Gamma-aminobutyric acid type b receptor nanobodies and methods of making and using the same

Abstract

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

Description

TECHNICAL FIELD

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

[0002] Gamma aminobutyric acid type B receptor (GABA B R), is a cell membrane surface receptor of gamma aminobutyric acid (GABA) which is an important neurotransmitter in the central nervous system of human, mouse and other higher animals, and belongs to the C family G protein-coupled receptor (GPCR). B GABA B R is widely present in nature, and the functional GABA B R in human, mouse and other mammals is a heterodimeric complex composed of GABA B R1 (referred to as GB1) subunit and GABA B R2 (referred to as GB2) subunit. Figure 1 The GB1 and GB2 can be divided into extracellular domain (ECD), heptahelical domain (HD) and intracellular domain from N-terminal to C-terminal of the peptide chain in terms of domain composition, and the ECD is similar to Venus flytrap and thus is called Venus flytrap domain (VFT). The VFT of GB1 can bind to endogenous ligand GABA or other artificial ligands (such as Baclofen, etc.), and then cause the conformational change of the HD domain of GB2, coupling with G protein trimer (Gα, Gβ, Gγ), controlling the downstream voltage-sensitive calcium ion channel, inward-rectifying potassium ion channel and adenylyl cyclase and other main effectors of the central nervous system. Unlike GB1, the VFT of GB2 cannot bind to endogenous ligand GABA or other artificial ligands.

[0003] Human GB1 includes two common splice variants, GB1a and GB1b. GB1a is a classical full-length receptor, which consists of 961 amino acids, and its ECD includes a Sushi domain and a VFT domain, wherein the Sushi domain is about 67 amino acids in length (30-96 amino acids); GB1b consists of 844 amino acids, and is different from GB1a in that its ECD does not contain a Sushi domain. Except for the Sushi domain, the amino acid sequences of the HD and intracellular side domains of GB1a and GB1b are completely identical. After being synthesized by ribosomes attached to the endoplasmic reticulum, GB1 is affected by an endoplasmic reticulum retention signal sequence (923RSR925) in its intracellular side domain, interacts with protein molecules such as PRAF2 in the endoplasmic reticulum, and is retained in the endoplasmic reticulum, and thus cannot be further sorted to the Golgi apparatus, and 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, release the endoplasmic reticulum retention of GB1, and allow GB1 and GB2 to form a heterodimer and be sorted and transported to the cell membrane.

[0004] Human GB2 consists of 1220 amino acids, and the amino acid sequence of the intracellular loop region of the HD thereof is responsible for coupling the G protein alpha subunit and mediating the downstream signal pathway. After being synthesized by ribosomes attached to the endoplasmic reticulum, GB2 can bind to the retained GB1 in the endoplasmic reticulum, so that GB1 and GB2 form a heterodimer and are sorted and transported to the cell membrane. If GB1 is not expressed in the cell, GB2 alone can also normally undergo sorting and transport to the cell membrane, but has no normal physiological functions such as signal transduction. For many years, there have been few reports of antibodies against GABA B Receptors, and therefore, there is a need to develop a gamma-aminobutyric acid type B receptor nanobody. SUMMARY

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

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

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

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

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

[0010] The sixth aspect of the present application aims to provide a vector.

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

[0012] The eighth aspect of the present application aims to provide a method for preparing the nanobody or antigen-binding fragment thereof of the first aspect, 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 of the present application.

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

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

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

[0016] The twelfth aspect of the present application aims to provide uses of the nanobody or antigen-binding fragment thereof of the first aspect, 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 of the present application.

[0017] To achieve the above-mentioned objects, the technical solutions adopted by the present application are as follows:

[0018] In a first aspect, the present application provides a nanobody or antigen-binding fragment thereof that specifically binds to a GABA type B receptor.

[0019] In some embodiments, the nanobody or antigen-binding fragment thereof that specifically binds to a GABA type B receptor comprises:

[0020] a CDR-H1, a CDR-H2 and a CDR-H3 comprised in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 10, or 15.

[0021] a nanobody or antigen-binding fragment thereof that specifically binds to a GABA type B receptor, the nanobody or antigen-binding fragment thereof that specifically binds to a GABA type B receptor comprising:

[0022] a1) a heavy chain variable region comprising the following 3 CDRs: a CDR-H1 having an amino acid sequence as set forth in SEQ ID NO: 7, a CDR-H2 having an amino acid sequence as set forth in SEQ ID NO: 8, and a CDR-H3 having an amino acid sequence as set forth in SEQ ID NO: 9; or

[0023] a2) a heavy chain variable region comprising CDR-H1 having an amino acid sequence as set forth in SEQ ID NO: 12, CDR-H2 having an amino acid sequence as set forth in SEQ ID NO: 13, and CDR-H3 having an amino acid sequence as set forth in SEQ ID NO: 14.

[0024] In some embodiments, the heavy chain variable region of the Nanobody or antigen binding fragment thereof that specifically binds to a GABA type B receptor further comprises a framework region of a heavy chain variable region.

[0025] In some embodiments, the framework region of the heavy chain variable region comprises a framework region of a heavy chain variable region of an immunoglobulin derived from a murine, primate, bovine, equine, bovine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervine, mink, chicken, duck, or goose, or a mutant thereof.

[0026] In some embodiments, the Nanobody or antigen binding fragment thereof that specifically binds to a GABA type B receptor comprises:

[0027] b1) a heavy chain variable region comprising an amino acid sequence as set forth in 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 an amino acid sequence as set forth 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] In a second aspect of the application, there is provided a heavy chain antibody or antigen binding fragment thereof that specifically binds to a GABA type B receptor, comprising an immunoglobulin Fc domain and a Nanobody or antigen binding fragment thereof of the first aspect of the application.

[0031] In some embodiments, the immunoglobulin Fc domain comprises an Fc domain of an immunoglobulin derived from a murine, primate, bovine, equine, bovine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervine, mink, chicken, duck, or goose, or a mutant thereof.

[0032] In a third aspect of the present application, there is provided a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, the antigen binding domain comprising a Nanobody or antigen binding fragment thereof of the first aspect of the present application or a heavy chain antibody or antigen binding fragment thereof of the second aspect of the present application.

[0033] In a fourth aspect of the present application, there is provided a multispecific antibody or antigen binding fragment thereof 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 the present application or a heavy chain antibody or antigen binding fragment thereof of the second aspect of the present application.

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

[0035] It will be understood by a person skilled in the art that nucleotides in the nucleic acid molecule can be substituted according to codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized.

[0036] In some embodiments, the nucleotide sequence encoding a Nanobody or antigen binding fragment thereof of the first aspect of the present application 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] In a sixth aspect of the present application, there is provided a vector comprising the nucleic acid molecule of the fifth aspect of the present application.

[0038] In some embodiments, the vector can be an expression vector. In some embodiments, the expression vector can comprise a eukaryotic cell expression vector and / or a prokaryotic cell expression vector. In some embodiments, the eukaryotic expression vector comprises, 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 can comprise, but is not limited to, a plasmid, a retroviral vector, a lentiviral vector, a bacteriophage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector.

[0039] In some embodiments, the vector can be selected from the group consisting of a nanoparticle, a liposome, an exosome, a microvesicle, or a gene gun.

[0040] In a seventh aspect of the present application, a cell comprising the Nanobody or antigen-binding fragment thereof of the first aspect, 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 is provided.

[0041] In some embodiments, the cell does not involve reproductive material.

[0042] In some embodiments, the cell can be a host cell routinely used in the art, as long as it can enable the expression vector to stably express the nucleic acid molecule carried thereby as the aforementioned Nanobody or antigen-binding fragment thereof, heavy chain antibody or antigen-binding fragment thereof, chimeric antigen receptor, or multispecific antibody or antigen-binding fragment thereof of the present disclosure. In some embodiments, the host cell can be a prokaryotic cell, such as E. coli, and / or a eukaryotic cell, such as CHO cell, HEK293 cell, BHK cell, NS0 cell, SP2 / 0 cell, YO myeloma cell, P3X63 mouse myeloma cell, PER cell, PER.C6 cell, HeLa cell, Vero cell, Expi293 cell, hybridoma cell, yeast cell, and insect cell.

[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 cell, NK cell, DC cell, and macrophage. In these embodiments, the immune cell can express the aforementioned chimeric antigen receptor of the present disclosure (i.e., a modified immune cell).

[0044] In an eighth aspect of the present application, a method for preparing the Nanobody or antigen-binding fragment thereof of the first aspect, 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 is provided, by culturing the cell of the seventh aspect of the present application.

[0045] In a ninth aspect of the present application, a conjugate comprising the Nanobody or antigen-binding fragment thereof of the first aspect, or the heavy chain antibody or antigen-binding fragment thereof of the second aspect; and, a conjugating moiety is provided.

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

[0047] In some embodiments, the detectable label can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, chemistry, etc. Such labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g.,3H,125I,35S,14C, or32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dot, or a cyanine dye derivative (e.g., Cy7, Alexa 750)), acridinium esters, magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for use with avidin (e.g., streptavidin) modified to bind the above labels. In some embodiments, such labels can be suitable for use in immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable label is selected from the group consisting of 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 linked to the above-mentioned nanobodies or antigen-binding fragments thereof, or heavy chain antibodies or antigen-binding fragments thereof of the present disclosure, or the conjugate of the present disclosure, through linkers of different lengths to reduce potential steric hindrance.

[0048] In some embodiments, the detectable label can include, but is not limited to, an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), a colored substance, biotin, etc.

[0049] In some embodiments, the therapeutic agent can include, for example, but is not limited to, a drug for preventing and / or treating a disease or disorder associated with a gamma-aminobutyric acid type B receptor.

[0050] In some embodiments, the conjugating moiety can be selected from a substance that can improve the biological properties of the antibody (e.g., increase the serum half-life), which can be, for example, a chemical group such as polyethylene glycol (PEG), a methyl group, an ethyl group, or a sugar group.

[0051] In a tenth aspect of the present disclosure, a pharmaceutical composition is provided, which comprises: the nanobody or antigen-binding fragment thereof of the first aspect, 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 can further comprise an additional pharmaceutically active agent.

[0053] In some embodiments, the additional pharmaceutically active agent can be a drug having a biological activity, for example, a drug capable of preventing and / or treating a disease or disorder associated with GABA type B receptor.

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

[0055] In some embodiments, the pharmaceutical composition can be administered by, for example, parenteral, subcutaneous injection, sublingual, rectal, nasal, intravenous injection, intramuscular injection, oral, ocular, topical, etc.

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

[0057] In an eleventh aspect of the present application, there is provided a diagnostic or therapeutic kit comprising: the Nanobody or antigen-binding fragment thereof of the first aspect, 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 can further comprise an instruction and / or a device for administration.

[0059] In some embodiments, the kit can be used for diagnosing a disease or disorder associated with GABA type B receptor.

[0060] In some embodiments, the kit can be used for preventing or treating a disease or disorder associated with GABA type B receptor.

[0061] In a twelfth aspect of the present application, there is provided use of the Nanobody or antigen-binding fragment thereof of the first aspect, 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 manufacture of a product for any one of c1) to c3):

[0062] c1) Diagnose diseases or conditions related to γ-aminobutyric acid type B receptor;

[0063] c2) Prevention or treatment of diseases or conditions related to γ-aminobutyric acid type B receptor;

[0064] c3) Detect the presence or level of γ-aminobutyric acid type B receptor in the sample.

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

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

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

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

[0069] In some implementations, the test subject includes mammals such as humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits).

[0070] In some implementations, the subject of the test includes humans.

[0071] In this invention, the γ-aminobutyric acid B receptor-related diseases or conditions are epilepsy, anxiety, depression, or cognitive impairment due to nerve damage.

[0072] The beneficial effects of this invention are:

[0073] This invention provides nanobodies or antigen-binding fragments thereof that specifically bind to the γ-aminobutyric acid (GABA) type B receptor. These nanobodies can specifically recognize and bind to the GABA type B receptor and have good affinity for it. They can be used to prepare products for the diagnosis, prevention or treatment of GABA type B receptor-related diseases or conditions, or for detecting the presence or level of GABA type B receptor in samples. Attached Figure Description

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

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

[0076] Figure 3The results of the antigen affinity detection of the nanobody NB-5E9 are shown in the schematic diagram. DETAILED DESCRIPTION

[0077] To make the objectives, technical solutions, and advantages of the present application clearer, the present application is further described in detail below with reference to embodiments. The specific embodiments described herein are intended for illustration only and are not intended to be limiting in any way. Moreover, in the following description, descriptions of well-known structures and techniques are omitted to avoid obscuring the concept of the present disclosure. Such structures and techniques are described in many publications.

[0078] Definitions

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied to the descriptions of the present application and throughout this document, and apply equally, where appropriate, to the singular as well as to the plural, or vice versa.

[0080] The expressions "a" and "an" as used herein include plural references unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents, and the like, as will be apparent to one of ordinary skill in the art.

[0081] As used herein, the term "about" means a range of ±20% of the value that follows. In some embodiments, the term "about" means a range of ±10% of the value that follows. In some embodiments, the term "about" means a range of ±5% of the value that follows.

[0082] K D values : dissociation constant (K D ) is a specific type of equilibrium constant that measures the tendency of a larger object to separate (dissociate) into smaller components, is the inverse of the association constant, and has units of mol / L (M) or nmol / L (nM). The smaller the K D value, the stronger the binding ability of the two substances.

[0083] NanobodyNatural missing light chain antibodies existing in peripheral blood of Camelidae, the antibodies only contain one heavy chain variable region (VHH) and two conventional CH2 region and CH3 region, but are not easy to adhere to each other, even aggregate into blocks, unlike artificially modified single-chain antibody fragments; the VHH structure cloned and expressed alone has structural stability comparable to the original heavy chain antibody and binding activity to antigens, and is the smallest unit known to bind target antigens; the VHH crystal is 2.5 nm long and 4 nm, and the molecular weight is only about 15 kD, so it is also called nanobody (Nb). Compared with traditional mice, rabbits and other animals that can only recognize the polypeptide on the flat surface of the antigen, the immune system in the camelidae can recognize the complex spatial structure on the surface of the antigen, and can produce highly specific and high-affinity nanobodies.

[0084] Unlike traditional technologies relying on classic model animals such as mice, rabbits, monkeys, and sheep, the technical solution of the present application is an antibody produced by the immune system of a llama, known as a "nanobody". Nanobodies are small antibody fragments isolated from immunoglobulins in animals such as camels, which have the same antigen binding capacity and structural stability as complete antibodies, and are the smallest units known to bind target antigens, with a relative molecular mass of only about 15 kD. Compared with traditional mice, rabbits and other animals that can only recognize the polypeptide on the flat surface of the antigen, the immune system in the camelidae can recognize the complex spatial structure on the surface of the antigen, and can produce highly specific and high-affinity nanobodies.

[0085] According to the technical solution of the present application, 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; Gin 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; Gin Val Ile; Leu Ile Leu; Val

[0088] In addition, due to the degeneracy of 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] The following examples and drawings are provided to help understand the present application. However, it should be understood that these examples and drawings are only used to illustrate the present application, but do not constitute any limitation. The actual protection scope of the present application is set forth in the claims. It should be understood that any modification and change can be made without departing from the spirit of the present application.

[0093] Example 1. Preparation of antigen

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

[0095] (2) After transfection, the cells were cultured for about 48 h to allow the GABA B receptor heterodimer to be fully expressed on the cell membrane surface;

[0096] (3) About 4*10 8 cells were collected, ultrasonically broken, homogenized, and ultracentrifuged, and the cell membrane extract was obtained, which was a suitable antigen; the preparation steps of the cell membrane extract were as follows:

[0097] 1) About 4*10 8 cells were taken out from -80°C, and an appropriate amount of buffer was added for thawing on ice.

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

[0099] 3) Homogenization grinding: for each construct, the cells were taken and transferred to a Dounce homogenizer pre-cooled in ice, and ground evenly for about 20 times.

[0100] 4) Ultrasonic disruption: the sample was transferred to a pre-cooled 15 mL tube, and ultrasonic disruption was performed using an ultrasonic rod with appropriate thickness and a suitable power size.

[0101] 5) The supernatant was collected by centrifugation and was recorded as supernatant 1;

[0102] 6) The sample was resuspended with 6 mL of Buffer and homogenized for about 20 times, and the supernatant was collected by centrifugation and was recorded as supernatant 2;

[0103] 7) The above step was repeated and was recorded as supernatant 3;

[0104] 8) Supernatants 1+2+3 were combined, and the total volume was about 15-20 mL, and ultracentrifugation was performed at 50000 g for 1-1.5 h.

[0105] 9) The supernatant was discarded, and the precipitate was resuspended with an appropriate amount of PBS to obtain the cell membrane extract.

[0106] 10) Mix all the cell membrane extracts of all the constructs evenly, control the total volume at about 4 mL, and aliquot into 1.5 mL EP tubes, 4 tubes, 1 mL per tube, and store at -80℃.

[0107] GABA B The receptor can be correctly expressed, present on the cell membrane surface, maintain the correct assembly, folding and conformational state, and especially maintain the complex spatial structure on the surface.

[0108] Example 2. Immunization of alpaca

[0109] In this example, the cell membrane extract of Example 1 (suitable antigen GABA B receptor) is used to immunize alpaca. The specific steps are as follows:

[0110] (1) Divide the antigen in Example 1 into 4 equal parts, about 0.5 mg each; immunize the alpaca 4 times, and inject the antigen subcutaneously into the animal, with the first immunization being day 1, and the subsequent immunizations being on day 11, day 21 and day 31;

[0111] (2) On day 30, before the fourth immunization, collect about 200 mL of alpaca peripheral venous blood;

[0112] (3) On day 45, 14 days after the fourth immunization, collect about 200 mL of alpaca peripheral venous blood.

[0113] Compared with the traditional immunization technology scheme of mouse, rabbit and other animal antibodies, the technical advantage of the present application is that a large amount of alpaca peripheral venous blood is collected, which is beneficial to the subsequent screening of highly diverse nanobodies.

[0114] Example 3. Construction of alpaca nanobody library

[0115] Two batches of alpaca peripheral venous blood collected in Example 2 are used as raw materials to construct a high-diversity nanobody library. The processing methods of the two batches of alpaca peripheral venous blood are the same, and the specific steps are as follows:

[0116] (1) Use density gradient centrifugation and other methods to separate lymphocytes from alpaca peripheral venous blood;

[0117] (2) Extract total mRNA from the lymphocytes and reverse transcribe it into cDNA;

[0118] (3) Use appropriate DNA primers (see Table 3 below), and use the above cDNA as a template to amplify the VHH fragments of alpaca immunoglobulin IgG2 and IgG3, i.e. the DNA fragments of nanobodies, by polymerase chain reaction (PCR);

[0119] Table 3. Primers used for constructing nanobody library

[0120]

[0121]

[0122] (4) Link the DNA of VHH to the phage surface display selection vector phen1 to form a VHH-His-pIII fusion protein expression vector plasmid library; where pIII is a protein present on the flagella on the surface of the phage, and His is a histidine tag (6 consecutive histidines, i.e. His-His-His-His-His-His).

[0123] (5) The DNA ligation product was transformed into TG1 competent Escherichia coli by electroporation. After appropriate culture, all colonies were collected to obtain the alpaca nanobody library.

[0124] Compared to traditional methods of isolating antibodies from the serum or lymphocytes of animals such as mice and rabbits, this invention 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.

[0125] Example 4. Displaying screening-specific nanobodies on the surface of bacteriophages

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

[0127] (1) Take an appropriate amount of frozen nanobody library, inoculate it into bacterial culture medium, and after appropriate culture, add an appropriate amount of helper phage (M13KO7 helper phage, NEB, N0315S) and continue to culture under appropriate conditions.

[0128] (2) The bacteriophages amplified in the bacterial culture supernatant were extracted by PEG-NaC method and frozen in an ultra-low temperature freezer at -80℃ for later use.

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

[0130] (4) Negative screening: Prepare approximately 5*10 7 HEK293T tool cells that were not transfected with any exogenous DNA vector plasmids were thawed, and the phages were incubated with the tool cells for 2 hours. After centrifugation, the supernatant was collected.

[0131] (5) Positive screening: Collect approximately 5*10 from (3). 7HEK293T cells overexpressing GABA B Incubate the supernatant obtained after centrifugation in (4) with the HEK293T cells overexpressing GABA receptor for 2 hours.

[0132] (6) Elute: Discard the phage, and then rinse the antigen cells with PBS buffer for 3 times. Elute and remove the phage that is not specifically combined with the antigen, and retain the phage that is specifically combined with the antigen.

[0133] (7) Elute: Treat the phage that is specifically combined with the antigen with an acidic glycine solution to separate the phage from the antigen and retain the phage.

[0134] At this point, the phage expressing the specific nanobody is obtained, and the phage can be subjected to the following technical operations:

[0135] (8) Convert into a specific nanobody library: Infect the phage into TG1 competent E. coli again for culture, but do not add the helper phage again. After the phage infection is complete, the specific nanobody exists in the E. coli in the form of a DNA plasmid. Collect all the E. coli, which becomes the antigen-specific nanobody library. The library can be used as raw material to return to step (1) for the next round of phage surface display screening;

[0136] (9) Convert into a monoclonal nanobody colony: Take a small amount (such as 0.5%) of the phage obtained in step (7), dilute it, and then infect TG1 competent E. coli again for culture, but do not add the helper phage again. After the phage infection is complete, evenly spread the E. coli on a bacterial culture dish, and then adjust the culture to obtain a monoclonal colony containing a nanobody DNA plasmid. Use the monoclonal colony as raw material to identify the positive monoclonal nanobody.

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

[0138] This example uses the bacterial culture dish with the monoclonal colony obtained in step (9) of Example 4 to identify the positive monoclonal nanobody. The specific steps are as follows:

[0139] (1) Pick the monoclonal colony on a microplate for culture;

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

[0141] (3) Use a liposome transfection reagent (Lipo3000, ThermoFisher) to co-transfect a DNA vector plasmid encoding human GB1a and GB2 into HEK293T tool cells. Continue to culture the cells for 24 hours after transfection;

[0142] (4) Collect the nanobody-containing bacterial culture supernatant obtained in (2) and incubate with the cells obtained in (3) for 2 hours, about 2*10^5 overexpressing GABA B receptors per well of the 96-well plate, centrifuge after the incubation, and discard the supernatant;

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

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

[0145] (7) Detect the distribution of Alexa Flour 647 fluorescence intensity on the cell surface using a flow cytometer to determine whether the nanobody can bind to the GABA B receptors on the cell surface.

[0146] (8) For the monoclonal nanobodies (NB-4C10, NB-5E9) that can specifically recognize and bind to antigens, the TG1 strain expressing the relevant monoclonal nanobody is cultured overnight at 37°C, the DNA plasmid is extracted and subjected to Sanger sequencing to obtain the nucleotide sequence of the nanobody, which is then translated to obtain the amino acid sequence of the nanobody. The specific sequences are shown in Tables 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 recombinant expression and purification of monoclonal nanobodies

[0153] (1) The monoclonal nanobodies obtained in Example 5 that can specifically recognize and bind to antigens are transformed into BL21(DE3) competent cells with the DNA plasmid encoding the nanobodies (NB-4C10, NB-5E9), and the nanobodies are expressed by means of E. coli recombinant expression. After bacterial lysis, histidine tag affinity chromatography, and gel filtration sequencing, the monoclonal nanobodies are obtained, with a batch production capacity of about several milligrams.

[0154] (2) Using flow cytometry analysis method, incubate nanobodies of different concentrations, and measure the binding ability of nanobodies to cells overexpressing human GABA B receptor (the construction method is the same as that in Example 1) to measure the affinity of nanobodies to antigens.

[0155] The detection results are shown in Table 1. Figures 2-3 The affinity values K D of the monoclonal nanobodies NB-4C10 and NB-5E9 are 3.969 nM and 6.148 nM, respectively.

[0156] The technical solutions of the present application are not limited to the above specific examples, and any technical variations made according to the technical solutions of the present application fall within the protection scope of the present application.

Claims

1. A Nanobody or an antigen-binding fragment thereof that specifically binds to a GABA type B receptor, comprising: a CDR-H1, a CDR-H2 and a CDR-H3 comprised in a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO: 10, or 15.

2. The Nanobody or the antigen-binding fragment thereof according to claim 1, wherein the Nanobody or the antigen-binding fragment thereof that specifically binds to a GABA type B receptor comprises a heavy chain variable region comprising: a1) a CDR-H1 of an amino acid sequence as set forth in SEQ ID NO: 7, a CDR-H2 of an amino acid sequence as set forth in SEQ ID NO: 8 and a CDR-H3 of an amino acid sequence as set forth in SEQ ID NO: 9; or a2) a CDR-H1 of an amino acid sequence as set forth in SEQ ID NO: 12, a CDR-H2 of an amino acid sequence as set forth in SEQ ID NO: 13 and a CDR-H3 of an amino acid sequence as set forth in SEQ ID NO:

14.

3. The Nanobody or the antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region of the Nanobody or the antigen-binding fragment thereof that specifically binds to a GABA type B receptor further comprises a framework region of the heavy chain variable region.

4. The Nanobody or the antigen-binding fragment thereof according to claim 3, wherein the framework region of the heavy chain variable region comprises a framework region of a heavy chain variable region of an immunoglobulin derived from a murine, a primate, a bovine, a horse, a porcine, an ovine, a caprine, a canine, a feline, a leporine, a camelid, an equid, a cervid, a mustelid, a chicken, a duck or a goose, or a mutant thereof.

5. The Nanobody or the antigen-binding fragment thereof according to claim 4, wherein the Nanobody or the antigen-binding fragment thereof that specifically binds to a GABA type B receptor comprises: b1) a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 10; or b2) a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

15.

6. The Nanobody or the antigen-binding fragment thereof according to claim 5, wherein the Nanobody or the antigen-binding fragment thereof that specifically binds to a GABA type B receptor comprises: b1) a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 10; or b2) a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:

15.

7. A heavy chain antibody or an antigen-binding fragment thereof that specifically binds to a GABA type B receptor, comprising an immunoglobulin Fc domain and a Nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 6.

8. A chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, the antigen binding domain comprising the Nanobody or antigen binding fragment thereof of any one of claims 1-6 or the heavy chain antibody or antigen binding fragment thereof of claim 7.

9. 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 of any one of claims 1-6 or the heavy chain antibody or antigen binding fragment thereof of claim 7.

10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the Nanobody or antigen binding fragment thereof of any one of claims 1-6, the heavy chain antibody or antigen binding fragment thereof of claim 7, the chimeric antigen receptor of claim 8, or the multispecific antibody or antigen binding fragment thereof of claim 9.

11. A vector comprising the nucleic acid molecule of claim 10.

12. A cell comprising the Nanobody or antigen binding fragment thereof of any one of claims 1-6, the heavy chain antibody or antigen binding fragment thereof of claim 7, the chimeric antigen receptor of claim 8, the multispecific antibody or antigen binding fragment thereof of claim 9, the nucleic acid molecule of claim 10, or the vector of claim 11.

13. A method of producing the Nanobody or antigen binding fragment thereof of any one of claims 1-6, the heavy chain antibody or antigen binding fragment thereof of claim 7, the chimeric antigen receptor of claim 8, or the multispecific antibody or antigen binding fragment thereof of claim 9, by culturing the cell of claim 12.

14. A conjugate comprising the Nanobody or antigen binding fragment thereof of any one of claims 1-6 or the heavy chain antibody or antigen binding fragment thereof of claim 7; and, a conjugating moiety.

15. The conjugate of claim 14, wherein, the conjugating moiety comprises a detectable label or a therapeutic agent; 16. The conjugate of claim 15, wherein, the detectable label comprises an enzyme, a radionuclide, a luminescent substance, a colored substance, and / or biotin; or the therapeutic agent comprises a drug that prevents and / or treats a disease or disorder associated with GABA type B receptors; the disease or disorder associated with GABA type B receptors is epilepsy, anxiety, depression, or reduced cognitive ability due to nerve damage.

17. The conjugate of claim 15, wherein, the detectable label comprises a fluorescent dye.

18. A pharmaceutical composition comprising: the Nanobody or antigen binding fragment thereof of any one of claims 1-6, the heavy chain antibody or antigen binding fragment thereof of claim 7, the chimeric antigen receptor of claim 8, the multispecific antibody or antigen binding fragment thereof of claim 9, the nucleic acid molecule of claim 10, the vector of claim 11, the cell of claim 12, or the conjugate of any one of claims 14-17; and, a pharmaceutically acceptable carrier.

19. The pharmaceutical composition of claim 18, wherein, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

20. A diagnostic or therapeutic kit comprising: The nanobody or antigen-binding fragment thereof of any one of claims 1-6, the heavy chain antibody or antigen-binding fragment thereof of claim 7, the chimeric antigen receptor of claim 8, the multispecific antibody or antigen-binding fragment thereof of claim 9, the nucleic acid molecule of claim 10, the vector of claim 11, the cell of claim 12, the conjugate of any one of claims 14-17, or the pharmaceutical composition of any one of claims 18-19.

21. The kit of claim 20, further comprising instructions and / or a device for administration.

22. Use of the nanobody or antigen-binding fragment thereof of any one of claims 1-6, the heavy chain antibody or antigen-binding fragment thereof of claim 7, the chimeric antigen receptor of claim 8, the multispecific antibody or antigen-binding fragment thereof of claim 9, the nucleic acid molecule of claim 10, the vector of claim 11, the cell of claim 12, the conjugate of any one of claims 14-17, or the pharmaceutical composition of any one of claims 18-19 for the manufacture of a product for any one of c1) - c3): c1) diagnosing a GABA type B receptor related disease or disorder; c2) preventing or treating a GABA type B receptor related disease or disorder; c3) detecting the presence or level of GABA type B receptor in a sample; the GABA type B receptor related disease or disorder is epilepsy, anxiety, depression or cognitive impairment due to nerve damage. ​

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