Nanobodies targeting human epidermal growth factor receptor-2 and uses thereof
By designing nanobodies targeting HER2, the limited efficacy of existing antibody drugs in the treatment of HER2-positive cancers has been addressed. This approach enables efficient binding and detection of the HER2 protein and has potential for clinical application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HARBIN MEDICAL UNIVERSITY
- Filing Date
- 2025-05-16
- Publication Date
- 2026-05-15
AI Technical Summary
Existing antibody drugs have limited efficacy in treating HER2-positive cancers, especially ovarian and endometrial cancers, and traditional antibodies are difficult to target the HER2 protein efficiently.
A nanobody targeting human epidermal growth factor receptor-2 was developed. Through specific amino acid sequence design and phage display technology, the nanobody capable of efficiently binding to the HER2 protein was prepared and purified for protein detection and treatment.
It achieves specific binding to HER2-positive cells, can be prepared in large quantities in vitro, and can be applied to the detection and treatment of HER2-expressing tumor tissues, which has significant commercial value.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology technology and relates to a nanobody and its application. Background Technology
[0002] Human epidermal growth factor receptor 2 (HER2) was discovered and named in the 1980s. This protein consists of a 1255-amino acid sequence with a molecular weight of approximately 185 kDa. It is a glycosylated transmembrane protein, and its encoding gene is located in a specific region (17q12) on the long arm of human chromosome 17. The extracellular region of HER2 contains ligand-binding domains LD1 and LD2, cysteine-rich structural units CR1 and CR2, and a transmembrane domain composed of hydrophobic amino acids that anchor to the cell membrane. The intracellular region contains a tyrosine kinase domain (TK), which can activate downstream signal transduction through autophosphorylation. The carboxy-terminal tail (CT) contains multiple tyrosine phosphorylation sites that regulate the continuous activation of the signaling pathway.
[0003] HER2 lacks a known direct activating ligand, allowing it to form homodimers or heterodimers without ligand binding. This characteristic makes it a key synergistic gene with other receptors in its family. The HER2-HER3 heterodimer is considered the most active signaling complex, activating the PI3K / AKT signaling pathway and promoting cell proliferation, survival, and metabolism. Studies have shown that HER2 is highly expressed on the surface of various cancer cells, making it a frequent target for cancer therapy. Abnormally high HER2 expression in breast and gastric malignancies has been proven to be a key prognostic indicator and therapeutic target. HER2-targeted therapy has significantly improved the prognosis of breast and gastric cancer patients, but its efficacy in solid tumors such as ovarian and endometrial cancer is limited. Currently, antibody drugs targeting HER2 include monoclonal antibodies and antibody-drug conjugates, primarily including trastuzumab, tyrosine kinase inhibitors (TKIs), and pertuzumab.
[0004] Nanobodies are a class of single-domain antibodies with unique structural advantages. Their core consists of a compact ellipsoidal structure formed by nine β-sheets, with a molecular weight of only 15 kDa (1 / 10 that of conventional antibodies). Stability is maintained through conserved disulfide and hydrogen bonds. Key features include an ultra-long CDR3 ring and a framework region enriched with hydrophilic amino acids. The former enhances affinity through extensive antigen contact, enabling them to bind to occult epitopes that are difficult for conventional antibodies to target, while the latter reduces aggregation tendency. Due to their small molecular weight, ease of engineering, and superior tissue penetration, nanobodies have been widely used in cancer imaging and therapy. They have become a powerful tool for developing efficient, rapid, and accurate in vitro diagnostic kits, bringing innovative opportunities to medical testing and disease diagnosis. Summary of the Invention
[0005] Taking advantage of the unique advantages of nanobodies in the prior art, this invention provides a nanobody that targets human epidermal growth factor receptor-2 and its application.
[0006] One of the objectives of this invention is to provide a nanobody that targets human epidermal growth factor receptor-2, wherein the amino acid sequence of the nanobody that targets human epidermal growth factor receptor-2 is shown in SEQ ID NO.1.
[0007] In a preferred embodiment of the present invention, the nanobody targeting human epidermal growth factor receptor-2 includes complementarity-determining regions CDR1, CDR2 and CDR3.
[0008] In a preferred embodiment of the present invention, the amino acid sequence of CDR1 is shown in SEQ ID NO.2, the amino acid sequence of CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of CDR3 is shown in SEQ ID NO.4.
[0009] In a preferred embodiment of the present invention, the nanobody targeting human epidermal growth factor receptor-2 further includes framework regions FR1, FR2, FR3 and FR4.
[0010] In a preferred embodiment of the present invention, the amino acid sequence of FR1 is shown in SEQ ID NO.5, the amino acid sequence of FR2 is shown in SEQ ID NO.6, the amino acid sequence of FR3 is shown in SEQ ID NO.7, and the amino acid sequence of FR4 is shown in SEQ ID NO.8.
[0011] A second objective of this invention is to provide a nucleic acid comprising a nucleic acid sequence or its complementary sequence encoding the aforementioned nanobody targeting human epidermal growth factor receptor-2.
[0012] A third objective of this invention is to provide an expression vector containing the aforementioned nucleic acid.
[0013] A fourth objective of this invention is to provide a host cell containing the aforementioned expression vector.
[0014] The fifth objective of this invention is to provide the application of the above-mentioned nanobody targeting human epidermal growth factor receptor-2 in the preparation of protein detection antibodies or therapeutic antibodies.
[0015] In a preferred embodiment of the present invention, the application refers to the detection of tumor cell lines or tumor tissues expressing human epidermal growth factor receptor-2.
[0016] The beneficial effects of this invention are as follows: This invention provides a nanobody targeting human epidermal growth factor receptor-2 (HER2). Performance data demonstrates that the HER2-targeting nanobody provided by this invention binds to HER2-positive 293T-vector cells but not to HER2-non-HER2-expressing 293T-HER2-KO cells. Simultaneously, this nanobody specifically binds to naturally HER2-expressing NCI-N87 gastric cancer cells but not to the HER2-non-HER2-expressing MDA-MB-468 cell line; this confirms the targeting binding ability of the HER2-targeting nanobody in natural cell models. The HER2-targeting nanobody provided by this invention can bind to the HER2 protein expressed on the surface of naturally occurring cells. It can be prepared through large-scale expression using engineered bacteria in vitro and can be used to prepare protein detection antibodies or therapeutic antibodies. It can be used for in vitro and in vivo detection of HER2-expressing tumor tissues, and has significant commercial value in clinical disease diagnosis and treatment. Attached Figure Description
[0017] Figure 1 This is a diagram showing the results of HER2 extracellular domain (HER2-ECD) cloning and eukaryotic expression vector construction in Example 1;
[0018] M1: DL2000 DNA molecular weight standard; Lane 1 shows the HER2-ECD PCR amplification result; Lane 2 shows the amplification result of the pCAGGS-TPA-HER2-his vector; Lanes 3-4 show the PCR identification result of the recombinant pCAGGS-TPA-HER2-ECD-his plasmid; M2: DL15000 DNA molecular weight standard;
[0019] Figure 2 This is a diagram showing the secretory expression and identification results of the HER2-ECD recombinant protein in Example 1;
[0020] Figure 3 Ni of the HER2-ECD recombinant protein in Example 1 2+ Affinity purification results (image);
[0021] Figure 4 This is a graph showing the antibody titer determination of alpaca immune serum in Example 2;
[0022] Figure 5 This is a graph showing the capacity detection of the cDNA library and phage library in Example 3;
[0023] Figure 6 This is a diagram showing the construction results of the HER2 knockout cell line in Example 4; M represents the 200 kDa protein molecular weight standard.
[0024] Figure 7 The following are flow cytometry results of the HER2 knockout cell lines in Example 4; where red represents the APC-anti-HER2 antibody group and blue represents the Blank group; A shows the binding results of APC-anti-HER2 antibody with the 293T cell line; B shows the binding results of APC-anti-HER2 antibody with the 293T-vector cell line; and C shows the binding results of APC-anti-HER2 antibody with the 293T-HER2-KO cell line.
[0025] Figure 8 This is a graph showing the recovery rate of the phage panning library in Example 4;
[0026] Figure 9 This is a graph showing the indirect ELISA identification results of the panning phages in Example 5;
[0027] Figure 10 Ni is the prokaryotic expression protein of the nanobody in Example 5. 2+ Affinity purification results (image);
[0028] Figure 11 The following are flow cytometry results of the binding of HER2 nanobody to naturally expressed or non-HER2 expressed cell lines in Example 6. In the figure, red represents the VHH group, blue represents the VHH-isotype control group, and yellow represents the Blank group. Figure A shows the binding results of VHH with the MDA-MB-468 cell line. Figure B shows the binding results of VHH with the NCI-N87 cell line. Detailed Implementation
[0029] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.
[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.
[0031] In the following examples, human epidermal growth factor receptor-2 will be referred to as HER2.
[0032] The Western blot used in this embodiment includes the following steps:
[0033] S1: Collect protein samples for SDS-PAGE polyacrylamide gel electrophoresis, and perform constant voltage electrophoresis at 120V.
[0034] S2: After electrophoresis in S1, the protein sample in the gel was transferred to a nitrocellulose membrane and transferred at a constant current of 230mA for 2 hours in an ice box.
[0035] S3: After the transfer is completed, place the membrane from S2 into the blocking solution (5% skim milk powder) and seal it at room temperature for 2 hours;
[0036] S4: Wash the membrane after blocking in S3 three times with 1×TBST solution, 10 min each time, then add a...
[0037] Anti-solvent, incubate at room temperature for 2 hours;
[0038] S5: Wash the membrane from S4 after primary antibody incubation three times with 1×TBST solution, 10 min each time, then add...
[0039] Add the secondary antibody solution and incubate at room temperature for 1 hour;
[0040] S6: The membrane after incubation with the secondary antibody in S5 was washed three times with 1×TBST solution for 10 min each time. Then, a developing solution was prepared for development, followed by development, photographing, and storage in a gel imaging system.
[0041] Example 1: Preparation of HER2-ECD recombinant immunogen
[0042] (1) The extracellular segment of the HER2 target gene (HER2-ECD) was amplified using pM18T-HER2 plasmid as a template;
[0043] (2) The vector pCAGGS-TPA-6His and the expression vector pCAGGS-TPA-HER2-ECD-his obtained in (1) were amplified by overlap extension PCR. The expression vectors obtained above were verified by PCR, and the results are as follows. Figure 1 As shown, the plasmids that tested positive were sequenced and analyzed. The sequencing results were consistent with the template sequence, indicating that the recombinant expression vector was successfully constructed. Subsequently, a large number of plasmids were extracted and stored for later use.
[0044] (3) The recombinant expression vector obtained in (2) was used to transfect 293T cells. After 5 days, the supernatant was collected for Western spectroscopy. The results are as follows: Figure 2 As shown, the target protein was expressed at 110 KD; subsequently, the recombinant protein was expressed by transfection into the HEK293 mammalian suspension expression system. The protein supernatant was bound to a Ni-NTA affinity chromatography column, and the bound protein was eluted and purified using glycine buffers with different pH gradients. After the protein concentration was determined, the HER2-ECD recombinant protein was obtained and stored at -80℃ for later use.
[0045] This embodiment describes the Ni process for the HER2-ECD recombinant protein obtained above. 2+ Affinity purification identification, results as follows Figure 3 As shown, the recombinant target protein was obtained at 110 kDa by pH elution, indicating that the HER2-ECD recombinant protein was successfully purified.
[0046] Example 2: Animal Immunization Procedure
[0047] (1) Mix 0.5 mg of the HER2-ECD recombinant protein obtained in Example 1 with 0.5 mL of Freund's adjuvant and emulsify to obtain a HER2-ECD recombinant protein mixture, and store at 4°C for later use;
[0048] (2) Select alpacas and record their ear numbers. Inject 0.4 mL of the HER2-ECD recombinant protein mixture obtained in (1) into the subcutaneous tissue of the alpaca's buttocks on both sides, with two injection points on each side. Observe for 30 minutes after the immunization injection to confirm that the alpaca is in good condition and has no discomfort symptoms. Immunize once every 2 weeks for a total of 4 immunizations. Before each immunization injection, collect blood from the alpaca's jugular vein, taking 10 mL of blood each time. Separate the serum and store it at -80℃ for later use. On the 5th day after the last immunization injection, draw 100 mL of peripheral blood from the alpaca's neck to obtain the immunized alpaca peripheral anticoagulated blood sample, i.e., the immune blood sample.
[0049] (3) Add 3 mL of cell separation solution to a 15 mL centrifuge tube, then slowly add 3 mL of the serially diluted immune blood sample obtained in (2). Pre-cool the centrifuge and centrifuge at 400 g for 30 min. Observe the blood separation in the centrifuge tube. Use a 200 μl pipette to aspirate the middle cotton-like upper layer of immune cells into a new 15 mL centrifuge tube and save the upper plasma into a new centrifuge tube. Store at -80℃. Add 10 mL of room temperature PBS buffer to the above centrifuge tube and centrifuge at 400 g for 20 min. Discard the supernatant. Then add 5 mL of room temperature PBS buffer and centrifuge at 400 g for 20 min. Count the number of cells using a hemocytometer, centrifuge and discard the supernatant. Use Trizol (Sigma) to dissolve and separate lymphocytes according to the number of cells and the instructions. Store at -80℃.
[0050] This embodiment detects antibody titers in both non-immunized and immunized sera, and the results are as follows: Figure 4 As shown, the antibody titer of the immune serum was higher than that of the non-immune serum at a serum dilution concentration of 1:64000, indicating that the alpaca immunization was successful.
[0051] Example 3: Preparation of a HER2-targeting nanobody library
[0052] Total RNA was extracted from the lymphocytes obtained in Example 2 using an RNA extraction kit (Invitrogen), and a cDNA library was generated using a reverse transcription kit (Invitrogen). Nested PCR was performed using alpaca heavy chain antibody and primers specific to the variable region of the heavy chain antibody to amplify the VHH gene fragment. The primer sequences were the upstream primer FR1-RSCF as shown in SEQ ID NO.9 and the downstream primer VHH-RSCB as shown in SEQ ID NO.10. The VHH gene fragment was ligated to the phage expression vector pCombo by enzyme digestion to construct a recombinant phage vector. The recombinant phage vector was then transformed into E. coli TG1 competent cells by electroporation for amplification. With the help of helper phages, VHH was displayed on the surface of the phages, forming a VHH phage library, which is a nanobody library targeting HER2.
[0053] This embodiment tests the capacity of the obtained cDNA library and phage library as follows: Figure 5 As shown, the cDNA library size is 9.17 × 10⁻⁶. 7 pfu / mL, phage library capacity 7.1×10 13 cfu / mL.
[0054] Example 4: Screening of HER2-targeting nanobodies
[0055] 1. Construction of HER2 knockout cell lines
[0056] The sgRNA targeting the HER2 gene was designed using the Benchling website. Two amplification primers were designed, with the primer sequences shown in SEQ ID NO.11 and SEQ ID NO.12. Each pair of oligonucleotides was phosphorylated and annealed, and then ligated into the lentiCRISPRv2 vector digested with BsmBI enzyme.
[0057] The recombinant plasmid was co-transfected with the packaging plasmids Gag-pol and VSV-G into 293T cells for viral packaging. The packaged viral solution was then used to infect 293T cells to construct the HER2 knockout cell line 293T-HER2-KO. Positive cell lines obtained through puromycin screening were then used for further processing.
[0058] In this embodiment, the HER2 knockout cell line 293T-HER2-KO obtained above was subjected to Western spectroscopy. The primary antibody used in the Western spectroscopy was a HER2 monoclonal antibody (Proteintech), and the secondary antibody was an HRP-labeled goat anti-mouse secondary antibody (Thermo). The results are as follows: Figure 6 As shown, no target band was detected in the knockout group at approximately 110KD compared to the control group, indicating that the HER2 knockout cell line 293T-HER2-KO was successfully constructed.
[0059] This embodiment further validated the HER2 knockout cell line 293T-HER2-KO using flow cytometry, and the results are as follows: Figure 7 As shown, compared with the 293T control group and the 293T-vector group, the 293T-HER2-KO cell line did not deviate, proving that the cell line was successfully constructed and can be used for subsequent screening.
[0060] 2. Selection of nanobodies based on phage display technology
[0061] (1) Negative washing
[0062] S1: Prepare negative washed cells of the 293T-HER2-KO cell line, count the cells, and use 3.5 × 10⁻⁶ cells each time. 8 One cell;
[0063] S2: Wash the cells in S1 twice with PBS, resuspending them with 30 mL of PBS each time. Centrifuge at 4℃ and 500g for 5 min, discard the supernatant, and resuspend them with 2 mL of PBS for the last time before placing them in cryovials.
[0064] S3: Centrifuge the resuspended cells obtained in S2 at 4℃ and 500g for 5 min, discard the supernatant, then add 500μL of phage-milk / PBS to resuspend, and incubate at room temperature for 30 min; then centrifuge at 13000rpm for 2 min, and collect the phage supernatant, i.e., phage elution buffer.
[0065] (2) Positive washing
[0066] S1: Prepare positively washed 293T-vector cell lines, count the cells, and use 5 × 10⁶ cells per wash. 8 One cell;
[0067] S2: Wash the cells in S1 twice with PBS, resuspending them with 30 mL of PBS each time. Centrifuge at 4℃ and 500g for 5 min, discard the supernatant, and resuspend them with 2 mL of PBS for the last time before placing them in cryovials.
[0068] S3: The resuspended cells obtained in S2 were centrifuged at 4°C and 500g for 5 min, the supernatant was discarded, and then 500 μL of the phage elution buffer collected by negative washing obtained in (1) was added, the cells were resuspended, and incubated at room temperature for 30 min; the cells were washed five times with PBS, centrifuged at 500g at room temperature for 2 min, and the PBS was removed; 150 μL of Elution Buffer (glycine solution, pH 2.2) was added, and the reaction was carried out at room temperature for 10 min; 10 μL of 2M Tris Base solution (glycine solution, pH 9.0) was added to neutralize; then the cells were centrifuged at 13000 rpm for 2 min, and the phage elution buffer was collected;
[0069] S4: Take 10 μL of the phage elution buffer obtained in S3 and dilute it 5 times (103). 1-5 (Dilution 1.5 times), add 90 μL OD to each EP tube. 600 The TG1 bacterial suspension was prepared at 0.5-0.6 and incubated at 37°C for 15 minutes. Then, the bacterial suspensions of five different gradients were spread onto 2×YT culture plates containing ampicillin resistance and incubated upside down overnight at 37°C. The number of single colonies on the culture plates was counted to calculate the titer.
[0070] S5: Take 10 mL of the remaining phage elution buffer from S3 and add it to the OD200. 600Add 0.5-0.6 μL of TG1 bacterial culture and let stand at room temperature for 30 min; add 0.1% Amp resistance and continue culturing at 37℃ and 250 rpm for 30 min; then add 100 μL of helper phage, let stand at room temperature for 30 min, centrifuge at 8000 rpm for 10 min, transfer the precipitate after centrifugation to 100 mL of 2×YT culture plate containing 0.1% ampicillin and kanamycin resistance, and incubate overnight at 37℃ and 220 rpm. The next day, concentrate the phage for later use.
[0071] (3) The VHH phage library obtained in Example 3 was incubated with the 293T-HER2-KO / 293T-vector cell line obtained in Example 4. After three rounds of negative and positive washing, VHH phages that specifically bind to HER2 membrane antigen and are highly enriched were obtained, i.e., positive phage screening library.
[0072] This embodiment detects the phage recovery rate of the obtained positive phage screening library, and the results are as follows: Figure 8 As shown, the amount of phage recovered increased progressively with each round, ultimately reaching a phage recovery rate of 2.3 × 10⁻⁶. -3 P / input.
[0073] Example 5: Identification of HER2-targeting nanobodies
[0074] 1. Indirect ELISA identification
[0075] Take the phage eluent obtained from the third round of negative and positive washes in Example 4, spread it on a plate, randomly select 96 bacterial clones, and culture them statically overnight at 37°C. Take 10 μL of each of the above bacterial solutions and transfer them to 2xYT medium in a 96-well plate (1 mL / well). Culture until the logarithmic phase. Add 50% sterile glycerol to the remaining bacterial solution, shake well, and store at -80°C. Add IPTG to a final concentration of 0.2 mM and induce expression for 12 h at 37°C and 220 rpm. Centrifuge at 4000 rpm and 4°C for 15 min, then place the bacterial cells at -20°C and freeze for 30 min. After returning to room temperature, resuspend the bacterial cells in PBS (1 mL / well) and shake at 300 rpm and 4°C for 30 min. Centrifuge at 4000 rpm and 4°C for 10 min to obtain the supernatant, which is the crude nanobody extract.
[0076] The HER2-ECD recombinant protein obtained in Example 1 was diluted with coating buffer and added to wells at 400 ng / well and 100 μL / well, respectively, and incubated overnight at 4°C. On the second day, the plates were washed three times with PBST for 2 min each time. 300 μL of 5% skim milk was added to each well to block the ELISA plate for 2 h. The plate was then washed three times with PBST for 2 min each time. 100 μL of the crude nanobody extract obtained above was added to each well as the primary antibody and incubated at 37°C for 2 h. The plate was washed three times with PBST for 2 min each time. 100 μL of Anti-HA-HRP antibody was added to each well as the secondary antibody and incubated at 37°C for 1 h. The plate was washed three times with PBST for 2 min each time. 100 μL of TMB chromogenic solution was added to each well and the reaction was carried out at 37°C. 50 μL of 2M sulfuric acid was added to terminate the reaction, and the OD was read. 450 nm value.
[0077] In Example 5, the crude extract of the induced nanobody was detected by indirect ELISA, and the identification results are as follows: Figure 9 As shown, OD 450 >1.0 was identified as a positive colony, and a total of fourteen colonies were sent for sequencing.
[0078] 2. Prokaryotic expression and purification of nanobodies
[0079] One strain with the positive sequence identified in Part 2 of this embodiment was cloned, and the prokaryotic expression vector PET22b-HER2-VHH-6his was constructed using overlap extension technology. The positive plasmid was transformed into Rosetta (DE3) competent cells for induced expression. The cells were picked and placed in 6 mL of LB liquid medium containing ampicillin, and cultured at 37°C and 220 rpm / min for 12-15 h to obtain a bacterial culture. The above 2 mL of bacterial culture was activated and placed in LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37°C and 220 rpm / min for 3-4 h. When the OD value of the bacterial culture reached 0.4, the above bacterial culture was induced to express for 12 h at 16°C using 0.2 mM IPTG. After centrifugation, the supernatant was discarded to obtain the bacterial cell precipitate, which is a nanobody solution containing HER2 targeting.
[0080] The nanobody obtained in Part 2 of this example was purified using an AKATA protein purification system via Ni Sepharose excel affinity chromatography column, followed by SDS-PAGE purification and identification. The results are as follows: Figure 10 As shown, the target band appears at around 15kD, indicating that this embodiment successfully obtained the nanobody protein targeting HER2.
[0081] Example 6: Application of HER2-targeting nanobodies
[0082] This embodiment uses the cell line NCI-N87, which naturally expresses HER2 membrane protein, and the cell line MDA-MB-468, which does not express HER2 membrane protein, as research subjects. The cultured cell lines were counted, and a sample of 1×10⁻⁶ cells was collected. 6 Cells were washed with 500 μL of PBS solution, centrifuged at 500 g for 5 min, and the supernatant was discarded. Then, 1 μg of the purified HER2-targeting nanobody from Example 5 was added as the primary antibody and incubated at 4 °C for 45 min. Then, 500 μL of PBS solution was added again, centrifuged at 500 g for 5 min, and the supernatant was discarded. The washing was repeated twice. Then, APC-anti-His flow cytometry antibody (Biolegend) was added as the secondary antibody and incubated at 4 °C for 45 min. Then, 500 μL of PBS solution was added again, centrifuged at 500 g for 5 min, and the supernatant was discarded. The cells were washed twice. The cells were resuspended in 1 mL of PBS solution and finally filtered through a 200-mesh filter cloth.
[0083] The filtered cells were identified using flow cytometry, and the results are as follows: Figure 11 As shown, when bound to the HER2 naturally expressing cell line NCI-N87, 99.8% of positive cells were deflected. When bound to the cell line MDA-MB-468, which does not express HER2 membrane protein, it did not bind. This indicates that the HER2-targeting nanobody provided by the present invention can specifically bind to the HER2 protein on the cell membrane surface. Therefore, it can be used as a subsequent detection and therapeutic antibody.
[0084] The contents not described in detail in this specification are well-known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A nanobody targeting human epidermal growth factor receptor-2, characterized in that, The amino acid sequence of the nanobody targeting human epidermal growth factor receptor-2 is shown in SEQ ID NO.1; The nanobody targeting human epidermal growth factor receptor-2 includes complementarity-determining regions CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO.2, the amino acid sequence of CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of CDR3 is shown in SEQ ID NO.4; The nanobody targeting human epidermal growth factor receptor-2 also includes the framework regions FR1, FR2, FR3 and FR4; The amino acid sequence of FR1 is shown in SEQ ID NO.5, the amino acid sequence of FR2 is shown in SEQ ID NO.6, the amino acid sequence of FR3 is shown in SEQ ID NO.7, and the amino acid sequence of FR4 is shown in SEQ ID NO.
8.
2. A nucleic acid, characterized in that, The nucleic acid is the nucleic acid sequence encoding the nanobody targeting human epidermal growth factor receptor-2 as described in claim 1.
3. An expression carrier, characterized in that, The expression vector contains the nucleic acid as described in claim 2.
4. A host cell, characterized in that, The host cell contains the expression vector as described in claim 3.
5. The application of the nanobody targeting human epidermal growth factor receptor-2 as described in claim 1 in the preparation of HER2 protein detection antibody reagent.
6. The application according to claim 5, characterized in that, The application refers to the detection of tumor cell lines or tumor tissues expressing human epidermal growth factor receptor-2.