Nanobodies targeting egfr / egfrviii and uses thereof

Abstract

The application discloses a nano antibody targeting EGFR / EGFRvIII and an application thereof, and belongs to the technical field of molecular biology. In view of the unique advantages of the nano antibody in the prior art, the application provides the nano antibody targeting EGFR / EGFRvIII, wherein the nano antibody targeting EGFR / EGFRvIII comprises CDR1 as shown in SEQ ID NO. 1, CDR2 as shown in SEQ ID NO. 2 and CDR3 as shown in SEQ ID NO. 3. The detection result shows that the nano antibody targeting EGFR / EGFRvIII provided by the application can be specifically combined with EGFR / EGFRvIII expressed on the cell membrane surface, and can be applied to preparation of a protein detection antibody or a therapeutic antibody, and has important commercial value in clinical disease diagnosis and treatment.

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] Epidermal growth factor receptor (EGFR) is a 170 kDa transmembrane glycoprotein and cell membrane receptor composed of an extracellular receptor domain (N-terminus), a transmembrane region (hydrophobic α-helix structure), and an intracellular receptor domain (C-terminus). It is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, and other cells. The EGFR signaling pathway plays a crucial role in physiological processes such as cell growth, proliferation, and differentiation. The extracellular domain of EGFR contains 618 amino acids and consists of four subdomains. A unique feature of the EGFR family is the tandem combination of two domains: domains I and III are compact, with β-helix folds, while domains II and IV are elongated and contain cysteine ​​residues.

[0003] EGFR was the first discovered tyrosine kinase receptor (RTK) and the first RTK associated with cancer, exhibiting high EGFR activity in many tumors, such as glioblastoma, non-small cell lung cancer, colorectal cancer, and head and neck cancer. Compared to small molecule TKIs (the tyrosine kinase binding region of EGFR) biologics, EGFR biologics currently have less development momentum. However, existing biologics targeting intracellular TKIs suffer from severe drug resistance and mutation issues. Extracellular antibody proteins can block ligand receptor binding and dimer formation, thereby inhibiting tyrosine receptor activation and blocking downstream signaling pathways. Multispecific antibodies targeting multiple targets can avoid tumor drug resistance mechanisms by acting on multiple signaling pathways. Currently, marketed biologics targeting EGFR include monoclonal antibodies, bispecific antibodies, and ADCs. Cetuximab sarotalocan is currently the only phototherapy drug globally, but only cetuximab and nimotuzumab are available in China. Furthermore, none of the EGFR biologics under development in China are in Phase III clinical trials. The indications for EGFR biologics cover solid tumors in multiple sites, including head and neck cancer, nasopharyngeal carcinoma, and colorectal cancer.

[0004] EGFRvIII is a mutant of the epidermal growth factor receptor (EGFR) expressed on the surface of tumor cells. Studies have found that 40%–60% of glomerular basement membranes significantly express EGFR, with EGFRvIII being the predominant mutant form. Compared to the complete EGFR structure, EGFRvIII has exons 2–7 deleted, resulting in a loss of 801 base pairs. The deleted ends are linked by a new glycine codon (GGT). After translation into protein, amino acids 6–273 of the extracellular region of EGFRvIII are replaced by a single glycine residue. EGFRvIII, lacking its extracellular ligand-binding site, can constitutively activate tyrosine kinases without ligand binding, leading to autophosphorylation, inducing downstream signal transduction pathways, triggering a cascade reaction, and affecting the biological behavior of tumor cells. Currently, there are no drugs specifically targeting EGFRvIII on the market; drugs under development are all in Phase I or II clinical trials, mainly CAR-T, monoclonal antibodies, and bispecific single-chain antibodies. In conclusion, EGFR / EGFRvIII has great potential as a therapeutic target for solid tumors and their metastases.

[0005] In 1989, researchers discovered a heavy-chain antibody derived from camels, naturally lacking the light chain. Its variable region had a molecular weight of only 15 kDa, one-tenth the molecular weight of a conventional antibody. Its protein crystal structure was 4 nm long and 2.5 nm in diameter, making it the smallest known antibody, termed a nanobody. Nanobodies can more easily cross cell membranes and tissue barriers, penetrating deep tissues and specific cells. Compared to traditional antibodies, nanobodies have longer active binding regions, containing 16-18 amino acids. They possess high affinity and specificity, binding to specific epitopes of target molecules for highly precise recognition and intervention, making them an important tool in research and therapy. Nanobodies also exhibit good stability, maintaining their structure and function under a wide range of environmental conditions. They demonstrate high thermal and chemical stability, making them suitable for various applications and less susceptible to damage during storage and transportation. In addition, unlike the traditional hybridoma cell preparation method for antibodies, the method for preparing nanobodies is the engineered bacteria expression method, which has the advantages of easy expression and easy genetic engineering modification, as well as relatively low production cost and easy large-scale production. Summary of the Invention

[0006] Taking advantage of the unique advantages of nanobodies in the prior art, this invention provides a nanobody targeting EGFR / EGFRvIII and its application.

[0007] One of the objectives of this invention is to provide a nanobody that targets EGFR / EGFRvIII, wherein the amino acid sequence of the nanobody that targets EGFR / EGFRvIII is shown in SEQ ID NO.8.

[0008] In a preferred embodiment of the present invention, the nanobody targeting EGFR / EGFRvIII includes complementarity-determining regions CDR1, CDR2 and CDR3.

[0009] In a preferred embodiment of the present invention, the amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.3.

[0010] In a preferred embodiment of the present invention, the nanobody targeting EGFR / EGFRvIII further includes framework regions FR1, FR2, FR3 and FR4.

[0011] In a preferred embodiment of the present invention, the amino acid sequence of FR1 is shown in SEQ ID NO.4, the amino acid sequence of FR2 is shown in SEQ ID NO.5, the amino acid sequence of FR3 is shown in SEQ ID NO.6, and the amino acid sequence of FR4 is shown in SEQ ID NO.7.

[0012] A second objective of this invention is to provide a nucleic acid comprising a nucleic acid sequence encoding the aforementioned nanobody targeting EGFR / EGFRvIII or its complementary sequence.

[0013] A third objective of this invention is to provide an expression vector containing the aforementioned nucleic acid.

[0014] A fourth objective of this invention is to provide a host cell containing the aforementioned expression vector.

[0015] The fifth objective of this invention is to provide the application of the above-mentioned EGFR / EGFRvIII-targeting nanobodies in the preparation of protein detection antibodies or therapeutic antibodies.

[0016] In a preferred embodiment of the present invention, the application refers to detecting tumor tissue expressing EGFR / EGFRvIII.

[0017] The beneficial effects of this invention are:

[0018] This invention provides a nanobody targeting EGFR / EGFRvIII. Flow cytometry results show that the nanobody targeting EGFR / EGFRvIII provided by this invention has a strong binding ability with U87 cells expressing EGFR / EGFRvIII. Biacore assay results show that the binding KD of the nanobody targeting EGFR / EGFRvIII provided by this invention with recombinant EGFR protein is 1.68 nM, and the binding KD with recombinant EGFRvIII protein is 1.53 nM.

[0019] The EGFR / EGFRvIII-targeting nanobodies provided by this invention can specifically bind to EGFR / EGFRvIII expressed on the cell membrane surface. Moreover, the above-mentioned nanobodies can be prepared by large-scale expression in vitro using engineered bacteria. They can be used to prepare protein detection antibodies or therapeutic antibodies, and have important commercial value in clinical disease diagnosis and treatment. Attached Figure Description

[0020] Figure 1 This is a diagram showing the construction results of the eukaryotic expression vector pCDNA3.4-SP-EGFR / EGFRvIII-6his in Example 1; M is the DL2000 DNA maker.

[0021] Figure 2 This is a graph showing the expression and identification of the EGFR / EGFRvIII-EC recombinant protein in Example 1; M represents the protein molecular weight standard.

[0022] Figure 3 The recombinant EGFR / EGFRvIII-EC protein Ni in Example 1 2+ Affinity purification identification diagram; A is the identification diagram of EGFR-EC recombinant protein; B is the identification diagram of EGFRvIII-EC recombinant protein; M is the protein molecular weight standard;

[0023] Figure 4 Figure A shows the serum antibody titer determination in Example 2; Figure B shows the EGFR serum antibody titer determination; Figure C shows the EGFRvIII serum antibody titer determination.

[0024] Figure 5 This is a graph showing the capacity detection of the cDNA library and phage library in Example 3;

[0025] Figure 6 This is a diagram showing the construction results of the EGFR / EGFRvIII membrane protein 293T overexpression cell line in Example 4; M represents the protein molecular weight standard.

[0026] Figure 7Figure A shows the recovery rate of the phage panning library in Example 4; Figure B shows the recovery rate of the EGFR phage panning library; Figure C shows the recovery rate of the EGFRvIII phage panning library.

[0027] Figure 8 Figure A shows the results of indirect ELISA identification in Example 5; Figure B shows the results of indirect ELISA identification of EGFR; Figure C shows the results of indirect ELISA identification of EGFR vIII.

[0028] Figure 9 Ni is the prokaryotic expression protein of the nanobody in Example 5. 2+ Affinity purification and identification diagram; M represents the protein molecular weight standard;

[0029] Figure 10 The nano-antibody Ni in Example 5 2+ Affinity-purified protein identification diagram; M represents the protein molecular weight standard;

[0030] Figure 11 Figure 1 shows the affinity test results for the binding of the nanobody to the EGFR / EGFRvIII-EC recombinant protein in Example 5; Figure 2 shows the affinity test result for EGFR; Figure 3 shows the affinity test result for EGFRvIII.

[0031] Figure 12 The images show the identification of the EGFR / EGFRvIII nanobody used as a flow cytometry detection antibody in Example 6; A is the identification image of U87 negative cells; B is the identification image of U87-EGFR cells; C is the identification image of U87-EGFRvIII cells; blue represents the control group, and red represents VHH-his. Detailed Implementation

[0032] Those skilled in the art can refer to the content of this document to appropriately improve the process parameters. 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.

[0033] 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 and accompanying drawings. 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.

[0034] EGFR: Epidermal Growth Factor Receptor, EGFRvIII;

[0035] The Western broach used in this embodiment includes the following steps:

[0036] S1: Collect protein samples for SDS-PAGE polyacrylamide gel electrophoresis, and perform constant voltage electrophoresis at 120V.

[0037] S2: After electrophoresis in S1, the protein sample in the gel was transferred to a nitrocellulose membrane and transferred under constant current at 230mA for 2 hours in an ice box.

[0038] S3: After the S2 transfer membrane is completed, place the membrane in the sealing solution (5% skim milk powder) and seal it at room temperature for 2 hours;

[0039] S4: Wash the membrane after blocking in S3 three times with 1×TBST solution for 5 min each time, then add primary antibody.

[0040] Incubate the solution at room temperature for 2 hours;

[0041] S5: Wash the membrane after primary antibody incubation in S4 three times with 1×TBST solution for 5 min each time, then add...

[0042] Secondary antibody solution, incubate at room temperature for 1 hour;

[0043] S6: The membrane after incubation with the secondary antibody in S5 was washed three times with 1×TBST solution for 5 minutes each time. Then, a developing solution was prepared for development, followed by development, photography, and storage in a gel imaging system.

[0044] Example 1: Preparation of EGFR / EGFRvIII-EC recombinant protein

[0045] (1) The extracellular segment of the target gene EGFR / EGFRvIII was amplified using pMD18-T-EGFR / EGFRvIII plasmid as a template to obtain EGFR / EGFRvIII-EC;

[0046] (2) The target gene obtained in (1) was obtained by overlapping extension PCR technology and the vector pcDNA3.4 was amplified to construct the eukaryotic expression vector pcDNA3.4-SP-EGFR / EGFRvIII-6his. The eukaryotic expression vector obtained above was verified by PCR, and the results are as follows: Figure 1 As shown in the figure; the PCR product was sequenced, and after successful sequencing, a large number of plasmids were extracted. PCR verification and sequencing results both proved that the eukaryotic expression vector was successfully constructed, and the eukaryotic expression vector pcDNA3.4-SP-EGFR / EGFRvIII-6his was obtained.

[0047] (3) The recombinant protein was expressed by transfecting the HEK293 mammalian suspension expression system with the plasmid obtained in (2). Then, the target protein was purified by using the Ni Sepharose excel affinity chromatography packing material from Cytiva and the AKATA instrument. After the protein concentration was determined, the EGFR / EGFRvIII-EC recombinant protein was obtained and stored at -80℃ for later use.

[0048] This embodiment describes the Western blot analysis of the obtained EGFR / EGFRvIII-EC recombinant protein. The primary antibody used in the Western blot analysis was an anti-His-tagged monoclonal antibody (Proteintech), and the secondary antibody was an HRP-labeled goat anti-mouse antibody (Thermo). The results are as follows: Figure 2 As shown, the EGFR / EGFRvIII-EC recombinant protein was successfully expressed.

[0049] This embodiment describes the Ni process for the EGFR / EGFRvIII-EC recombinant protein obtained above. 2+ Affinity purification identification, results as follows Figure 3 As shown in Figure A, EGFR-EC recombinant protein was obtained by elution at 100-130 KD using pH elution; as... Figure 3 As shown in Figure B, the recombinant EGFRvIII-EC protein was obtained at 55-70 KD, and the recombinant protein EGFR / EGFRvIII-EC was successfully purified.

[0050] Example 2: Animal Immunization Procedure

[0051] (1) Mix 0.5 mg of EGFR-EC and EGFRvIII-EC recombinant proteins obtained in Example 1 with 2 mL of Freund's adjuvant and emulsify to obtain an EGFR / EGFRvIII-EC recombinant protein mixture, and store at 4°C for later use;

[0052] (2) Select one alpaca and record the ear number. Inject the alpaca subcutaneously on both sides of the buttocks, with two injection points on each side. Inject 0.4 mL of the EGFR / EGFRvIII-EC recombinant protein mixture obtained in (1) at each point. 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 immunization injections. Before each immunization injection, collect blood from the alpaca’s jugular vein, take 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.

[0053] (3) Add 3 mL of cell separation solution to a 15 mL centrifuge tube, then slowly add 3 mL of the immune blood sample obtained in (2), serial dilution solution, pre-cool the centrifuge, 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, and store it at -80℃; add 10 mL of PBS buffer that has been placed at room temperature to the above centrifuge tube, centrifuge at 400 g for 20 min, discard the supernatant, and then add 5 mL of PBS buffer that has been placed at room temperature, centrifuge at 400 g for 20 min; use a hemocytometer to count the number of cells, centrifuge and discard the supernatant, use Trizol (Sigma) to dissolve and separate lymphocytes according to the number of cells and the instructions, and store them at -80℃.

[0054] This embodiment detects antibody titers in non-immunized serum and immunized serum, and the results are as follows: Figure 4 As shown in Figure A, the antibody titer of the EGFR-EC recombinant protein in immune serum is approximately 5 times that of non-immune serum at a serum dilution concentration of 1 / 6400; Figure 4 As shown in B, the antibody titer of the EGFRvIII-EC recombinant protein in immune serum is approximately 5 times that of non-immune serum at a serum dilution concentration of 1 / 6400. The antibody titer of the aforementioned immune serum is consistently higher than that of non-immune serum at serum dilution concentrations ranging from 1 / 1000 to 1 / 1024000, indicating that the immune serum library has been successfully constructed.

[0055] Example 3: Preparation of a nanobody library targeting EGFR / EGFRvIII

[0056] 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 pComb 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 EGFR / EGFRvIII.

[0057] This embodiment tests the capacity of the obtained cDNA library and phage library as follows: Figure 5 As shown, the cDNA library size is 1.56 × 10⁻⁶. 9 pfu / ml, phage library size 5.9×10 12 cfu / ml.

[0058] Example 4: Screening of nanobodies targeting EGFR / EGFRvIII

[0059] 1. Construction of EGFR / EGFRvIII overexpression cell lines

[0060] The lentiviral plasmid pLVSIN-EGFR / EGFRvIII, packaging plasmid Gag-pol, and VSV-G were transiently transfected into 293T cells for viral packaging. The packaged virus was then used to infect U87 cells to construct the EGFR / EGFRvIII overexpression cell line U87-EGFR / EGFRvIII.

[0061] This embodiment validates the EGFR / EGFRvIII overexpressing cell line U87-EGFR / EGFRvIII obtained above using Western blotting. The primary antibody used in the Western blotting was an EGFR monoclonal antibody (Abcam), and the secondary antibody was an HRP-labeled goat anti-mouse antibody (Thermo). The results are as follows: Figure 6 As shown, the EGFR / EGFRvIII overexpression cell line U87-EGFR / EGFRvIII was successfully constructed.

[0062] 2. Solid-phase panning of nanobodies based on phage display technology

[0063] (1) Coating: Dilute EGFR / EGFRvIII-ECD protein to different concentrations (10μg / 100μl, 5μg / 100μl, 1μg / 100μl) with coating solution and coat overnight at 4℃.

[0064] (2) Blocking: After washing the plate with 0.05% PBST solution, add 300 μl of 5% skim milk powder to each well and block at 37°C for 2 h.

[0065] (3) Binding: Wash the plate with 0.05% PBST solution, 1×10 11 PFU phage + 5% skim milk powder, 100 μl per well, incubated at 37°C for 2 h.

[0066] (4) Elution: Wash the plate with 0.05% PBST solution, add 100 μl of 100 mM triethylamine solution to each well, shake for 10 min, add 1 M Tris-HCl solution at a ratio of 1:1 to neutralize, gently pipette to mix, and collect in a 1.5 mL EP tube. The resulting eluent is called Output.

[0067] (5) Titration: Dilute the output sequentially with 2×YT, with a dilution factor of 10. 1 10 2 10 3 10 4 10 5 Take 10 μl of TG1 bacterial suspension in the logarithmic growth phase (OD600 of 0.6-0.8), let it stand in a 37℃ incubator for 30 min, then drop the bacterial suspension onto a 2×YT plate containing AMP and incubate it upside down in a 37℃ incubator overnight.

[0068] (6) Counting: Record the number of colonies, calculate the titer according to the formula, and the first round of screening is completed.

[0069] (7) Add 1 mL of the recovered bacterial culture to 4 mL of TG1 bacterial culture and shake well. Let stand for 30 min, then add 4 mL of 2×YT culture medium, place in a shaker (37℃, 200 rpm) and shake for 1 h. Add 1 mL of helper phage, shake well by hand and let stand at room temperature for 30 min. After standing, centrifuge at 3000 g and 4℃ for 10 min, discard the supernatant, resuspend the precipitate in SB culture medium, add ampicillin and kanamycin resistance, and culture overnight in a shaker (37℃, 200 rpm).

[0070] (8) The next day, the culture medium was poured into a 50 mL centrifuge tube and centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatant was collected. PEG / NaCl solution was added to the supernatant at a ratio of 1:6, and the mixture was mixed by inverting the tube. The mixture was then placed on ice for 2 h. After the ice bath, the mixture was centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatant was discarded, and the precipitate was resuspended in PBS. The precipitate was then transferred to a 1.5 mL EP tube, placed on ice for 10 min, and centrifuged at 12,000 rpm for 10 min at 4 °C.

[0071] (9) Transfer the supernatant to a new EP tube, add Glycercol and shake to mix. Serially dilute the amplified phage with 2×YT, spread it on 2×YT plates containing Amp resistance, incubate overnight at 37°C, and store the remaining bacterial culture at 4°C to obtain the second round of input.

[0072] (10) Solid-phase panning was completed through three rounds of "adsorption-elution-amplification" to obtain VHH phages that specifically bind to and are highly enriched with EGFR / EGFRvIII-EC antigen, which is the positive phage screening library.

[0073] This embodiment detects the phage recovery rate of the obtained positive phage screening library, and the results are as follows: Figure 7 As shown, the amount of phage recovered increased progressively with each round, ultimately reaching 1.25 × 10⁻⁶. 2 pfu(7A, EGFR) and 1.75×10 2 pfu(7B,EGFRvIII).

[0074] Example 5: Identification of nanobodies targeting EGFR / EGFRvIII

[0075] 1. Indirect ELISA identification

[0076] Take the plates from the third round of phage elution in Example 4, randomly select 48 bacterial clones, and incubate them statically overnight at 37°C. Take 10 μl of each of the above bacterial solutions and transfer them to 2×YT medium in a 96-well plate (1 mL / well). Incubate until the logarithmic phase. Add 50% sterile glycerol to the remaining bacterial solution, shake well, and store at -80°C. Add 0.2 mM IPTG to the plate 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.

[0077] The recombinant protein EGFR / EGFRvIII-EC 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.

[0078] In this embodiment, the crude extract of the induced nanobody was detected by indirect ELISA, and the identification results are as follows: Figure 8 As shown, OD 450 >1.0 was identified as a positive colony and sent to the company for sequencing.

[0079] 2. Prokaryotic expression, purification, specificity and affinity identification of nanobodies

[0080] The positive sequence for EGFR / EGFRvIII binding, identified by sequencing in Part 2 of this embodiment, was cloned. A prokaryotic expression vector, PET22b-EGFR / EGFRvIII-VHH-6his, was constructed using overlap extension technology. The positive plasmid was transformed into Rosetta (DE3) competent cells for induced expression. Bacteria were picked and placed in 6 mL of LB broth containing ampicillin, and cultured at 37°C and 220 rpm / min for 12-15 h to obtain a bacterial culture. 2 mL of the activated bacterial culture was placed in LB broth 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, expression was induced for 12 h at 16°C using 0.2 mM IPTG. After centrifugation and discarding the supernatant, the bacterial precipitate, i.e., the EGFR / EGFRvIII-targeting nanobody, was obtained. Subsequently, Ni... 2+ Affinity purification: The nanobodies obtained after imidazole elution were identified by SDS-PAGE, and the results are as follows: Figure 9 As shown, the target band appeared at 15 kDa, and Western blot analysis was performed. The primary antibody was an anti-His-tagged monoclonal antibody (Proteintech), and the secondary antibody was an HRP-labeled goat anti-mouse antibody (Thermo). The results are as follows. Figure 10 As shown, this indicates that VHH purification was successful.

[0081] In this embodiment, the purified nanobody protein was subjected to a SPR assay using a Biacore 8k instrument to verify the binding affinity between the purified nanobody and the EGFR / EGFRvIII-EC recombinant protein. The KD constant was calculated and analyzed, and the results are as follows: Figure 11 As shown, the binding KD of the nanobody targeting EGFR / EGFRvIII to recombinant EGFR-EC protein is 1.68 nM, and the binding KD of the nanobody to recombinant EGFRvIII-EC protein is 1.53 nM, which shows higher affinity compared to traditional antibodies.

[0082] Example 6: Application of nanobodies targeting EGFR / EGFRvIII

[0083] This embodiment uses U87, U87-EGFR, and U87-EGFRvIII cells as research subjects. The cultured cell lines were counted, and 1×10⁻⁶ cells were used. 6 Cells were washed with 500 μl of PBS solution, centrifuged at 500 g for 5 min, and the supernatant was discarded. Then, 2 μg of the purified EGFR / EGFRvIII 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 cells were washed 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.

[0084] The filtered stained cells were analyzed by flow cytometry, and the results are as follows: Figure 12 As shown, compared with the control group (12A), U87-EGFR cells overexpressing EGFR showed a 87.2% shift (12B), and U87-EGFRvIII cells overexpressing EGFRvIII showed a 95.8% shift (12C). This indicates that the EGFR / EGFRvIII-targeting nanobodies provided by the present invention can specifically bind to EGFR / EGFRvIII proteins on the cell membrane surface, and therefore can be used as a tool for detection and therapeutic antibodies.

[0085] 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 EGFR / EGFRvIII, characterized in that, The amino acid sequence of the nanobody targeting EGFR / EGFRvIII is shown in SEQ ID NO.8; The nanobody targeting EGFR / EGFRvIII includes complementarity-determining regions CDR1, CDR2, and CDR3; The amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.3; The nanobody targeting EGFR / EGFRVIII also includes the framework regions FR1, FR2, FR3 and FR4; The amino acid sequence of FR1 is shown in SEQ ID NO.4, the amino acid sequence of FR2 is shown in SEQ ID NO.5, the amino acid sequence of FR3 is shown in SEQ ID NO.6, and the amino acid sequence of FR4 is shown in SEQ ID NO.

7.

2. A nucleic acid, characterized in that, The nucleic acid encodes the nanobody targeting EGFR / EGFRvIII 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 use of the EGFR / EGFRvIII-targeting nanobody as described in claim 1 in the preparation of reagents for detecting EGFR / EGFRvIII.

6. The application according to claim 5, characterized in that, The application refers to the detection of EGFR / EGFRvIII expression in tumor tissues.

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