An anti-human notch2 single chain antibody, encoding gene, kit and use thereof

Abstract

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

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-human NOTCH2 single-chain antibody, its encoding gene, a reagent kit, and its uses. Background Technology

[0002] Cancer has long been a major challenge for humanity, leading to the development of various treatments, including surgical resection, chemotherapy, radiation therapy, and biotherapy. While surgical removal of malignant tumor tissue cannot completely eliminate it, approximately 50% of cancer patients who have had detectable malignant tumor tissue removed do not experience recurrence. Furthermore, surgical resection may extend life expectancy or reduce the incidence of cancer recurrence; therefore, it remains the most commonly used and effective treatment. Due to the importance of complete removal of malignant tumor tissue, diagnostic methods that ensure accurate and complete identification of malignant tumor tissue have immense practical value.

[0003] Gastric cancer is recognized globally as the fifth most common malignant tumor and the fourth leading cause of death. Despite significant advances in diagnostic techniques and treatments, only a small percentage of patients are diagnosed at an early stage. Therefore, the prognosis for advanced gastric cancer remains poor, necessitating further development of molecular diagnostics and treatments for this disease.

[0004] Despite the recognition of the importance of complete tumor removal and the availability of identification techniques for visualizing tumor masses, many malignant tumors still evade detection, leading to disease recurrence and ultimately death. Therefore, achieving more accurate and complete tumor identification is a core problem that urgently needs to be solved in this field. With the discovery and application of fluorescent dyes, an intraoperative navigation technique called "fluorescence-guided surgery" has gradually become a favorite in surgery. This technique uses specific wavelengths of excitation light to stimulate the tumor's own fluorescence, the fluorescence of retained fluorescent molecules, or the fluorescence emitted by exogenous fluorescent substances taken up by cells, thereby guiding surgeons to precisely remove the tumor. However, because fluorescent dyes themselves lack targeting, they accumulate not only in tumor tissue but also in normal tissue, potentially leading to over-removal and damage to surrounding healthy tissue. Therefore, there is a need to develop a targeted fluorescent probe.

[0005] Notch signaling is a tightly controlled and conserved pathway crucial for the normal morphological development of multicellular organisms, regulating interactions between interacting cells in a multicellular environment. Therefore, abnormal NOTCH signaling may lead to disease and cancer. Studies have shown that NOTCH2 is overexpressed in various types of tumors, including gastric cancer, lymphocytic malignancies, and brain tumors, and its enhanced activity plays a vital 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 stem cell-like proliferation of cancer cells, thereby contributing to disease progression. The four notch receptor subtypes play distinct roles in cancer development and progression. NOTCH2, a Notch receptor, is widely overexpressed in a range of cancers and is associated with a unique oncogenic mechanism. Overexpression of NOTCH2 or mutations exhibiting gain-of-function NOTCH2 have been found in a wide range of cancer types, and enhanced NOTCH2 activity plays a crucial role in tumor progression. Overactive NOTCH2 signaling is also associated with dysregulation of certain miRNAs, tumor-associated stromal cell infusion, and regulation of intracellular and external stimuli in tumor cells. Sustained NOTCH2 signaling promotes the stem cell-like ability of tumor cells to avoid apoptotic cell death while simultaneously promoting EMT (emergent stem cell mutation), and elevated NOTCH2 expression is associated with poor clinical prognosis in patients. NOTCH2 further increases the resistance of tumor cells to chemotherapy and radiation, making these cancers less sensitive to treatment. Successful efforts to block NOTCH2 signaling will depend on efficacy and specificity for patients.

[0007] Studies have found that anti-NOTCH monoclonal antibodies (brontictuzumab, Notch 1-specific monoclonal antibody; tarextumab, Notch 2 / 3-specific monoclonal antibody) were discontinued in clinical trials for oncology due to severe gastrointestinal side effects and lack of efficacy. However, while monoclonal antibodies exhibit high target specificity, they have low penetration into solid tumors, and their complex manufacturing processes and high production costs limit their application in cancer treatment. Summary of the Invention

[0008] To address the aforementioned shortcomings in the prior art, this invention provides an anti-human NOTCH2 single-chain antibody, its encoding gene, a kit, and its uses. This nanobody exhibits specific recognition and binding capabilities for NOTCH2 and holds promise as a diagnostic and therapeutic antibody for various cancers.

[0009] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[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 this 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 linked by linker peptides.

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

[0017] A vector comprising the aforementioned nucleic acid.

[0018] Cells containing 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 its use in the preparation of medicines, kits and / or devices for the prevention and / or treatment of NOTCH2 positive diseases or conditions.

[0021] The above-mentioned anti-human NOTCH2 single-chain antibody is used in the preparation of contrast agents.

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

[0023] Furthermore, the fluorescent molecules are near-infrared I region fluorescent molecules.

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

[0025]

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

[0027] Furthermore, the contrast agent is applied to one or more of the following tumors with upregulated NOTCH2 expression: gastric cancer, pancreatic cancer, cholangiocarcinoma, and colorectal cancer.

[0028] The beneficial effects of this invention are:

[0029] The anti-human NOTCH2 single-chain antibody provided by this invention, as well as ICG-SCFV, 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. Attached Figure Description

[0030] Figure 1 Antigen-based mouse immunization and ELISA detection of mouse hNOTCH2 antibody serum titer;

[0031] Figure 2 The image shows an agarose gel electrophoresis image after PCR amplification of the variable region sequence of B lymphocyte antibody; where a. is the amplification of the β-actin sequence using reverse transcribed cDNA, used to detect cDNA quality; b and c. are the amplification of the VH and VL variable region sequences of antibody using nested PCR with cDNA; d. is the amplification of the SCFV sequence.

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

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

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

[0035] Figure 6 Flow cytometry validation of the purified hNOTCH2 antibody;

[0036] Figure 7 The photophysical properties of the probe were detected; (a) is the absorption spectrum of the ICG-SCFV probe in the range of 400-1000 nm; (b) is the fluorescence emission spectrum of the ICG-SCFV probe in the range of 740-840 nm with an excitation wavelength of 790 nm.

[0037] Figure 8The experiment was designed to detect the binding and competition of ICG-SCFV with NOTCH2 in MKN45 cells; (a) was the ICG-SCFV binding assay; and (b) was the binding specificity assay.

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

[0039] Figure 10 Near-infrared imaging of MKN45 xenograft tumor mice in vivo / ex vivo and biodistribution map of ICG-SCFV. Detailed Implementation

[0040] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

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

[0042] 1. Detection of mouse serum titer using mouse immunization and enzyme-linked immunosorbent assay (ELISA)

[0043] (1) Cellular immunity experiments were performed using 6-8 week old Balb / C mice, and 2×10⁻⁶ mice were used. 7 Cells fixed with 4% paraformaldehyde were used to immunize mice for the first time via intraperitoneal injection. Ten days later, 1×10⁶ cells were collected. 7 A second immunization was performed on 10 cells, and 0.5 × 10⁻⁶ cells were collected 20 days later. 7 Each cell undergoes a third immunization.

[0044] (2) Blood was collected from the tail vein of mice 30 days after immunization, and incubated at room temperature for 1 hour, then overnight at 4°C. The supernatant was collected and centrifuged at 15,000 rpm for 15 minutes at 4°C; this process was repeated once. A portion of the serum was diluted 10,000 times with PBS for ELISA detection. The remaining portion could be stored at 4°C for a short time or at -80°C for long-term storage. Figure 1 )

[0045] (3) After immunization, the mice were euthanized 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 70μm cell sieve, add 6mL PBS, grind the spleen with the plunger of a syringe, transfer the cell filtrate to a sterile culture dish, after grinding, rinse the 70μm cell sieve again with 6mL PBS, collect all the filtrate into a 15mL centrifuge tube, centrifuge at 2000rpm for 5min, discard the supernatant; resuspend the cells in 10mL PBS, pass through the 70μm cell sieve again, and calculate the total number of cells.

[0048] (2) Centrifuge the sieved spleen cells at 2000 rpm for 5 min, discard the supernatant, resuspend in 500 μL PBS, transfer to a flow cytometer, separate B cells according to the instructions of the B cell sorting kit, and count the cells.

[0049] (3) Specific B cell sorting: B cells were transferred to flow cytometry tubes and 10 μg of 2.4G2 antibody was added for blocking at room temperature for 10-15 min. 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 experimental group, respectively, and incubated at 4℃ for 30 min. The cells were washed twice with 4 mL PBS (centrifuged at 1500 rpm for 5 min), the supernatant was discarded, and the cells were resuspended in 2 mL PBS and flow cytometry tubes for sorting.

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

[0051] (1) RNA extraction

[0052] This experiment used the RNeasy Mini Kit to extract RNA from flow cytometry-sorted mouse B cells. Mouse B cells were collected into sterile EP tubes, centrifuged at 8000 rpm for 2 min at 4°C, the supernatant was discarded, and the cells were washed with PBS before RNA extraction. Detailed extraction procedures are provided in the RNeasy Mini Kit instruction manual.

[0053] (2) Reverse transcription of RNA

[0054] This experiment uses SMARTScribe TM Reverse transcription is performed using the Reverse Transcriptase kit. For detailed steps, please refer to the SMARTScribe kit. TM Reverse Transcriptase Instructions.

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

[0056] Amplify the VH and VK sequences using the PCR reaction procedure in Table 1 with a 2× hot start Green master mix.

[0057] Table 1 First PCR Amplification Procedure

[0058]

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

[0060] Amplify the VH and VK sequences using the PCR reaction procedure in Table 2 with a 2× hot start Green master mix.

[0061] Table 2 Second PCR Amplification Program

[0062]

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

[0064] Following the PCR reaction procedure in Table 3, the SCFV fragment was amplified using KOD high-fidelity enzyme.

[0065] Table 3 Third PCR Amplification Program

[0066]

[0067] After the third PCR amplification, 10 μL of DNA loading buffer (6×) was added to the amplification product, mixed well, and then all the sample was loaded into the wells of a 1% agarose gel. Electrophoresis was performed at 150V for 30 min, and the gel was photographed and saved. The gel was then cut and the concentration was determined. Figure 2 )

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

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

[0070] (2) Using an electroporator, the ligation product was electroporated to DH5α, cultured and plasmid extracted. For plasmid purification steps, please refer to the "Large-scale Plasmid Extraction Kit Instruction Manual".

[0071] (3) An antibody display library stably expressing hNOTCH2-SCFV mix was constructed in 293T cells using a lentiviral packaging system.

[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) Extract the genome from the sorted positive SCFV 293T cells, amplify the SCFV mix sequence, clone the purified and recovered SCFV fragment into the SY002 expression vector, plate it on an agar plate to obtain positive single clones, pick 100 single clones, and extract plasmids for later use.

[0075] (3) SCFV expression plasmid was transfected into 293T cells. The cell expression supernatant was collected 24 h after transfection and centrifuged at 10000 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 supernatant was stored at 4 °C for later use.

[0076] (4) Flow cytometry analysis: 1×10⁻⁶ samples were taken from each group. 6 Notch2 overexpressing cells were placed in sterile EP tubes, and 100 μL of cell expression supernatant was added. The mixture was then incubated at 4°C for 30 min. After incubation, the cells were centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with Wash Buffer. 1 μL of secondary antibody (anti-mouse IgG APC) was added to the EP tubes, and the cells were incubated at 4°C for 30 min. After incubation, 1 mL of Wash Buffer was added, the mixture was centrifuged, the supernatant was discarded, and the washing was repeated twice. 300 μL of Wash Buffer was added to the EP tubes to resuspend the cells, and all cells were transferred to flow cytometry tubes for analysis. Positive clones were screened and sent to Xi'an Qingke Sequencing Center. Figure 4 , Figure 5 ).

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

[0078] (1) Adjusting cell density before transfection

[0079] The day before transfection, adjust the cell density to 2×10⁻⁶. 6 cells / mL (cells diluted to 2 × 10⁻⁶ with fresh culture medium) 6 The cells (density) were placed in a constant temperature shaker at 37°C, 8% CO2, and 110 rpm for incubation.

[0080] On the day of transfection, adjust the cell density to 3×10⁻⁶. 6 cells / mL (cells diluted to 3 × 10⁻⁶ with fresh culture medium) 6 (density of cells / mL), and the volume of each bottle of cell solution is 40mL;

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

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

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

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

[0085] Slowly add the contents of tube A into tube B, gently blow and aspirate 7-8 times, mix well, and let stand at room temperature for 15 minutes.

[0086] Add the PEI / DNA complex dropwise to the cell culture medium to be transfected, gently shaking the culture flask while adding. After mixing, return the flask to the shaker and continue culturing.

[0087] (3) Add VPA

[0088] 20 h after transfection, add 320 μL of VPA (500 mM) dropwise to a final concentration of 4 mM, while gently shaking the culture flask.

[0089] (4) Add feeding medium

[0090] 1.4 mL of SMS293-SUPI feed solution was added dropwise at 24 h post-transfection, while gently shaking the culture flask during the addition.

[0091] (5) Harvest cell supernatant

[0092] On days 4-5 after transfection (when the viability is between 50% and 70%), the cell expression supernatant was collected 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 twice. After centrifugation, the supernatant was filtered through a 0.22 μm filter membrane and stored at 4°C for purification.

[0093] 7. Affinity chromatography purification of antibodies

[0094] (1) Cell culture supernatant was filtered through a 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 into the collected 293F cell culture supernatant, let it flow through the purification column, collect the filtrate, and repeat this step to pass the collected filtrate through the purification column a second time.

[0099] Wash the purification column with 20 mL of binding buffer and detect the presence of contaminating proteins in the wash buffer with Coomassie Brilliant Blue. Discard the wash buffer.

[0100] The elution buffer was slowly passed through the purification column at a flow rate of 1 mL / min, and the eluent was collected. During elution, the protein concentration in the eluent was monitored using Coomassie Brilliant Blue reagent (100 μL of Coomassie reagent was added to a 96-well plate, and then 5 μL of the eluent was taken for testing) to ensure that there was almost no protein residue in the last fraction before washing. Finally, all the collected eluent was added to 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, add all the collected eluent containing the target protein into the ultrafiltration tube, and centrifuge at 5000xg for 15 min at a fixed angle in a centrifuge at 4°C; discard the liquid at the bottom of the ultrafiltration tube, mix the retained protein solution by pipetting, add 1×PBS to the top and centrifuge again; repeat the previous step once; mix the retained protein solution and collect it into a 1.5 mL centrifuge tube, store at 4°C for later analysis;

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

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

[0105] EVQLQESGPELVKSGASVKMSCKASGYTFTDYIINWVRQRTGQGLEWIG

[0106] EIYPGSGSTYHNEKFKGKATLTADKSSNTAYMQLSSLTSEDSAVYFCARRVYD

[0107] KAYAMDYWGQGTSLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDR

[0108] VTITCRASQNIKRFLAWYQQKPGKAPKLLIYGASTRESGVPSRFSGSGSGTDF

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

[0110] Its 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 testing

[0115] The titer of the purified hNOTCH2 monoclonal antibody in the culture supernatant was detected using an indirect ELISA. The specific steps are as follows:

[0116] (1) The purified SCFV protein was coated onto the microplate at 2 μg / mL, 50 pL / well and incubated overnight at 4℃.

[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 the plate, add serially diluted monoclonal antibodies (1000, 2000, 4000, 8000, 16000, 32000, 64000 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 sheep anti-mouse antibody and incubate at 37°C for 1 hour.

[0120] (5) Add 100 μL of TMB chromogenic 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 450nm using the yeast analyzer. 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 conjugated to the monoclonal antibody, and the specific process is as follows:

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

[0128] like Figure 7 As shown, 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. Assay of ICG-SCFV cell binding affinity and specificity, and cell viability.

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

[0131] (2) MKN45 cells were incubated with different concentrations of unlabeled SCFV (0, 0.1, 1, 10, and 100 μM), followed by incubation with 1 μM ICG-SCFV. The specificity of ICG-SCFV binding to NOTCH2 in MKN45 cells was assessed. Figure 8 )

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

[0133] In specific assays, the results showed a negative correlation between fluorescence intensity and unlabeled SCFV concentration. Compared to 0 μM (100%), the fluorescence intensities of unlabeled SCFV at 0.1, 1, 10, and 100 μM were 73%, 65%, 48%, and 26%, respectively. Figure 8b). These results indicate that ICG-SCFV has a strong specific binding affinity for NOTCH2 in 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 hours. Cell viability was then assessed using the WST-8 assay to evaluate the cytotoxicity of ICG-SCFV to MKN45 cells. Nonlinear regression analysis was used to determine the 2ICP-SCFV concentration. 50 value( Figure 9 ).

[0135] like Figure 9 As shown, ICG-SCFV and unlabeled SCFV have different effects on the ICG levels of MKN45 cells. 50 The values ​​were 1.13±0.02 and 0.92±0.05, respectively, indicating the IC50 of unlabeled SCFV in MKN45 cells. 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 a gastric cancer model mouse

[0137] Twelve five-week-old female homozygous nude mice (Charles River, NCI-Frederick, Frederick, MD) underwent subcutaneous implantation of MKN45 cells into the posterior aspect of their right upper limb. When the tumor diameter reached 10 mm, the mice were used for the following imaging studies. Mice with subcutaneous tumor xenografts were intravenously injected with ICG-SCFV (10 mg / kg). Fluorescence images were obtained 0–96 hours post-injection using an IVIS in vivo spectral imaging system (Perkin Elmer Inc., Waltham, MA). The filters used for ICG observation consisted of a 786 / 30 nm excitation filter and an 822 / 20 nm emission filter. Results are shown below. 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 near-infrared images of tumors, heart, liver, lung, spleen, kidney, stomach, small intestine, and head excised 36h and 120h after injection of ICG-SCFV (2mg / kg) and unlabeled SCFV (100ug / mouse). Data are presented as mean ± standard deviation (n=3). (c, e) show the mean fluorescence intensity of the resected tumors and organs in both groups at 36h and 120h.

[0138] Mice in the experimental group were administered ICG-SCFV via the tail vein, while mice in the competitive inhibition group were administered unlabeled SCFV and ICG-SCFV, respectively. Figure 10 As shown, a typical imaging mode is as follows Figure 10 As shown in Figure a. One hour after injection, no signal was observed at the tumor site. The fluorescence signal was significant 6 hours after ICG-SCFV administration, peaked at 36 hours, and remained visible at 120 hours. The fluorescence signal in the competitive inhibition group was weaker at 12 hours, and significantly weaker than the experimental group at 36 hours. After 96 hours, the signal at the tumor site was undetectable. 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 its peak at 36 hours after injection (32,430±6766 a.u, 16,365±2755 a.u), and then gradually decreased, maintaining at 28,800±2936 a.u and 11,134±1946 a.u, respectively, at 72 hours after injection.

[0139] At 36 hours, strong fluorescence signals were observed 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 almost no fluorescence signal was observed in the competitive inhibition group (Figure e). These experimental results indicate that ICG-SCFV, after being metabolized by the liver, can accumulate at the tumor site, thereby prolonging the retention time of ICG at the tumor site.

[0140] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An anti-human NOTCH2 single-chain antibody, characterized in that, The amino acid sequence of the heavy chain variable region of this 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.

2. The anti-human NOTCH2 single-chain antibody according to claim 1, characterized in that, The amino acid sequence of this anti-human NOTCH2 single-chain antibody is shown in SEQ ID NO.

3.

3. A nucleic acid encoding the anti-human NOTCH2 single-chain antibody of claim 2, characterized in that, Its nucleic acid sequence is shown in SEQ ID NO.

4.

4. A nucleic acid encoding the heavy chain variable region and the light chain variable region as described in claim 1, 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.

5. A carrier, characterized in that, It includes the nucleic acid as described in claim 3 or 4.

6. A reagent kit, characterized in that, Includes the anti-human NOTCH2 single-chain antibody as described in claim 1 or 2.

7. Use of the anti-human NOTCH2 single-chain antibody of claim 1 or 2, the nucleic acid of claim 3 or 4, or the vector of claim 5 in the preparation of a medicament for treating NOTCH2-positive diseases or conditions, wherein the NOTCH2-positive disease or condition is gastric cancer.

8. Use of the anti-human NOTCH2 single-chain antibody according to claim 1 or 2 in the preparation of a contrast agent.

9. The use according to claim 8, characterized in that, The anti-human NOTCH2 single-chain antibody is coupled with at least one fluorescent molecule.

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