CN112457404B - An anti-human EGFR nanobody and its application - Google Patents

Abstract

The invention discloses an anti-human EGFR nano antibody and application. The Nanobody is an aEG4D9 Nanobody whose amino acid sequence is shown in SEQ ID NO:4; or an aEG4D9 Nanobody whose amino acid sequence is shown in SEQ ID NO:4 and an aEG1B4 Nanobody whose amino acid sequence is shown in SEQ ID NO:1 An antibody formed by combining at least one of the aEG2C7 nanobody with the amino acid sequence shown in SEQ ID NO:2 and the aEG6B2 nanobody with the amino acid sequence shown in SEQ ID NO:5. The nanobody has high affinity, specificity and low immunogenicity, and has the advantages of good stability, simple structure, easy engineering transformation and the like, and can be better applied to the development of antibody drugs.

Description

An anti-human EGFR nanobody and its application

This application is a divisional application of the Chinese invention patent application with the application number of "201811129703.9" and the invention name of "anti-human EGFR nanobody and its application".

technical field

The invention belongs to the field of biotechnology, and relates to an anti-human EGFR nanobody and application.

Background technique

The term cancer was proposed in more than 400 BC, and it has not been conquered for more than 2,000 years. Today, the threat to human health is becoming more and more obvious. In recent years, studies have found that epidermal growth factor (EGFR) is overexpressed in a variety of solid tumor cells, and plays an important role in the occurrence and development of cancer, and is positively correlated with poor prognosis and drug resistance. Overexpression of EGFR in cancer cells can lead to the continuous activation of the EGFR/EGF signaling pathway, promote the proliferation, migration and invasion of cancer cells, and can also promote the secretion of VEGF factors, resulting in the formation of blood vessels in the cancer tissue microenvironment. At present, EGFR-targeting antibodies have achieved good results in the clinical treatment of non-small cell lung cancer, but because they are chimeric antibodies, they have certain immunogenicity, and due to their large molecular weight and poor permeability, they cannot be effectively removed tiny lesions.

Nanobody (Nb) is a genetically engineered antibody containing only a single domain, which can bind to antigen with high affinity and specificity. Due to the small molecular weight of antibodies, nanobodies are easy to express in E. coli, which greatly reduces the cost of antibody production. At the same time, the structure of the antibody is simple, which is conducive to its further modification. The small size of the nanobody molecule also improves the tissue penetration ability of the antibody during therapy. Therefore, anti-EGFR nanobodies have high medical application value in EGFR-targeted cancer therapy.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a human EGFR nanobody.

Another object of the present invention is to provide the application of the above-mentioned anti-human EGFR nanobody.

The object of the present invention is achieved by the following technical solutions: an anti-human EGFR nanobody, named aEG4D9 nanobody; An antibody formed by combining at least one of the Nanobodies;

The amino acid sequence of the aEG1B4 Nanobody is shown in SEQ ID NO: 1;

The amino acid sequence of the aEG2C7 Nanobody is shown in SEQ ID NO: 2;

The amino acid sequence of the aEG2E12 Nanobody is shown in SEQ ID NO: 3;

The amino acid sequence of the aEG4D9 Nanobody is shown in SEQ ID NO: 4;

The amino acid sequence of the aEG6B2 Nanobody is shown in SEQ ID NO:5.

The nucleotide sequence encoding the above-mentioned anti-human EGFR Nanobody is the nucleotide sequence encoding the aEG4D9 Nanobody; or the nucleotide sequence encoding the aEG4D9 Nanobody and the aEG1B4 Nanobody encoding At least one combination of the nucleotide sequence, the nucleotide sequence encoding the aEG2C7 Nanobody, the nucleotide sequence encoding the aEG2E12 Nanobody, and the nucleotide sequence encoding the aEG6B2 Nanobody The resulting nucleotide sequence.

The nucleotide sequence encoding the aEG1B4 Nanobody is preferably shown in SEQ ID NO:8.

The nucleotide sequence encoding the aEG2C7 Nanobody is preferably shown in SEQ ID NO:9.

The nucleotide sequence encoding the aEG2E12 Nanobody is preferably shown in SEQ ID NO:10.

The nucleotide sequence encoding the aEG4D9 Nanobody is preferably shown in SEQ ID NO:11.

The nucleotide sequence encoding the aEG6B2 Nanobody is preferably shown in SEQ ID NO:12.

The nucleotide sequences encoding the aEG1B4 Nanobodies, aEG2C7 Nanobodies, aEG2E12 Nanobodies, aEG4D9 Nanobodies and aEG6B2 Nanobodies respectively consist of 375, 393, 375, 381 and 372 bases, and the corresponding encoded amino acids are respectively 125, 131, 125, 127 and 124. The aEG1B4 antibody contains three complementary determinants (CDRs), wherein the amino acid encoding CDR1 is VNVSNEVMS, the amino acid encoding CDR2 is TIANHS, and the amino acid encoding CDR3 is TLYSGATKQLEY. The aEG2C7 antibody contains three complementary determinants, wherein the amino acid encoding CDR1 is FTFNNEIMA, the amino acid encoding CDR2 is SIAANN, and the amino acid encoding CDR3 is RYAAEPHTYSMGNKSLRY. The aEG2E12 antibody contains three complementary determinants, wherein the amino acid encoding CDR1 is YSSNNEFMA, the amino acid encoding CDR2 is AISTRN, and the amino acid encoding CDR3 is GVSYRRPQQLKY. The aEG4D9 antibody contains three complementary determinants, wherein the amino acid encoding CDR1 is DMLSPDNMT, the amino acid encoding CDR2 is TIHKTD, and the amino acid encoding CDR3 is GLRSRGLSSKYLEY. The aEG6B2 antibody contains three CDRs, wherein the amino acid encoding CDR1 is FNVNPKYMT, the amino acid encoding CDR2 is SIRSPG, and the amino acid encoding CDR3 is SVSRDEKYMRF.

The preparation method of the anti-human EGFR nanobody comprises the following steps: synthesizing the nucleotide encoding the anti-human EGFR nanobody by the method of gene (DNA) synthesis, then cloning it into an expression plasmid vector, and transforming it into The anti-human EGFR nanobody can be obtained by expressing and purifying in the host cell; the anti-human EGFR nanobody can also be directly synthesized by the method of polypeptide synthesis.

The application of the anti-human EGFR nanobody in the preparation of an antibody drug for treating EGFR overexpression as a characteristic disease.

The diseases characterized by EGFR overexpression are autoimmune diseases and cancers.

The cancer is a tumor with high EGFR expression.

The EGFR high expression tumor can specifically be lung cancer, head and neck cancer, colon cancer and brain tumor.

Compared with the prior art, the present invention has the following advantages and effects:

1. The nanobodies that interact with the EGFR extracellular domain (domain) III screened in the human nanobody library by the method of phage display of the present invention can obtain fully human monoclonal antibodies without using the antigen to immunize the human body. Clonal nanobodies, with a molecular weight of about 13kDa, contain only a single domain, have high affinity, specificity and low immunogenicity, and can significantly reduce the production cost of antibodies by expressing antibodies in prokaryotic hosts with high protein expression , to promote the application of antibodies.

2. The nanobody provided by the present invention has the advantages of good stability, easy transformation, and good tissue penetration ability.

3. The nanobody provided by the present invention is of human origin, so it has no immunogenicity in the human body, and can be better applied to the development of anti-tumor antibody drugs.

4. The prokaryotic host with high expression of the Nanobody binding protein provided by the present invention expresses the antibody, which can significantly reduce the production cost of the antibody and promote the application of the antibody.

Description of drawings

Figure 1 shows the results of screening and enrichment of the nanobody library analyzed by polyclonal ELISA; in which, the PBS well is a blank control, and the EGFR well is coated with synthetic EGFR polypeptide; the primary antibody is the polyclonal obtained by amplification and purification after each round of library screening Phage antibody, the secondary antibody is HRP-labeled anti-phage M13 antibody.

Figure 2 shows the results of monoclonal phage ELISA screening for positive clones that bind to EGFR polypeptide; wherein each monoclonal includes: PBS (blank control well), CXCR4 polypeptide (irrelevant antigen control well), EGFR polypeptide (target antigen well).

Figure 3 is an SDS-PAGE protein electrophoresis image after antibody protein expression and purification; wherein, Figure A is the electrophoresis image of aEG1B4, Figure B is the electrophoresis image of aEG2C7, Figure C is the electrophoresis image of aEG2E12, and Figure D is the electrophoresis image of aEG4D9. E is aEG6B2, in Figures A-E: lane 1 is marker, lane 2 is uninduced whole protein, lane 3 is induced whole protein, lane 4 is induced fragmentation pellet, lane 5 is induced fragmentation supernatant, lane 6 is After passing the column liquid protein, lane 7 is the washing liquid protein, and lanes 8-12 are the elution protein tubes.

Figure 4 is a graph showing the binding of purified anti-EGFR nanobodies to EGFR polypeptides by ELISA; wherein, each antibody includes blank control wells PBS, 7 irrelevant antigen control wells: VEGF, CAMPH, BMP2, ENDOF1, FGF21, HER2 and CXCR4 peptide, target antigen pore: EGFR peptide.

Figure 5 is a graph showing the binding of purified anti-EGFR nanobodies to the intact extracellular segment of EGFR by Western Blotting; wherein, Figure A is aEG1B4, Figure B is aEG2C7, Figure C is aEG2E12, Figure D is aEG4D9, and Figure E is aEG6B2, in Figures A-E: lane 1 is the protein marker, lane 2 is the complete extracellular segment of EGFR; the primary antibody is purified anti-EGFR antibody, and the secondary antibody is proteinA-HRP.

Figure 6 is a graph showing the effect of anti-EGFR nanobody on the proliferation of cancer cells detected by MTT method; wherein, Figure A is the result of the effect of Nanobody on the proliferation of A549 cells, and Figure B is the result of the effect of Nanobody on the proliferation of MCF-7 cells , Figure C is the result of the effect of Nanobodies on the proliferation of DU145 cells; aVE201 and aHer2-13C1 were used as negative control antibodies; *: p<0.05vs 0μg/mL, **: p<0.01vs 0μg/mL (n=3) .

Figure 7 shows the results of flow cytometry and Annexin V/PI double staining to detect the effect of nanobodies on cancer cell apoptosis; in which, Figures A, C, and E are the results of apoptotic A549, MCF-7 and DU145 cells, respectively. Two-dimensional scatter plot, Figures B, D, and F are the apoptotic ratios obtained from Figures A, C, and E, respectively; each cell includes a control group without antibody, and an experimental antibody group (50 μg/mL): aEG1B4, aEG2C7, aEG2E12, aEG4D9 and aEG6B2, negative control antibody group (50 μg/mL): aVE201 and aHer2-13C1; *: p<0.05 vs control, **: p<0.01 vs control (n=3).

Figure 8 is a photo of the results of the scratch healing method detecting the migration of nanobodies to cancer cells; wherein, Figures A to C are the photos of the migration results of A549, MCF-7 and DU145 cells at 0h and 24h, respectively.

Fig. 9 is a graph of the mobility results calculated according to the scratch length of Fig. 8; wherein, Figs A to C are A549, MCF-7 and DU145 cells respectively; *: p<0.05vs 0μg/mL, **:p <0.01 vs 0 μg/mL (n=3).

Figure 10 is a photo of the results of the Transwell method detecting the migration of nanobodies to cancer cells; among them, Figures A to C are the results of the migration of A549, MCF-7 and DU145 cells treated with nanobodies at various concentrations.

Figure 11 is the change trend diagram of antibody absorbance value obtained according to Figure 10; in which, Figures A to C are A549, MCF-7 and DU145 cells respectively; *: p<0.05vs 0μg/mL, **: p<0.01vs 0μg /mL (n=3).

Figure 12 is a photo of the results of Transwell assay detecting the effect of Nanobodies on cancer cell invasion; among them, Figures A to C are the results of A549, MCF-7 and DU145 cells treated with Nanobodies at various concentrations, respectively.

Figure 13 is the change trend diagram of antibody absorbance value obtained according to Figure 12; wherein, Figures A to C are A549, MCF-7 and DU145 cells respectively; *: p<0.05vs 0μg/mL, **: p<0.01vs 0μg /mL (n=3).

Figure 14 is a graph showing the results of using the lung cancer mouse model to verify the inhibitory effect of Nanobodies on tumor size; wherein, Figure A is the volume change curve of the tumor after administration, Figure B is the tumor size of each group at the end of the administration period, Figure 14 C is the tumor weight of each group after the end of the dosing cycle; *: p<0.05 vs 0 μg/mL, **: p<0.01 vs 0 μg/mL (n=4/n=5).

specific embodiment

The present invention will be described in further detail below with reference to specific embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1 Preparation of helper phage

(1) Take out the TG1 Escherichia coli cloned strain stored in glycerol from the -80°C refrigerator, spread it on a TYE non-resistant plate by the method of four-district streak, and cultivate it upside down at 37°C for 13 hours.

(2) Pick a TG1 monoclonal strain from the plate and inoculate it into 5mL of 2×TY non-resistance liquid medium, and cultivate at 37°C and 230rpm for 13h.

(3) The TG1 bacterial solution was transferred to 5 mL of 2×TY non-resistant liquid medium at a volume ratio of 1:100, and cultured at 37° C. and 230 rpm for 2 h (the bacterial solution OD ₆₀₀ = about 0.5).

(4) Take 200 μL of TG1 bacterial solution (OD ₆₀₀ = about 0.5) into a 1.5 mL centrifuge tube, add 10 μL of KM13 helper phage (1×10 ¹³ pfu/mL), and put it into preheated 37°C water for 30 min.

(5) After preparing soft agar and cooling it to about 40°C, pour the TG1 bacterial solution treated in step (4), mix, and then pour the pre-prepared TYE containing 50 μg/mL kanamycin resistance On a solid culture plate, stand at room temperature to solidify, and invert at 37°C for 13h.

(6) Pick a plaque, put it into 5 mL of TG1 bacterial solution (OD ₆₀₀ =0.5), and culture it on a shaker at 37° C. and 230 rpm for 2 hours.

(7) Transfer all the bacterial liquid obtained in step (6) to 500 mL of 2×TY medium, cultivate at 37° C., 230 rpm shaker for 2 hours, and add 500 μL of kanamycin (the concentration in the medium is 50 μg/mL) ), cultured at 30°C and 230rpm for 20h.

(8) Centrifuge the bacterial solution at 3300g for 20min, collect the supernatant into a sterile tube, mix the supernatant with 20% PEG/NaCl solution at a volume ratio of 1:4, and place on ice for 4h.

(9) Then centrifuge at 3300g for 30min, collect the precipitate, resuspend the precipitate with 1mL sterile PBS (pH=7.4, 0.01M) solution, centrifuge at 4000g for 5min, collect the supernatant, which is the helper phage.

Example 2 Amplification of Phage Nanobody Libraries

(1) Thaw the TG1 bacteria containing the antibody plasmid (i.e., the human nanobody phage library, SourceBioscience, London, UK) on ice, and transfer it to 500 mL of 2×TY liquid medium (containing 0.1% ampicillin in a mass-to-volume ratio). and 1% glucose in a mass-volume ratio), incubated at 37°C and 230rpm in a shaker for 2.5h (OD ₆₀₀ =0.5), and then added 2×10 ¹² of helper phage, and water bathed at 37°C for 30min.

(2) Divide the bacterial liquid into 250mL/bottle, centrifuge at 3200g for 10min, remove the supernatant, and use 500mL 2×TY medium (containing 0.1% ampicillin and 0.05% kanamycin in mass volume ratio) The pellet was resuspended and incubated at 25°C with shaking at 220rpm for 20h.

(3) Centrifuge the bacterial solution at 3200 g for 20 min, collect the supernatant, and purify the phage by the same method as steps (8)-(9) in Example 1, and store the prepared library phage at -80°C.

(4) Measure the titer of the antibody library. The first one: using a spectrophotometer to measure the light absorption value at 260 nm to estimate the phage titer in the prepared library, the library phage titer formula: the number of phages/mL=OD ₂₆₀ ×dilution factor×22.14×10 ¹⁰ . The second method: using cloning technique to estimate, after diluting the phage to a certain concentration, spread the TG1 bacteria that infect the logarithmic growth phase on a solid plate containing ampicillin, cultivate overnight at 37°C, and calculate by the number of clones. The titer of the amplified antibody library was 4.8×10 ¹³ pfu/mL.

Example 3 Screening of Nanobodies against EGFR Fragments from Phage Nanobody Libraries

(1) Screening of anti-EGFR nanobody phage

1) Coat the EGFR polypeptide (purchased from Shanghai Botai Biological Company, with the amino acid sequence shown in SEQ ID NO. 15) in an immune tube (nunc) (the first round: 0.1 mg/mL; the second and third rounds) : 0.05 mg/mL; the fourth and fifth rounds: 0.025 mg/mL; ), set a PBS (control) tube. 4°C overnight.

2) Pour out the coating solution, wash each tube with 4.5mL PBS three times (after each addition of liquid, directly discard it without stopping), add 4.5mL BSA blocking solution with a concentration of 2% by mass to each tube, and place at room temperature. 2h.

3) Pour off the BSA blocking solution, and wash each tube three times with 4.5 mL of PBS (after each addition of liquid, discard directly without stopping).

4) 4 mL of 2% BSA solution containing 5×10 ¹² pfu bacteriophage was added to each tube, and allowed to stand at room temperature for 1 h.

5) Wash 10 times with PBST (after each addition of liquid, discard directly without stopping).

6) Add 500 μL of 1 mg/mL trypsin solution to each tube, invert the digestion and elute for 10 min.

7) Collect the phage eluate into a sterile 1.5mL EP tube, take 125μL of the phage eluate, add it to 875μL of TG1 bacteria (OD ₆₀₀ = 0.5), mix well, and put it into a warm bath in preheated 37°C water 30min.

8) Dilute the mixed bacterial solution according to the experimental needs, and then take 2 μL of the infected bacterial solution and spread it on a TYE solid plate containing 1% glucose and 0.1% ampicillin for identification and screening results.

9) Coat the remaining infected bacterial liquid of the experimental group on the same TYE solid plate, and cultivate at 37°C overnight.

10) 2 mL of 2×TY (containing 15% glycerol) was added to the plate in step (9), and all bacteria were scraped off and collected into a sterile 1.5 mL centrifuge tube. Aspirate 50 μL of bacterial liquid from it and inoculate it into 50 mL of new 2×TY liquid medium (containing 1% glucose, 0.1% ampicillin), and cultivate at 37°C and 230 rpm on a shaker for about 2 hours to make the bacterial concentration reach OD ₆₀₀ = 0.5. After 2 hours, from Take 10 mL of bacterial solution from 50 mL into a 50 mL centrifuge tube, add helper phage to the centrifuge tube, and then put the centrifuge tube into a 37°C water bath for 30 minutes.

11) Centrifuge at 3000g for 15min, discard the supernatant, use 2×TY liquid medium (containing 0.1% glucose, 0.1% ampicillin, 0.05% kanamycin) to resuspend the cell pellet, and cultivate with shaking at 25°C and 230rpm for 20h .

12) Centrifuge at 3000g for 20min, take 40mL of supernatant and pour it into a sterile 50mL centrifuge tube, then add 10mL of 20% PEG/NaCl to the centrifuge tube, invert up and down and mix, and ice bath for 4h.

13) After 4h, centrifuge at 4000g at 4°C for 30min, and discard the supernatant. Add 1 mL of PBS to resuspend the pellet, transfer to a 1.5 mL centrifuge tube, and centrifuge again at 10,000g at 4°C for 10 min, collect the supernatant, and store at 4°C.

14) Repeat steps 1) to 13) for a total of 5 rounds of enrichment screening, and screen the phages obtained in the previous round in turn. The enrichment results are shown in Table 1. Enrichment ratio = number of clones in wells coated with EGFR antigen/number of clones in negative control wells

(2) Polyclonal phage ELISA

1) Take 0.2 μg of EGFR polypeptide to coat a 96-well immunoplate with 100 μL/well of the coating solution, and set a blank control well (PBS) at 4° C. overnight.

2) Pour off the coating solution. Wash 3 times with PBS (discard without stopping after each addition of liquid).

3) Then add 280 μL of BSA blocking solution with a concentration of 2% by volume, at room temperature for 2 hours.

4) Pour off all the 2% (w/v) BSA blocking solution. Wash 3 times with PBS (discard without stopping after each addition of liquid).

5) Take each round of phage solution prepared in step 13) as the primary antibody, mix with 2% (w/v) BSA blocking solution at a volume ratio of 1:3, and coat each well with 100 μL of the mixed solution. 1h at room temperature.

6) After 1 h, wash 4 times with PBST (after each addition of liquid, discard directly without stopping).

7) Add 100 μL of HRP-labeled phage M13 (M13 Bacteriophage-HRP) antibody (diluted with 2% (w/v) BSA blocking solution at a volume ratio of 1:10000) to each well as a secondary antibody for 1 h at room temperature.

8) After 1 h, wash 4 times with PBST and once with PBS (after each addition of liquid, discard directly without stopping).

9) Add 100 μL of TMB chromogenic solution (Biyuntian) to each well, and react at room temperature for 4 minutes in the dark.

10) For 4 min, add 50 μL of 1M H ₂ SO ₄ solution to each well.

11) Use a microplate reader to measure the absorbance at OD450nm.

The above results indicated that phage antibodies specific for EGFR were accumulated through 5 rounds of screening.

Table 1 Enrichment results of each round of screening of phage Nanobody library

Example 4 Picking monoclonal phage from the 5th round enriched library for ELISA validation

(1) Dilute the phage obtained in the fifth round of enrichment screening to 10 ⁸ -10 ¹⁰ times, take 100 μL of the dilution to infect the TG1 bacterial solution in the logarithmic growth phase, and randomly select 448 monoclonal strains from the plate and place them on the plate. Culture in a 96-well culture plate, 200 μL of 2×TY medium per well/one clone, 37° C., 230 rpm shaker for 13 h.

(2) Transfer 5 μL of overnight bacterial solution from each well to a new 96-well cell culture plate for culture, 200 μL of 2×TY medium per well/one clone, and culture at 37°C and 250 rpm for 2 hours. After aspirating 95 μL of bacterial liquid, 100 μL of glycerol aqueous solution with a concentration of 30% by volume was added to each well, and stored at -80°C.

(3) Transfer to a 1.5 mL sterile centrifuge tube after 2 hours, add 50 μL of 2×TY containing helper phage, mix well, and place in a 37°C water bath for 30 minutes.

(4) Centrifuge at 3000g for 10min, discard the supernatant, resuspend the cell pellet with 200μL 2×TY (containing 0.1% glucose, 0.1% ampicillin, 0.05% kanamycin), and add the bacterial solution from each tube to a new The 96-well plate was incubated at 25°C with shaking at 250rpm for 20h.

(5) Centrifuge at 3000g for 10min, collect the supernatant from each well into a 1.5mL centrifuge tube, and temporarily store at 4°C for later use.

(6) Take 0.2 μg of EGFR polypeptide to coat a 96-well immune plate, the coating solution is 100 μL/well, and set blank control wells (PBS) and CXCR4 polypeptide (purchased from Shanghai Botai Biotech Co., Ltd.) irrelevant antigen control wells, 4 ℃ overnight.

(7) Pour off the coating solution. Wash 3 times with PBS (discard without stopping after each addition of liquid).

(8) Then add 280 μL of BSA blocking solution with a concentration of 2% by mass and volume, and keep it at room temperature for 2 h.

(9) The 2% (w/v) BSA blocking solution was completely poured out. Wash 3 times with PBS (discard without stopping after each addition of liquid).

(10) Take the monoclonal phage solution prepared in step (5) as the primary antibody, and mix it with 2% (w/v) BSA blocking solution at a volume ratio of 1:3, with 100 μL of the mixed solution per coated well. 1h at room temperature.

(11) After 1 h, wash 4 times with PBST (after each addition of liquid, discard directly without stopping).

(12) Add 100 μL of HRP-labeled phage M13 (M13 Bacteriophage-HRP) antibody (diluted with 2% (w/v) BSA blocking solution at a volume ratio of 1:10000) to each well as secondary antibody, at room temperature for 1 h.

(13) After 1 h, wash 4 times with PBST and 1 time with PBS (after each addition of liquid, discard directly without stopping).

(14) Add 100 μL of TMB chromogenic solution (Biyuntian) to each well, and react at room temperature for 4 min in the dark.

(15) For 4 min, add 50 μL of 1M H ₂ SO ₄ solution to each well.

(16) Use a microplate reader to measure the absorbance at OD450nm. As a result, 25 positive clones were obtained. Figure 2 shows the ELISA results of 64 of the monoclonal phages.

(17) Send the positive clones to BGI for DNA sequencing. The sequenced DNA results were analyzed in NCBI OFR, and the same nucleotide sequences were excluded, and 5 sequences were obtained, which were named aEG1B4, aEG2C7, aEG2E12, aEG4D9 and aEG6B2. The sequences of the 5 Nanobodies are as follows:

aEG1B4：ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGAGTTAACGTTAGCAATGAGGTTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAACCATTGCTAACCATAGCGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGACACTTTATAGTGGTGCTACGAAGCAGTTGGAGTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA；

aEG2C7：ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTTACCTTTAACAATGAGATTATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAAGCATTGCGGCCAATAACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGAGATATGCGGCGGAGCCGCATACGTATTCGATGGGGAACAAGTCGCTGAGGTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA；

aEG2E12：ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATATAGCTCTAACAATGAGTTTATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAGCCATTTCTACGAGAAACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGGGTGTGTCTTATAGGAGGCCCCAGCAGTTGAAGTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA；

aEG4D9：ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGAGATATGCTTAGCCCTGACAATATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAACCATTCATAAGACTGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGGGATTGCGTAGTAGGGGGCTTAGTTCGAAGTACCTGGAGTATTGGGGTCAGGGAACCCCGGTCACCGTCTCGAGCGCGGCCGCA；

aEG6B2：ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTTAACGTTAACCCTAAGTATATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAAGCATTCGTAGCCCTGGCGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGAGTGTTAGTCGGGATGAGAAGTACATGCGCTTTTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA。

Example 5 Obtaining negative control Nanobodies

Negative control Nanobodies were obtained by the methods of Examples 1-4, and only the EGFR polypeptides in (1) and (6) in Example 3 were replaced by synthetic polypeptides of human epidermal growth factor receptor-2 (Her2) ( Shanghai Botai Biological Co., Ltd.) or vascular endothelial growth factor (VEGF) synthetic polypeptide (Shanghai Botai Biological Co., Ltd.), select 2 clones that do not bind to the antigen by ELISA to obtain negative control antibodies.

Nanobodies aHer2-13C1 (with Her2 as an antigen) and aVE201 (with VEGF as an antigen) as negative controls in the experimental example have the amino acid sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The nucleotide sequences encoding the aHer2-13C12 and aVE201 are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

Example 6 Prokaryotic expression and purification of nanobodies expressing anti-EGFR

(1) Preparation of BL21 competent cells

1) Pick a newly activated Escherichia coli DH5α and BL21(DE3) colonies from the LB plate respectively (BL21(DE3) was purchased from Tianjin Tianli Technology Co., Ltd.), inoculated in 5mL LB liquid medium, 37℃, 220rpm Culture overnight (about 12h or so).

2) Inoculate the bacterial suspension at a volume ratio of 1:100, transfer 500 μL of bacterial liquid to 50 mL of LB liquid medium (this step can be scaled up or down according to the required amount), and shake cultured at 37°C 2～3h, to about OD ₆₀₀ =0.5.

3) Transfer the bacterial solution to a 50mL centrifuge tube, place on ice for 10min, centrifuge at 3000g for 5min at 4°C, discard the supernatant, and gently suspend the bacterial cells with 10mL of 0.1mol/L _CaCl2 solution pre-cooled on ice. placed on the 30min.

4) Centrifuge at 3000g at 4°C for 5 min, discard the supernatant, add 3 mL of pre-cooled 0.1 mol/LCaCl ₂ solution, gently suspend the bacterial cells, and let stand on ice for 5 min to complete the preparation of competent cells.

5) Add 3 mL of sterilized and pre-cooled 30% glycerol aqueous solution by volume to 3 mL of the prepared competent cells (i.e. 1:1 volume, so that the final concentration of glycerol is 15%), mix gently, and divide into 100 μL per tube, quickly transferred to -80 ℃ refrigerator for storage.

(2) Construction of Nanobody Recombinant Vector

Prepare PCR reaction system: 10 μL of 5× PrimerStar buffer, 4 μL of dNTP mixture (2.5 mM each), 1 μL of aEG-F primer (10 μM, 5’-GATCCATGGCCCAGGTGCAGCTGT-3’), 1 μL of aEG-R primer (10 μM, 5’-TCTGCGGCCGCGCTCGAGAC -3') 1 μL, template 10 ng, PrimerStar DNA polymerase 0.5 μL, sterile deionized water to make up to 50 μL.

PCR reaction: 96 °C for 10 min; 95 °C for 10 sec, 60 °C for 10 sec, 72 °C for 30 sec, 30 cycles; 72 °C for 5 min.

The PCR product was purified by a PCR product purification kit.

The pET-22b vector (purchased from EMD Biosciences (Novagen)) and the PCR product were subjected to double digestion reaction according to the following scheme. The digestion system was as follows:

2 μg of pET-22b vector plasmid, 5 μL of digestion buffer, 2 μL of NotI, 2 μL of NcoI, and sterile water to make up to 50 μL.

1.4 μg of PCR product, 6 μL of digestion buffer, 2 μL of NotI, 2 μL of NcoI, and sterile water to make up to 60 μL.

The above reaction system was kept at 37°C for 15 minutes. That is, the pET-22b double-enzyme digestion product and the target fragment double-enzyme digestion product are obtained. The digestion product was purified by the digestion product purification kit.

Next, the ligation reaction was carried out, and the system was as follows: pET-22b double-enzyme digestion product 0.03 pmol, target fragment double-enzyme digestion product 0.3 pmol, enzyme ligation buffer 2.5 μL, T4 DNA ligase 1 μL, and sterile water to make up to 25 μL.

The ligation was performed overnight at 4°C to obtain the ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells, spread on the LB plate containing 100 μg/mL ampicillin, and then the colonies were initially screened by primers aEG-F and aEG-R, and the obtained positive clones were expanded and cultured. The plasmid was extracted, re-screened by enzyme digestion reaction, and the positive clones obtained by re-screening were sequenced to obtain the recombinant expression plasmid obtained by recombining the pET-22b vector and the nanobody.

(3) Transformation of competent cells with nanobody recombinant vector

1) Take out the frozen BL21 competent cells from the -80°C freezer and thaw them on ice.

2) Briefly centrifuge the recombinant expression plasmid, take 10 μL of the recombinant expression plasmid into a 1.5 mL EP tube and put it on ice, add 100 μL of competent cells to the EP tube, flick the bottom of the tube to mix well, and place on ice for 30 minutes.

3) Incubate the mixture in a constant temperature water bath at 42°C for 100s, and quickly transfer it to ice to cool for at least 2min. 900 μL of antibiotic-free LB medium was added to the EP tube, and the bacteria were shaken at 37° C. and 220 rpm for 1.5 h.

4) Centrifuge the bacterial solution at 8000g for 1 min to precipitate the bacteria to the bottom of the tube, remove 900 μL of the supernatant, resuspend the bacterial precipitate with the remaining 100 μL of the culture solution, and evenly spread the bacterial solution on a solution containing 0.1% ampicillin and 1% glucose. On LB solid medium, culture at 37°C for 12-16h.

(4) Prokaryotic expression and purification of Nanobodies

1) Pick a single clone of BL21(DE3) containing the recombinant expression plasmid vector and inoculate it into 5 mL of LB liquid medium containing 1% glucose and 100 μg/mL ampicillin, and culture it on a shaker at 37°C and 230 rpm for 13 hours.

2) Inoculate in a ratio of 1:100 by volume, transfer 4 mL of overnight bacterial solution to 400 mL of LB liquid medium containing 100 μg/mL ampicillin, and shake at 37°C and 220 rpm for 2-3 hours to make OD ₆₀₀ =0.6- 0.8.

3) Add 0.25mM IPTG to induce expression, shake and culture at 25°C, 230rpm for 6h, centrifuge at 5000g for 5min, discard the supernatant, resuspend the bacterial cell pellet in 40mL sterilization buffer, ultrasonically disrupt, 40% of ultrasonic power 650W, ultrasonically disrupt for 4s, Pause for 8s, protect the temperature at 10°C, and work for 40min.

4) Collect the broken liquid into a centrifuge tube, centrifuge at 15000g at 4°C for 30 minutes, collect the supernatant into a new centrifuge tube, filter with a 0.22 μm filter, and store at 4°C for purification (storage time should not exceed 4 days).

5) Purify the antibody protein in the crushed supernatant using a nickel column. The nickel column stored at 4° C. was taken out, the preservation solution (20% alcohol) was dropped, and 10 mL of ultrapure water was added for washing, and subsequent purification was started.

Step 1: Equilibrate the column with 10 mL of sterilization buffer.

Step 2: Pass the collected protein supernatant through the column to bind the target protein to the column.

The third step: use 20mL wash buffer (containing 0.02M imidazole) to wash off impurities.

Step 4: Use 5mL of elution buffer (containing 0.2M imidazole) to elute the target protein, and collect 5 tubes of eluate, 1mL/tube. At the same time, the permeate of each step was collected for subsequent detection.

6) Prepare SDS-PAGE electrophoresis loading buffer for different components of proteins in each step, and then perform SDS-PAGE electrophoresis detection respectively. The SDS-PAGE protein electrophoresis image after antibody protein expression and purification is shown in Figure 3.

7) All purified antibody solutions were dialyzed into PBS for cells (pH=7.4, 0.01M), sterilized by filtration using a 0.22 μm syringe filter, and all proteins were stored at -80°C.

8) The BCA method was used to determine the concentration of anti-EGFR antibody protein. The protein solution was adjusted to 1 mg/mL with PBS for subsequent experiments.

(5) Detecting the binding of purified anti-EGFR nanobodies to EGFR polypeptides by ELISA

1) Take 0.2 μg of VEGF, CAMPH, BMP2, ENDOF1, FGF21, HER2, CXCR4, and EGFR fragments (the above fragments are all provided by Shanghai Biotech Co., Ltd.) to coat a 96-well immunoplate, and the coating solution is 100 μL/well. Set up blank control wells (PBS) overnight at 4°C.

2) The remaining steps follow steps 2)-11) in the polyclonal phage ELISA process in Example 3, only replacing the HRP-labeled phage M13 in step 7) with proteinA-HRP protein (1:5000 dilution by volume). The results are shown in Figure 4.

(6) Western Blotting method was used to detect the binding of purified anti-EGFR nanobodies to the complete extracellular segment of EGFR

1) Take 0.5 μg of EGFR complete extracellular segment (purchased from Beijing Yiqiao Shenzhou Biological Co., Ltd.) for SDS-PAGE electrophoresis, transfer to PVDF membrane, and block with 5% nonfat milk powder.

2) Add purified aEG antibody, overnight at 4°C.

3) On the second day, wash with PBST 3 times, 6 min/time.

4) Add secondary antibody protein A-HRP (1:5000) for 1 h at room temperature.

5) Washing with PBST 3 times, 8 min each time.

6) Use ECL luminescent solution (volume ratio of solution A: solution B=1:1) to spread evenly on the PVDF membrane, and place it in a gel imaging system for imaging. The results are shown in Figure 5.

Experimental example 7

The inhibitory effect of nanobodies on the proliferation of human lung cancer cell A549, human breast cancer cell MCF-7 and human prostate cancer cell DU145 was detected by MTT method (A549, MCF-7 and DU145 were purchased from Shanghai Xinyu Biotechnology Co., Ltd.).

(1) 5000 cells were seeded into a 96-well plate (the number of replicates n=3), and cultured overnight in a cell culture incubator at 37°C with a CO ₂ concentration of 5%.

(2) Remove the old medium, add 100 μL of serum-free medium to each well (1640 for A549, DU145, DMEM for MCF-7), and culture for 4 h.

(3) The serum-free medium was replaced with a medium containing 1% FBS of antibodies, added to the corresponding cells, and cultured at 37° C., 5% CO ₂ for 72 h. Four concentrations of each antibody were set: 0 μg/mL, 25 μg/mL, 50 μg/mL and 100 μg/mL. AVE201 and aHer2-13C1 were used as negative control antibodies.

(4) After adding Nanobody for 72 hours, discard the old medium, add 120 μL of MTT-DMEM mixed solution (1 mg/mL MTT:DMEM=volume ratio 1:5) to each well, and incubate at 37°C for 4 hours.

(5) The MTT-DMEM mixture was aspirated, and the plate was air-dried. Add 150 μL DMSO to each well to dissolve the pellet. Place on a destaining shaker for 10 min.

(6) Use a microplate reader to measure the light absorption value OD of each well at a wavelength of 570 nm.

The results are shown in Figure 6. Compared with the group without antibody (0 μg/mL), all five anti-EGFR antibodies can significantly inhibit the proliferation of cancer cells. Among them, aEG2E12 had a significant inhibitory effect on A549 and DU145 at a concentration of 25μg/mL, while significantly inhibited the proliferation of MCF-7 at a concentration of 50μg/mL. The antibody concentration was positively correlated with the inhibitory effect on proliferation. The above results proved that the purified five anti-EGFR nanobodies could inhibit the proliferation of A549, MCF-7 and DU145 cells.

Experimental Example 8 Effect of Nanobodies on Apoptosis of A549, MCF-7 and DU145 Cells

(1) 250,000 cells/well (six-well plate) were seeded, and cultured at 37° C. and 5% CO ₂ overnight to make them adherent.

(2) Remove the old medium, add serum-free medium to each well, culture for 4 hours, add 1% (v/v) FBS medium, so that the medium contains anti-EGFR antibody with a final concentration of 50 μg/mL, using aVE201 and aHer2-13C1 were used as negative control antibodies, and were incubated at 37°C, 5% CO ₂ for 48 h.

(3) Remove the cells from the incubator, remove the old medium, wash once with sterile PBS, add 700 μL of 0.25% (w/v) trypsin solution for digestion, and add medium containing 10% FBS to stop Digestion. Gently pipetting, collect the cell suspension into a sterile 1.5mL EP tube, and centrifuge at 1050g for 5min.

(4) Add 1 mL of sterile PBS to the EP tube containing the cell pellet, resuspend the cells gently with a pipette tip, and centrifuge at 3000 g for 5 min. Repeat the operation once

(5) Resuspend the cells with 195 μL of binding buffer (Binding buffer, 1×), then add 5 μL of Annexin V-FITC, and incubate at room temperature for 15 min in the dark.

(6) Centrifuge at 4°C and 1000 rpm for 5 min, and discard the supernatant. Add 200 μL of binding buffer to resuspend the cells, centrifuge at 1000 g for 5 min at 4°C, and discard the supernatant.

(7) Resuspend the cells in 190 μL of binding buffer, then add 10 μL of propidium iodide, and detect on the machine within 4 hours.

The results are shown in Figure 7, in which, Figures A, C, and E are two-dimensional scatter plots of apoptotic A549, MCF-7, and DU145 cells, respectively. It can be seen that apoptotic cells in the figure are more than those of the control without antibody ( right half). Panels B, D, and F are the apoptotic ratios obtained from panels A, C, and E, respectively. The results showed that the purified five anti-EGFR nanobodies could promote the apoptosis of A549, MCF-7 and DU145 cells.

Experimental Example 9 Effect of Nanobodies on Migration of A549, MCF-7 and DU145 Cells

(1) Take a 12-well plate and draw two parallel lines with a distance of 5mm on the back of the well parallel to the long side. 500,000 cells/well were seeded and cultured overnight at 37°C in 5% CO ₂ to allow them to adhere.

(2) Remove the old medium (1640 for A549, DU145, DMEM for MCF-7), add serum-free medium to each well, and culture for 4 h. The cells were drawn with a 200 μL muzzle line perpendicular to the back of the plate, three gaps per well, and the exfoliated cells were washed with PBS.

(3) The medium containing 1% FBS of the antibody was added to the corresponding cells at the same time, and cultured at 37° C., 5% CO ₂ for 24 h. Four concentrations were set for each antibody: 0 μg/mL, 25 μg/mL, 50 μg/mL, and 100 μg/mL. aVE201 and aHer2-13C1 were used as negative control antibodies.

(4) Take pictures under an inverted microscope, measure and record the distance of the gap.

The results are shown in Figures 8 and 9. As the concentrations of the five Nanobodies increased, the migration rates of A549, MCF-7 and DU145 cells all decreased, and the decrease in the migration rates of A549 and DU145 cells was particularly significant. The migration rates were negatively correlated, and the results demonstrated that the purified five anti-EGFR nanobodies could inhibit the migration of A549, MCF-7 and DU145 cells.

Experimental Example 10 The effect of nanobodies on the migration of A549, MCF-7 and DU145 cells

(1) Take out the sterile cell culture Transwell chamber and place it in a 24-well plate, inoculate 200 μL (2% FBS) of the mixed suspension of the corresponding antibody and 50 million cells/well (24-well plate) in the upper chamber, and in the lower chamber 500 μL of medium (containing 10% FBS by volume) was added, and cultured at 37° C., 5% CO ₂ for 24 h. Four concentrations were set for each antibody: 0 μg/mL, 25 μg/mL, 50 μg/mL, and 100 μg/mL. aVE201 and aHer2-13C1 were used as negative control antibodies.

(2) Take out the culture plate, hold the chamber with tweezers, wash twice with PBS, and wash off the medium.

(3) 4% paraformaldehyde was fixed for 15 min, and then washed twice with PBS.

(4) 2% crystal violet was stained for 30 minutes in the dark, and then washed twice with PBS.

(5) Wipe off the cells inside the chamber with a cotton swab, and place the chamber under an inverted microscope to take pictures.

(6) 100 μL of 20% acetic acid solution was used to wash off the cell staining solution on the outside of each upper chamber.

(7) The eluate was sucked into the corresponding well of the 96-well plate, and the OD570 nm was measured by the microplate reader, and the measurement was repeated 3 times.

The results are shown in Figures 10 and 11, as the concentration of the five nanobodies increased, the migration rate of cancer cells decreased significantly. The above results proved that the purified five anti-EGFR nanobodies could inhibit the proliferation of A549, MCF-7 and DU145 cells. migrate.

Experimental Example 11 Effect of Nanobodies on Invasion of MCF-7, A549 and DU-145 Cells

(1) Place a sterile Transwell chamber into a 24-well plate, add 50 μL of Matrigel (1:9 dilution) to each well, gently shake the culture plate to cover the gel evenly, and incubate at 37°C overnight to allow the gel to solidify.

(2) Take out the culture plate covered with the gel, and inoculate the upper chamber with 50,000 cells/well of the corresponding antibody-treated cell suspension in 200 μL (2% FBS medium), each antibody is set to 4 concentrations: 0 μg/mL , 25 μg/mL, 50 μg/mL and 100 μg/mL, add 500 μL of medium (10% FBS) to the lower chamber, and culture at 37° C., 5% CO ₂ for 24 h. aVE201 and aHer2-13C1 were used as negative control antibodies.

(3) Take out the culture plate, hold the chamber with tweezers, and wash twice with PBS to wash off the medium.

(4) 4% paraformaldehyde was fixed for 15 minutes, 500 μL per well, and the upper chamber was washed twice with PBS after fixation.

(5) 2% crystal violet staining, protected from light for 30 min, and washed twice with PBS.

(6) Gently wipe off the cells on the inner side of the upper chamber with a cotton swab, and place the chamber under an inverted microscope to take pictures.

(7) 100 μL of 20% acetic acid solution to wash off the cell staining solution on the outside of each upper chamber.

(8) The eluate was sucked into the corresponding well of the 96-well plate, and the OD570 nm was measured with a microplate reader.

The results are shown in Figures 12-13. Compared with the group without antibody (0 μg/mL), the five anti-EGFR antibodies can significantly inhibit the invasion of cancer cells. Among them, aEG2E12 significantly inhibited the migration of three cancer cells at a concentration of 25 μg/mL, aEG4D9 significantly inhibited the migration of A549 and DU145 at a concentration of 25 μg/mL, and significantly inhibited the migration of MCF-7 at a concentration of 50 μg/mL. The above results demonstrated that the purified five anti-EGFR nanobodies could inhibit the invasion of A549, MCF-7 and DU145 cells.

Experimental Example 12 The effect of nanobodies on mouse lung cancer model

(1) A549 cells were digested with trypsin and centrifuged at 1050 g for 5 min.

(2) Discard the supernatant, resuspend the collected cells with PBS, and place in a centrifuge at 1050 g for 5 min.

(3) Repeat step (2). Discard the supernatant, add an appropriate amount of PBS to resuspend the cell pellet and adjust the cell density to 5×10 ⁷ /mL.

(4) Select 4-week-old male Balb/C nude mice (purchased from Guangdong Provincial Medical Laboratory Animal Center), use a 1 mL sterile syringe to draw 100 μL of cell suspension (containing 5×10 ⁶ A549 cells), and subcutaneously inject The cell suspension was injected into the right armpit of the mice for tumor formation. The mice were placed in an SPF animal laboratory and raised as usual, and the tumor volume of the mice was measured with a vernier caliper every three days.

(5) Select antibodies aEG2E12 and aEG4D9 with the best in vitro functional test results for animal experiments, and aHer2-13C1 and aVE201 as negative controls. After the tumor volume reached 100 mm ³ , the mice were randomly divided into 6 groups with 5 mice in each group. Group 1 was blank control group (PBS), group 1 was positive control group (cisplatin, DDP), group 2 was experimental antibody group (aEG2E12, aEG4D9), and group 2 was negative control antibody group (aHer2-13C1, aVE201) . Administer via the tail vein using an insulin syringe. The dose of each mouse in the experimental antibody group and the negative control nanobody group was 10 mg/kg, each in the positive control group was dosed with 2 mg/kg, and each in the blank control group was injected with 100 μL of PBS buffer for cells. The dosing period was 24 days, and the dosing frequency was 3 days/time, and the tumor volume of the mice in each group was recorded at the same time. Mice were sacrificed after administration, tumors were excised, photographed and weighed.

The results are shown in Figure 14. After administration, the increase of tumor volume in the experimental antibody group was significantly slower than that in the blank control group and the negative control antibody group, and the final volume was also significantly smaller, proving that the purified aEG2E12 and aEG4D9 nanobodies can inhibit A549 Tumor size in a cell-induced mouse lung cancer model.

The preparation method of the reagent used in the embodiment is as follows:

(1) PBS/PBST (pH 7.4): KH ₂ PO ₄ 0.24 g, NaCl 8 g, KCl 0.2 g, Na ₂ HPO ₄ ·12H ₂ O 9.07 g.

Weigh the reagent, add 900mL of deionized water to dissolve, and make up to 1L.

PBST: Add Tween-20 with a final concentration of 0.1% to the prepared PBS buffer, mix well, sterilize at 121°C, and store at 4°C.

(2) TBS/TBST: Tris base 6.05 g, NaCl 21.93 g.

Dissolve the reagent with 400 mL of deionized water, adjust the pH to 7.4 with dilute hydrochloric acid, and dilute to 500 mL.

TBST: Add 500 μL of Tween-20 to 500 mL of TBS buffer and mix well.

(3) LB liquid medium: 2 g of NaCl, 2 g of tryptone, 1 g of yeast extract, and 200 mL of ultrapure water.

LB solid medium: Add 4g agar powder to the LB liquid medium formula.

(4) 20% glucose: 200 g of glucose powder was weighed, dissolved in 1 L of deionized water, and sterilized by filtration using a 0.22 μM filter.

(5) 100 μg/mL ampicillin solution: Weigh 1 g of the powder, dissolve it in 10 mL of deionized water to prepare a solution of 100 mg/mL, filter and sterilize it with a 0.22 μM filter, divide it into 1 mL tubes, and store at -20°C.

(6) SDS-PAGE electrophoresis solution: 94 g of glycine, 30.2 g of Tris base, and 5 g of SDS.

Dissolve the reagent in 900 mL of deionized water and make up to 1 L. This reagent is a 5× electrophoresis solution and is stored at 4°C.

(7) SDS-PAGE transfer solution: Tris base 15.14g, glycine 72g.

Dissolve the reagent in 900 mL of deionized water, dilute to 1 L, and store at 4°C. This reagent is a 5× electrophoresis solution.

(8) 500M IPTG: 11.915 g of IPTG powder was weighed, dissolved in 100 mL of deionized water, and sterilized by filtration using a 0.2 μm filter.

(9) 1M H ₂ SO ₄ : 10 mL of concentrated sulfuric acid was slowly added to 187 mL of deionized water.

(10) 100×PMFS: Weigh 1.74 g of PMSF, dissolve in 100 mL of isopropanol, and store at -20°C.

(11) Protein expression purification buffer

Bacteria breaking buffer: Tris base 2.42g, NaCl 14.6g, 100×PMSF 10mL

Dissolve the reagent in 900 mL of ultrapure water, adjust the pH to 7.45 with dilute hydrochloric acid, dilute to 1 L, and store at 4°C.

Loading buffer: the same as sterilization buffer.

Washing buffer: take 20 mL of loading buffer and add 200 μL of imidazole stock solution (2M).

Elution buffer: take 9 mL of loading buffer and add 1 mL of imidazole stock solution (2M).

(12) 2% BSA: 2% bovine serum albumin powder (w/v) was added to PBS buffer.

(13) 30% glycerol solution (glycerol-PBS): measure 15 mL of glycerol, add 35 mL of PBS buffer (pH=7.4), filter and sterilize using a 0.22 μm filter, and store at 4°C.

(14) 10% ammonium persulfate (APS): 0.5 g of ammonium sulfate, 5 mL of ddH ₂ O. Aliquot into 500 μL tubes and store at -20°C.

(15) 1M Tris-HCl (PH=8.8/PH=6.8): Weigh 0.2g of Tris-Base powder, dissolve it in 900mL of deionized water, and use dilute hydrochloric acid to adjust the pH to 6.8 or 8.8. Dilute to 1L, 121 Sterilize at ℃ and store at 4℃.

(16) Coomassie brilliant blue staining solution: 1 g of Coomassie brilliant blue R-250, 250 mL of isopropanol, 100 mL of glacial acetic acid, and 650 mL of ddH ₂ O.

(17) Coomassie brilliant blue staining solution: 100 mL of glacial acetic acid, 50 mL of ethanol, and 850 mL of ddH ₂ O.

(18) 5× protein loading buffer: pH 6.8, 1M Tris-HCl 12.5mL, SDS 5g, bromophenol blue 0.25g, glycerol 25mL. Add ddH ₂ O to the reagent, dilute to 50 mL, and store at room temperature. Take 500 μL before use and optionally add 25 μL of β-mercaptoethanol.

(19) TYE solid medium (400 mL): 5 g of peptone, 2.5 g of yeast powder, 4 g of agar powder, and 400 mL of ddH ₂ O. Sterilize at 121°C, add 1% glucose solution and 100 μg/mL ampicillin when cooled to 50°C, mix well, pour the plate, and store in a refrigerator at 4°C.

(20) 2×TY medium (100 mL): 1.6 g of peptone, 1 g of yeast powder, 0.5 g of NaCl, and 100 mL of ddH ₂ O. Sterilize at 121°C.

(21) 20% PEG/NaCl solution (500 mL): PEG600 100 g, NaCl 73 g, ddH ₂ O 400 mL.

After dissolving in deionized water, dilute to 500 mL, sterilize at 121°C for 20 min under high temperature and high pressure, and store at room temperature.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

sequence listing

<110> Jinan University

<120> An anti-human EGFR nanobody and its application

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Asn Glu Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

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Ser Val Lys GlyArgPheThr Ile Ser Arg Asp Asn Ser Lys AsnThr

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cgtctctcctgtgcagcctccggagttaacgttagcaatgaggttatgagctgggtccgc 120

caggctccagggaagggtctagagtgggtatcaaccattgctaaccatagcggtagcaca 180

tactacgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacg 240

ctgtatctgcaaatgaacagcctgcgtgccgaggacaccgcggtatattattgcgcgaca 300

ctttatagtggtgctacgaagcagttggagtattggggtcagggaaccctggtcaccgtc 360

tcgagcgcgg ccgca 375

<210> 9

<211> 393

atggcccaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctg 60

cgtctctcctgtgcagcctccggatttacctttaacaatgagattatggcctgggtccgc 120

caggctccagggaagggtctagagtgggtatcaagcattgcggccaataacggtagcaca 180

tactacgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacg 240

ctgtatctgcaaatgaacagcctgcgtgccgaggacaccgcggtatattattgcgcgaga 300

tatgcggcggagccgcatacgtattcgatggggaacaagtcgctgaggtattggggtcag 360

ggaaccctggtcaccgtctcgagcgcggccgca 393

<210> 10

<211> 375

atggcccaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctg 60

cgtctctcctgtgcagcctccggatatagctctaacaatgagtttatggcctgggtccgc 120

caggctccagggaagggtctagagtgggtatcagccatttctacgagaaacggtagcaca 180

tactacgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacg 240

ctgtatctgcaaatgaacagcctgcgtgccgaggacaccgcggtatattattgcgcgggt 300

gtgtcttataggaggccccagcagttgaagtattggggtcagggaaccctggtcaccgtc 360

tcgagcgcgg ccgca 375

<210> 11

<211> 381

atggcccaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctg 60

cgtctctcctgtgcagcctccggagatatgcttagccctgacaatatgacctgggtccgc 120

caggctccagggaagggtctagagtgggtatcaaccattcataagactgacggtagcaca 180

tactacgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacg 240

ctgtatctgcaaatgaacagcctgcgtgccgaggacaccgcggtatattattgcgcggga300

ttgcgtagtagggggcttagttcgaagtacctggagtattggggtcagggaaccccggtc 360

accgtctcgagcgcggccgc a 381

<210> 12

<211> 372

atggcccaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctg 60

cgtctctcctgtgcagcctccggatttaacgttaaccctaagtatatgacctgggtccgc 120

caggctccagggaagggtctagagtgggtatcaagcattcgtagccctggcggtagcaca 180

tactacgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacg 240

ctgtatctgcaaatgaacagcctgcgtgccgaggacaccgcggtatattattgcgcgagt 300

gttagtcgggatgagaagtacatgcgcttttggggtcagggaaccctggtcaccgtctcg 360

agcgcggccg ca 372

<210> 13

<211> 387

atggcccaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctg 60

cgtctctcctgtgcagcctccggatatagcgttagctctgagaatatgggctgggtccgc 120

caggctccagggaagggtctagagtgggtatcaggcattttggcgggagacggtagcaca 180

tactacgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacg 240

ctgtatctgcaaatgaacagcctgcgtgccgaggacaccgcggtatattattgcgcgaga 300

tttacgtcgggtcaggggtcgttgcggtccgaccccatccggtcttggggtcagggaacc 360

ctggtcaccgtctcgagcgcggccgca 387

<210> 14

<211> 384

atggcccaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctg 60

cgtctctcctgtgcagcctccggagttagcgttagcaatgaggctatgggctgggtccgc 120

caggctccagggaagggtctagagtgggtatcaagcattactgaccaaagcggtagcaca 180

tactacgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacg 240

ctgtatctgcaaatgaacagcctgcgtgccgaggacaccgcggtatattattgcgcgaga 300

gggcagcgtcgtaggcagatgcattcgtacaaggtcagctcttggggtcagggaaccctg 360

gtcaccgtctcgagcgcggccgca 384

<210> 15

<211> 30

Gln Ala Trp Pro Glu AsnArgThr Asp Leu His Ala Phe Glu Asn Leu

1 5 10 15

Glu Ile IleArgGlyArgThr Lys Gln His Gly Gln Phe Ser

20 25 30

<210> 16

<211> 24

<223>aEG-F primer

gatccatggcccaggtgcagctgt 24

<210> 17

<211> 20

<223>aEG-R primer

tctgcggccg cgctcgagac 20

Claims (5)

1. A nanobody against human EGFR, characterized in that: the nano antibody for resisting the human EGFR is named as aEG4D9 nano antibody; or an antibody formed by combining an aEG4D9 nanobody with at least one of aEG1B4 nanobodies, aEG2C7 nanobodies and aEG6B2 nanobodies;

the amino acid sequence of the aEG1B4 nano antibody is shown as SEQ ID NO. 1;

the amino acid sequence of the aEG2C7 nano antibody is shown as SEQ ID NO. 2;

the amino acid sequence of the aEG4D9 nano antibody is shown as SEQ ID NO. 4;

the amino acid sequence of the aEG6B2 nano antibody is shown as SEQ ID NO. 5.

2. The nucleic acid encoding the nanobody against human EGFR according to claim 1, wherein: is nucleic acid encoding said aEG4D9 nanobody; or a nucleic acid formed by combining a nucleic acid encoding the aEG4D9 nanobody with at least one of a nucleic acid encoding the aEG1B4 nanobody, a nucleic acid encoding the aEG2C7 nanobody and a nucleic acid encoding the aEG6B2 nanobody.

3. The nucleic acid encoding the nanobody against human EGFR according to claim 2, wherein:

the sequence of the nucleic acid for coding the aEG1B4 nano antibody is shown as SEQ ID NO. 8;

the sequence of the nucleic acid for coding the aEG2C7 nano antibody is shown as SEQ ID NO. 9;

the sequence of the nucleic acid for coding the aEG4D9 nano antibody is shown as SEQ ID NO. 11;

the sequence of the nucleic acid for coding the aEG6B2 nano antibody is shown as SEQ ID NO. 12.

4. The method for preparing an anti-human EGFR nanobody according to claim 1, comprising the steps of: synthesizing nucleic acid for encoding the anti-human EGFR nano antibody by a gene synthesis method, then cloning the nucleic acid to an expression plasmid vector, and transforming the nucleic acid to a host cell for expression and purification to obtain the anti-human EGFR nano antibody; or the antihuman EGFR nano antibody is directly synthesized by a polypeptide synthesis method.

5. The use of the anti-human EGFR nanobody according to claim 1 for the preparation of an antibody drug for the treatment of diseases characterized by EGFR overexpression, characterized by: the disease characterized by EGFR overexpression is lung cancer, head and neck cancer, colon cancer, brain tumor, breast cancer or prostate cancer.

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