CN110964113A

CN110964113A - Single-domain antibody for mediated immunoglobulin binding, bifunctional antibody constructed by same and application thereof

Info

Publication number: CN110964113A
Application number: CN201911349057.1A
Authority: CN
Inventors: 叶青
Original assignee: Beijing Newanbo Biotechnology Co Ltd
Current assignee: Beijing Newanbo Biotechnology Co Ltd
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2020-04-07
Anticipated expiration: 2039-12-24
Also published as: CN110964113B

Abstract

The invention discloses a single-domain antibody mediated to be combined with immunoglobulin, a bifunctional antibody constructed by the single-domain antibody and application of the bifunctional antibody. The invention obtains the single domain antibody which can mediate and combine with the immunoglobulin from the single domain antibody gene library, the amino acid sequences of the single domain antibody are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, the single domain antibody can specifically combine with human IgG, IgA and mouse IgG, and the affinity is strong. The invention also discloses a bifunctional antibody obtained by fusing the single-domain antibody and a therapeutic antibody or polypeptide. Animal experiments prove that the single-domain antibody has the effects of prolonging the half-life time of the functional antibody or the therapeutic antibody in an animal body, effectively improving the bioavailability of the therapeutic functional antibody or the therapeutic antibody, remarkably enhancing the biological targeting treatment of the therapeutic antibody and the like.

Description

Single-domain antibody for mediated immunoglobulin binding, bifunctional antibody constructed by same and application thereof

Technical Field

The invention relates to a single domain antibody, in particular to a group of single domain antibodies mediating and binding immunoglobulin and bifunctional antibodies obtained by fusing the single domain antibodies and therapeutic single domain antibodies or functional antibodies, and further relates to application of the single domain antibodies and the therapeutic single domain antibodies in treatment or detection of diseases such as tumors, pathogenic microorganism infection and the like, belonging to the field of single domain antibodies mediating and binding immunoglobulin and application thereof.

Background

The research of Raymond Hamers et al by Belgian scientists finds that besides the ordinary four-chain antibody, the camel blood also has a naturally-existing heavy-chain antibody with a deleted light chain, and the heavy-chain antibody gene cuts off the gene of a CH1 region in a heavy-chain stable region during recombination, so that the expressed heavy chain lacks a CH1 region and cannot be connected with the light chain to form the ordinary light-heavy-chain four-chain antibody, and the heavy-chain antibody with only two heavy chains (without CH1) is formed. The variable region of the heavy chain antibody is designated as VHH to distinguish it from the variable region VH of a normal antibody. Through a molecular biological method, a VHH antibody gene library is prepared by using techniques such as phage display and the like for VHH, and the obtained VHH is screened and called nanobody (nano antibody), or called single domain antibody, or called antigen binding domain antibody fragment. The single domain antibody has a series of advantages of simple structure, strong penetrating power, easy expression and purification, high affinity and stability, no toxic or side reaction and the like, so that the single domain antibody becomes a hot topic for the research of the field of biological pharmacy.

The single domain antibody has advantages as described above, but is also rapidly cleared from blood circulation in an organism due to its small molecule, resulting in its short elimination half-life in an organism. The elimination half-life of single domain antibodies in organisms is one of the problems to be solved in the development of this particular biomolecular medicine. The Ablynx company of Belgium has developed anti-human albumin (HSA) nano antibody (see patent: CN200780043274) for the research and development of nano antibody candidate drugs of the company, and has developed bispecific nano antibody (2 or 3) drugs, for example, nano antibodies of anti-TNF, IL-6R and the like and genes of nano antibodies of anti-HSA are respectively cloned into expression bifunctional specific nano antibody fusion protein, and the bispecific specific targeting nano antibody has been used as a technical means for researching and developing specific targeting nano antibody treatment candidate drugs, so that the half life of a monomer nano antibody in an animal body is improved to 2-3 weeks from 15 minutes originally, and good effects are obtained, and a plurality of candidate drugs have entered clinical phase I and II tests.

Disclosure of Invention

It is an object of the present invention to provide single domain antibodies that mediate binding to immunoglobulins;

the second object of the present invention is to provide humanized anti-Her 2/neu single domain antibodies;

the third purpose of the invention is to fuse the single-domain antibody which mediates the combination of immunoglobulin with a therapeutic single-domain antibody to obtain a bifunctional single-domain antibody;

the fourth purpose of the invention is to apply the single-domain antibody and the bifunctional antibody which mediate and combine the immunoglobulin to the treatment of diseases such as tumors, pathogenic microorganism infection and the like or to detect or enhance the action effect of the therapeutic antibody or polypeptide and increase the stability of the therapeutic antibody or polypeptide.

The invention firstly provides a group of single domain antibodies sdAb-C3 and sdAb-F12 capable of specifically binding to immunoglobulins;

wherein the amino acid sequence of the single domain antibody sdAb-C3 comprises 3 Complementarity Determining Regions (CDRs) and 4 Framework Regions (FRs), wherein the amino acid sequences of the 3 complementarity determining regions are represented by SEQ ID No.5, SEQ ID No.6, and SEQ ID No.7, respectively; the amino acid sequences of the 4 framework regions are respectively shown as SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO. 14.

Further preferably, the amino acid sequence of the single domain antibody sdAb-C3 in the present invention is selected from any one of amino acid sequences (1) - (3):

(1) an amino acid sequence shown as SEQ ID NO. 1; or (2) a protein mutant obtained by deleting, substituting, inserting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.1, wherein the protein mutant has the same function as the protein before mutation; or (3) an amino acid sequence with at least 75 percent of identity with the amino acid sequence shown in SEQ ID NO. 1.

The invention further provides a coding gene of the single domain antibody sdAb-C3; the nucleotide sequence of the encoding gene is selected from any one of (1) to (3):

(1) a polynucleotide sequence shown as SEQ ID NO. 3; or (2) a polynucleotide sequence that hybridizes to the complement of the polynucleotide sequence set forth in SEQ ID No.3 under stringent hybridization conditions; or (3) a polynucleotide sequence having at least 75% identity to the polynucleotide sequence shown in SEQ ID NO. 3; preferably, the polynucleotide sequence has at least more than 80% identity with the polynucleotide sequence shown in SEQ ID NO. 3; further preferably, the polynucleotide sequence has at least more than 85% identity with the polynucleotide sequence shown in SEQ ID NO. 3; more preferably, the polynucleotide sequence has at least 95% identity with the polynucleotide sequence shown in SEQ ID NO. 3; most preferably, the polynucleotide sequence has more than 99% identity with the polynucleotide sequence shown in SEQ ID NO. 3.

The amino acid sequence of the single-domain antibody sdAb-F12 comprises 3 complementarity determining regions and 4 framework regions, wherein the amino acid sequences of the 3 complementarity determining regions are shown in SEQ ID No.8, SEQ ID No.9, and SEQ ID No.10, respectively; the amino acid sequences of the 4 framework regions are respectively shown as SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.1 and 7SEQ ID NO. 18.

Further preferably, the amino acid sequence of the single domain antibody sdAb-F12 in the present invention is selected from any one of amino acid sequences (1) - (3):

(1) an amino acid sequence shown as SEQ ID NO. 2; or (2) a protein mutant obtained by deleting, substituting, inserting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.2, wherein the protein mutant has the same function as the protein before mutation; or (3) an amino acid sequence with at least 75 percent of identity with the amino acid sequence shown in SEQ ID NO. 2.

The invention further provides a coding gene of the single domain antibody sdAb-F12; the nucleotide sequence of the encoding gene is selected from any one of (1) to (3):

(1) the polynucleotide sequence shown in SEQ ID NO. 4; or (2) a polynucleotide sequence that hybridizes to the complement of the polynucleotide sequence set forth in SEQ ID No.4 under stringent hybridization conditions; or (3) a polynucleotide sequence having at least 75% identity to the polynucleotide sequence shown in SEQ ID NO. 4; preferably, the polynucleotide sequence has at least more than 80% identity with the polynucleotide sequence shown in SEQ ID NO. 4; further preferably, the polynucleotide sequence has at least more than 85% identity with the polynucleotide sequence shown in SEQ ID NO. 4; more preferably, a polynucleotide sequence having at least 95% identity to the polynucleotide sequence shown in SEQ ID NO. 4; most preferably, the polynucleotide sequence has more than 99% identity with the polynucleotide sequence shown in SEQ ID NO. 4.

The invention also provides a humanized anti-Her 2/neu single-domain antibody (HerG1M), the amino acid sequence of which is selected from any one of the amino acid sequences (1) to (3):

(1) an amino acid sequence shown as SEQ ID NO. 19; or (2) a protein mutant obtained by deleting, substituting, inserting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.19, wherein the protein mutant has the same function as the protein before mutation; or (3) an amino acid sequence with at least 75 percent of identity with the amino acid sequence shown in SEQ ID NO. 19.

The invention also further provides a coding gene of the humanized anti-Her 2/neu single-domain antibody; the nucleotide sequence of the encoding gene is selected from any one of (1) to (3):

(1) the polynucleotide sequence shown in SEQ ID NO. 20; or (2) a polynucleotide sequence that hybridizes to the complement of the polynucleotide sequence set forth in SEQ ID No.20 under stringent hybridization conditions; or (3) a polynucleotide sequence having at least 75% identity to the polynucleotide sequence shown in SEQ ID NO. 20; preferably, the polynucleotide sequence has at least more than 80% identity with the polynucleotide sequence shown in SEQ ID NO. 20; further preferably, the polynucleotide sequence has at least more than 85% identity with the polynucleotide sequence shown in SEQ ID NO. 20; more preferably, a polynucleotide sequence having at least 95% identity to the polynucleotide sequence shown in SEQ ID NO. 20; most preferably, the polynucleotide sequence has more than 99% identity with the polynucleotide sequence shown in SEQ ID NO. 20.

The single domain antibodies sdAb-C3 and sdAb-F12 mediated and bound to immunoglobulin provided by the invention can be applied to preparation of reagents for diagnosing or detecting diseases caused by infection of tumor or pathogenic microorganisms or medicines for treating diseases caused by infection of tumor or pathogenic microorganisms, and are used for enhancing the action effect of therapeutic antibodies or polypeptides, increasing the stability of therapeutic antibodies or polypeptides and/or eliminating half-life in organisms. Preferably, the therapeutic antibody is an antibody against a virus, a bacterium or a tumor. More preferably, the therapeutic antibody is an anti-Her 2 single domain antibody.

Furthermore, the single domain antibody sdAb-C3 or sdAb-F12 mediated to bind immunoglobulin of the present invention can be fused with other therapeutic antibodies or polypeptides to obtain a bifunctional or multifunctional single domain antibody or polypeptide fusion protein, which is used for preparing drugs for treating diseases such as tumors and pathogenic microorganism infection.

As a preferred embodiment, the present invention provides a bifunctional single domain antibody,

the single-domain antibody sdAb-C3 is linked with a humanized anti-Her 2/neu single-domain antibody through a linker; the single domain antibody sdAb-C3 can be linked to the humanized anti-Her 2/neu single domain antibody using a linker peptide linker commonly used in the art to give a bifunctional antibody (or fusion protein); for reference, the amino acid sequence of the linker peptide of the present invention may be the amino acid sequence shown in SEQ ID NO.21 or SEQ ID NO. 22; the bifunctional antibody can be used for preparing a medicine for detecting and/or treating diseases caused by tumor or pathogenic microorganism infection.

The invention also provides an expression vector containing the coding gene of the single domain antibody and an expression vector containing the coding gene of the bifunctional antibody; the expression vector can be a prokaryotic expression vector, a eukaryotic expression vector or other expression vectors;

the invention also discloses a recombinant host cell containing the expression vector. Wherein, the host cell is prokaryotic expression cell, eukaryotic expression cell, fungal cell or yeast cell, and the eukaryotic expression cell is preferably CHO cell.

The invention also provides a method for preparing the single-domain antibody mediated to bind to the immunoglobulin. The method comprises the steps of immunizing alpaca with purified human IgG and IgA antigens, constructing a specific single domain antibody gene library (a nano antibody phage display gene library), screening the immune nano antibody gene library to obtain a specific nano antibody (or called single domain antibody fragment) resisting human Ig, connecting and recombining the group of genes (sdAb-C3 and sdAb-F12) and an expression vector to construct nano antibody strains sdAb-C3-pSJF2 and sdAb-F12-pSJF2 which can be efficiently expressed in escherichia coli, producing the nano antibody strains on a small scale, and purifying the nano antibody strains by Ni + resin gel column affinity chromatography to obtain the single domain antibody.

Different forms of single domain antibodies (monomer, dimer or multimer) that mediate binding of the immunoglobulin-binding single domain antibody sdAb-C3 of the present invention are prepared by reference to the following references: 1. tanha J, Dubuc G, Selection by pharmacological display of llama conditional V (H) fragments with heavy chain antibodies V (H) Hproperties. J Immunol methods.2002,263(1-2): 97-109); 2. gueorguieva D, LiS.identification of single-domain, Bax-specific intrablocks that is resistant to a mammalian cell against oxidative-stress-induced apoptosis. FASEB J.2006,20(14): 2636-8; 3. the patent: nanobodies or polypeptides against breast cancer Her/neu, patent No.: ZL 201110280031.3.

Compared with the prior art, the invention has the main beneficial effects that:

(1) single domain antibodies sdAb-C3 and sdAb-F12 capable of mediating and binding immunoglobulin are obtained through screening, and the single domain antibodies sdAb-C3 and sdAb-F12 can specifically bind human IgG, IgA and mouse IgG; the invention further constructs the bifunctional and multifunctional specific fusion protein by using the single domain antibodies sdAb-C3 and sdAb-F12 and other antibodies against tumors and virus pathogens or single domain antibodies (such as HerG1M, scfv, VL, VH, VHH and the like), so that the half-life in vivo of other functional antibodies or single domain antibodies is prolonged, the bioavailability is improved, and the biological targeting treatment effect is enhanced.

(2) The invention constructs a bispecific bifunctional single-domain antibody fusion protein of an anti-Ig single-domain antibody sdAb-C3 and a humanized anti-Her 2/neu single-domain antibody, the bispecific bifunctional single-domain antibody fusion protein enhances the effect of the anti-Her 2/neu single-domain antibody on killing breast cancer cells, prolongs the half-life time of the anti-Her 2/neu single-domain antibody in an animal body, and effectively improves the targeted therapeutic effect of the anti-Her 2/neu single-domain antibody.

The invention relates to the terms and definitions

The "substitution" as referred to in the present invention may be a conservative substitution, that is, a substitution of a specific amino acid residue for a residue having similar physicochemical characteristics. Non-limiting examples of conservative substitutions include substitutions between amino acid residues containing aliphatic groups (e.g., substitutions between Ile, Val, Leu, or Ala), substitutions between polar residues (e.g., substitutions between Lys and Arg, Glu and Asp, Gln and Asn), and the like. Mutants resulting from deletion, substitution, insertion and/or addition of amino acids can be made by, for example, site-directed mutagenesis (see, for example, Nucleic acid research, Vol.10, No.20, p.6487-6500, 1982, which is incorporated herein by reference in its entirety) as a well-known technique on DNA encoding a wild-type protein.

In the present specification, "one or more amino acids" refers to amino acids that can be deleted, substituted, inserted, and/or added by a site-directed mutagenesis method, and is not limited, but is preferably 20 or less, 15 or less, 10 or less, or 7 or less, and more preferably 5 or less. In the case of site-directed mutagenesis, for example, in addition to the desired variation, i.e., a specific mismatch, synthetic oligonucleotide primers complementary to the single-stranded phage DNA to be mutated can be used in the following manner. That is, a strand complementary to the phage is synthesized using the synthetic oligonucleotide as a primer, and the resulting double-stranded DNA is used to transform a host cell. The culture of the transformed bacteria was plated on agar and plaques were formed from phage-containing single cells. Then, plaques hybridized with the probe were collected and cultured to recover DNA. Further, there are methods of deleting, substituting, inserting and/or adding one or more amino acids from an amino acid sequence of a biologically active peptide such as an enzyme while maintaining its activity, and in addition to the above-mentioned site-directed mutagenesis, there are also methods of treating a gene with a mutagenesis source, and methods of selectively cleaving a gene, then deleting, substituting, inserting or adding a selected nucleotide, and then ligating it.

The term "single domain antibody (sdAb)" as used herein refers to a fragment comprising a single variable domain of an antibody, also known as a Nanobody. Like an intact antibody, it binds selectively to a particular antigen. The single domain antibody appears much smaller, approximately only 12-15 kDa, compared to the 150-160 kDa mass of the intact antibody. The first single domain antibody was artificially engineered from a camelid heavy chain antibody, referred to as a "VHH segment".

The term "identity" of sequences as used herein is used interchangeably with "identity" and refers to the degree of similarity between sequences as determined by sequence alignment software, such as BLAST. Methods and software for sequence alignment are well known to those skilled in the art. An engineered nucleotide sequence may be obtained by substitution, deletion and/or addition of one or several (e.g., 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more) amino acids or bases to a known sequence. For example, by conventional means (e.g., conservative substitutions, etc.), the sequences of SEQ ID NOs: 1-198, and can have greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% sequence identity thereto, and substantially the same properties, all within the scope of the present invention. Preferably, the present invention obtains sequence identity by conservative substitutions, but is not limited to conservative substitutions.

The term "complementary" as used herein refers to two nucleotide sequences comprising antiparallel nucleotide sequences capable of pairing with each other upon hydrogen bonding between complementary base residues of the antiparallel nucleotide sequences. It is known in the art that the nucleotide sequences of two complementary strands are reverse complementary to each other when the sequences are viewed in both 5 'to 3' directions. It is also known in the art that two sequences that hybridize to each other under a given set of conditions do not necessarily have to be 100% perfectly complementary.

The term "amino acid sequence" refers to the sequence of amino acids linked together to form a peptide chain (or polypeptide), and the amino acid sequence can only be read in one orientation. There are more than 100 different types of amino acids, 20 of which are commonly used, and the present invention does not exclude other substances such as saccharides, lipids, etc. from the amino acid chain, nor is the present invention limited to the amino acids commonly used in 20.

The term "nucleotide sequence" refers to the order of bases in DNA or RNA, i.e., A, T, G, C in DNA or A, U, G, C in mRNA, and also includes the order of bases in rRNA, tRNA and mRNA. It is understood that the antibody genes claimed in the present invention also encompass RNA (rRNA, tRNA, mRNA) and their complementary sequences in addition to DNA sequences.

The term "Expression vectors" refers to vectors in which Expression elements (e.g., promoter, RBS, terminator, etc.) are added to the basic backbone of a cloning vector to enable the Expression of a desired gene. The expression vector comprises four parts: target gene, promoter, terminator and marker gene. The present invention includes, but is not limited to, prokaryotic, eukaryotic, or other cellular expression vectors.

The term "Framework region", i.e., a Framework region, has a large variation of about 110 amino acid sequences near the N-terminus of H and L chains of an immunoglobulin, and the amino acid sequences of the other portions are relatively constant, whereby the light chain and the heavy chain can be distinguished into a variable region (V) and a constant region (C). The variable region includes the hypervariable region HVR (hypervariable region) or Complementarity determining region CDR (complementary-determining region) and FR framework regions. The "complementarity determining regions" in the antibodies described in the present invention are primarily responsible for antigen recognition.

The term "humanized" antibody refers to the Fr region portion of the variable region (VH or VHH), the constant region portion (i.e., the CH and CL regions) or all of the antibody being encoded by human antibody genes. Humanized antibodies can greatly reduce the immune side effects of heterologous antibodies on the human body. Humanized antibodies include chimeric antibodies, modified antibodies, fully humanized antibodies, and the like. It will be appreciated that those skilled in the art will be able to prepare suitable humanized forms of the single domain antibodies of the invention as required and within the scope of the invention.

The term "stringent hybridization conditions" means conditions of low ionic strength and high temperature as known in the art. Typically, a probe hybridizes to its target sequence to a greater extent (e.g., at least 2-fold over background) than to other sequences under stringent conditions. Stringent hybridization conditions are sequence dependent and will be different under different environmental conditions, with longer sequences specifically hybridizing at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the stringency of hybridization or wash conditions. For an exhaustive guidance of Nucleic acid Hybridization, reference is made to the literature (Tijssen, Techniques in biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of Hybridization and the" protocol of Nucleic acids analysis. 1993). More specifically, the stringent conditions are typically selected to be about 5-10 ℃ below the thermal melting point (Tm) of the specific sequence at a defined ionic strength pH. The Tm is the temperature (at a given ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because the target sequence is present in excess, 50% of the probes are occupied at Tm at equilibrium). Stringent conditions may be as follows: wherein the salt concentration is less than about 1.0M sodium ion concentration, typically about 0.01 to 1.0M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature is at least about 30 ℃ for short probes (including but not limited to 10 to 50 nucleotides) and at least about 60 ℃ for long probes (including but not limited to greater than 50 nucleotides). Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal can be at least two times background hybridization, optionally 10 times background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5 XSSC and 1% SDS, incubated at 42 ℃; or 5 XSSC, 1% SDS, incubated at 65 ℃, washed in 0.2 XSSC and washed in 0.1% SDS at 65 ℃. The washing may be for 5, 15, 30, 60, 120 minutes or more.

The terms "mutation" and "mutant" have their usual meanings herein, and refer to a genetic, naturally occurring or introduced change in a nucleic acid or polypeptide sequence, which has the same meaning as is commonly known to those of skill in the art.

The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce the recombinant host cell, e.g., direct uptake, transduction, f-pairing or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome.

Drawings

FIG. 1 shows the results of ELISA detection of the serum-specific antibody production levels before and after alpaca immunization (before and after Ig immunization, serum 10-fold serial dilutions ELISA were used to determine the antibody titer against Ig, and the abscissa is the

serum sample dilution

10²,10³,10⁴,10⁵,10⁶Multiple);

FIG. 2 shows the result of ELISA for detecting the titer of specific heavy chain antibody in immune serum separated by Protein G column (before Ig immunization and after 4 th immunization, the titer of heavy chain antibody obtained by diluting the serum by 500 times and then diluting it by 2 times in series);

FIG. 3 shows the first PCR amplification of heavy chain antibody gene and common heavy chain antibody (where M is marker with molecular weight of 100bp, 1,2,3,4 represent 4 different PCR reaction tubes, and the usage of 1,2,3,4 templates is 3,5,10, 15. mu.l, respectively);

FIG. 4 shows the second PCR amplification of VHH antibody target gene fragments (where M is marker with molecular weight of 100bp, and 1,2,3,4 represent 4 different PCR reaction tubes);

FIG. 5 shows SDS-PAGE results of proteins purified by Ni + ion affinity chromatography column for sdAb-C3-pSJF2 and sdAb-F12-pSJF 2;

FIG. 6 is a graph of the results of chromatography of sdAb-C3 on molecular sieve dextran gel (superG75) columns;

FIG. 7 bifunctional single domain antibody fusion protein HerG1M-sdAb-C3 via Ni⁺SDS-PAGE electrophoresis result of protein purified by the ion affinity chromatographic column;

FIG. 8 schematic representation of the linkage of the dual-function single-domain antibody fusion protein HerG 1M-sdAb-C3; 2 single-domain antibodies are connected by 2 different connecting peptides;

FIG. 9 shows the results of measuring the antibody titers of sdAb-C3, sdAb-F12 against human IgA, IgG and murine IgG by ELISA; wherein A is the antibody titer of sdAb-C3 against human IgA, IgG and murine IgG, and B is the antibody titer of sdAb-F12 against human IgA, IgG and murine IgG;

FIG. 10 shows the results of experiments on the inhibition of breast cancer cell growth by the dual-function single-domain antibody fusion protein HerG 1M-sdAb-C3;

FIG. 11 shows the results of ELISA assay for the change in antibody levels in mouse serum after administration.

Detailed Description

Embodiments of the present invention are illustrated by the following examples. However, embodiments of the invention are not limited to the specific details of these examples, as other variations will be known to those of ordinary skill in the art or will be apparent from the instant disclosure and the appended claims. Accordingly, all techniques implemented based on the above teachings of the present invention are within the scope of the present invention.

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biomaterials, if not specifically indicated, are commercially available.

EXAMPLE 1 mediated construction of Gene libraries for human immunoglobulin (Ig) -binding specific Single Domain antibodies

1. Alpaca immunization programs, monitoring of specific single domain antibody production

(1) Alpaca immunization program: selecting two adult sheep, performing subcutaneous multipoint injection on the back of the neck, preparing emulsion by isometric human IgG and IgA antigens and Freund's complete adjuvant for the first immunization, and preparing emulsion by isometric human IgG and IgA antigens and Freund's incomplete adjuvant for the 2 nd, 3 rd and 4 th immunizations; the 1 st immunization was 4 weeks apart, and the 2 nd, 3 rd, 4 th, and 5 th immunization were 3 weeks apart.

(2) After the 4 th immunization, the peripheral blood of alpaca (blood taken 1 st after the 4 th immunization) was collected 1 week after the 4 th immunization, and the antibody production level of the Ig antigen was measured by ELISA, and the results are shown in fig. 1. As is clear from FIG. 1, the serum titer was highest after the 5 th immunization (in the 2 nd blood collection). Separating heavy chain antibody in serum after 4 th immunization with Protein G, wherein the elution peak of 100mM acetate elution buffer is heavy chain antibody, the elution peak of 100mM glycine-hydrochloric acid elution buffer is common antibody, and the elution peak of pH2.7 is pH 3.5. The titer of the heavy chain antibody is detected, the specific result is shown in figure 2, and when the titer reaches more than 1 ten thousand times, 50ml of peripheral blood is collected and used for separating alpaca peripheral blood lymphocytes.

2. Alpaca peripheral blood lymphocyte separation and RNA purification

Extracting total RNA from separated alpaca peripheral blood lymph by using an RNA extraction kit of QIAGEN company according to the operation instruction of the kit, and obtaining the quality control of the RNA as follows: the OD260/280 ratio is equal to or more than 1.8, the concentration is more than 40ng/ul, and the extracted RNA is quickly used for a template for amplifying VHH by RT-PCR or is stored for standby at a temperature of-80 ℃ for a short time.

3. Amplification of alpaca heavy chain antibody variable region-VHH by RT-PCR and nested PCR method

Taking the RNA extracted in the step 2 as a template, adopting a cDNA synthesis kit of GE Life Science or Invitrogen company, and carrying out reverse transcription by using a primer pd (N)6 in the kit to synthesize cDNA; the specific process of reverse transcription was performed according to the instructions of the reverse transcription cDNA kit of GE Life science or Invitrogen.

The synthesized cDNA was used as a template for nested PCR amplification (PCR amplification reaction system (50. mu.l) was performed by using 3,5,10, 15. mu.l of cDNA template, 1. mu.l of upstream primer, 1. mu.l of downstream primer, 4. mu.l of 10mM dNTPs, 5. mu.l of 10-fold PCR buffer, 0.5. mu.l of Taq DNA polymerase and water to 50. mu.l, under PCR amplification conditions of 94 ℃ for 3 minutes, 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, 72 ℃ for 1 minute, 25-35 cycles of amplification and 72 ℃ for 7 minutes). The primers used for the first PCR amplification were: YT-15 'CGC CATCAA GGT ACC AGT TGA 3' and YTBN1:5 'GCC CAG CCG GCC ATG GCC SMK GTR CAG CTG GTGGAK TCT GGG GGA G3';

the amplified product is a common heavy chain (VH) gene segment with the length of more than 800bp, and a heavy chain antibody (VHH) gene segment with the length of 500-800 bp and a deleted light chain (figure 3); recovering and purifying heavy chain antibody (VHH) gene segments of the deleted light chain by adopting a gel recovery kit, and performing second PCR amplification by using the gene segments as templates and using VHH specific primers, wherein the primers adopted by the second PCR amplification are as follows: YTVHH-B2: 5 'CAT GTG TAG ATT CCT GGC CGG CCT GGC CTG AGG AGA CGG TGA CCTGG 3' and YTVHH-F2: 5 'CAT GTG CAT GGC CTA GAC TCG CGG CCC AGC CGG CCA TGG CC 3', the amplification product was a VHH of 500bp (FIG. 4).

4. Ligation of VHH fragments with phage display vectors and electrotransformation of TG1 competent

(1) SfI Single-digested VHH fragment: purifying the second PCR product (VHH) by using a QIAgen PCR kit, performing water bath at 50 ℃ for 3-5 hours by using restriction endonuclease sfII (New Biolabs), purifying and recovering the digested VHH fragment by using the QIAgen PCR kit, and storing at-20 ℃ for later use.

(2) The phage vector pHEN6 (modified from pHEN1, see Andrea Belllel. differential tumor-targeting antibodies of tumor single-domain antibodies formats. cancer Letters 289(2010) 81-90) was treated with restriction endonuclease sfII (New NewlandBiolabs) in a water bath at 50 ℃ for 3-5 hours. The digested pHEN6 vector was recovered by purification using QIAGEN PCR kit.

(3) Single domain antibody gene VHH and pHEN6 vectorAnd (3) a linking reaction: t is₄Mu.l ligase (NEB high concentration 5. mu.l), 3mmol cleaved VHH fragment, 1mmol cleaved pHEN6 vector, 4. mu.l ligase buffer, water make-up to 20. mu.l, ligation overnight at 16 ℃.

(4) And (3) bacterial transformation: the ligation reaction was purified, 1ul of the ligation reaction was added to E.coli TG1 competent cells, electrotransferred using an electrotransfer instrument (BioRad), plated on LB ampicillin plates, cultured overnight at 32 ℃ and the efficiency of single domain antibody gene VHH ligation was verified by colony PCR.

5. Identification and preservation of library capacity and diversity of VHH antibody gene libraries

The library volume of the VHH antibody gene library, which is approximately 10 volumes, was calculated from the titer determined after transformation multiplied by the total amount of transformation¹⁰Cloning of (4). In addition, after electric transformation, 30-50 positive clones growing on the plate are selected at random and screened, sequencing is carried out, and if no repeated sequence occurs, the diversity of the positive clones can be judged to be good. Adding a proper amount of 2YT/Amp culture medium into the residual bacterial liquid for 4 hours of culture, adding glycerol, subpackaging, and freezing at-80 ℃ to obtain the VHH antibody gene library for mediating and combining the immunoglobulin.

6. Titration of VHH antibody Gene libraries

The VHH antibody gene bank was rescued by adding the helper phage M13K07 (Invitrogen): mu. l M13K07 was added to infected cells containing 6mL of 2YT, 0.4mL of 20% glucose + 4. mu.l Amp (100mg/mL) medium, left to stand at 37 ℃ for 15min, this 6mL medium was added to a medium containing 92mL of 2YT, 92. mu.l AMP, left to stand for 15min, cultured at 37 ℃ at 250rmp for 1h, and then 100. mu.l Kan (50mg/mL) was added overnight at 37 ℃. After the rescue, 100 mul of supernatant is used for primary function identification of a VHH antibody gene bank, the remaining part is used for precipitating phage by PEG8000, the precipitate is collected by centrifugation under the condition of 4 ℃, the titer is determined, and the precipitate is stored at-80 ℃.

Example 2 mediated screening of immunoglobulin Single Domain binding antibodies

1. Screening for antibodies that mediate binding of immunoglobulin single domains (also known as anti-Ig specific single domain antibodies)

Coating of high adsorption of thermoelectric corporation with purified human IgG and IgA at 100ug/mlStyrene plate in micro well, 150 ul/well, 4 ℃ overnight. Removing the coating antigen by suction, adding 350ul 2% MPBST (2% skimmed milk, 0.05M, pH7.2-7.4PBS, 0.05% T20), sealing at room temperature for 2 hr, removing the sealing solution, adding phage 5 × 10 of the VHH antibody gene library¹¹Binding for 2 hours at room temperature, washing with PBST and PBS for 10 times respectively, adding TEA, standing at room temperature for 10min, eluting specifically bound phage, neutralizing TEA with 1M Tris-HCl (pH8.0Tris-HCl), and storing on ice. Infecting phage with TG1 growing in a semilog phase, diluting a proper amount of bacterial liquid, coating an AMP/LB plate, culturing at 32 ℃, determining the titer of eluent, performing amplification culture on the rest bacterial liquid, superinfecting with M13K07, performing shaking culture overnight, collecting supernatant on day 2, purifying with PEG, and concentrating to obtain a secondary phage antibody library for the next round of screening. After 3 rounds of screening, the titer of the specific phage antibody obtained after each round of screening is titrated, and the enrichment of the titer of the specific phage antibody eluted in the next round of screening is increased by 50-100 times compared with the previous round of screening, thereby showing that the screening is successful.

2. Selection of positive clones by phage ELISA

From the 3 rd round screening elution of specific phage titer AMP/LB plate, random picking single colony, inoculated in Amp 2YT liquid medium 96 hole culture plate culture, with helper phage infection induced expression phage antibody. And harvesting an expression supernatant, performing ELISA (enzyme-linked immunosorbent assay) by taking Ig as an antigen, selecting an Ig positive hole, and performing DNA sequencing to identify the gene sequence of the anti-Ig single-domain antibody clone to obtain a series of single-domain antibody gene sequences for further expressing and screening the specific and high-activity single-domain antibody.

EXAMPLE 3 construction of specific Single Domain antibody expression plasmid

PCR amplification was carried out using the gene sequence of the anti-Ig specific single domain antibody obtained in example 2 as a template to obtain PCR products carrying restriction endonuclease BbsI and BamHI sites, and the PCR products and pSJF2 vector (kim is. Biosic biochem.2002,66(5):1148-51, CN102321175A (ZL201110280031)) were treated with the restriction endonucleases BbsI and BamHI, respectively, and then subjected to T₄Ligase ligation and recombination are carried out to obtain plasmid sdAb-C3-pSJF2 which can be efficiently expressed in Escherichia coli, andgene sequencing to determine the correctness of its sequence. The sequences of the primers used for PCR were as follows:

a forward primer: 5 '-TATGAAGACACCAGGCCCAGGTRMAGCTGGWGGAGTCT-3';

reverse primer: 5'-gaagatctccggatccTGAGGAGACGGTGACCTGGGT-3' are provided.

Example 4 expression and purification of anti-Ig specific Single Domain antibodies

1. Expression, purification and identification of anti-Ig specific single domain antibody in Escherichia coli

(1) The strain containing plasmid sdAb-C3-pSJF2 described in example 3 was inoculated onto LB plates containing ampicillin and cultured overnight at 37 ℃. (2) Individual colonies were selected and inoculated into 12ml of LB medium containing ampicillin and shake-cultured overnight at 37 ℃. (3) Transferring 10ml of the seed into 1L 2YT culture solution containing ampicillin, carrying out shake culture at 37 ℃, carrying out 240 r/M, adding 0.1-0.5M IPTG when the OD value reaches 0.6-1.0, and continuing to culture overnight. (4) And (4) centrifuging and collecting bacteria. (5) Adding fusogenic enzyme to lyse bacteria, centrifuging, and collecting supernatant soluble single-domain antibody protein. (6) The soluble single-domain antibody protein is purified by a Ni + ion affinity chromatography column, and the SDS-PAGE result of the purified protein is shown in figure 5, and the purity of the purified protein can reach more than 95%.

2. Fast protein liquid chromatography

(1) Antigen and buffer preparation: the single domain antibody protein solution and PB buffer were filtered through a 0.22 μm microfiltration membrane. Preparing a chromatographic column: the Sephadex column (superG75) column was equilibrated with PB buffer (pH7.2) at a flow rate of 0.5 ml/min.

(2) Sample purification: injecting 1ml of filtered single-domain antibody sample into the sample ring by using a syringe, wherein the flow rate is 0.5ml/min, automatically measuring the A280nm value of the eluent by using an ultraviolet detector, and collecting the single-domain antibody required to be purified.

High pressure liquid phase analysis experiments by filtration through a molecular sieve sephadex column (superG75) showed that the time of appearance of the elution peak of the anti-Ig specific single domain antibody should be 15kd in peak molecular weight when compared with the standard molecular weight protein Marker (FIG. 6).

Sequencing to obtain the gene sequences of anti-Ig specific single domain antibodies sdAb-C3 and sdAb-F12, wherein the gene sequences are respectively shown in SEQ ID No.3 and SEQ ID No. 4; the amino acid sequences of the anti-Ig specific single domain antibodies sdAb-C3 and sdAb-F12 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

Example 5 construction of an anti-Ig Single Domain antibody sdAb-C3 and a humanized anti-Her 2/neu Single Domain antibody bispecific bifunctional Single Domain antibody fusion protein

1. Preparation of humanized anti-Her 2/neu Single Domain antibody

A humanized VH single-domain antibody gene library (NRC, Dr. Tanha J gift) is screened by a Her2/neu antigen (purchased from Beijing Yishenzhou biotechnology Co., Ltd.), and a humanized anti-Her 2/neu single-domain antibody (HerG1M) is obtained by the screening method as described in example 2, wherein the amino acid sequence of the humanized anti-Her 2/neu single-domain antibody is SEQ ID NO.19, and the gene sequence of the humanized anti-Her single-domain antibody is shown in SEQ ID NO. 20.

2. Fusion of anti-Ig single-domain antibody sdAb-C3 and humanized anti-Her 2/neu single-domain antibody (HerG1M) to construct bispecific bifunctional single-domain antibody fusion protein

The anti-Ig single-domain antibody sdAb-C3 and the humanized anti-Her 2/neu single-domain antibody (HerG1M) were constructed into a bi-specific bifunctional single-domain antibody fusion protein expression vector (HerG1M-sdAb-C3-pJSF2), and the expression and purification methods of the fusion protein were the same as those of example 4. Bifunctional antibody fusion protein (HerG1M-sdAb-C3) expressed by HerG1M-sdAb-C3-pJSF2 through Ni⁺The SDS-PAGE result after the purification of the ion affinity chromatographic column is shown in figure 7, the purity of the purified fusion protein can reach more than 90 percent, and the molecular weight of the fusion protein is about 31,000 kd.

The connection mode of the HerG1M-sdAb-C3 is shown in FIG. 8, and the single domain antibody HerG1M and the single domain antibody sdAb-C3 can be connected by a connecting peptide shown in any one of SEQ ID NO.21 or SEQ ID NO. 22. The amino acid sequence of linker peptide 1 is: GGGGSGGGGSGGGGS (SEQ ID NO. 21); the amino acid sequence of linker peptide 2 is: AEPKSCDKTHTCPPCPAEPEKSCDKTHTCPPCP (SEQ ID NO. 22).

Test example 1 anti-Ig specific Single Domain antibody titer (affinity) determination test

Human IgG, IgA and mouse IgG were coated onto ELISA plates at a concentration of 5. mu.g/ml overnight at 4 ℃. Blocking with 0.5% BSA-PBST was performed at 37 ℃ for 2 hours. A10-fold serial dilution of the anti-Ig single domain antibody (starting at 0.5mg/ml) was added at 37 ℃ for 2 hours, a 1: 3000-fold dilution of the HRP-anti-His 2 antibody was added at 37 ℃ for 1 hour, TMB was added thereto at room temperature for 15 minutes, and the OD450 value was measured. The final dilution factor with OD450 value greater than the control 2-fold value was used to calculate the antibody titer, and the detection results are shown in FIG. 9.

According to the test results, the single domain antibody sdAb-C3 and the single domain antibody sdAb-F12 obtained by screening have excellent affinity with human IgG, IgA and mouse IgG.

Experimental example 2 Dual-function bispecific Single Domain antibody fusion protein (HerG1M-sdAb-C3) inhibition of Breast cancer cell growth assay

1. Test method

BT474 cell culture: (1) preparing a constant-temperature water tank at 37 ℃; (2) preparing a fresh culture solution (an improved 1640 low-sugar culture medium + 10% FBS), centrifuging a tube; (3) taking out the frozen BT474 cells (a gift from a monoclonal antibody research laboratory, Beijing medical basic research institute) from a refrigerator at the temperature of-80 ℃, immediately putting the cells into a water bath at the temperature of 37 ℃, and quickly thawing the cells during activation so as to avoid the cell death caused by the recrystallization of ice crystals and the damage to the cells; (4) after thawing, sucking the cells into a centrifuge tube, and adding 2mL of fresh culture solution to reduce the damage of DMSO to the cells; (5) centrifuging the mixture in a low-speed centrifuge at 1000r/mim for 10 min; (6) the supernatant was aspirated, 4mL of fresh medium was added to the centrifuge tube, the cells were suspended by blowing and aspirated into a petri dish, and CO was added₂An incubator.

CCK8 experiments: (1) 100. mu.L of BT474 cell suspension was prepared in a 96-well plate to give a cell concentration of 10⁵Perml, 96-well plates were pre-incubated in an incubator for 24 hours (5% CO at 37 ℃)₂Under the conditions of (a); (2) adding 10 μ L of different concentrations of drugs (Herceptin, HerG1M-sdAb-C3, sdAb-C3) to 96-well plates, (3) incubating the 96-well plates in an incubator for 24h, 48h, 72 h; (4) add 10. mu.L of CCK solution to each well of a 96-well plate (care is taken not to generate bubbles in the wells which would affect the OD reading); (5) incubating the 96-well culture plate in an incubator for 2 hours; (6) the absorbance at 450nm was measured by a microplate reader, and the results are shown in FIG. 10.

2. Test results

As can be seen from FIG. 10, the bispecific single domain antibody fusion protein HerG1M-sdAb-C3 and the clinically applied antibody drug Herceptin all have an inhibitory effect on the growth of breast cancer cell BT474, and the bispecific single domain antibody fusion protein HerG1M-sdAb-C3 has about 50 times stronger inhibitory effect on breast cancer cell BT474 than Herceptin.

Experimental example 3 dynamic Change test of bispecific bifunctional Single Domain antibody fusion protein HerG1M-sdAb-C3 in animals

Purified bispecific bifunctional single domain antibody fusion proteins HerG1M-sdAb-C3 and anti-Her 2/neu single domain antibody (HerG1M) were injected into mice at 100ug/0.1ml PBS, respectively, via tail vein, 3 mice were injected each, and blood was collected at 15min, 30 min, 1 hr, 1 day, 3 days, and 7 days after injection. The dynamic changes of the fusion protein HerG1M-sdAb-C3 and the anti-Her 2/neu single domain antibody (HerG1M) in the collected mouse serum were measured by ELISA method, and the specific results are shown in FIG. 11. As can be seen from FIG. 11, compared with the single anti-Her 2/neu single domain antibody, the half-life time of the bifunctional antibody obtained by fusing the single domain antibody sdAb-C3 and the anti-Her 2/neu single domain antibody in the animal body is remarkably prolonged, which indicates that the single domain antibody sdAb-C3 of the invention has the effects of prolonging the half-life of the anti-Her 2/neu single domain antibody in the organism, improving the bioavailability of the anti-Her 2/neu single domain antibody and enhancing the biological targeting treatment effect of the anti-Her 2/neu single domain antibody.

SEQUENCE LISTING

<110> Beijing Newcastle Biotechnology Ltd

<120> Single domain antibody mediating binding to immunoglobulin, bifunctional antibody constructed therefrom and use thereof

<130>BJ-3038-191105A

<160>22

<170>PatentIn version 3.5

<210>1

<211>123

<212>PRT

<213>Artifical sequence

<400>1

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Arg Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Tyr Glu Leu Val

35 40 45

Ala Val Ile Thr Ser Ala Gly Ser Thr Asn Tyr Ala Asp Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

His Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala His Thr Asp Tyr Thr Asn Asp Asp Ala Val Pro Ile Ser Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>2

<211>118

<212>PRT

<213>Artifical sequence

<400>2

Gln Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Ala

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Arg Asp

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Asp Glu Glu Phe Val

35 40 45

Ala Ala Ile Asn Trp Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 7075 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Arg Cys

85 90 95

Ala Ala Glu Gly Arg Asn Gly Val Tyr Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser

115

<210>3

<211>369

<212>DNA

<213>Artifical sequence

<400>3

caggtaaagc tggaggagtc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60

tcctgtgcgg cctctggaag catcttcagt cgcaacgcca tgggctggta ccgacaggct 120

ccagggaagc agtacgagtt ggtcgcagtt attactagtg ctggtagcac aaactatgcg 180

gacttcgcga agggccgatt caccatcgcc agagacaatg ccaagaacac ggtgtatcta 240

cacatgaaca acctgaaacc tgaggacacg gccgtctatt actgtaatgc ccacacggac 300

tataccaacg atgatgccgt ccctatctcg gactactggg gccaggggac ccaggtcacc 360

gtctcctca 369

<210>4

<211>354

<212>DNA

<213>Artifical sequence

<400>4

caggtacagc tggaggagtc tgggggagga ttggtgcagg ctggggcctc tctgagactc 60

tcctgtgcag cctctggacg caccttcagt agagatgcca tgggctggtt ccgccaggct 120

ccagggaagg acgaagagtt tgtagcagct attaactgga gtggtggtag cacatcgtac 180

gcagactccg tgaagggccg attcgccatc tccagagaca acgccaagaa cacggtgtat 240

ctgcaaatga acaacctgaa acctgaggac acggccgttt atcggtgtgc agcagagggt 300

aggaacgggg tgtatgacta ctggggccag gggacccagg tcaccgtctc ctca 354

<210>5

<211>5

<212>PRT

<213>Artifical sequence

<400>5

Arg Asn Ala Met Gly

1 5

<210>6

<211>18

<212>PRT

<213>Artifical sequence

<400>6

Val Ala Val Ile Thr Ser Ala Gly Ser Thr Asn Tyr Ala Asp Phe Ala

1 5 10 15

Lys Gly

<210>7

<211>17

<212>PRT

<213>Artifical sequence

<400>7

Cys Asn Ala His Thr Asp Tyr Thr Asn Asp Asp Ala Val Pro Ile Ser

1 5 10 15

Asp

<210>8

<211>5

<212>PRT

<213>Artifical sequence

<400>8

Arg Asp Ala Met Gly

1 5

<210>9

<211>19

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<213>Artifical sequence

<400>9

Val Ala Ala Ile Asn Trp Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser

1 5 10 15

Val Lys Gly

<210>10

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<212>PRT

<213>Artifical sequence

<400>10

Cys Ala Ala Glu Gly Arg Asn Gly Val Tyr Asp

1 5 10

<210>11

<211>30

<212>PRT

<213>Artifical sequence

<400>11

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser

20 25 30

<210>12

<211>12

<212>PRT

<213>Artifical sequence

<400>12

Trp Tyr Arg Gln Ala Pro Gly Lys Gln Tyr Glu Leu

1 5 10

<210>13

<211>29

<212>PRT

<213>Artifical sequence

<400>13

Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu His

1 5 10 15

Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

20 25

<210>14

<211>12

<212>PRT

<213>Artifical sequence

<400>14

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210>15

<211>30

<212>PRT

<213>Artifical sequence

<400>15

Gln Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Ala

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser

20 25 30

<210>16

<211>12

<212>PRT

<213>Artifical sequence

<400>16

Trp Phe Arg Gln Ala Pro Gly Lys Asp Glu Glu Phe

1 5 10

<210>17

<211>29

<212>PRT

<213>Artifical sequence

<400>17

Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln

1 5 10 15

Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Arg

20 25

<210>18

<211>12

<212>PRT

<213>Artifical sequence

<400>18

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210>19

<211>125

<212>PRT

<213>Artifical sequence

<400>19

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Ile Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Ile Ile Asp Glu

20 25 30

Lys Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Ser Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ala Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Ala Val Lys Pro Lys Cys Asn Met Gln Asn Cys Arg Ala Ser Val

100 105 110

Asn Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>20

<211>375

<212>DNA

<213>Artifical sequence

<400>20

caggtgaagc tggaggagtc tgggggaggc ttgataaagc ctggggggtc ccttagactc 60

tcctgtgcag cctctggatt taggattatc gatgagaaga tggcctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtagta gtggtggtag cacatactac 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cgccgtgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtat attactgtgt ggcagtgaag 300

ccgaagtgta atatgcaaaa ttgcagggcg tcggttaact tttggggcca agggacccag 360

gtcaccgtct cctca 375

<210>21

<211>15

<212>PRT

<213>Artifical sequence

<400>21

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>22

<211>33

<212>PRT

<213>Artifical sequence

<400>22

Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Glu Pro Glu Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

2025 30

Pro

Claims

1. A single domain antibody that mediates binding to an immunoglobulin, wherein the single domain antibody is the single domain antibody sdAb-C3 or the single domain antibody sdAb-F12; wherein the amino acid sequence of the single domain antibody sdAb-C3 comprises 3 complementarity determining regions and 4 framework regions; the amino acid sequences of the 3 complementarity determining regions are respectively shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 7; the amino acid sequences of the 4 framework regions are respectively shown as SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO. 14;

the amino acid sequence of the single-domain antibody sdAb-F12 comprises 3 complementarity determining regions and 4 framework regions, wherein the amino acid sequences of the 3 complementarity determining regions are shown in SEQ ID No.8, SEQ ID No.9, and SEQ ID No.10, respectively; the amino acid sequences of the 4 framework regions are respectively shown as SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO. 18.

2. The single domain antibody according to claim 1, characterized in that the amino acid sequence of the single domain antibody sdAb-C3 is selected from any one of the amino acid sequences (1) - (3): (1) an amino acid sequence shown as SEQ ID NO. 1; or (2) a protein mutant obtained by deleting, substituting, inserting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.1, wherein the protein mutant has the same function with the protein before mutation; or (3) an amino acid sequence with at least 75 percent of identity with the amino acid sequence shown in SEQ ID NO. 1;

the amino acid sequence of the single-domain antibody sdAb-F12 is selected from any one of amino acid sequences (1) - (3):

3. The gene encoding the single domain antibody of claim 1 or 2, wherein the nucleotide sequence of the gene encoding the single domain antibody sdAb-C3 is selected from any one of (1) to (3):

(1) a polynucleotide sequence shown as SEQ ID NO. 3; or (2) a polynucleotide sequence that hybridizes to the complement of the polynucleotide sequence set forth in SEQ ID No.3 under stringent hybridization conditions; or (3) a polynucleotide sequence having at least 75% identity to the polynucleotide sequence shown in SEQ ID NO. 3; preferably, the polynucleotide sequence has at least more than 80% identity with the polynucleotide sequence shown in SEQ ID NO. 3; further preferably, the polynucleotide sequence has at least more than 85% identity with the polynucleotide sequence shown in SEQ ID NO. 3; more preferably, the polynucleotide sequence has at least 95% identity with the polynucleotide sequence shown in SEQ ID NO. 3; most preferably, a polynucleotide sequence having more than 99% identity to the polynucleotide sequence shown in SEQ ID NO. 3;

the nucleotide sequence of the gene encoding the single domain antibody sdAb-F12 is selected from any one of (1) to (3):

4. A humanized anti-Her 2/neu single domain antibody, characterized in that its amino acid sequence is selected from any one of the amino acid sequences (1) - (3):

5. The gene encoding the humanized anti-Her 2/neu single domain antibody of claim 4, wherein the nucleotide sequence encoding the gene is selected from the group consisting of any one of (1) - (3):

6. A bifunctional single domain antibody, which is obtained by linking the single domain antibody of claim 1 with the humanized anti-Her 2/neu single domain antibody of claim 4 by a linker; preferably, the amino acid sequence of the connecting peptide linker is the amino acid sequence shown in SEQ ID NO.21 or SEQ ID NO. 22.

7. An expression vector comprising the coding gene of claim 3 or 5.

8. Use of the coding gene of claim 3 or 5 in the preparation of a reagent for diagnosing or detecting a disease caused by infection with a tumor or a pathogenic microorganism or a medicament for treating a disease caused by infection with a tumor or a pathogenic microorganism; or in the preparation of medicaments for enhancing the action effect and the stability of the therapeutic antibody or the polypeptide.

9. Use of the single domain antibody of claim 1,2 or 4 for the preparation of an agent for diagnosing or detecting a disease caused by infection with a tumor or a pathogenic microorganism or a medicament for treating a disease caused by infection with a tumor or a pathogenic microorganism; or in the preparation of medicaments for enhancing the action effect and the stability of the therapeutic antibody or the polypeptide; preferably, the therapeutic antibody is an antibody against a virus, a bacterium or a tumor.

10. Use of the bifunctional antibody of claim 5 in the preparation of a reagent for diagnosing or detecting a disease caused by infection by a tumor or a pathogenic microorganism or a medicament for treating a disease caused by infection by a tumor or a pathogenic microorganism.