Anti-human growth hormone single domain antibody and application thereof

Abstract

The present invention relates to a single-domain antibody specifically binding to human growth hormone or an antigen binding fragment thereof, a 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 comprising the same, and related uses thereof. In addition, this invention relates to the detection of said conjugates or the purification use of human growth hormones.

Description

Anti-human growth hormone single-domain antibody and its application Technical Field

[0001] The present invention relates to single-domain antibodies or antigen-binding fragments thereof that specifically bind to human growth hormone, conjugates containing such single-domain antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding such single-domain antibodies or antigen-binding fragments thereof, host cells containing such nucleic acids, and related uses. Furthermore, this invention relates to the use of such conjugates for detecting or purifying human growth hormone. Background Art

[0002] Human growth hormone (hGH) is a protein secreted by pituitary cells and a peptide hormone. It is a single-chain protein hormone secreted by eosinophils in the anterior pituitary gland and 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 available for large-scale production in pharmaceutical factories in the 1980s, its clinical indications have continued to expand. It is widely used not only in promoting 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 immunity.

[0003] 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 naturally lacks 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 are also resistant to acid and alkali, high temperatures, and are easy to produce. They can be advantageously used in the immunoaffinity chromatography purification of human GH to replace the traditional cumbersome human GH purification method.

[0004] Summary of the Invention

[0005] After extensive research, the inventors of this application have screened and obtained a series of single-domain antibodies against human growth hormone (HGH) that exhibit high binding activity. Specifically, the single-domain antibodies of the present invention possess excellent resistance to high temperatures, acids, and alkalis, and exhibit excellent stability. They effectively bind to hGH under neutral conditions and dissociate from hGH under low pH conditions, making them advantageously applicable to affinity purification of HGH. Furthermore, these single-domain antibodies possess a small molecular weight and are easy to produce.

[0006] Based on this, the present application also provides a 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, a host cell containing the same, and related uses.

[0007] Single domain antibodies and antigen-binding fragments thereof

[0008] Therefore, in one aspect, the present application provides a single domain antibody or an antigen-binding fragment thereof that can specifically bind to human growth hormone (hGH), wherein the single domain antibody or the antigen-binding fragment thereof comprises:

[0009] (a) CDR1 having the structure shown as X1X2AX3G (Formula I, SEQ ID NO: 23);

[0010] (b) CDR2, which has X4IX5X6X7GX8X9TX 10 The structure shown in YADSVKG (Formula II, SEQ ID NO: 24);

[0011] (c) CDR3 having a CDR3 sequence such as AFSVTIPTRARHWVD (SEQ ID NO: 19) or ATX 11 YYSDYDVPX 12 X 13 SX 14 EX 15 DY (Formula III, SEQ ID NO: 25) or SRGDX 16 The structure shown in GILHGVVHY (Formula IV, SEQ ID NO: 26);

[0012] in,

[0013] X1 is selected from the group consisting of (i) amino acid residues A, S, N and (ii) amino acid residues that are conservative substitutions relative to (i);

[0014] X2 is selected from the group consisting of (i) amino acid residues R, F, Y and (ii) amino acid residues that are conservative substitutions relative to (i);

[0015] X3 is selected from (i) amino acid residues M, V and (ii) amino acid residues that are conservative substitutions relative to (i);

[0016] X4 is selected from (i) amino acid residues A, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0017] X5 is selected from the group consisting of (i) amino acid residues E, S, G and (ii) amino acid residues that are conservative substitutions relative to (i);

[0018] X6 is selected from (i) amino acid residues G, W and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0019] X7 is selected from (i) amino acid residues I, M, L and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0020] X8 is selected from the group consisting of (i) amino acid residues A, G and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0021] X9 is selected from the group consisting of (i) amino acid residues T, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0022] X 10 selected from the group consisting of (i) amino acid residues Y, V, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0023] X 11 selected from (i) amino acid residues S, N and (ii) amino acid residues that are conservative substitutions relative to (i);

[0024] X 12 selected from (i) amino acid residues V, A and (ii) amino acid residues that are conservative substitutions relative to (i);

[0025] X 13 selected from (i) amino acid residues R, T and (ii) amino acid residues that are conservative substitutions relative to (i);

[0026] X 14 selected from (i) amino acid residues N, D and (ii) amino acid residues that are conservative substitutions relative to (i);

[0027] X 15 selected from the group consisting of (i) amino acid residues Y, F and (ii) amino acid residues that are conservative substitutions relative to (i);

[0028] X 16 Selected from (i) amino acid residues F, Y and (ii) amino acid residues that are conservative substitutions relative to (i).

[0029] In certain embodiments, X1 is selected from amino acid residues A, S, N; X2 is selected from amino acid residues R, F, Y; X3 is selected from amino acid residues M, V; X4 is selected from amino acid residues A, S; X5 is selected from amino acid residues E, S, G; X6 is selected from amino acid residues G, W; X7 is selected from amino acid residues I, M, L; X8 is selected from amino acid residues A, G; X9 is selected from amino acid residues T, S; X 10 Selected from amino acid residues Y, V, S; X 11 Selected from amino acid residues S, N; X 12Selected from amino acid residues V, A; X 13 Selected from amino acid residues R, T; X 14 Selected from amino acid residues N, D; X 15 Selected from amino acid residues Y, F; X 16 Selected from amino acid residues F, Y.

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

[0031] (a) CDR1 having: a sequence as shown in any one of SEQ ID NOs: 17, 1, 5, 9, 13, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 17, 1, 5, 9, 13;

[0032] (b) CDR2 having: a sequence as shown in any one of SEQ ID NOs: 18, 2, 6, 10, 14, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 18, 2, 6, 10, 14; and

[0033] (c) CDR3 having: a sequence as shown in any one of SEQ ID NOs: 19, 3, 7, 11, 15, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 19, 3, 7, 11, 15.

[0034] In certain embodiments, the substitutions are conservative substitutions.

[0035] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises:

[0036] (1) CDR1 as set forth in SEQ ID NO: 17; CDR2 as set forth in SEQ ID NO: 18; and CDR3 as set forth in SEQ ID NO: 19;

[0037] (2) CDR1 as shown in SEQ ID NO: 1; CDR2 as shown in SEQ ID NO: 2; and CDR3 as shown in SEQ ID NO: 3;

[0038] (3) CDR1 as set forth in SEQ ID NO: 5; CDR2 as set forth in SEQ ID NO: 6; and CDR3 as set forth in SEQ ID NO: 7;

[0039] (4) CDR1 as set forth in SEQ ID NO:9; CDR2 as set forth in SEQ ID NO:10; and CDR3 as set forth in SEQ ID NO:11;

[0040] or,

[0041] (5) CDR1 as shown in SEQ ID NO: 13; CDR2 as shown in SEQ ID NO: 14; and CDR3 as shown in SEQ ID NO: 15.

[0042] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of:

[0043] (i) a sequence as shown in any one of SEQ ID NOs: 20, 4, 8, 12, and 16;

[0044] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 20, 4, 8, 12, 16; or

[0045] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 20, 4, 8, 12, 16.

[0046] In certain embodiments, the substitutions are conservative substitutions.

[0047] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof can specifically bind to hGH in a first solvent to form a complex; and the complex formed by the single-domain antibody or antigen-binding fragment thereof and hGH can dissociate in a second solvent;

[0048] The pH value of the first solvent is within the range of 6.5-8.5 (eg, 7.2-7.6); the pH value of the second solvent is within the range of 2.5-3.5 (eg, 2.8-3.2).

[0049] In certain embodiments, the first solvent is a phosphate buffer, a Tris-HCl buffer, or a HEPES buffer with a pH of 6.5-8.5 (eg, 7.2-7.6).

[0050] In certain embodiments, the second solvent is a citrate buffer, a Gly-HCl buffer, or an acetate buffer having a pH of 2.5-3.5 (eg, 2.8-3.2).

[0051] Isolated nucleic acid molecules

[0052] In another aspect, the present application also provides an isolated nucleic acid molecule encoding the single domain antibody or antigen-binding fragment thereof as described above.

[0053] carrier

[0054] In another aspect, the present application also provides a vector comprising the nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

[0055] host cells

[0056] On the other hand, the application also provides a host cell comprising a nucleic acid molecule or a vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as Escherichia 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.

[0057] Preparation method

[0058] The single-domain antibodies, polypeptide constructs, or fusion proteins 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 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 of the present invention.

[0059] 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.

[0060] On the other hand, the present application also provides a method for preparing the single-domain antibody or its antigen-binding fragment as described above, which comprises culturing the host cell as described above under conditions allowing protein expression, and recovering the single-domain antibody or its antigen-binding fragment from the cultured host cell culture.

[0061] Bispecific or multispecific antibodies

[0062] In another aspect, the present application also provides a bispecific or multispecific antibody comprising the single domain antibody or antigen-binding fragment thereof as described above.

[0063] In certain embodiments, the bispecific or multispecific antibody specifically binds hGH and additionally specifically binds one or more other targets.

[0064] In certain embodiments, the bispecific or multispecific antibody further comprises at least one second antibody with a second binding specificity for a second target.

[0065] The present application also provides the use of the single domain antibody or antigen binding fragment thereof in the purification or detection of hGH. In a first aspect, the present application provides the use of the single domain antibody or antigen binding fragment thereof in the purification of hGH.

[0066] Conjugate

[0067] In one aspect, the present application also provides a conjugate comprising the single-domain antibody or antigen-binding fragment thereof as described above, and a solid support linked to the single-domain antibody or antigen-binding fragment thereof.

[0068] In certain embodiments, the solid support is selected from the group consisting of magnetic beads, agarose microspheres, dextran microspheres, polymethacrylate, polystyrene, silica microspheres, and combinations thereof.

[0069] In certain embodiments, the solid support is selected from a porous material. For example, the solid support is selected from one porous material or a combination of multiple porous materials.

[0070] In certain embodiments, the conjugate can specifically bind to hGH in a first solvent to form a complex; and the complex formed between the conjugate and hGH can dissociate in a second solvent.

[0071] In certain embodiments, the first solvent and / or the second solvent are as defined above.

[0072] Methods for purifying hGH

[0073] In another aspect, the present application also provides a method for purifying hGH, which comprises using the conjugate as described above.

[0074] In certain embodiments, the method is a method comprising purifying hGH by affinity chromatography, comprising the steps of:

[0075] (1) allowing the conjugate to combine with the hGH to form a complex in a first solvent;

[0076] (2) dissociating the complex in a second solvent; and,

[0077] (3) collecting the dissociation product containing the hGH;

[0078] Wherein, the first solvent and the second solvent are as defined above.

[0079] In another aspect, the present application also provides use of the single-domain antibody or antigen-binding fragment or conjugate thereof as described above in preparing an hGH purification reagent.

[0080] In a second aspect, the present application provides the use of the single-domain antibody or antigen-binding fragment thereof in hGH detection.

[0081] Conjugate

[0082] In one aspect, the present application also provides a conjugate comprising the single domain antibody or antigen-binding fragment thereof as described above, and a detectable label linked to the single domain antibody or antigen-binding fragment thereof.

[0083] 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.

[0084] In another aspect, the present application also provides a method for detecting the presence or level of hGH in a sample, which comprises using the single domain antibody or antigen-binding fragment or conjugate thereof as described above.

[0085] In certain embodiments, the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic, non-diagnostic purposes.

[0086] 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.

[0087] In certain embodiments, the methods comprise use of a conjugate as described above.

[0088] In certain embodiments, the method comprises using a single domain antibody or antigen-binding fragment thereof as described above, and the method further comprises detecting the single domain antibody or antigen-binding fragment thereof 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).

[0089] In certain embodiments, the method comprises: (1) contacting the sample with a single domain antibody or antigen-binding fragment thereof or conjugate of the present invention; and (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of hGH.

[0090] The present application also provides a method for diagnosing a disease associated with abnormal hGH levels, comprising detecting the level of hGH in a sample from a subject using the method described above. In certain embodiments, when the level of hGH in the sample from the subject is significantly increased or decreased compared to a reference level (e.g., compared to a healthy control), it indicates that the subject suffers from a disease associated with abnormally high or low hGH levels (e.g., gigantism / dwarfism).

[0091] In another aspect, the present application also provides use of the single domain antibody or antigen-binding fragment or conjugate thereof as described above in preparing a detection reagent for detecting the presence or level of hGH in a sample and / or diagnosing a disease associated with abnormal hGH levels.

[0092] In certain embodiments, the detection reagent detects the presence or level of hGH in a sample by the methods described above.

[0093] Reagent test kit

[0094] In another aspect, the present application also provides a kit comprising the single domain antibody or antigen-binding fragment thereof as described above, the conjugate as described in the first aspect, or the conjugate as described in the second aspect.

[0095] In certain embodiments, the kit comprises the conjugate of the first aspect.

[0096] In certain embodiments, the kit comprises the conjugate of the second aspect.

[0097] In certain embodiments, the kit comprises a single domain antibody or an antigen-binding fragment thereof as described above, and a second antibody that specifically recognizes the single domain antibody or the antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0098] In certain embodiments, the kit further comprises a buffer (eg, a detection buffer, a protein purification buffer).

[0099] In certain embodiments, the kit comprises the single domain antibody or antigen-binding fragment thereof as described above or the conjugate as described in the first aspect, and a buffer for protein purification.

[0100] In certain embodiments, the kit comprises the single domain antibody or antigen-binding fragment thereof as described above or the conjugate as described in the second aspect, and a detection buffer.

[0101] Definition of terms

[0102] 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.

[0103] 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."

[0104] 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.

[0105] 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 nanobody consists of four framework regions and three complementary determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A nanobody can 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.

[0106] As used herein, the term "antigen-binding fragment" of a single-domain antibody refers to a polypeptide comprising a fragment of a single-domain antibody that retains the ability to specifically bind to the same antigen to which the single-domain antibody binds, and / or competes with the single-domain antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of the antibodies of the invention can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the nanobodies of the invention. In some embodiments, the "antigen-binding fragment" of the single-domain antibody may be truncated at the N-terminus or C-terminus compared to the full-length single-domain antibody so that it comprises only part of FR1 and / or FR4, or lacks one or both of those framework regions, as long as it substantially retains antigen binding and specificity.

[0107] Antigen-binding fragments of single-domain antibodies can be obtained from a given single-domain antibody (such as the Nanobodies provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and antigen-binding fragments of single-domain antibodies can be screened for specificity in the same manner as for intact Nanobodies.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0112] The determination of percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403.

[0113] 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.

[0114] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D , kon and kdis values. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0115] 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, Alexa 750)), 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.

[0116] 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.

[0117] 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.

[0118] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0119] 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.

[0120] Advantageous Effects of the Invention

[0121] The present application provides a series of single-domain antibodies against human growth hormone (hGH) that exhibit high binding activity to hGH. Specifically, the single-domain antibodies of the present invention possess excellent resistance to high temperatures, acids, and alkalis, and exhibit excellent stability. They effectively bind to hGH under neutral conditions and effectively dissociate from hGH under low pH conditions, making them advantageously applicable to affinity purification of hGH. Furthermore, these single-domain antibodies have the advantages of a small molecular weight and ease of production.

[0122] 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

[0123] FIG1 shows the results of ELISA assays for the binding of five single-domain antibodies (anti-hGH Nb-1 / 9 / 18 / 20 / 27) to hGH.

[0124] Figure 2 shows the ELISA results of three single-domain antibodies (anti-hGH Nb-1 / 9 / 18) binding to hGH after treatment at different high temperatures (45°C, 55°C) for different times (24 hours, 48 ​​hours). Figure 2A shows the results of anti-hGH Nb-1 binding to hGH, and Figure 2B shows the results of anti-hGH Nb-9 / 18 binding to hGH.

[0125] FIG3 shows the results of ELISA assay for the binding of three single-domain antibodies (anti-hGH Nb-1 / 9 / 18) to hGH after treatment at pH 3.0 for different time periods (0 h, 24 h, 48 h).

[0126] Figure 4 shows the non-reducing SDS-PAGE results of the anti-hGH single domain antibodies prepared in Example 4; Figure 4A shows the SDS-PAGE results of anti-hGH Nb-1 / 9 / 18, where lane M is a molecular weight marker, lane 1 is anti-hGH Nb-18, lane 2 is anti-hGH Nb-9, and lane 3 is anti-hGH Nb-1; Figure 4B shows the SDS-PAGE results of anti-hGH Nb-20 / 27, where lane M is a molecular weight marker, lane 1 is anti-hGH Nb-20, and lane 2 is anti-hGH Nb-27.

[0127] Figure 5 shows a trend graph of the relationship between flow-through rate and binding capacity of affinity media coupled with different single-domain antibodies (anti-hGH Nb-1 / 9 / 18 / 20 / 27); Figure 5A shows a trend graph of the relationship between flow-through rate and binding capacity of the affinity media coupled with anti-hGH Nb-1 / 9 / 18, where C represents the flow-through sample concentration and C0 represents the hGH stock solution loading concentration on the abscissa; Figure 5B shows a trend graph of the relationship between flow-through rate and binding capacity of the affinity media coupled with anti-hGH Nb-20 / 27.

[0128] Sequence information

[0129] A description of the sequences involved in this application is provided in the table below.

[0130] Table 1: Sequence information DETAILED DESCRIPTION

[0131] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit it.

[0132] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the product manufacturers. It will be appreciated by those skilled in the art that the examples illustrate the present invention by way of example and are not intended to limit the scope of the present invention.

[0133] Example 1 Construction of an Anti-recombinant Human Growth Hormone (hGH) Single Domain Antibody Immune Library

[0134] Alpacas (Vicugna pacos) were immunized subcutaneously with 2 mg of hGH protein (amino acid sequence shown in SEQ ID NO: 27) emulsified with complete Freund's adjuvant at multiple sites. Twenty days after the first immunization, 1.5 mg of hGH protein was administered with incomplete Freund's adjuvant. Subsequently, immunizations were repeated every 14 days, and venous blood was collected for serum titer determination using ELISA. After the seventh immunization, peripheral blood was collected for lymphocyte isolation and total RNA extraction.

[0135] RNA extraction was performed according to Invitrogen TRIzol TMFirst-strand cDNA was synthesized according to the TakaRa Reagent instructions using total RNA as a template and oligo dT and Random 6 as primers. Nested PCR was performed using NEB's 2×Q5 High-Fidelity Polymerase to obtain the variable region encoding the heavy chain antibody (primers are shown in Table 2).

[0136] Table 2 Primer names and sequences Note: R=A / G, Y=C / T, S=C / G.

[0137] In the first round of PCR, cDNA was amplified using primers Alpaca-CALL01-F and Alpaca-CALL02-R, respectively. The reaction conditions were 98°C, 30s; 98°C, 5s, 58°C, 15s, 72°C, 20s, 27 cycles; and extension at 72°C for 2min.

[0138] The first-round PCR products were electrophoresed on a 1.5% (w / v) agarose gel, and a DNA fragment of approximately 750 bp was recovered as a template for the second-round PCR. The second-round primers (an equimolar mixture of Alpaca-IGHV3S53 / 61-F, Alpaca-IGHV3-3-F, Alpaca-IGHV3S59 / 67-F, Alpaca-IGHV3S64 / 64-F, and Alpaca-IGHV3S65-F as forward primers, and an equimolar mixture of Alpaca-IGHJ1-R, Alpaca-IGHJ2 / 3 / 7-R, Alpaca-IGHJ5-Rnew, and Alpaca-IGHJ4 / 6-R as reverse primers) were used for amplification. The reaction conditions were: 98°C for 30 s; 98°C for 5 s, 58°C for 15 s, and 72°C for 20 s, for 27 cycles; and extension at 72°C for 2 min. Recovered and quantified using an Omega gel recovery kit and stored at -20°C until use. Phagemid vector pGS249-3 and PCR product were digested with Bgl I. Phagemid vector pGS249-3 was digested with Bgl I and then with Nsi I to prevent vector self-ligation. After column recovery and quantification using a gel recovery kit, the product was ligated at a 1:3 molar ratio at 22°C for 1 hour to obtain a ligation product.

[0139] After the ligation product was recovered using a gel extraction kit, it was dissolved in 50 μl of ultrapure water. 500 ng of the purified ligation product was electroporated into E. coli TG1 competent cells for a total of 16 electroporations. Immediately after electroporation, 37°C preheated SOC medium was added, mixed, and the cells were shaken at 37°C for 1 hour. After recovery, 50 μl of the bacterial solution from each cell was mixed, serially diluted, and plated on 2×YT plates (containing 100 μg / ml ampicillin). The remaining bacterial solution was plated on 150×20 mm 2×YT plates (containing 100 μg / ml ampicillin) and incubated inverted at 37°C overnight. The next day, colony PCR was performed using primers M13R (SEQ ID NO: 21) and 249R (SEQ ID NO: 22) to calculate the library size and library positive rate. The bacterial lawn on the plate was scraped and washed with 2×YT medium (containing 100 μg / ml ampicillin), and glycerol was added to a final concentration of 15% (V / V). The culture was aliquoted and stored at -80°C for later use.

[0140] According to the calculated storage capacity, 100 times the storage capacity of the bacterial solution was inoculated into 10 L of 2×YT medium (containing 2% (w / v) glucose, 100 μg / ml ampicillin) and cultured at 37°C and 200 rpm until the OD 600 = 0.5, add helper phage M13KO7 at a multiplicity of infection of 100:1, incubate at 37°C for 15 minutes, then incubate at 37°C with shaking at 200 rpm for 1 hour. The culture was centrifuged, and the pellet was resuspended in 5 L of 2×YT (containing 100 μg / ml ampicillin and 70 μg / ml kanamycin) and incubated at 30°C with shaking at 200 rpm overnight. The supernatant was collected by centrifugation, and 1 / 4 volume of PEG / NaCl was added. Mix well, incubate on ice for 1 hour, centrifuge, and resuspend the pellet in PBS to obtain the anti-hGH single-domain antibody immune library. A 20 μl aliquot was used for titer determination, and the remainder was added to a final concentration of 50% (v / v) glycerol, aliquoted, and stored at -80°C.

[0141] Example 2: Panning and Identification of Anti-hGH Single Domain Antibodies

[0142] Single domain antibodies against hGH were selected from the anti-hGH single domain antibody immune library obtained in Example 1 using solid phase panning. 1 ml of hGH protein was added to an immunotube and coated overnight at 4°C. The hGH coating concentrations for each round of panning were 20 μg / ml, 20 μg / ml, 10 μg / ml, and 10 μg / ml, respectively. A blank immunotube (as a control) was added with 5% (w / v) milk and blocked at 37°C for 2 h. The tube was washed three times with PBST (containing 0.05% (v / v) Tween-20). Phage (5×10 phage) that had been depleted of 5% milk was added to both immunotubes. 13pfu), incubated at room temperature for 1 h, washed 10 times with PBST (15 times in the last two rounds) to remove unbound phages, eluted phages adsorbed on the immunotube with 0.02 M citric acid-sodium citrate (ACID, pH 3.0), neutralized the eluate with 160 μl of 1 M Tris-HCl (pH 8.0), and took 100 μl of the infection OD 600 = 0.5 of TG1, the titer was determined, and the remaining eluate was amplified and used for the next round of panning.

[0143] After four rounds of selection, single clones were picked, cultured overnight at 37°C, and then transferred to grow the bacterial solution to OD 600 =0.5, IPTG was added to a final concentration of 1 mM for overnight induction at 30°C. The supernatant was collected by centrifugation and used for ELISA. The cells were coated with 1 μg / ml hGH in 5% milk overnight at 4°C, blocked at 37°C for 2 h, washed three times with PBST, and 50 μl of the induction supernatant was added. The cells were washed three times with PBST, and the anti-His-HRP secondary antibody was added for color development.

[0144] The ELISA-positive clones were sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing. After sequence analysis, five single-domain antibodies with good diversity were selected for subsequent eukaryotic expression.

[0145] Example 3 Preparation and physicochemical screening of anti-hGH single domain antibodies

[0146] 3.1 Preparation of anti-hGH single domain antibody and ELISA binding assay

[0147] The five single-domain antibody gene fragments were cloned into the eukaryotic expression vector pGS003, and the expression vector was successfully constructed after sequencing.

[0148] Extract plasmids and use FreeStyle TM 293E cells were transiently transfected and expressed in Freestyle medium. 24 hours before transfection, 0.5×10 625 ml of 293E cells at a concentration of 1 cell / ml were cultured in a 37°C, 5% CO2 incubator with a shaker at 130 rpm. For transfection, 60 μl of 293E Fectin was added to 1 ml of Opti-MEM, mixed thoroughly, and incubated at room temperature for 5 minutes. Simultaneously, 25 μg of recombinant vector total plasmid DNA was dissolved in 1 ml of Opti-MEM. The plasmid DNA and 293E Fectin were thoroughly mixed to a total volume of 2 ml and incubated at room temperature for 15 minutes. The entire mixture was then added to the cell culture wells, mixed thoroughly, and cultured in a 37°C, 5% CO2 incubator with a shaker at 130 rpm for 7 days. The culture medium was centrifuged at high speed, and the supernatant was vacuum filtered through a microporous filter. Purification of the single-domain antibody was performed using a nickel column according to the manufacturer's protocol.

[0149] The plates were coated with 0.5 μg / ml hGH (amino acid sequence such as SEQ ID NO: 27) at 50 μl / well at 4°C overnight, blocked with 250 μl / well 1% (w / v) BSA, and washed three times with PBST. The purified anti-hGH single domain antibody prepared above was added at a starting concentration of 10 μg / ml and diluted three times, for a total of 8 spots, at 50 μl / well. The plates were incubated at room temperature for 1 h, washed three times with PBST, and developed with Anti-His-HRP secondary antibody.

[0150] The results are shown in FIG1 , indicating that the five single-domain antibodies (anti-hGH Nb-1 / 9 / 18 / 20 / 27) all have high binding activity to hGH.

[0151] A simulated affinity purification step was used to verify that the antibody molecules could dissociate normally under the acidic conditions of 0.02M citric acid-sodium citrate (ACID, pH 3.0). The steps were as follows: 0.5μg / ml hGH was coated overnight at 4°C, 250μl / well of 1% (w / v) BSA was added for blocking, and then washed three times with PBST. 10μg / ml anti-hGH single-domain antibody was added at 50μl / well and incubated at room temperature for 1 hour. Washing was performed using ACID, and washing with PBST (pH 7.2-7.4) was used as a control. Finally, Anti-His-HRP secondary antibody was added and color was developed. The results are shown in Table 3.

[0152] Table 3

[0153] The results showed that the single-domain antibodies anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18, anti-hGH Nb-20, and anti-hGH Nb-27 could bind well to hGH under neutral conditions, but could dissociate well from hGH under low pH acidic conditions.

[0154] 3.2 Octet-based determination of the affinity of hGH and anti-hGH single-domain antibodies

[0155] First, equilibration buffer PBS, 30 μg / ml anti-hGH single-domain antibody, and hGH were added to a microplate. A baseline adjustment was performed, and the HIS1K probe was used to bind to the 30 μg / ml anti-hGH single-domain antibody. Then, a baseline adjustment was performed again, and the probe bound to the anti-hGH single-domain antibody was used to bind to hGH. Data analysis software was used to determine the affinity (M) for hGH and the anti-hGH single-domain antibody. The results are shown in Table 4.

[0156] Table 4 Affinity of single domain antibodies to hGH

[0157] 3.3 Stability Assessment of Anti-hGH Single Domain Antibodies

[0158] Four tubes (100 μg / tube) of purified single-domain antibodies (anti-hGH Nb-1, anti-hGH Nb-9, and anti-hGH Nb-18) were placed in a 45°C or 55°C water bath for 24 and 48 hours, respectively. ELISA assays were then performed according to step 3.1 above to verify the stability of the anti-hGH single-domain antibodies at high temperatures. The results are shown in Figures 2A and 2B, demonstrating that all three single-domain antibodies maintained their hGH binding activity after these treatments, demonstrating good stability.

[0159] Three tubes (100 μg / tube) of purified single-domain antibodies (anti-hGH Nb-1, anti-hGH Nb-9, and anti-hGH Nb-18) were incubated at pH 3.0 for 24 and 48 hours, respectively. ELISA assays were then performed to verify the stability of the anti-hGH single-domain antibodies under acidic conditions. The results, shown in Figure 3, demonstrate that all three single-domain antibodies maintained their hGH binding activity after the aforementioned treatment, demonstrating good stability.

[0160] Example 4 Preparation and Purification of Anti-hGH Single Domain Antibodies

[0161] Single domain antibodies anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18, anti-hGH Nb-20, and anti-hGH Nb-27 were prepared and purified.

[0162] 4.1 Preparation and purification of anti-hGH Nb-1, anti-hGH Nb-9, and anti-hGH Nb-18

[0163] The target protein was captured by ProA affinity chromatography. The specific preparation process and parameters of anti-hGH Nb-9 are shown in Table 5-1.

[0164] Table 5-1 ProA affinity chromatography process and parameters

[0165] The target protein was captured by Ni affinity chromatography. The specific preparation process and parameters of anti-hGH Nb-18 and anti-hGH Nb-1 are shown in Table 5-2.

[0166] Table 5-2 Ni affinity chromatography process and parameters

[0167] The purity of the prepared hGH single domain antibody candidate molecules was evaluated by SDS-PAGE. The SDS-PAGE results are shown in FIG4A . The preparation and collection information of each purified single domain antibody sample is shown in Table 6 .

[0168] Table 6 Sample preparation and collection information

[0169] According to the electrophoresis analysis of the samples collected for candidate molecule preparation, the purity of the three candidate molecules was greater than 95%, and they could be used to continue the preparation experiment of the affinity coupling medium.

[0170] 4.2 Preparation and purification of anti-hGH Nb-20 ​​and anti-hGH Nb-27

[0171] The target protein was captured by Ni affinity chromatography. The specific preparation process and parameters of anti-hGH Nb-20 ​​and anti-hGH Nb-27 are shown in Table 7.

[0172] Table 7 Ni affinity chromatography process and parameters

[0173] The purity of the prepared hGH single domain antibody candidate molecules was evaluated by SDS-PAGE. The SDS-PAGE results are shown in FIG4B . The preparation and collection information of each purified single domain antibody sample is shown in Table 8 .

[0174] Table 8 Sample preparation and collection information

[0175] According to the electrophoresis analysis of the samples collected for candidate molecule preparation, the purity of the two candidate molecules was greater than 95%, and they could be used to continue the preparation experiment of the affinity coupling medium.

[0176] Example 5 Preparation of anti-hGH single domain antibody affinity coupling medium

[0177] Affinity coupling media were prepared for single-domain antibodies anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18, anti-hGH Nb-20, and anti-hGH Nb-27.

[0178] 5.1 Preparation of affinity coupling media for anti-hGH Nb-1, anti-hGH Nb-9, and anti-hGH Nb-18

[0179] 5.1.1 Medium swelling

[0180] 1.5 g of dry medium was weighed for each of the candidate molecules, anti-hGH Nb-1, anti-hGH Nb-9, and anti-hGH Nb-18, purified as in Example 4. 10 ml of 1 mM HCl (pH 3.0) was added to each of the dry medium. The mixture was shaken at room temperature to ensure thorough contact and mixing. The mixture was allowed to swell for 2 h. The medium was then poured into a solid phase extraction column, the supernatant was discarded, and the column was rinsed with 1 mM HCl.

[0181] 5.1.2 Coupling

[0182] In a solid-phase extraction column, rinse the medium with coupling buffer (0.5M NaCl-0.1M NaHCO₃) (pH 7.7-8.3) until the pH at the outlet matches that of the coupling buffer. Drain the liquid. Pour the treated medium into a sterile centrifuge tube and pre-cool at 4°C for 0.5 h. Replace each hGH single-domain antibody with the coupling buffer, using a medium dosage of 1-10 μmol / ml to ensure a consistent total amount of each hGH single-domain antibody. Pour the medium into the centrifuge tube and mix thoroughly with the medium. Shake at room temperature for 2 h. Pour the coupled medium into the solid-phase extraction column, collect the supernatant, and rinse the medium with 0.5 CV of coupling buffer. Combine the rinses. Detect protein concentration using UV light to calculate protein coupling efficiency.

[0183] 5.1.3 Closure

[0184] The affinity coupling medium was blocked with 1M Tris-HCl-100mM EDTA (pH 7.8-8.2) at 4°C overnight.

[0185] 5.1.4 Elution

[0186] Sequentially wash the sample three times with Elution Solution A: 0.5M NaCl-0.1M Tris-HCl-10mM EDTA (pH 7.7-8.3), Elution Solution B: 20mM Citrate (pH 3.0), and Regeneration Solution C: 1M Acetic Acid. Collect the elution (regeneration) peak and monitor protein concentration under UV light. Repeat this process three times. Calculate the coupling efficiency of the affinity coupling medium.

[0187] Detailed information on affinity coupling experiments of candidate molecules is shown in Table 9.

[0188] Table 9 hGH single domain antibody candidate molecule coupling affinity medium information

[0189] The coupling ratios of candidate molecules anti-hGH Nb-18, anti-hGH Nb-9 and anti-hGH Nb-1 as ligands to CNBr activated Sepharose 4B medium were 15.92 mg / ml, 17.13 mg / ml and 15.83 mg / ml respectively.

[0190] 5.2 Preparation of affinity coupling media for anti-hGH Nb-20 ​​and anti-hGH Nb-27

[0191] 5.2.1 Medium swelling

[0192] 10 g and 2 g of dry medium were weighed for the candidate molecules anti-hGH Nb-20 ​​and anti-hGH Nb-27 purified as described in Example 4, respectively. 1 mM HCl (pH 3.0) was added to each at a ratio of 1:5 (w / v). The mixture was shaken at room temperature to ensure thorough contact and mixing. The mixture was allowed to swell for 2 h. The medium was then poured into a solid phase extraction column, the supernatant was discarded, and the column was rinsed with 1 mM HCl.

[0193] 5.2.2 Coupling

[0194] In a solid-phase extraction column, rinse the medium with coupling buffer (0.5M NaCl-0.1M NaHCO₃) (pH 7.7-8.3) until the pH at the outlet matches that of the coupling buffer. Drain the liquid. Pour the treated medium into a sterile centrifuge tube and pre-cool at 4°C for 0.5 h. Replace each hGH single-domain antibody with the coupling buffer, using a medium dosage of 1-10 μmol / ml to ensure a consistent total amount of each hGH single-domain antibody. Pour the medium into the centrifuge tube and mix thoroughly. Shake overnight at 2-8°C for 15 h. Pour the coupled medium into a solid-phase extraction column, collect the supernatant, and rinse the medium with 0.5 CV of coupling buffer. Combine the rinses. Detect protein concentration using UV light to calculate protein coupling efficiency.

[0195] 5.2.3 Closure

[0196] Block the affinity coupling medium with 1M Tris-HCl-100mM EDTA (pH 7.8-8.2) at 4°C for at least 3h.

[0197] 5.2.4 Medium flushing

[0198] Sequentially rinse the sample three times with a coupling equilibration solution (1): 0.5M NaCl-0.1M Tris-HCl-10mM EDTA (pH 7.7-8.3), an elution solution (2): 20mM citrate (pH 3.0), and a regeneration solution (3): 1M acetic acid. Collect the elution (regeneration) peak and monitor protein concentration under UV light. Repeat this process three times. Calculate the coupling efficiency of the affinity coupling medium.

[0199] Detailed information on affinity coupling experiments of candidate molecules is shown in Table 10.

[0200] Table 10 hGH single domain antibody candidate molecule coupled with affinity media information

[0201] The coupling ratios of candidate molecules anti-hGH Nb-20 ​​and anti-hGH Nb-27 as ligands to CNBr activated Sepharose 4B medium were 14.51 mg / ml and 16.18 mg / ml, respectively.

[0202] Example 6 Evaluation of anti-hGH single domain antibody affinity coupling media

[0203] 6.1 Evaluation of affinity coupling media for anti-hGH Nb-1, anti-hGH Nb-9, and anti-hGH Nb-18

[0204] The conjugated hGH single-domain antibody affinity matrix was loaded onto a Boglund 10 / 20 chromatography column with a column volume of 3.14 ml. Affinity conjugated matrices with different ligands were evaluated by dynamic binding capacity (DBC), effective elution, and purification performance (purity, associated proteins, etc.).

[0205] 6.1.1 Dynamic binding capacity determination of affinity media

[0206] Referring to the operating procedures and parameters in Table 11, an hGH stock solution (amino acid sequence such as SEQ ID NO: 27; diluted and loaded, concentration after dilution: 1.33 mg / ml) was overloaded (20 mg / ml) and loaded with a retention time of 10 min. The flow-through was collected and the concentration was determined. The dynamic binding capacity at a flow-through rate of 5% was calculated.

[0207] Table 11 Affinity chromatography capacity determination process and parameters

[0208] The flowthrough sample concentration was detected and the flowthrough rate was calculated to obtain the maximum binding capacity corresponding to the retention time at 5% sample flowthrough rate (see Table 12). The relationship between the flowthrough rate and binding capacity of the affinity medium coupled with different anti-hGH single domain antibodies is shown in Figure 5A.

[0209] Table 12 Maximum binding capacity corresponding to a retention time of 10 min at a sample flow-through rate of 5%

[0210] According to the above results, the maximum binding capacity of the candidate molecule anti-hGH Nb-18 affinity medium corresponding to a retention time of 10 minutes at a sample flow-through rate of 5% is 7.24 mg / ml. The maximum dynamic binding capacity of the anti-hGH Nb-9 affinity medium and the anti-hGH Nb-1 affinity medium are 4.65 mg / ml and 6.30 mg / ml, respectively.

[0211] 6.1.2 Evaluation of effective elution of affinity medium samples

[0212] Referring to the operating procedures and parameters shown in Table 13, different candidate molecules were loaded onto the affinity media at their maximum binding capacity. The loaded sample was hGH stock solution (the stock solution was diluted and loaded, and the diluted concentration was 1.33 mg / ml). The product yield of the sample was collected after purification to evaluate the effective elution of the affinity coupling media.

[0213] Table 13 Affinity chromatography effective elution process and parameters

[0214] The concentration of the collected samples was determined, and the yield of hGH samples eluted with each candidate molecule affinity medium at pH 3.0 was calculated based on the collected volume and other information. The results are shown in Table 14.

[0215] Table 14 Affinity chromatography product yield

[0216] According to the above results, under the harsh elution condition of pH 3.0, it can be seen that the hGH recovery rates of the candidate molecules anti-hGH Nb-18, anti-hGH Nb-9 and anti-hGH Nb-1 affinity media are 85.68%, 86.99% and 70.17%, respectively.

[0217] 6.1.3 Evaluation of sample purification effects using affinity media

[0218] Referring to the preparation process and parameters in Table 15, chromatography was performed across a range of loading capacities for various candidate molecules. hGH supernatant from an E. coli lysate (the concentration of the hGH supernatant was 1.26 mg / ml as determined by SDS-PAGE grayscale analysis) was loaded onto the purified hGH product to assess the quality of the affinity-coupled medium. The hGH purity and the content of related proteins (non-hGH proteins) were assessed by size exclusion chromatography (SEC).

[0219] The concentration of affinity collected samples was determined, and the SEC purity of hGH and the content of related proteins (non-hGH proteins) in the collected samples were detected. The results are shown in Table 15. The SEC purity of hGH was detected by SEC-HPLC, and the content of related proteins was detected by RP-HPLC.

[0220] Table 15 Product quality of hGH collected by affinity chromatography

[0221] The purification effect evaluation results in Table 15 show that, in terms of SEC purity, the purities of hGH samples purified using the affinity-coupled media for anti-hGH Nb-18, anti-hGH Nb-9, and anti-hGH Nb-1 candidate molecules can reach 94.58%, 92.40%, and 87.96%, respectively. In terms of impurities, the contents of related proteins are 12.17%, 12.59%, and 14.94%, respectively.

[0222] Based on the evaluation results of the dynamic loading capacity, effective elution and purification effect of the affinity media of different candidate molecules under this experimental condition, the affinity media of the three candidate molecules all performed well and can be used in subsequent production processes.

[0223] 6.2 Evaluation of anti-hGH Nb-20 ​​and anti-hGH Nb-27 affinity coupling media

[0224] The conjugated hGH single-domain antibody affinity matrix was loaded onto a Boglund 10 / 20 chromatography column with a column volume of 6.12 ml. Affinity conjugated matrices with different ligands were evaluated by dynamic binding capacity (DBC), effective elution, and purification performance (purity, related proteins, etc.).

[0225] 6.2.1 Dynamic binding capacity determination of affinity media

[0226] Referring to the operating procedures and parameters in Table 16, an hGH stock solution (amino acid sequence such as SEQ ID NO: 27; diluted and loaded to a concentration of 1-1.5 mg / ml) was overloaded (20 mg / ml) with a retention time of 5 min. The flow-through was collected and the concentration was determined. The dynamic binding capacity at a 10% flow-through rate was calculated.

[0227] Table 16 Affinity chromatography capacity determination process and parameters

[0228] The concentration of the flow-through sample was detected and the flow-through rate was calculated to obtain the maximum binding capacity corresponding to the retention time at 10% sample flow-through rate (see Table 17). The relationship between the flow-through rate and the binding capacity of the affinity medium coupled with different anti-hGH single domain antibodies is shown in Figure 5B.

[0229] Table 17 Maximum binding capacity corresponding to a retention time of 5 min at a sample flow-through rate of 10%

[0230] According to the above results, the maximum binding capacities of the affinity media for candidate molecules anti-hGH Nb-20 ​​and anti-hGH Nb-27 at a retention time of 5 min at a sample flow-through rate of 10% were 10.04 mg / ml and 11.15 mg / ml, respectively.

[0231] 6.2.2 Evaluation of effective elution of affinity medium samples

[0232] Referring to the operating procedures and parameters shown in Table 18, different candidate molecules were loaded onto affinity media according to their maximum binding capacity. The loaded samples were intermediate products from the hGH stock solution production process. The sample yields were collected after purification to evaluate the purification effect of the affinity coupling media.

[0233] Table 18 Affinity coupling medium purification effect evaluation process and parameters

[0234] The concentration of the collected samples was determined, and the yield of hGH samples eluted with each candidate molecular affinity medium at pH 3.0 was calculated based on the collected volume and other information. The results are shown in Table 19.

[0235] Table 19 Affinity chromatography product yield

[0236] According to the above results, under the relatively harsh elution condition of pH 3.0, it can be seen that the hGH recovery rates of the candidate molecules anti-hGH Nb-20 ​​and anti-hGH Nb-27 affinity media are 93.48% and 95.98%, respectively.

[0237] 6.2.3 Evaluation of sample purification effects using affinity media

[0238] Referring to the preparation process and parameters in Table 20, chromatography was performed across a range of loading capacities for various candidate molecules. hGH supernatant from an E. coli lysate (the concentration of the hGH supernatant was 1.26 mg / ml as determined by SDS-PAGE grayscale analysis) was loaded onto the purified hGH product to assess the quality of the affinity-coupled medium. The hGH purity and the content of related proteins (non-hGH proteins) were assessed by size exclusion chromatography (SEC).

[0239] The concentration of affinity collected samples was determined, and the SEC purity of hGH and the content of related proteins (non-hGH proteins) in the collected samples were detected. The results are shown in Table 20. The SEC purity of hGH was detected by SEC-HPLC, and the content of related proteins was detected by RP-HPLC.

[0240] Table 20 Product quality of hGH collected by affinity chromatography

[0241] The purification effect evaluation results in Table 20 show that, in terms of SEC purity, the purities of hGH samples purified using the affinity-coupled media for anti-hGH Nb-20 ​​and anti-hGH Nb-27 candidate molecules can reach 99.84% and 99.39%, respectively. In terms of impurities, the contents of related proteins are 7.33% and 7.32%, respectively.

[0242] Based on the evaluation results of the dynamic loading capacity, effective elution and purification effect of the affinity media of different candidate molecules under this experimental condition, the affinity media of the two candidate molecules performed well and can be used in subsequent production processes.

[0243] 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 antigen-binding fragment thereof capable of specifically binding to human growth hormone (hGH), the single-domain antibody or antigen-binding fragment thereof comprising: (a) CDR1 having the structure shown as X1X2AX3G (Formula I, SEQ ID NO: 23); (b) CDR2, which has X4IX5X6X7GX8X9TX 10 The structure shown in YADSVKG (Formula II, SEQ ID NO: 24); (c) CDR3 having a CDR3 sequence such as AFSVTIPTRARHWVD (SEQ ID NO: 19) or ATX 11 YYSDYDVPX 12 X 13 SX 14 EX 15 DY (Formula III, SEQ ID NO: 25) or SRGDX 16 The structure shown in GILHGVVHY (Formula IV, SEQ ID NO: 26); in, X1 is selected from the group consisting of (i) amino acid residues A, S, N and (ii) amino acid residues that are conservative substitutions relative to (i); X2 is selected from the group consisting of (i) amino acid residues R, F, Y and (ii) amino acid residues that are conservative substitutions relative to (i); X3 is selected from (i) amino acid residues M, V and (ii) amino acid residues that are conservative substitutions relative to (i); X4 is selected from the group consisting of (i) amino acid residues A, S and (ii) amino acid residues that are conservative substitutions relative to (i); X5 is selected from the group consisting of (i) amino acid residues E, S, G and (ii) amino acid residues that are conservative substitutions relative to (i); X6 is selected from (i) amino acid residues G, W and (ii) an amino acid residue that is a conservative substitution relative to (i); X7 is selected from (i) amino acid residues I, M, L and (ii) an amino acid residue that is a conservative substitution relative to (i); X8 is selected from the group consisting of (i) amino acid residues A, G and (ii) an amino acid residue that is a conservative substitution relative to (i); X9 is selected from the group consisting of (i) amino acid residues T, S and (ii) amino acid residues that are conservative substitutions relative to (i); X 10 selected from the group consisting of (i) amino acid residues Y, V, S and (ii) amino acid residues that are conservative substitutions relative to (i); X 11 selected from (i) amino acid residues S, N and (ii) amino acid residues that are conservative substitutions relative to (i); X 12 selected from (i) amino acid residues V, A and (ii) amino acid residues that are conservative substitutions relative to (i); X 13 selected from (i) amino acid residues R, T and (ii) amino acid residues that are conservative substitutions relative to (i); X 14 selected from (i) amino acid residues N, D and (ii) amino acid residues that are conservative substitutions relative to (i); X 15 selected from the group consisting of (i) amino acid residues Y, F and (ii) amino acid residues that are conservative substitutions relative to (i); X 16 selected from (i) amino acid residues F, Y and (ii) amino acid residues that are conservative substitutions relative to (i); Preferably, X1 is selected from amino acid residues A, S, N; X2 is selected from amino acid residues R, F, Y; X3 is selected from amino acid residues M, V; X4 is selected from amino acid residues A, S; X5 is selected from amino acid residues E, S, G; X6 is selected from amino acid residues G, W; X7 is selected from amino acid residues I, M, L; X8 is selected from amino acid residues A, G; X9 is selected from amino acid residues T, S; X 10 Selected from amino acid residues Y, V, S; X 11 Selected from amino acid residues S, N; X 12 Selected from amino acid residues V, A; X 13 Selected from amino acid residues R, T; X 14 Selected from amino acid residues N, D; X 15 selected from amino acid residues Y, F; X 16 Selected from amino acid residues F, Y.

2. The single-domain antibody or antigen-binding fragment thereof according to claim 1, comprising: (a) CDR1 having: a sequence as shown in any one of SEQ ID NOs: 17, 1, 5, 9, 13, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 17, 1, 5, 9, 13; (b) CDR2 having: a sequence as shown in any one of SEQ ID NOs: 18, 2, 6, 10, 14, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 18, 2, 6, 10, 14; and (c) CDR3 having: a sequence as shown in any one of SEQ ID NOs: 19, 3, 7, 11, 15, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the sequence shown in any one of SEQ ID NOs: 19, 3, 7, 11, 15; Preferably, the substitution is a conservative substitution; preferably, the single-domain antibody or antigen-binding fragment thereof comprises: (1) CDR1 as set forth in SEQ ID NO: 17; CDR2 as set forth in SEQ ID NO: 18; and CDR3 as set forth in SEQ ID NO: 19; (2) CDR1 as shown in SEQ ID NO: 1; CDR2 as shown in SEQ ID NO: 2; and CDR3 as shown in SEQ ID NO: 3; (3) CDR1 as set forth in SEQ ID NO: 5; CDR2 as set forth in SEQ ID NO: 6; and CDR3 as set forth in SEQ ID NO: 7; (4) CDR1 as set forth in SEQ ID NO:9; CDR2 as set forth in SEQ ID NO:10; and CDR3 as set forth in SEQ ID NO:11; or, (5) CDR1 as shown in SEQ ID NO: 13; CDR2 as shown in SEQ ID NO: 14; and CDR3 as shown in SEQ ID NO:

15.

3. The single-domain antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising an amino acid sequence selected from the group consisting of: (i) a sequence as shown in any one of SEQ ID NOs: 20, 4, 8, 12, and 16; (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 20, 4, 8, 12, 16; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 20, 4, 8, 12, 16; Preferably, the substitutions are conservative substitutions.

4. The single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The single-domain antibody or antigen-binding fragment thereof can specifically bind to hGH in a first solvent to form a complex; and the complex formed by the single-domain antibody or antigen-binding fragment thereof and hGH can dissociate in a second solvent; Wherein, the pH value of the first solvent is in the range of 6.5-8.5 (e.g., 7.2-7.6); the pH value of the second solvent is in the range of 2.5-3.5 (e.g., 2.8-3.2); Preferably, the first solvent is a phosphate buffer, a Tris-HCl buffer, or a HEPES buffer with a pH of 6.5-8.5 (e.g., 7.2-7.6); Preferably, the second solvent is a citrate buffer, a Gly-HCl buffer, or an acetate buffer with a pH of 2.5-3.5 (eg, 2.8-3.2).

5. An isolated nucleic acid molecule encoding the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. A vector comprising the nucleic acid molecule according to claim 5; preferably, the vector is a cloning vector or an expression vector.

7. A host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.

8. A method for preparing the single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising culturing the host cell according to claim 7 under conditions that allow protein expression, and recovering the single-domain antibody or antigen-binding fragment thereof from the cultured host cell culture.

9. A bispecific or multispecific antibody comprising the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; Preferably, the bispecific or multispecific antibody specifically binds hGH and additionally specifically binds one or more other targets; Preferably, the bispecific or multispecific antibody further comprises at least one second antibody with a second binding specificity for a second target.

10. A conjugate comprising the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and a solid support linked to the single domain antibody or antigen-binding fragment thereof; Preferably, the solid support is selected from the group consisting of: magnetic beads, agarose microspheres, dextran microspheres, polymethacrylate, Polystyrene, silica microspheres and combinations thereof; Preferably, the solid support is selected from a porous material, for example, selected from one porous material or a combination of multiple porous materials; Preferably, the conjugate can specifically bind to hGH in a first solvent to form a complex; and the complex formed by the conjugate and hGH can dissociate in a second solvent; Preferably, the first solvent and / or the second solvent is as defined in claim 4.

11. A method for purifying hGH comprising using the conjugate of claim 10; Preferably, the method is a method comprising purifying hGH by affinity chromatography, comprising the following steps: (1) allowing the conjugate to bind to the hGH in a first solvent to form a complex; (2) dissociating the complex in a second solvent; and, (3) collecting the dissociation product containing the hGH; Wherein, the first solvent and the second solvent are as defined in claim 4.

12. Use of the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the conjugate according to claim 10 in the preparation of an hGH purification reagent.

13. A conjugate comprising the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and a detectable label linked to the single domain antibody or antigen-binding fragment thereof; Preferably, the detectable label is selected from 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.

14. A method for detecting the presence or level of hGH in a sample, comprising using the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the conjugate according to claim 13; Preferably, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g. ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay; Preferably, the method comprises using the conjugate of claim 13; Preferably, the method comprises using the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and the method further comprises using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin) to detect the single domain antibody or antigen-binding fragment thereof.

15. The method of claim 14, comprising: (1) contacting the sample with the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the conjugate according to claim 13; (2) Detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex, wherein the formation of the immune complex indicates the presence of hGH.

16. The method according to claim 14 or 15, wherein The method is used to diagnose a disease associated with abnormal hGH levels; and the method comprises detecting the level of hGH in a sample from a subject; Preferably, the method comprises detecting the level of hGH in a sample from a subject, and comparing the level with a reference level (e.g., compared with a healthy control); wherein, when the level of hGH in the sample from the subject is significantly increased or decreased, it indicates that the subject suffers from a disease associated with abnormally high or low hGH levels.

17. Use of the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the conjugate according to claim 13 in the preparation of a detection reagent for detecting the presence or level of hGH in a sample and / or diagnosing a disease associated with abnormal hGH levels.

18. A kit comprising the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the conjugate according to claim 10, or the conjugate according to claim 13.

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