Anti-human NOTCH2 single-chain antibody, coding gene, kit and application of anti-human NOTCH2 single-chain antibody

Abstract

The invention discloses an anti-human NOTCH2 single-chain antibody, a coding gene, a kit and application of the anti-human NOTCH2 single-chain antibody, and belongs to the technical field of biological medicine. The amino acid sequence of the anti-human NOTCH2 single-chain antibody is shown as SEQ ID NO.3, the amino acid sequence of a heavy chain variable region of the anti-human NOTCH2 single-chain antibody is shown as SEQ ID NO.1, and the amino acid sequence of a light chain variable region of the anti-human NOTCH2 single-chain antibody is shown as SEQ ID NO.2. The anti-human NOTCH2 single-chain antibody and the ICG-SCFV provided by the invention have specific recognition and binding capacities on NOTCH2, and are expected to be used as diagnostic and therapeutic antibodies for diagnosis and treatment of tumors such as gastric cancer and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-human NOTCH2 single-chain antibody, an encoding gene, a kit and uses thereof. Background Art

[0002] Cancer has been a century-old problem plaguing humanity, necessitating the development of a variety of treatments, including surgical resection, chemotherapy, radiotherapy, and biological therapy. Although surgical resection of malignant tumor tissue cannot completely eliminate it, approximately 50% of cancer patients who have detectable malignant tumor tissue removed experience no recurrence of the disease, and surgical resection can extend life expectancy or reduce the incidence of cancer recurrence. Therefore, surgical resection remains the most commonly used and effective treatment. Precisely because of the importance of complete resection of malignant tumor tissue, diagnostic methods that can ensure accurate and complete identification of malignant tumor tissue are of great application value.

[0003] Gastric cancer is recognized as the fifth most common malignancy worldwide and ranks fourth in mortality. Despite significant advances in diagnostic techniques and treatment modalities, only a small fraction of patients are diagnosed at an early stage. As a result, the prognosis for advanced gastric cancer remains poor, necessitating further developments in molecular diagnostics and treatments for this disease.

[0004] Although people have recognized the importance of completely removing tumors and the availability of some identification technologies in visualizing tumor masses, many malignant tumor tissues still escape detection, leading to disease recurrence and ultimately death. Therefore, how to achieve more accurate and complete tumor identification is a core issue that needs to be urgently addressed in this field. With the discovery and application of fluorescent dyes, an intraoperative navigation technology called "fluorescence-guided surgery" has gradually become a "darling" in surgical operations. This technology uses excitation light of a specific wavelength to stimulate the tumor's own fluorescence, retained fluorescent molecules, or exogenous fluorescent substances taken up by cells to emit fluorescence, thereby guiding doctors to accurately remove the tumor. However, since the fluorescent dye itself is not targeted, it not only accumulates in tumor tissue, but also accumulates in normal tissue, which may lead to excessive resection and damage surrounding normal tissues. Therefore, it is necessary to develop a targeted fluorescent probe.

[0005] Notch signaling is a tightly controlled and conserved pathway that is crucial for the normal morphological development of multicellular organisms, controlling interactions between interacting cells in a multicellular environment. Therefore, abnormalities in NOTCH signaling may lead to disease and cancer. Studies have shown that NOTCH2 is overexpressed in different types of tumors, including gastric cancer, lymphocytic malignancies, and brain tumors, and its enhanced activity plays a crucial role in tumor progression. NOTCH2 overexpression is significantly associated with poor prognosis in patients with diffuse gastric cancer.

[0006] Four Notch receptors (Notch1-4) and five Notch ligands (Delta-like 1, 3, 4, and Jagged 1-2) have been identified in mammals. Dysregulated Notch signaling is considered a key factor in promoting the stem-like proliferation of cancer cells, thereby promoting disease progression. The four Notch receptor isoforms play distinct roles in the development and progression of cancer. NOTCH2 is a Notch receptor that is commonly overexpressed in a range of cancers and is associated with a unique oncogenic mechanism. A wide range of cancer types have been found to overexpress NOTCH2 or exhibit gain-of-function mutations, and enhanced NOTCH2 activity plays a crucial role in tumor progression. Overactive NOTCH2 signaling is also associated with the dysregulation of certain miRNAs, tumor-associated stromal cell input, and modulation of internal and external stimuli in tumor cells. Sustained NOTCH2 signaling promotes the stem-like ability of tumor cells to avoid apoptotic cell death while self-renewing and promoting EMT. Elevated NOTCH2 expression is associated with poor clinical prognosis in patients. NOTCH2 further increases chemotherapy and radioresistance in tumor cells, thereby making these cancers less sensitive to treatment. Successful efforts to block NOTCH2 signaling will depend on being effective and specific for patients.

[0007] Research has found that clinical trials of anti-NOTCH monoclonal antibodies (brontictuzumab, a Notch 1-specific monoclonal antibody; tarextumab, a Notch 2 / 3-specific monoclonal antibody) for cancer treatment were terminated due to severe gastrointestinal side effects and lack of efficacy. However, monoclonal antibodies have high target specificity but low penetration into solid tumors. Furthermore, the manufacturing process is complex and the production cost is high, which limits their application in cancer treatment. Summary of the Invention

[0008] In response to the above-mentioned deficiencies in the prior art, the present invention provides an anti-human NOTCH2 single-chain antibody, encoding gene, kit and use thereof. The nanobody has specific recognition and binding ability to NOTCH2 and is expected to be used as a diagnostic and therapeutic antibody for various cancers.

[0009] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0010] An anti-human NOTCH2 single-chain antibody, the amino acid sequence of which is shown in SEQ ID NO.3.

[0011] Furthermore, the amino acid sequence of the heavy chain variable region of the anti-human NOTCH2 single-chain antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2.

[0012] Furthermore, the heavy chain variable region and the light chain variable region are connected via a linker peptide.

[0013] Furthermore, the heavy chain CDR and light chain CDR sequences are as follows, wherein the heavy chain CDR and light chain CDR sequences are as follows, wherein the amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO.7, the amino acid sequence of CDR2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR3 is shown in SEQ ID NO.9;

[0014] The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.10, the amino acid sequence of CDR2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR3 is shown in SEQ ID NO.12.

[0015] A nucleic acid encoding the above-mentioned anti-human NOTCH2 single-chain antibody, the nucleic acid sequence of which is shown in SEQ ID NO.4.

[0016] A nucleic acid encoding the heavy chain variable region and light chain variable region, wherein the nucleic acid sequence encoding the heavy chain variable region is shown as SEQ ID NO.5, and the nucleic acid sequence encoding the light chain variable region is shown as SEQ ID NO.6.

[0017] A vector comprising the above nucleic acid.

[0018] A cell comprising or producing the above-mentioned anti-human NOTCH2 single-chain antibody.

[0019] A kit comprising the above-mentioned anti-human NOTCH2 single-chain antibody.

[0020] The above-mentioned anti-human NOTCH2 single-chain antibody, nucleic acid, vector, cell, or use in the preparation of a medicament, kit and / or device for preventing and / or treating NOTCH2-positive diseases or disorders.

[0021] Use of the above-mentioned anti-human NOTCH2 single-chain antibody in the preparation of a developer.

[0022] Furthermore, the anti-human NOTCH2 single-chain antibody is coupled with at least one fluorescent molecule.

[0023] Furthermore, the fluorescent molecule is a near-infrared region I fluorescent molecule.

[0024] Furthermore, the near-infrared region I fluorescent molecule is an indocyanine green activated ester (ICG-NHS) fluorescent molecule, and its structure is shown below:

[0025]

[0026] Furthermore, when the substance targeting NOTCH2 is a single-chain antibody (SCFV), and the antibody is directly coupled to a fluorescent molecule, it is expressed as an ICG-SCFV antibody-coupled fluorescent molecular probe, wherein the carboxylic acid group on ICG-NHS can directly react with the amino group of SCFV to form a stable amide bond.

[0027] Furthermore, the imaging agent is applied to tumors with upregulated NOTCH2 expression, such as gastric cancer, pancreatic cancer, bile duct cancer, and colorectal cancer.

[0028] Beneficial effects of the present invention:

[0029] The anti-human NOTCH2 single-chain antibody and ICG-SCFV provided by the present invention have specific recognition and binding capabilities to NOTCH2, and are expected to be used as diagnostic and therapeutic antibodies for the diagnosis and treatment of tumors such as gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Immunization of mice with antigens and ELISA detection of mouse hNOTCH2 antibody serum titers;

[0031] Figure 2 Figure 1 is an agarose gel electrophoresis diagram of PCR-amplified B lymphocyte antibody variable region sequences. Figure a shows the amplification of the β-actin sequence using reverse-transcribed cDNA, used to test cDNA quality. Figures b and c show nested PCR amplification of the antibody VH and VL variable region sequences using cDNA. Figure d shows the amplification of the SCFV sequence.

[0032] Figure 3 is the agarose gel electrophoresis image of LentiCMV-hNOTCH2-SCFV-puro plasmid;

[0033] Figure 4 To sort positive 293T cells using flow cytometer;

[0034] Figure 5 High-throughput screening of hNOTCH2-positive SCFV sequences;

[0035] Figure 6 hNOTCH2 antibody was purified and validated by flow cytometry;

[0036] Figure 7 Detection of the photophysical properties of the probe; wherein, (a) is the absorption spectrum of the ICG-SCFV probe at 400-1000 nm; (b) is the fluorescence emission spectrum of the ICG-SCFV probe at 740-840 nm with an excitation wavelength of 790 nm;

[0037] Figure 8Binding and competition assays of ICG-SCFV and NOTCH2 on MKN45 cells; (a) is an ICG-SCFV binding assay; (b) is a binding specificity assay;

[0038] Figure 9 For the cytotoxicity assay of labeled SCFV and ICG-SCFV on mkn45 cells, the data represent the mean ± SD (n = 3);

[0039] Figure 10 In vivo / ex vivo near-infrared imaging and biodistribution of ICG-SCFV in mice bearing MKN45 xenograft tumors. DETAILED DESCRIPTION

[0040] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0041] Example 1 Preparation of anti-human NOTCH2 single-chain antibody

[0042] 1. Mouse immunization and enzyme-linked immunosorbent assay (ELISA) to detect mouse serum titer

[0043] (1) Cell-mediated immunization experiments were performed using 6-8 week-old Balb / C mice. 2×10 7 The cells were fixed with 4% paraformaldehyde and injected into the peritoneum for the first time to immunize mice. Ten days later, 1×10 7 cells for secondary immunization, and 0.5×10 7 The third immunization was performed with 10 cells.

[0044] (2) After selecting mice that have been immunized for 30 days, collect blood from the tail vein, let it stand at room temperature for 1 hour, and then store it at 4°C overnight. Aspirate the upper serum, centrifuge it at 15,000 rpm at 4°C for 15 minutes, and take the supernatant; repeat once. Take part of the serum and dilute it 10,000 times with PBS for Elisa test. The rest can be stored at 4°C for a short time or at -80°C for a long time. Figure 1 )

[0045] (3) After immunization, the mice were killed by cervical dislocation, and the spleen was removed for the next experiment.

[0046] 2. Enrichment of positive B lymphocytes

[0047] (1) Place the mouse spleen in a 70 μm cell sieve, add 6 mL of PBS, grind the spleen with the tip of a syringe, and transfer the cell filtrate to a sterile culture dish. After grinding, rinse the 70 μm cell sieve again with 6 mL of PBS, collect all the filtrate into a 15 mL centrifuge tube, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant; resuspend the cells in 10 mL of PBS, pass through the 70 μm cell sieve again, and count the total number of cells.

[0048] (2) The sieved spleen cells were centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 500 μL PBS and transferred to a flow cytometry tube. B cells were isolated according to the instructions of the B cell isolation kit and the cells were counted.

[0049] (3) Specific B cell sorting: B cells were transferred to a flow cytometry tube and blocked at room temperature for 10-15 min with 10 μg of 2.4G2 antibody. After blocking, 0.5 μg of FITC anti-mCD19, PE-Cy7 anti-mIgM, PE-Cy7 anti-mIgD, and 1 μg of hNOTCH2-hFC recombinant protein were added to the control group and the experimental group, respectively, and incubated at 4°C for 30 min. The cells were washed twice with 4 mL of PBS (centrifuged at 1500 rpm for 5 min), the supernatant was discarded, and the cells were resuspended in 2 mL of PBS and transferred to a flow cytometry tube for flow cytometry sorting.

[0050] 3. PCR amplification of positive B cell SCFV sequences

[0051] (1) RNA extraction

[0052] In this experiment, RNA was extracted from flow cytometry-sorted mouse B cells using the RNeasy Mini Kit. Mouse B cells were collected into sterile EP tubes and centrifuged at 8000 rpm at 4°C for 2 minutes. The supernatant was discarded and the cells were washed with PBS before RNA extraction. For detailed extraction procedures, please refer to the RNeasy Mini Kit Instructions.

[0053] (2) RNA reverse transcription

[0054] This experiment uses SMARTScribe TM Reverse transcription was performed with the Reverse Transcriptase Kit. For details, see the SMARTScribe TM Reverse Transcriptase Instructions.

[0055] (3) First PCR amplification of VH and VK sequences

[0056] According to the PCR reaction program in Table 1, VH and VK sequences were amplified using 2× hot start Green master mix.

[0057] Table 1 First PCR amplification program

[0058]

[0059] (4) Second PCR amplification of VH and VK sequences (using the first PCR product as a template)

[0060] According to the PCR reaction program in Table 2, VH and VK sequences were amplified using 2× hot start Green master mix.

[0061] Table 2 Second PCR amplification program

[0062]

[0063] (5) Third PCR amplification of SCFV fragment

[0064] According to the PCR reaction program in Table 3, the SCFV fragment was amplified by KOD high-fidelity enzyme.

[0065] Table 3 Third PCR amplification program

[0066]

[0067] After the third PCR amplification is completed, add 10 μL DNA loading buffer (6×) to the amplified product, mix well and apply all the samples to the wells of 1% agarose gel, electrophorese at 150V for 30 minutes, take pictures and save them under a gel imager, cut the gel and recover it, and determine the concentration. Figure 2 )

[0068] 4. Construction of 293T cell antibody display library

[0069] (1) Using the Gibson assembly system, the hNOTCH2-SCFV mix fragment recovered after amplification was cloned into the restriction enzyme-cut vector LentiCMV-DP3-HA-Puro.

[0070] (2) Use an electroporator to electrotransfer the ligation product to DH5α, shake the cells and extract the plasmid. For details on the plasmid purification steps, please refer to the "Instructions for the Large-Scale Plasmid Extraction Kit".

[0071] (3) Using the lentiviral packaging system, an antibody display library stably expressing hNOTCH2-SCFV mix was constructed in 293T cells.

[0072] 5. High-throughput screening of hNOTCH2 monoclonal antibody SCFV sequences

[0073] (1) A positive SCFV antibody display library was obtained by flow cytometry sorting.

[0074] (2) The genome of the sorted positive SCFV 293T cells was extracted, the SCFV mix sequence was amplified, and the purified and recovered SCFV fragment was cloned into the SY002 expression vector. After coating the agar plate, positive single clones were obtained, and 100 single clones were picked and the plasmid was extracted for later use.

[0075] (3) The SCFV expression plasmid was transfected into 293T cells. The cell expression supernatant was collected 24 h after transfection and centrifuged at 10,000 rpm for 10 min at 4°C. The precipitate was discarded and the supernatant was transferred to a new centrifuge tube and centrifuged again. The tube was stored at 4°C until use.

[0076] (4) Flow cytometry analysis: 1×10 6 100 μL of cell expression supernatant was added to a sterile EP tube, and the mixture was mixed by pipetting. The tube was incubated at 4°C for 30 min. After the incubation, the tube was centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. The tube was washed twice with Wash Buffer. 1 μL of secondary antibody (anti-mouse IgG APC) was added to the EP tube, and the tube was incubated at 4°C for 30 min. After the incubation, 1 mL of Wash Buffer was added to the tube, and the mixture was mixed by pipetting and centrifuged. The supernatant was discarded. The washing was repeated twice. 300 μL of Wash Buffer was added to the EP tube to resuspend the cells and the cells were transferred to a flow cytometry tube for flow cytometry analysis. Positive clones were screened and sent to Xi'an Qingke for sequencing ( Figure 4 、 Figure 5 ).

[0077] 6. Transfection of 293F cells with PEI transfection reagent

[0078] (1) Adjust cell density before transfection

[0079] One day before transfection, adjust the cell density to 2×10 6 cells / mL (dilute the cells to 2×10 6 cells / mL) and cultured in a constant temperature shaker at 37°C, 8% CO2, and 110 rpm;

[0080] On the day of transfection, the cell density was adjusted to 3 × 10 6 cells / mL (dilute the cells to 3×10 6 cells / mL), and the volume of each bottle of cell solution was 40 mL;

[0081] (2) Preparation of PEI / DNA complex

[0082] First, prepare PEI (1 mg / mL), recombinant protein expression plasmid, and pre-chilled SMM293-TII medium. Add the complexes to centrifuge tubes at the following ratio (PEI: DNA = 3 μL: 1 μg). After mixing, let tubes A and B stand at room temperature for 5 minutes each.

[0083] Tube A: culture medium + 40 μg recombinant expression plasmid (gently mix), total volume 2 mL

[0084] Tube B: culture medium + 120 μL PEI transfection reagent (mix gently), total volume 2 mL

[0085] Aspirate tube A and slowly add it to tube B, pipette gently 7-8 times, mix well and let it stand at room temperature for 15 minutes;

[0086] Add the PEI / DNA complex dropwise to the cell culture medium to be transfected, gently shake the culture bottle while adding, shake well, and return it to the shaker to continue culturing;

[0087] (3) Add VPA

[0088] At 20 h after transfection, 320 μL of VPA (500 mM) was added dropwise to a final concentration of 4 mM, and the culture flask was gently shaken while adding dropwise;

[0089] (4) Add feed medium

[0090] 24 h after transfection, add 1.4 mL of SMS293-SUPI feed solution dropwise while gently shaking the culture flask;

[0091] (5) Harvest cell supernatant

[0092] On the 4th to 5th day after transfection (viability between 50% and 70%), the cell expression supernatant was collected and centrifuged at 10,000 rpm for 10 min in a 4°C centrifuge. The precipitate was discarded and the supernatant was transferred to a new centrifuge tube and centrifuged twice. After centrifugation, the supernatant was filtered with a 0.22 μm filter membrane and stored at 4°C for purification.

[0093] 7. Antibody purification by affinity chromatography

[0094] (1) Cell culture supernatant was passed through the column

[0095] Set the flow rate: Connect 1 mL of Protein A HP cloμLns to the purification pump and set it to the minimum flow rate (approximately 1 mL / min).

[0096] Wash the purification column with 20 mL of elution buffer and discard the washing solution;

[0097] Wash the purification column with 20 mL of binding buffer and discard the washing solution;

[0098] Place the inlet tube in the collected 293F cell culture supernatant, pass it through the purification column, collect the filtrate, and repeat this step to pass the collected filtrate through the purification column for the second time;

[0099] Wash the purification column with 20 mL of binding buffer, detect the presence of foreign proteins in the washing solution with Coomassie Brilliant Blue, and discard the washing solution;

[0100] Elution buffer was slowly passed through the purification column at a flow rate of 1 mL / min and the eluate was collected. During the elution process, the protein concentration in the eluate was monitored using Coomassie Brilliant Blue reagent (100 μL of Coomassie reagent was added to a 96-well plate, and then 5 μL of eluate was taken for separate testing) to ensure that there was almost no protein residue in the last fraction before washing. Finally, all the collected eluates were added with neutralization buffer at a ratio of 10:1 and stored at 4°C for ultrafiltration.

[0101] (2) Ultrafiltration concentration

[0102] Select an ultrafiltration tube of appropriate molecular weight and add all the collected eluate containing the target protein to the ultrafiltration tube. Centrifuge at 5000xg in a fixed-angle centrifuge at 4°C for 15 minutes. Discard the liquid at the bottom of the ultrafiltration tube, pipette to mix the retained protein solution, fill with 1× PBS, and continue centrifugation. Repeat the previous step once. Mix the retained protein solution and collect it in a 1.5mL centrifuge tube. Store at 4°C until assayed.

[0103] (3) Identification of protein concentration and purity. Figure 6 )

[0104] Finally, the antibody was sequenced and its amino acid sequence was as follows:

[0105] EVQLQESGPELVKSGASVKMSCKASGYTFTDYIINWVRQRTGQGLEWIG

[0106] EIYPGSGSTYHNEKFKGKATLTADKSSNTAYMQLSSLTSEDSAVYFCARRVYD

[0107] KAYAMDYWGQGTSLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDR

[0108] VTITCRASQNIKRFLAWYQQKPGKAPKLLIYGASTRESGVPSRFSGSGSGTDF

[0109] TLTISSLQPEDFATYYCQQYYRSPHTFGQGTKVEIKR (SEQ ID NO. 3).

[0110] The heavy chain sequence is as follows:

[0111] EVQLQESGPELVKSGASVKMSCKASGYTFTDYIINWVRQRTGQGLEWIG EIYPGSGSTYHNEKFKGKATLTADKSSNTAYMQLSSLTSEDSAVYFCARRVYD KAYAMDYWGQGTSLTVSS (SEQ ID NO. 1).

[0112] The light chain sequence is as follows:

[0113] DIQMTQSPSSSLSASVGDRVTITCRASQNIKRFLAWYQQKPGKAPKLLIYGASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYRSPHTFGQGTKV EIKR (SEQ ID NO. 2).

[0114] 8. Antibody titer detection

[0115] Indirect ELISA was used to detect the titer of hNOTCH2 monoclonal antibody purified from culture supernatant. The specific steps were as follows:

[0116] (1) The purified SCFV protein was coated on an ELISA plate at 2 μg / mL, 50 μL / well, and incubated at 4°C overnight.

[0117] (2) Wash the coated plate, add 200 μL / well of PBS containing 2% BSA, and block at 37°C for 2 h.

[0118] (3) After washing, add monoclonal antibodies at serial dilutions (1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold, 32000-fold, 64000-fold) and PBS wells as controls, and incubate at 37°C for 1 h.

[0119] (4) After washing the plate, add 1:5000 horseradish peroxidase-labeled goat anti-mouse antibody and incubate at 37°C for 1 h.

[0120] (5) Add 100 μL of TMB colorimetric substrate to each well and react for 5 min.

[0121] (6) Add 50 μL of stop solution to terminate the reaction.

[0122] (7) Read the OD value of each well at 450 nm using a biomarker. The test results are shown in Table 4.

[0123] Table 4 Anti-hNOTCH2 antibody titer

[0124] Antibody dilution factor <![CDATA[OD 450 ]]> NC-hNOTCH2 0.1585 PC-hNOTCH2 2.479 hNOTCH2-Ab-1 / 1000 0.9545 hNOTCH2-Ab-1 / 2000 0.8978 hNOTCH2-Ab-1 / 4000 0.7745 hNOTCH2-Ab-1 / 8000 0.637 hNOTCH2-Ab-1 / 16000 0.4425 hNOTCH2-Ab-1 / 32000 0.2829 hNOTCH2-Ab-1 / 64000 0.1623

[0125] Example 2 Construction of ICG-SCFV probe

[0126] 1. The dye is coupled to the monoclonal antibody. The specific process is as follows:

[0127] SCFV (1 mg, 6.8 nmol) was incubated with ICG (34.2 nmol, 10 mmol / L, DMF) in 0.1 mol / L Na2HPO4 (pH 8.5) at 4°C overnight. The reaction was purified using a gel filtration column (Sephadex G25 column, PD-10, GE Healthcare, Piscataway, NJ). The photophysical properties of the probe were determined using a microplate reader ( Figure 7 ).

[0128] like Figure 7 As shown in Figure 3, the maximum absorbance wavelength of ICG-SCFV in PBS buffer is 820 nm, and the maximum emission wavelength is 790 nm. These results are generally similar to the photophysical properties of ICG.

[0129] 2. ICG-SCFV cell binding affinity and specificity, and cell viability determination

[0130] (1) The fluorescence intensity of ICG-SCFV at different concentrations of 0.01, 0.1, 1, 10, and 100 uM after incubation with MKN45 cells was detected to detect the binding affinity of ICG-SCFV and NOTCH2 on MKN45 cells.

[0131] (2) Unlabeled SCFV at different concentrations of 0, 0.1, 1, 10, and 100 μM was added to MKN45 cells for incubation, followed by incubation with 1 μM ICG-SCFV. The binding specificity of ICG-SCFV to NOTCH2 on MKN45 cells was detected. Figure 8 )

[0132] like Figure 8 As shown in a, in the range of 0 to 10 μM, the near-infrared fluorescence intensity of ICG-SCFV was positively correlated with the probe concentration, and the maximum fluorescence intensity was 29.3 ± 1.7 au at 10 μM.

[0133] In the specificity test, the results showed that the fluorescence intensity was negatively correlated with the concentration of unlabeled SCFV. Compared with 0 μM (100%), the fluorescence intensity of unlabeled SCFV at 0.1, 1, 10 and 100 μM was 73%, 65%, 48% and 26% ( Figure 8b) These results indicate that ICG-SCFV has a strong and specific binding affinity for NOTCH2 on MKN45 cells.

[0134] (3) MKN45 cells were incubated with unlabeled SCFV and ICG-SCFV at concentrations of 0, 6.25, 12.5, 25, 50, and 100 nM for 72 h. Cell viability was then determined using the WST-8 assay to assess the toxicity of ICG-SCFV on MKN45 cells. Nonlinear regression analysis was used to determine the 2IC 50 value( Figure 9 ).

[0135] like Figure 9 As shown, the IC values of ICG-SCFV and unlabeled SCFV on MKN45 cells were 50 The values were 1.13 ± 0.02 and 0.92 ± 0.05, respectively, and the IC values of unlabeled SCFV in MKN45 cells were 50 The value was lower than that of ICG-SCFV (P<0.01). Therefore, the cytotoxicity of ICG-SCFV is comparable to that of unlabeled SCFV.

[0136] Example 3 In vivo and in vitro fluorescence imaging of gastric cancer model mice

[0137] Twelve five-week-old female homozygous nude mice (Charles River, NCI-Frederick, Frederick, MD) were implanted subcutaneously with MKN45 cells on the posterior aspect of the right upper limb. When tumors reached 10 mm in diameter, the mice were used for the following imaging studies. Mice bearing subcutaneous tumor xenografts were intravenously injected with ICG-SCFV (10 mg / kg). Fluorescence images were obtained 0-96 hours after injection using an IVIS Spectrum in vivo imaging system (Perkin Elmer Inc., Waltham, MA). The filters used to observe ICG consisted of a 786 / 30 nm excitation filter and an 822 / 20 nm emission filter. The results are shown in Figure 10 In the figure, (a) shows in vivo near-infrared imaging of the experimental group and the competitive inhibition group. (b) shows the mean fluorescence intensity of the tumor ROI at 1h, 6h, 12h, 24h, 36h, 48h, 72h, 96h, and 120h after injection. (d) shows the near-infrared images of the tumor, heart, liver, lung, spleen, kidney, stomach, small intestine, and head in vitro 36h and 120h after injection of ICG-SCFV (2mg / kg) and unlabeled SCFV (100ug / mouse). Data are mean ± SD (n = 3). (c, e) show the mean fluorescence intensity of the excised tumors and organs in the two groups at 36h and 120h.

[0138] The mice in the experimental group were given ICG-SCFV via the tail vein, and the mice in the competitive inhibition group were given unlabeled SCFV and ICG-SCFV, respectively. Figure 10 As shown, typical imaging modes are as follows Figure 10 As shown in a. One hour after injection, no signal was observed at the tumor site. Fluorescence signal was evident six hours after ICG-SCFV administration, peaked at 36 hours, and was still visible at 120 hours. Fluorescence signal in the competitive inhibition group was weak at 12 hours and significantly weaker than that in the experimental group at 36 hours. After 96 hours, signal at the tumor site was no longer detectable. Figure 10 b shows the changes in tumor fluorescence signal intensity at different time points in the experimental group and the competitive inhibition group. The mean fluorescence intensity (±SD) of the tumor reached a peak at 36 hours after injection (32,430±6766 a.u, 16,365±2755 a.u), then gradually decreased, and maintained at 28,800±2936 a.u and 11,134±1946 a.u, respectively, 72 hours after injection.

[0139] At 36 hours, strong fluorescence signals appeared in the liver, tumor, kidney, stomach, and small intestine of the experimental group. The fluorescence intensity of the tumor was 14538±5018 a.u., while the signals from the liver, kidney, stomach, and small intestine were 26588±6792 a.u., 18342±5436 a.u., 23183±6540 a.u., and 16009±3997 a.u., respectively. The organ signals in the competitive inhibition group were significantly weaker than those in the experimental group (Figure c). At 120 hours, fluorescence signals were still observed in the tumors of the experimental group, while there was almost no fluorescence signal in the competitive inhibition group (Figure e). The above experimental results indicate that ICG-SCFV is metabolized by the liver and can accumulate in the tumor site, thereby prolonging the retention time of ICG in the tumor site.

[0140] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. An anti-human NOTCH2 single-chain antibody, characterized in that Its amino acid sequence is shown in SEQ ID NO.

3.

2. The anti-human NOTCH2 single-chain antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the anti-human NOTCH2 single-chain antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

3. The anti-human NOTCH2 single-chain antibody according to claim 1 or 2, characterized in that The heavy chain CDR and light chain CDR sequences are as follows, wherein the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO.7, the amino acid sequence of CDR2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR3 is shown in SEQ ID NO.9; The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.10, the amino acid sequence of CDR2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

12.

4. A nucleic acid encoding the anti-human NOTCH2 single-chain antibody according to claim 1, characterized in that: Its nucleic acid sequence is shown in SEQ ID NO.

4.

5. A nucleic acid encoding the heavy chain variable region and light chain variable region according to claim 2, characterized in that: The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.5, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO.

6.

6. A carrier, characterized in that It comprises the nucleic acid according to claim 4 or 5.

7. A kit, characterized in that The present invention comprises the anti-human NOTCH2 single-chain antibody according to any one of claims 1 to 3.

8. Use of the anti-human NOTCH2 single-chain antibody according to any one of claims 1 to 3, the nucleic acid according to claim 4 or 5, the vector according to claim 6, or the kit according to claim 7 in the preparation of a medicament, kit and / or device for preventing and / or treating a NOTCH2-positive disease or condition.

9. Use of the anti-human NOTCH2 single-chain antibody according to any one of claims 1 to 3 in the preparation of a developer.

10. The use according to claim 9, characterized in that The anti-human NOTCH2 single-chain antibody is coupled to at least one fluorescent molecule.

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