High-affinity nano antibody for resisting nervous centralis specific protein S100beta or antigen binding fragment of high-affinity nano antibody and application of high-affinity nano antibody or antigen binding fragment

Abstract

The invention discloses a high-affinity nano antibody for resisting a nervous centralis specific protein S100beta or an antigen binding fragment of the high-affinity nano antibody and application of the high-affinity nano antibody, and belongs to the technical field of antibodies. The amino acid sequence of the nano antibody is as shown in SEQ ID NO: 2, and the nucleotide sequence of the nano antibody is as shown in SEQ ID NO: 1. The invention provides a high-affinity antibody with EC50 reaching 4.0 nM and a recombinant expression scheme capable of realizing large-scale production, and provides a high-affinity and low-cost S100beta nano antibody by reasonably designing a four-round screening strategy.

Description

Technical Field

[0001] This invention belongs to the technical field of antibodies, specifically relating to a high-affinity nanobody against the central nervous system-specific protein S100β or its antigen-binding fragment and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] S100β, a central nervous system-specific protein belonging to the EF-hand calcium-binding protein family, is primarily secreted by astrocytes and plays a crucial role in central nervous system development, cell proliferation and differentiation, damage repair, and the regulation of inflammatory responses. S100β binds to receptors on the cell surface, initiating downstream signaling pathways and participating in pathophysiological processes such as glial scar formation, inflammatory cytokine release, and neuronal damage. In central nervous system diseases such as Alzheimer's disease, traumatic brain injury, brain tumors, and multiple sclerosis, abnormally high expression of S100β can exacerbate pathological damage and lead to adverse consequences such as neurological dysfunction. S100β antagonists can block the interaction between S100β and its receptors, thereby neutralizing the pathological function of S100β. Currently, S100β antagonists used clinically to treat S100β-related diseases suffer from limitations such as large molecular weight (150 kDa), complex structure, difficulty in efficiently penetrating the blood-brain barrier, complex production processes requiring mammalian cell expression, and high production costs, which restrict their widespread application.

[0004] Nanobodies (VHH), as single-domain antibodies derived from camel-derived heavy-chain antibodies, have a molecular weight of only about 15 kDa and possess significant advantages such as high stability, strong affinity, good tissue penetration, and efficient expression in prokaryotic cells. However, natural camel-derived nanobodies may trigger an immune response in humans, and the affinity and specificity of existing S100β nanobodies still need improvement. Therefore, developing a highly efficient, low-cost, and high-affinity S100β nanobody has significant clinical application value and market potential. Summary of the Invention

[0005] To address the insufficient affinity of existing S100β nanobodies, the present invention aims to provide a high-affinity nanobodiment or its antigen-binding fragment against the central nervous system-specific protein S100β, and its applications. This invention provides a high-affinity antibody with an EC50 of 4.0 nM and a recombinant expression scheme suitable for scalable production. Therefore, through a rationally designed four-round screening strategy, a high-affinity, low-cost S100β nanobodiment is provided.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a high-affinity nanobody against the central nervous system-specific protein S100β or an antigen-binding fragment thereof, wherein the VHH chain of the nanobody includes a complementarity-determining region and a framework region; the complementarity-determining region includes CDR1 shown in SEQ ID NO:12, CDR2 shown in SEQ ID NO:14, and CDR3 shown in SEQ ID NO:16.

[0008] The nanobodies described herein typically comprise a VHH consisting of four framework regions (FRs) and three complementarity-determining regions (CDRs), referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The antigen-binding fragment contains at least a portion of the nanobody, sufficient to confer the fragment the ability to specifically bind S100β. In some embodiments, the nanobodies of the present invention may be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to omit one or both of those framework regions, as long as they substantially maintain antigen binding and specificity.

[0009] In some embodiments, the nanobody comprises a heavy chain framework region derived from the central nervous system-specific protein S100β (e.g., a heavy chain framework region contained in the amino acid sequence encoded by the antibody gene).

[0010] The frame region includes: FR1 shown in SEQ ID NO:4, FR2 shown in SEQ ID NO:6, FR3 shown in SEQ ID NO:8, and FR4 shown in SEQ ID NO:10; Preferably, the nanobody comprises the sequence shown in SEQ ID NO:2.

[0011] The amino acid sequence of CDR1 is shown in SEQ ID NO:12, and its nucleotide sequence is shown in SEQ ID NO:11; the amino acid sequence of CDR2 is shown in SEQ ID NO:14, and its nucleotide sequence is shown in SEQ ID NO:13; the amino acid sequence of CDR3 is shown in SEQ ID NO:16, and its nucleotide sequence is shown in SEQ ID NO:15.

[0012] The amino acid sequence of FR1 is shown in SEQ ID NO:4, and its nucleotide sequence is shown in SEQ ID NO:3; the amino acid sequence of FR2 is shown in SEQ ID NO:6, and its nucleotide sequence is shown in SEQ ID NO:5; the amino acid sequence of FR3 is shown in SEQ ID NO:8, and its nucleotide sequence is shown in SEQ ID NO:7; the amino acid sequence of FR4 is shown in SEQ ID NO:10, and its nucleotide sequence is shown in SEQ ID NO:9.

[0013] Furthermore, the amino acid sequence of the heavy chain variable region of the nanobody is shown in SEQ ID NO:2, and its nucleic acid sequence is shown in SEQ ID NO:1.

[0014] Furthermore, the amino acid sequence of the heavy chain variable region of the nanobody has at least 80% homology with SEQ ID NO:2; more preferably, at least 90% homology; most preferably, at least 95% homology; such as at least 96%, 97%, 98%, or 99% homology.

[0015] It should be noted that the "antigen-binding fragment" of the antibody of the present invention is a part of the antibody, that is, a molecule corresponding to a part of the structure of the antibody of the present invention. These antigen-binding fragments can also be referred to as functional fragments of the antibody. Such fragments exhibit the same or substantially the same antigen-binding specificity and affinity for the said antigen.

[0016] Secondly, the present invention provides a nucleic acid molecule encoding the above-described nanobody or its antigen-binding fragment, the sequence of which is shown in SEQ ID NO:1.

[0017] Thirdly, the present invention provides an expression vector containing the above-mentioned nucleic acid molecules.

[0018] Specifically, the expression vector is obtained by effectively linking the above-mentioned nucleic acid with the expression vector. The expression vector can be selected from: DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or combinations thereof; preferably, the expression vector includes viral vectors, such as lentivirus, adenovirus, AAV virus, retrovirus, or combinations thereof.

[0019] Preferably, the expression vector includes pET26b(+).

[0020] Fourthly, the present invention provides a host cell. It comprises the above-described expression vector, or comprises the above-described nucleic acid molecule integrated into the host cell genome; or, the host cell expresses the above-described antibody or its antigen-binding fragment.

[0021] The host cell includes cells derived from prokaryotes and eukaryotes. Among them, prokaryotes are preferably *Escherichia coli*, and eukaryotes include yeast (Pichia pastoris), plants (or cells), insects (or cells), and mammals (or cells). Preferably, the host cell includes *Escherichia coli* BL21.

[0022] Fifthly, the present invention provides a method for preparing the above-mentioned antibody or its antigen-binding fragment, comprising culturing the above-mentioned host cells.

[0023] Specifically: (I) culturing the host cells described above under conditions suitable for antibody production to obtain a culture containing the antibody or its antigen-binding fragment; and, (II) Isolate or recover the antibody or its antigen-binding fragment from the culture.

[0024] The preparation method of anti-S100β nanobody includes the following steps: (a) Four rounds of phage screening; (II) ELISA validation of screening effectiveness; (iii) ELISA screening and sequencing of positive clones; (iv) Plasmid construction and chemical transformation of nanobodies; (v) Expression and purification of nanobodies; (vi) ELISA verification of nanobody affinity.

[0025] Phage display technology is a core platform for screening nanobodies, but traditional three-round screening often leads to the omission of high-affinity clones, while overly stringent screening strategies (such as high-concentration competitive elution) reduce clonal diversity. To address these issues, this invention employs a rationally designed four-round screening process to produce high-affinity S100β nanobodies.

[0026] In a sixth aspect, the present invention provides a recombinant polypeptide conjugate comprising the antibody or its antigen-binding fragment described in the present invention, and a conjugation portion, wherein the conjugation portion is a detectable marker, drug, toxin, cytokine, viral capsid protein, or VLP, etc.

[0027] The detectable markers include radioactive isotopes, fluorescent substances, chemiluminescent substances, colored substances, or enzymes.

[0028] The drug may be a cytotoxic drug, such as an anti-tubulin drug, DNA minor groove binder, DNA replication inhibitor, alkylating agent, antibiotic, folic acid antagonist, antimetabolite, chemotherapy sensitizer, topoisomerase inhibitor, vinca alkaloid, etc.

[0029] The toxins may include tastatins, chlortetracycline, phenytoin, acetaminophen, ethidium bromide, mitomycin, diphtheria toxin, absinthecin, white tree toxin, metoprolol, localized aspergillin, phenolmycin, jatropha toxin, croton toxin, chalcogenide, glucocorticoids, etc.

[0030] The recombinant polypeptide conjugate contains a polyvalent (e.g., divalent) antibody as described in this invention. The polyvalent meaning refers to the presence of multiple repeating antibodies as described in this invention within the amino acid sequence of the recombinant polypeptide conjugate.

[0031] In a seventh aspect, the present invention provides a reagent or kit comprising the high-affinity nanobody or its antigen-binding fragment described above, and / or the conjugate described above; the kit can be used to detect the presence or concentration of an antibody in a sample.

[0032] Preferably, the kit further includes a second antibody that specifically recognizes the antibody; optionally, the second antibody further includes a detectable marker, such as a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme.

[0033] In an eighth aspect, the present invention provides a pharmaceutical composition comprising the antibody or its antigen-binding fragment described above, or the recombinant polypeptide conjugate described above.

[0034] The pharmaceutical composition may also contain at least one inactive pharmaceutical ingredient.

[0035] The inactive components of the drug can be pharmaceutically commonly used carriers, excipients, and diluents. Furthermore, according to conventional methods, it can be formulated into oral, topical, suppository, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays.

[0036] The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0037] The carrier, excipients, and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, etc.

[0038] The pharmaceutical compositions of the present invention can be administered into the body in known ways. For example, they can be delivered to the tissue of interest via intravenous systemic delivery or local injection. Alternatively, administration can be made via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via a single dose or multiple doses. Those skilled in the art will understand that the actual dose to be administered in the present invention can vary considerably depending on a variety of factors, such as platelets, target cells, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0039] In a ninth aspect, the present invention provides the use of the above-described high-affinity nanobody, the above-described reagent or kit, the above-described recombinant polypeptide conjugate, and the above-described pharmaceutical composition in any one or more of the following: A1: To develop products for the detection, monitoring, diagnosis and / or prognosis of central nervous system diseases; A2: To develop products for the prevention and / or treatment of central nervous system diseases.

[0040] This includes diagnostic reagents. The central nervous system diseases mentioned include Alzheimer's disease, traumatic brain injury, brain tumors, multiple sclerosis, etc.

[0041] In a tenth aspect, the present invention provides a method for detecting, monitoring, and / or diagnosing S100β protein in a sample, comprising: a) Under conditions sufficient to allow an antibody / antigen binding reaction to occur, the S100β protein antigen in the test sample is contacted with the antibody, reagent, or kit described above to form an immune complex. b) Detect the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

[0042] The present invention also provides a method for preventing and / or treating central nervous system diseases, the method comprising: administering the above-mentioned antibody or its antigen-binding fragment, the above-mentioned immunoconjugate, or the above-mentioned pharmaceutical composition to the subject.

[0043] The “individual” or “subject” can be a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual or subject is a human.

[0044] The term "pharmaceutical composition" refers to a formulation in which the biological activity of the active ingredient contained therein is effective and which does not contain any other ingredients that would have unacceptable toxicity to a subject who would receive the composition.

[0045] The term "treatment / prevention" refers to an attempt to alter the natural course of a disease in an individual, and can be a clinical intervention implemented for prevention or during the course of clinicopathological processes. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and eliminating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or slow the progression of symptoms.

[0046] As used herein, the term "nanobody" has the meaning commonly understood by those skilled in the art as an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camel or shark antibody). Typically, a nanobody consists of four framework regions and three complementarity-determining regions, having a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to omit one or both of those framework regions, as long as they substantially maintain antigen binding and specificity. Nanobodies are also called single-domain antibodies (sdAbs), and the two terms are used interchangeably.

[0047] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residue in the variable region of an antibody responsible for antigen binding. The nanobody contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given nanobody, those skilled in the art will readily identify the CDR defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefrance et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0048] As used herein, the term “frame region” or “FR” residues refer to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0049] One or more of the above technical solutions have the following advantages or beneficial effects: (1) High screening efficiency: Through four rounds of optimized phage screening, the enrichment efficiency of high affinity antibodies was significantly improved, and the high affinity positive clone rate reached 12.9%.

[0050] (2) Strong antibody affinity: The EC50 of the obtained D3 nanobody reached 4.0 nM, and the affinity was better than most existing anti-S100β antibodies.

[0051] (3) High efficiency of expression system: The pET26b(+)-BL21 expression system is adopted, which greatly increases the yield and significantly reduces the production cost.

[0052] (4) Broad application prospects: This nanobody can be used to prepare diagnostic reagents for S100β-related diseases, and has extremely high practical application value. Attached Figure Description

[0053] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0054] Figure 1 A curve validating the screening effect of S100β nanobodies; Figure 2 Gel electrophoresis image of linearized pET26b(+) plasmid; Figure 3 This is a gel electrophoresis image of the antibody gene amplified by PCR. Figure 4 This is a protein gel electrophoresis image showing the entire process of expression and purification of the S100β nanobody (D3). Figure 5 This is an affinity curve of the S100β nanobody. Detailed Implementation

[0055] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0056] Table 1. Reagent Sources name brand Item number S100β antigen Righteousness and righteousness in China 10181-H07E Phage library Chengdu critical point AIpSDAb-P TG1 E. coli cells Chengdu critical point P008 Top 10 Cheminable Cells Sangon Biotech B528412 BL21 chemocompetent cells Sangon Biotech B528414 M13KO7 helper phage HonorGene VSW0920 pET26b(+) plasmid HonorGene HG-VYN0166 Bovine serum albumin (BSA) Aladdin B265993 High-purity ovalbumin (OVA) Solarborg 9006-59-1 Carbenicillin Genha R20018 Kanamycin Aladdin K331597 2xYT medium Qingdao Haibo Biotechnology HBDC003 SOC culture medium Qingdao Haibo Biotechnology HBDC002 LB solid culture medium Qingdao Haibo Biotechnology HB0129 LB medium Qingdao Haibo Biotechnology HB0128 Agar BioFroxx 8211GR500 Polyethylene glycol 8000 Genha S30193 Sodium chloride Sinopharm Group 10019308 1M Tris-HCl (pH=8.0) Azure Sky ST780-500ml IPTG Solarborg I8070 sucrose Sinopharm Reagent 20059026 Tris Sangon Biotech A100826-0100 HEPES Aladdin H109406-100g imidazole Solarborg I8090 Ni-NTA resin Yisheng Bio 20502ES10 NcoI restriction endonuclease MCE HY-KE7025 XhoI restriction endonuclease MCE HY-KE7045 Plasmid mini-prep kit QIAGEN 27104 GeneJET Gel Recovery Kit thermo fisher K0692 2×PCR Master Mix thermo fisher K0172 GenBuilder 2x Master Mix Genscript Biotech L00701 anti-M13 Bacteriophage-HRP secondary antibody Chengdu critical point 052-201-005 anti-His-HRP secondary antibody Proteintech Group HRP-66005 To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0057] Example 1 This invention provides a method for preparing anti-S100β nanobodies, comprising the following steps: (a) Four rounds of phage screening Plate coating: Coat 5 μg / mL S100β antigen onto an ELISA plate, and coat the blank group with PBS buffer. Incubate overnight at 4°C. Blocking the wells: Discard the coating solution and block the ELISA plate at room temperature for 2 hours using 3% BSA-PBS (changing the blocking solution every time can greatly reduce the enrichment of phages against a specific blocking solution). Blocking the library: Mix 90 μL of phage library, 180 μL of PBS and 30 μL of 30% BSA-PBS solution, shake at 220 rpm and block at room temperature for 1 hour; Binding: Discard the blocking solution, shake the microplate dry, add the phage library, shake at 220 rpm and incubate at 37°C for 2 hours; Washing: Pour out the unbound phages from the ELISA plate, wash 5 times with PBST, and wash 3 times with PBS; Elution: Add 200 μL HCl (0.1 M, pH=2.2) to the enzyme-labeled wells, shake at 220 rpm and incubate at 37°C for 10 min to elute the bound phages; Neutralization: Neutralize using 1M Tris-HCl (pH=8.0); Amplification: Using 400 μL of the neutralized solution eluted from the OD, infect 4 mL of OD. 600 =0.5% TG1 Escherichia coli cells, then cultured at 37°C with shaking for 1 hour; then add M13KO7 helper phage, and culture at 37°C with shaking for 1 hour; finally transfer to 50 mL 2×YT / Carb / Kan medium and culture overnight at 30°C; Titer determination (performed simultaneously with the amplification steps above): The bacterial culture after infecting TG1 cells was serially diluted 10× with 2×YT (8 concentrations). 10 μL of each gradient was dropped into 2×YT / Carb solid medium and incubated overnight at 37°C. The bacterial culture medium in PBS wells was used as a negative control. Phage purification: The amplified phages were purified using the PEG / NaCl precipitation method. The phage concentration was calibrated by measuring the OD 268nm absorbance using a Nanodrop 2000c before proceeding to the next round of screening. The key conditions for each round of screening were modified according to this procedure, and a total of four rounds of phage screening were performed.

[0058] The conditions and enrichment rates for the four rounds of screening are shown in Table 2 below.

[0059] Table 2 Screening rounds Antigen concentration (μg / mL) Sealing liquid Combined with time (h) PBST washing times Enrichment rate (%) Round 1 5 BSA 2 5 1.2 Second round 3 OVA 1.5 10 4.5 Third round 1 BSA 1 15 2.0 Fourth round 1 OVA 0.5 20 1.0 (II) ELISA Validation of Screening Results The purified phage libraries from each round were used to validate the screening effect via ELISA. The specific process is as follows: Plate coating: Coat an enzyme-labeled plate with 1 μg / mL S100β antigen and incubate overnight at 4°C; Blocking: Discard the coating solution and block the ELISA plate with 1% BSA-PBS at room temperature for 2 hours; Diluting the phage library: Dilute the phage library to 1.0 OD in each round and perform a 3× serial dilution with PBS (8 dilutions). Binding: Discard the blocking solution, shake the ELISA plate to dry, add the serially diluted phage to the ELISA plate, and incubate at 37°C for 30 min; Cleaning: Wash 5 times with PBST; Add secondary antibody: Add anti-M13-HRP secondary antibody (1:5000 dilution) to the microplate and incubate at 37°C for 1 h; Further washing: Wash 5 times with PBST; Color development: Add 100 μL of TMB color development solution, place at 37℃, and develop for 15 min; Termination: The reaction was terminated by adding 50 μL of 2M sulfuric acid; Measurement: The absorbance at 450 nm was measured using an ELISA reader.

[0060] The measured OD values ​​were combined with phage OD values ​​to plot a fitted curve. The point where the strongest signal was halfway down was identified; the corresponding phage OD value is the EC50. EC50 is defined as the concentration at which 50% efficacy or binding is achieved. The curves showing the effects of the four rounds of screening and the EC50 results are shown below. Figure 1 .

[0061] (III) ELISA screening and sequencing of positive clones 1. After the fourth round of screening, 480 single colonies were randomly selected and inoculated into 96-well plates. Each well contained 400 μL of 2×YT / Carb and 4 μL of M13K07 helper phage. The plates were incubated overnight at 37°C and centrifuged at 3000 rpm to obtain the phage supernatant. The specific process of monoclonal ELISA screening is as follows: Plate coating: Coat the microplate with 1 μg / mL S100β antigen, and coat the blank group with PBS buffer. Incubate overnight at 4°C. Blocking: Discard the coating solution and block the ELISA plate with 1% BSA-PBS at room temperature for 2 hours; Dilute the phage supernatant: Mix the phage supernatant with ELISA buffer at a 1:1 ratio until homogeneous; Binding: Discard the blocking solution, shake the ELISA plate to dry it, add the diluted phage supernatant to the ELISA plate, and incubate at 37°C for 1 h; Cleaning: Wash 5 times with PBST; Add secondary antibody: Add anti-M13-HRP secondary antibody (1:5000 dilution) to the microplate and incubate at 37°C for 1 h; Further washing: Wash 5 times with PBST; Color development: Add 100 μL of TMB color development solution, place at 37℃, and develop for 15 min; Termination: The reaction was terminated by adding 50 μL of 2M sulfuric acid; Measurement: The absorbance at 450 nm was measured using an ELISA reader.

[0062] Clones with an OD value of ≥2 for binding to S100β and an OD value of ≥2 for binding to BSA are defined as positive clones. Clones with an OD value >1.8 for binding to S100β and an OD value <0.3 for binding to BSA, and a ratio ≥5, are defined as high-affinity positive clones.

[0063] The statistical table of positive clones screened by ELISA is shown in Table 3 below.

[0064] Table 3 name quantity Detecting clones 480 <![CDATA[Positive clone (OD TNF / OD BSA ≥ 2)]]> 323 <![CDATA[High affinity clone (OD TNF > 1.8, OD BSA < 0.3, OD TNF / OD BSA ≥ 5)]]> 61 2. Positive clones were selected for sequencing (primers: 5'-gttgtgtggaattgtgagcgg-3'). Through sequence alignment and cluster analysis, the high-affinity candidate sequence D3 was identified for further research. The antibody sequences are shown in Tables 4 and 5.

[0065] Table 4 name nucleotide sequence amino acid sequence D3 CAATTACAGCTAGTAGAATCAGGTGGAGGGGTGGTTCAGGCGGGCGGTTCGCTGCGTCTGAGCTGTGCAGCGAGCGGTCGCACCTTTGCAGGCTACAACATGGCGTGGTTCCGCCAAGCTCCGGTAAAGAGCGCGAGTTCGTGGCCGATTTCGTGCCGGCTACTAGGTCTACTAG CGCTGATAGCATGAAAGGTCGTTTTACCATCAGCCGTGACAACGCGAAGAACACCGTGTATCTGCAGATGAATTCCTTGAAGTTCGAAGATACTGCGGTTTATTACTGCGGTGGCCGTGCGCGTGACGCTACCATGACCTACGGCAGACAAGGTACGCAGGTTACCGTCAGCTCC(SEQ ID NO:1) QLQLVESGGGVQAGGSLRLSCAASGRTFAGYNMAWFRQAPGKEREFVAAISRAGAYTYYADSSMKGRFTISRDNAKNTVYLQMNSLKFEDTAVYYCGGRARDATMTYGRQGTQVTVSS(SEQ ID NO:2) D3-FR1 CAATTACAGCTAGTAGAATCAGGTGGAGGGGTGGTTCAGGCGGGCGGTTCGCTGCGTCTGAGCTGTGCAGCGAGC(SEQID NO:3) QLQLVESGGGVVQAGGSLRLSCAAS(SEQ ID NO:4) Table 5 D3-FR2 ATGGCGTGGTTCCGCCAAGCTCCGGGTAAAGAGCGCGAGTTCGTGGCCGCG(SEQ ID NO:5) MAWFRQAPGKEREFVAA(SEQ ID NO:6) D3-FR3 TACTACGCTGATAGCATGAAAGGTCGTTTTACCATCAGCCGTGACAACGCGAAGAACACCGTGTATCTGCAGATTCCCTTGAAGTTCGAAGATACTGCGGTTTATTACTGC(SEQ ID NO:7) YYADSMCGRFTISRDNAKNTVYLQMNSLKFEDTAVYYC(SEQ ID NO:8) D3-FR4 GGCAGACAAGGTACGCAGGTTACCGTTCAGCTCC(SEQ ID NO:9) GRQGTQVTVSS(SEQ ID NO:10) D3-CDR1 GGTCGCACCTTTGCAGGCTACAAC(SEQ ID NO:11) GRTFAGYN (SEQ ID NO:12) D3-CDR2 ATTTCTCGTGCCGGCGCATATACG(SEQ ID NO:13) ISRAGAYT (SEQ ID NO:14) D3-CDR3 GGTGGCCGTGCGCGTGACGCTACCATGACCTAC(SEQ ID NO:15) GGRARDATMTY (SEQ ID NO:16) (iv) Plasmid construction and chemical transformation of nanobodies 1. Expression plasmid construction First, the pET26b(+) plasmid was linearized by enzyme digestion; then, using the positive clone plasmid as a template, the gene fragment containing the nanobody was amplified by PCR, and homologous arms of the pET26b(+) plasmid were introduced at both ends of the antibody gene; finally, the linearized plasmid and the nanobody gene fragment were ligated into a complete plasmid through homologous recombination.

[0066] (1) Linearized plasmids The pET26b(+) plasmid was amplified in Top10 cells, extracted using a mini-prep kit, and its concentration was determined using Nanodrop2000c. The pET26(+) plasmid was digested with NcoI and XhoI restriction endonucleases and incubated at 37°C for 2 hours. The preparation of the digestion mixture is shown in Table 6.

[0067] Table 6 Reagent Name volume Reagent Name volume pET26b(+) plasmid 20μL 10× enzyme digestion buffer 4μL NcoI restriction endonuclease 4μL <![CDATA[ddH2O]]> 8μL XhoI restriction endonuclease 4μL DNA agarose gel electrophoresis was performed, confirming that the target product was approximately 5 kbp. (See attached image.) Figure 2 Finally, the linearized plasmid was recovered from the gel and its concentration was calibrated using a Nanodrop 2000c.

[0068] (2) PCR amplification of the target antibody gene: The preparation of the PCR mixture is shown in Table 7.

[0069] Table 7 Reagent Name volume 2×PCR master Mix 25μL upstream primer 1μL Downstream primer 1μL Target antibody plasmid 1μL <![CDATA[ddH2O]]> 22μL The PCR thermal program settings are shown in Table 8.

[0070] Table 8

[0071] DNA agarose gel electrophoresis confirmed that the target product was approximately 400 bp. (See attached image) Figure 3 Finally, the PCR product was recovered to obtain the target product, and its concentration was determined using Nanodrop 2000c.

[0072] (3) Homologous recombination: The linearized pET26b(+) plasmid and the PCR-amplified target antibody gene were used to prepare the ligation mixture according to Table 9.

[0073] Table 9 Reagent Name Added amount GenBuilder 2x Master Mix 10μL Linearized pET26b(+) plasmid 0.033 pmol Target antibody gene 0.1 pmol <![CDATA[ddH2O]]> Add to 20μL The ligation mixture was incubated at 50°C for 15 minutes for chemical transformation.

[0074] 2. Chemical transformation: Add 5 μL of the homologous recombination product to 50 μL of chemocompetent BL21 cells and incubate on ice for 30 minutes. After a heat shock at 42°C for 30 seconds, quickly return to ice and incubate for 2 minutes. Add 950 μL of SOC medium and incubate at 37°C and 220 rpm for 1 hour. Spread 100 μL of the culture medium onto LB / Kan solid medium and incubate overnight at 37°C. The next day, clones grew on the antibiotic-containing medium, confirming successful plasmid homologous recombination.

[0075] (V) Expression and purification of nanobodies The target nanobody was induced to express in the BL21 host and the antibody was purified using a Ni-NTA column.

[0076] 1. Antibody expression: BL21 cells containing the target antibody were seeded into 5 mL of LB / Kan medium and cultured overnight at 37°C and 200 rpm. The next day, 5 mL of cell culture medium was transferred to 500 mL of LB / Kan medium and cultured at 37°C and 200 rpm until OD500. 600 = 0.6, add IPTG to 1mM, and incubate at 25℃ and 200 rpm for about 16 hours to induce antibody expression. On the third day, centrifuge the induced culture medium at 5000 rpm and harvest the cell pellet.

[0077] 2. Antibody purification: The cell pellet was resuspended in 100 mL of lysis buffer (0.5 M sucrose, 0.2 M Tris, pH 8, 0.5 mM EDTA), 200 mL of deionized water was added, a magnetic stir bar was added, and the mixture was stirred at 4 °C for 45 minutes to release the nanobody.

[0078] Centrifuge at 12000 rpm for 20 minutes and collect the supernatant. Add the supernatant to a 3 mL Ni-NTA resin-assembled filter column and discard the filtrate. Wash the column with approximately 100 mL of high-salt washing buffer (20 mM HEPES, 500 mM sodium chloride, 20 mM imidazole, pH 7.5), then wash the column again with approximately 100 mL of washing buffer (20 mM HEPES, 100 mM sodium chloride, 20 mM imidazole, pH 7.5). Finally, elute the nanobody bound to the nickel column with 30 mL of elution buffer (20 mM HEPES, 100 mM sodium chloride, 400 mM imidazole, pH 7.5). See the protein gel electrophoresis results for the entire process of D3 nanobody expression and purification. Figure 4 .

[0079] After dialyzing the eluent in PBS, 12% SDS-PAGE protein gel electrophoresis was performed to confirm that the nanobody was approximately 15 kDa, and the nanobody concentration was determined using Nanodrop 2000c. Finally, 50% glycerol was added, and the nanobody was aliquoted and stored at -20°C.

[0080] (vi) ELISA validation of nanobody affinity Plate coating: Coat an enzyme-labeled plate with 1 μg / mL S100β antigen and incubate overnight at 4°C; Blocking: Discard the coating solution and block the ELISA plate with 1% BSA-PBS at room temperature for 2 hours; Diluting the antibody: Dilute the D3 nanobody to 2 μM and perform a 3× serial dilution with PBS (8 concentrations). Binding: Discard the blocking solution, shake the ELISA plate to dry it, add the diluted nanobody to the ELISA plate, and incubate at 37°C for 1 h; Cleaning: Wash 5 times with PBST; Add secondary antibody: Add anti-His-HRP secondary antibody (1:5000 dilution) to the microplate and incubate at 37°C for 1 h; Further washing: Wash 5 times with PBST; Color development: Add 100 μL of TMB color development solution, place at 37℃, and develop for 15 min; Termination: The reaction was terminated by adding 50 μL of 2M sulfuric acid; Measurement: The absorbance at 450 nm was measured using an ELISA reader.

[0081] Record the obtained OD values, plot the fitted curve in conjunction with the antibody concentration, and calculate the EC50 value of the antibody. The nanobody affinity results are shown below. Figure 5 .

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A high-affinity nanobody or antigen-binding fragment thereof against the central nervous system-specific protein S100β, characterized in that, The VHH chain of the nanobody includes a complementarity-determining region and a framework region; The complementary decision regions include CDR1 shown in SEQ ID NO:12, CDR2 shown in SEQ ID NO:14, and CDR3 shown in SEQ ID NO:

16.

2. The high-affinity nanobody or its antigen-binding fragment according to claim 1, characterized in that, The frame region includes: FR1 shown in SEQ ID NO:4, FR2 shown in SEQ ID NO:6, FR3 shown in SEQ ID NO:8, and FR4 shown in SEQ ID NO:10; Preferably, the nanobody comprises the sequence shown in SEQ ID NO:

2.

3. A nucleic acid molecule encoding a high-affinity nanobody or an antigen-binding fragment thereof of the anti-central nervous system specific protein S100β as described in claim 1 or 2.

4. An expression vector containing the nucleic acid molecule of claim 3.

5. A host cell, characterized in that, It comprises the expression vector of claim 4, or the nucleic acid molecule of claim 3 integrated into the host cell genome; or the host cell expresses the high-affinity nanobody against the central nervous system-specific protein S100β of claim 1 or 2, or its antigen-binding fragment.

6. A method for preparing a high-affinity nanobody against the central nervous system-specific protein S100β as described in claim 1 or 2, or an antigen-binding fragment thereof, characterized in that, This includes culturing the host cells as described in claim 5.

7. A recombinant polypeptide conjugate, characterized in that, It includes the high-affinity nanobody against the central nervous system-specific protein S100β as described in claim 1 or 2, or its antigen-binding fragment, and the conjugated portion.

8. A reagent or kit, characterized in that, The reagents or kits comprise the high-affinity nanobody against the central nervous system-specific protein S100β as described in claim 1 or 2, or its antigen-binding fragment, and / or the recombinant polypeptide conjugate as described in claim 7.

9. A pharmaceutical composition, characterized in that, It includes the high-affinity nanobody against the central nervous system-specific protein S100β as described in claim 1 or 2, or its antigen-binding fragment, or the recombinant polypeptide conjugate as described in claim 7.

10. The use of a high-affinity nanobody against central nervous system-specific protein S100β as described in claim 1 or 2, or an antigen-binding fragment thereof, the recombinant polypeptide conjugate as described in claim 7, the reagent or kit as described in claim 8, and / or the pharmaceutical composition as described in claim 9, in any one or more of the following: A1: To develop products for the detection, monitoring, diagnosis and / or prognosis of central nervous system diseases; A2: To prepare products for the prevention and / or treatment of central nervous system diseases; Preferably, central nervous system diseases include Alzheimer's disease, traumatic brain injury, brain tumors, and multiple sclerosis.

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