Gamma-aminobutyric acid type B receptor nano antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a gamma-aminobutyric acid type B receptor nano antibody as well as a preparation method and application thereof. The nano antibody can specifically recognize and bind the gamma-aminobutyric acid type B receptor, has good affinity with the gamma-aminobutyric acid type B receptor, and can be used for preparing products for diagnosing, preventing or treating gamma-aminobutyric acid type B receptor related diseases or symptoms or detecting the existence or level of the gamma-aminobutyric acid type B receptor in a sample.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a nanobody against gamma-aminobutyric acid type B receptor, and a preparation method and application thereof. Background Art

[0002] Gamma-aminobutyric acid type B receptor (hereinafter referred to as GABA B receptor, or GABA B R) is a cell membrane surface receptor of gamma-aminobutyric acid (GABA), an important neurotransmitter in the central nervous system of higher animals such as humans and mice, and belongs to the C family of G protein-coupled receptors (GPCR). GABA B receptors are widely present in nature. Functional GABA B receptors in mammals such as humans and mice are heterodimeric complexes composed of GABA B R1 (hereinafter referred to as GB1) subunit and GABA B R2 (hereinafter referred to as GB2) subunit ( Figure 1 ). GB1 and GB2 can be divided into an extracellular domain (ECD), a heptahelical domain (HD), and an intracellular domain from the N-terminus to the C-terminus of the peptide chain in terms of domain composition. The ECD is shaped like a Venus flytrap and is therefore called the Venus flytrap domain (VFT). The VFT of GB1 can bind to the endogenous ligand GABA or other artificial ligands (such as Baclofen, etc.), thereby causing a conformational change in the HD domain of GB2 and coupling the intracellular G protein trimer (Gα, Gβ, Gγ), and controlling various main effectors such as voltage-sensitive calcium channels, inwardly rectifying potassium channels, and adenylate cyclase in the downstream of the central nervous system. Different from GB1, the VFT of GB2 cannot bind to the endogenous ligand GABA or other artificial ligands.

[0003] Human GB1 includes two common splice forms, GB1a and GB1b. Among them: GB1a is a classic full-length receptor, composed of 961 amino acids, and its ECD includes the Sushi domain and the VFT domain, of which the Sushi domain is about 67 amino acids in length (amino acids 30-96); GB1b is composed of 844 amino acids, and the difference between it and GB1a is that its ECD does not contain the Sushi domain. In addition to the Sushi domain, the amino acid sequences of the HD and intracellular domains of GB1a and GB1b are exactly the same. After GB1 is synthesized by ribosomes attached to the endoplasmic reticulum, it is affected by an endoplasmic reticulum retention signal sequence (923RSR925) in its intracellular domain, and interacts with protein molecules in the endoplasmic reticulum such as PRAF2 and is retained in the endoplasmic reticulum, unable to be further sorted to the Golgi apparatus, and therefore cannot be subsequently sorted and transported to the cell membrane. In this state, unless GB2 is also expressed in the cell, GB2 will competitively bind to GB1, relieving GB1's endoplasmic reticulum retention, allowing GB1 and GB2 to combine to form a heterodimer and be sorted and transported to the cell membrane.

[0004] Human GB2 is composed of 1220 amino acids. The amino acid sequence of the intracellular ring region of HD is responsible for coupling the G protein α subunit and mediating the downstream signaling pathway. After GB2 is synthesized by ribosomes attached to the endoplasmic reticulum, it can bind to the retained GB1 in the endoplasmic reticulum, allowing GB1 and GB2 to combine to form a heterodimer and be sorted and transported to the cell membrane. If GB1 is not expressed in the cell, a single GB2 can also undergo normal sorting and transport to the cell membrane, but it has no normal physiological functions such as signal transduction. For many years, research on GABA B There are few reports on antibodies against receptors, therefore, it is necessary to develop a γ-aminobutyric acid type B receptor nanobody. Summary of the invention

[0005] The purpose of the first aspect of the present invention is to provide a nanobody or an antigen-binding fragment thereof that specifically binds to the γ-aminobutyric acid type B receptor.

[0006] The second aspect of the present invention aims to provide a heavy chain antibody or an antigen-binding fragment thereof that specifically binds to γ-aminobutyric acid type B receptor.

[0007] The third aspect of the present invention aims to provide a chimeric antigen receptor.

[0008] The fourth aspect of the present invention aims to provide a multispecific antibody or an antigen-binding fragment thereof.

[0009] The fifth aspect of the present invention aims to provide an isolated nucleic acid molecule.

[0010] The sixth aspect of the present invention aims to provide a carrier.

[0011] The seventh aspect of the present invention aims to provide a cell.

[0012] The purpose of the eighth aspect of the present invention is to provide a method for preparing the nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, or the multispecific antibody or antigen-binding fragment thereof of the fourth aspect.

[0013] The ninth aspect of the present invention aims to provide a conjugate.

[0014] The tenth aspect of the present invention aims to provide a pharmaceutical composition.

[0015] The eleventh aspect of the present invention aims to provide a diagnostic or therapeutic kit.

[0016] The object of the twelfth aspect of the present invention is to provide the use of the nanoantibody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, the nucleic acid molecule of the fifth aspect, the vector of the sixth aspect, the cell of the seventh aspect, the conjugate of the ninth aspect, or the pharmaceutical composition of the tenth aspect.

[0017] In order to achieve the above object, the technical solution adopted by the present invention is:

[0018] In a first aspect of the present invention, a Nanobody or an antigen-binding fragment thereof that specifically binds to a γ-aminobutyric acid type B receptor is provided.

[0019] In some embodiments, the Nanobody or antigen-binding fragment thereof that specifically binds to the γ-aminobutyric acid type B receptor comprises:

[0020] The CDR-H1, CDR-H2 and CDR-H3 included in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 10 or 15.

[0021] A nanobody or an antigen-binding fragment thereof that specifically binds to a γ-aminobutyric acid type B receptor, wherein the nanobody or an antigen-binding fragment thereof that specifically binds to a γ-aminobutyric acid type B receptor comprises:

[0022] a1) a heavy chain variable region comprising 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; or

[0023] a2) a heavy chain variable region comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 12, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 13, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 14.

[0024] In some embodiments, the heavy chain variable region of the Nanobody or its antigen-binding fragment that specifically binds to γ-aminobutyric acid type B receptor also includes a framework region of the heavy chain variable region.

[0025] In some embodiments, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region of an immunoglobulin derived from mouse, primate, bovine, horse, cattle, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof.

[0026] In some embodiments, the Nanobody or antigen-binding fragment thereof that specifically binds to the γ-aminobutyric acid type B receptor comprises:

[0027] b1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, 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 thereto; or

[0028] b2) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:15, 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 thereto.

[0029] In some embodiments, the GABA type B receptor comprises human GB1a and human GB2.

[0030] The second aspect of the present invention provides a heavy chain antibody or an antigen-binding fragment thereof that specifically binds to a γ-aminobutyric acid type B receptor, which comprises an immunoglobulin Fc domain and a Nanobody or an antigen-binding fragment thereof according to the first aspect of the present invention.

[0031] In some embodiments, the immunoglobulin Fc domain comprises an Fc domain of an immunoglobulin from mouse, primate, bovine, horse, cattle, porcine, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof.

[0032] The third aspect of the present invention provides a chimeric antigen receptor, which includes an antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain includes the nanobody or antigen binding fragment thereof of the first aspect of the present invention or the heavy chain antibody or antigen binding fragment thereof of the second aspect.

[0033] A fourth aspect of the invention provides a multispecific antibody or antigen-binding fragment thereof, which comprises two or more (e.g., three or four) antigen-binding domains, wherein one antigen-binding domain comprises the nanobody or antigen-binding fragment thereof of the first aspect of the invention or the heavy chain antibody or antigen-binding fragment thereof of the second aspect.

[0034] The fifth aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the Nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, or the multispecific antibody or antigen-binding fragment thereof of the fourth aspect.

[0035] Those skilled in the art will appreciate that nucleotides in a nucleic acid molecule may be substituted based on codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon optimized.

[0036] In some embodiments, the nucleotide sequence encoding the Nanobody or antigen-binding fragment thereof of the first aspect of the invention comprises: SEQ ID NO: 11, or 16, 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 thereto.

[0037] The sixth aspect of the present invention provides a vector comprising the nucleic acid molecule of the fifth aspect of the present invention.

[0038] In some embodiments, the vector may be an expression vector. In some embodiments, the expression vector may include a eukaryotic expression vector and / or a prokaryotic expression vector. In some embodiments, the eukaryotic expression vector includes, for example, but is not limited to, a yeast expression vector, a mammalian expression vector, and an insect expression vector. For example, the expression vector may include, but is not limited to, a plasmid, a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector.

[0039] In some embodiments, the carrier can be selected from nanoparticles, liposomes, exosomes, microbubbles or gene guns.

[0040] The seventh aspect of the present invention provides a cell, which includes the nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, the nucleic acid molecule of the fifth aspect, or the vector of the sixth aspect.

[0041] In some embodiments, the cells are not involved in propagation material.

[0042] In some embodiments, the cell can be a host cell conventionally used in the art, as long as the expression vector can stably express the carried nucleic acid molecule as the above-mentioned nano antibody or antigen-binding fragment thereof, heavy chain antibody or antigen-binding fragment thereof, chimeric antigen receptor or multispecific antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell, the prokaryotic cell can include, for example, Escherichia coli, the eukaryotic cell can include, for example, CHO cells, HEK293 cells, BHK cells, NS0 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.

[0043] In some embodiments, the cell can be an immune cell. In some embodiments, the immune cell can include, but is not limited to, T cells, NK cells, DC cells and macrophages. In these embodiments, the immune cell can express the above-mentioned chimeric antigen receptor of the present disclosure (i.e., a modified immune cell).

[0044] The eighth aspect of the present invention provides a method for preparing the nanoantibody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, or the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, which is obtained by culturing the cells of the seventh aspect of the present invention.

[0045] A ninth aspect of the present invention provides a conjugate comprising the Nanobody or antigen-binding fragment thereof of the first aspect of the present invention, or the heavy chain antibody or antigen-binding fragment thereof of the second aspect; and a coupling portion.

[0046] In some embodiments, the conjugated moiety may include, but is not limited to, a detectable label or a therapeutic agent.

[0047] In some embodiments, the detectable marker can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, chemistry, etc. Such labels are well known in the art, and examples thereof 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)), acridinium ester compounds, magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned markers. In some embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In some embodiments, the detectable label is selected from a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme. In some embodiments, the detectable label as described above can be connected to the above-mentioned nanobody or its antigen-binding fragment, or heavy chain antibody or its antigen-binding fragment of the present disclosure by linkers of different lengths to reduce potential steric hindrance.

[0048] In some embodiments, the detectable marker may include, but is not limited to, an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), a colored substance, biotin, and the like.

[0049] In some embodiments, the therapeutic agent may include, for example, but not limited to, a drug for preventing and / or treating a GABA type B receptor-related disease or disorder.

[0050] In some embodiments, the coupling moiety is selected from substances that can improve the biological properties of the antibody (eg, increase serum half-life), for example, it can be a chemical group such as polyethylene glycol (PEG), methyl, ethyl or sugar group.

[0051] The tenth aspect of the present invention provides a pharmaceutical composition, which includes: the nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, the nucleic acid molecule of the fifth aspect, the vector of the sixth aspect, the cell of the seventh aspect or the conjugate of the ninth aspect; and a pharmaceutically acceptable carrier.

[0052] In some embodiments, the pharmaceutical composition may further include additional pharmaceutically active agents.

[0053] In some embodiments, the additional pharmaceutically active agent may be a biologically active drug, such as a drug capable of preventing and / or treating a GABA type B receptor-associated disease or disorder.

[0054] In some embodiments, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as mixed components.

[0055] In some embodiments, the pharmaceutical composition can be administered, for example, parenterally, subcutaneously, sublingually, rectally, nasally, intravenously, intramuscularly, orally, ophthalmically, topically, or the like.

[0056] In some embodiments, the pharmaceutical composition is in the form of, for example, an aqueous solution, suspension, powder, tablet, capsule, granule, powder, pill, disintegrant, syrup, spray, gel, emulsion, injection, elixir, lozenge, suppository, etc.

[0057] The eleventh aspect of the present invention provides a diagnostic or therapeutic kit, which comprises: the nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, the nucleic acid molecule of the fifth aspect, the vector of the sixth aspect, the cell of the seventh aspect, the conjugate of the ninth aspect, or the pharmaceutical composition of the tenth aspect.

[0058] In some embodiments, the kit may further include instructions and / or an administration device.

[0059] In some embodiments, the kit can be used to diagnose GABA type B receptor-related diseases or disorders.

[0060] In some embodiments, the kit can be used to prevent or treat GABA type B receptor-related diseases or disorders.

[0061] The twelfth aspect of the present invention provides the use of the Nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, the nucleic acid molecule of the fifth aspect, the vector of the sixth aspect, the cell of the seventh aspect, the conjugate of the ninth aspect, or the pharmaceutical composition of the tenth aspect in the preparation of a product, wherein the product is used for any one of c1) to c3):

[0062] c1) diagnosing GABA type B receptor related diseases or disorders;

[0063] c2) preventing or treating diseases or conditions associated with GABA type B receptor;

[0064] c3) detecting the presence or level of GABA type B receptor in the sample.

[0065] In some embodiments, the sample is selected from at least one of body fluids, tissues, cells, and excrement of the subject to be tested.

[0066] In some embodiments, the body fluid comprises at least one of blood and lymph.

[0067] In some embodiments, the blood comprises at least one of serum, plasma, dried blood spots, and whole blood.

[0068] In some embodiments, the excreta comprises at least one of urine, feces, and tears.

[0069] In some embodiments, the subject to be tested comprises mammals, such as humans, non-human primates (such as gorillas, apes), rodents (such as rats, mice, guinea pigs), pets (such as cats, dogs), and livestock (such as horses, cows, sheep, pigs, rabbits).

[0070] In some embodiments, the subject comprises a human.

[0071] In the present invention, the γ-aminobutyric acid type B receptor-related disease or condition is epilepsy, anxiety, depression, or cognitive impairment caused by nerve damage.

[0072] The beneficial effects of the present invention are:

[0073] The present invention provides a nanobody or an antigen-binding fragment thereof that specifically binds to a γ-aminobutyric acid type B receptor, which can specifically recognize and bind to the γ-aminobutyric acid type B receptor and has good affinity thereto, and can be used to prepare a product for diagnosing, preventing or treating a γ-aminobutyric acid type B receptor-related disease or condition, or detecting the presence or level of the γ-aminobutyric acid type B receptor in a sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 GABA B Schematic diagram of the receptor heterodimer structure.

[0075] Figure 2 A schematic diagram showing the results of affinity testing between nanobody NB-4C10 and antigen is shown.

[0076] Figure 3Shows a schematic diagram of the detection result of the affinity between the nanobody NB-5E9 and the antigen. Detailed implementation mode

[0077] To make the objectives, technical solutions and advantages of the present invention more clearly understood, the following further describes the present invention in detail in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies have also been described in many publications.

[0078] definition

[0079] Unless otherwise defined, all technical terms and scientific and technological terms used in the present invention have the same meanings as those commonly used in the field to which the present invention belongs. For the purpose of explaining this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form and vice versa.

[0080] Unless the context clearly indicates otherwise, the expressions "a" and "an" used herein include plural referents. For example, referring to "a cell" includes multiple such cells and equivalents known to those skilled in the art and the like.

[0081] The term "about" used herein represents a range of ±20% of the value thereafter. In some embodiments, the term "about" represents a range of ±10% of the value thereafter. In some embodiments, the term "about" represents a range of ±5% of the value thereafter.

[0082] K D value : Dissociation constant (K D ) is a specific type of equilibrium constant used to measure the tendency of a larger object to separate (dissociate) from another smaller component, and is the reciprocal of the association constant, with the unit of mol / L (M) or nmol / L (nM). K D The smaller the K value, the stronger the binding ability of the two substances.

[0083] Nanobodies: An antibody that naturally lacks light chains and exists in the peripheral blood of camelids. This antibody contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but it is not as easy to stick to each other or even aggregate into clumps as artificially modified single-chain antibody fragments; the VHH structure cloned and expressed separately has the same structural stability and antigen binding activity as the original heavy chain antibody, and is the smallest unit known to bind to the target antigen; the VHH crystal is 2.5nm, 4nm long, and has a molecular weight of only about 15kD, so it is also called a nanobody (Nanobody, Nb). Compared with traditional animals such as mice and rabbits that can only recognize flat polypeptides on the surface of antigens, the immune system in camelids can recognize the complex spatial structure of the antigen surface and can produce highly specific and high-affinity nanobodies.

[0084] Unlike traditional technologies that rely on classic model animals such as mice, rabbits, monkeys, and sheep, the technical solution of the present invention relies on antibodies produced by the immune system of alpacas, which are called "nanoantibodies". Nanoantibodies are tiny antibody fragments separated from immunoglobulins in animals such as camels. They have the same antigen binding ability and structural stability as complete antibodies. They are the smallest units that can bind to target antigens, with a relative molecular mass of only about 15kD. Compared with traditional animals such as mice and rabbits that can only recognize flat polypeptides on the surface of antigens, the immune systems in animals such as alpacas can recognize the complex spatial structure of the antigen surface and can produce highly specific and high-affinity nanoantibodies.

[0085] According to the technical solution of the present invention, certain amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, as shown in Table 1 below:

[0086] Table 1

[0087] Residue Conservative substitution Residue Conservative substitution Ala Ser Leu Ile; Val Arg Lys Lys Arg; Gln Asn Gln; His Met Leu; Ile Asp Glu Phe Met; Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu Asp Trp Tyr Gly Pro Tyr Trp; Phe His Asn; Gln Val Ile; Leu Ile Leu; Val

[0088] In addition, due to the degeneracy of the bases, the bases of the polynucleotide sequence can be substituted without changing the activity or function of the polynucleotide sequence, as shown in Table 2 below:

[0089] Table 2

[0090]

[0091]

[0092] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0093] Example 1. Preparation of antigens

[0094] (1) The DNA vector plasmid (pRK5) encoding human GB1a (gene ID: 2550) and the DNA vector plasmid (pRK5) encoding GB2 (gene ID: 9568) were co-transfected into HEK293T tool cells using liposome transfection reagent (Lipo3000, ThermoFisher);

[0095] (2) Continue to culture cells for about 48 hours after transfection to allow GABA B Receptor heterodimers are fully expressed on the cell membrane surface;

[0096] (3) Collect about 4*10 8 After ultrasonic disruption, homogenization, and ultracentrifugation, the cell membrane extract is taken out as a suitable antigen; wherein the preparation steps of the cell membrane extract are as follows:

[0097] 1) Collect about 4*10 8 Add appropriate amount of buffer and let stand on ice to thaw.

[0098] 2) Add about 6 mL of Buffer (Buffer: PBS pH 7.4, cocktail) to each construct.

[0099] 3) Homogenization: For each construction, transfer the cells to a pre-cooled Dulbecco homogenizer and grind them evenly up and down for about 20 times.

[0100] 4) Ultrasonic disruption: Transfer the sample to a pre-cooled 15 mL tube and use an ultrasonic rod of appropriate thickness and power for ultrasonic disruption.

[0101] 5) Collect the supernatant by centrifugation and record it as supernatant 1;

[0102] 6) Resuspend with 6 mL of buffer, grind the homogenate about 20 times, and collect the supernatant by centrifugation, which is recorded as supernatant 2;

[0103] 7) Repeat the previous step and record it as supernatant 3;

[0104] 8) Combine supernatants 1+2+3 to a total volume of about 15-20 mL, and ultracentrifuge at 50,000 g for 1-1.5 h.

[0105] 9) Discard the supernatant and resuspend the precipitate with an appropriate amount of PBS to obtain the cell membrane extract.

[0106] 10) Mix all the cell membrane extracts constructed uniformly, control the total volume to about 4 mL, divide into 1.5 mL EP tubes, divide into 4 tubes, 1 mL per tube, and freeze at -80°C.

[0107] GABA B The receptors can be correctly expressed and exist on the surface of the cell membrane, maintaining the correct assembly, folding and conformational state, and especially maintaining the complex spatial structure of the surface.

[0108] Example 2. Alpaca Immunization

[0109] In this example, the cell membrane extract of Example 1 (suitable antigen GABA B The specific steps are as follows:

[0110] (1) The antigen in Example 1 was evenly divided into 4 portions, each portion was about 0.5 mg; the alpacas were immunized 4 times in total, and the antigen was injected subcutaneously into the animal body, with the first immunization being recorded as day 1, and the subsequent immunizations were recorded on days 11, 21, and 31 respectively;

[0111] (2) On day 30, before the fourth immunization, approximately 200 mL of peripheral venous blood was collected from the alpaca;

[0112] (3) On day 45, i.e. 14 days after the fourth immunization, approximately 200 mL of peripheral venous blood was collected from the alpaca.

[0113] Compared with traditional immunization technology solutions for animal antibodies such as mice and rabbits, the technical advantage of the present invention lies in the collection of a large amount of alpaca venous peripheral blood, which is conducive to subsequent screening to obtain highly diverse nano-antibodies.

[0114] Example 3. Construction of Alpaca Nanobody Library

[0115] The two batches of alpaca venous peripheral blood collected in Example 2 were used as raw materials to construct a highly diverse nanoantibody library. The two batches of alpaca venous peripheral blood were processed in the same way, and the specific steps were as follows:

[0116] (1) Lymphocytes were isolated from alpaca venous peripheral blood using density gradient centrifugation and other methods;

[0117] (2) extracting total mRNA from lymphocytes and reverse transcribed into cDNA;

[0118] (3) using appropriate DNA primers (see Table 3 below) and the above cDNA as a template, amplifying the VHH fragments of alpaca immunoglobulins IgG2 and IgG3, i.e., the DNA fragments of the nanobody, by polymerase chain reaction (PCR);

[0119] Table 3. Primers used to construct the nanobody library

[0120]

[0121]

[0122] (4) Connecting the VHH DNA to the phage surface display screening vector phen1 to construct a VHH-His-pIII fusion protein expression vector plasmid library; wherein pIII is a protein present on the flagella on the phage surface, and His is a histidine tag (six consecutive histidines, i.e., His-His-His-His-His-His);

[0123] (5) The DNA ligation product is transformed into TG1 competent Escherichia coli by electroporation. After appropriate cultivation, all colonies are collected to obtain the nanoantibody library of the alpaca.

[0124] Compared with the traditional method of isolating antibodies from the serum or lymphocytes of animals such as mice and rabbits, the present invention can preserve all the nano-antibody fragments (i.e., the library) of alpacas for a long time, and can continuously support the subsequent screening and development of nano-antibodies.

[0125] Example 4. Screening of specific nanobodies by phage surface display

[0126] This example uses the nanobody library obtained in Example 3 as a source, and obtains antigen-specific nanobodies through phage surface display screening. The specific steps are as follows:

[0127] (1) Taking an appropriate amount of frozen nanobody library, inoculating it into bacterial culture medium, adding an appropriate amount of helper phage (M13KO7 helper phage, NEB, N0315S) after appropriate cultivation, and continuing to culture under appropriate conditions;

[0128] (2) extracting the amplified phages from the bacterial culture supernatant by the PEG-NaC method and freezing them in a -80°C ultra-low temperature freezer for future use;

[0129] (3) DNA vector plasmids encoding human GB1a and GB2 were co-transfected into HEK293T tool cells using liposome transfection reagent (Lipo3000, ThermoFisher), and the cells were cultured for 24 hours after transfection;

[0130] (4) Negative screening: prepare about 5*10 7 HEK293T tool cells that have not been transfected with any exogenous DNA vector plasmids, thaw the phage, incubate the phage and tool cells for 2 hours, centrifuge and retain the supernatant;

[0131] (5) Positive screening: Collect about 5*10 7Overexpression of GABA B The recipient HEK293T cells were incubated with the supernatant obtained after centrifugation in (4) for 2 hours.

[0132] (6) Washing: Discard the phages and rinse the antigen cells three times with PBS buffer to wash and remove the phages that non-specifically bind to the antigen and retain the phages that specifically bind to the antigen.

[0133] (7) Elution: Treat the phages that specifically bind to the antigen with an acidic glycine solution to dissociate the phages from the antigen and retain them.

[0134] At this point, phages expressing specific nanobodies have been obtained, and these phages can be used for the following technical operations:

[0135] (8) Transformation into a specific nanobody library: The phage is infected again and cultured to TG1 competent E. coli, but no helper phage is added. After the phage infection is complete, the specific nanobody exists in the E. coli in the form of a DNA plasmid. Collect all these E. coli to form an antigen-specific nanobody library. This library can be used as a raw material and return to step (1) for the next round of phage surface display screening;

[0136] (9) Transformation into monoclonal nanoantibody colonies: Take a small amount of the phage obtained in step (7) (e.g., 0.5%), dilute it, and infect and culture it again until it reaches TG1 competent E. coli, but no helper phage is added. After the phage infection is complete, the E. coli are evenly spread on a bacterial culture dish, and monoclonal colonies containing nanoantibody DNA plasmids can be obtained under appropriate conditions. These monoclonal colonies are used as raw materials to identify positive monoclonal nanoantibodies.

[0137] Example 5. Identification of positive monoclonal nanobodies and nanobody sequencing

[0138] This example uses step (9) of Example 4 to obtain a bacterial culture dish with monoclonal colonies, and conducts identification of positive monoclonal nanoantibodies. The specific steps are as follows:

[0139] (1) Pick a single clone and culture it in a microplate;

[0140] (2) adding IPTG to induce the expression of VHH-pIII (i.e., the fusion protein containing the nanobody);

[0141] (3) DNA vector plasmids encoding human GB1a and GB2 were co-transfected into HEK293T tool cells using liposome transfection reagent (Lipo3000, ThermoFisher), and the cells were cultured for 24 hours after transfection;

[0142] (4) Collect the bacterial culture supernatant containing nanobodies obtained in (2) and incubate with the cells obtained in (3) for 2 hours. Approximately 2*10^5 cells overexpressing GABA are used in each well of a 96-well plate. B After the incubation, the recipient cells were centrifuged and the supernatant was discarded;

[0143] (5) Incubate the cells obtained in (4) above with His tag-specific mouse monoclonal antibody labeled with Alexa Flour 647 (brand: Research & Development, IC0501R) for 2 hours;

[0144] (6) Centrifuge, discard the supernatant, and resuspend the cells with PBS buffer. Resuspend each well of the 96-well plate with 100 μL of PBS solution;

[0145] (7) Use flow cytometry to detect the fluorescence intensity distribution of Alexa Flour 647 on the cell surface to determine whether the nanobody can bind to GABA on the cell surface B Receptor binding.

[0146] (8) For monoclonal nanobodies (NB-4C10, NB-5E9) that can specifically recognize and bind to antigens, the TG1 strain expressing the relevant monoclonal nanobodies was cultured at 37°C overnight, the DNA plasmid was extracted and Sanger sequencing was performed to obtain the nucleotide sequence of the nanobody, and then the amino acid sequence of the nanobody was obtained after translation, as shown in Table 4-5.

[0147] Table 4: Amino acid sequence and nucleotide sequence of NB-4C10

[0148]

[0149]

[0150] Table 5: Amino acid sequence and nucleotide sequence of NB-5E9

[0151]

[0152] Example 6. Small batch monoclonal nanobody recombinant expression and purification

[0153] (1) Example 5 obtained a monoclonal nanobody that can specifically recognize and bind to an antigen. The DNA plasmid encoding the above nanobodies (NB-4C10, NB-5E9) was transformed into BL21 (DE3) competent cells, and the nanobodies were recombinantly expressed in Escherichia coli. The monoclonal nanobodies were obtained through bacterial lysis, histidine tag affinity chromatography, gel filtration sequence and other steps. The batch production capacity is about several milligrams.

[0154] (2) Using flow cytometry analysis, different concentrations of nanoantibodies were incubated to determine the relative expression of nanoantibodies with overexpressed human GABA. B The binding ability of the cells to the receptor (constructed in the same manner as in Example 1) measures the affinity of the nanobody to the antigen.

[0155] Test results such as Figure 2-3 As shown, the affinity values ​​K of monoclonal nanoantibodies NB-4C10 and NB-5E9 D They are 3.969nM and 6.148nM respectively.

[0156] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A nanobody or an antigen-binding fragment thereof that specifically binds to a γ-aminobutyric acid type B receptor.

2. The nanobody or antigen-binding fragment thereof according to claim 1, characterized in that The nanobody or antigen-binding fragment thereof that specifically binds to the γ-aminobutyric acid type B receptor includes: CDR-H1, CDR-H2 and CDR-H3 included in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 10 or 15; Preferably, the Nanobody or antigen-binding fragment thereof that specifically binds to the γ-aminobutyric acid type B receptor comprises a heavy chain variable region, and the heavy chain variable region comprises: a1) CDR-H1 having the amino acid sequence of SEQ ID NO:7, CDR-H2 having the amino acid sequence of SEQ ID NO:8, and CDR-H3 having the amino acid sequence of SEQ ID NO:9; or a2) CDR-H1 having the amino acid sequence of SEQ ID NO: 12, CDR-H2 having the amino acid sequence of SEQ ID NO: 13, and CDR-H3 having the amino acid sequence of SEQ ID NO: 14; Preferably, the heavy chain variable region of the Nanobody or antigen-binding fragment thereof that specifically binds to the γ-aminobutyric acid type B receptor further comprises a framework region of the heavy chain variable region; Preferably, the framework region of the heavy chain variable region comprises a framework region of a heavy chain variable region of an immunoglobulin derived from mouse, primate, bovine, horse, cattle, porcine, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof; Preferably, the Nanobody or antigen-binding fragment thereof that specifically binds to γ-aminobutyric acid type B receptor comprises: b1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, 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 thereto; or b2) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:15, 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 thereto.

3. A heavy chain antibody or antigen-binding fragment thereof that specifically binds to a γ-aminobutyric acid type B receptor, comprising an immunoglobulin Fc domain and a nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 2.

4. A chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain comprises the nanobody or antigen binding fragment thereof according to any one of claims 1 to 2 or the heavy chain antibody or antigen binding fragment thereof according to claim 3.

5. A multispecific antibody or antigen-binding fragment thereof, comprising two or more antigen-binding domains, wherein one antigen-binding domain comprises the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2 or the heavy chain antibody or antigen-binding fragment thereof according to claim 3.

6. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, or the multispecific antibody or antigen-binding fragment thereof according to claim 5. A vector comprising the nucleic acid molecule according to claim 6.

8. A cell comprising the nanobody or antigen-binding fragment thereof according to any one of claims 1-2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or antigen-binding fragment thereof according to claim 5, the nucleic acid molecule according to claim 6, or the vector according to claim 7.

9. A method for preparing the nanobody or antigen-binding fragment thereof according to any one of claims 1-2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, or the multispecific antibody or antigen-binding fragment thereof according to claim 5, which is obtained by culturing the cell according to claim 8.

10. A conjugate comprising the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, or the heavy chain antibody or antigen-binding fragment thereof according to claim 3; and a coupling portion; Preferably, the conjugated moiety comprises a detectable label or a therapeutic agent; Preferably, the detectable marker comprises an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, a colored substance, and / or biotin; Preferably, the therapeutic agent includes a drug for preventing and / or treating a γ-aminobutyric acid type B receptor-related disease or disorder.

11. A pharmaceutical composition comprising: The nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or antigen-binding fragment thereof according to claim 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the cell according to claim 8 or the conjugate according to claim 10; and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

12. A diagnostic or therapeutic kit comprising: The nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or antigen-binding fragment thereof according to claim 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the cell according to claim 8, the conjugate according to claim 10, or the pharmaceutical composition according to claim 11; Preferably, the kit further comprises instructions and / or an administration device.

13. Use of the Nanobody or antigen-binding fragment thereof according to any one of claims 1-2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or antigen-binding fragment thereof according to claim 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the cell according to claim 8, the conjugate according to claim 10, or the pharmaceutical composition according to claim 11 in the preparation of a product, wherein the product is used for any one of c1)-c3): c1) diagnosing GABA type B receptor related diseases or disorders; c2) preventing or treating diseases or conditions associated with GABA type B receptor; c3) detecting the presence or level of GABA type B receptor in the sample; Preferably, the γ-aminobutyric acid type B receptor-related disease or disorder is epilepsy, anxiety, depression, or cognitive impairment caused by nerve damage.

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