Strep-tag II specific antibodies and their applications

Abstract

The present disclosure provides a Nanobody or an antigen-binding fragment thereof specifically binding to Strep-Tag II, which can specifically recognize and bind to Strep-Tag II and has good affinity therewith.

Description

Technical Field

[0001] This disclosure belongs to the field of biotechnology, specifically relating to Strep-tag II specific antibodies and their applications. Background Technology

[0002] Strep-Tag II is an 8-amino acid polypeptide (WSHPQFEK), which is associated with streptavidin or its mutants (such as...). Specific binding. Strepavidin is a tetrameric protein derived from Streptomyces that exhibits a strong binding affinity for biotin. To utilize this strong interaction between streptavidin and biotin for recombinant protein purification, researchers aimed to design a peptide that, upon fusion with a recombinant protein, would bind to the biotin-binding pocket of streptavidin, thus serving as a purification tag. Strep-tag technology (IBA LifeScience) is based on the highly selective and easily controllable interaction between the Strep-tag II peptide and engineered streptavidin. Strep-tag II specifically binds to Strep-Tactin by occupying the binding pocket of the natural biotin ligand. This interaction can be easily reversed by overdosing on competitive substrates such as the natural biotin ligand, allowing for the elution and purification of recombinant proteins fused with Strep-tag II. Since the relative molecular mass of Strep-tag II is only about 1.06 kDa, it does not affect the structure and function of the fused protein, thus eliminating the need to remove the tag. Strep-tag II has been rapidly and widely used due to its outstanding features such as high specificity, high purity in single-step purification, mild purification conditions, and the ability to fuse both ends of the protein. Summary of the Invention

[0003] This disclosure provides a specific antibody against STEP-TAG II. Using the specific antibody provided in this disclosure, it is possible to specifically recognize and bind to STEP-TAG II, and it exhibits good affinity for it.

[0004] According to the first aspect of this disclosure, a nanobody that specifically binds to STREP-TAG II or an antigen-binding fragment thereof is provided.

[0005] In some embodiments, the nanobody that specifically binds to STREP-TAG II or its antigen-binding fragment includes CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region having the amino acid sequences shown in SEQ ID NO: 19-22.

[0006] In some implementations, each CDR is defined by any numbering system commonly used by those skilled in the art. Exemplary numbering systems include, but are not limited to, Kabat, AbM, Chothia, Contact, IMGT, or combinations thereof.

[0007] In some embodiments, a nanobody or antigen-binding fragment thereof specifically binds to STEP-TAG II, wherein the nanobody or antigen-binding fragment specifically binding to STEP-TAG II comprises:

[0008] a1) includes the heavy chain variable regions of the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 7, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 8, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 9;

[0009] a2) includes the heavy chain variable regions of the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 10, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 11, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 12;

[0010] a3) includes the heavy chain variable regions of the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 13, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 14, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 15;

[0011] a4) includes the heavy chain variable regions of the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 16, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 17, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 18.

[0012] In some embodiments, the heavy chain variable region of the nanobody that specifically binds to STREP-TAG II or its antigen-binding fragment further includes a framework region.

[0013] In some embodiments, the frame region includes the frame region of an immunoglobulin derived from a mouse, primate, cow, horse, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, or goose, or a mutant thereof.

[0014] In some embodiments, the nanobody that specifically binds to STREP-TAG II or its antigen-binding fragment comprises:

[0015] b1) Heavy chain variable region, comprising the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0016] b2) Heavy chain variable region, comprising the amino acid sequence shown in SEQ ID NO: 20, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0017] b3) Heavy chain variable region, comprising the amino acid sequence shown in SEQ ID NO: 21, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0018] b4) Heavy chain variable region, which includes the amino acid sequence shown in SEQ ID NO: 22, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0019] In some embodiments, the STEP-TAG II is the amino acid sequence shown in SEQ ID NO: 66.

[0020] According to a second aspect of this disclosure, a heavy chain antibody or antigen-binding fragment thereof that specifically binds to STREP-TAG II is provided, comprising an immunoglobulin Fc domain and a nanobody or antigen-binding fragment thereof of the first aspect of this disclosure.

[0021] In some embodiments, the immunoglobulin Fc domain includes the Fc domain of an immunoglobulin derived from a mouse, primate, cow, horse, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, or goose, or a mutant thereof.

[0022] According to a third aspect of this disclosure, a chimeric antigen receptor is provided, comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof of the first aspect of this disclosure or a heavy chain antibody or an antigen-binding fragment thereof of the second aspect.

[0023] According to a fourth aspect of this disclosure, a multispecific antibody or antigen-binding fragment thereof is provided, comprising two or more (e.g., three or four) antigen-binding domains, wherein one antigen-binding domain comprises a nanobody or antigen-binding fragment thereof of the first aspect of this disclosure or a heavy chain antibody or antigen-binding fragment thereof of the second aspect.

[0024] According to a fifth aspect of this disclosure, an isolated nucleic acid molecule is provided, comprising a nucleotide sequence encoding a nanobody or antigen-binding fragment thereof of the first aspect of this disclosure, a heavy chain antibody or antigen-binding fragment thereof of the second aspect, a chimeric antigen receptor of the third aspect, or a multispecific antibody or antigen-binding fragment thereof of the fourth aspect.

[0025] Those skilled in the art will understand that nucleotides in nucleic acid molecules can be substituted based on codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized.

[0026] In some embodiments, the nucleotide sequence encoding the nanobody or its antigen-binding fragment of the first aspect of this disclosure includes: SEQ ID NO: 19-22, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0027] According to the sixth aspect of this disclosure, a carrier is provided that includes the nucleic acid molecule of the fifth aspect of this disclosure.

[0028] In some embodiments, the vector may be an expression vector. In some embodiments, the expression vector may include eukaryotic cell expression vectors and / or prokaryotic cell expression vectors. In some embodiments, the eukaryotic expression vector includes, for example, but not limited to, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0029] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0030] According to a seventh aspect of this disclosure, a cell is provided that includes a nanobody or antigen-binding fragment thereof of the first aspect of this disclosure, a heavy chain antibody or antigen-binding fragment thereof of the second aspect, a chimeric antigen receptor of the third aspect, a multispecific antibody or antigen-binding fragment thereof of the fourth aspect, a nucleic acid molecule of the fifth aspect, or a carrier of the sixth aspect.

[0031] In some embodiments, the cells do not involve reproductive material.

[0032] In some embodiments, the cell can be a host cell conventionally used in the art, as long as the expression vector stably expresses the carried nucleic acid molecule as the nanobody or its antigen-binding fragment, heavy chain antibody or its antigen-binding fragment, chimeric antigen receptor or multispecific antibody or its antigen-binding fragment disclosed herein. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell. The prokaryotic cell may include, for example, *Escherichia coli*, and the eukaryotic cell may include, for example, CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, Vero cells, Expi293 cells, hybridoma cells, yeast cells, and insect cells.

[0033] In some embodiments, the cells may be immune cells. In some embodiments, the immune cells may include, but are not limited to, T cells, NK cells, dendritic cells (DCs), and macrophages. In these embodiments, the immune cells may express the chimeric antigen receptors described above (i.e., modified immune cells).

[0034] According to the eighth aspect of this disclosure, a method for preparing a nanobody or antigen-binding fragment thereof of the first aspect of this disclosure, a heavy chain antibody or antigen-binding fragment thereof of the second aspect, a chimeric antigen receptor of the third aspect, or a multispecific antibody or antigen-binding fragment thereof of the fourth aspect is provided, obtained by culturing cells according to the seventh aspect of this disclosure.

[0035] According to the ninth aspect of this disclosure, a conjugate is provided, comprising a nanobody or an antigen-binding fragment thereof of the first aspect of this disclosure, or a heavy chain antibody or an antigen-binding fragment thereof of the second aspect of this disclosure; and a conjugation portion.

[0036] In some implementations, the coupling portion may include, but is not limited to, a detectable marker or a therapeutic agent.

[0037] In some embodiments, the detectable marker can be any substance detectable by means of fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, chemistry, etc. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 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 (e.g., Cy7, Alexa 750)), acridine esters, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable markers described above can be linked to the nanobodies or their antigen-binding fragments, or heavy chain antibodies or their antigen-binding fragments, of this disclosure using linkers of different lengths to reduce potential steric hindrance.

[0038] In some embodiments, the detectable marker may include, but is not limited to, enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), colored substances, streptavidin and / or biotin, etc.

[0039] In some embodiments, the coupling portion is selected from substances that can improve the biological properties of the antibody (e.g., increase serum half-life), such as chemical groups, such as polyethylene glycol (PEG), methyl, ethyl, or glycosyl groups.

[0040] According to the tenth aspect of this disclosure, a detection or purification kit is provided, comprising: a nanobody or antigen-binding fragment thereof of the first aspect of this disclosure, a heavy chain antibody or antigen-binding fragment thereof of the second aspect, a chimeric antigen receptor thereof of the third aspect, a multispecific antibody or antigen-binding fragment thereof of the fourth aspect, a nucleic acid molecule thereof of the fifth aspect, a carrier thereof of the sixth aspect, a cell thereof of the seventh aspect, or a conjugate thereof of the ninth aspect.

[0041] In some embodiments, the kit can be used to detect or purify proteins carrying STREP-TAG II.

[0042] In some embodiments, the kit may also include instructions.

[0043] In some implementations, the detection includes an immune detection.

[0044] In some embodiments, the immunoassay includes one or more of the following: immunochromatographic assay, enzyme-linked immunosorbent assay (ELISA), or chemiluminescent immunoassay.

[0045] In some embodiments, the immune detection includes one or more of the following: Western blotting, immunofluorescence detection, or immunoprecipitation.

[0046] In some embodiments, the purification includes affinity chromatography.

[0047] The beneficial effects of this disclosure are:

[0048] This disclosure provides nanobodies or antigen-binding fragments thereof that specifically bind to STEP-TAG II, which can specifically recognize and bind to STEP-TAG II and have good affinity for it. Attached Figure Description

[0049] Figure 1 The ELISA assay demonstrates the affinity of 1C11 nanobody for STREP-TAG II.

[0050] Figure 2 The ELISA assay demonstrates the affinity of 1F9 nanobody for STREP-TAG II.

[0051] Figure 3 The ELISA assay demonstrates the affinity of 1C9 nanobody for STREP-TAG II.

[0052] Figure 4 The ELISA assay demonstrates the affinity of 3B9 nanobody for STEP-TAG II. Detailed Implementation

[0053] The present invention aims to develop nanobodies based on the alpaca immune system, design and implement effective and feasible nanobodies screening and preparation technology schemes, and obtain nanobodies that can specifically recognize Strep-tag II.

[0054] definition

[0055] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0056] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references.

[0057] The term "about" as used herein is as understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is unfamiliar with the use of this term in the context in which it is used, "about" will mean a particular value plus or minus 10%.

[0058] As used herein, the term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in the binding of an antigen-binding molecule to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain is sufficient to confer antigen-binding specificity. The term "variable" in this invention refers to the fact that certain segments of the variable domain are generally sequence-differentiated between antibodies. The FR domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed throughout the variable domain. Instead, it is concentrated in three segments within the variable domains of both the light and heavy chains, called hypervariable regions (HVRs). The more highly conserved portions of the variable domain are called frame regions (FRs). The variable domains of the natural heavy and light chains each contain four FRs, mostly in a β-sheet configuration, linked by three HVRs that form loops and, in some cases, form part of a β-sheet structure. The HVRs in each chain are tightly held together by the FR regions and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site. The constant domain does not directly participate in antibody-antigen binding but has other effector functions, such as participating in antibody-dependent cytotoxicity.

[0059] The term "nanobody" used in this article can also be referred to as a single-domain antibody (sdAb), a heavy-chain single-domain antibody (VHH), or a camelid antibody. It refers to a naturally occurring antibody lacking a light chain, found in the peripheral blood of camels. This antibody contains only one heavy-chain variable region (VH) and two conventional CH2 and CH3 regions. The heavy-chain variable region includes four conserved framework regions (FRs) and three hypervariable regions (HVRs), or complementarity-determining regions (CDRs). Unlike artificially engineered single-chain antibody fragments, nanobodies do not easily adhere to each other or aggregate. Individually cloned and expressed VHH structures possess structural stability and antigen-binding activity comparable to the original heavy-chain antibody, and are the smallest known unit capable of binding target antigens. VHH crystals are 2.5 nm in size, 4 nm in length, and have a molecular weight of only about 15 kDa, hence the name nanobody (Nb). Compared to traditional animals such as mice and rabbits, which can only recognize flat peptides on the surface of antigens, the immune system in camels can recognize the complex spatial structure on the surface of antigens, and can produce highly specific and high-affinity nanobodies.

[0060] Unlike traditional technologies that rely on classic model animals such as mice, rabbits, monkeys, and sheep, the disclosed technology utilizes antibodies produced by the alpaca's immune system, known as "nanobodies." Nanobodies are tiny antibody fragments isolated from immunoglobulins in animals such as camels. They possess the same antigen-binding ability and structural stability as intact antibodies and are the smallest existing units capable of binding target antigens, with a relative molecular mass of only about 15 kDa. Compared to traditional methods where mice and rabbits can only recognize flat polypeptides on the surface of antigens, the immune systems in animals like alpacas can recognize the complex spatial structures on the antigen surface, enabling the production of highly specific and high-affinity nanobodies.

[0061] According to the technical solution of this disclosure, certain amino acids in the amino acid sequence can be conservedly substituted without changing the activity or function of the protein, as shown in Table 1 below:

[0062] Table 1

[0063]

[0064]

[0065] In this disclosure, the term "affinity" or "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Binding affinity is typically expressed as a dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constants (koff and kon, respectively). Therefore, equivalent affinity can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured using conventional methods known in the art, such as surface plasmon resonance (sPR). The smaller the equilibrium dissociation constant, the more tightly the antibody or its antigen-binding fragment binds to STREP-TAGII according to this disclosure.

[0066] In this disclosure, the term "specific binding" refers to binding selectivity for an antigen, which can be distinguished from unwanted or nonspecific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (sPR) techniques and conventional binding assays. In one embodiment, for example, as measured by SPR, the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen.

[0067] As used herein, the terms “% sequence identity” or “sequence identity” are used in the context of this invention to describe the degree of similarity between two nucleotide sequences or two amino acid sequences, and have the same meaning as “percentage identity”. The percentage homology of two sequences can be calculated by dividing the number of identical residue positions by the total length of the aligned sequence after alignment, and then multiplying by 100%. Methods and tools for aligning two amino acid sequences or nucleotide sequences are well known in the art, such as the BLAST kit available on the NCBI website (Altschul, SF et al. (1990) J. Mol. Biol. 215: 403-410). The phrase "at least 80% sequence identity" as used herein refers to having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence.

[0068] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. The actual scope of protection of this invention is set forth in the claims. In the following description, descriptions of well-known structures and techniques are omitted to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications. Unless otherwise specified, the equipment, instruments, reagents, and / or kits used in the following embodiments are commercially available or obtained through conventional methods known to those skilled in the art.

[0069] Example

[0070] Example 1: Preparation of Antigen

[0071] (1) Peptide synthesis: Strep-tag II peptide was synthesized based on the amino acid sequence (sEQ ID NO: 66: WSHPQFEK) (Nanjing Genscript).

[0072] (2) Antigen preparation: BSA conjugation kit (AAT Bioquest, ReadiLink) was used. TM The BSA Conjugation Kit conjugates Strep-tag II peptides with fetal bovine serum albumin (BSA) according to the instructions, resulting in BSA-Strep-tag II, which is the antigen.

[0073] Example 2. Alpaca Immunization

[0074] The specific technical solution is as follows:

[0075] (1) Alpacas were immunized a total of 4 times using the BSA-Strep-tag II (antigen) obtained in Example 1, with 0.2 mg of antigen injected subcutaneously into the animals each time. The first immunization was recorded as day 1, and the subsequent immunizations were recorded on day 11, day 21, and day 31, respectively;

[0076] (2) On day 30, before the fourth immunization injection, about 200 mL of alpaca peripheral blood was collected from the vein;

[0077] (3) On day 45, that is, 14 days after the fourth immunization, about 200 mL of alpaca peripheral blood was collected.

[0078] Compared to traditional immunization techniques using animal antibodies from mice and rabbits, the advantage of this invention lies in the collection of a large amount of peripheral blood from alpacas, which facilitates subsequent screening to obtain highly diverse nanobodies.

[0079] Example 3. Construction of an alpaca nanobody library

[0080] Using two batches of alpaca peripheral blood collected in Example 2 as raw materials, a highly diverse nanobody library was constructed. The processing methods for the two batches of alpaca peripheral blood were the same, and the specific technical solution is as follows:

[0081] (1) Lymphocytes were isolated from peripheral blood of alpaca veins using density gradient centrifugation and other methods.

[0082] (2) Extract total mRNA from lymphocytes and reverse transcribe it into cDNA.

[0083] (3) Using the DNA primers shown in Table 2, the above cDNA was used as a template to amplify the VHH fragments of alpaca immunoglobulins IgG2 and IgG3 by polymerase chain reaction (PCR), which are the DNA fragments of nanobodies.

[0084] Table 2. DNA Primers

[0085]

[0086] (4) The DNA of VHH was ligated into the phage surface display and selection vector (pHEN1 with his and c-myc tag nucleic acid sequences) to form a VHH-pIII fusion protein expression vector plasmid library. Among them, pIII is a protein present on the flagella of the phage surface.

[0087] (5) The DNA ligation product was transformed into TG1 competent bacteria by electroporation. After appropriate culture, all colonies were collected to form the alpaca nanobody library.

[0088] Compared to traditional methods of isolating antibodies from the serum or lymphocytes of animals such as mice and rabbits, the method disclosed herein can preserve all nanobody fragments (i.e., libraries) of alpacas for a long time, which can continuously support the subsequent screening and development of nanobodies.

[0089] Example 4. Displaying screening-specific nanobodies on the surface of bacteriophages

[0090] Using the nanobody library obtained in Example 3 as the source, antigen-specific nanobodies were obtained through phage surface display screening. The specific technical solution is as follows:

[0091] (1) Take an appropriate amount of frozen nanobody library and inoculate it into LB medium containing host Escherichia coli TG1. After appropriate culture, add an appropriate amount of helper phage (M13KO7 helper phage, NEB, N0315S) and continue to culture under appropriate conditions.

[0092] (2) Phages amplified in bacterial culture supernatant were extracted using the PEG-NaC method;

[0093] (3) Incubate the phage with the antigen (BSA-Strep-tagII antigen prepared in Example 1) appropriately. The STREP-TAG II antigen is pre-fixed in an immunoassay tube (Maxisorp immunoassay tube, ThermoFisher Scientific).

[0094] (4) Washing. Discard the phages, then wash the antigen with PBS buffer an appropriate number of times (3-5 times) to remove the phages that are not specifically bound to the antigen, and retain the phages that are specifically bound to the antigen.

[0095] (5) Elution. Incubate with 0.1M hydrochloric acid solution (pH adjusted to 2.2 with glycine and containing 0.1% BSA) at room temperature for 30 minutes to obtain phages that have dissociated from the antigen. Immediately add an equal volume of 2M Tris buffer (pH 8.0) to dissociate the phages from the antigen and retain them.

[0096] At this point, bacteriophages expressing specific nanobodies have been obtained, and these bacteriophages can be used for the following technical operations:

[0097] (6) Transformation into a specific nanobody library. Phages are re-infected into TG1 E. coli, but no helper phages are added. After complete phage infection, the specific nanobodies exist in the E. coli in the form of DNA plasmids. Collecting all these E. coli samples yields an antigen-specific nanobody library. This library serves as the raw material for returning to step (1) for the next round of phage surface display screening.

[0098] (7) Transform into monoclonal nanobody colonies. Take a small amount of the phage obtained in step (5), dilute it, and re-infect E. coli cultured to a suitable state, but do not add helper phage. After the phage infection is complete, spread these E. coli evenly on bacterial culture dishes and culture to obtain monoclonal colonies containing nanobody DNA plasmids. Use these monoclonal colonies as raw materials for the following experiments.

[0099] Example 5. Identification of positive monoclonal nanobodies.

[0100] Bacterial culture dishes with monoclonal colonies were obtained through step (7) of Example 4, and positive monoclonal nanobodies were identified. The specific technical solution is as follows:

[0101] (1) Pick single colonies and culture them in microplates;

[0102] (2) Adding IPTG (isopropyl-β-D-thiogalactoside) to induce the expression of VHH-pIII (i.e., the fusion protein containing nanobodies);

[0103] (3) Collect the bacterial culture supernatant containing nanobodies and incubate it with the antigen STREP-TAG II. The antigen is pre-fixed in a 96-well microplate (Maxisorp transparent microplate, ThermoFisher Scientific).

[0104] (4) Use enzyme-linked immunosorbent assay (ELISA) to detect whether monoclonal nanobodies bind to the STREP-TAG II antigen.

[0105] The main experimental steps are as follows:

[0106] 1) Coating: Dilute the antigen with PBS to 5 μg / mL, 50 μL / well, and incubate overnight at 4°C with shaking;

[0107] 2) Blocking: On the second day, discard the antigen and add 100 μL / well of PBS-2% BSA. Incubate at room temperature with shaking for 1 hour.

[0108] 3) Washing: 3 times with PBST, 3 times with PBS, 150 μL / well;

[0109] 4) Add culture supernatant, 50 μL / well, and incubate with shaking at room temperature for 1-2 hours;

[0110] 5) Washing: 3 times with PBST, 3 times with PBS, 150 μL / well;

[0111] 6) Add diluted anti-myc HRP and incubate at room temperature for 1 hour;

[0112] 7) Washing: 3 times with PBST, 3 times with PBS, 150 μL / well;

[0113] 8) Add ELISA substrate and incubate at room temperature in the dark for 30 min;

[0114] 9) Read OD 450nm .

[0115] (5) For microbial colonies of monoclonal nanobodies that can bind to antigens, after appropriate reculturing, DNA plasmids are extracted and DNA sequencing is performed to obtain the nanobody nucleic acid sequence, as shown in Table 4; after translation, the complete amino acid sequence of the nanobody is obtained, as shown in Table 3. The amino acid sequences of the CDR and FR regions of the nanobody are shown in Tables 5-6 (IMGT numbers), and the corresponding nucleotide sequences of each region are shown in Table 7.

[0116] Table 3. Amino acid sequence of the variable region of the heavy chain of nanobody

[0117]

[0118] Table 4. Nucleotide sequences of the variable region of the heavy chain of nanobody

[0119]

[0120] Table 5. Amino acid sequences of nanobodies CDR1-2 and FR1-2

[0121]

[0122] Table 6. Amino acid sequences of nanobodies CDR3 and FR3-4

[0123]

[0124] Table 7. Nucleotide sequences (5′-3′) of nanobodies CDR and FR

[0125]

[0126] Example 6. Recombinant Expression and Purification of Small-Batch Monoclonal Nanobodies

[0127] (1) In Example 5, a monoclonal nanobody that can specifically recognize and bind to the antigen was obtained. The DNA plasmid of the nanobody was transformed into BL21(DE3) competent cells, and then monoclonal cells were picked and cultured at 37°C with shaking.

[0128] (2) IPTG (isopropyl-β-D-thiogalactoside) was added and cultured at 30°C to induce the expression of VHH-pIII (i.e., the fusion protein containing nanobodies);

[0129] (3) Collect all bacteria, resuspend the bacteria in TBS solution containing 25% sucrose (mass / volume, g / mL), and incubate at 4°C with shaking for 30 minutes to fully lyse the outer membrane of Escherichia coli while keeping the inner membrane of Escherichia coli intact. That is, extract nanobodies from the periplasmic cavity between the inner and outer membranes of Escherichia coli to ensure that the nanobodies can fold correctly and maintain their activity.

[0130] (4) After lysis, centrifuge and collect the supernatant;

[0131] (5) Histidine-tagged affinity chromatography: The supernatant obtained after centrifugation was incubated with nickel affinity chromatography packing material, and then the nanobodies with histidine tags bound to the nickel packing material were eluted.

[0132] (6) Gel filtration chromatography: The product obtained after the above affinity chromatography is concentrated and then purified monoclonal nanobody is obtained by gel filtration chromatography.

[0133] Example 7. Affinity Detection of Monoclonal Nanobodies

[0134] Using the ELISA method, nanobodies of different concentrations were incubated, and the affinity between the nanobodies and the antigen was measured based on the binding ability of the nanobodies to STREP-TAG II.

[0135] The main experimental steps are as follows:

[0136] 1) Coating: The antigen (BSA-Strep-tag II antigen prepared in Example 1) was diluted with PBS to 5 μg / mL, 50 μL / well, and incubated overnight at 4°C with shaking;

[0137] 2) Blocking: On the second day, discard the antigen and add 100 μL / well of PBS-2% BSA. Incubate at room temperature with shaking for 1 hour.

[0138] 3) Washing: 3 times with PBST, 3 times with PBS, 150 μL / well;

[0139] 4) Add different concentrations of nanobodies, 50 μL / well, and incubate at room temperature with shaking for 1-2 hours;

[0140] 5) Washing: 3 times with PBST, 3 times with PBS, 150 μL / well;

[0141] 6) Add diluted anti-myc HRP and incubate at room temperature for 1 hour;

[0142] 7) Washing: 3 times with PBST, 3 times with PBS, 150 μL / well;

[0143] 8) Add ELISA substrate and incubate at room temperature in the dark for 30 min;

[0144] 9) Read OD450nm.

[0145] The results are as follows Figures 1-4 As shown. From Figures 1-4 The results show that the KD values ​​of the antibodies screened in this publication that bind to STEP-TAGII are all below 200 nM.

[0146] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A nanobody that specifically binds to STREP-TAG II, characterized in that, The nanobodies that specifically bind to STEP-TAG II include: The CDRs are CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region of the amino acid sequences shown in SEQ ID NO: 19-22, respectively; each CDR is defined by the numbering system of Kabat, AbM, Chothia, Contact, or IMGT.

2. The nanobody that specifically binds to STREP-TAG II according to claim 1, characterized in that, The nanobodies that specifically bind to STEP-TAG II include: a1) The heavy chain variable regions of the following three CDRs: CDR-H1 with amino acid sequence as shown in SEQ ID NO: 7, CDR-H2 with amino acid sequence as shown in SEQ ID NO: 8, and CDR-H3 with amino acid sequence as shown in SEQ ID NO: 9; a2) The heavy chain variable regions of the following three CDRs: CDR-H1 with amino acid sequence as shown in SEQ ID NO: 10, CDR-H2 with amino acid sequence as shown in SEQ ID NO: 11, and CDR-H3 with amino acid sequence as shown in SEQ ID NO: 12; a3) The heavy chain variable regions of the following three CDRs: CDR-H1 with amino acid sequence as shown in SEQ ID NO: 13, CDR-H2 with amino acid sequence as shown in SEQ ID NO: 14, and CDR-H3 with amino acid sequence as shown in SEQ ID NO: 15; a4) The heavy chain variable regions of the following three CDRs: CDR-H1 with amino acid sequence as shown in SEQ ID NO: 16, CDR-H2 with amino acid sequence as shown in SEQ ID NO: 17, and CDR-H3 with amino acid sequence as shown in SEQ ID NO:

18.

3. The nanobody that specifically binds to STREP-TAG II according to claim 1, characterized in that, The heavy chain variable region of the nanobody that specifically binds to STEP-TAG II also includes a framework region.

4. The nanobody that specifically binds to STREP-TAG II according to claim 3, characterized in that, The frame region includes the frame regions of immunoglobulins derived from mice, primates, horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

5. The nanobody that specifically binds to STREP-TAG II according to claim 1, characterized in that, The nanobodies that specifically bind to STEP-TAG II include: b1) Heavy chain variable region, comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 19; b2) Heavy chain variable region, comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; b3) Heavy chain variable region, which includes an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 21; b4) Heavy chain variable region, which includes an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

22.

6. A heavy chain antibody that specifically binds to STREP-TAG II, comprising an immunoglobulin Fc domain and a nanobody as described in any one of claims 1-5.

7. An isolated nucleic acid molecule encoding the nucleotide sequence of the nanobody according to any one of claims 1-5 or the heavy chain antibody according to claim 6.

8. A carrier comprising the nucleic acid molecule of claim 7.

9. A cell comprising the nanobody of any one of claims 1-5, the heavy chain antibody of claim 6, the nucleic acid molecule of claim 7, or the carrier of claim 8.

10. The method for preparing the nanobody according to any one of claims 1-5, or the heavy chain antibody according to claim 6, is obtained by culturing the cells according to claim 9.

11. A conjugate comprising a nanobody according to any one of claims 1-5 or a heavy chain antibody according to claim 6; and a conjugation portion, said conjugation portion being a detectable marker.

12. The coupling according to claim 11, characterized in that, The detectable markers include enzymes, radionuclides, luminescent substances, colored substances, streptavidin, and / or biotin.

13. The coupling according to claim 11, characterized in that, The detectable markers include fluorescent dyes.

14. A detection or purification kit, comprising: The nanobody according to any one of claims 1-5, the heavy chain antibody according to claim 6, the cell according to claim 9, or the conjugate according to any one of claims 11-13.

15. The detection or purification kit according to claim 14, characterized in that, The kit can be used to detect or purify proteins containing STREP-TAG II.

16. The detection or purification kit according to claim 14, characterized in that, The kit may also include instructions.

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