CN111499749B

CN111499749B - Cetuximab mutant and application thereof

Info

Publication number: CN111499749B
Application number: CN202010174651.8A
Authority: CN
Inventors: 庄鑫磊; 王哲; 赵文彬; 潘利强; 周展; 陈枢青
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-03-13
Filing date: 2020-03-13
Publication date: 2021-11-09
Anticipated expiration: 2040-03-13
Also published as: CN111499749A

Abstract

The invention discloses a cetuximab mutant and application thereof. The amino acid sequence of the heavy chain variable region of the cetuximab mutant is shown in any one of SEQ ID No. 1-6. The Cetuximab mutant with the amino acid sequence of the heavy chain variable region of the antibody shown in SEQ ID No.1-5 is effective to EGFR S492R, and the Cetuximab mutant with the amino acid sequence of the heavy chain variable region of the antibody shown in SEQ ID No.1 and 2 has better effect than Panitumumab. The Cetuximab mutant with the amino acid sequence of the heavy chain variable region of the antibody shown in SEQ ID NO.6 is effective to EGFR G465R and has obvious effect. The Cetuximab mutant of the amino acid sequence of the heavy chain variable region of the antibody obtained by the invention is shown in SEQ ID NO.1, 2 and 6, and still maintains the affinity to the wild type EGFR.

Description

Cetuximab mutant and application thereof

Technical Field

The invention relates to the technical field of antibody medicines, in particular to a cetuximab mutant and application thereof.

Background

Cancer has become one of the major diseases seriously harming human health, and in China, 1 million people have been diagnosed with cancer every day, and on average 7 people get cancer every minute. Therefore, the development of anticancer drugs has become an urgent need for national health. Chemotherapy, although one of the important anticancer approaches including surgery, radiotherapy, and targeted therapy, limits the wide clinical application of these anticancer compounds due to the great toxic side effects due to the small difference between cancer cells and normal cells. The antibody drug has the incomparable advantages of the traditional chemotherapy because of the characteristics of strong targeting property, high specificity, low toxic and side effects and the like, and is an important treatment means for resisting tumors. Antibody drugs have played an important role in the treatment of cancer for the past 20 years, but their therapeutic effects are also plagued by drug resistance.

The human Epidermal Growth Factor Receptor (EGFR) is an important target for antibody drugs, and is overexpressed in many tumors, such as breast cancer, lung cancer, colorectal cancer, head and neck tumors, bladder cancer, ovarian cancer, prostate cancer and the like, and the overexpression of the EGFR has correlation with poor prognosis, shortened tumor-free survival and drug resistance generation. Researches show that EGFR (epidermal growth factor receptor) which is overexpressed in tumor tissues can effectively inhibit tumor cell proliferation, promote apoptosis and inhibit metastasis and invasion, so that EGFR is an important target point for treating cancers. The monoclonal antibody drug can inhibit the activation of EGFR by blocking the homodimerization of EGFR, thereby achieving the aim of resisting cancer. Monoclonal antibody drugs against EGFR targets that have been approved by the FDA for marketing include: cetuximab (Cetuximab, Erbitux) and Panitumumab (Panitumumab, Vectibix), the indication being metastatic colorectal cancer. Cetuximab is a specific EGFR human mouse chimeric monoclonal antibody, Panitumumab is a specific EGFR fully humanized monoclonal antibody, and both can be specifically combined with EGFR extracellular regions expressed on the surfaces of normal cells and various cancer cells and competitively block EGF and TGF-alpha. The combination of the EGFR inhibitor and EGFR can stimulate endocytic degradation of the latter, so that the expression of EGFR is reduced, and tyrosine kinase phosphorylation and intracellular signal transduction pathways of EGFR are blocked, thereby inhibiting the proliferation of cancer cells and inducing the apoptosis of the cancer cells.

However, the drug resistance problem is an inevitable problem in the use process of the monoclonal antibody drug. Cetuximab and Panitumumab, when used, require patients not to have KRAS gene mutations that would otherwise result in drug resistance. However, in recent years, a number of researchers have reported that patients carrying wild-type KRAS gene still have drug resistance after treatment with Cetuximab. In this case, Spanish researchers first found that The Mutation of serine to arginine at The 492 th site of The Extracellular Domain of EGFR (S492R) resulted in The failure of Cetuximab to bind to The mutated EGFR, i.e., The failure of Cetuximab to block The binding of The mutated EGFR to its ligand activates EGFR downstream signaling pathways and inhibits tumor Growth, proliferation, invasion and metastasis, thereby resulting in acquired Resistance to Cetuximab (Montagut, C., et al., Identification of A Mutation in The Extracellular Growth Factor Receptor, structural center Resistance in The biological cancer. Nat., 2012.18(2): p. 221-3.). However, this mutation of EGFR is not on the binding epitope of Panitumumab, i.e., does not confer resistance to Panitumumab. In addition to resistance of S492R to Cetuximab, studies have shown that mutation of glycine to arginine at position 465 of the extracellular domain of EGFR (G465R) also results in failure of Cetuximab to bind to Cetuximab, and that the mutation of G465R has resistance to both Cetuximab and Panitumumab because the mutation of G465R is also on the binding epitope of Panitumumab.

ASPECCT reported by The American hospitalization company as a clinical three-phase study, S492R point mutation was detected in 46 of 285 patients with cetuximab, and The mutation frequency of S492R was about 16% (New hall K, Price T, Peeters M, et al. frequency of S492R Mutations in The epidemal Growth FaVector Receptor: Analysis of Plasma DNA from Metastatic Cancer Patients Treated with Panitumumab or Cetuximab Monotherapy Ann Oncol,2014,25: ii 109.). Researchers at medical center of university of hamburger, germany reported that 2 of 9 patients with Cetuximab detected a G465R point mutation. The mutation frequency of G465R was about 22% (Braig F,

m, Schieferdecker A, et al. epidemic Growth Factor Receptor Mutation media Cross-Resistance to Panitumumab And Cetuximab in synergistic cancer. oncotarget 2015,6(14): 12035.). Current research to overcome the two high frequency mutations S492R and G465R is mainly directed to the use of mixed antibodies of non-overlapping epitopes, such as Sym004 and MM-151([1 ]]Pedersen,M.W.,et al.,Sym004:A Novel Synergistic Anti-Epidermal Growth Factor Receptor Antibody Mixture with Superior Anticancer Efficacy.Cancer Res,2010.70(2):p.588-97.[2]Kearns, J.D., et al, Enhanced Targeting of The EGFR Network with MM-151, An Oligclonal Anti-EGFR Antibody therapeutic. mol Cancer Ther,2015.14(7): p.1625-36.), The principle is to bind mutated EGFR by avoiding The site of The mutation. Sym004 was a mixture of two monoclonal antibodies targeting non-overlapping EGFR antigens, MM-151 was a mixture of three monoclonal antibodies targeting non-overlapping EGFR antigens, all of which were able to bind to different EGFR epitopes simultaneously, and these binding epitopes were all spared the mutation site. However, the methods for overcoming the drug resistance point mutation are based on the existing antibody capable of avoiding the mutation site, so that the problem of drug resistance of the antibody caused by the point mutation is fundamentally solved, and the development of the second-generation antibody by directional modification starting from the structure of the antibody is a good strategy for solving the drug resistance of the antibody caused by the point mutation.

The research on the directional modification of antibodies by experts and scholars at home and abroad is already available, the technical basis of the directional modification of antibodies is laid by the invention of the technology for displaying the polypeptide and the protein phage from the end of 80 to 90 years in the 20 th century, and the polypeptide and antibody phage display technology awarded to George P.Smith and Sir Gregory P.winter B in half of the Nobel chemical prize in 2018 is used for highlighting the contribution of the experts and scholars to the directional evolution of macromolecular proteins. The phage display technology is characterized in that a gene sequence for coding foreign protein is inserted into a structural gene of phage coat protein, proper insertion position is selected without influencing normal functional activity of the coat protein, the protein expressed by the foreign gene is expressed by attaching to the expression of the coat protein, and finally the target protein and the coat protein are fused and displayed on the surface of the phage. The protein in the display form can keep relatively correct spatial conception and functional activity, and related target proteins can be selected for screening, so that proteins such as antibodies and the like capable of specifically binding with the target proteins can be identified. The directional modification of antibody by using phage display technology firstly needs to insert diversified antibody gene fragments obtained by artificial synthesis or amplification into a phage genome to construct a phage display library for displaying the diversified antibody fragments. And then, carrying out co-incubation on the target antigen and the constructed phage display library for a certain time to remove unbound and non-specifically bound phage, eluting the phage specifically bound with the target protein, infecting host cells with the eluted phage for propagation and amplification for the next round of screening, and screening to obtain the candidate antibody specifically bound with the target protein after two to five rounds of the same process. However, the mutation sites of the antibody fragments in the traditional phage antibody library are random, so that the library capacity is large, the screening work is complicated, the screening period is long, the screening efficiency is greatly reduced, the screening cost is increased, and the potential of the phage display technology in the application of antibody directional modification is limited.

With the rise of computational biology and the development of protein structure prediction, the antibody directional modification based on the simulation prediction of the three-dimensional structure of protein can effectively improve the antibody directional modification efficiency and shorten the screening period. The simulation prediction of the three-dimensional structure of the antibody-antigen complex protein is based on protein-protein interactions (PPI), and the focal point of PPI research is usually protein interface, and thus is crucial to the study of antibody and antigen binding interface. Rosetta is a de novo protein structure prediction software in which an InterfaceAnalyzer module is a program for protein interface analysis, and can calculate a plurality of indicators for evaluating the quality of a binding interface, including dG _ separated, packstat, and the like. Cetuximab was redesigned against the S492R and G465R point mutations EGFR, and the experimentally found phenomenon of antibody affinity decrease due to antigen mutations was first explained by computational simulation. Antibody interface residues were then single point mutated using the Rosetta program and binding free energy calculations were performed using the InterfaceAnalyzer method. The distribution of the combined free energy difference before and after mutation is counted, effective mutation sites are determined, a phage antibody library is established according to the effective mutation sites, the directionally-modified antibody is screened, the antibody capable of overcoming the drug resistance point mutation is rapidly obtained, and two basic problems of time and benefit in antibody directional modification are solved.

Disclosure of Invention

The invention provides a Cetuximab mutant and application thereof, and aims to solve the problem of acquired drug resistance caused by S492R and G465R point mutation of EGFR after long-term clinical Cetuximab administration.

Aiming at the problems, the invention adopts computer simulation prediction, antibody-receptor complex crystal structure analysis and phage antibody display technology to directionally modify Cetuximab, thereby obtaining the Cetuximab mutant capable of overcoming the S492R or G465R point mutation. Wherein the amino acid sequence of the heavy chain variable region of the antibody of the Cetuximab mutant capable of overcoming the S492R point mutation is shown as SEQ ID NO. 1-5; the amino acid sequence of the heavy chain variable region of the antibody of the Cetuximab mutant capable of overcoming the G465R point mutation is shown as SEQ ID NO. 6.

In the invention, random mutation is carried out on potential effective mutation sites of Cetuximab to construct a phage display library for directionally transforming the Cetuximab. We first predicted a potentially useful mutation site for Cetuximab after mutation against EGFR S492R using Rosetta software. We found that N32, W94 and T96 on the Cetuximab light chain and V50, D58 and Y104 residues on the heavy chain might be better mutation sites. We also predicted, using Rosetta software, a potential effective mutation site for Cetuximab following mutation against EGFR G465R. We found that residues V50 and W52 on the Cetuximab heavy chain might be better mutation sites. Based on the above findings we performed site-directed random mutagenesis of these sites.Aiming at S492R mutation, random mutation is introduced into the CDR2 region V50 position, D58 position, the CDR3 region Y104 position, the CDR1 region N32 position, the CDR3 region W94 position and the T96 position of the Cetuximab heavy chain variable region by NNS degenerate codon so as to obtain (Gly₄Ser)₃For the linker, a mutant gene library of the scFv version of Cetuximab was constructed. For the G465R mutation, we introduced random mutations at V50 and W52 positions of CDR2 region of the Cetuximab heavy chain variable region by NNS degenerate codon to (Gly₄Ser)₃For the linker, a mutant gene library of the scFv version of Cetuximab was constructed. And (3) displaying the scFv mutant by using a phage display technology to obtain a phage display library. Then, we screened mutants of the scFv form of Cetuximab that overcome the S492R and G465R point mutations of EGFR using model cells that highly express wild type, S492R or G465R mutant EGFR. Mutants of Cetuximab were obtained by substituting the altered amino acids in the scFv mutants into the heavy chain variable region of Cetuximab.

The invention firstly provides a cetuximab mutant, and the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID No. 1-6.

The invention also provides a gene for coding the cetuximab mutant.

The invention also provides a single-chain antibody, which comprises a light chain variable region of cetuximab and a heavy chain variable region with mutation, wherein the amino acid sequence of the heavy chain variable region with mutation is shown in any one of SEQ ID No. 1-6.

The invention also provides a gene encoding the single-chain antibody.

The invention also provides a Fab fragment antibody, which comprises a light chain of cetuximab, a CH1 region of a heavy chain and a heavy chain variable region with mutation, wherein the amino acid sequence of the heavy chain variable region with mutation is shown as any one of SEQ ID No. 1-6.

The invention also provides a gene encoding the Fab fragment antibody.

The invention also provides a (Fab)2 fragment antibody, wherein each molecule of antibody contains two molecules of the Fab fragment antibody.

The invention also provides application of the cetuximab mutant, the single-chain antibody, the Fab fragment antibody or the (Fab)2 fragment antibody in preparation of a medicament targeting EGFR.

Preferably, when the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID Nos. 1 to 5, the EGFR carries the mutation S492R; when the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.6, EGFR carries the G465R mutation.

Preferably, the disease to which the drug is directed is colorectal cancer.

The currently marketed monoclonal antibody drug Panitumumab against colorectal cancer is still effective against mutations in EGFR S492R and not EGFR G465R. The Cetuximab mutant with the amino acid sequence of the heavy chain variable region of the antibody shown in SEQ ID No.1-5 is effective to EGFR S492R, and the Cetuximab mutant with the amino acid sequence of the heavy chain variable region of the antibody shown in SEQ ID No.1 and 2 has better effect than Panitumumab. The Cetuximab mutant with the amino acid sequence of the heavy chain variable region of the antibody shown in SEQ ID NO.6 is effective to EGFR G465R and has obvious effect. The Cetuximab mutant of the amino acid sequence of the heavy chain variable region of the antibody obtained by the invention is shown in SEQ ID NO.1, 2 and 6, and still maintains the affinity to the wild type EGFR.

Drawings

FIG. 1 is a graph of the results of ELISA screening of monoclonal IPTG-induced supernatants for candidate clones.

FIG. 2 is a diagram showing the results of SDS-PAGE analysis of antibody proteins.

FIG. 3 is a graph showing the results of an ELISA assay for the affinity of the mutant for S492R mutant EGFR, wherein hulgG control is nonspecific IgG without any labeling as a control.

FIG. 4 is a graph of the results of ELISA testing for the affinity of the mutants for G465R mutant EGFR.

FIG. 5 is a graph showing the results of ELISA for detecting the affinity of the mutant for wild-type EGFR.

Detailed Description

Example 1

Construction of wild-type, S492R and G465R mutant EGFR high expression monoclonal cell lines.

Wild-type EGFR expression plasmids were obtained from beijing yi qiao shenzhou technologies ltd. Amplifying the full length of a wild type EGFR gene sequence (the GenBank number is AY888105.1, wherein the last base T is also added with GA to form a TGA stop codon) by PCR, connecting the amplified EGFR gene sequence to a pMD18-T vector, introducing an S492R point mutation (1476C in the EGFR gene sequence is more than A) and a G465R point mutation (1393G in the EGFR gene sequence is more than C) into the EGFR gene sequence by a circular point mutation PCR method, and finally connecting correctly sequenced S492R and G465R mutant EGFR genes back to a pCMV3 eukaryotic expression vector to obtain S492R and G465R mutant EGFR eukaryotic expression plasmids. The constructed wild type, S492R and G465R mutant EGFR expression plasmids are transfected into mouse fibroblast NIH3T3 (original NIH3T3 cells are purchased from Shanghai cell bank) respectively through liposomes, and after administration, screening and flow sorting, an NIH3T3 monoclonal cell line which can stably express wild type (the amino acid sequence of the wild type EGFR is shown as SEQ ID No. 7), S492R and G465R mutant EGFR proteins is selected.

NIH3T3 cells highly expressing wild type EGFR are called WT-EGFR-NIH3T3 cells for short;

the NIH3T3 cells highly expressing the S492R mutant EGFR are called S492R-EGFR-NIH3T3 cells for short;

the high expression of G465R mutant EGFR NIH3T3 cells are abbreviated as G465R-EGFR-NIH3T3 cells.

Example 2

And (3) expressing and purifying wild type, S492R and G465R mutant EGFR extracellular region Fc fusion proteins.

Wild type, S492R and G465R mutant EGFR extracellular region sequences and human IgG1 type Fc segment sequences (namely Fc for Cetuximab antibody, and coding sequence is 555-1347bp coding the Cetuximab antibody heavy chain gene sequence) are respectively amplified by PCR, and then the wild type, S492R and G465R mutant EGFR extracellular region sequences and the human IgG1 type Fc segment sequences are connected by overlapping PCR reaction and are connected to a pMH3 eukaryotic expression vector after double enzyme digestion reaction. The ligation product was transformed into DH 5. alpha. competent colonies, and single colonies were picked. Sequencing the selected monoclonal bacterial liquid, expanding and culturing strains with correct sequencing, and extracting plasmids from the strains to obtain eukaryotic expression plasmids of wild type, S492R and G465R mutant EGFR extracellular region Fc fusion proteins. Respectively transferring eukaryotic expression plasmids of wild type, S492R and G465R mutant EGFR extracellular region Fc fusion proteins into HEK-293F cells, culturing for 4 days in a 37 ℃ cell shaker, purifying proteins to obtain wild type, S492R and G465R mutant EGFR extracellular region Fc fusion proteins,

the wild type EGFR extracellular region Fc fusion protein is abbreviated as: WT-EGFR-ECD-Fc;

S492R mutant EGFR extracellular region Fc fusion protein abbreviation: S492R-EGFR-ECD-Fc;

G465R mutant EGFR extracellular region Fc fusion protein abbreviation: G465R-EGFR-ECD-Fc.

Example 3

Computer modeling predicts effective mutation sites for Cetuximab.

The focus of research on protein-protein interactions (PPIs) is generally on the protein interface, whereas the study of the EGFR-Cetuximab system belongs to PPI studies, and thus the study of the EGFR and Cetuximab binding interface is a key breakthrough. The InterfaceAnalyzer module in Rosetta is a program for protein interface analysis and can calculate a plurality of indicators for evaluating the quality of binding interfaces including dG _ separated, packstat, etc. Among them, dG _ separated is an indicator of similar binding energy, and is the difference in energy between protein interface separation and binding. We chose this index to evaluate the effect of mutations on Cetuximab affinity.

Redesigning Cetuximab for EGFR S492R point mutation first needs to consider whether the phenomenon of drug resistance caused by experimentally found mutations can be explained by computational simulation. Binding free energy is a quantitative indicator for investigating affinity, and a smaller value indicates stronger binding, whereas a weaker value indicates weaker binding. Therefore, we generated 100S 492R mutant complex structures by Rosetta program using EGFR extracellular region and Cetuximab complex crystal (PDB number 1YY9) as initial structures, and calculated Δ Δ G values by the InterfaceAnalyzer method were mostly distributed between 0 and 100Rosetta Energy units, and the rest were even far over 100Rosetta Energy units, which indicated that affinity of Cetuximab and EGFR after mutation decreased sharply, consistent with experimental conclusions, indicating that the InterfaceAnalyzer method is suitable for the study of this system. According to the delta G obtained by the InterfaceAnalyzer method, the first 30 EGFR S492R-cetuximab complexes are taken according to the descending order of the delta G value for cluster analysis, and 3 representative complex structures are obtained. The interfacial residues were identified and then single point mutations were performed on cetuximab interfacial residues using the Rosetta program and binding free energy calculations were performed using the interfaceAnalyzer method. And (4) counting the distribution of the combined free energy difference before and after mutation, and determining effective mutation sites.

By combining the statistics of the interfacial residue mutation scans and the free energy changes in binding, we predicted that the C chain of Cetuximab, i.e., N32, W94, and T96 on the antibody light chain, and the D chain, i.e., V50, D58, and Y104 residues on the antibody heavy chain, might be better mutation sites.

Similarly, the effective mutation site of the newly designed Cetuximab aiming at the G465R point mutation of the EGFR is the possible better mutation site of the Cetuximab D chain, namely the V50 and W52 sites on the heavy chain of the antibody.

Example 4

And (5) constructing a phage display library.

The Cetuximab gene sequence is divided into a light chain sequence and a heavy chain sequence, wherein the light chain sequence is shown as SEQ ID No.10, and the heavy chain sequence is shown as SEQ ID No.11 (wherein the first 357 bases are heavy chain variable regions).

For the mutation of S492R, we introduced random mutations by PCR with NNS (N ═ A, T, C, G; S ═ C or G) degenerate codons at V50, D58, Y104 in CDR3, N32 in CDR1, W94 in CDR3 and T96 in the heavy chain variable region CDR2, and light chain variable region CDR1 of Cetuximab gene sequence to (Gly)₄Ser)₃For the linker, a mutant of Cetuximab in the form of scFv was constructed and ligated into the phage display plasmid pCANTAB-5E via the SfiI and NotI cleavage sites to form a gene library. For G465R mutation, we introduced random mutations by PCR using NNS degenerate codon at V50 and W52 in CDR2 region of heavy chain variable region of Cetuximab gene sequence to (Gly₄Ser)₃For the linker, a mutant of Cetuximab in the form of scFv was constructed and ligated into the phage display vector pCANTAB-5E via the SfiI and NotI cleavage sites to form a gene library. At the same time, we constructed pCANTAB-5E plasmid in the form of scFv of wild type Cetuximab without introducing any mutation. After that we will put two groups separatelyXL1Blue competent cells were electroporated with the library and pCANTAB-5E plasmid in the form of scFv of wild-type Cetuximab, and a phage display library and a strain displaying scFv of wild-type Cetuximab were prepared. The method comprises the following specific steps:

(1) electric rotary cup treatment: soaking in 75% ethanol electric revolving cup, washing with 100% ethanol, oven drying at 50 deg.C, and pre-cooling at 4 deg.C.

(2) 100ng of plasmid was added to 1 tube of competence, and the total volume after plasmid addition was 75-90. mu.L.

(3) Mix gently and stand on ice for 5 min. Simultaneously, 1mL SOC medium (2% peptone, 0.5% yeast extract, 0.05% NaCl, 2.5mM KCl, 10mM MgCl) was taken₂20mM glucose) was preheated at 37 ℃.

(4) The total volume of the bacterial solution added with the ligation product transferred to the gap of the electric rotating cup can not exceed 90 mu L.

(5) Setting parameters of the electrotransport instrument: 1800V voltage, 250 omega resistance, 1mm thick electrical rotor and 25 muF capacitance.

(6) Wiping the periphery of the electric rotating cup with water, putting the electric rotating cup into an electric rotating instrument, opening the cover of the electric rotating cup, electrically shocking the electric rotating cup after the cover of the electric rotating cup is opened, and immediately adding 1mL of preheated SOC culture medium to mix uniformly after the electric shock is finished.

(7) The bacterial solution was transferred to a 15mL gauze-wrapped test tube and shake-cultured at 37 ℃ for 1 h.

(8) Electric conversion efficiency verification: 2TY + G + A solid culture plates (1.6% peptone, 1% yeast extract, 0.5% NaCl, 2% agar, 2.5% glucose, 0.1mg/mL ampicillin) preheated at 37 ℃ and SOC medium were mixed with 10. mu.L of shake-up bacterial solution in 90. mu.L of SOC medium, diluted 10-fold as described above, and subjected to 6 gradients. For each gradient, 5. mu.L of bacteria were dropped onto the plate in 3 parallel groups. After the liquid was dried, it was cultured overnight at 37 ℃ in an inverted state. The next day, a certain countable dilution gradient is selected, the clone number is read, and the electric conversion efficiency is calculated according to the dilution ratio.

(9) 10 μ L of the bacterial solution was removed for confirmation of the electrotransformation efficiency, and the remaining bacterial solution was applied to a 2TY + G + A + tet solid culture plate (1.6% peptone, 1% yeast extract, 0.5% NaCl, 2% agar, 2.5% glucose, 0.1mg/mL ampicillin, 0.05mg/mL tetracycline), dried, and cultured overnight in an inverted state at 37 ℃.

(10) After the plate-coating culture is carried out overnight for about 12 hours, the bacteria on the plate are scraped and subpackaged in an EP tube by using a 2TY culture medium (1.6 percent peptone, 1 percent yeast extract and 0.5 percent NaCl) containing 15 percent glycerol, and the glycerol bacteria bank is obtained after the bacteria liquid is frozen at the temperature of minus 20 ℃ and then transferred to the temperature of minus 80 ℃ for storage.

(11) A defined amount of the strain was taken from the glycerol pool and inoculated into 2TY + G + A + tet medium (1.6% peptone, 1% yeast extract, 0.5% NaCl, 2.5% glucose, 0.1mg/mL ampicillin, 0.05mg/mL tetracycline).

(12) Incubation at 37 ℃ 200rpm/min for >1h allowed full expression of bacterial flagella, but at the same time the OD600 should be < 0.5.

(13) Adding helper phage, helper phage: the number of bacteria is about 10: superinfection is carried out at 1, 37 ℃ and 200rpm/min for 30-60 min.

(14) After superinfection was completed, superinfection efficiency was verified by titration on 2TY + G + A + tet solid culture plates (1.6% peptone, 1% yeast extract, 0.5% NaCl, 2% agar, 2.5% glucose, 0.1mg/mL ampicillin, 0.05mg/mL tetracycline) (the solid culture plates counting the total number of bacteria) and 2TY + G + K + tet solid culture plates (1.6% peptone, 1% yeast extract, 0.5% NaCl, 2% agar, 2.5% glucose, 0.05mg/mL kanamycin, 0.05mg/mL tetracycline) (the solid culture plates counting the number of bacteria of superinfection helper phage), respectively, and superinfection efficiency was calculated (superinfection efficiency ═ number of bacteria of infection helper phage/total number of bacteria, should be > 50%).

(15)3000 Xg, centrifuged for 15min, the supernatant was removed, and 2 volumes of 2TY + A + K + tet medium (1.6% peptone, 1% yeast extract, 0.5% NaCl, 2.5% glucose, 0.1mg/mL ampicillin, 0.05mg/mL kanamycin, 0.05mg/mL tetracycline) were added to the supernatant for overnight culture at 30 ℃ at 200 rpm/min.

(16) Centrifuging at 4 deg.C for 10min at 10000 Xg the next day, and collecting supernatant; add 1/5 volumes of PEG/NaCl (20% PEG8000, 2.5M NaCl) to the supernatant and precipitate the phage for 1h at 4 ℃.

(17)4500 Xg, 4 ℃, 15min centrifugation, remove supernatant. Using 1/20 volumes of supernatantLeft and right sterilization, PBS (137mM NaCl, 2.683mM KCl, 8.1mM Na) at 4 deg.C in cold₂HPO₄，1.76mM KH₂PO₄) Resuspending the pellet.

(18) Centrifuging at 12000 Xg and 4 ℃ for 10min, and taking the supernatant to obtain the phage-antibody solution.

(19) Phage concentration was roughly estimated by absorbance (OD268 ═ 1.0 for 5 × 10 per mL¹²Individual phage).

(20) Subpackaging and storing at-80 ℃ to obtain the phage display library. Both phage display libraries against S492R and G465R and strains displaying scFv of wild-type Cetuximab were prepared using the methods described above.

Example 5

1. Screening of phage antibody display libraries

(1) Cell pretreatment: trypsinizing NIH3T3 cells, NIH3T3 cells highly expressing wild type EGFR (WT-EGFR-NIH3T3 cells) and NIH3T3 cells highly expressing S492R mutant EGFR (S492R-EGFR-NIH3T3 cells) or NIH3T3 cells highly expressing G465R mutant EGFR (G465R-EGFR-NIH3T3 cells), and counting trypan blue stained live cells after complete culture and basic suspension. 300 Xg, 4 ℃, 5min after centrifugation, 10mL precooled PBS heavy suspension; each is 1 × 10⁷The cells of (4) were subjected to the next procedure.

(2) Non-specific screening: phage-antibody 100 times larger than the storage volume is added into 1mL of blocking solution and mixed for 1h at 4 ℃ in a rotating way. 5mL of blocking solution was added to NIH3T3 cells and vortexed at 4 ℃ for 1 h. Cells were centrifuged at 300 Xg for 5min at 4 ℃. The supernatant was removed and the phage-antibody containing solution was vortexed at 4 ℃ for 2 h. Centrifuge at 300 Xg for 5min at 4 ℃ and transfer the supernatant to a new centrifuge tube.

(3) And (3) specific screening: 5mL of each blocking solution was added to S492R-EGFR-NIH3T3 and G465R-EGFR-NIH3T3 cells, respectively, and mixed by rotation at 4 ℃ for 1 h. Centrifuge at 300 Xg, 4 ℃ for 5min and discard the supernatant. Adding the supernatant obtained in (2) into a closed S492R/G465R-EGFR-NIH3T3 cell, and rotating for 2h at 4 ℃. Centrifuge at 300 Xg, 4 ℃ for 5min and discard the supernatant. Cells were washed 20 times with 1mL PBS. Adding 500 μ L of eluent, standing for about 10min, and collecting supernatant. The neutralizing solution was added to the elution supernatant, and the pH was adjusted to about 7.2. 5mL of blocking solution was added to WT-EGFR-NIH3T3 cells and vortexed at 4 ℃ for 1 h. Centrifuge at 300 Xg, 4 ℃ for 5min and discard the supernatant. The above neutralized supernatant was added to blocked WT-EGFR-NIH3T3 cells and spun at 4 ℃ for 2 h. Centrifuge at 300 Xg, 4 ℃ for 5min and discard the supernatant. Cells were washed 20 times with 1mL PBS. Adding 500 μ L of eluent, standing for about 10min, and collecting supernatant. The neutralizing solution was added to the elution supernatant, and the pH was adjusted to about 7.2. 10 μ L of the neutralized supernatant was titrated and the phage concentration was roughly calculated using OD268 absorbance values, followed by storage at 4 ℃ for the next round of screening.

(4) Sorting in different rounds: inoculating 10mL of XL-1Blue Escherichia coli into a 2TY + G + tet culture medium, shaking until OD600 is 0.5, adding the supernatant containing the phage obtained by screening in (3) into XL-1Blue Escherichia coli, and culturing at 37 ℃ and 200rpm/min for 1 h. Adding 10 times of the bacterial amount of the helper phage, and superinfecting for 1h at 37 ℃ and 200 rpm/min. Centrifuge at 3000 Xg for 10min at 25 ℃ and discard the supernatant. 20mL of 2TY + A + K + tet medium was added, and the mixture was shaken at 200rpm/min at 30 ℃ overnight. And preparing a secondary phage antibody library according to the phage display library preparation method, and carrying out the next round of screening. The screening method is the same as the first round, and the identification is started after three rounds of screening. On the last screening round, XL-1Blue E.coli was prepared in advance and shaken to OD600 of 0.1-0.5. The phage-containing supernatant obtained by the screening was added to the prepared XL-1Blue E.coli, and cultured at 37 ℃ and 200rpm/min for 1 hour. Centrifuging at 3000 Xg for 10min, leaving a proper amount of supernatant to resuspend the thalli, coating the thalli on a solid culture plate of 2TY + G + A + tet, culturing overnight in an inverted mode, and picking out a monoclonal for subsequent identification.

2. Identification of Positive clones

After three rounds of screening, the scFv mutants capable of overcoming the point mutation of EGFR S492R or G465R were identified by ELISA, which comprises the following steps:

(1) culturing of monoclonal strains: single clones were randomly picked from the solid culture plates of the third round of screening into 2mL EP tubes containing 900. mu.L of 2TY + G + A + tet medium, while XL-1Blue displaying the scFv form of wild-type Cetuximab and a blank were set. The EP tube was placed in an incubator at 37 ℃ and 220rpm/min to achieve an OD600 of about 0.5.

(2) IPTG-induced secretory expression of scFv: 3000 Xg, 10min centrifugation, supernatant removal, addition of new 2TY + A + tet medium (1.6% peptone, 1% yeast extract, 0.5% NaCl, 0.1mg/mL ampicillin, 0.05mg/mL tetracycline) and 1mM IPTG, 30 ℃, 180rpm/min overnight induction of secretory expression.

(3) The next day, 4 ℃, 12000 × g, 10min centrifugation, and supernatant taken for ELISA and flow cytometry identification.

(4) Antigen coating: the WT-EGFR-ECD-Fc and S492R-EGFR-ECD-Fc or G465R-EGFR-ECD-Fc antigenic proteins were coated onto ELISA plates at 1. mu.g/mL, 100. mu.L/well and coated overnight at 4 ℃.

(5) And (3) sealing: the overnight-coated antigen was discarded, the ELISA plates were washed three times with PBST, blotted dry, and 200. mu.L/well blocking solution was added to the ELISA plates, incubated at 37 ℃ for 1 h.

(6) Primary antibody incubation: the blocking solution in the ELISA plate was discarded, patted dry, and 100. mu.L/well of the collected induction supernatant was added, and incubated at 37 ℃ for 1 hour.

(7) And (3) secondary antibody incubation: the primary antibody in the ELISA plate was discarded, the ELISA plate was washed five times with PBST, patted dry, and 100. mu.L/well of Mouse-anti-E-tag monoclonal antibody was added, and the secondary antibody was diluted in 1% skim milk at a ratio of 1:1000 and incubated at 37 ℃ for 1 h.

(8) And (3) incubation of three antibodies: the secondary antibody in the ELISA plate was discarded, the ELISA plate was washed five times with PBST, blotted dry, and then 100. mu.L/well of HRP-conjugated-Goat-anti-Mouse IgG (H + L) polyclonal antibody was added, and the tertiary antibody was diluted in 1% skim milk at a ratio of 1:1000 and incubated at 37 ℃ for 1H.

(9) Color development: discarding the primary antibody in the ELISA plate, washing the ELISA plate with PBST five times, beating to dry, adding 100 mu L/hole TMB color development solution, carrying out dark reaction in an incubator at 37 ℃ for 15-30min, measuring the light absorption value (A405) of each monoclonal hole at 405nm by using an enzyme-labeling instrument, and determining the positive candidate mutant clone according to the signal values of the antigen hole and the control hole. The results of the partial ELISA are shown in FIG. 1, with 5 strong positive clones for the S492R point mutation and 1 strong positive clone for the G465R point mutation.

Expression and purification of Cetuximab, Panitumumab, and Cetuximab mutants

The Cetuximab mutant full-antibody form heavy chain plasmid is constructed by sequencing and replacing the amino acids changed in the heavy chain variable region cloned from the positive candidate mutant into the heavy chain variable region of Cetuximab. The Cetuximab (the light chain amino acid sequence is shown as SEQ ID No.8, the heavy chain amino acid sequence is shown as SEQ ID No. 9), Panitumumab and the whole antibody form heavy chain plasmid and the light chain plasmid of the Cetuximab mutant are transferred into HEK-293F cells instantly, cell culture solution supernatant is collected after culturing for 4 days in a cell shaker at 37 ℃, Protein is purified by a Protein A affinity column to obtain Cetuximab, Panitumumab and the Cetuximab mutant with the heavy chain variable region amino acid sequence shown as SEQ ID No.1-6, six Cetuximab mutants respectively correspond to VY in figure 2 (the amino acid sequence of the heavy chain is shown as SEQ ID No.1, the base sequence is mutated from GTA coding V50 bit to CAG, the TAC coding Y104 bit to GTG, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.2, the base sequence is mutated from TAC coding Y104 bit to GAT, the base sequence of the TAC coding Y104 bit is shown as GTG, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.3, the base sequence is mutated from TAC at the position of encoding Y104 to AAC), Y104C (the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.4, the base sequence is mutated from TAC at the position of encoding Y104 to TGC), Y104V (the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.5, the base sequence is mutated from TAC at the position of encoding Y104 to GTG), and W52D (the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.6, and the base sequence is mutated from TGG at the position of encoding W52 to GAC). SDS-PAGE analysis of the collected Cetuximab, Panitumumab and 6 Cetuximab mutant antibody proteins revealed that the desired bands appeared at positions around 55kDa and 28kDa under reducing conditions, indicating that the Cetuximab, Panitumumab and 6 Cetuximab mutants were successfully expressed.

The AKTA protein purifier was operated as follows:

(1) preparing a related solution for purifying a ProteinA affinity column, and filtering the solution by using a 0.22 mu m filter membrane to remove impurities;

(2) starting an AKTA protein purification instrument, setting various parameters and pressure upper limit: 0.15MPa, flow rate: 3 mL/min;

(3) putting the pump head into pure water, and starting a cleaning program;

(4) loading Protein A affinity column on a Protein purifier, and flushing with pure water to balance;

(5) washing the column with an elution buffer and a binding buffer in sequence to equilibrium;

(6) sampling;

(7) after loading is complete, the column is washed to equilibrium with binding buffer;

(8) washing with elution buffer, collecting protein, and identifying protein purity by SDS-PAGE;

(9) sequentially washing the column with pure water and 20% ethanol until the column is balanced;

(10) collecting the column, and storing in 20% ethanol at 4 deg.C.

The target protein is replaced by PBS buffer solution by an ultrafiltration tube with the membrane molecular weight cutoff of 30KDa, and the protein concentration is measured by NanoDrop and stored at-80 ℃.

ELISA determination of affinity of Each Cetuximab mutant

Each Cetuximab mutant was tested by ELISA to verify its affinity function. The ELISA procedure was as follows:

(1) coating antigen: diluting the wild type, or S492R mutant, or G465R mutant EGFR extracellular region Fc fusion protein to 1 mu G/mL by using a coating solution, adding 100 mu L of the diluted protein into a 96-well enzyme label plate per well, and standing overnight at 4 ℃;

(2) washing the plate: throwing off liquid in the holes, adding 250 mu L of PBST in each hole, slightly shaking for a plurality of times, then discarding the liquid, and repeatedly washing for 5 times;

(3) and (3) sealing: adding 200 mu L of confining liquid into each hole, and incubating for 1h at 37 ℃;

(4) incubating primary antibody: removing liquid, adding 100 μ L of antibody diluted in gradient of 7 concentrations from 100nM to 10 times per well, and incubating at 37 deg.C for 1 h;

(5) washing the plate for 5 times, and performing the same step (2);

(6) hatching a secondary antibody: mu.L of goat anti-human Kappa light chain secondary antibody labeled with HRP diluted with 1:250 blocking solution was added to each well and incubated at 37 ℃ for 1 h;

(7) washing the plate for 5 times, and performing the same step (2);

(8) color development: adding 100 μ L of color development solution into each well, and incubating at 37 deg.C in dark for 20-30 min;

(9) and (4) terminating: adding 100 mu L of stop solution into each hole, and slightly flapping the 96-hole plate to uniformly mix the solution;

(10) reading: the OD450 value of each well was read with a microplate reader.

(11) The GraphPad Prism5 plots affinity curves.

ELISA affinity detection experiments were performed by gradient dilution of antibody concentrations to further compare the affinities of Cetuximab, Panitumumab, VY, Y104D, Y104N, Y104C, Y104V with S492R mutant EGFR extracellular region Fc fusion protein (S492R-EGFR-ECD-Fc), the affinity curves for the S492R mutant EGFR extracellular region are shown in fig. 3, the affinity curves for Panitumumab, VY, Y104D, Y104N, Y104C, Y104V are clearly concentration dependent compared to the affinity curve for cetitumumab, and the effect of VY, Y104D is not weak and is even better than Panitumumab. The results show that Cetuximab cannot be combined with S492R mutant EGFR, Panituximab and 5 Cetuximab mutants of VY, Y104D, Y104N, Y104C and Y104V can be combined with S492R mutant EGFR, and the two Cetuximab mutants of VY and Y104D have no weak effect or even better effect than Panitumumab.

ELISA affinity detection experiments were performed by gradient dilution of antibody concentrations to further compare the affinity of Cetuximab, Panitumumab, and W52D to the Fc fusion protein of the extracellular region of G465R mutant EGFR (G465R-EGFR-ECD-Fc), and the affinity curve for the extracellular region of G465R mutant EGFR is shown in FIG. 4, where the affinity curve for W52D appears significantly concentration-dependent compared to the affinity curves for Cetuximab and Panitumumab. It is shown that Cetuximab and Panitumumab cannot bind to G465R mutant EGFR, and W52D Cetuximab mutant can bind to G465R mutant EGFR.

The affinity of Cetuximab, Panitumumab, VY, Y104D, W52D to the Fc fusion protein of the extracellular domain of wild-type EGFR (WT-EGFR-ECD-Fc) was further verified by performing ELISA affinity assay experiments by diluting antibody concentrations in a gradient, the affinity curves for the extracellular domain of wild-type EGFR are shown in FIG. 5, and the affinity curves for VY, Y104D, W52D, Cetuximab and Panitumumab all show concentration dependence. It was demonstrated that the three Cetuximab mutants, VY, Y104D, W52D, did not affect binding to wild-type EGFR.

Sequence listing

<110> Zhejiang university

<120> cetuximab mutant and application thereof

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<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Gln Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Val Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 2

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Asp Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 3

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Asn Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 4

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Cys Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 5

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Val Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 6

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Asp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 7

<211> 1210

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 7

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln

20 25 30

Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe

35 40 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr

370 375 380

Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu

385 390 395 400

Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln

420 425 430

His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu

465 470 475 480

Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro

500 505 510

Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn

515 520 525

Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly

530 535 540

Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620

Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly

625 630 635 640

Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu

645 650 655

Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His

660 665 670

Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu

675 680 685

Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu

690 695 700

Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser

705 710 715 720

Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu

725 730 735

Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser

740 745 750

Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser

755 760 765

Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser

770 775 780

Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp

785 790 795 800

Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn

805 810 815

Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg

820 825 830

Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro

835 840 845

Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala

850 855 860

Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp

865 870 875 880

Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp

885 890 895

Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser

900 905 910

Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu

915 920 925

Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr

930 935 940

Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys

945 950 955 960

Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln

965 970 975

Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro

980 985 990

Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp

995 1000 1005

Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe Phe

1010 1015 1020

Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu Ser Ala

1025 1030 1035 1040

Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn Gly Leu Gln

1045 1050 1055

Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg Tyr Ser Ser Asp

1060 1065 1070

Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Asp Thr Phe Leu Pro

1075 1080 1085

Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg Pro Ala Gly Ser

1090 1095 1100

Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro Ser

1105 1110 1115 1120

Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala Val Gly Asn Pro

1125 1130 1135

Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn Ser Thr Phe Asp

1140 1145 1150

Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln Ile Ser Leu Asp

1155 1160 1165

Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn

1170 1175 1180

Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val

1185 1190 1195 1200

Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala

1205 1210

<210> 8

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 9

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 11

<211> 642

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gacatcctgc tgacccagtc tccagtaatc ctgtctgtgt cccctggtga acgcgtcagc 60

ttttcttgcc gcgcgtccca aagcattggt accaacattc actggtacca gcagcgtacc 120

aacggttccc cgcgtctgct gatcaagtac gcatctgaaa gcatttccgg catcccgtcc 180

cgtttctctg gcagcggttc cggcaccgat ttcactctga gcatcaactc tgttgagtct 240

gaagatatcg ctgactacta ttgtcagcag aacaataact ggccgaccac tttcggtgcc 300

ggcacgaaac tggaactgaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642

<210> 11

<211> 1347

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

caggttcaac tgaaacagtc cggtccgggt ctggtccagc cgtctcaatc cctgtctatt 60

acctgtacgg ttagcggttt ctctctgact aactacggcg tgcactgggt gcgtcagagc 120

cctggcaaag gcctggaatg gctgggtgta atctggagcg gcggtaacac cgactacaat 180

accccattca cctcccgtct gtccatcaac aaggacaact ctaaatctca ggttttcttt 240

aaaatgaaca gcctgcagtc taacgatact gcgatctatt actgcgcccg cgctctgacc 300

tattacgatt acgagttcgc gtattggggt cagggcactc tggttaccgt atccgcagct 360

agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg 1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200

gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1260

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320

aagagcctct ccctgtctcc gggtaaa 1347

<210> 12

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Claims

1. The cetuximab mutant is characterized by comprising a cetuximab light chain and a heavy chain with mutation, wherein the mutation of the heavy chain is positioned in a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID No. 1-6.

2. A gene encoding the cetuximab mutant of claim 1.

3. A single-chain antibody is characterized by comprising a light chain variable region of cetuximab and a heavy chain variable region with mutation, wherein the amino acid sequence of the heavy chain variable region with mutation is shown in any one of SEQ ID No. 1-6.

4. A gene encoding the single-chain antibody of claim 3.

5. A Fab fragment antibody, which is characterized by comprising a cetuximab light chain, a CH1 region of a heavy chain and a heavy chain variable region with mutation, wherein the amino acid sequence of the heavy chain variable region with mutation is shown as any one of SEQ ID No. 1-6.

6. A gene encoding the Fab fragment antibody of claim 5.

7. A (Fab)2 fragment antibody, comprising two molecules of the Fab fragment antibody of claim 5 per molecule of the antibody.

8. Use of the cetuximab mutant according to claim 1, the single-chain antibody according to claim 3, the Fab fragment antibody according to claim 5 or the (Fab)2 fragment antibody according to claim 7 for the preparation of a medicament targeting EGFR.

9. The use according to claim 8, wherein the EGFR carries the mutation S492R when the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID Nos. 1 to 5; when the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.6, EGFR carries the G465R mutation.

10. The use of claim 8, wherein the disease to which the medicament is directed is colorectal cancer.