A bivalent nanobody against EGFR and its preparation method and application

Abstract

The present invention provides a bivalent anti-EGFR nanobody and a preparation method and application thereof, wherein the bivalent nanobody is formed by connecting two monovalent anti-EGFR nanobodies in series, and the amino acid sequence of the monovalent nanobody is shown in SEQ ID NO: 1. The present invention improves the affinity of the antibody and further expands the application potential of the antibody.

Description

Technical Field

[0001] The present invention relates to the technical field of nano antibodies, and in particular to an anti-EGFR bivalent nano antibody and a preparation method and application thereof. Background Art

[0002] Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor and a member of the ErbB family. The ErbB family includes four members: ErbB1 (also known as EGFR / HER1), ErbB2 (also known as Neu / HER2), ErbB3 (HER3), and ErbB4 (HER4). Each ErbB family member contains an extracellular ligand binding region, a transmembrane region, and an intracellular tyrosine kinase region (except ErbB3, which has weak or no tyrosine kinase activity). The ErbB family is a group of transmembrane receptor tyrosine kinases that play a key role in many cellular processes, especially cell growth and differentiation. EGFR plays a key role in a variety of cell biological processes, such as proliferation, migration, differentiation, and survival.

[0003] In many tumors, EGFR is overexpressed, amplified, or mutated, which leads to its continuous activation, which in turn stimulates tumor growth, invasion, and metastasis. For example, in non-small cell lung cancer, liver cancer, colorectal cancer, and head and neck cancer. EGFR promotes tumor expression mainly in the following ways: (1) Promoting cell proliferation: After EGFR activation, it promotes cell entry into the cell cycle and proliferation through the MAPK and PI3K / Akt signaling pathways. (2) Inhibiting cell apoptosis: Through the PI3K / Akt signaling pathway, EGFR activation can enhance anti-apoptotic signals and help tumor cells escape normal programmed death mechanisms. (3) Promoting tumor angiogenesis: EGFR activation can enhance the expression and secretion of VEGF (vascular endothelial growth factor), thereby promoting the formation of new blood vessels in the tumor and providing nutrition for rapidly growing tumors. (4) Promoting tumor invasion and metastasis: EGFR signals can enhance the ability of cells to degrade the extracellular matrix and promote cell migration and invasion. In addition, EGFR is also associated with epithelial to mesenchymal transition (EMT), a process that plays a key role in tumor metastasis. It is precisely because of these important functions of EGFR protein that it has become a popular target for the treatment of tumor diseases.

[0004] Nanobodies are the variable regions of heavy chain antibodies that naturally lack light chains and exist in camelids and sharks, discovered by Belgian scientists in 1993. They are also called single-domain antibodies (sdAb), or VHH antibodies. VHH crystals are 2.5nm, 4nm long, and have a molecular weight of only 15KDa, which is about 1 / 10 of the molecular weight of monoclonal antibodies. Therefore, they are called nanobodies (Nb). They are the smallest units known to bind to target antigens. Compared with ordinary four-chain antibodies and single-chain antibodies, nanobodies have the advantages of small molecular weight, high solubility, high stability, easy expression, ability to bind to gap epitopes, and low immunogenicity. Therefore, nanobodies have broad application prospects in the field of new drug development. However, nanobodies often have insufficient binding force because they only have one structural domain. Summary of the invention

[0005] The present invention aims to provide a bivalent anti-EGFR nanobody and a preparation method and application thereof, thereby improving the affinity of the antibody and further expanding the application potential of the antibody.

[0006] The technical solution adopted by the present invention to achieve the above technical purpose is:

[0007] The present invention provides a bivalent anti-EGFR nanobody, wherein the bivalent nanobody is formed by connecting two monovalent anti-EGFR nanobodies in series, and the amino acid sequence of the monovalent nanobody is shown in SEQ ID NO:1.

[0008] Preferably, two anti-EGFR monovalent Nanobodies are connected in series using a linker, and the amino acid sequence of the linker is shown in SEQ ID NO:3.

[0009] Preferably, the amino acid sequence of the bivalent Nanobody is as shown in SEQ ID NO:2.

[0010] The present invention also provides a method for preparing the above-mentioned anti-EGFR bivalent nanobody, comprising:

[0011] The sequences of two anti-EGFR monovalent nanobodies are expressed in series using a linker, and the amino acid sequence of the linker is shown in SEQ ID NO: 3, which is a bivalent nanobody.

[0012] The present invention further provides the use of the above-mentioned anti-EGFR bivalent nanobody in the preparation of a drug for treating tumor diseases associated with EGFR.

[0013] The beneficial effects of the present invention are:

[0014] The present invention provides a bivalent anti-EGFR nanobody, which improves the affinity of the antibody and further expands the application potential of the antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The agarose gel electrophoresis diagram of two rounds of nested PCR products. Lane M: DL5000 DNA marker, lane E is the first round of PCR products, and lanes E-1 and E-2 are the second round of PCR products.

[0016] Figure 2 OD values ​​of samples screened for the display library of EGFR protein.

[0017] Figure 3 These are the affinity test results of the E1 nanobody and the bivalent nanobody (E2). DETAILED DESCRIPTION

[0018] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0019] In the following examples, unless otherwise specified, the reagents used are all commercially available products, and the operations used can also be achieved by conventional technical means.

[0020] Example

[0021] 1. Alpaca immunization and antibody library construction of EGFR protein

[0022] (1) Dissolve 500 μg of EGFR protein (purchased from Beijing Sino Biological Technology Co., Ltd., catalog number: 10001-H08H) in 500 μL of PBS and mix with 500 μL of Freund's complete adjuvant (1:1);

[0023] (2) A healthy adult alpaca was selected and immunized by multiple subcutaneous injections at the back at 6-7 μg / kg for a total of three immunizations, each with an interval of 2 weeks. 30 mL of peripheral blood was collected one week after the third immunization;

[0024] (3) PBMC cells were extracted from alpaca peripheral blood using a camel peripheral blood mononuclear cell isolation kit (Beijing Solebow Technology Co., Ltd., catalog number P5350). A total of 5 × 10 7 PBMC cells, and 20 μg of total RNA was extracted from the extracted PBMC cells;

[0025] (4) According to the instructions of the reverse transcription kit (Roche

[0026] Total RNA was reverse transcribed into cDNA in the procedure of transcripor first stand cDNA synthesis KIT;

[0027] (5) The cDNA obtained by reverse transcription was used as a template for PCR reaction. Two rounds of amplification were performed. The primer sequences for the first round of PCR were as follows:

[0028] CALL001:GTCCTGGCTGCTCTTCTACAAGG(SEQ ID NO:4),

[0029] CALL002:GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO:5);

[0030] The PCR reaction conditions and procedures are: 95°C for 5 minutes; 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds, 30 cycles; 72°C for 7 minutes. Use an agarose gel recovery kit to recover a band of about 700 bp, and finally adjust the nucleic acid concentration to 5 ng / μl with water. The primer sequences for the second round of PCR are as follows:

[0031] VHH-Back: GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO: 6),

[0032] VHH-For:CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT(SEQ ID NO:7);

[0033] The PCR reaction conditions and procedures were: 95°C for 5 minutes; 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, 15 cycles; 72°C for 7 minutes. The PCR product was purified using a PCR product recovery kit. The two rounds of PCR products were subjected to agarose gel electrophoresis. The results were as follows: Figure 1 ;

[0034] (6) The amplified VHH gene was inserted into the pComb3XSS plasmid and then transferred into TG1 Escherichia coli to construct an anti-EGFR nanoantibody gene library with a library capacity of 3.2×10 8 ;

[0035] (7) 10 mL of the anti-EGFR nanobody gene library was added to 40 mL of LB medium and cultured at 37°C for 2.5 h. After that, 50 μL of helper phage (M13KO7) was added and cultured at 30°C, 220 rpm overnight.

[0036] (8) The next day, centrifuge at 10,000 g for 10 min, take the supernatant, add 10 mL of PEG-NaCl solution, place at 4 °C for 8 h, and then centrifuge. Resuspend the precipitate with 1 mL of PBS to obtain an anti-EGFR nanobody display library with a library capacity of 4.6 × 10 12 .

[0037] 2. Construction of EGFR protein display library and antibody screening

[0038] (1) Add 5 μg of biotinylated EGFR protein and 0.5 mL of anti-EGFR nanobody display library into a 1.5 mL EP tube, incubate at 37°C for 1 h, then add magnetic beads containing streptomycin and incubate again for 30 min;

[0039] (2) Place the EP tube on the magnetic rack. You can see that the magnetic beads are enriched and adsorbed to the tube wall. After removing the solution in the tube, take it off the magnetic rack and add 1 mL of PBST solution. Use a pipette to blow the magnetic beads evenly.

[0040] (3) Place the EP tube on the magnetic rack. You can see that the magnetic beads are enriched and adsorbed to the tube wall. After removing the solution in the tube, take the EP tube off the magnetic rack and add 1 mL of PBST solution. Use a pipette to blow the magnetic beads evenly. Repeat this operation and wash with PBST 3 times in total.

[0041] (4) After washing three times with PBST, the magnetic beads were added to 5 mL of TG1 E. coli and cultured at 37°C for 2.5 h. After that, 10 μL of the bacterial solution was taken out and coated on an ampicillin-resistant plate to measure the titer. The remaining bacterial solution was added to 45 mL of LB medium and 10 μL of helper phage and cultured at 30°C, 220 rpm overnight.

[0042] (5) On the second day, centrifuge at 10,000 g for 10 min, take the supernatant, add 5 mL of PEG-NaCl solution, place at 4 °C for 8 h, and then centrifuge. Resuspend the precipitate with 1 mL of PBS to obtain the first round of screening display library of anti-EGFR nanoantibodies;

[0043] (6) Repeat the above screening steps for the first round of anti-EGFR nanoantibody screening display library to form the second round of anti-EGFR nanoantibody screening display library, and repeat again to form the third round of EGFR nanoantibody screening display library;

[0044] (7) 24 single clones were selected from the plate used for the third round of screening and titer determination and added to 1 mL of LB medium. After culturing at 37°C for 2.5 h, 1 μL of helper phage was added and cultured overnight at 30°C and 220 rpm.

[0045] (8) On the second day, centrifuge at 10,000 g for 10 min, collect the supernatant, and number them 1-24;

[0046] (9) Dilute the EGFR protein to 5 μg / mL and coat the ELISA plate, with 100 μL coated per well;

[0047] (10) The next day, the EGFR protein solution on the ELISA plate was discarded and the plate was washed twice with PBST solution. 100 μL of skim milk was added to each well and the plate was incubated at 37°C for 2 h.

[0048] (11) Remove skim milk and add 200 μL of the yeast monoclonal display solution in step (7) to each well and place at 37°C for 1 h;

[0049] (12) After washing with PBST for 5 times, add anti-M13 protein antibody with HRP and incubate at 37°C for 1 h;

[0050] (13) After washing with PBST for 5 times, TMB colorimetric solution was added and the mixture was incubated at 37°C for 5 min before adding ELISA stop solution;

[0051] (14) Read the ELISA plate at OD450. Figure 2 As shown, the monoclonal yeast solutions of Nos. 3, 5, 12, and 19 were sent for sequencing to obtain a monovalent nanobody sequence, whose sequence was QLQESGGGLVQPGGSLRLSCAASGFTLDDYAVGWFRQAPGKEREGVSCISSSG TSTNYADSVKGRFTIARDNDKNTVYLQMNSLKPEDTAVYYCATRTHCMLLQPSSNDYWGQGTQVTVSS (SEQ ID NO: 1), named E1 nanobody.

[0052] 3. Preparation of bivalent anti-EGFR nanobodies

[0053] The anti-EGFR bivalent nanobody was sent to Beijing Qingke Biotechnology Co., Ltd. for synthesis. The two E1 nanobody sequences were expressed in tandem, with the intermediate linker being GGGGSGGGGSGGGGS (SEQ ID NO: 3), i.e., the bivalent nanobody E2, whose sequence is

[0054] QLQESGGGLVQPGGSLRLSCAASGFTLDDYAVGWFRQAPGKEREGVSCISSSGTSTNYADSVKGRFTIARDNDKNTVYLQMNSLKPEDTAVYYCATRTHCMLLQPSSNDYWGQGTQVTVSSGGGGSGGGG SGGGGSQLQESGGGLVQPGGSLRLSCAASGFTLDDYAVGWFRQAPGKEREGVSCISSSGTSTNYADSVKGRFTIARDNDKNTVYLQMNSLKPEDTAVYYCATRTHCMLLQPSSNDYWGQGTQVTVSS(SEQ ID NO:2).

[0055] 4. Comparison of expression and affinity between E1 nanobody and bivalent nanobody (E2)

[0056] (1) The E1 nanobody sequence and the E2 nanobody sequence were synthesized into the pET28a plasmid, named E1-pET28a and E2-pET28a, 2 ng of E1-pET28a and E2-pET28a were transformed into BL21 competent cells, and the kanamycin plates were inverted and cultured overnight;

[0057] (2) On the second day, a single clone was picked from the plate and placed in 5 mL LB-kanana liquid medium. After shaking at 37°C and 220 rpm for 8 h, 1 L LB-Amp liquid medium was added and cultured in a shaker at 37°C and 220 rpm for 4 h.

[0058] (3) Add 1 mL of 0.1 M IPTG and culture overnight at 30°C and 180 rpm;

[0059] (4) The next day, the bacterial solution was collected by centrifugation and resuspended in 50 mL PBS and then ultrasonically disrupted at 150 W for 10 s and rest for 10 s.

[0060] (5) collecting the supernatant by centrifugation, passing through a nickel column, and then passing through a nickel column with 20 mL of a PBS solution containing 50 mM imidazole, and then adding 10 mL of a PBS solution containing 300 mM imidazole to pass through a nickel column to elute the nanoantibody, desalting the obtained nanoantibody solution, and eluting with PBS to obtain E1 and E2 nanoantibodies dissolved in PBS. The concentration of the nanoantibodies was determined by the BCA method, and the concentration of E1 was 4.3 mg / mL, and the concentration of E2 was 1.7 mg / mL;

[0061] (6) Dilute BSA protein and EGFR protein to 5 μg and coat the plate with 100 μL per well, 20 wells per plate, and place at 4°C overnight;

[0062] (7) The next day, the protein solution on the ELISA plate was removed, the plate was washed twice with PBST, 100 μL of skim milk was added to each well, and the plate was placed at 37°C for 2 h.

[0063] (8) Remove skim milk, add 40 μg / mL E1 and E2 nanoantibody solutions to the first well of the coating, then dilute them in multiples and place at 37°C for 1 h;

[0064] (9) After washing five times with PBST, add anti-His tag antibody with HRP and incubate at 37°C for 1 h;

[0065] (10) After washing with PBST for 5 times, TMB colorimetric solution was added and the mixture was incubated at 37°C for 5 min before adding ELISA stop solution;

[0066] (11) The analysis data showed that the data of the EGFR protein-coated wells formed a concentration-dependent curve with the added E1 and E2 nanoantibodies, and the data of the BSA protein-coated wells showed that the E1 and E2 nanoantibodies were basically not bound, proving that the E1 and E2 nanoantibodies specifically bound to EGFR. At the same time, it can be concluded that the affinity of E2 to EGFR is greater than that of E1 to EGFR. Figure 3 shown.

[0067] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A bivalent anti-EGFR nanobody, wherein the bivalent nanobody is composed of two anti-EGFR monovalent nanobodies connected in series, and the amino acid sequence of the monovalent nanobody is shown in SEQ ID NO:

1.

2. The bivalent Nanobody according to claim 1, characterized in that Two anti-EGFR monovalent nanobodies are connected in series using a linker, and the amino acid sequence of the linker is shown in SEQ ID NO:

3.

3. The bivalent Nanobody according to claim 1, characterized in that The amino acid sequence of the bivalent nanobody is shown in SEQ ID NO:

2.

4. A method for preparing a bivalent nanobody according to claim 1 or 3, comprising: The sequences of two monovalent anti-EGFR nanobodies are expressed in series using a linker, and the amino acid sequence of the linker is shown in SEQ ID NO: 3, which is a bivalent nanobody.

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