A sv2 receptor ectodomain nanobody and uses thereof

Abstract

Neuroendocrine tumors (NETs) are a class of highly heterogeneous tumors derived from neuroendocrine cells. Synaptic vesicle 2 (SV2) protein is a novel marker of NETs, and there are three subtypes of SV2A, SV2B and SV2C, wherein the structure of SV2C is more stable than the other two, and the largest cavity ring L4 thereof is the most important binding area with toxins. The present application obtains a nanobody of the extracellular soluble part L4 of SV2 by screening through phage display technology, and the nanobody labeled with a fluorescein, a small molecule of a chemical toxin or a nuclide is used to prepare a probe molecule, and the result can be specifically combined with neuroendocrine tumor cells. The present application provides a nanobody of the extracellular soluble part L4 of SV2, and the nanobody can be applied in the preparation of diagnostic and therapeutic preparations for neuroendocrine tumors.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and provides SV2 extracellular soluble fraction nanobodies for use in the preparation of neuroendocrine tumor nuclear imaging probe molecules and small molecule conjugates of radioactive or chemical toxins targeting neuroendocrine tumors. Background Technology

[0002] Radiopharmaceuticals are an important component of nuclear medicine, widely used in cancer diagnosis and treatment, myocardial imaging and cardiac disease diagnosis, and monitoring of neurodegenerative diseases. Radiopharmaceuticals can be categorized into diagnostic radiopharmaceuticals and therapeutic radiopharmaceuticals based on their application. Diagnostic radiopharmaceuticals can be further divided into single-photon radiopharmaceuticals and positron emission tomography (PET) radiopharmaceuticals. These can be combined with single-photon computed tomography (SPECT) or positron emission tomography (PET) to study the function and metabolic processes of drugs in vivo at the molecular level. This enables rapid, non-invasive, and real-time imaging of physiological and pathological processes, providing new methods and tools for truly early diagnosis and timely treatment, and offering potential pathways to preventive medicine, translational medicine, and personalized medicine. [1] .

[0003] Neuroendocrine tumors (NETs) consist of a group of malignant tumors that may originate from neuroendocrine cells throughout the body. These tumors are characterized by their ability to produce peptides that lead to a characteristic hormone syndrome. The disease is primarily characterized by the excessive secretion of neurotransmitters and hormones, causing related clinical symptoms such as flushing, rash, diarrhea, and heart failure. Neuroendocrine tumors can occur in many parts of the body; neuroendocrine cells can form neuroendocrine tumors in many organs. Female patients are more likely to have primary tumors in the lungs, stomach, appendix, or cecum, while male patients are more likely to have primary tumors in the thymus, duodenum, pancreas, jejunum / ileum, or rectum. The prognosis and mortality rate depend on the degree of differentiation of the NETs; patients with poorly differentiated malignant tumors have a median life expectancy of less than one year. [2] .

[0004] Neuroendocrine tumors can be divided into two main categories based on whether the substances secreted by the tumor can cause typical clinical symptoms: functional and non-functional. Functional neuroendocrine tumors often present with symptoms caused by excessive secretion of tumor-related substances, while non-functional neuroendocrine tumors do not cause corresponding clinical manifestations even with elevated hormone or peptide concentrations. Due to the lack of typical clinical manifestations, early detection is often difficult. Patients often only seek medical attention when the tumor is large enough to be palpable in the abdomen or when the tumor has caused related complications, often by which time metastasis has already occurred. Therefore, somatostatin receptor imaging (SRI) is commonly used clinically to accurately locate the tumor. However, studies have shown that some patients are negative for somatostatin receptor imaging, which makes SRI a limitation. Therefore, there is an urgent clinical need for more effective molecular probes for NET nuclear imaging to more accurately determine the size of NETs and their metastases. [3] .

[0005] Synaptic vesicle 2 (SV2) protein is abundantly expressed in NET tissues and has been identified as a novel biomarker for NETs. SV2 acts as a Ca2+ receptor antagonist. 2+ There are three different subtypes of triggered extraosmotic regulators (SV2A / B / C). [4-6] Because exocytosis plays a crucial role in regulating external messenger interactions and internal signal transduction, SV2 may promote cellular mechanisms of antigen processing and presentation in cancerous tumors to evade the immune system. In cancer, SV2 may also be involved in regulating membrane markers, transdifferentiation, and tumor angiogenesis, as well as inhibiting apoptosis. [7] .

[0006] Nanobodies (Nb) are a special type of antibody lacking a light chain structure. They were first discovered in camel-like animals by Belgian scientist C. Hamers-Casterman in 1993, and subsequently found in some deep-sea fish such as nurse sharks and chinchillas. [8] Nanobody structures contain only one heavy chain variable region and two heavy chain constant regions, exhibiting advantages such as low susceptibility to sticking together, structural stability, and high hydrophilicity. [9] Nanobodies share up to 80% homology with the heavy chain variable region (VH) of human antibodies.

[10] It has low immunogenicity and a long complementarity determining region (CDR), among which the CDR3 region helps to recognize some antigenic epitopes that conventional antibodies cannot recognize.

[11] Based on these unique properties and advantages, nanobodies have a wide range of research and applications in fields such as disease diagnosis and treatment, and new drug development.

[0007] Phage display technology was first developed by Nobel laureate George P. Smith in 1985. This technology uses molecular cloning to ligate a obtained DNA fragment into the gene encoding the coat protein of a bacteriophage. This allows the exogenous DNA to be expressed on the phage surface along with the phage's proliferation, thus enabling the identification of the encoding gene through phenotypic selection.

[12] Antibody libraries constructed using phage display technology have significantly increased capacity. Furthermore, because phage display can be performed during bacterial proliferation, the required cycle is short, which is beneficial for antibody production.

[13] Based on the above advantages and characteristics, a phage display library can be constructed to screen for antibodies corresponding to target molecules. Further analysis of the sequence information of the nanobodies obtained from the screening can then be performed to construct expression vectors and express the nanobodies.

[0008] This invention expresses and purifies the extracellular soluble fraction of the SV2 receptor, uses it as an antigen to prepare and screen nanobodies, and ultimately provides a highly efficient SV2 nanobodies as ligands targeting the SV2 target in neuroendocrine malignancies. These nanobodies can be prepared using conventional methods employing radionuclides. 68 Ga 99 Tc, 131 I, 89 Zr, 111 In, 90 Y, 18 F and other substances can be labeled to make them nuclear imaging probe molecules or nuclear drug molecules with therapeutic effects; small chemical toxin molecules such as auristatin, maytansine and their analogues, calicheamicin, duocarmycins, anthracyclines, pyrrolobenzodiazepine dimers, amatoxin and quinoline alkaloid (SN-38) can also be used to conjugate them to make them nanobody chemical toxin molecule conjugates; fluorescent dyes can also be used to label them to make them fluorescent imaging probe molecules, which can be used as research tools for studying neuroendocrine tumors.

[0009] References

[0010] [1] Wang Zheng, Xu Jianfeng, Cai Yuting, Dai Juan, Li Shihong, Luo Zhigang. Current status and development trend of radiopharmaceuticals in China [J]. China Food and Drug Administration, 2018; (7): 44-49.

[0011] [2] Massironi S, Rossi RE, Casazza G, Conte D, Ciafardini C, Galeazzi M, et al. Chromogranin A in diagnosing and monitoring patients with gastroenteropancreatic neuroendocrine neoplasms: a large series from a singleinstitution. Neuroendocrinology. 2014; 100:240-249.

[0012] [3] Lu Tingting, Wang Xiaohui, Yang Guoren, Huo Zongwei. (99) Study on the diagnostic value of Tcm-octreotide SPECT / CT imaging for neuroendocrine tumors. Chinese Journal of Cancer Prevention and Treatment, 2020; 27(21):74-78.

[0013] [4]Portela-Gomes GM, Lukinius A, Grimelius L. Synaptic vesicle protein2, Anew neuroendocrine cell marker. Am J Pathol. 2000; 157:1299-1309.

[0014] [5]Jakobsen AM, Ahlman H, Wangberg B, Kolby L, Bengtsson M, NilssonO. Expression of synaptic vesicle protein 2(SV2) in neuroendocrine tumours of the gastrointestinal tract and pancreas. J Pathol. 2002; 196:44-50.

[0015] [6] Nilsson O, Jakobsen AM, Kolby L, Bernhardt P, Forssell Aronsson E, Ahlman H. Importance of vesicle proteins in the diagnosis and treatment of neuroendocrine tumors. AnnNYAcad Sci. 2004; 1014:280-283.

[0016] [7]Bandala C et al. Botulinum neurotoxin type A inhibits synapticvesicle 2expression in breast cancer cell lines. International Journal of Clinical and Experimental Pathology, 2015; 8(7):8411-8418.

[0017] [8] van Bockstaele F, Holz JB, Revets H. The development of nanobodies for therapeutic applications. Curr Opin Investig Drugs, 2009; 10(11): 1212-1224.

[0018] [9] Muyldermans S, Baral TN, Retamozzo VC, et al. Camelid immunoglobulins and nanobody technology. Vet Immunol Immunopathol, 2009; 128(1 / 3):178-183.

[12] Harmsen MM, HaardHJD. Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol,2007;77(1):13-22.

[0019]

[10] Wu Yue, Hao Xiujing, Li Min. Application of nanobodies in cancer therapy. Chinese Journal of Biotechnology, 2017; 33(7):1085-1090.

[0020]

[11] Dumoulin M, Conrath K, Van Meirhaeghe A, et al. Single-domain antibody fragments with high conformational stability. Protein Sci, 2002; 11(3): 500-515.

[0021]

[12] Besnerais M, VeyradierA, BenhamouY, et al. Caplacizumab: a change in the paradigm of thrombotic thrombocytopenic purpura treatment. Expert Opin BiolTher, 2019; 19(11): 1127-1134.

[0022]

[13] Pardon Els et al. A general protocol for the generation of Nanobodies for structural biology. Nature protocols, 2014; 9(3): 674-693. Summary of the Invention

[0023] The purpose of this invention is to provide a variety of SV2 extracellular soluble fraction nanobodies for use in the preparation of neuroendocrine tumor nuclear imaging probe molecules and small molecule conjugates of radioactive or chemical toxins targeting neuroendocrine tumors.

[0024] This invention constructs the pGEX-4T-1-SV2C-L4 recombinant plasmid. The SV2C-L4 gene sequence fragment was synthesized by Genewiz, outsourced to Genewiz, and inserted into the pGEX-4T-1 plasmid. Sequencing and quality control confirmed the SV2C-L4 gene sequence was Seq ID No. 1, and the protein sequence was Seq ID No. 14. Further, pGEX-4T-1-SV2C-L4 was transformed into *E. coli* BL21 competent cells using the heat shock method. The cells were plated on LB agar plates containing 100 μg / mL ampicillin and cultured overnight at 37°C. Single colonies were picked and cultured in 20 mL of LB liquid medium containing 100 μg / mL ampicillin, and then cultured overnight at 37°C with shaking at 200 rpm as the bacterial strain. The bacterial strain was inoculated at 1% in LB medium containing 100 μg / mL ampicillin and cultured at 37°C until the OD600 value was approximately 0.6. IPTG was then added to a final concentration of 0.4 mmol / L, and the culture was induced at 16°C for 20 h. The bacterial cells were harvested by centrifugation at 8000 rpm for 5 min, and 30 times the weight of the bacterial cells were added to Binding Buffer (20 mM PB, pH 7.4, 150 mM NaCl). The cells were homogenized at 800 bar until the culture was clear. 10% Triton X-100 was added, and the culture was incubated at 4°C for 30 min, followed by centrifugation at 10000 rpm for 10 min. The supernatant was purified by GSH affinity chromatography. The chromatography packing material was equilibrated with 20 column volumes of Binding Buffer. After loading the sample, the sample was washed with Binding Buffer until the UV detector reading reached baseline. Then, the sample was eluted in stages with Elution Buffer. The Elution Buffer formulations were: Elution Buffer 1: 50 mM Tris-HCl, pH 8.0, 5 mM glutathione; Elution Buffer 2: 50 mM Tris-HCl, pH 8.0, 10 mM glutathione. The target protein was present in the elution fraction Elution 1. Elution sample 1 was collected and desalted using a desalting column. The desalting buffer formulation was: 50 mM NaCl, 20 mM PB, pH 7.4. After desalting, 5% glycerol was added to the collected protein, and the sample was rapidly frozen with liquid nitrogen and stored at -80°C.

[0025] The purified pGEX-4T-1-SV2C-L4 protein was used as an antigen and sent to Shanxi Nami Biotechnology Development Co., Ltd. in dry ice for outsourced alpaca immunization. After four immunizations and passing quality testing of the alpaca antiserum, alpaca blood was collected to isolate lymphocytes. Total RNA was extracted from the returned alpaca lymphocytes, followed by RNA reverse transcription, cDNA amplification, and PCR to construct the pMES4-VHH plasmid. After electroporation, an SV2C-L4 nanobody phage library was established. The nanobody phage library was then screened using phage display methods. Finally, after screening for nanobody binding affinity, nanobodies with strong binding affinity were selected and outsourced to Genewiz for sequencing, resulting in 12 nanobody gene sequences with different sequences, numbered as: 1-A4, 1-A5, 1-A10, 1-C6, 1-F8, 1-H9, 2-A2, 2-A5, 2-C4, 2-F1, 2-G11, and 2-H4.

[0026] The selected SV2C-L4 nanobody strain was inoculated at 1% in LB medium and cultured at 37°C until the OD600 value reached approximately 0.6. IPTG was then added to a final concentration of 0.5 mmol / L, and the culture was induced at 20°C for 16 h. The cells were then harvested. Binding Buffer (20 times its weight) was added to the cells, and the cells were homogenized and centrifuged at 10,000 rpm for 10 min. The supernatant was purified using a Ni column (equilibrated with 20 column volumes of Binding Buffer: 20 mM PB, pH 7.4, 20 mM NaCl, 100 mM imidazole; Elution Buffer: 20 mM PB, pH 7.4, 100 mM NaCl, 500 mM imidazole). The target protein was present in the Elution sample.

[0027] The purified protein was dialyzed, and the buffer was replaced with 0.1M NaHCO3 solution. Super Flour 680SE dye solution was slowly added to 1 mL of protein solution for labeling, using a mass ratio of 10–200 μg Super Flour SE per mg of IgG antibody. The solution was then slowly stirred at room temperature in the dark for 1 hour. The labeled protein solution was purified using a Sephadex G25 column, eluted with PBS, and the eluted labeled protein solution was collected based on the UV detector reading.

[0028] The labeled nanobody solution was co-incubated with Neuro-2a cells to verify its cell targeting. Most of the labeled protein bound to the receptors on the Neuro-2a cell membrane, so red fluorescence could be observed on the cell membrane. However, no receptors bound to the nanobody were observed on the negative control HeLa cells, and no fluorescence was observed.

[0029] The SV2 extracellular soluble nanobody provided by this invention is an antibody that is naturally missing a light chain in alpaca peripheral blood. It has the advantages of small molecular weight, ability to penetrate the blood-brain barrier, high expression obtained using Escherichia coli, high specificity, high affinity, and weak immunogenicity to humans.

[0030] Furthermore, different radionuclides can be used to label the extracellular soluble portion of SV2 nanobodies. Radionuclide-labeled proteins can then become nuclear imaging probe molecules or radiopharmaceutical molecules with therapeutic effects, which is understandable to those skilled in the art. Generally, 89 Zr、 68 Ga、 99 Tc, 111 In、 90 Nuclide Y and others are used to label the extracellular soluble portion of SV2 nanobodies with bifunctional chelating agents such as DOPA, DTPA, and HYNIC. 18 F is synthesized first. 18 F precursor molecules were used to label the extracellular soluble portion of SV2 nanobodies; 131 I labeled the extracellular soluble fraction of SV2 nanobodies using the chloramine-T method.

[0031] Using conventional chemical methods, toxin molecules can be conjugated to the extracellular soluble fraction of SV2 nanobodies. These toxin-conjugated SV2 extracellular soluble fraction nanobodies can become nuclear drug molecules with therapeutic activity, a concept easily understood by those skilled in the art. Such toxin molecules include: microtubule formation inhibitors such as auristatin, maytansine, and their analogues; DNA damage factors such as calicheamicin, duocarmycins, anthracyclines, and pyrrolobenzodiazepine dimers; and DNA transcription inhibitors such as amatoxin and quinoline alkaloid (SN-38). Attached Figure Description

[0032] Figure 1 The results of protein induction of .pGEX-4T-1-SV2C-L4 are shown in the figure. M is the Protein Ladder, lane 1 is the uninduced sample, and lane 2 is the induced sample.

[0033] Figure 2The results of .pGEX-4T-1-SV2C-L4 protein purification are shown in the figure. M is the Protein Ladder. Lane 1 is the precipitate sample after centrifugation of the lysate. Lane 2 is the pre-column sample of the lysate supernatant. Lane 3 is the chromatographic breakthrough sample. Lane 4 is the washed sample. Lane 5 is the sample with elution peak 1. Lane 6 is the sample with elution peak 2.

[0034] Figure 3 ELISA affinity assay results for the selected nanobodies;

[0035] Figure 4 Sequence alignment results of the screened nanobody proteins;

[0036] Figure 5 The purified nanobody results obtained from the screening show that lanes 1-13 contain nanobody bands numbered 1-A4, 1-A5, 1-A10, 2-A5, 1-H9, 2-F1, 1-C6, 1-F8, 2-A2, 2-C4, 2-H4, 2-G11, and 2-A5, respectively.

[0037] Figure 6 SF-680SE-labeled nanobodies bind to N2a cells. Specific Implementation

[0038] Example 1: Expression and purification of pGEX-4T-1-SV2C-L4 protein

[0039] 1.1 Materials

[0040] 2×Hieff Gold PCR Master Mix high-fidelity enzyme premix (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10149ES03), agarose (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10208ES60), low-melting-point agarose (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10214ES60), nucleic acid dye (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10202ES76), 1kb DNA ladder (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10510-A), 6× DNA loading buffer (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10213ES08), SpeedyCut BamHⅠ rapid digestion enzyme, EcoRⅠ endonuclease, and NdeⅠ endonuclease were purchased from Sangon Biotech (Shanghai) Co., Ltd. SanPrep column DNA gel extraction kit (Sangon Biotech, catalog number B518131), SanPrep column plasmid DNA mini-extraction kit (Sangon Biotech, catalog number B518191), ampicillin sodium (Sangon Biotech, catalog number A610028-0025), kanamycin sulfate (Sangon Biotech, catalog number A506636-0025), IPTG (Sangon Biotech, catalog number A600168-0025), and imidazole (Sangon Biotech, catalog number A600277-) were also available. 0100), SDS (Sangon Biotech Co., Ltd., catalog number A600485-0100), glycine (Sangon Biotech Co., Ltd., catalog number A610235-0005), Tris (Sangon Biotech Co., Ltd., catalog number A610195-0005), Coomassie Brilliant Blue R-250 (Sangon Biotech Co., Ltd., catalog number A610037), Sephadex G-25 Resin chromatography medium (Sangon Biotech Co., Ltd., catalog number C510060-0026), BL21(DE3) E. coli competent cells (Sangon Biotech Co., Ltd., catalog number B528414), GST-Sefinose™ Resin (Settled) Resin (Sangon Biotech Co., Ltd., catalog number C600031-0005), T4 DNA Ligase (Beijing Baori Biotechnology Co., Ltd., catalog number 2011A), Triton X-100, agar powder (Beijing Solarbio Science & Technology Co., Ltd., catalog number A8190), TEMED (Beijing Solarbio Science & Technology Co., Ltd., catalog number T8090), bromophenol blue (Beijing Solarbio Science & Technology Co., Ltd., catalog number B8120), sodium chloride, sodium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, methanol, glacial acetic acid, and anhydrous ethanol were purchased from Tianjin Baishi Chemical Co., Ltd., and tryptone (Thermo Fisher Scientific Co., Ltd.).The following ingredients are listed: LP0042B (product number), yeast extract (Thermo Fisher Scientific, product number LP0021), ammonium persulfate (Beijing Jinming Biotechnology Co., Ltd., product number 7727-54-0), acrylamide (Beijing Jinming Biotechnology Co., Ltd., product number 79-06-1), N,N-methylenebisacrylamide (Beijing Jinming Biotechnology Co., Ltd., product number 110-26-9), M5 HiClear Prestained Plus ProteinLadder (10-250kDa) (Beijing Jumei Biotechnology Co., Ltd., product number MF028-plus-01), Ni Sepharose. TM 6Fast Flow (GE Healthcare, catalog number 17-5318-01).

[0041] 1.2 Implementation Steps

[0042] 1.2.1 Construction of pGEX-4T-1-SV2C-L4 plasmid

[0043] The SV2C-L4 gene sequence fragment was synthesized by Genewiz and inserted into the pGEX-4T-1 plasmid. Sequencing and quality control were performed, and the SV2C-L4 gene sequence is Seq ID No.:1, while the protein sequence is Seq ID No.:14. Upstream and downstream PCR primers were designed, SV2C-F:

[0044] CGCGGATCCTTTCCGGATGTGATTAAACCGC; SV2C-R:

[0045] CCGGAATTCTTACGCGCTATAATCGTCATCAAAGG, using these two primers from pET-28 a+ The SV2C-L4 sequence was amplified on the SV2C-L4 plasmid. The PCR system was prepared according to Table 1, and the PCR program was as shown in Table 2.

[0046] Table 1. PCR system based on SV2C-L4 sequence

[0047]

[0048] Table 2. PCR Procedure for SV2C-L4 Sequence

[0049]

[0050] Following the supplier's instructions, the PCR product was double-digested with EcoRI and BamHI, and the double-digested fragment was purified by gel extraction. Then, the double-digested PCR product was ligated with the pGEX-4T-1 vector digested with the same two enzymes at a molar ratio of vector:fragment = 1:10 overnight at 16°C to obtain the recombinant plasmid pGEX-4T-1-SV2C-L4.

[0051] The ligation product was transformed into competent E. coli BL21 cells using the conventional heat shock transformation method. The cells were then plated on LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37°C. Positive clones were picked from the plates, and plasmids were extracted using a plasmid extraction kit according to the kit instructions. The plasmids were then sent for testing. Clones whose target gene sequences matched the theoretical sequences were selected. These single clones were inoculated into 20 mL of LB liquid medium containing 100 μg / mL ampicillin and incubated overnight at 37°C. The bacterial strain was then preserved in 20% glycerol at -80°C.

[0052] 1.2.2 pGEX-4T-1-SV2C-L4 protein expression

[0053] The bacterial strain was removed from the -80℃ ultra-low temperature freezer and inoculated (5 μL) into 20 mL of LB broth containing 100 μg / mL ampicillin. The culture was incubated overnight at 37℃ to obtain the bacterial strain. BL21 bacteria carrying the recombinant plasmid pGEX-4T-1-SV2C-L4 were inoculated at 1% in LB broth containing 100 μg / mL ampicillin and incubated at 37℃ until the OD600 value reached approximately 0.6. The uninduced sample was collected, and IPTG was added to a final concentration of 0.4 mmol / L. Induction was performed at 16℃ for 20 h, and the induced sample was collected. The uninduced and induced samples were centrifuged and prepared for SDS-PAGE electrophoresis to detect the expression of pGEX-4T-1-SV2C-L4 protein. Results are shown below. Figure 1 The induced sample showed a distinct induced band at approximately 42 kDa, the size of which was consistent with the theoretical size of pGEX-4T-1-SV2C-L4.

[0054] 1.2.3 pGEX-4T-1-SV2C-L4 protein purification

[0055] Add 30 times its weight of Binding Buffer to the bacterial cells, homogenize using an 800 bar homogenizer until the bacterial solution is clear, add 10% Triton X-100, incubate at 4°C for 30 min, and centrifuge at 10,000 rpm for 10 min. Collect the supernatant and purify using a GSH affinity chromatography column, equilibrate with 20 column volumes of Binding Buffer (20 mM PB, pH 7.4, 150 mM NaCl); elute using a gradient of elution buffers: Elution Buffer 1: 50 mM Tris-HCl, pH 8.0, 5 mM glutathione; Elution Buffer 2: 50 mM Tris-HCl, pH 8.0, 10 mM glutathione. The target protein was mainly found in Elution 1. SDS-PAGE electrophoresis results are shown below. Figure 2 The collected samples were desalted using a desalting column (desalting buffer: 50mM NaCl; 20mM PB, pH 7.4). After desalting, 5% glycerol was added to the collected target protein, and the sample was rapidly frozen with liquid nitrogen and stored at -80°C.

[0056] Example 2: Establishment of SV2C-L4 nanobody phage library

[0057] 2.1 Materials

[0058] Stripwell TM Microplate (COSTAR, catalog number 42592), Tween 20 (purchased from Tianjin Bailunsi Biotechnology Co., Ltd.), Total RNA Extraction Reagent (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10606ES60), AEBSF (Beijing Solarbio Technology Co., Ltd., catalog number IA0110). (NEB Corporation, part number R3140V) (NEB, catalog number R3162V), pMES4 phage vector (Fenghui Biotechnology Co., Ltd., catalog number ZT642), TG1 Escherichia coli cell line (Ruichu Biotechnology Co., Ltd., catalog number R09003), SanPrep column PCR product purification kit (Sangon Biotech Co., Ltd., catalog number B518141-0100).

[0059] 2.2 Implementation Steps

[0060] 2.2.1 Total RNA extraction from alpaca immune lymphocytes

[0061] The purified pGEX-4T-1-SV2C-L4 protein was used as an antigen and sent to Shanxi Nami Biotechnology Development Co., Ltd. in dry ice for outsourced alpaca immunization. After four immunizations, the alpaca antiserum passed quality testing and achieved a titer of 10. 5 At that time, Ficoll was used to separate lymphocytes from alpaca blood, and each tube of alpaca immune lymphocytes contained approximately 5 × 10⁶ cells. 7 Extract total RNA using the following steps:

[0062] 1. Every 5 × 10 6 -1×10 7 Add 1 mL of Total RNA Extraction Reagent to each cell and repeatedly pipette.

[0063] 2. After vigorously shaking the homogenized sample, let it stand at room temperature for 5 minutes to allow the ribosomes to completely dissociate.

[0064] 3. Add 1 / 5 volume of chloroform to the above lysis buffer (e.g., 0.2 mL of chloroform per 1 mL Total RNA Extraction Reagent). Tightly cap the centrifuge tube, shake vigorously for 15 seconds, and let stand at room temperature for 2-3 minutes.

[0065] Centrifuge at 4.4℃, 12000g for 10-15 minutes.

[0066] After centrifugation, the mixture separates into three layers: an upper colorless aqueous layer, a middle layer, and a lower red organic phenol-chloroform layer. RNA is present in the aqueous layer, and the upper layer accounts for approximately 50-60% of the total volume of the added Total RNA Extraction Reagent.

[0067] 5. Carefully aspirate the upper aqueous phase into a new centrifuge tube, add 1 / 2 volume of isopropanol, invert to mix, and let stand at room temperature for 10 minutes.

[0068] Centrifuge at 6.4℃, 12000g for 10-15 minutes.

[0069] 7. Carefully discard the supernatant and add an equal volume of 75% ethanol (prepared with DEPC water, i.e., 1 mL of 75% ethanol per 1 mL Total RNA Extraction Reagent). Vortex thoroughly to wash, and gently tap the bottom of the tube to suspend the precipitate.

[0070] Centrifuge at 8.4℃ and 7500g for 5 minutes, discard the supernatant, and carefully aspirate the supernatant to prevent loss of RNA precipitate.

[0071] 9. Repeat steps 7 and 8 twice, that is, wash the precipitate twice.

[0072] 10. Allow to air dry at room temperature for 10 minutes. Add 20 μL of RNase-free water to dissolve the RNA. After complete dissolution, take a small amount for testing, and store the remaining solution at -70°C.

[0073] 11. RNA Integrity Detection: Add 2 μL of RNA to 2 μL of 2×RNAloading buffer and mix well. Perform electrophoresis. (The appearance of three clear bands indicates good RNA integrity.)

[0074] 12. RNA purity detection: Detect OD values ​​at 260nm and 280nm, and calculate the A260 / A280 ratio. The ratio for pure RNA should be around 2.0.

[0075] 2.2.2 RNA Reverse Transcription

[0076] Add reactants to the reverse transcription system as shown in Table 3, and follow the reverse transcription procedure as shown in Table 4.

[0077] Table 3 Reverse Transcription Reaction System

[0078]

[0079] Table 4 Reverse Transcription Procedure

[0080] temperature time 25℃ 5min 55℃ 15min 85℃ 5min

[0081] 2.2.3 cDNA amplification

[0082] The amplification primer sequences used are

[13] :

[0083] CALL001 Primer:GTCCTGGCTGCTCTTCTACAAGG;

[0084] CALL002 Primer:GGTACGTGCTGTTGAACTGTTCC.

[0085] The cDNA amplification system should be prepared according to Table 5, and the cDNA amplification procedure should be prepared according to Table 6.

[0086] Table 5 cDNA amplification system

[0087]

[0088] Table 6 cDNA amplification program

[0089]

[0090] 2.2.4 PCR - Step 2

[0091] The cDNA amplification products were extracted using the gel extraction kit according to the instructions. The extracted products were then used for PCR-Step 2. The primer sequences used were: VHH-Back: GATGTGCAGCTGCAGGAGTCTGGRGGAGG; VHH-For:

[0092] CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT.

[0093] Add the reactants to the PCR-Step 2 system as shown in Table 7, and follow the PCR-Step 2 procedure as shown in Table 8.

[0094] Table 7 PCR-Step 2 System

[0095]

[0096] Table 8 PCR-Step 2 Procedure

[0097] 2.2.5 Phage Plasmid Construction

[0098] The PCR-Step 2 amplification products were recovered using the gel recovery kit according to the instructions. The recovered products, namely the VHH fragment and pMES4 vector, were then subjected to double enzyme digestion.

[0099] For the VHH fragment double digestion system, add the reactants as shown in Table 9; for the pMES4 vector double digestion system, add the reactants as shown in Table 10.

[0100] Table 9. Double enzyme digestion system for VHH fragment

[0101]

[0102] Table 10 pMES4 vector double digestion system

[0103]

[0104] After digestion at 37℃ for 1 hour, the enzyme was then digested overnight at 4℃.

[0105] The double-digested VHH fragment was ligated to the pMES4 vector at a molar ratio of 3:1. The reactants for the pMES4-VHH ligation system were added according to Table 11.

[0106] Table 11 pMES4-VHH Connection System

[0107]

[0108]

[0109] Ligation was performed overnight at 16°C. The ligation product was purified using the SanPrep column PCR product purification kit according to the manufacturer's instructions. Elution was performed with 30 μL of sterile water. The purified product was then mixed with TG1 competent cells and electroporated under the following conditions: Geminni X2 electroporator, 1 cm electroporation cuvette, 1.8 kV, 25 μF, and 200 ohms. After electroporation, the cells were added to 1 mL of SOC medium and incubated at 37°C for 1 hour with shaking. The mixture was then diluted 1000-fold, plated onto LB agar plates containing 100 μg / mL ampicillin and 2% glucose, and incubated overnight at 37°C.

[0110] Fifteen single clones were randomly selected and colony PCR was performed to verify the insertion rate. Theoretically, it should be above 75%, and the insertion rate of this invention reached 93.3%. The primer sequences used for PCR verification were: MP57: TTATGCTTCCGGCTCGTATG; GIII: CCACAGACAGCCCTCATAG.

[0111] Add the reactants to the colony PCR system according to Table 12, and follow the colony PCR procedure in Table 13.

[0112] Table 12 Colony PCR System

[0113]

[0114] Table 13 Colony PCR Procedure

[0115]

[0116] After the insertion rate test was verified to be qualified, the constructed pMES4-VHHs plasmid was then subjected to multiple rounds of electroporation to construct a phage display library.

[0117] In the first round of electroporation, 2 μL of plasmid (14.91 ng / μL) and 25 μL of competent cells were used. After a 1000-fold dilution, only 81 clones grew, and the transformation efficiency was calculated to be approximately 2.7 × 10⁻⁶. 6 cfu / μg.

[0118] The second round of electroporation, under the same conditions as the first round, with a 1000-fold dilution, resulted in the growth of 114 clones, with a calculated conversion rate of approximately 3.82 × 10⁻⁶. 6 cfu / μg.

[0119] The third round of electroporation was conducted under the same conditions as above. After a 1000-fold dilution, 247 clones grew, and the calculated conversion rate was approximately 8.28 × 10⁻⁶. 6 cfu / μg.

[0120] Through three rounds of cumulative database construction via electronic transfer, at least 10 databases are required for database construction, based on the minimum requirements outlined in the literature. 7 A separate transformed library that meets the requirements for library construction.

[0121] Example 3: Expansion and Screening of SV2C-L4 Nanobody Library

[0122] 3.1 Materials

[0123] Bovine serum albumin V (Beijing Solarbio Science & Technology Co., Ltd., catalog number A8020), high-efficiency RIPA tissue / cell rapid lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., catalog number R0010), TMB single-component chromogenic solution (Beijing Solarbio Science & Technology Co., Ltd., catalog number PR1200), 1kD dialysis bag (Beijing Solarbio Science & Technology Co., Ltd., catalog number YA1047), Anti-6×His TagRabbit pAb (Saiwell Biotechnology Co., Ltd., catalog number GB111251), HRP×GoatAnti Rabbit IgG (H+L) (Immuno Way, catalog number RS0002), Bovine Thrombin (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 20402ES03).

[0124] 3.2 Experimental Procedure

[0125] The total number of *E. coli* cells in the construct constructed in this invention was counted to be 4.8 × 10⁶ per 100 μL. 9 Each cell.

[0126] 3.2.1 Amplification of the phage library:

[0127] Inoculation: Take 50 mL of 2×YT and inoculate 100 μL of total library, add 100 μg / mL ampicillin and 2% glucose solution, incubate at 37℃ and 200 rpm until the OD600 value is about 0.5.

[0128] Add 20 mL of the above bacterial culture to a 50 mL centrifuge tube, then add 50 μL of 4 × 10⁻⁶ HCl. 10 PFU helper phage VCSM13 was incubated at 37°C for 30 min. Centrifuged at 2800g, 20°C for 10 min. The supernatant was discarded, and the phage was resuspended in 100 mL of 2×YT medium. Ampicillin and kanamycin were added to a final concentration of 100 μg / mL, and the mixture was incubated overnight at 37°C and 200 rpm.

[0129] 3.2.2 Enrichment of the phage library:

[0130] 1. Centrifuge 5000g of the overnight cultured bacterial solution at 4℃ for 15 minutes.

[0131] 2. Transfer the supernatant to a new centrifuge tube, add 10 mL of pre-cooled PEG6000, invert the tube 5 times to mix, and then incubate on ice for 30 min.

[0132] Centrifuge at 3200g, 4℃ for 10 min, discard the supernatant, resuspend each tube in 1mL of ice-cold 1×PBS, transfer to a 1.5mL centrifuge tube, centrifuge at 20000g, 4℃ for 1 min.

[0133] 4. Transfer the supernatant to a new 1.5 mL centrifuge tube, add 250 μL PEG6000, invert and mix 10 times, and incubate on ice for 10 min.

[0134] Centrifuge at 5.4℃, 20000g for 15 min, discard the supernatant, aspirate clean, and resuspend in 1mL PBS.

[0135] Centrifuge at 6.4℃, 20000g for 1 min, and transfer the supernatant to a 1.5mL centrifuge tube to obtain phage concentrate.

[0136] 3.2.3 Determination of phage concentrate titer:

[0137] TG1 was inoculated into LB medium and cultured until the OD600 value was 0.5.

[0138] Prepare 15 200μL tubes and add 90μL of 1×PBS. Add 10μL of the prepared phage concentrate to the first tube, mix well, and then take 10μL and add it to the next tube, diluting sequentially. Take the samples from the last 8 tubes.

[0139] Prepare eight 200μL tubes, add 90μL of TG1, and then add the samples from the previous eight tubes one by one to these eight tubes. Incubate at 37℃ for 15min. Then divide the plate into nine regions on a 100μg / mL ampicillin and 2% glucose LB plate, add 5μL to each region, and incubate overnight at 37℃.

[0140] The titer of the phage concentrate was measured to be 10. 12 Those meeting or exceeding the screening standards.

[0141] 3.2.4 SV2C-L4 protein coating

[0142] The previously purified GST-SV2C-L4 antigen protein was digested with 50U of thrombin per 1mL of protein at 25°C for 4 hours. The GST tag was removed by GSH affinity chromatography, and the tag-free SV2C-L4 protein was obtained in the breakthrough peak.

[0143] Dilute the SV2C-L4 protein (without the GST tag) to 6 μg / μL with PBS. Add 100 μL of 1×PBS to the control wells and 100 μL of the diluted protein to the experimental wells. Cover the enzyme strips and store overnight at 4°C.

[0144] 3.2.5 Screening

[0145] Wash the coated plate three times with 250 μL of PBST per well, then add 250 μL of 5% BSA to each well and seal at 700 rpm for 2 hours.

[0146] Wash 5 times with 250 μL PBST, then add 100 μL of incubated phage to each control and experimental well. Shake at 700 rpm for 2 h, then wash 15 times with 250 μL PBST. Phage incubation: 50 μL phage concentrate, 50 μL 5% BSA, 400 μL PBS, shake at 700 rpm for 0.5 h.

[0147] Use 0.25% trypsin diluted 10 times, add 100 μL to each well, and shake at 700 rpm for 0.5 h.

[0148] Prepare four 200 μL tubes, each pre-filled with 5 μL of LAEBSF. Transfer the samples from the control wells and experimental wells into the prepared tubes and label them clearly.

[0149] The titer of bacteriophages obtained after trypsin digestion was determined. The titers of bacteriophages in the experimental and control groups were determined according to the aforementioned method for determining the titer of bacteriophage concentrate.

[0150] While determining the titer, prepare three 50 mL centrifuge tubes, add 3 mL of TG1 with an OD600 value of 0.5 to each tube, and add 50 μL of each of the three experimental groups to the corresponding tubes. Incubate at 37 °C for 30 min. Add LB to a final volume of 10 mL, add 10 μL of 100 μg / mL ampicillin and 2% glucose, and incubate overnight at 37 °C with 200 rpm. Add 20% glycerol and store the strains obtained in the first round of screening at -80 °C.

[0151] Using the bacterial strains screened in the previous round of experimental groups, a second round of screening was conducted, and so on, until the screening results reached the level where the experimental group should be two orders of magnitude higher than the control group. Three rounds of screening yielded a phage sublibrary enriched with strong affinity for SV2C-L4 after screening #1 to #3.

[0152] Example 4: Screening of Nanobody Binding Ability

[0153] 4.1 Preparation of Nanoparticles

[0154] 1. Coating board

[0155] Take 10 μL of the phage sublibrary obtained above, add 90 μL of LB culture medium, and serially dilute to 10⁻⁶. -4 and 10 -5 Spread the mixture onto LB agar plates containing 100 μg / mL ampicillin and 2% glucose, and incubate overnight at 37°C.

[0156] 2. Making the mother plate

[0157] Take a 96-well plate and add 100 μL of culture medium (2×YT, 100 μg / mL ampicillin, 2% glucose, 10% glycerol) to each well. Pick single colonies from the plate and add them to each well sequentially, leaving 4 wells as controls to detect contamination. Seal the plate with sealing film and incubate overnight at 37°C and 200 rpm.

[0158] 3. Induction

[0159] Add 1 mL of culture medium (2×YT, 100 μg / mL ampicillin, 2% glucose) to a 1.5 mL EP tube, and add 10 μL of the bacterial culture from the corresponding well on the mother plate to each tube. Incubate at 37 °C and 200 rpm for 6 h.

[0160] Add 1 μL of 1M IPTG to each tube and incubate overnight at 37°C and 200 rpm.

[0161] Centrifuge at 3200g, 20℃ for 10 min, discard the supernatant, add 250 μL LRIPA lysis buffer (per 100 μL LRIPA + 1 μL PMSF) to each tube, shake to mix, place on ice and shake for 1 h.

[0162] Centrifuge at 10000 rpm and 4℃ for 5 minutes.

[0163] Take 200 μL of supernatant and place it in a new 1.5 mL EP tube. The resulting nanobody is the nanobody.

[0164] 4.2 ELISA detection:

[0165] 1. Coat SV2C-L4 protein at a concentration of 0.5 μg / mL (diluted with PBS) and incubate overnight at 4°C.

[0166] 2. Wash each well 5 times with 250 μL PBST, add 250 μL 5% BSA, and incubate at 700 rpm for 2 hours.

[0167] 3. After blocking, wash each well 5 times with 250 μL of PBST, then add 50 μL of buffer (5% BSA:PBST = 1:20) to each well. Add 50 μL of the corresponding nanoparticles to each experimental well. Add 50 μL of RIPA buffer (PMSF:RIPA = 1:100) to each control well. Shake at 700 rpm for 1 hour.

[0168] 4. Wash each well 5 times with 250 μL PBST. Add 100 μL of buffer (5% BSA:PBST = 1:20) to the control wells without primary antibody. Add 100 μL of Anti-6×His Tag RabbitpAb primary antibody to the remaining wells. Dilute the primary antibody 1:2000 with buffer and shake at 700 rpm for 1 h.

[0169] 5. Wash each well 5 times with 250 μL PBST. Add 100 μL buffer (5% BSA:PBST = 1:20) to the control wells without secondary antibody. Add 100 μL HRP×GoatAnti Rabbit IgG (H+L) secondary antibody to the remaining wells. Dilute the secondary antibody 1:10000 with buffer and shake at 700 rpm for 1 h.

[0170] 6. Wash each well with 250 μL PBST 10 times, wipe the bottom of the microplate clean, and place it on aluminum foil.

[0171] 7. Add 100 μL of TMB colorimetric solution to each well, wrap it with aluminum foil, and let it stand in the dark for 30 minutes.

[0172] 8. Add 50 μL of 2M H2SO4 to each well before testing to terminate the reaction.

[0173] 9. Measure the absorbance at 450 nm using an ELISA reader and record the sample number with the highest absorbance value.

[0174] After screening all samples in the master plate, 33 samples with high absorbance values ​​were selected for PCR verification and sent for testing. The bacterial culture PCR system was prepared according to Table 14, and the bacterial culture PCR procedure was as shown in Table 15.

[0175] Table 14 Bacterial PCR System

[0176]

[0177] Table 15 Bacterial PCR Procedure

[0178]

[0179] Sequencing of 33 samples yielded 12 nanobody protein genes with distinct sequences, numbered 1-A4, 1-A5, 1-A10, 1-C6, 1-F8, 1-H9, 2-A2, 2-A5, 2-C4, 2-F1, 2-G11, and 2-H4. The OD450 affinity assay results for these 12 nanobody proteins are shown below. Figure 3 All were significantly higher than the blank control. Bacterial cultures containing these 12 nanoantibody plasmids were ex vivo sequenced by Genewiz. The computer-readable nucleic acid sequences are shown in SeqID No. 2-13, and the protein sequence alignments are shown in [link to SeqID]. Figure 4 The computer-readable sequence is SeqID No. 15-26. Example 5: Purification of SV2C-L4 Nanobody

[0180] 5.1 Materials

[0181] MEM (containing NEAA) basal medium (Pronoss, catalog number 22E1061), FBS (gibco, catalog number 10099-141C), DMEM / HIGH GLUCOSE (Cytiva, catalog number SH30022.01), Trypsin-EDTA solution (Soluble Biotech, catalog number T1320), penicillin-streptomycin mixture (100X) (Soluble Biotech, catalog number P1400), DMSO (Soluble Biotech, catalog number D8371).

[0182] 5.2 Induced expression of SV2C-L4 nanobody

[0183] The 12 selected SV2C-L4 nanobody strains were inoculated at 1% in LB medium and cultured at 37℃ and 200rpm until the OD600 value was approximately 0.6. Uninduced samples were collected, and IPTG was added to a final concentration of 0.5mmol / L. Induction was performed at 20℃ for 16h, and induced samples were collected. The cells were collected by centrifugation at 8000rpm for 5min.

[0184] After centrifugation, the uninduced and induced samples were prepared and subjected to SDS-PAGE electrophoresis. The results showed that the expression of SV2C-L4 nanobody was successfully induced in both cases.

[0185] 5.3 Purification of SV2C-L4 nanobody

[0186] Add 20 times its weight of Binding Buffer to the collected bacterial cells, homogenize at 800 bar until the cells are clear, and centrifuge at 10,000 rpm for 10 min. Collect the supernatant and purify by nickel affinity chromatography (equilibrate with 20 column volumes of Binding Buffer; Binding Buffer: 20 mM PB, pH 7.4, 20 mM NaCl, 100 mM imidazole; Elution Buffer: 20 mM PB, pH 7.4, 100 mM NaCl, 500 mM imidazole). The target protein was present in the elution sample. SDS-PAGE results are shown below. Figure 5 .

[0187] Example 6: SF-680SE-labeled SV2C-L4 nanobody

[0188] (1) Protein dissolution: Replace the buffer of the protein solution with 0.1M NaHCO3 using a concentration tube.

[0189] (2) Dye dissolution: After the activated Super Flour SE dye solid powder was placed in a desiccator from the refrigerator and slowly heated to room temperature, the dye was prepared to a concentration of 1 mg / mL using anhydrous DMSO.

[0190] (3) Labeling: Slowly add an appropriate amount of dye solution to 1 mL of protein solution (protein:dye molar ratio = 1:20-50; the mass ratio can be optimized within the range of 10-200 μg Super Flour SE per mg of IgG antibody). Simultaneously, stir slowly at room temperature in the dark for 1 hour. Alternatively, the protein solution can be directly added to the reagent bottle containing the solid dye powder, while simultaneously stirring slowly at room temperature in the dark for 1 hour.

[0191] (4) Purification: The product was purified using Sephadex G25 dextran.

[0192] 8 mL of Sephadex G25 dextran column was packed with the column and equilibrated with PBS solution. The labeled SV2C nanobody protein solution was then purified using a Sephadex G25 column to wash away excess unlabeled fluorescent dye from the SV2C nanobody. The eluted labeled protein solution was eluted with PBS solution, and the eluted fractions were collected based on the UV detector reading. The eluted fractions were collected in fractions and analyzed by SDS-PAGE. The highest concentration purified fractions were collected for later use.

[0193] Example 7: SV2C-L4 nanobody targeting N2a cells after labeling

[0194] Remove the frozen N2a cell line from liquid nitrogen and thaw it as quickly as possible in a 37°C water bath by rapid shaking, following the correct "slow freeze, rapid thaw" cell resuscitation principle. Transfer approximately 1 mL of the cell cryopreservation solution from the cryovial to a 5 mL centrifuge tube, add 2 mL of standard cell culture medium, mix well by pipetting, and centrifuge at 900 rpm for 3 min. After centrifugation, aspirate the supernatant, add another 1 mL of standard cell culture medium, and pipette the N2a cells to mix. Then, pipette the cell suspension into a 10 cm diameter culture dish containing 12 mL of cell culture medium. Gently shake the suspension from side to side, and then carefully incubate it in a 37°C cell culture incubator with 5% CO2 and 90% humidity.

[0195] When the cells reach approximately 80% confluence, they can be passaged. Use a pipette to transfer the old culture medium into a waste bottle. Wash the cultured cells 2-3 times with 2 mL of sterile PBS solution. Then, add 1 mL of 0.25% trypsin to digest the cells. Note that the trypsin can be aspirated onto the wall of the culture dish to allow it to flow smoothly down, avoiding cell damage. Gently shake the culture dish to disperse the trypsin evenly on the surface of the cells to be digested. Place the culture dish in a cell culture incubator for 1-2 minutes. Use a pipette to inject 1 mL of culture medium containing fetal bovine serum to neutralize the digestive effect of the trypsin. Carefully pipette the cells; once digestion is complete, they can be easily pipetted off. Transfer the cell digest to a 10 mL centrifuge tube and centrifuge the cell suspension at 900 rpm for 3 minutes. After centrifugation, remove the supernatant and resuspend the cells in 1 mL of culture medium. After carefully mixing the cells by pipetting, transfer the cell suspension to a 10 cm diameter culture dish containing 12 mL of cell culture medium at a ratio of 1:4.

[0196] While passaged, N2a cells were seeded in 24-well plates, with 10 cells per well. 5 Labeled SV2C-L4 nanobodies were added to each well of N2a cells to a final protein concentration of 2 μM. The cells were co-incubated overnight, and the binding of N2a cells to the labeled SV2C-L4 nanobodies was observed under a laser confocal microscope. Most of the labeled protein bound to receptors on the N2a cell membrane, resulting in observed red fluorescence. In contrast, no receptors for nanobodies were observed on the negative control HeLa cells, and no fluorescence was observed. Figure 6 ).

[0197] The SV2 extracellular soluble fraction nanobody provided by this invention, as a fluorescent probe molecule, can serve as a research tool for studying neuroendocrine tumors. Generally, depending on research needs, the SV2 extracellular soluble fraction nanobody can be conjugated with any other fluorescent probe according to the methods provided by the dye supplier, which is understandable to those skilled in the art.

[0198] Obviously, 89 Zr、 68 Ga、 99 Tc, 111 In、 90 Nuclide Y and others are used to label the extracellular soluble portion of SV2 nanobodies with bifunctional chelating agents such as DOPA, DTPA, and HYNIC. 18 F is synthesized first. 18 F precursor molecules were used to label the extracellular soluble portion of SV2 nanobodies; 131I. SV2 extracellular soluble fraction nanobodies were labeled using the chloramine-T method. The use of radionuclides to label SV2 extracellular soluble fraction nanobodies allows them to function as nuclear imaging probe molecules or radiopharmaceutical molecules with therapeutic effects, a process understood by those skilled in the art.

[0199] Using conventional chemical methods, toxin molecules can be conjugated to the extracellular soluble fraction of SV2 nanobodies. These toxin-conjugated SV2 extracellular soluble fraction nanobodies can become nuclear drug molecules with therapeutic activity, a concept easily understood by those skilled in the art. Such toxin molecules include: microtubule formation inhibitors such as auristatin, maytansine, and their analogues; DNA damage factors such as calicheamicin, duocarmycins, anthracyclines, and pyrrolobenzodiazepine dimers; and DNA transcription inhibitors such as amatoxin and quinoline alkaloid (SN-38).

[0200] The nucleotide sequence involved in this invention is:

[0201] Seq ID No:1

[0202] SV2C-L4:

[0203] CATATGTTTTCCGGATGTGATTAAACCGCTGCAGAGCGATGAATATGCGCTGCTGACCCGCAACGTGGAACGCGATAAATATGCGAACTTTACCATTAACTTTACCATGGAAAATCAGATTCATACCGGCATGGAATATGATAACGGCCGCTTTATTGGCGTGAAATTTAAAAGCGTGACCTTTAAAGATAGCGTGT TTAAAAGCTGCACCTTTGAAGATGTGACGAGCGTGAACACCTATTTTAAAACTGCACCTTTATTGATACCGTGTTTGATAACACCGATTTTGAACCGTATAAATTTATTGATAGCGAATTTAAAAATTGCAGCTTTTTTCATAACAAAACCGGCTGTCAGATTACCTTTGATGACGATTATAGCGCGTAAGAATTC

[0204] Seq ID No:2

[0205] 1-A4:

[0206] CTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGGTCTCTAGGACTCTCCTGTGCAGCCTCTGGACGCACCATCAGTAGTTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAACTTGTCGCAGTTATCAATTGGAGTGGTGACAGAACATACTATGGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGTGTATCTGCGAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGTCCTAAGGTCATTTAATTCGCGCTCTGATCCCCTTTACAACTGGGGCCAGGGGACCCAGGTCACC

[0207] Seq ID No:3

[0208] 1-A5:

[0209] CTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAATCAGAATCAGTAACTATGCCATGGGCTGGTATCGCCAGGCTCCAGGAAAACAGCGCGAGTTGGTCGCACGTATTAATAGTGTTGGAGATTTAGACTATGCAGACTCCCTCAAGGGCCGATTCACCATCTCCAGAGACAGCGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCGGAGGACACGGCCCTCTATTACTGTAATACAGCCATGGGGGCCTGGAAACTCCCGGGTGTCAAGGAGCCCTGGGGCCAGGGGACCCAGGTCACC

[0210] Seq ID No:4

[0211] 1-A10:

[0212] CTGCAGGAGTCTGGAGGAGGCTTGGTGCATGGTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTCCGTCCATCTTCAGTGGCTTGTCCATGGCCTGGTACCGCCAGGCTCCAGGGGAGGAGCGCGAATTGGTCGCACGAATTAGTCGTGATGGAAGTACAAACTATGCAGACACCGTACAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGTGTATCTCCAAATGAACAGCCTGAAACCAGAGGACGCGGGCGTCTATTTCTGTAATAGAGGAATAGGTGCTAGAACCTTTTGGGGCCGGGGGACCCAGGTCACC

[0213] Seq ID No:5

[0214] 1-C6:

[0215] CTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAATCATAATCAGTAACTATGCCATGGGCTGGTATCGCCAGGCTCCAGGAAAACAGCGCGAGTTGGTCGCACGTATTAATAGTGTTGGAGATTTAGACTATGCAGACTCCCTCAAGGGCCGATTCACCATCTCCAGAGACAGCGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCGGAGGACACGGCCCTCTATTACTGTAATACAGCCATGGGGGCCTGGAAACTCCCGGGTGTCAAGGAGCCCTGGGGCCAGGGGACCCAGGTCACC

[0216] Seq ID No:6

[0217] 1-F8:

[0218] CTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGCTCTCTGAGTCTCTCCTGTACAGCCTCTACAATCAGAATCAGTAACTATGCCATGGGCTGGTATCGCCAGGCTCCAGGAAAACAGCGCGACTTGGTCGCTCGGATTAGTAGTGATGGTAGGTTAGACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGCGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCCTCTATTACTGTAATACAGCCATGGGGGCCTGGAAACTCCCGAGTGCAAAGGAATCCTGGGGCCAGGGGACCCGGGTCACC

[0219] Seq ID No:7

[0220] 1-H9:

[0221] CTGCAGGAGTCTGGAGGAGAATTGGTGCAGTTTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTATAATCAGGATAAGTAGCTATTCCATGGGCTGGTATCGCCAGGCTCCAGGAAAACAGCGCGAGTTGGTCGCACGTATTAGTAGTGATGGCAGCACACGCTATTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGCGCCACGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCCTCTATTACTGTAATACAGCCATGGGGGCCTGGCAACTCCCAAGTGCCAAGGAACCCTGGGGCCCGGGGACCCAGGTCACC

[0222] Seq ID No:8

[0223] 2-A2:

[0224] CTGCAGGAGTCTGGAGGAGGCCAGGTGCATGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTCCGTCCATCTTCAGTGGCTTGTCCATGGCCTGGTACCGCCAGGCTCCAGGGGAGGAGCGCGAATACGTCGCACGAATTAGTCGTGATGGAAGTACAGCCTATGCAGACACCGTACAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTCCAAATGAACAGCCTGAAACCAGAGGACACGGGCGTCTATTTCTGTAATAGAGGAGTAGGTGCTAGAACCTTTTGGGGCCGGGGGACCCAGGTCACC

[0225] Seq ID No:9

[0226] 2-A5:

[0227] CTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCGTGGGGGGTCTCTGAGACTCTCCTGT

[0228] GCAGCCTCTCCGTCCATCTTCAGTGGCTTGTCCATGGCCTGGTACCGCCAGGCTCCAGG

[0229] GGAGGAGCGCGAATTGGTCGCACGAATTAGTCGTGATGGAAGTACAAAGTATGCAGAC

[0230] ACCGTACAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGTGTATCTCC

[0231] AAATGAACAGCCTGAAACCAGAGGACGCGGGCGTCTATTTCTGTAATAGAGGAATAGG

[0232] TGCTAGAACCTTTTGGGGCCAGGGGACCCAGGTCACCSeq ID No:10

[0233] 2-C4:

[0234] CTGCAGGAGTCTGGGGGAGAATTGGTGCAGCCTGGGGGGTCTCTGACACTCTTCCTGTG

[0235] CTGCCTCTGGAATCAGAATCAGTAACTATGCCATGGGCTGGTATCGCCAGGCTCCAGGA

[0236] AAACAGCGCGACTTGGTCGCACGTATTAGTAGTGACGGTAGTACACGCTATGCAGACT

[0237] CCGTGAACGGCCGATTCACCATCTCCAGAGACAGCGCCAAGAACACGGTGTTTCTGCA

[0238] AATGAACAGCCCTGAAACCTGAGGACACGGCCCTCTATTACTGTAATACAGCCATGGGG

[0239] GCCTGGAAACTCCCGACTACCAAGGAATCCTGGGGCCAGGGGACCCAGGTCACCSeq ID No:11

[0240] 2-F1:

[0241] CTGCAGGAGTCTGGAGGAGGCCAGGTGCATGCTGGGGGGTCTCTGAGACTCTTCCTGT

[0242] GCAGCCTCTCCGTCCATCTTCAGTGGCTTGTCCATGGCCTGGTACCGCCAGGCTCCAGG

[0243] GGAGGAGCGCGAATACGTCGCACGAATTAGTCGTGATGGAAATACAGCCTATGCAGAC

[0244] ACCGTACAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTCC

[0245] AAATGAACAGCCCTGAAACCAGAGGACACGGGCGTCTATTTCTGTAATAGAGGAGTAGG

[0246] TGCTAGAACCTTTTGGGGCCGGGGGACCCAGGTCACCSeq ID No:12

[0247] 2-G11:

[0248] CTGCAGGAGTCTGGGGGAGAATTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGT

[0249] GCAGCCTCTGGAATCAGAATCAGTAACTATGCCATGGGCTGGTATCGCCAGGCTCCAG

[0250] GAAAACAGCGCGACTTGGTCGCACGTATTAGTAGTAGTGGTGATACACGCTATGCAGA

[0251] CTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGCGCCAAGAACACGGTGTATCTG

[0252] CAAATGAATAGCCTGAAACCTGAGGACACGGCCCTCTATTACTGTAATACAGCCATGGG

[0253] GGCCTGGAAAGTCCCGAGTGCCCAGGAATCCTGGGGCCAGGGGACCCAGGTCACCSeq ID No:13

[0254] 2-H4:

[0255] CTGCAGGAGTCTGGAGGAGGCCAGGTGCATGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTCCGTCCATCTTCAGTGGCTTGTCCATGGCCTGGTACCGCCAGGCTCCAGGGGAGGAGCGCGAATACGTCGCACGAATTAATCGTGATGGAAATACAGCCTATGCAGACACCGTACAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTCCAAATGAACAGCCTGAAACCAGAGGACACGGGCGTCTATTTCTGTAATAGAGGAGTAGGTGCTAGAACCTTTTGGGGCCGGGGGACCCAGGTCACC

[0256] Protein sequences related to the present invention:

[0257] Seq ID No:14

[0258] SV2C-L4:

[0259] HMFPDVIKPLQSDEYALLTRNVERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFKNCSFFHNKTGCQITFDDDYSA*EF

[0260] Seq ID No:15

[0261] 1-A4:

[0262] LQESGGGLVQAGGSLGLSCAASGRTISSYAMAWFRQAPGKERELVAVINWSGDRTYYGDSVKGRFTISRDDAKNTVYLRMNSLKPEDTAVYYCNVLRSFNSRSDPLYNWGQGTQVT

[0263] Seq ID No:16

[0264] 1-A5:

[0265] LQESGGGLVQPGGSLRLSCAASGIRISNYAMGWYRQAPGKQRELVARINSVGDLDYADSLKGRFTISRDSAKNTVYLQMNSLKPEDTALYYCNTAMGAWKLPGVKEPWGQGTQVT

[0266] Seq ID No:17

[0267] 1-A10:

[0268] LQESGGGLVHGGGSLRLSCAASPSIFSGLSMAWYRQAPGEERELVARISRDGSTNYADTVQGRFTISRDDAKNTVYLQMNSLKPEDAGVYFCNRGIGARTFWGRGTQVT

[0269] Seq ID No:18

[0270] 1-C6:

[0271] LQESGGGLVQPGGSLRLSCAASGIIISNYAMGWYRQAPGKQRELVARINSVGDLDYADSLKGRFTISRDSAKNTVYLQMNSLKPEDTALYYCNTAMGAWKLPGVKEPWGQGTQVT

[0272] Seq ID No:19

[0273] 1-F8:

[0274] LQESGGGLVQPGGSLSLSCTASTIRISNYAMGWYRQAPGKQRDLVARISSDGRLDYADSV

[0275] KGRFTISRDSAKNTVYLQMNSLKPEDTALYYCNTAMGAWKLPSAKESWGQGTRVTSeq ID No:20

[0276] 1-H9:

[0277] LQESGGELVQFGGSLRLSCAASIIRISSYSMGWYRQAPGKQRELVARISSDGSTRYSDSV

[0278] KGRFTISRDSATNTVYLQMNSLKPEDTALYYCNTAMGAWQLPSAKEPWGPGTQVTSeq ID No:21

[0279] 2-A2:

[0280] LQESGGGQVHAGGSLLRLSCAASPSIFSGLSMAWYRQAPGEEREYVARISRDGSTAYADTV

[0281] QGRFTISRDNAKNTVYLQMNSLKPEDTGVYFCNRGVGARTFWGRGTQVTSeq ID No:22

[0282] 2-A5:

[0283] LQESGGGLVQRGGSLRLSCAASPSIFSGLSMAWYRQAPGEERELVARISRDGSTKYADTV

[0284] QGRFTISRDDAKNTVYLQMNSLKPEDAGVYFCNRGIGARTFWGQGTQVTSeq ID No:23

[0285] 2-C4:

[0286] LQESGGELVQPGGSLTLSCAASGIRISNYAMGWYRQAPGKQRDLVARISSDGSTRYADSV

[0287] NGRFTISRDSAKNTVFLQMNSLKPEDTALYYCNTAMGAWKLPTTKESWGQGTQVTSeq ID No:24

[0288] 2-F1:

[0289] LQESGGGQVHAGGSLRLSCAASPSIFSGLSMAWYRQAPGEEREYVARISRDGNTAYADTV

[0290] QGRFTISRDNAKNTVYLQMNSLKPEDTGVYFCNRGVGARTFWGRGTQVTSeq ID No:25

[0291] 2-G11:

[0292] LQESGGELVQPGGSLRLSCAASGIRISNYAMGWYRQAPGKQRDLVARISSSGDTRYADSV

[0293] KGRFTISRDSAKNTVYLQMNSLKPEDTALYYCNTAMGAWKVPSAQESWGQGTQVTSeq ID No:26

[0294] 2-H4:

[0295] LQESGGGQVHAGGSLRLSCAASPSIFSGLSMAWYRQAPGEEREYVARINRDGNTAYADTV

[0296] QGRFTISRDNAKNTVYLQMNSLKPEDTGVYFCNRGVGARTFWGRGTQVT

Claims

1. A nanobody with an extracellular soluble fraction of SV2, characterized in that, The protein sequence of the nanobody is shown in Seq ID No:20; the protein sequence of the extracellular soluble portion of SV2 is shown in Seq ID No:

14.

2. The SV2 extracellular soluble nanobody according to claim 1, characterized in that, The nanobody-conjugated fluorescent dye Super Flour 680SE.

3. The application of the SV2 extracellular soluble fraction nanobody as described in claim 1 in the preparation of a neuroendocrine tumor nuclear imaging probe molecule.

4. The application of the SV2 extracellular soluble fraction nanobody as described in claim 1 in the preparation of fluorescently labeled probe molecular tools targeting neuroendocrine tumors.

5. The application of the SV2 extracellular soluble fraction nanobody as described in claim 1 in the preparation of enzyme-labeled probe molecular tools targeting neuroendocrine tumors.

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