultaneously or sequentially) another pharmaceutically active agent.

[0232] In certain embodiments, the other pharmaceutically active agent is selected from an agent for the treatment of a disease associated with GH (growth hormone), IGF-1 (insulin-like growth factor-1) and / or GHRH (growth hormone releasing hormone) deficiency.

[0233] In a thirteenth 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 any one of the first to third aspects, the polypeptide construct of the fourth aspect, the isolated nucleic acid molecule of the fifth aspect, the vector of the sixth aspect, the host cell of the seventh aspect, the bispecific or multispecific molecule of the ninth aspect, the immunoconjugate of the tenth aspect, or the pharmaceutical composition of the eleventh aspect.

[0234] In certain embodiments, the prevention and / or treatment of the disease associated with GHR signaling would benefit from agonism of GHR signaling.

[0235] In certain embodiments, the disease associated with GHR signaling is selected from a disease associated with GH (growth hormone), IGF-1 (insulin-like growth factor-1), and / or GHRH (growth hormone releasing hormone) deficiency.

[0236] In certain embodiments, the disease associated with GHR signaling is selected from: growth hormone deficiency (GHD), kidney dysfunction (CKD), Turner syndrome, Prader-Willi syndrome, small for gestational age infant (SGA), idiopathic short stature (ISS), SHOX gene deficiency, Noonan syndrome, pediatric short stature, severe burn injury, adult growth hormone deficiency, adult short bowel syndrome, HIV wasting syndrome, aging, reproductive disorders, Laron syndrome.

[0237] In certain embodiments, the growth hormone deficiency (GHD) is growth hormone deficiency due to hypothalamic-pituitary disease, the pediatric short stature is pediatric short stature due to achondroplasia (ACH), and / or the reproductive disorder is a reproductive disorder associated with the growth hormone-growth hormone receptor signaling pathway (e.g., gonadal dysgenesis due to growth impairment in Turner syndrome).

[0238] In certain embodiments, the subject is a mammal, e.g., a human or a murine.

[0239] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof, the polypeptide construct, the isolated nucleic acid molecule, the vector, the host cell, the bispecific or multispecific molecule, the immunoconjugate, or the pharmaceutical composition is used alone, or is administered in any combination thereof, or is administered in combination (e.g., simultaneously or sequentially) with another pharmaceutically active agent.

[0240] In certain embodiments, the additional pharmaceutically active agent is selected from an agent for treating a disease associated with GH (growth hormone), IGF-1 (insulin-like growth factor-1), and / or GHRH (growth hormone releasing hormone) deficiency.

[0241] The single-domain antibody or antigen-binding fragment thereof or polypeptide construct or pharmaceutical composition of the present application can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The single-domain antibody or antigen-binding fragment thereof or polypeptide construct or pharmaceutical composition of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the single-domain antibody or antigen-binding fragment thereof or polypeptide construct or pharmaceutical composition of the present application in the required amount in an appropriate solvent with one or more of the other ingredients, as described below, as desired, followed by filtered sterilization. Furthermore, sterile injection solutions can be prepared as sterile lyophilized powders for reconstitution with a suitable vehicle, e.g., water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), dextrose solution (e.g., 5% dextrose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered saline), Ringer's solution, and any combination thereof, prior to use.

[0242] The single-domain antibody or antigen-binding fragment thereof or polypeptide construct or pharmaceutical composition of the present application can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracerebroventricular, inguinal, intravesical, local (e.g., powder, salve, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or means of administration will vary depending on the intended purpose. In certain embodiments, the single-domain antibody or antigen-binding fragment thereof or polypeptide construct or pharmaceutical composition of the present application is administered by intravenous injection or bolus.

[0243] In a fourteenth aspect, the present application provides a conjugate comprising the single domain antibody or antigen binding fragment thereof of any one of the first to third aspects or the polypeptide construct of the fourth aspect, and a detectable label linked to the single domain antibody or antigen binding fragment thereof or the polypeptide construct.

[0244] In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0245] In a fifteenth aspect, the present application provides a kit comprising the single domain antibody or antigen binding fragment thereof of any one of the first to third aspects, the polypeptide construct of the fourth aspect, or the conjugate of the fourteenth aspect.

[0246] In certain embodiments, the kit comprises a detection buffer.

[0247] In certain embodiments, the kit comprises the conjugate of the fourteenth aspect.

[0248] In certain embodiments, the kit comprises the single domain antibody or antigen binding fragment thereof of any one of the first to third aspects or the polypeptide construct of the fourth 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 esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0249] In a sixteenth aspect, the present application provides a method for detecting the presence or level of GHR in a sample, comprising using the single domain antibody or antigen binding fragment thereof of any one of the first to third aspects, the polypeptide construct of the fourth aspect, or the conjugate of the fourteenth aspect.

[0250] In certain embodiments, the method is for therapeutic purposes, diagnostic purposes, or non-therapeutic non-diagnostic purposes.

[0251] In certain embodiments, the method is an immunological detection, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.

[0252] In certain embodiments, the method comprises using the conjugate of the fourteenth aspect.

[0253] In certain embodiments, the method comprises using the single domain antibody or antigen binding fragment thereof of any one of the first to third aspects or the polypeptide construct of the fourth 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 reagent (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.

[0254] In certain embodiments, the method comprises: (1) contacting the sample with the single domain antibody or 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.

[0255] The present application also provides a method of diagnosing a disease associated with GHR (e.g. a disease associated with abnormal level or structure of GHR), which comprises detecting the presence or level of GHR in a sample from a subject using the method of the sixteenth aspect. In certain embodiments, when GHR is present or the level of GHR is increased as compared to a reference level (e.g. as compared to a healthy control), it indicates that the subject has a disease associated with GHR (e.g. a disease associated with abnormal level or structure of GHR).

[0256] In certain embodiments, the disease associated with GHR is selected from Laron syndrome.

[0257] In certain embodiments, the GHR is human GHR or murine GHR.

[0258] In certain embodiments, the subject is a mammal (preferably a human or a murine).

[0259] In certain embodiments, the sample is a tissue sample or a cell sample from a subject (e.g. a mammal, preferably a human or a murine).

[0260] In a seventeenth aspect, the present application provides use of the single domain antibody or antigen binding fragment thereof of any one of the first to third aspects, the polypeptide construct of the fourth aspect, or the conjugate of the fourteenth aspect in the manufacture of a detection reagent for detecting the presence or level of GHR in a sample and / or diagnosing a disease associated with GHR.

[0261] In certain embodiments, the detection reagent detects the presence or level of GHR in a sample by the method of the sixteenth aspect.

[0262] In certain embodiments, the detection reagent detects the presence or level of GHR in a sample by the method of the sixteenth aspect to thereby diagnose a GHR-associated disease (e.g., a disease associated with abnormal level or structure of GHR). In certain embodiments, when GHR is present or the level of GHR is increased as compared to a reference level (e.g., as compared to a healthy control), it indicates that the subject has a GHR-associated disease (e.g., a disease associated with abnormal level or structure of GHR).

[0263] In certain embodiments, the GHR-associated disease is selected from Laron syndrome.

[0264] In certain embodiments, the GHR is human GHR or murine GHR.

[0265] In certain embodiments, the subject is a mammal (preferably a human or a murine).

[0266] In certain embodiments, the sample is a tissue sample or a cell sample from a subject (e.g., a mammal, preferably a human or a murine).

[0267] Definitions of terms

[0268] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the laboratory procedures of virology, biochemistry, immunology followed herein are conventional procedures well established in the respective fields. Also, for better understanding of the present application, the definitions and explanations of the relevant terms are provided below.

[0269] When the terms "for example", "for instance", "such as", "including", "containing" or variations thereof are used in this document, these terms are not to be interpreted in an exclusive sense unless otherwise indicated.

[0270] Unless otherwise indicated herein, or in the context of a contradiction with the application as a whole, the terms "a" and "an" and the like are to be construed to cover both singular and plural references. It is contemplated that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0271] As used herein, the term "single-domain antibody" (sdAb) has the meaning generally understood by those skilled in the art, and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region, VHH), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or shark antibody). Single-domain antibodies are also known as nanobodies, and the two terms are used interchangeably. Typically, a nanobody consists of 4 framework regions and 3 complementarity determining regions, with the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or to lack one or both of those framework regions, so long as antigen binding and specificity are substantially maintained.

[0272] 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 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., 2nded. Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of the nanobodies of the application can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the nanobodies of the application. In some embodiments, the "antigen-binding fragment" of the single-domain antibody can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or to lack one or both of those framework regions, compared to the full-length single-domain antibody, so long as antigen binding and specificity are substantially maintained.

[0273] Antigen-binding fragments of single-domain antibodies can be obtained from a given single-domain antibody (e.g., a nanobody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and screened for specificity in the same manner as for the intact nanobody.

[0274] Herein, unless the context clearly indicates otherwise, when referring to the term "single-domain antibody", this includes not only intact single-domain antibodies, but also antigen-binding fragments of single-domain antibodies.

[0275] 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. There are three CDRs in a nanobody, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, e.g., as defined in 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 skilled person will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to the skilled person (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In the present application, the CDRs contained by the single-domain antibody or antigen-binding fragment thereof of the application can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by the single-domain antibody or antigen-binding fragment thereof of the application are preferably determined by the Kabat, Chothia, or IMGT numbering system.

[0276] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody that are not CDR residues as defined above.

[0277] The term "polypeptide construct" is used herein in its broadest sense. Generally, the polypeptide construct is generally used to denote a construct comprising one or more polypeptide or protein components, which can each independently have different origins or different biological activities or functions, and are linked by covalent and / or non-covalent means (e.g., covalent linkage by covalent bonds comprising peptide bonds, isopeptide bonds, and / or disulfide bonds, and / or non-covalent linkage by hydrogen bonds). The polypeptide construct of the present application is not limited in the number of molecular chains (e.g., peptide chains) it contains, for example, the polypeptide construct of the present application can contain only one molecular chain (e.g., peptide chain), or contain two or more molecular chains (e.g., peptide chains) covalently and / or non-covalently linked (e.g., covalent linkage by covalent bonds comprising peptide bonds, isopeptide bonds, and / or disulfide bonds, and / or non-covalent linkage by hydrogen bonds) between them. Likewise, it is readily understood by one skilled in the art that in embodiments comprising multiple polypeptide or protein components, the multiple polypeptide or protein components contained in the polypeptide construct of the present application can be located in the same molecular chain (e.g., peptide chain) in whole or in part, or can each be located in a different molecular chain (e.g., peptide chain).

[0278] As used herein, the term "Fc peptide", "Fc", "Fc fragment", or "Fc region" means an antibody fragment formed from the second constant region (CH2), the third constant region (CH3) of an immunoglobulin heavy chain. The Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding. For example, the Fc peptide can mediate the binding of immunoglobulins 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 between the CH1 region and the CH2 region. In certain embodiments, the Fc peptide does not comprise a hinge region between the CH1 region and the CH2 region.

[0279] As used herein, the term "hinge peptide" or "hinge region" means a hinge peptide of an immunoglobulin heavy chain, which is located at the C-terminus of the CH1 region of an immunoglobulin heavy chain for connecting the CH2 region of an immunoglobulin heavy chain. The hinge peptide usually comprises a cysteine, which can be used to form a disulfide bond between two heavy chains comprising the hinge peptide of an immunoglobulin, to mediate dimerization between two Fc peptides contained in the two heavy chains.

[0280] As used herein, the term "heavy chain antibody" (HCAb), or "heavy chain only antibody," means an antibody that lacks a light chain of a conventional antibody. In certain embodiments, the heavy chain antibody includes, but is not limited to, a dimeric antibody comprising a VHH antigen binding domain and CH2 and / or CH3 constant domains, 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.

[0281] As used herein, the term "bispecific antibody" or "bispecific molecule" refers to an antibody having binding specificity for two different antigens (or epitopes). The term "multispecific antibody" or "multispecific molecule" refers to an antibody having binding specificity for at least two more (e.g., three or four) different antigens (or epitopes). Bispecific antibodies or multispecific antibodies comprise multiple antigen binding domains having binding specificity for different antigens (or epitopes), enabling binding to at least two different binding sites and / or target molecules.

[0282] The term "antibody", as used herein, refers to a molecule derived from an immunoglobulin that is capable of specifically binding to a target antigen through at least one antigen-combining site located in its variable region. An "intact antibody" typically comprises two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define a different class of antibody, as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable region and the constant region are joined by a "J" region of about 12 or more amino acids, and the heavy chain further includes a "D" region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains are not involved directly in binding of an antibody to an antigen, but exhibit various effector functions, such as mediating immunoglobulin- dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) form the antigen binding site. Assignment of amino acids to each region or domain 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.

[0283] As used herein, the term "identity" is used in reference to the matching of sequences between two polypeptides or between two nucleic acids. 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., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a 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, then 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 x 100%). In certain embodiments, the two sequences are the same length.

[0284] Determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0285] As used herein, the term "variant", in the context of a polypeptide (including a polypeptide), also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by the introduction of an amino acid residue substitution, deletion, or addition. In certain instances, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not by way of limitation, a polypeptide can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivatized polypeptide or peptide can be produced by chemical modification using techniques known to those of ordinary skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Furthermore, a variant has similar, the same, or improved function as the polypeptide or peptide from which it is derived.

[0286] As used herein, the term "specifically binds" refers to a nonrandom binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be quantified by the equilibrium dissociation constant (Kd) of the interaction. A Kd of 10"7M or less, 10"8M or less, 10"9M or less, 10"10M or less, 10"11M or less, or 10"12M or less indicates a high affinity interaction. D) indicates. In the present application, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0287] 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 antigen binding site / antigen complexes. Both the "association rate constant" (kaor kon) and the "dissociation rate constant" (kdisor koff) can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361 : 186-187). The ratio of kdis / konis equal to the dissociation constant K D (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K D , konand kdisvalues can be measured using any effective method. 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.

[0288] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of the inserted polynucleotide, the vector is referred to as an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the host cell takes up (i.e., assimilates) and expresses the genetic material elements carried by the vector. 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, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (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. In addition, a vector can contain a replication origin.

[0289] 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 E. 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 fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0290] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a 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 an amino acid residue with an amino acid residue having a similar side chain, e.g., substitutions that take place within a family of amino acid residues that are physicochemically or functionally similar, e.g., have similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc. Families of amino acid residues having 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, a conservative substitution preferably replaces a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods of 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).

[0291] The writing of the twenty conventional amino acids referred to herein follows conventional usage. See, e.g., 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 application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by their single and three letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0292] Unless otherwise indicated herein, or otherwise clear from context, "A, B and / or C" or similar expressions are to be understood to mean "A, B, C, or any combination thereof", e.g., it is to be understood to mean any one selected from among A, B, C, A and B, A and C, B and C, A and B and C.

[0293] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible, physiologically and / or pharmacologically, with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to retard absorption, preservatives, stabilizers. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Adjuvants include, but are not limited to, inorganic adjuvants (such as aluminum hydroxide, vanadate), biological adjuvants (such as Mycobacterium tuberculosis, BCG, Corynebacterium parvum, Bordetella pertussis, Gram-negative bacterial endotoxins, B subunit of cholera toxin, muramyl dipeptide, cytokines), synthetic adjuvants (such as double-stranded polyribonucleotides, uridine acid), oil agents (such as Freund's complete adjuvant, peanut oil emulsion), and nano-adjuvants, etc. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl and the like. Agents to retard 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), etc. Preservatives include, but are not limited to, various antibacterial agents and antifungal agents, such as thiomersal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which 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 carriers or excipients include sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). 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), solutions containing surfactants (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), Ringer's solution, and any combination thereof.

[0294] As used herein, the term "prevent" refers to a method taking place in order to stop or delay the occurrence of a disease or disorder or a symptom in a subject. As used herein, the term "treat" refers to a method taking place in order to obtain a beneficial or desired clinical result. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Moreover, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0295] As used herein, the term "subject" refers to a mammal, for example, a human. In certain embodiments, the subject (e.g., human) has 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)) or is at risk of having a disease described above.

[0296] As used herein, the term "effective amount" refers to an amount that is sufficient to achieve or at least partially achieve a 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)) refers to an amount that is sufficient to prevent, stop, or delay the occurrence of the disease; an effective amount for treating a disease refers to an amount that is sufficient to cure or at least partially stop the disease and its complications in a patient already having the disease. Determining such effective amounts is well within the capability of those skilled in the art. For example, an amount effective for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, body weight, and sex, the mode of administration of a drug, and other therapies being administered concurrently, etc.

[0297] Advantages of the Invention

[0298] Compared to existing GHR agonists (e.g., GH or its modifications), the agonistic single-domain antibody or polypeptide construct comprising the same provided herein specifically binding to GHR has at least one or more of the following advantages:

[0299] 1) Existing GHR agonists based on modification of GH itself (e.g., GH-Fc fusion protein-based GHR agonists) are not ideal for patients with mutations (e.g., point mutations) in GHR or presence of GH-neutralizing antibodies, while the agonistic antibody provided herein is theoretically effective for both types of patients;

[0300] 2) The agonistic antibodies provided by the present application do not interact with prolactin receptors, and there are no adverse reactions associated with the mammary glands;

[0301] 3) The polypeptide constructs provided by the present application have a long half-life as general antibodies in vivo, which can greatly improve patient compliance compared to existing GHR agonistic preparations; in some embodiments, the in vivo efficacy of the polypeptide constructs provided by the present application can be 4 times that of PEG-GH, and it is expected to be administered to subjects as a monthly preparation.

[0302] 4) The agonistic single-domain antibodies or polypeptide constructs provided by the present application stimulate IGF-1 levels more gently than GH, avoiding potential safety hazards that can occur with high-dose use;

[0303] 5) The polypeptide constructs provided by the present application are heavy chain antibodies, which have high stability and drugability.

[0304] Embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present application, and are not limiting the scope of the present application. According to the following detailed description of the preferred embodiments and the accompanying drawings, the various objects and advantages of the present application will become apparent to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0305] Figure 1: Structural schematic diagram of an exemplary Fc fusion protein molecule of the present application.

[0306] Figure 2A: SZ0357-SZ0364, SZ0366, SZ0367 and hGHBP-His binding activity detection results; wherein the blue line is pH7.4 dissociation, and the red line is pH5.5 dissociation.

[0307] Figure 2B: SZ0368-SZ0374, SZ0376-SZ0383 and hGHBP-His binding activity detection results; wherein the blue line is pH7.4 dissociation, and the red line is pH5.5 dissociation.

[0308] Figure 2C: SZ0406-SZ0410, SZ0413, SZ0414, SZ0416-SZ0422 and hGHBP-His binding activity detection results; wherein the blue line is pH7.4 dissociation, and the red line is pH5.5 dissociation.

[0309] Figure 2D: SZ0424, SZ0426-SZ0438 and hGHBP-His binding activity detection results; wherein the blue line is pH7.4 dissociation, and the red line is pH5.5 dissociation.

[0310] Figure 3A: Results of single concentration activity detection of SZ0357-SZ0364, SZ0366-SZ0374, SZ0376-SZ0383 promoting Baf3-hGHR cell proliferation.

[0311] Figure 3B: Results of single concentration activity detection of SZ0406-SZ0410, SZ0413, SZ0414, SZ0416-SZ0422, SZ0424, SZ0426-SZ0438 promoting Baf3-hGHR cell proliferation.

[0312] Figure 4A: Results of multi-concentration activity detection of SZ0357, SZ0360, SZ0362-SZ0364 promoting Baf3-hGHR cell proliferation.

[0313] Figure 4B: Results of multi-concentration activity detection of SZ0366, SZ0368, SZ0372, SZ0373, SZ0377, SZ0383 promoting Baf3-hGHR cell proliferation.

[0314] Figure 4C: Results of multi-concentration activity detection of SZ0407, SZ0414, SZ0418, SZ0419 promoting Baf3-hGHR cell proliferation.

[0315] Figure 4D: Results of multi-concentration activity detection of SZ0420, SZ0421, SZ0424 promoting Baf3-hGHR cell proliferation.

[0316] Figure 4E: Results of multi-concentration activity detection of SZ0428-SZ0430, SZ0432, SZ0433, SZ0437 promoting Baf3-hGHR cell proliferation.

[0317] Figure 5: Body weight change of each group of animals after administration.

[0318] Figure 6: Serum IGF-1 content change of each group of animals after administration.

[0319] Figure 7: Body length of each group of animals after administration.

[0320] Figure 8: Tibia length of each group of animals after administration.

[0321] Figure 9A: Results of SZ0513-SZ0521 binding activity with hGHBP-His; wherein the blue line is pH7.4 dissociation, and the red line is pH5.5 dissociation.

[0322] Figure 9B: Results of SZ0586-SZ0592 binding activity with hGHBP-His; wherein the blue line is pH7.4 dissociation, and the red line is pH5.5 dissociation.

[0323] Figure 10: Results of multi-concentration activity detection of SZ0514, SZ0515, SZ0517, SZ0518, SZ0586-SZ0592 in promoting Baf3-hGHR cell proliferation.

[0324] Figure 11: Body weight change of each group of animals after administration.

[0325] Figure 12: Serum IGF-1 content change of each group of animals after administration.

[0326] Figure 13: Body weight change of each group of animals after administration.

[0327] Figure 14: Serum IGF-1 content change of each group of animals after administration.

[0328] Figure 15: Tibia length of each group of animals after administration.

[0329] Figure 16: Tail length of each group of animals after administration.

[0330] Figure 17: Body length of each group of animals after administration.

[0331] Figure 18: Cynomolgus monkey PK results.

[0332] Figure 19: SZ0055 variable region amino acid residue positions defined by IMGT numbering system. DETAILED DESCRIPTION

[0333] The present application will now be described with reference to the following examples which are intended to be illustrative, and not limiting, of the present application.

[0334] Unless otherwise indicated, the molecular biology and immunological techniques utilized in the present application are performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc., 1995. Those skilled in the art know that the examples describe the present application by way of illustration, and are not intended to limit the scope of the claimed application.

[0335] The present application is based on the pH-dependent modification of an agonistic parent nanobody that specifically binds to GHR (i.e., the nanobody comprised in the construct SZ0055 provided in the Chinese patent application with the application number CN202410020660.X (the same family PCT international application of which has the application number PCT / CN2025 / 070359)) to obtain a nanobody and an Fc fusion protein having an extended half-life compared to the parent nanobody. An exemplary structure of the Fc fusion protein is shown in Figure 1.

[0336] The parent nanobody amino acid sequence contained in the construct SZ0055 is shown as SEQ ID NO: 63, and the IMGT definition positions of CDR1-3 and FR1-4 are shown in FIG. 19.

[0337] Reagent preparation and experimental method

[0338] 1. Protein sample preparation

[0339] The amino acid sequences (SEQ ID NO: 95, 98, 120) of the proteins to be prepared (hGHBP-His, rGHBP-His, human GH) were back translated into nucleotide sequences, eukaryotic expression vectors were constructed, and CHO or 293 cells were transiently transfected. The target protein was purified by affinity chromatography (protein A, protein G or nickel column), and the purified protein was subjected to SDS-PAGE, SEC-HPLC and endotoxin detection before being used in subsequent experiments. Among them, hGHBP is the extracellular domain of human GHR, and rGHBP is the extracellular domain of rat GHR.

[0340] 2. Cell strain construction

[0341] The gene encoding human GHR protein (Uniprot accession number: P10912) was ligated into a plasmid to construct a lentivirus plasmid for overexpression of hGHR protein, lentivirus packaging was performed, and lentivirus solution was obtained after concentration. Baf3 cells were infected with the lentivirus solution. After infection, the infected cells were subjected to pressure screening by puromycin (Puro), and a resistant cell pool (Pool) was obtained. The expression of GHR protein was detected by flow cytometry. The identified positive pool (Pool) cells were subjected to monoclonalization by limiting dilution method, and after expansion culture and identification, a Baf3 cell strain overexpressing human GHR protein was obtained, which was named Baf3-hGHR cell strain.

[0342] 3. Cell proliferation activity detection

[0343] The Baf3-hGHR cell proliferation activity detection was performed using a cell proliferation-toxicity detection kit (Dingren Chemical): the detection cells Baf3-hGHR were washed twice with experimental medium (RPMI-1640+3% fetal bovine serum+50 μM mercaptoethanol), and the cells were counted after 4 h of starvation treatment, 40,000 cells / 50 μL / well were plated; the experimental medium was diluted to 25 nM in GH concentration, and the starting concentration of the test sample was 300 nM, which was diluted 4 times, 11 gradients, 50 μL / well, 3 replicate wells; 50 μL of cells were plated, 50 μL of test sample was added, and the total system was 100 μL. Incubate for 18 hours. Add detection reagent CCK8 (5 times dilution) 50 μL / well, and after 3 h, detect the OD450 value by enzyme-labeled instrument.

[0344] 4. Rat growth experiment

[0345] The animals are rGHR (rat GHR) knockout and hGHR (human GHR) knock-in rats, which can only partially activate hGHR with rGH, showing GH deficiency characteristics, and the animals are smaller and lighter than wild-type SD rats of the same age. hGHR transgenic rats 7-9 weeks old were randomly divided into groups according to body weight, 6-8 rats per group. The administration method is subcutaneous injection in the neck, and the negative control group is subcutaneously injected with the same volume of solvent. PEG-GH is prepared according to the method in CN101385858B, diluted with solvent according to the administration dose, and subcutaneously injected with the same volume.

[0346] The single-dose experiment is daily weighing after administration for 14-24 days, and the rats are selected at an appropriate time point for blood collection for IGF-1 level detection, and body length, tail length and tibia length are measured at the end of the experiment. Among them, the body length refers to the distance from the rat's nose to the anus; the tail length refers to the distance from the rat's anus to the tail tip.

[0347] According to the blood collection plan, the neck vein is collected using a syringe, and 100-150 μL of whole blood is collected from each animal. The collected whole blood is placed in a coagulation tube, and after 2-8°C for 2 hours, it is centrifuged at 4°C, 1500g, and centrifuged for 10 minutes twice. The collected serum is transferred to a new labeled another centrifuge tube and stored at -70°C or below.

[0348] Serum IGF-1 levels were determined by commercially available ELISA kits (R&D Systems; Cat. No: SMG100). The assay is a sandwich ELISA using IGF-1 specific polyclonal antibodies as capture agent and horseradish peroxidase labeled high affinity polyclonal antibodies as detection agent.

[0349] Example 1: SZ0055 pH-dependent modification

[0350] Receptor-mediated endocytosis may be the main way of SZ0055 metabolism, which binds to GHR, functions, and is endocytosed by the receptor, and finally enters the lysosomal pathway for degradation. In order to as far as possible improve the in vivo half-life of SZ0055, make SZ0055 after GHR binding can return to the extracellular, so the pH-dependent modification of SZ0055 is carried out, which can bind to GHR under neutral pH 7.4 conditions, activate downstream signals, but when the intracellular body gradually changes to an acidic environment (pH 5.5-pH 6.0), SZ0055 dissociates from GHR, then is captured by FcRn, and finally returns to the extracellular.

[0351] 1. Library construction and screening

[0352] In the experiment, phage display method is adopted, trimer primer synthesis method (Jinsuibiological) is used to construct phage display library, phage is combined with biotinylated human GHBP under pH 7.4, and pH-dependent clones are enriched by elution under pH 5.5.

[0353] 3CDRs are all numbered by IMGT, CDR regions are determined, and all CDR regions and the first amino acid of FR4 region (amino acid at position 118 defined by IMGT numbering system) are introduced with a certain proportion of histidine mutations, trimer primer synthesis method is used, fragments are amplified, and enzyme digestion is performed to connect into phagemid vector, E. coli is electrotransformed, and phage display library is constructed. After the library is packaged into phage, biotinylated human GHBP is used as an antigen to combine under pH 7.4, and then eluted in pH 5.5 PBS buffer. After elution, the phage re-infests E. coli TG1.

[0354] After infection, single colonies are picked for soluble expression, and periplasmic cavity proteins are extracted. Octet HTX molecular interaction instrument (Sartorius) is used, SA Biosensor is used, biotinylated human GHBP (hGHBP-His) is fixed on the SA Biosensor, and phage periplasmic cavity proteins are used as analytes for pH-dependent detection. After combining under pH 7.4, it is dissociated under pH 7.4 and pH 5.5, respectively; Octet Analysis Studio is used for data analysis. After the selected clones are sequenced, redundancies are removed, and part of the sequences are used to construct Fc fusion proteins for mammalian cell expression.

[0355] Fc fusion protein molecules SZ0357-SZ0364, SZ0366-SZ0374, SZ0376-SZ0383 and SZ0406-SZ0410, SZ0413, SZ0414, SZ0416-SZ0422, SZ0424, SZ0426-SZ0438 are obtained by the above method, the variable region amino acid sequences are respectively shown as SEQ ID NO:64-71, 73-94, 96, 97, 99-119, the hinge region amino acid is shown as SEQ ID NO:137, and the Fc region amino acid sequence is shown as SEQ ID NO:138.

[0356] The mutations contained in the variable region of Fc fusion protein molecules SZ0357-SZ0364, SZ0366-SZ0374, SZ0376-SZ0383, SZ0406-SZ0410, SZ0413, SZ0414, SZ0416-SZ0422, SZ0424, SZ0426-SZ0438 compared to SZ0055 are shown in Table 1:

[0357] Mutation types of each molecule in Table 1

[0358] Note: The positions of mutations are defined by IMGT numbering system. For example, the mutation "V112.2H" means that the residue V at position 112.2 in the variable region defined by IMGT numbering system is replaced by residue H.

[0359] The sequence information of CDRs and FRs defined by IMGT numbering system of Fc fusion protein molecules SZ0357-SZ0364, SZ0366-SZ0374, SZ0376-SZ0383, SZ0406-SZ0410, SZ0413, SZ0414, SZ0416-SZ0422, SZ0424, SZ0426-SZ0438 are shown in Table 2:

[0360] 2. Affinity identification

[0361] In this experiment, Octet HTX molecular interaction instrument (Sartorius) was used, protein A Biosensor was used, the antibody was fixed on the protein A Biosensor, hGHBP-His was used as the analyte for pH-dependent detection, after binding at pH 7.4, it was dissociated at pH 7.4 and pH 5.5, respectively; Octet Analysis Studio was used for data analysis.

[0362] The results of affinity identification are shown in Figures 2A-2D and Table 3.

[0363] Affinity data of each molecule in Table 3

[0364] 3. Cell proliferation activity

[0365] 3.1 Single concentration test

[0366] Baf3-hGHR cell proliferation experiment condition: GH and test sample concentration is 1 nM, other experimental conditions are the same as described in reagent preparation and experimental method. Each sample is repeated 3 times, OD450 is measured and the average value is calculated, and the blank control (Blank) is blank medium. The results are shown in Figures 3A-3B and Tables 4.1 and 4.2.

[0367] Table 4.1 Cell proliferation activity of each molecule

[0368] Table 4.2 Cell proliferation activity of each molecule

[0369] 3.2 Multi-concentration test

[0370] The experimental conditions are described in reagent preparation and experimental method. The results are shown in Figures 4A-4E and Tables 5.1 and 5.2.

[0371] Table 5.1 Cell proliferation activity of each molecule

[0372] Table 5.2 Cell proliferation activity of each molecule

[0373] The results show that the test molecules have good cell proliferation activity.

[0374] 4. Rat growth experiment

[0375] In vivo animal experiment number P091M22313. hGHR transgenic male rats 7-9 weeks old were randomly grouped according to body weight. The dosing information is shown in Table 6.

[0376] Table 6 Dosing information of each group

[0377] The body weight, IGF-1 level, body length and tibia length were detected respectively, and the results are shown in Figures 5-8 respectively.

[0378] The results show that the pH-dependent modified series of molecules provided by the application can significantly increase the body weight, body length and tibia length of animals, and compared with PEG-GH, the IGF-1 level has a long duration but a lower peak value, and the body weight growth can be improved to a maximum of 12 days of rapid growth compared with 3 days of PEG-GH.

[0379] Example 2: SZ0357 back mutation

[0380] In vivo results showed that SZ0357 had excellent performance, the rats' weight gain time could last for 12 days, much higher than 6 days of parent SZ0055 and 3 days of PEG-GH. Since SZ0357 had 5 histidine mutations compared with parent SZ0055, in order to verify whether all histidines were necessary, it was decided to perform back mutation on SZ0357 to find the minimum number of histidine mutations required to maintain in vitro and in vivo activity. At the same time, reference was made to other molecules, and possible mutation combinations were introduced at other positions.

[0381] 1. Molecular design

[0382] Back mutation was performed on SZ0357 to obtain Fc fusion proteins SZ0513-SZ0521 and SZ0586-SZ0592, the variable region amino acid sequences of which are shown in SEQ ID NO: 121-136, the hinge region amino acid is shown in SEQ ID NO: 137, and the Fc region amino acid sequence is shown in SEQ ID NO: 138.

[0383] The mutations contained in the variable region of Fc fusion protein molecules SZ0513-SZ0521 and SZ0586-SZ0592 compared with SZ0055 are shown in Table 7.

[0384] Table 7 Mutation types of each molecule

[0385] Note: Among them, the position of the mutation is defined by the IMGT numbering system. For example, the mutation "G112H" means that the residue G at position 112 defined by the IMGT numbering system in the variable region is replaced by residue H.

[0386] The CDRs and FRs sequence information of Fc fusion protein molecules SZ0513-SZ0521 and SZ0586-SZ0592 defined by the IMGT numbering system are shown in Table 8.

[0387] 2. Affinity identification

[0388] In this experiment, Octet HTX molecular interaction instrument (Sartorius) was used, protein A Biosensor was used, antibodies were fixed on protein A Biosensor, hGHBP-His was used as analyte for pH-dependent detection, after binding at pH 7.4, dissociation was performed at pH 7.4 and pH 5.5, respectively; Octet Analysis Studio was used for data analysis. The results are shown in Figures 9A-9B and Table 9.

[0389] Table 9 Affinity data of each molecule

[0390] 3. Cell proliferation experiment

[0391] The experimental conditions are as described in the reagent preparation and experimental methods. The results are shown in Figure 10 and Table 10.

[0392] Table 10 Cell proliferation activity of each molecule

[0393] The results show that all the test clones have good cell proliferation activity.

[0394] 4. Species cross-reactivity

[0395] Using a Biacore molecular interaction analyzer (cytiva, Biacore 8K+), according to the anti-Human capture kit, type 2 (cytiva, 29234600) operation instruction, the Anti-Human antibody was coupled to the CM-5 chip (cytiva, 29149603), and then the chip was used to capture the test molecules, and human GHBP-His and rat GHBP-His were used as analytes, respectively, flowing through the chip, binding for 120 s, dissociating for 180 s, and according to the collected data, the kinetic analysis was performed by Biacore Insight Evaluation Software 3.0.12 software, and the results are shown in Table 11.

[0396] Table 11 Affinity data of each molecule

[0397] The results show that the modified molecules are similar to the original molecule SZ0055, and both have human-rat cross-reactivity.

[0398] 5. In vivo efficacy experiment

[0399] 5.1 Single dose of back-mutation molecules

[0400] The in vivo animal experiment number is P091M22428. Male hGHR transgenic rats 7-9 weeks old were randomly grouped according to body weight. The dosing information is shown in Table 12.

[0401] Table 12 Dosing information of each group

[0402] The body weight and IGF-1 level were detected, and the results are shown in Figures 11 and 12, respectively.

[0403] The results show that the series of back-mutation molecules of SZ0357 can significantly increase the body weight of animals, and are significantly better than PEG-GH. Compared with PEG-GH for 3 days, the series of molecules can improve the rapid growth for at least 12 days, and some molecules still have the effect of promoting the body weight growth at the end of the 15-day experiment. Compared with PEG-GH, the series of molecules have a long duration on IGF-1 level, but a lower peak, and some molecules can still maintain a high IGF-1 level at the end of the 15-day experiment, in which SZ0586 performs the best.

[0404] 5.2 In vivo experiment of SZ0586 multiple doses

[0405] The in vivo animal experiment number is P091M22431. The hGHR transgenic male rats 7-9 weeks old are randomly grouped according to the body weight. The administration information is shown in Table 13.

[0406] Table 13 Administration information of each group

[0407] The body weight, IGF-1 level, tibia length, tail length and body length are detected respectively, and the results are shown in Figures 13-17 respectively.

[0408] The results show that the body weight of animals in each group is continuously increased after a single subcutaneous administration of SZ0586; the animals in different dose groups of SZ0586 have a dose-linear relationship in body weight, tibia length, tail length and body length with the increase of dose. SZ0586 can improve the rapid growth for up to 20 days on the animals.

[0409] 6. Cynomolgus monkey PK

[0410] The Fc region of Fc fusion protein molecules SZ0357 (SEQ ID NO: 141), SZ0368 (SEQ ID NO: 142) and SZ0586 (SEQ ID NO: 145) is replaced by IgG1 Fc (AA, YTE) (shown in SEQ ID NO: 139) respectively, to obtain Fc fusion protein molecules SZ0509 (SEQ ID NO: 143), SZ0510 (SEQ ID NO: 144) and SZ0924 (SEQ ID NO: 146) respectively, and the pharmacokinetic characteristics of the molecules are evaluated.

[0411] Taking SZ0357 and SZ0509 (SZ0357-YTE) as examples, the method is as follows:

[0412] After single subcutaneous injection of SZ0357 and SZ0357-YTE (i.e. SZ0509) in male cynomolgus monkeys, the serum of cynomolgus monkeys at each time point was collected to investigate the change of SZ0357 and SZ0357-YTE molecular exposure in male cynomolgus monkeys after administration, to evaluate and compare the pharmacokinetic characteristics of the two molecules of SZ0357 and SZ0357-YTE.

[0413] 6.1 Preparation of formulation

[0414] The test samples were SZ0357 and SZ0357-YTE subcutaneous injection (S.C) liquid formulations provided by Changchun Jin Saisai Pharmaceutical Co., Ltd. According to Table 14, the test samples were diluted to the corresponding concentration (5 mg / mL) with the formulation vehicle (no drug) and then administered.

[0415] 6.2 Experimental animals

[0416] The experimental animals were bred in the animal house of Suzhou Xihua New Drug Development Co., Ltd. (Animal Use License No. SYXK (Su) 2021-0018). The source and number of animals used in this experiment are shown in Table 14.

[0417] Table 14 Source and number of experimental animals

[0418] 6.3 Experimental design

[0419] 6.3.1 Grouping of experimental animals

[0420] The administration scheme is shown in Table 15.

[0421] Table 15 Grouping of animals

[0422] 6.3.2 Sample collection and processing

[0423] The collection time was 0 h before administration (within 10 min before administration), 1 h, 2 h, 4 h, 8 h, 12 h, 18 h, 24 h (D1), 36 h, 48 h (D2), 72 h (D3), 96 h (D4), 120 h (D5), 144 h (D6), 168 h (D7), 192 h (D8), 216 h (D9), 240 h (D10), 264 h (D11), 288 h (D12), 312 h (D13), 336 h (D14), 384 h (D16), 432 h (D18), 480 h (D20), 576 h (D24), 672 h (D28).

[0424] 6.4 Experimental data

[0425] The results are shown in Figure 18. The results show that the Fc fusion protein molecule SZ0357-YTE (i.e. SZ0509) constructed with the Fc segment comprising the YTE mutation has a significantly prolonged PK compared to SZ0357.

[0426] While the specific embodiments of the application have been described in detail, those skilled in the art will appreciate that various modifications and alterations to the details can be made within the scope of the application as disclosed in the above teachings and as defined in the following claims. The entire contents of the specification and the appended claims are hereby expressly incorporated by reference as part of this application.

Claims

1. A single-domain antibody or antigen-binding fragment thereof that specifically binds to a growth hormone receptor (GHR), wherein, one or more of the amino acid residues in the single domain antibody at positions 27, 28, 29, 35, 36, 37, 58, 62, 65, 106, 108, 111, 111.1, 112.2, 112.1, 112, 113, 114, 115, 116, 117 are histidine residues; wherein the positions are defined by the IMGT numbering system. one or more of the amino acid residues in the single domain antibody at positions 27, 28, 29, 35, 36, 37, 58, 62, 65, 106, 108, 111, 111.1, 112.2, 112.1, 112, 113, 114, 115, 116, 117 are histidine residues; 2. The single domain antibody or antigen-binding fragment thereof of claim 1, wherein, wherein the positions are defined by the IMGT numbering system. Preferably, one or more of the amino acid residues in the single domain antibody at positions 112.2, 112, 114, 115 are histidine residues. wherein the positions are defined by the IMGT numbering system.

3. The single domain antibody or antigen binding fragment thereof of claim 1 or 2, which possesses one or more of the features selected from the group consisting of: (1) the amino acid residue at position 30 is the amino acid residue F; (2) the amino acid residue at position 38 is the amino acid residue A; (3) the amino acid residue at position 56 is the amino acid residue I; (4) the amino acid residue at position 57 is the amino acid residue T; (5) the amino acid residue at position 59 is the amino acid residue L; (6) the amino acid residue at position 63 is the amino acid residue G; (7) the amino acid residue at position 64 is the amino acid residue H; (8) the amino acid residue at position 105 is the amino acid residue H; (9) the amino acid residue at position 107 is the amino acid residue A; (10) the amino acid residue at position 109 is the amino acid residue Y; (11) the amino acid residue at position 110 is the amino acid residue Y. The single domain antibody or antigen binding fragment thereof comprises: (i) a CDR1 having the structure as set forth in X1X2X3FX4X5X6A (Formula I, SEQ ID NO: 18); (ii) a CDR2 having the structure as set forth in ITX7LX8GHX9 (Formula II, SEQ ID NO: 53); 4. A single-domain antibody or antigen-binding fragment thereof that specifically binds to growth hormone receptor (GHR), wherein, and wherein the CDRs are defined by the IMGT numbering system; and, 6. The single domain antibody or antigen binding fragment thereof of any one of claims 1-5, which comprises: (i) a CDR1 having the structure as set forth in X1X2X3FX4X5X6A (Formula I, SEQ ID NO: 18); (iii) CDR3 has the structure as set forth in HX 10 AX 11 YYX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 ( Formula III, SEQ ID NO: 72). (ii) a CDR2 having the structure as set forth in ITX7LX8GHX9 (Formula II, SEQ ID NO: 53); X1is an amino acid residue H or G; X2is an amino acid residue H or F; X3is an amino acid residue H or T; X4is an amino acid residue H or I; X5is an amino acid residue H or N; X6is an amino acid residue H or Y; X7is an amino acid residue H or S; X8is an amino acid residue H or G; X9is an amino acid residue H or T; X 10 is an amino acid residue H or A; X 11 is an amino acid residue H or Q; X 12 is an amino acid residue H or R; X 13 is an amino acid residue H or E; X 14 is an amino acid residue H or V; X 15 is an amino acid residue H or L; X 16 is an amino acid residue H or G; X 17 is an amino acid residue H or G; X 18 is an amino acid residue H or Q; X 19 is an amino acid residue H or Y; X 20 is an amino acid residue H or D; X 21 is an amino acid residue H or Y; Preferably, at least one of the amino acid residues of X1to X 21 H.

5. The single domain antibody or antigen-binding fragment thereof of claim 4, wherein, X1is an amino acid residue G; X2is an amino acid residue H or F; X3is an amino acid residue T; X4is an amino acid residue I; X5is an amino acid residue N; X6is an amino acid residue Y; X7is an amino acid residue S; X8is an amino acid residue G; X9is an amino acid residue T; X 10 is an amino acid residue A; X 11 is an amino acid residue Q; X 12 is an amino acid residue R; X 13 is an amino acid residue E; X 14 is an amino acid residue H; X 15 is an amino acid residue L; X 16 is an amino acid residue H or G; X 17 is an amino acid residue G; X 18 is an amino acid residue H or Q; X 19 is an amino acid residue H or Y; X 20 is an amino acid residue D; X 21 is an amino acid residue H or Y; Preferably, X1is an amino acid residue G; X2is an amino acid residue F; X3is an amino acid residue T; X4is an amino acid residue I; X5is an amino acid residue N; X6is an amino acid residue Y; X7is an amino acid residue S; X8is an amino acid residue G; X9is an amino acid residue T; X 10 is an amino acid residue A; X 11 is an amino acid residue Q; X 12 is an amino acid residue R; X 13 is an amino acid residue E; X 14 is an amino acid residue H; X 15 is an amino acid residue L; X 16 is an amino acid residue H or G; X 17 is an amino acid residue G; X 18 is an amino acid residue H or Q; X 19 is an amino acid residue H or Y; X 20 is an amino acid residue D; X 21 is an amino acid residue Y. and wherein the CDRs are defined by the IMGT numbering system; and, 6. The single domain antibody or antigen binding fragment thereof of any one of claims 1-5, which comprises: a CDR1 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a CDR2 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, and a CDR3 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises a CDR1, a CDR2, and a CDR3 as contained in a VHH as shown in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136; Preferably, the CDRs are defined by the IMGT, Kabat or Chothia numbering system; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises: a CDR1 as shown in any one of SEQ ID NOs: 2-12; a CDR2 as shown in any one of SEQ ID NOs: 14, 15, 17, 19; and a CDR3 as shown in any one of SEQ ID NOs: 16, 21-52, 54-60; and the single-domain antibody or antigen-binding fragment thereof does not simultaneously comprise: a CDR1 as shown in SEQ ID NO: 2; a CDR2 as shown in SEQ ID NO: 14; and a CDR3 as shown in SEQ ID NO: 23; wherein the CDRs are defined by the IMGT numbering system; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises: (1) a CDR1 as shown in SEQ ID NO: 2; a CDR2 as shown in SEQ ID NO: 14; and a CDR3 as shown in SEQ ID NO: 21; (2) a CDR1 as shown in SEQ ID NO: 2; a CDR2 as shown in SEQ ID NO: 14; and a CDR3 as shown in SEQ ID NO: 22; (3) a CDR1 as shown in SEQ ID NO: 2; a CDR2 as shown in SEQ ID NO: 15; and a CDR3 as shown in SEQ ID NO: 23; (4) a CDR1 as shown in SEQ ID NO: 2; a CDR2 as shown in SEQ ID NO: 14; and a CDR3 as shown in SEQ ID NO: 24; (5) a CDR1 as shown in SEQ ID NO: 3; a CDR2 as shown in SEQ ID NO: 14; and a CDR3 as shown in SEQ ID NO: 23; (6) a CDR1 as shown in SEQ ID NO: 4; a CDR2 as shown in SEQ ID NO: 14; and a CDR3 as shown in SEQ ID NO: 23; (7) a CDR1 as set forth in SEQ ID NO: 5; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 23; (8) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 25; (9) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 26; (10) a CDR1 as set forth in SEQ ID NO: 6; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 27; (11) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 28; (12) a CDR1 as set forth in SEQ ID NO: 7; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 29; (13) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 29; (14) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 30; (15) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 31; (16) a CDR1 as set forth in SEQ ID NO: 4; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 31; (17) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 15; and, a CDR3 as set forth in SEQ ID NO: 32; (18) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 27; (19) a CDR1 as set forth in SEQ ID NO: 8; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 33; (20) a CDR1 as set forth in SEQ ID NO: 7; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 34; (21) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 34; (22) a CDR1 as set forth in SEQ ID NO: 9; a CDR2 as set forth in SEQ ID NO: 17; and, a CDR3 as set forth in SEQ ID NO: 35; (23) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 33; (24) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 36; (25) a CDR1 as set forth in SEQ ID NO: 9; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 37; (26) a CDR1 as set forth in SEQ ID NO: 10; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 23; (27) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 38; (28) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 39; (29) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 40; (30) a CDR1 as set forth in SEQ ID NO: 6; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 35; (31) a CDR1 as set forth in SEQ ID NO: 4; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 34; (32) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 41; (33) a CDR1 as set forth in SEQ ID NO: 6; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 42; (34) a CDR1 as set forth in SEQ ID NO: 9; a CDR2 as set forth in SEQ ID NO: 19; and, a CDR3 as set forth in SEQ ID NO: 34; (35) a CDR1 as set forth in SEQ ID NO: 6; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 43; (36) a CDR1 as set forth in SEQ ID NO: 6; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 44; (37) a CDR1 as set forth in SEQ ID NO: 9; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 42; (38) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 45; (39) a CDR1 as set forth in SEQ ID NO: 9; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 46; (40) a CDR1 as set forth in SEQ ID NO: 6; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 23; (41) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 47; (42) a CDR1 as set forth in SEQ ID NO: 4; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 43; (43) a CDR1 as set forth in SEQ ID NO: 6; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 36; (44) a CDR1 as set forth in SEQ ID NO: 11; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 48; (45) a CDR1 as set forth in SEQ ID NO: 6; a CDR2 as set forth in SEQ ID NO: 19; and, a CDR3 as set forth in SEQ ID NO: 34; (46) a CDR1 as set forth in SEQ ID NO: 2; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 49; (47) a CDR1 as set forth in SEQ ID NO: 12; a CDR2 as set forth in SEQ ID NO: 17; and, a CDR3 as set forth in SEQ ID NO: 50; (48) a CDR1 as set forth in SEQ ID NO: 6; a CDR2 as set forth in SEQ ID NO: 14; and, a CDR3 as set forth in SEQ ID NO: 51; (49) a CDR1 as depicted in SEQ ID NO: 4; a CDR2 as depicted in SEQ ID NO: 14; and, a CDR3 as depicted in SEQ ID NO: 52; (50) a CDR1 as depicted in SEQ ID NO: 6; a CDR2 as depicted in SEQ ID NO: 14; and, a CDR3 as depicted in SEQ ID NO: 48; (51) a CDR1 as depicted in SEQ ID NO: 2; a CDR2 as depicted in SEQ ID NO: 14; and, a CDR3 as depicted in SEQ ID NO: 54; (52) a CDR1 as depicted in SEQ ID NO: 2; a CDR2 as depicted in SEQ ID NO: 14; and, a CDR3 as depicted in SEQ ID NO: 55; (53) a CDR1 as depicted in SEQ ID NO: 2; a CDR2 as depicted in SEQ ID NO: 14; and, a CDR3 as depicted in SEQ ID NO: 56; (54) a CDR1 as depicted in SEQ ID NO: 2; a CDR2 as depicted in SEQ ID NO: 14; and, a CDR3 as depicted in SEQ ID NO: 57; (55) a CDR1 as depicted in SEQ ID NO: 2; a CDR2 as depicted in SEQ ID NO: 14; and, a CDR3 as depicted in SEQ ID NO: 58; (56) a CDR1 as depicted in SEQ ID NO: 2; a CDR2 as depicted in SEQ ID NO: 14; and, a CDR3 as depicted in SEQ ID NO: 59; (57) a CDR1 as depicted in SEQ ID NO: 2; a CDR2 as depicted in SEQ ID NO: 14; and, a CDR3 as depicted in SEQ ID NO: 60; (58) a CDR1 as depicted in SEQ ID NO: 2; a CDR2 as depicted in SEQ ID NO: 14; and, a CDR3 as depicted in SEQ ID NO: 16; (59) a CDR1 as depicted in SEQ ID NO: 9; a CDR2 as depicted in SEQ ID NO: 14; and, a CDR3 as depicted in SEQ ID NO: 60; (60) a CDR1 as depicted in SEQ ID NO: 6; a CDR2 as depicted in SEQ ID NO: 14; and, a CDR3 as depicted in SEQ ID NO: 60; or, (61) a CDR1 as depicted in SEQ ID NO: 4; a CDR2 as depicted in SEQ ID NO: 14; and, a CDR3 as depicted in SEQ ID NO: 60; wherein the CDRs are defined by the IMGT numbering system.

7. The single-domain antibody or antigen-binding fragment thereof of any one of claims 1-6, which comprises a heavy chain framework region derived from an immunoglobulin. Preferably, the heavy chain framework regions do not comprise a histidine residue, or, the heavy chain framework regions optionally comprise one or more histidine residues; Preferably, the amino acid residue at position 118 in the heavy chain framework region is amino acid residue H or R; wherein, The positions are defined by the IMGT numbering system.

8. The single domain antibody or antigen binding fragment thereof of any one of claims 1-7, which comprises a heavy chain framework region derived from a human immunoglobulin; Preferably, the single domain antibody or antigen binding fragment thereof comprises a heavy chain framework region in an 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 an amino acid sequence encoded by a human IGHV gene selected from IGHV3-23, IGHV3-30, IGHV3-34, IGHV3-7 and IGHV3-74.

9. The single-domain antibody or antigen-binding fragment thereof of any one of claims 1-7, comprising: FR1 as set forth in SEQ ID NO: 1, FR2 as set forth in SEQ ID NO: 13, FR3 as set forth in SEQ ID NO: 20, and / or, FR4 as set forth in SEQ ID NO: 61 or 62; wherein, The FRs are defined by the IMGT numbering system.

10. The single domain antibody or antigen binding fragment thereof of any one of claims 1-9, which comprises: (i) a sequence as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136; (ii) a sequence having one or several (e.g., 1, 2, 3, 4 or 5) amino acid substitutions, deletions or additions compared to the sequence as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136; 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 set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136; Preferably, the substitutions are conservative substitutions.

11. A polypeptide construct comprising the single domain antibody or antigen binding fragment thereof of any one of claims 1-10, and an Fc peptide and optionally a hinge peptide derived from an immunoglobulin; Preferably, the single domain antibody or antigen binding fragment thereof is covalently linked to the Fc peptide and the optional hinge peptide (e.g., by a covalent bond comprising a peptide bond, an isopeptide bond and / or a disulfide bond); Preferably, the polypeptide construct comprises the single domain antibody or antigen binding fragment thereof of any one of claims 1-10, 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 by a covalent bond comprising a peptide bond; Preferably, the polypeptide construct comprises a first peptide chain comprising: a first single-domain antibody or antigen binding fragment thereof, a first hinge peptide, and a first Fc peptide, wherein, the first single domain antibody or antigen binding fragment thereof is selected from the single domain antibody or antigen binding fragment thereof of any one of claims 1-10; preferably, the first peptide chain comprises, in the order from N-terminus to C-terminus: the first single domain antibody or antigen binding fragment thereof, the first hinge peptide, and the first Fc peptide; Preferably, the polypeptide construct further comprises a second peptide chain comprising a second hinge peptide and a second Fc peptide; preferably, the second peptide chain further comprises a second single-domain antibody or antigen-binding fragment thereof; preferably, the second single-domain antibody or antigen-binding fragment thereof is selected from the single-domain antibodies or antigen-binding fragments thereof that specifically bind to GHR; preferably, the second single-domain antibody or antigen-binding fragment thereof is selected from the single-domain antibodies or antigen-binding fragments thereof of any one of claims 1-10; Preferably, the second peptide chain comprises, in order 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; Preferably, the polypeptide construct possesses one or more of the features selected from the following: (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; (ii) the first hinge peptide and the second hinge peptide are derived from the same or different immunoglobulin; (iii) the first Fc peptide and the second Fc peptide are derived from the same or different immunoglobulin; (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) the first peptide chain and the second peptide chain are connected by disulfide bond formation between them.

12. The polypeptide construct of claim 11, 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 hinge peptide comprises a sequence as set forth in SEQ ID NO:

137.

13. The polypeptide construct of claim 11 or 12, wherein, The Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a 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 a 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 a Fc peptide derived from a human immunoglobulin IgG1 or IgG4.

14. The polypeptide construct of claim 13, wherein, The Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from a 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 variants thereof; wherein the Fc peptide variant has reduced Fc effector function (e.g., has reduced ADCC, CDC and / or ADCP activity) and / or prolonged half-life as compared to the Fc peptide of wild-type human immunoglobulin IgG1 ; Preferably, the Fc peptide variant has reduced FcyR binding activity, reduced serum complement molecule (C1q) binding activity, and / or enhanced FcRn binding activity as compared to the Fc peptide of wild-type human immunoglobulin IgG1 ; Preferably, the Fc peptide variant comprises mutations selected from the group consisting of L234A, L235A, M252Y, S254T, T256E, and any combination thereof (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)) as compared to the Fc peptide of wild-type human immunoglobulin IgG1 ; Preferably, the Fc peptide variant comprises mutations L234A and L235A; preferably, the Fc peptide variant further comprises mutations M252Y, S254T and T256E as compared to the Fc peptide of wild-type human immunoglobulin IgG1 ; Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) comprises a sequence as set forth in any one of SEQ ID NOs: 138-140.

15. The polypeptide construct of any one of claims 11-14, comprising a single-domain antibody or antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in any one of SEQ ID NOs: 138-140; Preferably, the polypeptide construct comprises a first peptide chain and, optionally, a second peptide chain; wherein, the first peptide chain comprises, in order from N-terminus to C-terminus: a single-domain antibody or antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in any one of SEQ ID NOs: 138-140; and / or, the second peptide chain comprises, in order from N-terminus to C-terminus: a single-domain antibody or antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in any one of SEQ ID NOs: 138-140; For example: (1) the first peptide chain comprises, in N-terminal to C-terminal order: a single-domain antibody or antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in SEQ ID NO: 138; and / or, the second peptide chain comprises, in N-terminal to C-terminal order: a single-domain antibody or antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in SEQ ID NO: 138; (2) the first peptide chain comprises, in N-terminal to C-terminal order: a single-domain antibody or antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in SEQ ID NO: 139; and / or, the second peptide chain comprises, in N-terminal to C-terminal order: a single-domain antibody or antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in SEQ ID NO: 139; or, (3) the first peptide chain comprises, in N-terminal to C-terminal order: a single-domain antibody or antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in SEQ ID NO: 140; and / or, the second peptide chain comprises, in N-terminal to C-terminal order: a single-domain antibody or antigen-binding fragment thereof as set forth in any one of SEQ ID NOs: 64-71, 73-94, 96, 97, 99-119, 121-136, a hinge peptide as set forth in SEQ ID NO: 137, and an Fc peptide as set forth in SEQ ID NO: 140; Preferably, (i) the first peptide chain and the second peptide chain comprise the same or different single-domain antibody or antigen-binding fragment thereof, and / or, (ii) the first peptide chain and the second peptide chain comprise the same or different Fc peptide.

16. The polypeptide construct of any one of claims 11-15, comprising a sequence as set forth in any one of SEQ ID NOs: 141-146; Preferably, the polypeptide construct comprises the first peptide chain and, optionally, the second peptide chain; wherein, the first peptide chain comprises a sequence as set forth in any one of SEQ ID NOs: 141-146, and / or, the second peptide chain comprises a sequence as set forth in any one of SEQ ID NOs: 141-146; Preferably, the polypeptide construct comprises the first peptide chain and, optionally, the second peptide chain; wherein: (1) the first and second peptide chains each independently comprise a sequence as set forth in SEQ ID NO: 141; (2) the first and second peptide chains each independently comprise a sequence as set forth in SEQ ID NO: 142; (3) the first and second peptide chains each independently comprise a sequence as set forth in SEQ ID NO: 143; (4) the first and second peptide chains each independently comprise a sequence as set forth in SEQ ID NO: 144; (5) the first and second peptide chains each independently comprise a sequence as set forth in SEQ ID NO: 145; or (6) the first and second peptide chains each independently comprise a sequence as set forth in SEQ ID NO:

146.

17. An isolated nucleic acid molecule encoding the single-domain antibody or antigen-binding fragment thereof of any one of claims 1-10, or the polypeptide construct of any one of claims 11-16.

18. A vector comprising the isolated nucleic acid molecule of claim 17; preferably, the vector is a cloning vector or an expression vector.

19. A host cell comprising the isolated nucleic acid molecule of claim 17 or the vector of claim 18.

20. A method of making the single-domain antibody or antigen-binding fragment thereof of any one of claims 1-10, or the polypeptide construct of any one of claims 11-16, comprising culturing the host cell of claim 19 under conditions that allow 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.

21. A bispecific or multispecific molecule comprising the single-domain antibody or antigen-binding fragment thereof of any one of claims 1-10, or the polypeptide construct of any one of claims 11-16; preferably, the bispecific or multispecific molecule specifically binds GHR and additionally specifically binds one or more target of interest; preferably, the bispecific or multispecific molecule further comprises at least one molecule (e.g., a second antibody or antigen-binding fragment thereof) having a second binding specificity for a second target.

22. An immunoconjugate comprising the single-domain antibody or antigen-binding fragment thereof of any one of claims 1-10, or the polypeptide construct of any one of claims 11-16, and a therapeutic agent linked to the single-domain antibody or antigen-binding fragment thereof or the polypeptide construct; preferably, the immunoconjugate is an antibody-drug conjugate (ADC).

23. A pharmaceutical composition comprising the single-domain antibody or antigen-binding fragment thereof of any one of claims 1-10, the polypeptide construct of any one of claims 11-16, the isolated nucleic acid molecule of claim 17, the vector of claim 18, the host cell of claim 19, the bispecific or multispecific molecule of claim 21, or the immunoconjugate of claim 22, and a pharmaceutically acceptable carrier and / or excipient; preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

24. Use of a single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, of a polypeptide construct according to any one of claims 11 to 16, of an isolated nucleic acid molecule according to claim 17, of a vector according to claim 18, of a host cell according to claim 19, of a bispecific or multispecific molecule according to claim 21, of an immunoconjugate according to claim 22, or of a pharmaceutical composition according to claim 23, for the manufacture of a medicament for the prevention and / or treatment of a disease associated with GHR signaling in a subject; Preferably, said prevention and / or treatment of a disease associated with GHR signaling would benefit from an agonism of GHR signaling; Preferably, said disease associated with GHR signaling is selected from a disease associated with GH (growth hormone), IGF-1 (insulin-like growth factor-1) and / or GHRH (growth hormone releasing hormone) deficiency; Preferably, said disease associated with GHR signaling is selected from: growth hormone deficiency (GHD), kidney dysfunction (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, Laron syndrome; Preferably, said growth hormone deficiency (GHD) is growth hormone deficiency due to hypothalamic-pituitary disease, said pediatric short stature is pediatric short stature due to achondroplasia (ACH), and / or said reproductive disorder is a reproductive disorder associated with the growth hormone-growth hormone receptor signaling pathway (such as gonadal dysgenesis due to growth impairment in Turner syndrome); Preferably, said subject is a mammal, such as a human or a murine; Preferably, said single-domain antibody or antigen-binding fragment thereof, polypeptide construct, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule, immunoconjugate or pharmaceutical composition is used alone, or in any combination thereof, or in combination with (e.g. concomitantly or sequentially) another pharmaceutically active agent.

25. A method for preventing and / or treating a disease associated with GHR signaling in a subject in a subject, comprising: administering to a subject in need thereof an effective amount of a single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, of a polypeptide construct according to any one of claims 11 to 16, of an isolated nucleic acid molecule according to claim 17, of a vector according to claim 18, of a host cell according to claim 19, of a bispecific or multispecific molecule according to claim 21, of an immunoconjugate according to claim 22, or of a pharmaceutical composition according to claim 23; Preferably, said prevention and / or treatment of a disease associated with GHR signaling would benefit from an agonism of GHR signaling; Preferably, said disease associated with GHR signaling is selected from a disease associated with GH (growth hormone), IGF-1 (insulin-like growth factor-1) and / or GHRH (growth hormone releasing hormone) deficiency; Preferably, said disease associated with GHR signaling is selected from: growth hormone deficiency (GHD), kidney dysfunction (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, Laron syndrome; Preferably, said growth hormone deficiency (GHD) is growth hormone deficiency due to hypothalamic-pituitary disease, said pediatric short stature is pediatric short stature due to achondroplasia (ACH), and / or said reproductive disorder is a reproductive disorder associated with the growth hormone-growth hormone receptor signaling pathway (such as gonadal dysgenesis due to growth impairment in Turner syndrome); Preferably, said subject is a mammal, such as a human or a murine; Preferably, said single-domain antibody or antigen-binding fragment thereof, polypeptide construct, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule, immunoconjugate or pharmaceutical composition is used alone, or in any combination thereof, or in combination with (e.g. concomitantly or sequentially) another pharmaceutically active agent. Preferably, said disease related to GHR signaling is selected from the group consisting of growth hormone deficiency (GHD), renal insufficiency (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, Laron syndrome; Preferably, said growth hormone deficiency (GHD) is growth hormone deficiency due to hypothalamo-pituitary disease, said pediatric short stature is pediatric short stature due to achondroplasia (ACH), and / or said reproductive disorder is a reproductive disorder related to the growth hormone-growth hormone receptor signaling pathway (such as gonadal dysgenesis due to growth impairment in Turner syndrome); Preferably, said subject is a mammal, such as a human or a murine; Preferably, said single-domain antibody or antigen-binding fragment thereof, polypeptide construct, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule, immunoconjugate or pharmaceutical composition is used alone, or is administered in any combination thereof, or is administered in combination (e.g., simultaneously or sequentially) with another pharmaceutically active agent.

26. A conjugate comprising a single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or a polypeptide construct according to any one of claims 11 to 16, and a detectable label linked to said single-domain antibody or antigen-binding fragment thereof or said polypeptide construct; Preferably, said detectable label is selected from the group consisting of an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as an acridinium ester compound, luminol and derivatives thereof, or a ruthenium derivative), a fluorescent dye (such as fluorescein or a fluorescent protein), a radionuclide or biotin.

27. A kit comprising a single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, a polypeptide construct according to any one of claims 11 to 16, or a conjugate according to claim 26; Preferably, said kit comprises a conjugate according to claim 26; Preferably, said kit comprises a single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or a polypeptide construct according to any one of claims 11 to 16, and a second antibody specifically recognizing said single-domain antibody or antigen-binding fragment thereof or said polypeptide construct; optionally, said second antibody further comprises a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as an acridinium ester compound, luminol and derivatives thereof, or a ruthenium derivative), a fluorescent dye (such as fluorescein or a fluorescent protein), a radionuclide or biotin.

28. A method for detecting the presence or level of GHR in a sample, comprising the use of a single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, a polypeptide construct according to any one of claims 11 to 16, or a conjugate according to claim 26. Preferably, the method is an immunological detection, such as immunoblotting, enzyme immunoassay (e.g. ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay; Preferably, the method comprises the use of a conjugate of claim 26; Preferably, the method comprises the use of a single-domain antibody or antigen-binding fragment thereof of any one of claims 1 to 10 or a polypeptide construct of any one of claims 11 to 16, and the method further comprises the use of a second antibody carrying a detectable label (such as an enzyme (e.g. horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g. acridinium esters, luminol and its derivatives, or ruthenium derivatives), 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; Preferably, the method comprises: (1) contacting the sample with the single-domain antibody or antigen-binding fragment thereof or the polypeptide construct or the conjugate; (2) detecting the formation of antigen-antibody immune complexes or detecting the amount of said immune complexes.

29. Use of a single-domain antibody or antigen-binding fragment thereof of any one of claims 1 to 10, a polypeptide construct of any one of claims 11 to 16, or a conjugate of claim 26 for the manufacture of a detection reagent for detecting the presence or level of GHR in a sample and / or diagnosing a disease associated with GHR.

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