WO2023231888A1

WO2023231888A1 - Anti-human growth hormone single-domain antibody and use thereof

Info

Publication number: WO2023231888A1
Application number: PCT/CN2023/096259
Authority: WO
Inventors: 王思勤; 梁阳秋; 刘艳双; 刘晓慧; 刘爽; 张睿; 宗毅; 王涛; 金磊
Original assignee: 长春金赛药业有限责任公司
Priority date: 2022-05-31
Filing date: 2023-05-25
Publication date: 2023-12-07

Abstract

The present invention relates to a single-domain antibody or an antigen-binding fragment thereof specifically binding to human growth hormone, 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 and a host cell containing same, and the related use thereof. In addition, the present invention relates to the use of the conjugate in the detection or purification of human growth hormone.

Description

Anti-human growth hormone single domain antibody and its application

Technical field

The present invention relates to single-domain antibodies or antigen-binding fragments thereof that specifically bind to human growth hormone, conjugates containing the single-domain antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the single-domain antibodies or antigen-binding fragments thereof, and Host cells containing the same, and related uses. Furthermore, the invention relates to the use of said conjugates for detection or purification of human growth hormone.

Background technique

Human growth hormone (hGH) is a protein secreted by adenohypophysis cells and a peptide hormone. It is a single peptide chain protein hormone secreted by eosinophils in the human anterior lobe of the pituitary gland. It is a peptide hormone composed of 191 amino acids. Growth hormone has been used clinically in 1958 as a specific drug for the treatment of dwarfism. After it was mass-produced by pharmaceutical companies in the 1980s, its clinical indications continued to expand. It is not only widely used clinically in promoting human body growth, but also in anti-tissue and organ failure, improving the nutrition of critically ill patients, treating cachexia (nutritional failure), anti-infection and inflammation, promoting the healing of wounds and burns, and improving the body's immunity. Has wide application.

Nanobodies are a type of special antibodies derived from camelids. In 1993, research by Hamers-Casterman and others showed that there is a type of antibody in camel-derived animals that naturally lacks the light chain and only contains heavy chains, which is called a heavy chain antibody. By cloning the variable region gene of a 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 fragments that can bind to antigens. In addition, nanobodies also have the characteristics of acid and alkali resistance, high temperature resistance and easy production. They can be advantageously used in the immunoaffinity chromatography purification of human GH to replace the cumbersome traditional human GH purification method.

Contents of the invention

The inventor of the present application has screened and obtained a series of single domain antibodies against human growth hormone through extensive research. The single domain antibodies have high binding activity to human growth hormone. In particular, the single domain antibody of the present invention has excellent high temperature resistance, acid and alkali resistance, and excellent stability. It can effectively bind to hGH under neutral conditions, and can effectively dissociate from hGH under low pH acidic conditions. It can be advantageously applied to the affinity purification of human growth hormone. In addition, the single domain antibody also has the characteristics of small molecular weight and easy production.

Based on this, the present application also provides conjugates containing the single domain antibody or antigen-binding fragment thereof, nucleic acid molecules encoding the single-domain antibody or antigen-binding fragment thereof, host cells containing the same, and related uses.

Single domain antibodies and antigen-binding fragments thereof

Therefore, in one aspect, the application provides a single domain antibody or an antigen-binding fragment thereof capable of specifically binding to human growth hormone (hGH), the single-domain antibody or an antigen-binding fragment thereof comprising:

(a) CDR1, which has a structure shown as X ₁ X ₂ AX ₃ G (Formula I, SEQ ID NO: 23);

(b) CDR2 having the structure shown as X ₄ IX ₅ X ₆ X ₇ GX ₈ X ₉ TX ₁₀ YADSVKG (Formula II, SEQ ID NO: 24);

(c) _CDR3 having, for example _, AFSVTIPTRARHWVD (SEQ ID NO: 19 ₎ or _ATX ₁₁ YYSDYDVPX ₁₂ 26) The structure shown;

in,

X ₁ is selected from (i) amino acid residues A, S, N and (ii) amino acid residues that are conservatively substituted relative to (i);

X ₂ is selected from (i) amino acid residues R, F, Y and (ii) amino acid residues that are conservatively substituted relative to (i);

X ₃ is selected from (i) amino acid residues M, V and (ii) amino acid residues that are conservatively substituted relative to (i);

X ₄ is selected from (i) amino acid residues A, S and (ii) amino acid residues that are conservatively substituted relative to (i);

X ₅ is selected from (i) amino acid residues E, S, G and (ii) amino acid residues that are conservatively substituted relative to (i);

X ₆ is selected from (i) amino acid residues G, W and (ii) amino acid residues that are conservatively substituted relative to (i);

X ₇ is selected from (i) amino acid residues I, M, L and (ii) amino acid residues that are conservatively substituted relative to (i);

X ₈ is selected from (i) amino acid residues A, G and (ii) amino acid residues that are conservatively substituted relative to (i);

X ₉ is selected from (i) amino acid residues T, S and (ii) amino acid residues that are conservatively substituted relative to (i);

X ₁₀ is selected from (i) amino acid residues Y, V, S and (ii) amino acid residues that are conservatively substituted relative to (i);

X ₁₁ is selected from (i) amino acid residues S, N and (ii) amino acid residues that are conservatively substituted relative to (i);

X ₁₂ is selected from (i) amino acid residues V, A and (ii) amino acid residues that are conservatively substituted relative to (i);

X ₁₃ is selected from (i) the amino acid residue R, T and (ii) the amino acid residue that is a conservative substitution relative to (i);

X ₁₄ is selected from (i) amino acid residues N, D and (ii) amino acid residues that are conservatively substituted relative to (i);

X ₁₅ is selected from (i) amino acid residues Y, F and (ii) amino acid residues that are conservatively substituted relative to (i);

X ₁₆ is selected from (i) amino acid residues F, Y and (ii) amino acid residues that are conservatively substituted relative to (i).

In certain embodiments, X ₁ is selected from amino acid residues A, S, N; X ₂ _is selected from amino acid residues R, F, Y; X ₃ is selected from amino acid residues M, V; Base A, S; X ₅ is selected from amino _acid residues E, S, G; X ₆ is selected from amino acid residues G, W; X ₇ is selected from amino acid residues I, M, L; , G; X ₉ is selected from amino acid residues T, _S ; X ₁₀ is selected from amino acid residues Y, V, S; X ₁₁ is selected from amino acid residues S, N; ₁₃ is selected from amino acid residues R, T; X ₁₄ is selected from amino acid residues N, D; X ₁₅ is selected from amino acid residues Y, F; X ₁₆ is selected from amino acid residues F, Y.

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

(a) CDR1, which has: a sequence as shown in any one of SEQ ID NOs: 17, 1, 5, 9, 13, or a sequence as shown in any one of SEQ ID NOs: 17, 1, 5, 9, 13 The sequence is compared with a sequence having one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions);

(b) CDR2, which has: a sequence as shown in any one of SEQ ID NOs: 18, 2, 6, 10, 14, or a sequence as shown in any one of SEQ ID NOs: 18, 2, 6, 10, 14 The sequence is compared to a sequence having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions); and

(c) CDR3, which has: a sequence as shown in any one of SEQ ID NOs: 19, 3, 7, 11, 15, or a sequence as shown in any one of SEQ ID NOs: 19, 3, 7, 11, 15 The sequence is compared to a sequence having one or several amino acid substitutions, deletions, or additions (eg, 1, 2, or 3 amino acid substitutions, deletions, or additions).

In certain embodiments, the substitutions are conservative substitutions.

(1) CDR1 as shown in SEQ ID NO:17; CDR2 as shown in SEQ ID NO:18; and CDR3 as shown 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 shown in SEQ ID NO:5; CDR2 as shown in SEQ ID NO:6; and CDR3 as shown in SEQ ID NO:7;

(4) CDR1 as shown in SEQ ID NO:9; CDR2 as shown in SEQ ID NO:10; and CDR3 as shown 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.

In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from:

(i) The sequence shown in any one of SEQ ID NOs: 20, 4, 8, 12, and 16;

(ii) Compared with the sequence shown in any one of SEQ ID NOs: 20, 4, 8, 12, 16, one or several amino acids are substituted, deleted or added (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(iii) At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least Sequences that have 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

In certain embodiments, the substitutions are conservative substitutions.

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

Wherein, the pH value of the first solvent is in the range of 6.5-8.5 (for example, 7.2-7.6); the pH value of the second solvent is in the range of 2.5-3.5 (for example, 2.8-3.2).

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

In certain embodiments, the second solvent is citrate buffer, Gly-HCl buffer, acetate buffer with a pH of 2.5-3.5 (eg, 2.8-3.2).

isolated nucleic acid molecules

In another aspect, the application also provides isolated nucleic acid molecules encoding single domain antibodies or antigen-binding fragments thereof as described above.

carrier

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

host cell

In another aspect, the application also provides a host cell comprising a nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., small mouse cells, human cells, etc.). In certain embodiments, the host cell is a microorganism.

Preparation

The single domain antibody or polypeptide construct or fusion protein of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, the DNA molecule encoding the single domain antibody of the present invention is obtained through chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector, and then the host cell is transformed/transfected. Then, the transformed/transfected host cells are cultured under specific conditions and express the single domain antibody of the invention.

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) ). Alternatively, these antigen-binding fragments can also be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )). Those of ordinary skill in the art are well aware of other techniques for preparing such antigen-binding fragments.

In another aspect, the present application also provides a method for preparing a single domain antibody or an antigen-binding fragment thereof as described above, which includes culturing the host cell as described above under conditions that allow protein expression, and from the cultured host The single domain antibody or antigen-binding fragment thereof is recovered in cell culture.

Bispecific or multispecific antibodies

In another aspect, the application also provides bispecific or multispecific antibodies comprising the single domain antibodies or antigen-binding fragments thereof as described above.

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

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

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

conjugate

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

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

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

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

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

Methods for purifying hGH

In another aspect, the present application also provides a method of purifying hGH, which includes using the conjugate as described above.

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

(1) In the first solvent, the conjugate is combined with the hGH to form a complex;

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

(3) Collect the dissociation product containing the hGH;

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

In another aspect, the present application also provides the use of the single domain antibody or antigen-binding fragment or conjugate thereof as described above in the preparation of hGH purification reagents.

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

conjugate

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

In certain embodiments, the detectable label is selected from the group consisting of enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent proteins), radionuclides or biotin.

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

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

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

In certain embodiments, the methods include using a conjugate as described above.

In certain embodiments, the methods include the use of a single domain antibody, or antigen-binding fragment thereof, as described above, and the methods further include the use of an enzyme carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin) Secondary antibodies are used to detect the single domain antibodies or antigen-binding fragments thereof.

In certain embodiments, the method includes: (1) contacting the sample with a single domain antibody of the invention or an antigen-binding fragment or conjugate thereof; (2) detecting the formation of an antigen-antibody immune complex or The amount of immune complexes is detected. The formation of immune complexes indicates the presence of hGH.

The present application also provides a method of diagnosing a disease associated with abnormal hGH levels, comprising detecting the level of hGH in a sample from a subject using the method as described above. In certain embodiments, when the level of hGH 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 disorder associated with an abnormality in hGH levels. Disorders related to height or height (e.g., gigantism/dwarfism).

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

In certain embodiments, the detection reagent detects the presence or level of hGH in a sample by a method as described above.

Reagent test kit

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.

In certain embodiments, the kit includes a conjugate as described in the first aspect.

In certain embodiments, the kit includes a conjugate as described in the second aspect.

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 an antigen-binding fragment thereof; optionally, the The secondary antibody may also include a detectable label such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives ), fluorescent dyes (such as fluorescein or fluorescent proteins), radionuclides or biotin.

In certain embodiments, the kit further includes a buffer (eg, detection buffer, protein purification buffer).

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

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

Definition of Terms

In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the virology, biochemistry, and immunology laboratory procedures used in this article are routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

When the terms "such as," "such as," "such as," "including," "including," or variations thereof are used herein, these terms will not be considered limiting terms and will instead be interpreted to mean "without limitation ” or “without limitation.”

Unless otherwise indicated herein or clearly contradicted by context, the terms "a" and "an" as well as "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 Singular and plural.

As used herein, the term "single domain antibody" has the meaning commonly 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 such as a camelid antibody or a shark antibody. Typically, Nanobodies are composed of four framework regions and three complementarity determining regions, with the structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N- or C-terminus so that they contain only part of FR1 and/or FR4, or lack one or both of those backbone regions, as long as they substantially retain antigen binding and specificity. Single domain antibodies are also called nanobodies, and the two are used interchangeably.

As used herein, the term "antigen-binding fragment" of a single domain antibody refers to a polypeptide comprising a fragment of a single domain antibody that retains the ability to specifically bind the same antigen to which the single domain antibody binds, and/or that is consistent with the single domain antibody. Antibodies compete for specific binding to an antigen, which is also known as the "antigen-binding moiety." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), adopted in its entirety This reference is incorporated herein 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 Nanobodies of the invention. In some embodiments, the "antigen-binding fragment" of the single domain antibody can be truncated at the N- or C-terminus compared to the full-length single domain antibody so that it contains only a portion of FR1 and/or FR4, or lacks those One or both of the backbone regions are sufficient as long as they substantially maintain antigen binding and specificity.

Antigen-binding fragments of a single domain antibody can be obtained from a given single domain antibody (such as a Nanobody provided by the invention) using conventional techniques known to those skilled in the art (for example, recombinant DNA technology or enzymatic or chemical fragmentation methods), And the antigen-binding fragments of single domain antibodies are screened for specificity in the same way as for intact Nanobodies.

As used herein, when the term "single domain antibody" is mentioned, it includes not only intact single domain antibodies but also antigen-binding fragments of single domain antibodies, unless the context clearly indicates otherwise.

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. Nanobodies contain three CDRs, named CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as 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), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature342:878-883) or IMGT numbering system (Lefranc et al ., Dev. Comparat. Immunol. 27:55-77, 2003). For a given Nanobody, one 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 (for example, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). Herein, the CDRs of Nanobodies are preferably determined by the Kabat numbering system.

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.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the first amino acid sequence or nucleic acid sequence to match the second amino acid sequence or nucleic acid sequence). best comparison). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. Molecules are identical 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 percent identity between two sequences is a function of the number of identical positions shared by the sequences (ie, percent identity = number of identical overlapping positions/total number of positions x 100%). In certain embodiments, both sequences are the same length.

Determination of percent identity between two sequences can also be accomplished using mathematical algorithms. One non-limiting example of a mathematical algorithm for comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as in Karlin and Altschul, 1993, Proc. Acad.Sci. Improved from USA90:5873-5877. Such algorithms were integrated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K _D ) of the interaction. In the present invention, the term " _KD " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

The specific binding properties between two molecules can be determined using methods known in the art. One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate. 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 kdis/kon is equal to the dissociation constant _KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). K _D , kon and kdis values can be measured by any valid method. In certain embodiments, dissociation constants can be measured in Biacore using surface plasmon resonance (SPR). Alternatively, bioluminescence interferometry or Kinexa can be used to measure dissociation constants.

As used herein, a detectable label of the invention may be any substance detectable 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, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides fluorescein (e.g., ^3H , ^125I , ^35S , ^14C , or ^32P ), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC) , phycoerythrin (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa 750)), luminescent substances (such as chemiluminescent substances, such as acridinium ester compounds, Lu mino and its derivatives, ruthenium derivatives such as ruthenium terpyridine), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and avidin modified to bind the above labels (e.g., streptavidin ) of biotin.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, polyomavacuolating virus (such as SV40). A vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication site.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. 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.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter 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 those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including 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 those 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), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids. Therefore, 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 substitutions of amino acids 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 is incorporated herein by reference).

The twenty conventional amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

Beneficial effects of the invention

This application provides a series of single domain antibodies against human growth hormone, which have high binding activity to human growth hormone. In particular, the single domain antibody of the present invention has excellent high temperature resistance, acid and alkali resistance, and excellent stability. It can effectively bind to hGH under neutral conditions, and can effectively dissociate from hGH under low pH acidic conditions. can be beneficially applied to life Affinity purification of long hormone. In addition, the single domain antibody also has the characteristics of small molecular weight and easy production.

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of preferred embodiments.

Description of the drawings

Figure 1 shows the experimental results of ELISA detection of the binding of five single domain antibodies (anti-hGH Nb-1/9/18/20/27) to hGH.

Figure 2 shows the experimental results of ELISA detection of the binding of three single domain antibodies (anti-hGH Nb-1/9/18) to hGH after being treated at different high temperatures (45°C, 55°C) for different times (24h, 48h); Among them, Figure 2A shows the experimental results of anti-hGH Nb-1 binding to hGH, and Figure 2B shows the experimental results of anti-hGH Nb-9/18 binding to hGH.

Figure 3 shows the experimental results of ELISA detection of the binding of three single domain antibodies (anti-hGH Nb-1/9/18) to hGH after treatment at pH 3.0 for different times (0h, 24h, 48h).

Figure 4 shows the non-reducing SDS-PAGE results of the anti-hGH single domain antibody prepared in Example 4; wherein, Figure 4A is the SDS-PAGE result of anti-hGH Nb-1/9/18, and lane M is the molecular weight Marker. 1 is anti-hGH Nb-18, lane 2 is anti-hGH Nb-9, lane 3 is anti-hGH Nb-1, Figure 4B is the SDS-PAGE result of anti-hGH Nb-20/27, lane M is the molecular weight Marker, lane 1 is anti-hGH Nb-20, lane 2 is anti-hGH Nb-27.

Figure 5 shows the trend chart of the relationship between the flow rate and loading capacity of affinity media coupled with different single domain antibodies (anti-hGH Nb-1/9/18/20/27); Figure 5A shows the relationship between affinity media coupled with Trend chart of the relationship between flow-through rate and loading capacity of the affinity medium of anti-hGH Nb-1/9/18. In the abscissa, C represents the concentration of the flow-through sample, and C0 represents the loading concentration of hGH stock solution; Figure 5B shows the coupling Trend chart of the relationship between flow rate and loading capacity of anti-hGH Nb-20/27 affinity media.

sequence information

A description of the sequences covered by this application is provided in the table below.

Table 1: Sequence information

Detailed ways

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

Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method was carried out as described in F.M. Ausubel et al., Compiled Experimental Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the product manufacturer. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

Example 1 Construction of anti-recombinant human growth hormone (hGH) single domain antibody immune library

After emulsifying 2 mg of hGH protein (amino acid sequence such as SEQ ID NO: 27) with Freund's complete adjuvant, alpacas (Vicugna pacos) were immunized by subcutaneous multi-point injection. Use 1.5mg hGH protein and Freund's incomplete adjuvant for the second immunization 20 days after the first immunization. After that, immunize every 14 days and collect intravenous blood. ELISA method is used to detect the serum titer. After the seventh immunization, peripheral blood is collected to isolate lymphocytes. , extract total RNA.

RNA extraction was performed according to the instructions of Invitrogen TRIzol ^TM Reagent. Using total RNA as the template, oligo dT and Random 6 as primers, the first-strand cDNA was synthesized according to the instructions of the TakaRa reverse transcription kit. use NEB's 2×Q5 high-fidelity polymerase was used to obtain the variable region encoding genes of heavy chain antibodies through nested PCR (see Table 2 for primers).

Table 2 Primer names and sequences

Note: R=A/G, Y=C/T, S=C/G.

The first round of PCR used primers Alpaca-CALL01-F and Alpaca-CALL02-R to amplify cDNA respectively. The reaction conditions were: 98°C, 30s; 98°C, 5s, 58°C, 15s, 72°C, 20s, 27 cycles; Extend at 72°C for 2 minutes.

The first-round PCR product was electrophoresed on a 1.5% (w/v) agarose gel to recover a DNA fragment of about 750 bp, which was used as a template for the second-round PCR, using the second-round primer (Alpaca-IGHV3S53/61-F , Alpaca-IGHV3-3-F, Alpaca-IGHV3S59/67-F, Alpaca-IGHV3S64/64-F, Alpaca-IGHV3S65-F are mixed in equal moles as forward primers, Alpaca-IGHJ1-R, Alpaca-IGHJ2/3/ 7-R, Alpaca-IGHJ5-Rnew, and Alpaca-IGHJ4/6-R were mixed in equal moles as reverse primers) for amplification. The reaction conditions were: 98°C, 30s; 98°C, 5s, 58°C, 15s, 72°C. , 20s, 27 cycles; extension at 72℃ for 2min. Recover and quantify using Omega gel recovery kit, and store at -20°C for later use. The phagemid vector pGS249-3 and the PCR recovery product were digested with BglⅠ. After the phagemid vector pGS249-3 was digested with BglⅠ, it was then digested with NsiⅠ to avoid self-ligation of the vector. The gel recovery kit was passed through the column and recovered. After quantification, ligation was performed at a molar ratio of 1:3 at 22°C for 1 hour to obtain the ligation product.

After the ligation product is recovered by the gel recovery kit, it is dissolved in 50 μl of ultrapure water. 500ng of the purified ligation product is electroporated into Escherichia coli TG1 competent cells. A total of 16 cells are electroporated. After electroporation, preheated SOC at 37°C is immediately added for culture. Base, mix well, and resuscitate on a 37°C shaker for 1 hour. After resuscitation, take 50 μl of bacterial liquid from each tube, mix, gradient dilute, and spread. 2×YT plate (containing 100 μg/ml ampicillin), and all the remaining bacterial liquid was spread on a 150×20 mm 2×YT plate (containing 100 μg/ml ampicillin), and incubated overnight at 37°C with inversion. The next day, colony PCR was performed using M13R (SEQ ID NO: 21) and 249R (SEQ ID NO: 22) primers to calculate the library capacity and library positivity rate. Use 2×YT medium (containing 100 μg/ml ampicillin) to scrape the bacterial lawn on the plate, add glycerol with a final concentration of 15% (V/V), aliquot, and store at -80°C for later use.

According to the calculated storage capacity, inoculate 100 times of the storage capacity into 10L of 2×YT culture medium (containing 2% (w/v) glucose, 100 μg/ml ampicillin), and culture at 37°C and 200 rpm until 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. Centrifuge the culture, resuspend the pellet in 5L of 2×YT (containing 100 μg/ml ampicillin and 70 μg/ml kanamycin), and culture overnight at 30°C with shaking at 200 rpm. Collect the supernatant by centrifugation, add 1/4 volume of PEG/NaCl to the supernatant, mix, incubate on ice for 1 hour, centrifuge, and resuspend the pellet in PBS to obtain the anti-hGH single domain antibody immune library. Take 20 μl to measure the titer. Add the remaining glycerol to a final concentration of 50% (V/V), aliquot, and store at -80°C.

Example 2 Panning and identification of anti-hGH single domain antibodies

Single domain antibodies against hGH were panned from the anti-hGH single domain antibody immune library obtained in Example 1 using a solid phase panning method. Add 1ml hGH protein to the immune tube and coat at 4°C overnight. The coating concentrations of hGH for each round of panning are 20 μg/ml, 20 μg/ml, 10 μg/ml, and 10 μg/ml respectively. Take a blank immune tube ( As a control), add 5% (w/v) milk, block at 37°C for 2 hours, wash 3 times with PBST (containing 0.05% (V/V) Tween-20), add 5% Milk-bound phage (5×10 ¹³ pfu) was incubated at room temperature for 1 h, washed 10 times with PBST (15 times in the last 2 rounds), washed away unbound phage, and washed with 0.02M citric acid-sodium citrate (ACID). , pH 3.0) to elute the phage adsorbed on the immune tube, neutralize the eluate with 160 μl of 1M Tris-HCl (pH 8.0), take 100 μl of TG1 infected with OD ₆₀₀ = 0.5, determine the titer, and elute the rest After amplification, the samples were used for the next round of panning.

After four rounds of panning, single clones were picked, cultured at 37°C overnight, and then transferred. When the bacterial concentration reached OD ₆₀₀ = 0.5, IPTG with a final concentration of 1mM was added for induction at 30°C overnight, and the supernatant was collected by centrifugation. , used in ELISA experiments. Coat with 1 μg/ml hGH and 5% milk overnight at 4°C, block for 2 hours at 37°C, wash 3 times with PBST, add 50 μl induction supernatant, wash 3 times with PBST, add Anti-His-HRP secondary antibody, and develop color.

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.

Example 3 Preparation and physical and chemical screening of anti-hGH single domain antibodies

3.1 Preparation of anti-hGH single domain antibody and ELISA binding experiment

Five single-domain antibody gene fragments were cloned into the eukaryotic expression vector pGS003, and after sequencing, the expression vector was successfully constructed.

The plasmid was extracted and transiently expressed in FreeStyle ^TM 293E cells in Freestyle medium. 24 hours before transfection, inoculate 25 ml of 293E cells at 0.5×10 ⁶ cells/ml in a 125 ml Erlenmeyer flask, and culture on a 130 rpm shaker in a 37°C 5% CO ₂ incubator. During transfection, first add 60 μl of 293E Fectin to 1 ml of Opti-MEM, mix thoroughly, and incubate at room temperature for 5 minutes; at the same time, dissolve 25 μg of the total plasmid DNA of the recombinant vector in 1 ml of Opti-MEM. Then, plasmid DNA and 293E Fectin were thoroughly mixed, with a total volume of 2 ml, and incubated at room temperature for 15 minutes. Then the entire mixture was added to the cell culture wells, mixed, and cultured on a 130 rpm shaker in a 37°C 5% _CO2 incubator for 7 days. Centrifuge the culture solution at high speed and take the supernatant for vacuum filtration through a microporous membrane. Purify using a nickel column according to the operating methods provided by the manufacturer to obtain purified single domain antibodies.

Coat 0.5μg/ml hGH (amino acid sequence such as SEQ ID NO: 27) 50μl/well, overnight at 4°C, add 250μl/well 1% (w/v) BSA to block, then wash with PBST 3 times; add 10μg/well Starting from ml, dilute three times, a total of 8 spots of purified anti-hGH single domain antibody prepared as above, 50 μl/well, incubate at room temperature for 1 hour, wash with PBST 3 times, add Anti-His-HRP secondary antibody, and develop color.

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

Simulate the affinity purification step to verify whether the antibody molecules can be dissociated normally under the acidic conditions of 0.02M citric acid-sodium citrate (ACID, pH3.0). The steps are as follows: Coat with 0.5 μg/ml hGH, keep overnight at 4°C, add 250 μl/well 1% (w/v) BSA to block, then wash 3 times with PBST, add 10 μg/ml anti-hGH single domain antibody, 50 μl/well , incubate at room temperature for 1 hour, wash with ACID, wash with PBST (pH 7.2-7.4) as a control, and finally add Anti-His-HRP secondary antibody to develop color. The results are shown in Table 3.

table 3

The results show 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 work better with hGH under neutral conditions. Combined with hGH, it can be better dissociated from hGH under low pH acidic conditions.

3.2 Octet determines the affinity of hGH and anti-hGH single domain antibodies

First add equilibrium buffer PBS, 30 μg/ml anti-hGH single domain antibody and hGH to the microwell plate. First of all, basic Line adjustment was performed using a HIS1K probe conjugated to 30 μg/ml anti-hGH single domain antibody. Baseline adjustment was then performed again, using a probe conjugated with an anti-hGH single domain antibody to bind to hGH. Use data analysis software to obtain the affinity (M) of hGH and anti-hGH single domain antibodies. The results are shown in Table 4.

Table 4 Affinity of single domain antibodies to hGH

3.3 Evaluation of stability of anti-hGH single domain antibodies

Take 4 tubes of 100μg/tube purified single domain antibodies (anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18), place them in 45℃ and 55℃ water baths for 24h and 48h respectively, and then follow the instructions Perform the ELISA experiment in step 3.1 above to verify whether the anti-hGH single domain antibody is stable at high temperature. The specific results are shown in Figure 2A and Figure 2B. The results show that the three single domain antibodies all maintain the binding activity to hGH after the above treatment and have good stability.

Take 3 tubes of 100μg/tube purified single domain antibodies (anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18), place them under pH 3.0 conditions for 24h and 48h respectively, and then perform ELISA Experiment to verify whether the anti-hGH single domain antibody is stable under acidic conditions. The specific results are shown in Figure 3. The results show that the three single domain antibodies all maintain the binding activity to hGH after the above treatment and have good stability.

Example 4 Preparation and Purification of Anti-hGH Single Domain Antibody

Preparation and purification of single domain antibodies anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18, anti-hGH Nb-20, and anti-hGH Nb-27.

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

The target protein is captured through ProA affinity chromatography. The specific preparation process and parameters of anti-hGH Nb-9 are shown in Table 5-1.

Table 5-1 ProA affinity chromatography process and parameters

The target protein is captured through Ni affinity chromatography. The specific preparation procedures and parameters of anti-hGH Nb-18 and anti-hGH Nb-1 are shown in Table 5-2.

Table 5-2 Ni affinity chromatography process and parameters

The purity of the prepared hGH single domain antibody candidate molecules was evaluated by SDS-PAGE. The SDS-PAGE results are shown in Figure 4A. The preparation and collection information of each purified single domain antibody sample is shown in Table 6.

Table 6 Sample preparation collection information

According to the analysis of the electrophoresis results of the samples collected during the preparation of the candidate molecules, the purity of the three candidate molecules was greater than 95% and can be used to continue the preparation experiment of the affinity coupling medium.

4.2 Preparation and purification of anti-hGH Nb-20 and anti-hGH Nb-27

The target protein is captured through Ni affinity chromatography. The specific preparation procedures and parameters of anti-hGH Nb-20 and anti-hGH Nb-27 are shown in Table 7.

Table 7 Ni affinity chromatography process and parameters

The purity of the prepared hGH single domain antibody candidate molecules was evaluated by SDS-PAGE. The SDS-PAGE results are shown in Figure 4B. The preparation and collection information of each purified single domain antibody sample is shown in Table 8.

Table 8 Sample preparation collection information

According to the analysis of the electrophoresis results of the samples collected during the preparation of the candidate molecules, the purity of the two candidate molecules is greater than 95% and can be used to continue the preparation experiment of the affinity coupling medium.

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

Prepare affinity coupling media 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.

5.1 Preparation of anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18 affinity coupling media

5.1.1 Medium swelling

Weigh 1.5g of dry medium as the candidate molecules anti-hGH Nb-1, anti-hGH Nb-9, and anti-hGH Nb-18 purified in Example 4, add 10ml of 1mM HCl (pH 3.0) to each, room temperature, and shake appropriately. Shake to fully contact and mix, swell for 2 hours, pour the medium into the solid phase extraction column, discard the supernatant, and rinse with 1mM HCl.

5.1.2 Coupling

The medium is in the solid-phase extraction column, and the coupling buffer 0.5M NaCl-0.1M NaHCO ₃ (pH 7.7~8.3) is used to flush the medium until the pH of the outflow end is consistent with the coupling buffer, and then the liquid is drained. Pour the processed medium into a sterile centrifuge tube and pre-cool at 4°C for 0.5h. Replace each hGH single domain antibody into the coupling buffer. Refer to the dosage of 1 to 10 μmol/ml medium to ensure that each hGH single domain antibody is used. When the total amount of antibody molecules is consistent, pour it into a centrifuge tube and mix with the medium. Shake at room temperature for 2 hours. Finally, pour the coupled medium into the solid-phase extraction column, collect the supernatant after coupling, rinse the medium with 0.5CV coupling buffer, and combine the rinses. Use UV to detect protein concentration to calculate protein coupling efficiency.

5.1.3 Closure

1M Tris-HCl-100mM EDTA (pH 7.8~8.2) blocks the affinity coupling medium and incubates at 4°C overnight.

5.1.4 Elution

Use 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, rinse sequentially 3 Double the volume, collect the elution (regeneration) peaks, and detect the protein concentration with UV light. Repeat three times. Calculate the coupling ratio of the affinity coupling medium.

The detailed information of the candidate molecule affinity coupling experiments is shown in Table 9.

Table 9 hGH single domain antibody candidate molecule coupling affinity media information

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 are 15.92 mg/ml, 17.13 mg/ml and 15.83 mg/ml respectively. ml.

5.2 Preparation of anti-hGH Nb-20 and anti-hGH Nb-27 affinity coupling media

5.2.1 Medium swelling

Weigh 10g and 2g of dry medium respectively for the candidate molecules anti-hGH Nb-20 and anti-hGH Nb-27 purified in Example 4, and add 1mM HCl (pH3.0) in a ratio of 1:5 (w/v). , room temperature, shake appropriately to fully contact and mix, swell for 2 hours, pour the medium into the solid phase extraction column, discard the supernatant, and rinse with 1mM HCl.

5.2.2 Coupling

The medium is in the solid-phase extraction column, and the coupling buffer 0.5M NaCl-0.1M NaHCO ₃ (pH 7.7~8.3) is used to flush the medium until the pH of the outflow end is consistent with the coupling buffer, and then the liquid is drained. Pour the processed medium into a sterile centrifuge tube and pre-cool at 4°C for 0.5h. Replace each hGH single domain antibody into the coupling buffer. Refer to the dosage of 1 to 10 μmol/ml medium to ensure that each hGH single domain antibody is used. When the total amount of antibody molecules is consistent, pour it into a centrifuge tube and mix with the medium. Shake overnight at 2-8°C for 15 hours. Finally, pour the coupled medium into the solid-phase extraction column, collect the supernatant after coupling, rinse the medium with 0.5CV coupling buffer, and combine the rinses. Use UV to detect protein concentration to calculate protein coupling efficiency.

5.2.3 Closure

1M Tris-HCl-100mM EDTA (pH 7.8~8.2) blocks the affinity coupling medium at 4℃ for at least 3h.

5.2.4 Media flushing

Use coupling equilibrium solution 1: 0.5M NaCl-0.1M Tris-HCl-10mM EDTA (pH7.7~8.3), elution solution 2: 20mM citrate (pH3.0) and regeneration solution 3: 1M acetic acid, Wash 3 times the volume sequentially, collect the elution (regeneration) peaks, and detect the protein concentration with UV light. Repeat three times. Calculate the coupling ratio of the affinity coupling medium.

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

Table 10 hGH single domain antibody candidate molecule coupling affinity media information

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

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

6.1 Evaluation of anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18 affinity coupling media

The coupled hGH single domain antibody affinity medium was loaded into the Borgron 10/20 chromatography column, with a column volume of 3.14 ml. Affinity coupling media with different ligands are evaluated through dynamic binding capacity (DBC), effective elution and purification effects (purity, related proteins, etc.).

6.1.1 Determination of dynamic capacity of affinity media

Refer to the operating procedures and parameters in Table 11. Under the condition of retention time of 10 minutes, hGH stock solution (amino acid sequence such as SEQ ID NO: 27; load after dilution, concentration after dilution is 1.33mg/ml) overload (20mg/ml) and load the sample. The flow-through fluid was collected and the concentration was determined, and the dynamic binding capacity at 5% flow-through rate was calculated.

Table 11 Affinity chromatography capacity determination process and parameters

Detect the concentration of the flow-through sample, calculate the flow-through rate, and then obtain the maximum binding capacity corresponding to the retention time at 5% sample flow-through rate (see Table 12). The relationship between affinity medium flow rate and loading capacity coupled with different anti-hGH single domain antibodies is shown in Figure 5A.

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

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

6.1.2 Evaluation of effective elution of affinity medium samples

Referring to the operating procedures and parameters shown in Table 13, different candidate molecule affinity media were loaded according to their maximum binding capacity. The sample was hGH stock solution (the stock solution was diluted and loaded, and the diluted concentration was 1.33 mg/ml). The sample was tested and purified. Sample product yields were then collected to evaluate the effective elution of the affinity coupling medium.

Table 13 Effective elution process and parameters of affinity chromatography

Determine the concentration of the collected sample, and calculate the yield of the hGH sample eluted by each candidate molecule affinity medium under pH 3.0 based on the collection volume and other information. The results are shown in Table 14.

Table 14 Affinity chromatography product yield table

According to the above results, it can be seen that the candidate molecules anti-hGH Nb-18, anti-hGH Nb-9 and anti-hGH Nb-1 affinity media elute under the harsh conditions of pH 3.0 for hGH. The yields were 85.68%, 86.99% and 70.17% respectively.

6.1.3 Evaluation of purification effect of affinity medium samples

Referring to the preparation process and parameters in Table 15, perform chromatography operations within the loading range of different candidate molecules. Use the hGH E. coli lysate supernatant (the concentration of the hGH lysate supernatant is 1.26 mg/ml after SDS-PAGE grayscale detection) ), and test the product quality of purified hGH to evaluate the purification effect of the affinity coupling medium. The test items are the SEC (size exclusion chromatography) purity of hGH and the content of related proteins (non-hGH proteins).

Determine the concentration of the affinity collection samples, and detect the SEC purity of hGH and the content of related proteins (non-hGH proteins) in the collected samples. The results are shown in Table 15. Among them, the SEC purity of hGH was detected by SEC-HPLC, and the content of related proteins passed RP-HPLC was used for detection.

Table 15 Product quality table of hGH collected by affinity chromatography

It can be seen from the purification effect evaluation results in Table 15 that in terms of SEC purity, the hGH samples purified by anti-hGH Nb-18, anti-hGH Nb-9 and anti-hGH Nb-1 candidate molecule affinity coupling media are The purity can reach 94.58%, 92.40% and 87.96% respectively; in terms of impurities, the related protein contents are 12.17%, 12.59% and 14.94% respectively.

Based on the evaluation results of dynamic loading, 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.

6.2 Evaluation of anti-hGH Nb-20 and anti-hGH Nb-27 affinity coupling media

Load the coupled hGH single domain antibody affinity medium into the Borgron 10/20 chromatography column, with a column volume of 6.12 ml. Affinity coupling media with different ligands are evaluated through dynamic binding capacity (DBC), effective elution and purification effects (purity, related proteins, etc.).

6.2.1 Determination of dynamic capacity of affinity media

Refer to the operating procedures and parameters in Table 16. Under the condition of retention time of 5 minutes, hGH stock solution (amino acid sequence such as SEQ ID NO: 27; load after dilution, concentration after dilution 1-1.5mg/ml) is overloaded (20mg/ml). Sample, collect the flow-through fluid and measure the concentration, and calculate the dynamic binding capacity at 10% flow-through rate.

Table 16 Affinity chromatography capacity determination process and parameters

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

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

According to the above results, the maximum binding capacities of candidate molecules anti-hGH Nb-20 and anti-hGH Nb-27 affinity media corresponding to a retention time of 5 minutes at 10% sample flow-through rate are 10.04mg/ml and 11.15mg/ml respectively. .

6.2.2 Evaluation of effective elution of affinity medium samples

Referring to the operating procedures and parameters shown in Table 18, different candidate molecule affinity media are loaded according to their maximum binding capacity. The loaded sample is an intermediate product in the hGH stock solution production process. The sample product yield is collected after purification to evaluate the affinity. Purification effect of coupling media.

Table 18 Affinity coupling medium purification effect evaluation process and parameters

Determine the concentration of the collected sample, and calculate the yield of the hGH sample eluted by each candidate molecule affinity medium under pH 3.0 based on the collection volume and other information. The results are shown in Table 19.

Table 19 Affinity chromatography product yield table

According to the above results, it can be seen that the yields of the candidate molecules anti-hGH Nb-20 and anti-hGH Nb-27 affinity media for hGH are 93.48% and 93.48%, respectively, when eluting under the harsh conditions of pH 3.0. 95.98%.

6.2.3 Evaluation of purification effect of affinity medium samples

Refer to the preparation process and parameters in Table 20, perform chromatography operations within the loading range of different candidate molecules, and use the hGH E. coli lysate supernatant (the concentration of the hGH lysate supernatant is 1.26 mg/ml after SDS-PAGE grayscale detection) ), and test the product quality of purified hGH to evaluate the purification effect of the affinity coupling medium. The test items are the SEC (size exclusion chromatography) purity of hGH and the content of related proteins (non-hGH proteins).

Determine the concentration of the affinity collection sample, and detect the SEC purity of hGH and the content of related proteins (non-hGH proteins) in the collected samples. The results are shown in Table 20. Among them, the SEC purity of hGH was detected by SEC-HPLC, and the content of related proteins passed RP-HPLC was used for detection.

Table 20 Product quality table of hGH collected by affinity chromatography

It can be seen from the purification effect evaluation results in Table 20 that in terms of SEC purity, the purity of hGH samples purified by anti-hGH Nb-20 and anti-hGH Nb-27 candidate molecule affinity coupling media can reach 99.84% and 99.84%, respectively. 99.39%; in terms of impurities, the related protein contents were 7.33% and 7.32% respectively.

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

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

Claims

A single domain antibody or an antigen-binding fragment thereof capable of specifically binding to human growth hormone (hGH), the single-domain antibody or an antigen-binding fragment thereof comprising:

(a) CDR1, which has a structure shown as X 1 X 2 AX 3 G (Formula I, SEQ ID NO: 23);

(b) CDR2 having the structure shown as X 4 IX 5 X 6 X 7 GX 8 X 9 TX 10 YADSVKG (Formula II, SEQ ID NO: 24);

(c) CDR3 having, for example , AFSVTIPTRARHWVD (SEQ ID NO: 19 ) or ATX 11 YYSDYDVPX 12 26) The structure shown;

in,

X 1 is selected from (i) amino acid residues A, S, N and (ii) amino acid residues that are conservatively substituted relative to (i);

X 2 is selected from (i) amino acid residues R, F, Y and (ii) amino acid residues that are conservatively substituted relative to (i);

X 3 is selected from (i) amino acid residues M, V and (ii) amino acid residues that are conservatively substituted relative to (i);

X 4 is selected from (i) amino acid residues A, S and (ii) amino acid residues that are conservatively substituted relative to (i);

X 5 is selected from (i) amino acid residues E, S, G and (ii) amino acid residues that are conservatively substituted relative to (i);

X 6 is selected from (i) amino acid residues G, W and (ii) amino acid residues that are conservatively substituted relative to (i);

X 7 is selected from (i) amino acid residues I, M, L and (ii) amino acid residues that are conservatively substituted relative to (i);

X 8 is selected from (i) amino acid residues A, G and (ii) amino acid residues that are conservatively substituted relative to (i);

X 9 is selected from (i) amino acid residues T, S and (ii) amino acid residues that are conservatively substituted relative to (i);

X 10 is selected from (i) amino acid residues Y, V, S and (ii) amino acid residues that are conservatively substituted relative to (i);

X 11 is selected from (i) amino acid residues S, N and (ii) amino acid residues that are conservatively substituted relative to (i);

X 12 is selected from (i) amino acid residues V, A and (ii) amino acid residues that are conservatively substituted relative to (i);

X 13 is selected from (i) the amino acid residue R, T and (ii) the amino acid residue that is a conservative substitution relative to (i);

X 14 is selected from (i) amino acid residues N, D and (ii) amino acid residues that are conservatively substituted relative to (i);

X 15 is selected from (i) amino acid residues Y, F and (ii) amino acid residues that are conservatively substituted relative to (i);

X 16 is selected from (i) amino acid residues F, Y and (ii) amino acid residues that are conservatively substituted relative to (i);

Preferably, X 1 is selected from amino acid residues A, S, N; X 2 is selected from amino acid residues R, F, Y; X 3 is selected from amino acid residues M, V; ; X 5 is selected from amino acid residues E, S , G; X 6 is selected from amino acid residues G, W; X 7 is selected from amino acid residues I, M, L; 9 is selected from amino acid residues T, S; X 10 is selected from amino acid residues Y, V, S; X 11 is selected from amino acid residues S, N; X 12 is selected from amino acid residues V, A; Residues R, T; X 14 is selected from amino acid residues N, D; X 15 is selected from amino acid residues Y, F; X 16 is selected from amino acid residues F, Y.
The single domain antibody or antigen-binding fragment thereof of claim 1, comprising:

(a) CDR1, which has: a sequence as shown in any one of SEQ ID NOs: 17, 1, 5, 9, 13, or a sequence as shown in any one of SEQ ID NOs: 17, 1, 5, 9, 13 The sequence is compared with a sequence having one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions);

(b) CDR2, which has: a sequence as shown in any one of SEQ ID NOs: 18, 2, 6, 10, 14, or a sequence as shown in any one of SEQ ID NOs: 18, 2, 6, 10, 14 The sequence is compared to a sequence having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions); and

(c) CDR3, which has: a sequence as shown in any one of SEQ ID NOs: 19, 3, 7, 11, 15, or a sequence as shown in any one of SEQ ID NOs: 19, 3, 7, 11, 15 The sequence is compared with a sequence having one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions);

Preferably, the substitution is a conservative substitution; preferably, the single domain antibody or antigen-binding fragment thereof contains:

(1) CDR1 as shown in SEQ ID NO:17; CDR2 as shown in SEQ ID NO:18; and CDR3 as shown 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 shown in SEQ ID NO:5; CDR2 as shown in SEQ ID NO:6; and CDR3 as shown in SEQ ID NO:7;

(4) CDR1 as shown in SEQ ID NO:9; CDR2 as shown in SEQ ID NO:10; and CDR3 as shown 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.
The single domain antibody or antigen-binding fragment thereof according to claim 1 or 2, which comprises an amino acid sequence selected from the following:

(i) The sequence shown in any one of SEQ ID NOs: 20, 4, 8, 12, and 16;

(ii) Compared with the sequence shown in any one of SEQ ID NOs: 20, 4, 8, 12, 16, one or several amino acids are substituted, deleted or added (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(iii) At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, or at least the sequence shown in any one of SEQ ID NOs: 20, 4, 8, 12, 16 Sequences that have 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

Preferably, the substitutions are conservative substitutions.
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 the first solvent to form a complex; and, the single domain antibody or antigen-binding fragment thereof can specifically bind to hGH in the first solvent; and, the single domain antibody or antigen-binding fragment thereof The complex formed by the domain antibody or its antigen-binding fragment and hGH can be dissociated in the second solvent;

Wherein, the pH value of the first solvent is in the range of 6.5-8.5 (for example, 7.2-7.6); the pH value of the second solvent is in the range of 2.5-3.5 (for example, 2.8-3.2);

Preferably, the first solvent is phosphate buffer, Tris-HCl buffer, or HEPES buffer with a pH of 6.5-8.5 (for example, 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 (for example, 2.8-3.2).
An isolated nucleic acid molecule encoding the single domain antibody or antigen-binding fragment thereof according to any one of claims 1-4.
A vector comprising the nucleic acid molecule of claim 5; preferably, the vector is a cloning vector or an expression vector.
A host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.
A method for preparing the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which includes culturing the host cell according to claim 7 under conditions that allow protein expression, and from the cultured host cell The single domain antibody or antigen-binding fragment thereof is recovered in culture.
Bispecific or multispecific antibodies, which comprise the single domain antibody or antigen-binding fragment thereof according to any one of claims 1-4;

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 having a second binding specificity for a second target.
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 connected to the single domain antibody or antigen-binding fragment thereof;

Preferably, the solid phase support is selected from: magnetic beads, agarose microspheres, dextran microspheres, polymethacrylate, Polystyrene, silica microspheres and combinations thereof;

Preferably, the solid support is selected from porous materials, for example, selected from one porous material or a combination of multiple porous materials;

Preferably, the conjugate can specifically bind to hGH in the first solvent to form a complex; and, the complex formed by the conjugate and hGH can dissociate in the second solvent;

Preferably, the first solvent and/or the second solvent are as defined in claim 4.
A method of purifying hGH comprising using the conjugate of claim 10;

Preferably, the method is a method comprising purifying hGH by affinity chromatography, which includes the following steps:

(1) In the first solvent, the conjugate is combined with the hGH to form a complex;

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

(3) Collect the dissociation product containing the hGH;

Wherein, the first solvent and the second solvent are as defined in claim 4.
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 hGH purification reagent.
A conjugate comprising the single domain antibody or antigen-binding fragment thereof according to any one of claims 1-4, and a detectable label connected to the single domain antibody or antigen-binding fragment thereof;

Preferably, the detectable label is selected from enzymes (such as horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives). ), fluorescent dyes (such as fluorescein or fluorescent proteins), radionuclides or biotin.
A method for detecting the presence of hGH in a sample or its level, comprising using the single domain antibody or antigen-binding fragment thereof according to any one of claims 1-4 or the conjugate according to claim 13;

Preferably, the method is an immunological detection, such as Western blotting, enzyme immunoassay (such as ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay;

Preferably, the method comprises using the conjugate of claim 13;

Preferably, the method includes using the single domain antibody or antigen-binding fragment thereof according to any one of claims 1-4, and the method further includes using an antibody carrying a detectable label (such as an enzyme (such as horseradish peroxide) enzyme or alkaline phosphatase), chemiluminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin ) second antibody to detect the single domain antibody or antigen-binding fragment thereof.
The method of claim 14, comprising: (1) contacting the sample with the single domain antibody or antigen-binding fragment thereof of any one of claims 1-4 or the conjugate of claim 13; (2) Detect the formation of antigen-antibody immune complexes or detect the amount of said immune complexes, the formation of which indicates the presence of hGH.
The method of claim 14 or 15, wherein the method is used to diagnose a disease associated with abnormal hGH levels; and the method includes detecting the level of hGH in a sample from the subject;

Preferably, the method comprises detecting the level of hGH in a sample from the subject and comparing the level to a reference level (e.g. compared to a healthy control); wherein when the level of hGH in the sample from the subject A significant increase or decrease indicates that the subject suffers from a disease associated with abnormally high or low hGH levels.
The 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 of hGH in a sample or its levels and/or diagnosis of diseases associated with abnormal hGH levels.
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.