CN112521504A

CN112521504A - Anti-human EGFR nano antibody and application thereof

Info

Publication number: CN112521504A
Application number: CN202011382799.7A
Authority: CN
Inventors: 魏星; 刘雪; 陈涛
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2018-09-27
Filing date: 2018-09-27
Publication date: 2021-03-19
Anticipated expiration: 2038-09-27
Also published as: CN112457405B; CN112457404A; CN112457405A; CN112552402B; CN109336976A; CN112521504B; CN109336976B; CN112457404B; CN112552402A

Abstract

The invention discloses a nano antibody for resisting human EGFR and application thereof. The nano antibody is aEG1B4 nano antibody with the amino acid sequence shown as SEQ ID NO. 1; or an antibody formed by combining aEG1B4 nano antibody with an amino acid sequence shown as SEQ ID NO.1, aEG2C7 nano antibody with an amino acid sequence shown as SEQ ID NO. 2, aEG2E12 nano antibody with an amino acid sequence shown as SEQ ID NO. 3, aEG4D9 nano antibody with an amino acid sequence shown as SEQ ID NO. 4 and aEG6B2 nano antibody with an amino acid sequence shown as SEQ ID NO. 5. The nano antibody has the advantages of high affinity, specificity and low immunogenicity, better stability, simple structure, easy engineering modification and the like, and can be better applied to the development of antibody drugs.

Description

Anti-human EGFR nano antibody and application thereof

The application is a divisional application of Chinese patent application with the application number of 201811129703.9 and the name of anti-human EGFR nano antibody and application thereof.

Technical Field

The invention belongs to the technical field of biology, and relates to a nano antibody for resisting human EGFR and application thereof.

Background

The term cancer was proposed over 400 years before the era and has not been overcome over 2000, and the threat to human health is now increasingly apparent. In recent years, studies have found that Epidermal Growth Factor (EGFR) is overexpressed in various solid tumor cells and plays an important role in the development and progression of cancer, and is positively correlated with poor prognosis and drug resistance. Overexpression of EGFR in cancer cells can result in sustained activation of the EGFR/EGF signaling pathway, promoting the proliferative, migratory and invasive activities of cancer cells, and can also promote the secretion of VEGF factors, resulting in the vascularization of the cancer tissue microenvironment. At present, an antibody targeting EGFR has a good effect on clinical treatment of non-small cell lung cancer, but a chimeric antibody has certain immunogenicity, and the micro focus cannot be effectively eliminated due to the poor permeability of the large molecular weight.

A Nanobody (Nb) is a genetically engineered antibody containing only a single domain, and can bind to an antigen with high affinity and specificity. The nano antibody has small molecular weight, so that the nano antibody is convenient to express in escherichia coli, and the production cost of the antibody is greatly reduced. Meanwhile, the antibody has a simple structure and is beneficial to further modification. The characteristic of small molecular volume of the nano antibody also improves the tissue penetration capability of the antibody in the treatment process. Therefore, the anti-EGFR nano antibody has high medical application value in EGFR targeted cancer treatment.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a human EGFR nano antibody.

The invention also aims to provide application of the anti-human EGFR nanobody.

The purpose of the invention is realized by the following technical scheme: a nanometer antibody for resisting human EGFR is named as aEG1B4 nanometer antibody; or an antibody formed by combining aEG1B4 nanobody with at least one of aEG2C7 nanobody, aEG2E12 nanobody, aEG4D9 nanobody and aEG6B2 nanobody;

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 aEG2E12 nano antibody is shown as SEQ ID NO. 3;

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.

The nucleotide sequence of the nano antibody for encoding the anti-human EGFR is the nucleotide sequence of the aEG1B4 nano antibody; or a nucleotide sequence formed by combining the nucleotide sequence coding the aEG1B4 nano antibody with at least one of the nucleotide sequence coding the aEG2C7 nano antibody, the nucleotide sequence coding the aEG2E12 nano antibody, the nucleotide sequence coding the aEG4D9 nano antibody and the nucleotide sequence coding the aEG6B2 nano antibody.

The nucleotide sequence of the aEG1B4 nano antibody is preferably shown as SEQ ID NO. 8.

The nucleotide sequence of the aEG2C7 nano antibody is preferably shown as SEQ ID NO. 9.

The nucleotide sequence of the aEG2E12 nano antibody is preferably shown as SEQ ID NO. 10.

The nucleotide sequence of the aEG4D9 nano antibody is preferably shown as SEQ ID NO. 11.

The nucleotide sequence of the aEG6B2 nano antibody is preferably shown as SEQ ID NO. 12.

The nucleotide sequences for coding the aEG1B4 nanobody, the aEG2C7 nanobody, the aEG2E12 nanobody, the aEG4D9 nanobody and the aEG6B2 nanobody respectively consist of 375, 393, 375, 381 and 372 bases, and correspondingly coded amino acids are 125, 131, 125, 127 and 124. The aEG1B4 antibody contains 3 complementarity determining groups (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 3 complementarity determining groups, 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 3 complementarity determining groups, 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 3 complementarity determining groups, 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 3 CDRs, of which the amino acid encoding CDR1 is FNVNPKYMT, the amino acid encoding CDR2 is SIRSPG, and the amino acid encoding CDR3 is SVSRDEKYMRF.

The anti-human EGFR nano antibody contains the same framework region: the amino acid encoding FR1 was MAQVQLLESGGGLVQPGGSLRLSCAASG, the amino acid encoding FR2 was WVRQAPGKGLEWVS, the amino acid encoding FR3 was GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA, and the amino acid encoding FR4 was WGQGTLVTVSSAAA.

The preparation method of the anti-human EGFR nano antibody comprises the following steps: synthesizing the nucleotide for coding the anti-human EGFR nano antibody by a gene (DNA) synthesis method, then cloning the nucleotide onto an expression plasmid vector, and transforming the expression plasmid vector into host cells for expression and purification to obtain the anti-human EGFR nano antibody; the antihuman EGFR nano antibody can also be directly synthesized by a polypeptide synthesis method.

The anti-human EGFR nano antibody is applied to the preparation of antibody drugs for treating EGFR overexpression characteristic diseases.

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

The cancer is an EGFR high expression tumor.

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

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

1. the invention can obtain the fully human monoclonal nano antibody without using antigen to immunize human body by the nano antibody which is screened from the human nano antibody library by a phage display method and interacts with the EGFR extracellular part structural domain (domain) III, the molecular weight of the nano antibody is about 13kDa, only the nano antibody only contains a single structural domain, and the nano antibody has high affinity, specificity and low degree of immunogenicity.

2. The nano antibody provided by the invention has the advantages of good stability, easiness in modification, better tissue infiltration capacity and the like.

3. The nano antibody provided by the invention is humanized, so that the nano antibody has no immunogenicity in a human body and can be better applied to the development of anti-tumor antibody medicines.

4. The nano antibody provided by the invention can be used for binding the prokaryotic host with high expression of protein to perform antibody expression, so that the production cost of the antibody can be obviously reduced, and the application of the antibody is promoted.

Drawings

FIG. 1 is a diagram showing the screening and enrichment results of polyclonal ELISA analysis of a nanobody library; wherein, the PBS hole is a blank control, and the EGFR hole is coated with the synthesized EGFR polypeptide; the primary antibody is a polyclonal phage antibody obtained by amplification and purification after screening of each round of libraries, and the secondary antibody is an antibody of an HRP-labeled anti-phage M13.

FIG. 2 is a graph showing the results of monoclonal phage ELISA screening positive clones that bind to EGFR polypeptide; wherein each monoclonal comprises: PBS (blank control wells), CXCR4 polypeptide (irrelevant antigen control wells), EGFR polypeptide (antigen wells of interest).

FIG. 3 is an electrophoresis chart of SDS-PAGE proteins after antibody protein expression and purification; wherein panel a is an electropherogram of aEG1B4, panel B is an electropherogram of aEG2C7, panel C is an electropherogram of aEG2E12, panel D is an electropherogram of aEG4D9, panel E is aEG6B2, and in panels a-E: lane 1 is Marker, lane 2 is uninduced whole protein, lane 3 is induced whole protein, lane 4 is induced disrupted precipitate, lane 5 is induced disrupted supernatant, lane 6 is column-over protein, lane 7 is wash-off protein, and lanes 8-12 are elution protein tubes.

FIG. 4 is a graph showing the detection of the binding of purified anti-EGFR nanobody to EGFR polypeptide by ELISA; each antibody included a blank control well PBS, 7 irrelevant antigen control wells: VEGF, CAMPH, BMP2, endf 1, FGF21, HER2, and CXCR4 peptide fragments, antigen wells of interest: an EGFR polypeptide.

FIG. 5 is a diagram of the Western Blotting method for detecting the binding between the purified anti-EGFR nanobody and the EGFR whole extracellular domain; wherein, panel a is aEG1B4, panel B is aEG2C7, panel C is aEG2E12, panel D is aEG4D9, panel E is aEG6B2, in panels a-E: lane 1 is protein marker, lane 2 is EGFR whole extracellular domain protein; the primary antibody was a purified anti-EGFR antibody and the secondary antibody was proteinA-HRP.

FIG. 6 is a graph showing the results of MTT method for detecting the effect of anti-EGFR nanobody on cancer cell proliferation; wherein, panel A is a graph showing the effect of nanobodies on the proliferation of A549 cells, panel B is a graph showing the effect of nanobodies on the proliferation of MCF-7 cells, and panel C is a graph showing the effect of nanobodies on the proliferation of DU145 cells; aVE201 and aHer2-13C1 as negative control antibodies; *: p <0.05vs 0 μ g/mL,.: p <0.01vs 0 μ g/mL (n ═ 3).

FIG. 7 is a diagram showing the effect of detecting the nano-antibody on cancer cell apoptosis by flow cytometry and Annexin V/PI double staining method; wherein, FIG. A, C, E is a two-dimensional scattergram of apoptotic A549, MCF-7 and DU145 cells, respectively, and FIG. B, D, F is the apoptosis ratio derived from FIG. A, C, E, respectively; each cell included no antibody control (control), experimental antibody (50. mu.g/mL): aEG1B4, aEG2C7, aEG2E12, aEG4D9, and aEG6B2, negative control antibody group (50 μ g/mL): aVE201 and aHer2-13C 1; *: p <0.05vs control,.: p <0.01vs control (n ═ 3).

FIG. 8 is a photograph showing the results of detecting the migration of cancer cells by nanobody in the scratch healing process; FIGS. A to C are photographs showing migration results of A549 cells, MCF-7 cells and DU145 cells, 0h and 24h, respectively.

FIG. 9 is a graph of mobility results calculated from the scratch lengths of FIG. 8; wherein panels A-C are A549, MCF-7 and DU145 cells, respectively; *: p <0.05vs 0 μ g/mL,.: p <0.01vs 0 μ g/mL (n ═ 3).

FIG. 10 is a photograph showing the results of detecting the migration of cancer cells by nanobody using the Transwell method; in which, panels A-C are photographs showing the migration results of A549 cells, MCF-7 cells and DU145 cells treated with nanobodies at various concentrations, respectively.

FIG. 11 is a graph of the trend of the absorbance of the antibody obtained from FIG. 10; wherein panels A-C are A549, MCF-7 and DU145 cells, respectively; *: p <0.05vs 0 μ g/mL,.: p <0.01vs 0 μ g/mL (n ═ 3).

FIG. 12 is a photograph showing the effect of the Transwell method on the invasion of cancer cells by detecting the effect of the nanobody; in these graphs, A to C are photographs showing the results of A549 cells, MCF-7 cells and DU145 cells treated with nanobodies at respective concentrations.

FIG. 13 is a graph of the trend of the absorbance of the antibody obtained from FIG. 12; wherein panels A-C are A549, MCF-7 and DU145 cells, respectively; *: p <0.05vs 0 μ g/mL,.: p <0.01vs 0 μ g/mL (n ═ 3).

FIG. 14 is a graph showing the results of the tumor size inhibition of the nanobody using a mouse model of lung cancer; wherein panel a is a plot of the change in volume of the tumor after administration, panel B is the tumor size of each group at the end of the administration cycle, and panel C is the tumor weight of each group after the end of the administration cycle; *: p <0.05vs 0 μ g/mL,.: p <0.01vs 0 μ g/mL (n-4/n-5).

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

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

EXAMPLE 1 preparation of helper phage

(1) The glycerol-preserved TG1 E.coli cloned strain was removed from the-80 ℃ freezer, smeared onto TYE non-resistant plates by four-zone streaking, and cultured for 13h at 37 ℃ in an inverted manner.

(2) A single clone of TG1 was picked from the plate and inoculated into 5mL of 2 XTY non-resistant liquid medium and cultured at 230rpm for 13 hours at 37 ℃.

(3) TG1 bacterial liquid was added in volumeTransferring the strain into 5mL of 2 XTY non-resistant liquid medium at a ratio of 1:100, culturing at 37 deg.C and 230rpm for 2h (bacterial liquid OD)₆₀₀About 0.5).

(4) 200 μ L of TG1 bacterial liquid (OD)₆₀₀Ca. 0.5) into a 1.5mL centrifuge tube, 10 μ L of KM13 helper phage (1 × 10) was added¹³pfu/mL), put into preheated 37 ℃ water for warm bath for 30 min.

(5) Preparing soft agar, cooling to about 40 ℃, pouring the TG1 bacterial liquid treated in the step (4) into the soft agar, mixing, pouring the mixture into a prepared TYE solid culture plate containing 50 mu g/mL kanamycin resistance, standing at room temperature to solidify the mixture, and carrying out inverted culture at 37 ℃ for 13 hours.

(6) Selecting a plaque, and adding 5ml of strain G1 (OD)₆₀₀0.5), shaking at 230rpm and 37 deg.C for 2 h.

(7) The whole amount of the bacterial suspension obtained in step (6) was transferred to 500mL of 2 XTY medium, shake-cultured at 37 ℃ and 230rpm for 2 hours, and then 500. mu.L of kanamycin (concentration in the medium is 50. mu.g/mL) was added, and culture was carried out at 30 ℃ and 230rpm for 20 hours.

(8) Centrifuging the bacterial solution at 3300g for 20min, collecting supernatant, mixing with 20% PEG/NaCl solution at a volume ratio of 1:4, and standing on ice for 4 h.

(9) Then, the mixture was centrifuged at 3300g for 30min, and the precipitate was collected, resuspended in 1mL of sterile PBS (pH 7.4, 0.01M), centrifuged at 4000g for 5min, and the supernatant was collected as helper phage.

Example 2 amplification of phage Nanobody libraries

(1) TG1 bacteria (human nanobody phage library, Source bioscience, London, UK) containing antibody plasmids were thawed on ice, transferred to 500mL of 2 XTY liquid medium (containing ampicillin 0.1% by mass/volume and glucose 1% by mass/volume), and shake-cultured at 37 ℃ and 230rpm for 2.5h (OD)₆₀₀0.5), and then added thereto in an amount of 2 × 10¹²The helper phage was washed in water at 37 ℃ for 30 min.

(2) The bacterial liquid is subpackaged into 250 mL/bottle, 3200g is centrifuged for 10min, the supernatant is removed, 500mL of 2 XTY culture medium (containing ampicillin with the mass volume ratio of 0.1 percent and kanamycin with the mass volume ratio of 0.05 percent) is used for resuspension and precipitation, and the shaking culture is carried out at 25 ℃ and 220rpm for 20 h.

(3) The bacterial liquid 3200g was centrifuged for 20min, the supernatant was collected, phages were purified by the same method as in steps (8) to (9) of example 1, and the prepared library phages were stored at-80 ℃.

(4) And (5) measuring the titer of the antibody library. The first method comprises the following steps: the phage titer in the prepared pool was estimated using a spectrophotometer to measure the light absorbance at 260nm, the pool phage titer formula: number of phage/mL ═ OD₂₆₀X dilution factor X22.14X 10¹⁰. And the second method comprises the following steps: using the cloning technique, after the phage was diluted to a certain concentration, TG1 bacteria infecting the logarithmic growth phase were spread on a solid plate containing ampicillin, cultured overnight at 37 ℃ and estimated by the number of clones. The titer of the amplified antibody library is 4.8 multiplied by 10¹³pfu/mL。

Example 3 screening of Nanobodies against EGFR fragments from phage Nanobody libraries

(1) Screening of anti-EGFR Nanobody phage

1) EGFR polypeptides (purchased from shanghai potita bio, having the amino acid sequence shown in SEQ ID No. 15) were coated in immune tubes (nunc) (first round: 0.1 mg/mL; second and third wheels: 0.05 mg/mL; fourth, fifth round: 0.025 mg/mL; ) PBS (control) tubes were set. 4 ℃ overnight.

2) The coating solution was poured out, washed three times with 4.5mL of PBS per tube (after each addition of the solution, the solution was discarded without retention), 4.5mL of BSA blocking solution with a concentration of 2% by mass/volume was added to each tube, and the tube was left at room temperature for 2 hours.

3) The BSA blocking solution was decanted and each tube was washed three times with 4.5mL PBS (after each addition of liquid, discarded without retention).

4) 4mL of a solution containing 5X 10¹²pfu phage in 2% BSA, room temperature standing for 1 h.

5) PBST was used for 10 washes (after each addition of liquid, discarded without retention).

6) Add 500. mu.L of 1mg/mL trypsin solution (trypsin solution) per tube and elute 10min with inverted digestion.

7) Collecting the phage eluateIn a sterile 1.5ml EP tube, 125. mu.L of phage eluate was taken and added to 875. mu.L of TG1 bacteria (OD)₆₀₀0.5), mixing, and putting into preheated 37 ℃ water for warm bath for 30 min.

8) Diluting the mixed bacterial liquid according to the experiment requirement, and then coating 2 mu L of the infected bacterial liquid on a TYE solid plate containing 1% of glucose and 0.1% of ampicillin for identifying and screening results.

9) The remaining experimental infection was spread on the same other TYE solid plate and incubated overnight at 37 ℃.

10)2mL of 2 XTY (containing 15% glycerol) was added to the plate from step (9), and all bacteria were scraped off and collected in a sterile 1.5mL centrifuge tube. Then, 50. mu.L of the culture broth was inoculated into 50mL of a fresh 2 XTY liquid medium (containing 1% glucose and 0.1% ampicillin) and cultured with shaking at 37 ℃ and 230rpm for about 2 hours to adjust the bacterial concentration to OD₆₀₀0.5. After 2h, 10mL of the bacterial solution is taken from 50mL into a 50mL centrifuge tube, then the helper phage is added into the centrifuge tube, and then the centrifuge tube is placed into a standing water bath at 37 ℃ for 30 min.

11) Centrifuging at 3000g for 15min, discarding supernatant, resuspending the bacterial pellet in 2 XTY liquid medium (containing 0.1% glucose, 0.1% ampicillin, 0.05% kanamycin), and shake-culturing at 25 deg.C and 230rpm for 20 h.

12) Centrifuging at 3000g for 20min, pouring 40mL of the supernatant into a sterile 50mL centrifuge tube, adding 10mL of 20% PEG/NaCl into the centrifuge tube, turning upside down, mixing, and ice-cooling for 4 h.

13) After 4h, the mixture was centrifuged at 4000g for 30min at 4 ℃ and the supernatant was decanted. Adding 1mL PBS to resuspend the precipitate, transferring to a 1.5mL centrifuge tube, centrifuging again at 4 ℃ and 10000g for 10min, collecting the supernatant, and storing at 4 ℃.

14) Repeating the steps 1) to 13), carrying out enrichment screening for 5 times in total, and sequentially carrying out screening from the phages obtained in the previous round. The enrichment results are shown in table 1. Enrichment rate-number of clones in wells coated with EGFR antigen/number of clones in negative control wells

(2) Polyclonal phage ELISA

1) A96-well immunoplate was coated with 0.2. mu.g of EGFR polypeptide at 100. mu.L/well and a blank control well (PBS) was set at 4 ℃ overnight.

2) The coating liquid is poured off. Wash 3 times with PBS (after each addition of liquid, discard directly without retention).

3) Then, 280. mu.L of BSA blocking solution with a concentration of 2% by mass/volume was added thereto at room temperature for 2 hours.

4) The 2% (w/v) BSA blocking solution was poured off. Wash 3 times with PBS (after each addition of liquid, discard directly without retention).

5) Taking each round of phage solution prepared in the step 13) as a primary antibody, mixing the primary antibody with 2% (w/v) BSA blocking solution uniformly according to the volume ratio of 1:3, and coating each hole with 100 mu L of mixed solution. Room temperature 1 h.

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

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

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

9) Adding 100 μ L of TMB color developing solution (Biyunyan day) into each well, and reacting at room temperature in dark for 4 min.

10)4min, 50. mu.L of 1M H was added to each well₂SO₄And (3) solution.

11) The absorbance of OD450nm was measured using a microplate reader.

The above results show that phage antibodies specific to anti-EGFR were accumulated by 5 rounds of screening.

TABLE 1 enrichment results of phage Nanobody library for each round of screening

Example 4 selection of monoclonal phages from the enriched library of round 5 for ELISA validation

(1) Diluting the phage obtained from the 5 th round enrichment screening to 10⁸-10¹⁰Doubling, 100 mul of diluent is used for infecting TG1 bacteria liquid in logarithmic phase to be coated on a plate, 448 monoclonal strains are randomly selected from the plate and placed in 96 holesThe plate was incubated with 200. mu.L of 2 × TY medium per clone in wells, shaking at 230rpm for 13h at 37 ℃.

(2) mu.L of overnight inoculum was aspirated from each well and transferred to a new 96-well cell culture plate for culture, 200. mu.L of 2 × TY medium/clone per well, cultured at 37 ℃ and 250rpm for 2 hours, and 95. mu.L of inoculum was aspirated from each well of the old plate, and then 100. mu.L of 30% by volume glycerol aqueous solution was added to each well and stored at-80 ℃.

(3) After 2h, the mixture was transferred to a 1.5mL sterile centrifuge tube, 50. mu.L of helper phage-containing 2 XTY was added, mixed well and placed in a 37 ℃ water bath for 30 min.

(4) Centrifuging at 3000g for 10min, discarding supernatant, resuspending the pellet with 200 μ L2 × TY (containing 0.1% glucose, 0.1% ampicillin, 0.05% kanamycin), adding the bacterial solution to a new 96-well plate, and shake culturing at 25 deg.C and 250rpm for 20 h.

(5) Centrifuging at 3000g for 10min, collecting the supernatant of each well into a 1.5mL centrifuge tube, and storing at 4 ℃ for later use.

(6) A96-well immunoplate was coated with 0.2. mu.g of EGFR polypeptide at 100. mu.L/well, and blank control wells (PBS) and CXCR4 polypeptide (purchased from Shanghai Polyalty, Inc.) independent antigen control wells were set and incubated overnight at 4 ℃.

(7) The coating liquid is poured off. Wash 3 times with PBS (after each addition of liquid, discard directly without retention).

(8) Then, 280. mu.L of BSA blocking solution with a concentration of 2% by mass/volume was added thereto at room temperature for 2 hours.

(9) The 2% (w/v) BSA blocking solution was poured off. Wash 3 times with PBS (after each addition of liquid, discard directly without retention).

(10) And (3) taking the monoclonal phage solution prepared in the step (5) as a primary antibody, and uniformly mixing the primary antibody with 2% (w/v) BSA blocking solution in a volume ratio of 1:3, wherein each coating hole is 100 mu L of mixed solution. Room temperature 1 h.

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

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

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

(14) Adding 100 μ L of TMB color developing solution (Biyunyan day) into each well, and reacting at room temperature in dark for 4 min.

(15)4min, 50. mu.L of 1M H was added to each well₂SO₄And (3) solution.

(16) The absorbance of OD450nm was measured using a microplate reader. The results demonstrated that 25 positive clones were obtained. FIG. 2 shows the ELISA results of 64 monoclonal phages.

(17) The positive clones were sent to Huada Gene Co for DNA sequencing. The sequenced DNA results were analyzed in NCBI OFR, excluding the same nucleotide sequence, to obtain 5 sequences, named aEG1B4, aEG2C7, aEG2E12, aEG4D9 and aEG6B2, and the sequences of 5 nanobodies were 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 method of examples 1 to 4, replacing only the EGFR polypeptide of example 3 (1) and example 4 (6) with a synthetic polypeptide of human epidermal growth factor receptor-2 (Her2) (shanghaita bio) or a synthetic polypeptide of Vascular Endothelial Growth Factor (VEGF) (shanghaita bio), and selecting 2 clones that did not bind to the antigen by ELISA to obtain negative control antibodies.

The nanobodies ahher 2-13C1 (with Her2 as antigen) and aVE201 (with VEGF as antigen) used as negative controls in the experimental examples had amino acid sequences shown in SEQ ID NO:6 and SEQ ID NO:7, respectively. The nucleotide sequences for coding the aHer2-13C12 and aVE201 are shown as 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) A newly activated colony of E.coli DH 5. alpha. and BL21(DE3) (BL21(DE3) from Tianjin advantageous science and technology Co., Ltd.) was picked from each LB plate, inoculated into 5mL of LB liquid medium, and cultured overnight (about 12 hours) at 37 ℃ and 220 rpm.

2) Inoculating the strain suspension at a volume ratio of 1:100, transferring 500 μ L of the strain suspension into 50mL LB liquid medium (which can be scaled up or down according to the required amount), and performing shake culture at 37 deg.C for 2-3h to OD₆₀₀About 0.5.

3) Transferring the bacterial liquid into a 50mL centrifuge tube, standing on ice for 10min, centrifuging at 4 deg.C for 5min at 3000g, discarding supernatant, and pre-cooling with 10mL0.1mol/L CaCl on ice₂The solution gently suspended bacterial cells and left on ice for 30 min.

4) Centrifuging at 4 deg.C for 5min at 3000g, discarding the supernatant, and adding 3mL of precooled 0.1mol/LCaCl₂The solution gently suspends the bacterial cells, and stands on ice for 5min to complete the preparation of competent cells.

5)3mL of sterilized and precooled 30% glycerol aqueous solution (i.e., 1:1 volume to a final glycerol concentration of 15%) was added to 3mL of the prepared competent cells, mixed gently, split-packed into 100. mu.L per tube, and rapidly transferred to a refrigerator at-80 ℃ for storage.

(2) Construction of Nanobody recombinant vector

Preparing a PCR reaction system: 5 XPrimerStar buffer 10. mu. L, dNTP mix (2.5 mM each) 4. mu. L, aEG-F primer (10. mu.M, 5'-GATCCATGGCCCAGGTGCAGCTGT-3') 1. mu. L, aEG-R primer (10. mu.M, 5'-TCTGCGGCCGCGCTCGAGAC-3') 1. mu.L, template 10ng, primerStar DNA polymerase 0.5. mu.L, sterile deionized water to make up to 50. mu.L.

And (3) PCR reaction: 10min at 96 ℃; 30 cycles of 95 ℃ 10sec, 60 ℃ 10sec, 72 ℃ 30 sec; 5min at 72 ℃.

The PCR product was purified by PCR product purification kit.

The pET-22b vector (purchased from EMD Biosciences (Novagen)) and the PCR product were subjected to a double digestion reaction according to the following protocol:

pET-22b vector plasmid 2 mug, digestion buffer 5 mug L, NotI2 mug L, NcoI2 mug, sterile water make up to 50 mug.

1.4. mu.g of PCR product, 6. mu. L, NotI 2. mu. L, NcoI 2. mu.L of enzyme digestion buffer solution, and sterile water to make up to 60. mu.L.

The reaction was incubated at 37 ℃ for 15 minutes. Thus obtaining pET-22b double enzyme digestion products and target fragment double enzyme digestion products. And purifying the enzyme digestion product by using an enzyme digestion product purification kit.

Then, the ligation reaction was carried out as follows: 0.03pmol of pET-22b double-restriction enzyme product, 0.3pmol of target fragment double-restriction enzyme product, 1 μ L of enzyme linked buffer 2.5 μ L, T4 DNA ligase, and sterile water to make up to 25 μ L.

Ligation was performed overnight at 4 ℃ to give a ligation product. And transforming the ligation product into escherichia coli DH5 alpha competent cells, coating the cells on an LB plate containing 100 mu g/mL ampicillin, then primarily screening colonies through primers aEG-F and aEG-R to obtain positive clones, carrying out expanded culture on the obtained positive clones, extracting plasmids, carrying out enzyme digestion reaction rescreening on the positive clones, and sequencing the positive clones obtained through rescreening to obtain a recombinant expression plasmid obtained by recombining a pET-22b vector and a nano antibody.

(3) Transformation of competent cells by nano antibody recombinant vector

1) The frozen BL21 competent cells were removed from the freezer at-80 ℃ and thawed on ice.

2) The recombinant expression plasmid was centrifuged briefly, 10. mu.L of the recombinant expression plasmid was placed in a 1.5mL EP tube on ice, 100. mu.L of competent cells was added to the EP tube, the tube bottom was flicked with a finger and mixed well, and the mixture was placed on ice for 30 min.

3) The mixture was incubated in a thermostatic water bath at 42 ℃ for 100s and quickly transferred to ice for cooling for at least 2 min. The EP tube was filled with 900. mu.L of LB medium without antibiotics, shaken at 220rpm at 37 ℃ for 1.5 hours.

4) Centrifuging 8000g of bacteria solution for 1min to precipitate thallus to the bottom of the tube, removing 900 μ L of supernatant, resuspending the bacteria precipitate with the remaining 100 μ L of culture solution, uniformly coating the bacteria solution on LB solid culture medium containing 0.1% ampicillin and 1% glucose, and culturing at 37 deg.C for 12-16 h.

(4) Prokaryotic expression and purification of nano antibody

1) BL21(DE3) containing the recombinant expression plasmid vector was picked up and inoculated into 5mL of LB liquid medium containing 1% glucose and 100. mu.g/mL ampicillin, and shake-cultured at 230rpm at 37 ℃ for 13 hours.

2) By volumeInoculating at a ratio of 1:100, inoculating 4mL of overnight bacterial liquid into 400mL LB liquid medium containing 100. mu.g/mL ampicillin, culturing at 37 deg.C and 220rpm for 2-3h with shaking to OD₆₀₀＝0.6-0.8。

3) Adding 0.25mM IPTG to induce expression, carrying out shake culture at 25 ℃ and 230rpm for 6h, centrifuging for 5min at 5000g, discarding supernatant, resuspending thalli precipitation in 40mL of bacteria breaking buffer solution, carrying out ultrasonic breaking, wherein the ultrasonic power is 40% of 650W, carrying out ultrasonic breaking for 4s, stopping for 8s, and working at 10 ℃ for 40 min.

4) Collecting the crushed solution in a centrifuge tube, centrifuging at 4 deg.C and 15000g for 30min, collecting supernatant in a new centrifuge tube, filtering with 0.22 μm filter membrane, and storing at 4 deg.C for purification (the storage time is not more than 4 days).

5) The antibody protein in the disruption supernatant was purified by a nickel column. The nickel column stored at 4 ℃ was taken out, the storage solution (20% ethanol) was dropped, and 10mL of ultrapure water was added thereto for washing to start the subsequent purification.

The first step is as follows: the column was equilibrated with 10mL of lysis buffer.

The second step is that: the collected protein supernatant is passed through a column to allow the target protein to bind to the column.

The third step: the contaminating proteins were washed off using 20mL of wash buffer (containing 0.02M imidazole).

The fourth step: the protein of interest was eluted using 5mL elution buffer (containing 0.2M imidazole) and 5 tubes of eluate, 1 mL/tube, were collected. And simultaneously collecting the column passing liquid of each step for subsequent detection.

6) SDS-PAGE electrophoresis loading buffer of different component proteins in each step is prepared, and then SDS-PAGE electrophoresis detection is carried out respectively. The electrophoresis chart of SDS-PAGE protein after antibody protein expression and purification is shown in FIG. 3.

7) All antibody solutions obtained from the purification were dialyzed into PBS (pH 7.4, 0.01M) and sterile filtered using a 0.22 μ M syringe filter, and all proteins were stored at-80 ℃.

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

(5) Detection of binding of purified anti-EGFR nanobody to EGFR polypeptide by ELISA method

1) Separately, 0.2. mu.g of VEGF, CAMPH, BMP2, ENDOF1, FGF21, HER2, CXCR4 and EGFR fragments (all provided by Shanghai Betay Co., Ltd.) were coated on a 96-well immunoplate in an amount of 100. mu.L/well in a blank control well (PBS) at 4 ℃ overnight.

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

(6) Method for detecting binding of purified anti-EGFR nano antibody and EGFR complete extracellular domain by WesternBlotting method

1) 0.5. mu.g of the EGFR whole extracellular domain (from Beijing Yiwangshu Biotech) was subjected to SDS-PAGE, transferred to a PVDF membrane, and blocked with 5% nonfat dry milk.

2) Purified aEG antibody was added overnight at 4 ℃.

3) The next day, PBST was washed 3 times for 6 min/time.

4) Secondary antibody protein A-HRP (1:5000) was added at room temperature for 1 h.

5) PBST was washed 3 times for 8min each.

6) An ECL luminescent solution (the volume ratio of the solution A to the solution B is 1:1) is uniformly coated on the PVDF membrane, and the PVDF membrane is placed in a gel imaging system for imaging. The results are shown in FIG. 5.

Experimental example 7

The MTT method is used for detecting the inhibition effect of the nano antibody on the proliferation of human lung cancer cells A549, human breast cancer cells MCF-7 and human prostate cancer cells DU145 (the A549, the MCF-7 and the DU145 are purchased from Shanghai Xinyu biological science and technology Limited company).

(1) 5000 cells were seeded in a 96-well plate (repetition number n-3), CO at 37 ℃₂The cells were cultured overnight in a 5% cell culture chamber.

(2) Old medium was removed, and 100. mu.L of serum-free medium (1640 for A549 and DU145 and DMEM for MCF-7) was added to each well and cultured for 4 hours.

(3) Replacing serum-free medium with antibody-containing 1% FBS medium, adding into corresponding cells, and culturing at 37 deg.C with 5% CO₂And culturing for 72 h. Each antibody was set at 4 concentrations: 0. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, and 100. mu.g/mL. aVE201 and aHer2-13C1 were used as negative control antibodies.

(4) After adding the nanobody for 72 hours, the old medium was discarded, and 120. mu.L of MTT-DMEM mixed solution (1mg/mL MTT: DMEM: 1:5 in volume) was added to each well, and the mixture was incubated at 37 ℃ for 4 hours.

(5) The MTT-DMEM mixture was aspirated and the plates were air dried. The pellet was dissolved by adding 150. mu.L of DMSO to each well. Placing in decolorizing shaking table for 10 min.

(6) The light absorption OD of each well at a wavelength of 570nm was measured by a microplate reader.

The results are shown in fig. 6, and 5 anti-EGFR antibodies were able to significantly inhibit cancer cell proliferation compared to the group without antibody (0 μ g/mL). Wherein aEG2E12 has obvious inhibition effect on A549 and DU145 at the concentration of 25 mu g/mL, while MCF-7 proliferation is obviously inhibited at the concentration of 50 mu g/mL, and the antibody concentration and the proliferation inhibition effect are positively correlated. The results prove that the purified 5 anti-EGFR nano-antibodies can inhibit the proliferation of A549 cells, MCF-7 cells and DU145 cells.

Experimental example 8 Effect of Nanobody on apoptosis of A549, MCF-7 and DU145 cells

(1) Inoculating 25 ten thousand cells/well (six-well plate), 37 deg.C, 5% CO₂Incubate overnight to allow for adherence.

(2) Removing old medium, adding serum-free medium to each well, culturing for 4h, adding 1% (v/v) FBS medium to make the medium contain anti-EGFR antibody at final concentration of 50 μ g/mL, using aVE201 and aHer2-13C1 as negative control antibodies at 37 deg.C and 5% CO₂Culturing for 48 h.

(3) The cells were removed from the incubator, the old medium was removed, washed once with sterile PBS, digested with 700. mu.L of 0.25% (w/v) pancreatin solution, and the digestion was stopped with medium containing 10% FBS. Gently blow and collect the cell suspension into a sterile 1.5mL EP tube, centrifuge at 1050g for 5 min.

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

(5) Cells were resuspended in 195. mu.L of Binding buffer (1X), then 5. mu.L of Annexin V-FITC was added and incubated at room temperature in the dark for 15 min.

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

(7) mu.L of binding buffer resuspended cells, then 10. mu.L of Propidium Iodide (Propidium Iodide) was added and the assay was performed on the machine within 4 h.

The results are shown in FIG. 7, where FIG. A, C, E is a two-dimensional scatter plot of apoptotic A549, MCF-7 and DU145 cells, respectively, showing an increase in apoptotic cells over the no antibody control (right half). FIG. B, D, F is the apoptosis ratios obtained from FIG. A, C, E, respectively. The results prove that the purified 5 anti-EGFR nano-antibodies can promote the apoptosis of A549 cells, MCF-7 cells and DU145 cells.

Experimental example 9 Effect of Nanobody on cell migration of A549, MCF-7 and DU145

(1) A12-well plate was taken and two parallel lines were drawn parallel to the long side at a distance of 5mm on the back of the well. Inoculating 50 ten thousand cells/well, 37 deg.C, 5% CO₂Incubate overnight to allow for adherence.

(2) Old medium was removed (A549, DU145 using 1640, MCF-7 using DMEM), and serum-free medium was added to each well for 4 hours. The cells were scribed with a 200 μ L line perpendicular to the back of the plate at the muzzle, three spaces per well, and the exfoliated cells were washed off with PBS.

(3) The antibody-containing 1% FBS medium was added simultaneously to the corresponding cells at 37 ℃ with 5% CO₂And culturing for 24 h. Each antibody was set at 4 concentrations: 0. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, and 100. mu.g/mL. aVE201 and aHer2-13C1 were used as negative control antibodies.

(4) The photographs were taken under an inverted microscope and the distance to the gap was measured and recorded.

The results are shown in fig. 8 and 9, as the concentration of the 5 nano-antibodies is increased, the mobilities of the A549 cells, the MCF-7 cells and the DU145 cells are all reduced, the reduction of the mobilities of the A549 cells and the DU145 cells is particularly obvious, the concentration of the nano-antibodies is inversely related to the mobilities of the cells, and the results prove that the purified 5 anti-EGFR nano-antibodies can inhibit the migration of the A549 cells, the MCF-7 cells and the DU145 cells.

Experimental example 10 Effect of Nanobody on cell migration of A549, MCF-7 and DU145

(1) The sterile cell culture Transwell chamber was removed and placed in a 24-well plate, the upper chamber was inoculated with 200. mu.L (2% FBS) of a mixed suspension of the corresponding antibody and 5000 ten thousand cells/well (24-well plate), and the lower chamber was charged with 500. mu.L of a medium (containing 10% by volume of FBS), 37 ℃ and 5% CO₂And culturing for 24 h. Each antibody was set at 4 concentrations: 0. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, and 100. mu.g/mL. aVE201 and aHer2-13C1 were used as negative control antibodies.

(2) The plate was removed, the chamber was clamped with forceps, washed twice with PBS, and the medium was washed away.

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

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

(5) The cells inside the chamber were wiped off with a cotton swab and the chamber was placed under an inverted microscope for photography.

(6)100 u L20% acetic acid solution washed away each upper chamber outside the cell stain.

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

Results as shown in fig. 10 and 11, the mobility of cancer cells was significantly reduced as the concentration of 5 nanobodies was increased, and the above results demonstrate that the purified 5 anti-EGFR nanobodies can inhibit the migration of a549, MCF-7 and DU145 cells.

Experimental example 11 Effect of Nanobody on MCF-7, A549 and DU-145 cell invasion

(1) Sterile Transwell chambers were placed in 24-well plates, 50. mu.L of matrigel (1:9 dilution) was added to each well, the plates were gently shaken to allow the gel to spread evenly, and the gel was allowed to set overnight in an incubator at 37 ℃.

(2) The plates were removed and the upper chamber was inoculated with 200 μ L of a cell mixed suspension of 5 ten thousand/well of corresponding antibody-treated cells (2% FBS medium), each antibody set at 4 concentrations: 0. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL and 100. mu.g/mL, 500. mu.L of a medium (10% FBS) was added to the lower chamber at 37 ℃ and 5 ℃％CO₂And culturing for 24 h. aVE201 and aHer2-13C1 were used as negative control antibodies.

(3) The plate was removed, the chamber was held by forceps, and washed twice with PBS to wash out the medium.

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

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

(6) The cells inside the upper chamber were gently wiped off with a cotton swab and the chamber was placed under an inverted microscope for photography.

(7)100 u L20% acetic acid solution washed away each upper chamber outside the cell stain.

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

The results are shown in fig. 12-13, and 5 anti-EGFR antibodies were able to significantly inhibit cancer cell invasion compared to the group without antibody (0 μ g/mL). Wherein aEG2E12 can obviously inhibit the migration of three cancer cells at the concentration of 25 mug/mL, aEG4D9 can obviously inhibit the migration of A549 and DU145 at the concentration of 25 mug/mL, and MCF-7 can obviously inhibit the migration at the concentration of 50 mug/mL. The results prove that the purified 5 anti-EGFR nano antibodies can inhibit the invasion of A549 cells, MCF-7 cells and DU145 cells.

Experimental example 12 Effect of Nanobody on mouse Lung cancer model

(1) Pancreatin A549 cells, 1050g, and 5min centrifugation.

(2) The supernatant was discarded, and the collected cells were resuspended in PBS and centrifuged at 1050g for 5min in a centrifuge.

(3) And (4) repeating the step (2). The supernatant was discarded, an appropriate amount of PBS was added to resuspend the cell pellet and adjust the cell density to 5X 10⁷/mL。

(4) Male Balb/C nude mice (purchased from Guangdong provincial animal center for medical laboratory) at 4 weeks were selected and 100. mu.L of the cell suspension (containing 5X 10 cells) was aspirated using a 1mL sterile syringe⁶A549 cells), injecting the cell suspension into the right axilla of the mouse in a subcutaneous injection mode for tumor formation, placing the mouse in an SPF animal laboratory to be fed according to a conventional method, and measuring the tumor volume of the mouse by using a vernier caliper once every three days.

(5) The antibodies aEG2E12 and aEG4D9 with the best results of in vitro functional experiments were selected for animal experiments, and aHer2-13C1 and aVE201 were used as negative controls. When the tumor volume reaches 100mm³Thereafter, the mice were randomly divided into 6 groups of 5 mice each. Wherein group 1 is blank control group (PBS), group 1 is positive control group (cisplatin, DDP), group 2 is experimental antibody group (aEG2E12, aEG4D9), and group 2 is negative control antibody group (aHer2-13C1, aVE 201). Administration was via tail vein using insulin syringe. Wherein the dosage of each mouse of the experimental antibody group and the negative control nano antibody group is 10mg/kg, the dosage of each positive control group is 2mg/kg, and each blank control group is injected with 100 mu L of PBS buffer solution for cells. The dosing period was 24 days, the dosing frequency was 3 days/time, and the tumor volume was recorded for each group of mice. After dosing, mice were sacrificed, tumors were stripped, photographed and weighed.

The results are shown in fig. 14, the increase of the tumor volume of the experimental antibody group after administration is obviously slower than that of the blank control group and the negative control antibody group, and the final volume is also obviously smaller, thus proving that the purified aEG2E12 and aEG4D9 nanobodies can inhibit the tumor size of the mouse lung cancer model induced by a549 cells.

The formulation of the reagents used in the examples were as follows:

(1)PBS/PBST(pH7.4)：KH₂PO₄ 0.24g、NaCl 8g、KCl 0.2g、Na₂HPO₄·12H₂O 9.07g。

weighing the reagent, adding 900mL of deionized water for dissolving, and fixing the volume to 1L.

PBST: adding Tween-20 with final concentration of 0.1% into the prepared PBS buffer solution, mixing, sterilizing at 121 deg.C, and storing at 4 deg.C.

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

The reagent was dissolved in 400mL of deionized water, the pH was adjusted to 7.4 with dilute hydrochloric acid, and the volume was adjusted to 500 mL.

TBST: add 500. mu.L Tween-20 to 500mL TBS buffer and mix well.

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

LB solid medium: 4g of agar powder is added into the LB liquid culture medium.

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

(5) 100. mu.g/mL ampicillin solution: 1g of the powder was weighed, dissolved in 10mL of deionized water to prepare a 100mg/mL solution, sterilized by filtration using a 0.22. mu.M filter, and dispensed into 1mL tubes and stored at-20 ℃.

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

The reagent was dissolved in 900mL of deionized water to a volume of 1L. The reagent is 5 Xelectrophoresis solution formula, and is stored at 4 ℃.

(7) SDS-PAGE membrane-transfer solution: 15.14g of Tris alkali and 72g of glycine.

The reagent is dissolved by 900mL of deionized water, the volume is constant to 1L, and the solution is stored at 4 ℃. The reagent is a 5X electrophoresis solution formula.

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

(9)1M H₂SO₄:10 mL of concentrated sulfuric acid was slowly added to 187mL of deionized water.

(10)100 × PMFS: 1.74g of PMSF was weighed out and dissolved in 100mL of isopropanol and stored at-20 ℃.

(11) Protein expression purification buffer

And (3) breaking the bacteria buffer solution: tris base 2.42g, NaCl 14.6g, 100 XPSF 10mL

The reagent was dissolved in 900mL of ultrapure water, the pH was adjusted to 7.45 with dilute hydrochloric acid, the volume was adjusted to 1L, and the solution was stored at 4 ℃.

Loading buffer solution: and (4) breaking the bacteria in the buffer solution.

Washing with a miscellaneous buffer solution: 20mL of the loading buffer was added with 200. mu.L of imidazole stock solution (2M).

Elution buffer: 9mL of the loading buffer was added with 1mL of imidazole mother liquor (2M).

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

(13) 30% glycerol solution (glycerol-PBS): 15mL of glycerol was measured, and 35mL of PBS buffer (pH 7.4) was added thereto, followed by filtration sterilization using a 0.22 μm filter and storage at 4 ℃.

(14) 10% Ammonium Persulfate (APS): ammonium sulfate 0.5g, ddH₂And O5 mL. Subpackaging into 500 μ L tubes, and storing at-20 deg.C.

(15)1M Tris-Hcl (PH 8.8/PH 6.8): weighing 0.2g Tris-Base powder, dissolving in 900mL deionized water, adjusting pH to 6.8 or 8.8 with dilute hydrochloric acid, diluting to 1L, sterilizing at 121 deg.C, and storing at 4 deg.C.

(16) Coomassie brilliant blue dye liquor: coomassie brilliant blue R-2501 g, isopropanol 250mL, glacial acetic acid 100mL, ddH₂O 650mL。

(17) Coomassie brilliant blue staining destaining solution: glacial acetic acid 100mL, ethanol 50mL, ddH₂O 850mL。

(18)5 × protein loading buffer: pH 6.8, 1M Tris-HCl 12.5mL, SDS 5g, bromophenol blue 0.25g, glycerol 25 mL. ddH was added to the reagent₂And O, diluting to 50mL, and storing at room temperature. Before use, 500. mu.L of beta-mercaptoethanol is added selectively.

(19) TYE solid medium (400 mL): 5g of peptone, 2.5g of yeast powder, 4g of agar powder and ddH₂O400 mL. Sterilizing at 121 deg.C, cooling to 50 deg.C, adding 1% glucose solution and 100 μ g/mL ampicillin, mixing, pouring into flat plate, and storing in refrigerator at 4 deg.C.

(20)2 × TY Medium (100 mL): peptone 1.6g, yeast powder 1g, NaCl 0.5g, ddH₂O100 mL. Sterilizing at 121 deg.C.

(21) 20% PEG/NaCl solution (500 mL): PEG 600100 g, NaCl 73g, ddH₂O 400mL。

Dissolving in deionized water, diluting to 500mL, sterilizing at 121 deg.C for 20min, and storing at room temperature.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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Met Ala Gln Val Gln Leu Leu Glu Ser GlyGlyGly Leu Val Gln Pro

1 5 10 15

GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Val Ser

20 25 30

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

35 40 45

Trp Val Ser Gly Ile Leu Ala Gly Asp Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

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

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala ArgPheThr Ser Gly Gln Gly Ser Leu Arg Ser Asp Pro

100 105 110

Ile Arg Ser TrpGly Gln GlyThr Leu Val Thr Val Ser Ser Ala Ala

115 120 125

Ala

<210> 7

<211> 128

<213> human (Homo sapiens)

Met Ala Gln Val Gln Leu Leu Glu Ser GlyGlyGly Leu Val Gln Pro

1 5 10 15

GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ser Val Ser

20 25 30

Asn Glu Ala Met GlyTrp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Ser Ile Thr Asp Gln Ser Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

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

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala ArgGly Gln ArgArgArg Gln Met His Ser Tyr Lys Val

100 105 110

Ser SerTrpGly Gln GlyThr Leu Val Thr Val Ser Ser Ala AlaAla

115 120 125

<210> 8

<211> 375

atggcccaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctg 60

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

ctgtatctgcaaatgaacagcctgcgtgccgaggacaccgcggtatattattgcgcggga 300

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

1. A nanobody against human EGFR, characterized in that: the nano antibody for resisting the human EGFR is named as aEG1B4 nano antibody; or an antibody formed by combining aEG1B4 nanobody with at least one of aEG2C7 nanobody, aEG2E12 nanobody, aEG4D9 nanobody and aEG6B2 nanobody;

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 aEG2E12 nano antibody is shown as SEQ ID NO. 3;

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 nucleotide sequence encoding the nanobody against human EGFR of claim 1, characterized in that: is a nucleotide sequence for coding the aEG1B4 nano antibody; or a nucleotide sequence formed by combining the nucleotide sequence coding the aEG1B4 nano antibody with at least one of the nucleotide sequence coding the aEG2C7 nano antibody, the nucleotide sequence coding the aEG2E12 nano antibody, the nucleotide sequence coding the aEG4D9 nano antibody and the nucleotide sequence coding the aEG6B2 nano antibody.

3. The coding nucleotide sequence of the anti-human EGFR nanobody according to claim 2, characterized in that:

the nucleotide sequence of the aEG1B4 nano antibody is shown as SEQ ID NO. 8;

the nucleotide sequence of the aEG2C7 nano antibody is shown as SEQ ID NO. 9;

the nucleotide sequence of the aEG2E12 nano antibody is shown as SEQ ID NO. 10;

the nucleotide sequence of the aEG4D9 nano antibody is shown as SEQ ID NO. 11;

the nucleotide sequence of 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 the nucleotide for coding the anti-human EGFR nano antibody by a gene synthesis method, then cloning the nucleotide onto an expression plasmid vector, and transforming the expression plasmid vector into host cells 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. Use of the anti-human EGFR nanobody according to claim 1 for the preparation of an antibody medicament for the treatment of diseases characterized by EGFR overexpression.

6. The use of the anti-human EGFR nanobody according to claim 5 for the preparation of an antibody medicament for the treatment of diseases characterized by EGFR overexpression, characterized in that: the diseases characterized by EGFR overexpression are autoimmune diseases and cancers.

7. The use of the anti-human EGFR nanobody according to claim 6 for the preparation of an antibody medicament for the treatment of diseases characterized by EGFR overexpression, characterized in that: the cancer is an EGFR high expression tumor.

8. The use of the anti-human EGFR nanobody according to claim 7 for the preparation of an antibody drug for the treatment of diseases characterized by EGFR overexpression, characterized by: the EGFR high expression tumor is lung cancer, head and neck cancer, colon cancer or brain tumor.