Anti-gh single-domain antibody and use thereof

Abstract

Provided are a single-domain antibody specifically binding to a growth hormone (e.g., a human growth hormone) or an antigen-binding fragment thereof, a polypeptide construct and conjugate containing the single-domain antibody or the antigen-binding fragment thereof, a nucleic acid molecule encoding the single-domain antibody or the antigen-binding fragment thereof, a host cell containing same, and the related use. In addition, further provided is the use of the single-domain antibody or the antigen-binding fragment thereof, the polypeptide construct or the conjugate in the purification and / or detection of the growth hormone (e.g., the human growth hormone).

Description

Anti-GH single domain antibody and its application

[0001] This application is based on the CN application with application number 202311630170.3 and application date November 30, 2023, and the CN application with application number 202410938901.9 and application date July 12, 2024, and claims its priority. The disclosed content of the CN application is hereby introduced into this application as a whole. Technical Field

[0002] The present invention relates to single-domain antibodies or antigen-binding fragments thereof that specifically bind to growth hormone (e.g., human growth hormone), polypeptide constructs and conjugates containing the domain antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the single-domain antibodies or antigen-binding fragments thereof, host cells containing the same, and related uses. In addition, the invention relates to the use of the single-domain antibodies or antigen-binding fragments thereof, polypeptide constructs or conjugates for the purification and / or detection of growth hormone (e.g., human growth hormone). Background Art

[0003] Human growth hormone (hGH) is a protein secreted by pituitary cells and a peptide hormone. It is a single peptide chain protein hormone secreted by eosinophils in the anterior pituitary gland of the human body. It is a peptide hormone composed of 191 amino acids. Growth hormone has been used clinically as a specific drug for the treatment of dwarfism since 1958. After it became mass-produced by pharmaceutical companies in the 1980s, its clinical indications have been continuously expanded. It is widely used not only in promoting human height growth, but also in combating tissue and organ failure, improving nutrition in critically ill patients, treating cachexia (nutritional failure), combating infection and inflammation, promoting wound and burn healing, and enhancing the body's immunity.

[0004] Nanobodies are a special type of antibody derived from camelids. In 1993, Hamers-Casterman et al. showed that camel-derived animals have a type of antibody that is naturally devoid of light chains and contains only heavy chains, which is called a heavy chain antibody. By cloning the variable region gene of the heavy chain antibody, a single-domain antibody consisting of only one heavy chain variable region can be obtained, which is called a VHH antibody. In the crystal structure of the VHH antibody, its diameter is only 2.5nm and its length is 4nm, so it is also called a nanobody. Nanobodies are only one-tenth the size of traditional IgG antibodies and are the smallest naturally occurring fragment that can bind to antigens. In addition, nanobodies also have characteristics such as easy production and can be advantageously used in the immunoaffinity chromatography purification of human GH to replace the traditional cumbersome human GH purification method.

[0005] Developed GH immunoaffinity chromatography media are being optimized and iterated, placing increasing demands on the heat and alkali resistance of the media's ligand proteins. Therefore, there is an urgent need to develop antibodies that specifically bind to GH and possess excellent heat and alkali resistance for the purification and / or detection of GH. Summary of the Invention

[0006] After extensive research, the inventors of this application have obtained a series of single-domain antibodies against growth hormone (e.g., human growth hormone). In particular, these single-domain antibodies not only have high binding activity with growth hormone (e.g., human growth hormone) but also have significantly excellent heat and alkali resistance, and can be advantageously used in the affinity purification of growth hormone (e.g., human growth hormone). For example, compared to existing anti-GH antibodies, the immunoaffinity medium prepared using the single-domain antibodies provided in this application is easier to clean, has a longer service life, and has a longer shelf life at room temperature. In addition, these single-domain antibodies also have the characteristics of small molecular weight and easy production.

[0007] Based on this, the present application also provides a polypeptide construct or conjugate containing the single-domain antibody or its antigen-binding fragment, a nucleic acid molecule encoding the single-domain antibody or its antigen-binding fragment and a host cell containing the same, as well as related uses.

[0008] Therefore, in a first aspect, the present application provides a single domain antibody or an antigen-binding fragment thereof that can specifically bind to growth hormone (GH), wherein the single domain antibody or the antigen-binding fragment thereof comprises a complementarity determining region (CDR) and a framework region (FR), and has one or more of the following characteristics:

[0009] (i) the amino acid residue at position 102 is Y;

[0010] (ii) the amino acid residue at position 105 is Q;

[0011] (iii) the amino acid residue at position 48 is V;

[0012] (iv) the amino acid residue at position 11 is K;

[0013] (v) the amino acid residue at position 100g is F;

[0014] (vi) the amino acid residue at position 101 is D;

[0015] (vii) the amino acid residue at position 28 is T;

[0016] (viii) the amino acid residue at position 19 is T;

[0017] (ix) the amino acid residue at position 56 is K;

[0018] (x) the amino acid residue at position 100e is E;

[0019] (xi) the amino acid residue at position 55 is D;

[0020] (xii) the amino acid residue at position 76 is S;

[0021] (xiii) the amino acid residue at position 14 is P;

[0022] Wherein, the positions are defined by the Kabat numbering system.

[0023] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3.

[0024] The positions of amino acid residues in the light chain variable region (VL) and heavy chain variable region (VH) of antibodies are defined according to the Kabat numbering system, which is well known to those 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 following amino acids: amino acid residues corresponding to positions 31 to 35 or positions 31 to 35c in the heavy chain variable domain (CDRH1), amino acid residues corresponding to positions 50 to 65 (CDRH2), and amino acid residues corresponding to positions 95 to 102 (CDRH3), see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991), which is incorporated herein by reference in its entirety.

[0025] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof has one or more of the following characteristics:

[0026] (a) the amino acid residue at position 102 is Y;

[0027] (b) the amino acid residue at position 105 is Q;

[0028] (c) the amino acid residue at position 48 is V;

[0029] (d) the amino acid residue at position 100g is F;

[0030] (e) the amino acid residue at position 28 is T;

[0031] (f) the amino acid residue at position 19 is T;

[0032] (g) the amino acid residue at position 100e is E;

[0033] (h) the amino acid residue at position 55 is D;

[0034] (i) the amino acid residue at position 14 is P;

[0035] Wherein, the positions are defined by the Kabat numbering system.

[0036] In certain embodiments, the single domain antibody or antigen-binding fragment thereof has a feature selected from the group consisting of:

[0037] (1) the amino acid residue at position 55 is D, and the amino acid residue at position 100e is E;

[0038] (2) the amino acid residue at position 28 is T, and the amino acid residue at position 55 is D;

[0039] (3) the amino acid residue at position 14 is P, and the amino acid residue at position 28 is T;

[0040] (4) the amino acid residue at position 14 is P, and the amino acid residue at position 105 is Q;

[0041] (5) the amino acid residue at position 14 is P, and the amino acid residue at position 19 is T;

[0042] (6) the amino acid residue at position 14 is P, and the amino acid residue at position 100e is E;

[0043] (7) the amino acid residue at position 55 is D, the amino acid residue at position 100e is E, and the amino acid residue at position 105 is Q;

[0044] (8) the amino acid residue at position 55 is D, the amino acid residue at position 100g is F, and the amino acid residue at position 102 is Y;

[0045] (9) the amino acid residue at position 55 is D, the amino acid residue at position 100e is E, and the amino acid residue at position 102 is Y;

[0046] (10) the amino acid residue at position 55 is D, and the amino acid residue at position 100g is F;

[0047] (11) the amino acid residue at position 55 is D, the amino acid residue at position 100e is E, and the amino acid residue at position 100g is F;

[0048] (12) the amino acid residue at position 55 is D, and the amino acid residue at position 102 is Y;

[0049] (13) the amino acid residue at position 48 is V, the amino acid residue at position 55 is D, and the amino acid residue at position 100e is E;

[0050] (14) the amino acid residue at position 55 is D, the amino acid residue at position 100e is E, and the amino acid residue at position 105 is Q;

[0051] (15) the amino acid residue at position 55 is D, the amino acid residue at position 100e is E, the amino acid residue at position 100g is F, and the amino acid residue at position 105 is Q;

[0052] Wherein, the positions are defined by the Kabat numbering system.

[0053] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof has CDR1, CDR2, and CDR3 as shown in any of the following:

[0054] (1) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:43; and CDR3 as set forth in SEQ ID NO:47;

[0055] (2) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:43; and CDR3 as set forth in SEQ ID NO:48;

[0056] (3) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:43; and CDR3 as set forth in SEQ ID NO:49;

[0057] (4) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:44; and CDR3 as set forth in SEQ ID NO:46;

[0058] (5) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:43; and CDR3 as set forth in SEQ ID NO:50;

[0059] (6) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:45; and CDR3 as set forth in SEQ ID NO:46;

[0060] (7) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:45; and CDR3 as set forth in SEQ ID NO:50;

[0061] (8) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:45; and CDR3 as set forth in SEQ ID NO:51;

[0062] (9) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:45; and CDR3 as set forth in SEQ ID NO:52;

[0063] (10) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:45; and CDR3 as set forth in SEQ ID NO:48;

[0064] (11) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:45; and CDR3 as set forth in SEQ ID NO:53;

[0065] or,

[0066] (12) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:47.

[0067] In certain embodiments, the single-domain antibody has one or more substitution mutations selected from the following compared to the sequence shown in SEQ ID NO: 37:

[0068] D102Y, P105Q, L48V, L11K, W100gF, V101D, P28T, R19T, T56K, R100eE, A55D, N76S, A14P;

[0069] The position of the mutation is defined by the Kabat numbering system.

[0070] In certain embodiments, the single-domain antibody has one or more substitution mutations selected from the following compared to the sequence shown in SEQ ID NO: 37:

[0071] D102Y, P105Q, L48V, W100gF, P28T, R19T, R100eE, A55D, A14P;

[0072] The position of the mutation is defined by the Kabat numbering system.

[0073] In certain embodiments, the single domain antibody comprises the following sequence:

[0074] (1) A at position 55 is substituted with D, and R at position 100e is substituted with E (A55D and R100eE);

[0075] (2) P at position 28 is substituted with T, and A at position 55 is substituted with D (P28T and A55D);

[0076] (3) A at position 14 is substituted with P, and P at position 28 is substituted with T (A14P and P28T);

[0077] (4) A at position 14 is substituted with P, and P at position 105 is substituted with Q (A14P and P105Q);

[0078] (5) A at position 14 is substituted with P, and R at position 19 is substituted with T (A14P and R19T);

[0079] (6) A at position 14 is substituted with P, and R at position 100e is substituted with E (A14P and R100eE);

[0080] (7) A at position 55 is substituted with D, R at position 100e is substituted with E, and P at position 105 is substituted with Q (A55D, R100eE, and P105Q);

[0081] (8) A at position 55 is substituted with D, W at position 100g is substituted with F, and D at position 102 is substituted with Y (A55D, W100gF, and D102Y);

[0082] (9) A at position 55 is substituted with D, R at position 100e is substituted with E, and D at position 102 is substituted with Y (A55D, R100eE, and D102Y);

[0083] (10) A at position 55 is substituted with D, and W at position 100g is substituted with F (A55D and W100gF);

[0084] (11) A at position 55 is substituted with D, R at position 100e is substituted with E, and W at position 100g is substituted with F (A55D, R100eE, and W100gF);

[0085] (12) A at position 55 is substituted with D, and D at position 102 is substituted with Y (A55D and D102Y);

[0086] (13) L at position 48 is substituted with V, A at position 55 is substituted with D, and R at position 100e is substituted with E (L48V, A55D, and R100eE);

[0087] (14) A at position 55 is substituted with D, R at position 100e is substituted with E, and P at position 105 is substituted with Q (A55D, R100eE, and P105Q);

[0088] or,

[0089] (15) A at position 55 is substituted with D, R at position 100e is substituted with E, W at position 100g is substituted with F, and P at position 105 is substituted with Q (A55D, R100eE, W100gF, and P105Q);

[0090] Wherein, the positions are defined by the Kabat numbering system.

[0091] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of:

[0092] (1) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 2;

[0093] (2) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 3;

[0094] (3) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 4;

[0095] (4) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 5;

[0096] (5) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 6;

[0097] (6) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 7;

[0098] (7) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 8;

[0099] (8) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 9;

[0100] (9) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 10;

[0101] (10) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 11;

[0102] (11) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 12;

[0103] (12) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 13;

[0104] (13) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 15;

[0105] (14) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 16;

[0106] (15) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 17;

[0107] (16) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 18;

[0108] (17) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 19;

[0109] (18) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 20;

[0110] (19) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 21;

[0111] (20) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 22;

[0112] (21) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 23;

[0113] (22) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 24;

[0114] (23) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 25;

[0115] (24) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 26;

[0116] (25) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 27;

[0117] (26) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 28;

[0118] (27) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 29;

[0119] (28) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 30;

[0120] (29) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 31;

[0121] (30) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 32;

[0122] (31) the amino acid sequence shown at positions 2 to 125 or positions 1 to 125 of SEQ ID NO: 33;

[0123] (32) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 34;

[0124] (33) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 35;

[0125] (34) The amino acid sequence shown at positions 2-125 or 1-125 of SEQ ID NO: 36.

[0126] In certain embodiments, the GH is human GH.

[0127] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof has better thermal stability and / or alkali stability than the antibody shown in SEQ ID NO: 1 or 37.

[0128] In a second aspect, the present application provides a single-domain antibody or an antigen-binding fragment thereof that can specifically bind to growth hormone (GH), wherein the single-domain antibody or the antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, and the single-domain antibody has one or more substitution mutations selected from the following compared to the sequence shown in SEQ ID NO: 37:

[0129] (i) D at position 114 of SEQ ID NO: 37 is substituted with Y;

[0130] (ii) P at position 117 of SEQ ID NO: 37 is substituted with Q;

[0131] (iii) L at position 49 of SEQ ID NO: 37 is substituted with V;

[0132] (iv) L at position 12 of SEQ ID NO: 37 is substituted with K;

[0133] (v) W at position 112 of SEQ ID NO: 37 is substituted with F;

[0134] (vi) V at position 113 of SEQ ID NO: 37 is substituted with D;

[0135] (vii) P at position 29 of SEQ ID NO: 37 is substituted with T;

[0136] (viii) R at position 20 of SEQ ID NO: 37 is substituted with T;

[0137] (ix) T at position 58 of SEQ ID NO: 37 is substituted with K;

[0138] (x) R at position 110 in SEQ ID NO: 37 is substituted with E;

[0139] (xi) the A at position 57 of SEQ ID NO: 37 is substituted with D;

[0140] (xii) N at position 78 of SEQ ID NO: 37 is substituted with S;

[0141] (xiii) A at position 15 of SEQ ID NO: 37 is substituted with P.

[0142] In certain embodiments, the single-domain antibody has one or more substitution mutations selected from the following compared to the sequence shown in SEQ ID NO: 37:

[0143] (a) D at position 114 of SEQ ID NO: 37 is substituted with Y;

[0144] (b) P at position 117 of SEQ ID NO: 37 is substituted with Q;

[0145] (c) L at position 49 of SEQ ID NO: 37 is substituted with V;

[0146] (d) W at position 112 of SEQ ID NO: 37 is substituted with F;

[0147] (e) P at position 29 of SEQ ID NO: 37 is substituted with T;

[0148] (f) R at position 20 of SEQ ID NO: 37 is substituted with T;

[0149] (g) R at position 110 of SEQ ID NO: 37 is substituted with E;

[0150] (h) A at position 57 of SEQ ID NO: 37 is substituted with D;

[0151] (i) A at position 15 of SEQ ID NO: 37 is substituted with P.

[0152] In certain embodiments, the single domain antibody comprises the following sequence:

[0153] (1) A at position 57 of SEQ ID NO: 37 is substituted with D, and R at position 110 of SEQ ID NO: 37 is substituted with E;

[0154] (2) P at position 29 of SEQ ID NO: 37 is substituted with T, and A at position 57 of SEQ ID NO: 37 is substituted with D;

[0155] (3) A at position 15 of SEQ ID NO: 37 is substituted with P, and P at position 29 of SEQ ID NO: 37 is substituted with T;

[0156] (4) A at position 15 of SEQ ID NO: 37 is substituted with P, and P at position 117 of SEQ ID NO: 37 is substituted with Q;

[0157] (5) A at position 15 of SEQ ID NO: 37 is substituted with P, and R at position 20 of SEQ ID NO: 37 is substituted with T;

[0158] (6) A at position 15 of SEQ ID NO: 37 is substituted with P, and R at position 110 of SEQ ID NO: 37 is substituted with E;

[0159] (7) A at position 57 of SEQ ID NO: 37 is substituted with D, R at position 110 of SEQ ID NO: 37 is substituted with E, and P at position 117 of SEQ ID NO: 37 is substituted with Q;

[0160] (8) A at position 57 of SEQ ID NO: 37 is substituted with D, W at position 112 of SEQ ID NO: 37 is substituted with F, and D at position 114 of SEQ ID NO: 37 is substituted with Y;

[0161] (9) A at position 57 of SEQ ID NO: 37 is substituted with D, R at position 110 of SEQ ID NO: 37 is substituted with E, and D at position 114 of SEQ ID NO: 37 is substituted with Y;

[0162] (10) A at position 57 of SEQ ID NO: 37 is substituted with D, and W at position 112 of SEQ ID NO: 37 is substituted with F;

[0163] (11) A at position 57 of SEQ ID NO: 37 is substituted with D, R at position 110 of SEQ ID NO: 37 is substituted with E, and W at position 112 of SEQ ID NO: 37 is substituted with F;

[0164] (12) A at position 57 of SEQ ID NO: 37 is substituted with D, and D at position 114 of SEQ ID NO: 37 is substituted with Y;

[0165] (13) L at position 49 of SEQ ID NO: 37 is substituted with V, A at position 57 of SEQ ID NO: 37 is substituted with D, and R at position 110 of SEQ ID NO: 37 is substituted with E;

[0166] (14) A at position 57 of SEQ ID NO: 37 is substituted with D, R at position 110 of SEQ ID NO: 37 is substituted with E, and P at position 117 of SEQ ID NO: 37 is substituted with Q;

[0167] or,

[0168] (15) A at position 57 of SEQ ID NO: 37 was substituted with D, R at position 110 of SEQ ID NO: 37 was substituted with E, W at position 112 of SEQ ID NO: 37 was substituted with F, and P at position 117 of SEQ ID NO: 37 was substituted with Q.

[0169] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of:

[0170] (1) the amino acid sequence as shown in positions 2-125 or positions 1-125 of SEQ ID NO: 2;

[0171] (2) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 3;

[0172] (3) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 4;

[0173] (4) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 5;

[0174] (5) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 6;

[0175] (6) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 7;

[0176] (7) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 8;

[0177] (8) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 9;

[0178] (9) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 10;

[0179] (10) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 11;

[0180] (11) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 12;

[0181] (12) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 13;

[0182] (13) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 15;

[0183] (14) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 16;

[0184] (15) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 17;

[0185] (16) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 18;

[0186] (17) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 19;

[0187] (18) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 20;

[0188] (19) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 21;

[0189] (20) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 22;

[0190] (21) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 23;

[0191] (22) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 24;

[0192] (23) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 25;

[0193] (24) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 26;

[0194] (25) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 27;

[0195] (26) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 28;

[0196] (27) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 29;

[0197] (28) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 30;

[0198] (29) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 31;

[0199] (30) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 32;

[0200] (31) the amino acid sequence shown at positions 2 to 125 or positions 1 to 125 of SEQ ID NO: 33;

[0201] (32) the amino acid sequence set forth at positions 2-125 or positions 1-125 of SEQ ID NO: 34; (33) the amino acid sequence set forth at positions 2-125 or positions 1-125 of SEQ ID NO: 35; (34) the amino acid sequence set forth at positions 2-125 or positions 1-125 of SEQ ID NO: 36. In certain embodiments, the GH is human GH.

[0202] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof has better thermal stability and / or alkali stability than the antibody shown in SEQ ID NO: 1 or 37.

[0203] In a third aspect, the present application provides a single-domain antibody or an antigen-binding fragment thereof that can specifically bind to growth hormone (GH), wherein the single-domain antibody or the antigen-binding fragment thereof comprises:

[0204] (1) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:43; and CDR3 as set forth in SEQ ID NO:47;

[0205] (2) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:43; and CDR3 as set forth in SEQ ID NO:48;

[0206] (3) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:43; and CDR3 as set forth in SEQ ID NO:49;

[0207] (4) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:44; and CDR3 as set forth in SEQ ID NO:46;

[0208] (5) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:43; and CDR3 as set forth in SEQ ID NO:50;

[0209] (6) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:45; and CDR3 as set forth in SEQ ID NO:46;

[0210] (7) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:45; and CDR3 as set forth in SEQ ID NO:50;

[0211] (8) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:45; and CDR3 as set forth in SEQ ID NO:51;

[0212] (9) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:45; and CDR3 as set forth in SEQ ID NO:52;

[0213] (10) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:45; and CDR3 as set forth in SEQ ID NO:48;

[0214] (11) CDR1 as set forth in SEQ ID NO:42; CDR2 as set forth in SEQ ID NO:45; and CDR3 as set forth in SEQ ID NO:53;

[0215] or,

[0216] (12) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:47.

[0217] In certain embodiments, the CDRs are defined by the Kabat numbering system.

[0218] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of:

[0219] (i) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 2;

[0220] (ii) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 6;

[0221] (iii) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 7;

[0222] (iv) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 10;

[0223] (v) the amino acid sequence as shown in positions 2 to 125 or positions 1 to 125 of SEQ ID NO: 11;

[0224] (vi) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 12;

[0225] (vii) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 16;

[0226] (viii) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 17;

[0227] (ix) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 23;

[0228] (x) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 24;

[0229] (xi) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 25;

[0230] (xii) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 26;

[0231] (xiii) the amino acid sequence as shown in positions 2 to 125 or positions 1 to 125 of SEQ ID NO: 27;

[0232] (xiv) the amino acid sequence shown in positions 2 to 125 or positions 1 to 125 of SEQ ID NO: 28;

[0233] (xv) the amino acid sequence shown in positions 2 to 125 or positions 1 to 125 of SEQ ID NO: 29;

[0234] (xvi) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 30;

[0235] (xvii) the amino acid sequence shown in positions 2 to 125 or positions 1 to 125 of SEQ ID NO: 31;

[0236] (xviii) the amino acid sequence as shown in positions 2 to 125 or positions 1 to 125 of SEQ ID NO: 32;

[0237] (xix) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 33;

[0238] (xx) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 34;

[0239] (xxi) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 35;

[0240] (xxii) the amino acid sequence shown at positions 2 to 125 or positions 1 to 125 of SEQ ID NO: 36.

[0241] In certain embodiments, the GH is human GH.

[0242] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof has better thermal stability and / or alkali stability than the antibody shown in SEQ ID NO: 1 or 37.

[0243] In a fourth aspect, the present application provides a polypeptide construct capable of specifically binding to growth hormone (GH), comprising the single domain antibody or antigen-binding fragment thereof as described in the first, second or third aspect, and another polypeptide.

[0244] In certain embodiments, the polypeptide construct is a fusion protein.

[0245] In certain embodiments, the additional polypeptide is linked to the N-terminus or C-terminus of the single domain antibody or antigen-binding fragment thereof, optionally via a linker.

[0246] In certain embodiments, the additional polypeptide is selected from a protein tag (e.g., a His tag, such as HHHHHH or HHHHHHHHH), a protease recognition sequence (e.g., an EK protease recognition sequence, such as DDDDK), a peptide linker (e.g., a flexible peptide linker; for example, a peptide linker comprising one or more glycine (G) and / or serine (S)), or any combination thereof.

[0247] In certain embodiments, the polypeptide construct comprises, from N-terminus to C-terminus, a single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, an EK protease recognition sequence (such as DDDDK), a flexible peptide linker (for example, a peptide linker as shown in SEQ ID NO: 40), and an optional His tag (such as HHHHHH or HHHHHHHH).

[0248] In certain embodiments, the polypeptide construct comprises an amino acid sequence selected from the group consisting of:

[0249] (1) the amino acid sequence as set forth in SEQ ID NO: 2, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 2;

[0250] (2) the amino acid sequence as set forth in SEQ ID NO: 3, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 3;

[0251] (3) the amino acid sequence as set forth in SEQ ID NO: 4, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 4;

[0252] (4) the amino acid sequence as set forth in SEQ ID NO: 5, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 5;

[0253] (5) the amino acid sequence as set forth in SEQ ID NO: 6, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 6;

[0254] (6) the amino acid sequence as set forth in SEQ ID NO: 7, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 7;

[0255] (7) the amino acid sequence as set forth in SEQ ID NO: 8, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 8;

[0256] (8) the amino acid sequence as set forth in SEQ ID NO: 9, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 9;

[0257] (9) the amino acid sequence as set forth in SEQ ID NO: 10, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 10;

[0258] (10) the amino acid sequence as set forth in SEQ ID NO: 11, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 11;

[0259] (11) the amino acid sequence as set forth in SEQ ID NO: 12, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 12;

[0260] (12) the amino acid sequence as set forth in SEQ ID NO: 13, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 13;

[0261] (13) the amino acid sequence as set forth in SEQ ID NO: 15, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 15;

[0262] (14) the amino acid sequence as set forth in SEQ ID NO: 16, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 16;

[0263] (15) the amino acid sequence as set forth in SEQ ID NO: 17, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 17;

[0264] (16) the amino acid sequence as set forth in SEQ ID NO: 19, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 19;

[0265] (17) the amino acid sequence as set forth in SEQ ID NO: 20, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 20;

[0266] (18) the amino acid sequence as set forth in SEQ ID NO: 21, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 21;

[0267] (19) the amino acid sequence as set forth in SEQ ID NO: 23, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 23;

[0268] (20) the amino acid sequence as set forth in SEQ ID NO: 24, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 24;

[0269] (21) the amino acid sequence as set forth in SEQ ID NO: 25, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 25;

[0270] (22) the amino acid sequence as set forth in SEQ ID NO: 27, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 27;

[0271] (23) the amino acid sequence as set forth in SEQ ID NO: 29, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 29;

[0272] (24) the amino acid sequence as set forth in SEQ ID NO: 31, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 31;

[0273] (25) The amino acid sequence as shown in SEQ ID NO: 34, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 34.

[0274] In certain embodiments, the GH is human GH.

[0275] In certain embodiments, the construct has better thermal stability and / or alkali stability than the antibody shown in SEQ ID NO: 1 or 37.

[0276] In a fifth aspect, the present application provides a polypeptide construct capable of specifically binding to growth hormone (GH), comprising a single domain antibody or an antigen-binding fragment thereof capable of specifically binding to GH, and a polypeptide as shown in SEQ ID NO: 41.

[0277] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof that can specifically bind to GH is selected from: (i) the single-domain antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect or the third aspect, and (ii) the single-domain antibody or antigen-binding fragment thereof as shown in SEQ ID NO: 37.

[0278] In certain embodiments, the polypeptide construct is a fusion protein.

[0279] In certain embodiments, the polypeptide construct comprises a polypeptide as shown in SEQ ID NO: 41 at the C-terminus of the single-domain antibody or antigen-binding fragment thereof that specifically binds to GH.

[0280] In certain embodiments, the polypeptide construct further comprises an EK protease recognition sequence (e.g.,

[0281] DDDDK) and / or a His tag (e.g., HHHHHH or HHHHHHHH).

[0282] In certain embodiments, the polypeptide construct comprises, from N-terminus to C-terminus, the single-domain antibody or antigen-binding fragment thereof that can specifically bind to GH, the EK protease recognition sequence, such as the polypeptide shown in SEQ ID NO: 41, and optionally the His tag.

[0283] In certain embodiments, the polypeptide construct comprises an amino acid sequence selected from the group consisting of:

[0284] (1) the amino acid sequence as set forth in SEQ ID NO: 14, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 14;

[0285] (2) the amino acid sequence as set forth in SEQ ID NO: 18, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 18;

[0286] (3) the amino acid sequence as set forth in SEQ ID NO: 22, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 22;

[0287] (4) the amino acid sequence as set forth in SEQ ID NO: 26, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 26;

[0288] (5) the amino acid sequence as set forth in SEQ ID NO: 28, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 28;

[0289] (6) the amino acid sequence as set forth in SEQ ID NO: 30, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 30;

[0290] (7) the amino acid sequence as set forth in SEQ ID NO: 32, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 32;

[0291] (8) the amino acid sequence as set forth in SEQ ID NO: 33, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 33;

[0292] (9) the amino acid sequence as set forth in SEQ ID NO: 35, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 35;

[0293] (10) The amino acid sequence as shown in SEQ ID NO: 36, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 36.

[0294] In certain embodiments, the GH is human GH.

[0295] In certain embodiments, the construct has better thermal stability and / or alkali stability than the antibody shown in SEQ ID NO: 1 or 37.

[0296] In a sixth aspect, the present application provides an isolated nucleic acid molecule encoding the single domain antibody or antigen-binding fragment thereof of the first, second or third aspect, or the polypeptide construct of the fourth or fifth aspect.

[0297] In a seventh aspect, the present application provides a vector comprising the isolated nucleic acid molecule of the sixth aspect. In certain embodiments, the vector is a cloning vector or an expression vector.

[0298] In an eighth aspect, the present application provides a host cell comprising the isolated nucleic acid molecule of the sixth aspect or the vector of the seventh aspect. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as E. coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell is a microorganism.

[0299] The single-domain antibodies or polypeptide constructs of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant techniques. For example, a DNA molecule encoding the single-domain antibody or construct of the present invention is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector, and then transformed / transfected into a host cell. The transformed / transfected host cell is then cultured under specific conditions to express the single-domain antibody or construct of the present invention.

[0300] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact nanobody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In addition, these antigen-binding fragments can also be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). Those skilled in the art are fully aware of other techniques for preparing these antigen-binding fragments.

[0301] In the ninth aspect, the present application provides a method for preparing the single domain antibody or antigen binding fragment thereof of the first, second or third aspect or the polypeptide construct of the fourth or fifth aspect, comprising culturing the host cell of the eighth aspect under conditions allowing protein expression, and recovering the single domain antibody or antigen binding fragment thereof or the polypeptide construct from the cultured host cell culture.

[0302] In the tenth aspect, the present application provides a conjugate comprising the single domain antibody or antigen-binding fragment thereof of the first, second or third aspect or the polypeptide construct of the fourth or fifth aspect, and a solid support connected to the single domain antibody or antigen-binding fragment thereof or the polypeptide construct.

[0303] In certain embodiments, the solid support is selected from the group consisting of magnetic beads, agarose microspheres, polymer matrices (e.g., dextran microspheres, polymethacrylate, polystyrene, polystyrene-divinylbenzene, polymethyl methacrylate), silica matrices (e.g., silica microspheres), graphitized carbon matrices, alumina matrices, zirconia matrices, and any combination thereof.

[0304] In certain embodiments, the solid support is selected from a porous material, for example, selected from one porous material or a combination of multiple porous materials.

[0305] In the eleventh aspect, the present application provides a method for purifying GH, which comprises using the conjugate of the tenth aspect.

[0306] In certain embodiments, the method is a method comprising purifying GH by affinity chromatography.

[0307] In certain embodiments, the method comprises the steps of:

[0308] (1) allowing the conjugate to bind to the GH in a first solvent to form a complex;

[0309] (2) dissociating the complex in a second solvent; and,

[0310] (3) collecting the dissociation product containing the GH;

[0311] The first solvent is a solvent suitable for forming a complex between the conjugate and the GH, and the second solvent is a solvent capable of dissociating the complex.

[0312] In some embodiments, the conductivity of the first solvent is 10-25 mS / cm (e.g., 15-19 mS / cm, 10-20 mS / cm, 15-25 mS / cm), and / or the conductivity of the second solvent is 0.3-5.0 mS / cm (e.g., 0.5-1.0 mS / cm, 0.5-3.0 mS / cm, 0.5-5.0 mS / cm, 1.0-3.0 mS / cm, 1.0-5.0 mS / cm).

[0313] In certain embodiments, the pH of the first solvent is 6.0-8.5 (e.g., 7.3-7.7, 6.0-8.0, 6.5-7.7, 6.5-8.5, 7.0-7.7, 7.0-8.5), and / or the pH of the second solvent is 3.0-4.5 (e.g., 3.4-3.8, 3.0-3.8, 3.0-4.2, 3.4-4.2, 3.4-4.5).

[0314] In certain embodiments, the GH is human GH.

[0315] In the twelfth aspect, the present application provides use of the single domain antibody or antigen-binding fragment thereof of the first, second or third aspect, or the polypeptide construct of the fourth or fifth aspect, or the conjugate of the tenth aspect in preparing a GH purification reagent.

[0316] In certain embodiments, the GH is human GH.

[0317] In the thirteenth aspect, the present application provides a conjugate comprising the single domain antibody or antigen-binding fragment thereof of the first, second or third aspect or the polypeptide construct of the fourth or fifth aspect, and a detectable label linked to the single domain antibody or antigen-binding fragment thereof or the polypeptide construct.

[0318] In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0319] In the fourteenth aspect, the present application provides a kit comprising the single domain antibody or antigen-binding fragment thereof of the first, second or third aspect, or the polypeptide construct of the fourth or fifth aspect, or the conjugate of the thirteenth aspect.

[0320] In certain embodiments, the kit comprises the conjugate of the thirteenth aspect.

[0321] In certain embodiments, the kit comprises the single domain antibody or antigen-binding fragment thereof of the first, second or third aspect or the polypeptide construct of the fourth or fifth 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 (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

[0322] In the fifteenth aspect, the present application provides a method for detecting the presence or level of GH in a sample, which comprises using the single domain antibody or antigen-binding fragment thereof of the first, second or third aspect, or the polypeptide construct of the fourth or fifth aspect, or the conjugate of the thirteenth aspect.

[0323] In certain embodiments, the method is an immunological assay, such as immunoblotting, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0324] In certain embodiments, the method comprises use of the conjugate of the thirteenth aspect.

[0325] In certain embodiments, the method comprises using the single domain antibody or antigen-binding fragment thereof of the first, second or third aspect or the polypeptide construct of the fourth or fifth aspect, and the method further comprises using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide or biotin) to detect the single domain antibody or antigen-binding fragment thereof or the polypeptide construct.

[0326] In certain embodiments, the method comprises: (1) contacting the sample with the single domain antibody or antigen-binding fragment thereof of the first, second, or third aspect, or the construct of the fourth or fifth aspect, or the conjugate of the thirteenth aspect; and (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of GH.

[0327] In certain embodiments, the GH is human GH.

[0328] In certain embodiments, the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic, non-diagnostic purposes.

[0329] In the sixteenth aspect, the present application also provides a method for diagnosing a disease associated with abnormal GH levels, which comprises detecting the level of GH in a sample from a subject using the method of the fifteenth aspect.

[0330] In certain embodiments, when the level of GH in a sample from a subject is significantly increased or decreased compared to a reference level (e.g., compared to a healthy control), it indicates that the subject has a disease associated with abnormally high or low GH levels (e.g., gigantism / dwarfism).

[0331] In certain embodiments, the disease associated with abnormal GH levels is selected from the group consisting of: endogenous GH deficiency, Noonan short stature, SHOX short stature / disorder, achondroplasia, Turner syndrome, small for gestational age SGA, and disease / stimulated GH deficiency.

[0332] In certain embodiments, the GH is human GH.

[0333] In the seventeenth aspect, the present application provides use of the single domain antibody or antigen-binding fragment thereof of the first, second or third aspect, or the polypeptide construct of the fourth or fifth aspect, or the conjugate of the thirteenth aspect in the preparation of a detection reagent, wherein the detection reagent is used to detect the presence or level of GH in a sample and / or diagnose a disease associated with abnormal GH levels.

[0334] In certain embodiments, the detection reagent detects the presence or level of GH in a sample using the method of the fifteenth aspect.

[0335] In certain embodiments, the disease associated with abnormal GH levels is selected from the group consisting of: endogenous GH deficiency, Noonan short stature, SHOX short stature / disorder, achondroplasia, Turner syndrome, small for gestational age SGA, and disease / stimulated GH deficiency.

[0336] In certain embodiments, the sample is a body fluid sample from a subject (eg, a mammal, preferably a human).

[0337] In certain embodiments, the GH is human GH.

[0338] Definition of terms

[0339] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0340] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0341] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0342] As used herein, the term "antibody" is used in the broadest sense and includes, but is not limited to, monoclonal antibodies (including human antibodies, humanized antibodies, or chimeric antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments thereof, so long as they exhibit the desired biological activity (e.g., antigen-binding activity).

[0343] As used herein, "antibody fragments" comprise a portion of an intact antibody that partially or completely retains the antigen-binding activity of the antibody from which it is derived. Antibody fragments preferably include the antigen-binding region or variable region of an intact antibody. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng., 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0344] As used herein, the term "single domain antibody" 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), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, a single domain antibody consists of four framework regions and three complementarity determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A single domain antibody may be truncated at the N-terminus or C-terminus so that it contains only part of FR1 and / or FR4, or lacks one or two of those framework regions, as long as it substantially maintains antigen binding and specificity. Single domain antibodies are also referred to as nanobodies, and the two are used interchangeably.

[0345] As used herein, the term "antigen-binding fragment" of a single-domain antibody refers to a polypeptide comprising a fragment of a single-domain antibody that retains the ability to specifically bind to the same antigen to which the single-domain antibody binds, and / or competes with the single-domain antibody for specific binding to the antigen, and is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of the antibodies of the invention can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the single-domain antibodies of the invention. In some embodiments, the "antigen-binding fragment" of the single-domain antibody can be truncated at the N-terminus or C-terminus compared to the full-length single-domain antibody so that it only comprises part of FR1 and / or FR4, or lacks one or both of those framework regions, as long as it substantially retains antigen binding and specificity.

[0346] Antigen-binding fragments of single-domain antibodies can be obtained from a given single-domain antibody (such as the Nanobodies provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and antigen-binding fragments of single-domain antibodies can be screened for specificity in the same manner as for intact single-domain antibodies.

[0347] Herein, unless the context clearly indicates otherwise, when referring to the term "single domain antibody", it includes not only intact single domain antibodies, but also antigen-binding fragments of single domain antibodies.

[0348] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. In nanobodies, there are three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given Nanobody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In this context, the CDRs of Nanobodies are preferably determined by the Kabat numbering system.

[0349] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

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

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

[0352] As used herein, the detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa750)), luminescent substances (e.g., chemiluminescent substances, such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0353] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0354] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

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

[0356] Advantageous Effects of the Invention

[0357] The series of single-domain antibodies against growth hormone (e.g., human growth hormone) provided in the present application have high binding activity to growth hormone (e.g., human growth hormone) and also have significantly excellent heat resistance and alkali resistance (e.g., able to withstand 0.3M or 0.5M alkali treatment for 24 hours, and / or, compared with the parent antibody, its T mThe single-domain antibody provided herein can be advantageously applied to affinity purification of growth hormone (e.g., human growth hormone) by increasing the temperature by 5-10°C. For example, compared to existing anti-GH antibodies, the immunoaffinity medium prepared using the single-domain antibody provided herein is easier to clean, has a longer service life, and can be stored at room temperature for a longer period. Furthermore, the single-domain antibody has the advantages of a small molecular weight and ease of production.

[0358] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0359] Figure 1: ELISA results of 45 single-point mutants treated with 0.3 M NaOH for 24 h.

[0360] Figure 2: ELISA test results of 14 single-point mutants treated with 0.5 M NaOH for 24 h.

[0361] Figure 3: SDS-PAGE detection results of 10 single-point mutants after 0.5M alkali treatment.

[0362] Figure 4: Tm value detection results of 10 single-point mutants.

[0363] Figure 5: Alkali-resistant SDS-PAGE electrophoresis results of 40 multi-point mutants (Figure 5A) and corresponding statistical results (Figure 5B).

[0364] Figure 6: EC of 21 multi-point mutants 50 Absolute value.

[0365] Figure 7: Tm value detection results of 21 multi-point mutants.

[0366] Figure 8: Electrophoresis detection results after treatment with preferred molecular bases.

[0367] Figure 9: Alkali resistance test results of 6 preferred molecular coupling media.

[0368] Sequence information

[0369] A description of the sequences involved in this application is provided in the table below.

[0370] Table 1: Sequence information DETAILED DESCRIPTION

[0371] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0372] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Molecular Biology: A Compendium of Laboratory Manuals, 3rd edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0373] Example 1: Sample preparation

[0374] 1.1 Protein design and preparation

[0375] Point mutations were designed for an anti-human growth hormone single-domain antibody (SEQ ID NO: 37, see patent application PCT / CN2023 / 096259) screened by Changchun Jinsai Pharmaceutical Co., Ltd., and heat and alkali resistance were optimized. Furthermore, to facilitate protein identification and / or purification, a His tag (SEQ ID NO: 39) and an EK protease recognition sequence (SEQ ID NO: 38) were fused to the C-terminus of the antibody. The EK protease recognition sequence and the His tag were connected via a linker (e.g., a linker as shown in SEQ ID NO: 40 or 41).

[0376] After protein design and single- or multi-site mutations, the sequence is artificially synthesized and constructed, and then used to transform E. coli host cells to express the target protein. After fermentation and culture, the host strain is disrupted by ultrasound and affinity purified using a nickel column to obtain the target protein solution. Ultrafiltration and centrifugation are then performed using ultrafiltration tubes to obtain a protein concentrate. The protein is then renatured with urea and a redox agent to renature its disulfide bonds, followed by a secondary purification step. The purified protein is then used as the target protein for analysis and modification.

[0377] 1.2 Experimental design

[0378] The target protein was measured by DSF differential scanning m The protein was treated with alkali, and the treated protein was pH neutralized. The affinity EC of the protein before and after treatment was measured using the ELISA method. 50 Under this fermentation preparation scale condition, the data of the modified protein before and after treatment were compared with the data of the unmodified GH single domain antibody molecule (amino acid sequence such as SEQ ID NO: 1).m The increase indicates that the thermal stability of the protein is improved, and the reduction of degradation bands in the electrophoresis results after alkali treatment indicates that the alkali resistance is enhanced. 50 Equivalent or decreased affinity indicates equivalent or improved affinity. Based on the heat and alkali resistance of the optimized protein, the optimal GH single domain antibody iterative molecule was screened.

[0379] Example 2: Alkali resistance study of a designed protein (single point mutant):

[0380] Untreated samples were subjected to ELISA affinity testing. Alkali resistance was also tested for 45 single-point mutants treated with 0.3M sodium hydroxide. The mutants' affinity was then tested using ELISA. Mutants with improved alkali resistance were then treated with 0.5M sodium hydroxide and tested again. The proportion of electrophoretic degradation bands observed for the selected proteins after 0.5M treatment was also examined.

[0381] 2.1 Antibody ELISA affinity detection

[0382] Dilute rhGH protein (see Uniprot: P01241) to 5 μg / ml, add 50 μl / well, and coat overnight; block; dilute the sample with PBS to a concentration of 40 μg / ml, and add 2-fold serial dilutions to 12 concentration points. Add each sample to the ELISA at 50 μl / well and incubate at room temperature for 1 hour; dilute Anti-6*His HRP secondary antibody at 1:5000 in PBS, add 50 μl / well, and incubate at room temperature for 1 hour; develop color and stop by adding 2 M sulfuric acid; read the plate at OD450.

[0383] Result processing: Draw ELISA curves of each sample according to OD450, and compare ELISA curves and EC 50 The alkaline stability of single-domain antibodies was evaluated by comparing the changes in the

[0384] 2.2 Antibody ELISA Alkali Resistance Test

[0385] Sample alkaline treatment: Dilute the sample to 2 mg / ml with PBS; mix the diluted sample solution with 0.6 M NaOH / 1 M NaOH solution at a ratio of 1:1 (V / V), mix gently, and place in a refrigerator at 2-8°C. Samples were taken at 24 h after treatment, and the treatment was terminated.

[0386] End of sample treatment: Add HCl solution to half the sample volume, mix gently until neutral, and store at -80°C until ready for use. The detection method is the same as the ELISA affinity test.

[0387] 2.3 Antibody SDS-PAGE detection

[0388] Alkali-treated samples were analyzed by SDS-PAGE: the sample was diluted with ultrapure water, 5X protein buffer was added at a ratio of 4:1, and the sample was heated at 95°C for 10 minutes. Electrophoresis was performed on a 15% SDS-PAGE gel at 120V for 65 minutes, followed by staining and destaining. A constant loading of 4 μg of protein was used for the control and 24-hour alkali-treated proteins. Relative quantification of small protein bands from the 24-hour treated proteins was performed using ImageJ and Imagelab software to determine protein cleavage and provide a more intuitive understanding of the protein's alkali resistance.

[0389] 2.4 Results

[0390] The results of ELISA detection of 45 single-point mutants treated with 0.3 M NaOH for 24 h are shown in Figure 1.

[0391] Two types of single-point mutants were screened using the following criteria: (1) affinity greater than or equal to WT in the untreated state and greater than or equal to WT after alkali treatment; (2) affinity weaker than WT in the untreated state but greater than or equal to WT after alkali treatment. Based on this screening criteria, 14 excellent single-point mutants were screened from 45 mutants, namely M8, M11, M20, M22, M24, M25, M27, M28, M29, M31, M33, M34, M45, and M50.

[0392] The 14 single-point mutants were further subjected to ELISA test using 0.5 M sodium hydroxide as the base, and the results are shown in FIG2 .

[0393] The results showed that there were 10 single-point mutants with stronger alkali resistance at 0.5M, namely M11, M20, M22, M24, M27, M28, M33, M34, M45 and M50. 50 The value can be increased by about 50% compared with WT. Based on the above results, it was preliminarily confirmed that 10 mutants among the 45 proteins expressed in the early stage can maintain good stability after treatment with 0.5M NaOH.

[0394] The results of SDS-PAGE analysis and calculations for the above 10 mutants are shown in Figure 3. Based on the calculation results, the mutant proteins can maintain good stability. The proportion of their small molecule fragmentation bands in the total protein is reduced by 30-50% compared with the WT.

[0395] Example 3: One round of heat resistance study of designed proteins (single point mutants):

[0396] Previous literature reports indicate that protein stability is uniform; improving stability in one specific environment can lead to varying degrees of improvement in stability in other environments. Therefore, we tested the Tm values ​​of several alkali-resistant proteins to demonstrate their improved stability.

[0397] 3.1 Detection of Antibody Heat Resistance

[0398] Sample preparation: Take an appropriate amount of sample, dilute the sample with buffer to the same concentration, mix well and set aside.

[0399] Tm DSF assay experimental steps: The above samples were tested using a DSF differential scanning calorimeter. The test protein was diluted to a concentration of 0.1 mg / mL using PB buffer. 100× Spyro Orange dye was diluted to 20× using PB buffer. The protein and dye were mixed at a ratio of 19:1 (total volume 20 μL), with three replicates per sample, and PB buffer was used as a blank control. The samples were vortexed and then detected using a qPCR instrument (Jena qTower3, filter set: 470 nm excitation, 625 nm emission). The temperature range was 25 to 95°C, the heating rate was 0.5°C / min, and the equilibrium time was 5 seconds at each temperature. Finally, the Tm value was approximately calculated by fitting the Boltzmann equation.

[0400] 3.2 Results

[0401] The results of the heat resistance test are shown in Figure 4. The Tm values ​​of the above 10 single-point mutants (M11, M20, M22, M24, M27, M28, M33, M34, M45, and M50) are all improved to varying degrees compared to the WT type. This means that the heat resistance of these mutants has also been improved to varying degrees.

[0402] Example 4: Alkali resistance study of the second round of designed proteins (multi-point mutants):

[0403] For multi-point mutants, the main basis for alkali resistance screening was the reduction of degradation bands after alkali treatment while maintaining strong affinity. The ELISA affinity detection method, alkali resistance detection method and SDS-PAGE detection method were the same.

[0404] Example 2.

[0405] With protein fragmentation after alkali treatment as the primary screening target, 40 multi-point mutants were subjected to alkali treatment (0.5 M NaOH for 24 h), and the SDS-PAGE results are summarized in Figure 5. For 21 mutants (M78, M74, M69, M53, M67, M88, M68, M82, M89, M66, M76, M64, M83, M81, M77, M71, M79, M70, M72, M75, and M59) with less than 50% degradation bands, affinity and alkali resistance for 24 h were compared by ELISA, and the results are shown in Figure 6.

[0406] By comparing the absolute values ​​of the ELISA test values ​​of the above-mentioned multi-point mutants after alkali treatment, it can be determined that 21 multi-point mutants still have binding ability, among which 8 multi-point mutants (M67, M68, M78, M64, M66, M69, M72 and M59) still maintain strong affinity after alkali treatment.

[0407] Example 5: Investigation of the heat resistance of the second round of designed proteins (multi-point mutants):

[0408] The heat resistance of 21 multi-point mutants was investigated. The heat resistance detection method is described in Example 3. As shown in Figure 7, the Tm values ​​of the above 21 multi-point mutants were improved to varying degrees compared with the WT, that is, the heat resistance of the above mutants was also improved to varying degrees, which indirectly indicates that the stability of the multi-point mutants was further improved.

[0409] Example 6: Preparation of preferred molecular samples and affinity media coupled thereto

[0410] After screening the molecular alkali resistance and heat resistance results of the preferred proteins in the first and second rounds of sequence design, we selected six preferred molecules, including the first round preferred molecules: M20 molecule, M45 molecule, and the second round preferred molecules: M67 molecule, M68 molecule, M74 molecule and M78 molecule, for further verification.

[0411] After protein sequence design and site mutation, the sequence is synthesized and constructed, used to transform host cells to express the target protein. After fermentation, nickel affinity chromatography, and ultrafiltration centrifugation, the protein is treated with urea and redox agents to renature its disulfide bonds, followed by fine purification. The purified protein is used as the target protein for analysis and modification. The protein is tested for purity, and samples with a purity >90% undergo verification experiments.

[0412] The protein Tm was determined by differential scanning calorimetry (DSC). The protein was incubated with 0.1M NaOH and 0.5M NaOH, then pH-neutralized and centrifuged for purity analysis using SDS-PAGE. The protein was then provided to the company that commissioned the development of the affinity matrix for the optimized molecule to be prepared. The matrix was then incubated with 0.1M NaOH and 0.5M NaOH for varying periods of time, and the dynamic binding capacity at 10% flow-through was evaluated before and after alkaline treatment. Simultaneously, the unmodified protein was treated and the matrix prepared, and the results were compared with those of the optimized molecule. An increase in the Tm of the optimized molecule indicates improved thermal stability of the protein. A decrease in degradation bands or smearing on the electrophoresis results after alkaline treatment indicates enhanced alkaline resistance. The reduced percentage of 10% dynamic binding capacity reduction in the optimized molecule coupling matrix after alkaline treatment indicates improved alkaline resistance, effectively enhancing the alkaline resistance of the affinity matrix.

[0413] Example 7: Investigation of Alkali Resistance of Preferred Molecules

[0414] The proteins before and after modification were incubated with 0.1M NaOH and 0.5M NaOH. The 0.1M NaOH system was incubated for 0 and 24 hours; the 0.5M NaOH system was incubated for 0, 6, 16, and 24 hours. After incubation, the pH was neutralized with acetic acid. Centrifuge at 4000 RPM for 20 minutes at 4°C, collect the supernatant, determine the concentration, and perform electrophoresis to determine purity. The alkaline-treated proteins were analyzed by SDS-PAGE to provide the most intuitive visualization of the protein's alkaline resistance.

[0415] Samples were diluted with ultrapure water and 2× non-reducing sample buffer was added at a 1:1 ratio. The samples were heated in a metal bath at 60°C for 5 min. Electrophoresis was performed using a 12% precast gel at a constant voltage of 100 V until bromophenol blue migrated to the lowest point of the gel (the edge of the film). Staining and destaining were performed. A constant total protein load of 5 μg was used for detection of the control and proteins treated with different alkali treatment times. The films were scanned and analyzed using the appropriate scanning instrument, and the area normalization method was used for calculation.

[0416] The protein electrophoresis results and corresponding purity changes are shown in Figure 8. Based on these results, after 24 hours of 0.1M NaOH treatment, proteins such as M45, M74, and M78 showed less degradation of the target band, indicating increased alkaline resistance compared to the unmodified proteins. After 0.5M NaOH treatment, the target protein bands of selected mutant proteins, such as M20, M74, and M78, increased in proportion compared to the unmodified proteins. Alkali treatment reduced the degree of diffusion, indicating increased alkaline stability compared to the unmodified proteins. Data from SDS-PAGE purity analyzer analysis have limitations and are for reference only. The electropherogram can intuitively reflect molecules with better alkaline resistance.

[0417] Example 8: Investigation of heat resistance of preferred molecules

[0418] The heat resistance was detected by DSC differential scanning calorimetry.

[0419] Preparation of test sample: Take an appropriate amount of test sample, dilute the sample with buffer to the same concentration, mix well and set aside; parameter settings: starting temperature; ending temperature; heating rate; equilibrium time; number of pumping times; sample volume.

[0420] The results are shown in Table 2. The Tm values ​​of the above six preferred molecules are comparable to or improved to varying degrees compared to WT.

[0421] Table 2 DSC verification results of preferred molecules

[0422] Example 9: Investigation of Alkali Resistance of Preferred Molecular Coupling Medium

[0423] The prepared preferred molecule was provided to the media manufacturer, who then prepared affinity media for the preferred molecule using the same coupling method and consistent protein coupling ratio. The prepared media was incubated with 0.1M NaOH for 24 and 32 hours, and 0.5M alkali for 6 and 24 hours. The dynamic binding capacity (DBC) at 10% flow-through was measured using GH stock solution before and after alkali incubation.

[0424] Experimental sample: GH stock solution, diluted to 5 mg / ml. Chromatography column model: Boglund 10 / 20, chromatography medium volume: 7.85 ml. Chromatographic experiment retention time: 5 min. The loading capacity determination process is as follows:

[0425] Table 3 Alkali resistance of coupling medium and loading determination process

[0426] Table 4 Dynamic binding capacity under 10% flow-through conditions after incubation with 0.1 M NaOH for 24 h and 32 h

[0427] Table 5 Dynamic binding capacity under 0.5 M NaOH incubation conditions of 6 h and 24 h with 10% flow-through

[0428] The results, as shown in Figure 9 and Tables 4-5, show that the six preferred molecular coupling media exhibited varying degrees of increased binding capacity compared to the WT after alkaline incubation. After 32 hours of treatment with 0.1M NaOH, DBC decay decreased from 35% to 0-14%, with the top three molecules, M20, M74, and M78, decreasing to no more than 6%. After 24 hours of treatment with 0.5M NaOH, the residual DBC decreased from 85% to 44-77%, with the top three molecules decreasing by no more than 49%. This experiment primarily considered the DBC decline trend; since the media coupling process was not optimized, the absolute DBC value at time 0 is not meaningful.

[0429] After verification of the alkali resistance and stability of the selected 6 molecules and molecular coupling media, the alkali resistance and heat resistance of the modified protein were improved compared with the WT molecule.

[0430] Summarize:

[0431] Sequence optimization was performed based on the second round of molecules and the first round of preferred molecules. The following 35 preferred molecules were sorted out, and the sequence information of the 35 preferred mutants and the control (WT) are sorted out in Table 6.

[0432] Table 6 Sequence information of WT molecules and preferred molecules

[0433] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A single domain antibody or an antigen binding fragment thereof that can specifically bind to growth hormone (GH), wherein the single domain antibody or the antigen binding fragment thereof comprises a complementarity determining region (CDR) and a framework region (FR), and has one or more of the following characteristics: (i) the amino acid residue at position 102 is Y; (ii) the amino acid residue at position 105 is Q; (iii) the amino acid residue at position 48 is V; (iv) the amino acid residue at position 11 is K; (v) the amino acid residue at position 100g is F; (vi) the amino acid residue at position 101 is D; (vii) the amino acid residue at position 28 is T; (viii) the amino acid residue at position 19 is T; (ix) the amino acid residue at position 56 is K; (x) the amino acid residue at position 100e is E; (xi) the amino acid residue at position 55 is D; (xii) the amino acid residue at position 76 is S; (xiii) the amino acid residue at position 14 is P; in, The positions are defined by the Kabat numbering system.

2. The single domain antibody or antigen binding fragment thereof according to claim 1, which has one or more of the following characteristics: (a) the amino acid residue at position 102 is Y; (b) the amino acid residue at position 105 is Q; (c) the amino acid residue at position 48 is V; (d) the amino acid residue at position 100g is F; (e) the amino acid residue at position 28 is T; (f) the amino acid residue at position 19 is T; (g) the amino acid residue at position 100e is E; (h) the amino acid residue at position 55 is D; (i) the amino acid residue at position 14 is P; in, The positions are defined by the Kabat numbering system; Preferably, the single domain antibody or antigen binding fragment thereof has a feature selected from the following: (1) the amino acid residue at position 55 is D, and the amino acid residue at position 100e is E; (2) the amino acid residue at position 28 is T, and the amino acid residue at position 55 is D; (3) the amino acid residue at position 14 is P, and the amino acid residue at position 28 is T; (4) the amino acid residue at position 14 is P, and the amino acid residue at position 105 is Q; (5) the amino acid residue at position 14 is P, and the amino acid residue at position 19 is T; (6) the amino acid residue at position 14 is P, and the amino acid residue at position 100e is E; (7) the amino acid residue at position 55 is D, the amino acid residue at position 100e is E, and the amino acid residue at position 105 is Q; (8) the amino acid residue at position 55 is D, the amino acid residue at position 100g is F, and the amino acid residue at position 102 is Y; (9) the amino acid residue at position 55 is D, the amino acid residue at position 100e is E, and the amino acid residue at position 102 is Y; (10) the amino acid residue at position 55 is D, and the amino acid residue at position 100g is F; (11) the amino acid residue at position 55 is D, the amino acid residue at position 100e is E, and the amino acid residue at position 100g is F; (12) the amino acid residue at position 55 is D, and the amino acid residue at position 102 is Y; (13) the amino acid residue at position 48 is V, the amino acid residue at position 55 is D, and the amino acid residue at position 100e is E; (14) the amino acid residue at position 55 is D, the amino acid residue at position 100e is E, and the amino acid residue at position 105 is Q; (15) the amino acid residue at position 55 is D, the amino acid residue at position 100e is E, the amino acid residue at position 100g is F, and the amino acid residue at position 105 is Q; Wherein, the positions are defined by the Kabat numbering system.

3. The single domain antibody or antigen binding fragment thereof according to claim 1 or 2, wherein: The single-domain antibody or antigen-binding fragment thereof has any of the following CDR1, CDR2, and CDR3: (1) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:43; and CDR3 as shown in SEQ ID NO:47; (2) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:43; and CDR3 as shown in SEQ ID NO:48; (3) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:43; and CDR3 as shown in SEQ ID NO:49; (4) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:44; and CDR3 as shown in SEQ ID NO:46; (5) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:43; and CDR3 as shown in SEQ ID NO:50; (6) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:46; (7) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:50; (8) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:51; (9) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:52; (10) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:48; (11) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:53; or, (12) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:

47.

4. The single domain antibody or antigen binding fragment thereof according to claim 1, wherein: The single-domain antibody has one or more substitution mutations selected from the following compared to the sequence shown in SEQ ID NO: 37: D102Y, P105Q, L48V, L11K, W100gF, V101D, P28T, R19T, T56K, R100eE, A55D, N76S, A14P; Wherein, the position of the mutation is defined by the Kabat numbering system; Preferably, the single domain antibody has one or more substitution mutations selected from the following compared to the sequence shown in SEQ ID NO: 37: D102Y, P105Q, L48V, W100gF, P28T, R19T, R100eE, A55D, A14P; Wherein, the position of the mutation is defined by the Kabat numbering system; Preferably, the single domain antibody is compared with the sequence shown in SEQ ID NO: 37: (1) A at position 55 is substituted with D, and R at position 100e is substituted with E (A55D and R100eE); (2) P at position 28 is substituted with T, and A at position 55 is substituted with D (P28T and A55D); (3) A at position 14 is substituted with P, and P at position 28 is substituted with T (A14P and P28T); (4) A at position 14 is substituted with P, and P at position 105 is substituted with Q (A14P and P105Q); (5) A at position 14 is substituted with P, and R at position 19 is substituted with T (A14P and R19T); (6) A at position 14 is substituted with P, and R at position 100e is substituted with E (A14P and R100eE); (7) A at position 55 is substituted with D, R at position 100e is substituted with E, and P at position 105 is substituted with Q (A55D, R100eE, and P105Q); (8) A at position 55 is substituted with D, W at position 100g is substituted with F, and D at position 102 is substituted with Y (A55D, W100gF, and D102Y); (9) A at position 55 is substituted with D, R at position 100e is substituted with E, and D at position 102 is substituted with Y (A55D, R100eE, and D102Y); (10) A at position 55 is substituted with D, and W at position 100g is substituted with F (A55D and W100gF); (11) A at position 55 is substituted with D, R at position 100e is substituted with E, and W at position 100g is substituted with F (A55D, R100eE, and W100gF); (12) A at position 55 is substituted with D, and D at position 102 is substituted with Y (A55D and D102Y); (13) L at position 48 is substituted with V, A at position 55 is substituted with D, and R at position 100e is substituted with E (L48V, A55D, and R100eE); (14) A at position 55 is substituted with D, R at position 100e is substituted with E, and P at position 105 is substituted with Q (A55D, R100eE, and P105Q); or, (15) A at position 55 is substituted with D, R at position 100e is substituted with E, W at position 100g is substituted with F, and P at position 105 is substituted with Q (A55D, R100eE, W100gF, and P105Q); Wherein, the positions are defined by the Kabat numbering system.

5. The single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 4, comprising an amino acid sequence selected from the group consisting of: (1) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 2; (2) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 3; (3) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 4; (4) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO:5; (5) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 6; (6) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO:7; (7) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 8; (8) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO:9; (9) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 10; (10) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 11; (11) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 12; (12) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 13; (13) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 15; (14) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 16; (15) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 17; (16) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 18; (17) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 19; (18) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 20; (19) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 21; (20) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 22; (21) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 23; (22) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 24; (23) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 25; (24) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 26; (25) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 27; (26) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 28; (27) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 29; (28) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO:30; (29) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO:31; (30) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 32; (31) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 33; (32) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 34; (33) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO:35; (34) The amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO:

36.

6. A single domain antibody or antigen binding fragment thereof capable of specifically binding to growth hormone (GH), the single domain antibody or antigen binding fragment thereof comprising: (1) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:43; and CDR3 as shown in SEQ ID NO:47; (2) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:43; and CDR3 as shown in SEQ ID NO:48; (3) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:43; and CDR3 as shown in SEQ ID NO:49; (4) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:44; and CDR3 as shown in SEQ ID NO:46; (5) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:43; and CDR3 as shown in SEQ ID NO:50; (6) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:46; (7) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:50; (8) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:51; (9) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:52; (10) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:48; (11) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:53; or, (12) CDR1 as shown in SEQ ID NO:42; CDR2 as shown in SEQ ID NO:45; and CDR3 as shown in SEQ ID NO:47; Preferably, the CDRs are defined by the Kabat numbering system; Preferably, the single domain antibody or antigen binding fragment thereof comprises an amino acid sequence selected from the group consisting of: (i) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 2; (ii) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 6; (iii) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO:7; (iv) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 10; (v) the amino acid sequence as shown in positions 2 to 125 or positions 1 to 125 of SEQ ID NO: 11; (vi) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 12; (vii) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 16; (viii) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 17; (ix) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 23; (x) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO: 24; (xi) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO:25; (xii) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO:26; (xiii) the amino acid sequence as shown in positions 2 to 125 or positions 1 to 125 of SEQ ID NO: 27; (xiv) the amino acid sequence as shown in positions 2 to 125 or 1 to 125 of SEQ ID NO: 28; (xv) the amino acid sequence as shown in positions 2 to 125 or positions 1 to 125 of SEQ ID NO: 29; (xvi) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO:30; (xvii) the amino acid sequence as shown in positions 2 to 125 or 1 to 125 of SEQ ID NO: 31; (xviii) the amino acid sequence as shown in positions 2 to 125 or positions 1 to 125 of SEQ ID NO: 32; (xix) the amino acid sequence as shown in positions 2 to 125 or positions 1 to 125 of SEQ ID NO: 33; (xx) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO:34; (xxi) the amino acid sequence as shown in positions 2-125 or 1-125 of SEQ ID NO:35; (xxii) the amino acid sequence shown at positions 2 to 125 or 1 to 125 of SEQ ID NO:

36.

7. A polypeptide construct capable of specifically binding to growth hormone (GH), comprising the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6, and another polypeptide; Preferably, the polypeptide construct is a fusion protein; Preferably, the additional polypeptide is linked to the N-terminus or C-terminus of the single domain antibody or antigen-binding fragment thereof, optionally via a linker; Preferably, the additional polypeptide is selected from a protein tag (e.g., a His tag, such as HHHHHH or HHHHHHHH), a protease recognition sequence (e.g., an EK protease recognition sequence, such as DDDDK), a peptide linker (e.g., a flexible peptide linker; such as a peptide linker comprising one or more glycine (G) and / or serine (S)), or any combination thereof; Preferably, the polypeptide construct comprises, from N-terminus to C-terminus, in sequence: the single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, an EK protease recognition sequence (such as DDDDK), a flexible peptide linker (for example, a peptide linker as shown in SEQ ID NO: 40), and an optional His tag (such as HHHHHH or HHHHHHHH); Preferably, the polypeptide construct comprises an amino acid sequence selected from the group consisting of: (1) the amino acid sequence as shown in SEQ ID NO: 2, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 2; (2) the amino acid sequence as shown in SEQ ID NO: 3, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 3; (3) the amino acid sequence as shown in SEQ ID NO: 4, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 4; (4) the amino acid sequence as shown in SEQ ID NO: 5, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 5; (5) the amino acid sequence as shown in SEQ ID NO: 6, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 6; (6) the amino acid sequence as shown in SEQ ID NO:7, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO:7; (7) the amino acid sequence as shown in SEQ ID NO: 8, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 8; (8) the amino acid sequence as shown in SEQ ID NO:9, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO:9; (9) the amino acid sequence as shown in SEQ ID NO: 10, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 10; (10) the amino acid sequence as shown in SEQ ID NO: 11, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 11; (11) the amino acid sequence as shown in SEQ ID NO: 12, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 12; (12) the amino acid sequence as shown in SEQ ID NO: 13, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 13; (13) the amino acid sequence as shown in SEQ ID NO: 15, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 15; (14) the amino acid sequence as shown in SEQ ID NO: 16, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 16; (15) the amino acid sequence as shown in SEQ ID NO: 17, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 17; (16) the amino acid sequence as shown in SEQ ID NO: 19, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 19; (17) the amino acid sequence as shown in SEQ ID NO: 20, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 20; (18) the amino acid sequence as shown in SEQ ID NO:21, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO:21; (19) the amino acid sequence as shown in SEQ ID NO: 23, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 23; (20) the amino acid sequence as shown in SEQ ID NO: 24, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 24; (21) the amino acid sequence as shown in SEQ ID NO: 25, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 25; (22) the amino acid sequence as shown in SEQ ID NO: 27, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO: 27; (23) the amino acid sequence as set forth in SEQ ID NO:29, or the amino acid sequence as set forth in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO:29; (24) the amino acid sequence as shown in SEQ ID NO:31, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO:31; (25) The amino acid sequence as shown in SEQ ID NO:34, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 136, or positions 2 to 142 of SEQ ID NO:

34.

8. A polypeptide construct capable of specifically binding to growth hormone (GH), comprising a single domain antibody or an antigen-binding fragment thereof capable of specifically binding to GH, and a polypeptide as shown in SEQ ID NO:

41.

9. The polypeptide construct of claim 8, wherein The single domain antibody or antigen binding fragment thereof capable of specifically binding to GH is selected from: (i) a single domain antibody or antigen binding fragment thereof as described in any one of claims 1 to 6, and (ii) a single domain antibody or antigen binding fragment thereof as shown in SEQ ID NO:

37.

10. The polypeptide construct of claim 8 or 9, wherein The polypeptide construct is a fusion protein; Preferably, the polypeptide construct comprises a polypeptide as shown in SEQ ID NO: 41 at the C-terminus of the single-domain antibody or antigen-binding fragment thereof capable of specifically binding to GH; Preferably, the polypeptide construct further comprises an EK protease recognition sequence (e.g., DDDDK) and / or a His tag (e.g., HHHHHH or HHHHHHHH); Preferably, the polypeptide construct comprises, from N-terminus to C-terminus, in order: the single-domain antibody or antigen-binding fragment thereof capable of specifically binding to GH, the EK protease recognition sequence, such as the polypeptide shown in SEQ ID NO: 41, and optionally the His tag; Preferably, the polypeptide construct comprises an amino acid sequence selected from the group consisting of: (1) the amino acid sequence as shown in SEQ ID NO: 14, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 14; (2) the amino acid sequence as shown in SEQ ID NO: 18, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 18; (3) the amino acid sequence as shown in SEQ ID NO: 22, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 22; (4) the amino acid sequence as shown in SEQ ID NO: 26, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 26; (5) the amino acid sequence as shown in SEQ ID NO: 28, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO: 28; (6) the amino acid sequence as shown in SEQ ID NO:30, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO:30; (7) the amino acid sequence as shown in SEQ ID NO:32, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO:32; (8) the amino acid sequence as shown in SEQ ID NO:33, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO:33; (9) the amino acid sequence as shown in SEQ ID NO:35, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO:35; (10) The amino acid sequence as shown in SEQ ID NO:36, or the amino acid sequence as shown in positions 1 to 136, or positions 2 to 142, or positions 2 to 136 of SEQ ID NO:

36.

11. An isolated nucleic acid molecule encoding the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6, or the polypeptide construct according to any one of claims 7 to 10.

12. A vector comprising the isolated nucleic acid molecule of claim 11; preferably, the vector is a cloning vector or an expression vector.

13. A host cell comprising the isolated nucleic acid molecule of claim 11 or the vector of claim 12.

14. A method for preparing a single domain antibody or an antigen binding fragment thereof according to any one of claims 1 to 6 or a polypeptide construct according to any one of claims 7 to 10, comprising culturing a host cell according to claim 13 under conditions allowing protein expression, and recovering the single domain antibody or an antigen binding fragment thereof or the polypeptide construct from the cultured host cell culture.

15. A conjugate comprising the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6 or the polypeptide construct according to any one of claims 7 to 10, and a solid support connected to the single domain antibody or antigen binding fragment thereof or the polypeptide construct; Preferably, the solid support is selected from the group consisting of: magnetic beads, agarose microspheres, polymer matrices (e.g., dextran microspheres, polymethacrylate, polystyrene, polystyrene-divinylbenzene, polymethyl methacrylate), silica matrices (e.g., silica microspheres), graphitized carbon matrices, alumina matrices, zirconia matrices, and any combination thereof; Preferably, the solid support is selected from porous materials, for example, selected from one porous material or a combination of multiple porous materials.

16. A method for purifying GH, comprising using the conjugate of claim 15; Preferably, the method is a method comprising purifying GH by affinity chromatography; Preferably, the method comprises the following steps: (1) combining the conjugate with the GH to form a complex in a first solvent; (2) dissociating the complex in a second solvent; and, (3) collecting the dissociation product containing the GH; The first solvent is a solvent suitable for forming a complex between the conjugate and the GH, and the second solvent is a solvent capable of dissociating the complex.

17. Use of the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6, or the polypeptide construct according to any one of claims 7 to 10, or the conjugate according to claim 15 in the preparation of a GH purification reagent.

18. A conjugate comprising the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6 or the polypeptide construct according to any one of claims 7 to 10, and a detectable label linked to the single domain antibody or antigen binding fragment thereof or the polypeptide construct; Preferably, the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

19. A kit comprising the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6, or the polypeptide construct according to any one of claims 7 to 10, or the conjugate according to claim 18; Preferably, the kit comprises the conjugate of claim 18; Preferably, the kit comprises a single domain antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6 or a polypeptide construct according to any one of claims 7 to 10, and a second antibody that specifically recognizes the single domain antibody or an antigen-binding fragment thereof or the polypeptide construct; optionally, the second antibody further comprises a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

20. A method for detecting the presence or level of GH in a sample, comprising using the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6 or the polypeptide construct according to any one of claims 7 to 10, or the conjugate according to claim 18; Preferably, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (eg ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay; Preferably, the method comprises using the conjugate of claim 18; Preferably, the method comprises using the single domain antibody or antigen binding fragment thereof of any one of claims 1 to 6 or the polypeptide construct of any one of claims 7 to 10, and the method further comprises using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin) to detect the single domain antibody or antigen binding fragment thereof or the polypeptide construct.

21. Use of the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6, or the polypeptide construct according to any one of claims 7 to 10, or the conjugate according to claim 18 in the preparation of a detection reagent for detecting the presence or level of GH in a sample and / or diagnosing a disease associated with abnormal GH levels; Preferably, the detection reagent detects the presence or level of GH in a sample by the method of claim 20; Preferably, the sample is a body fluid sample from a subject (eg, a mammal, preferably a human).

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