Screening, Preparation And Characterization Of Humanized Nanobodies Targeting The Dimer Interface Of Epidermal Growth Factor Receptor (EGFR)

Purpose:Epidermal growth factor?EGFR?,a member of the ErbB family,is one of the most famous cell signaling receptors in cellular receptors.As the first receptor identified as a protein-tyrosine kinase,EGFR plays an important role in regulating apoptosis,cell cycle progression,differentiation and transcription.Mutation and over-expression of EGFR are associated with the occurrence,development and prognosis of many cancers,and have become one of the hot targets for the treatment of many types of malignant tumors such as non-small cell lung cancer,breast cancer,stomach cancer,head and neck cancer and colorectal cancer.The currently marketed drugs targeting EGFR show low clinical response rates and drug resistance.Hereditary and pathological mutations on the surface of EGFR molecules and heterodimerization between EGFR and other family receptors are two main reasons.Therefore,the highly conserved EGFR dimerization interface on the target structure may be an effective strategy to solve the problem of hereditary and acquired resistance.The laboratory used the EGFR dimerization interface to directly participate in the dimerization.The?-loop polypeptide EGFR^237-267 was used as an antigen to construct a polypeptide vaccine and a monoclonal antibody targeting the EGFR dimer interface,and the feasibility of the strategy was preliminarily demonstrated by in vitro and in vivo experiments.Nanobodies have great advantages in size,water solubility,stability and preparation.They will focus on the problem of heterodimerization and the characteristics of nanobodies.This study intends to use the above antigen peptides to screen targets from humanized nanobody display libraries.Nanobodies at the EGFR dimerization interface were established with E.coli high-efficiency expression system,and the corresponding in vitro anti-tumor activity was studied,which laid a foundation for further development of therapeutic nanobodies targeting the EGFR dimer interface.Method:1.Phage display technology screening nanobodyThe short peptide was synthesized by the company's patented sequence for the EGFR²37-26737-267 dimerization interface.A synthetic humanized nanobody library using a synthetic antigen peptide as an antigen coated with an aminase target,using a VH3-23 heavy chain fragment to mutate the bases of the CDR1,CDR2 and CDR3 regions by PCR.According to the phage display screening humanized antibody fragment manipulation manual,three rounds of phage panning were performed.Finally,a highly specific nanobody targeting the EGFR dimerization interface was obtained by ELISA.The naonbodies gene sequence is finally obtained by gene sequencing.2.Construction of prokaryotic expression system of nanobodiesThe target gene sequence was designed according to the E.coli preference codon,and the recognition sequences of NdeI and HindIII were added to the front and rear ends of the target gene sequence,and the HIS-tag sequence was added before the 3'end restriction site.The designed target gene sequence was entrusted to the company for synthesis and cloned into the E.coli expression vector pET21b NdeI-HindIII site for efficient expression under the T7 promoter.The target gene expression vector was introduced into the host strain E.coli ShuffleT7-B to achieve soluble expression of the target protein.3.Nanobody expression and purificationBy reducing the temperature,the nanobodies can be expressed in a soluble manner,using a CM Sepharose column?10 mm×150 mm?and the desired product was purified by a Ni-NTA column?10 mm×150 mm?chromatography.4.Detection of surface specific binding of nanobodies and tumor cells over-expressing EGFREGFR over-expressing human epidermal cancer cells A431 were incubated with different concentrations of nanobodies and different concentrations of EGF,and specific binding of EGFR and nanobodies on the cell surface was detected by flow cytometry.5.Detection of the inhibition rate of nanobodies against EGFR over-expressing tumor cells by MTT assayThe MTT assay was used to compare the monoclonal antibody EGFR Dimer 5G9 prepared in our laboratory to the inhibition rate of EGFR over-expressing tumor cells A431?human epidermal carcinoma?and H292?human lung cancer?according to the corresponding concentration.Result:1.Phage display technology for screening nanobodiesFinally,11 positive clones were screened,and 4 different V-region sequences of nanobodies were obtained by sequencing,and the rest were completely consistent with the four V-region sequences.Four different positive clones capable of expressing nanobodies targeting the EGFR dimerization interface were obtained and designated as EGFR dimer Nb77,EGFR dimer Nb80,EGFR dimer Nb96,EGFR dimer Nb104.2.Nanobody expression and purificationE.coli ShuffleT7-B engineered bacteria containing EGFR dimer Nb77and EGFR dimer Nb96 recombinant plasmid were successfully constructed and expressed at 30°C.The expressed product was purified by cation exchange and nickel column affinity chromatography.Protein purity was more than 98%.3.Detection of surface specific binding of nanobodies and tumor cells over-expressing EGFRThe nanobody EGFR dimer Nb77 detected specifically binds to A431cells,and the specific binding is stronger as the nanobody concentration and the concentration of EGF increase.It is shown that A431 tumor cells are concentration dependent on the nanobody EGFR dimer Nb77 and EGF.4.Detection of the inhibition rate of nanobodies against EGFR over-expressing tumor cells by MTT assayEGFR dimer Nb77 has inhibitory activity against EGFR over-expressing cell lines A431 and H292,and the inhibition rate of nanobody EGFR dimer Nb77 on EGFR over-expressing cell line A431increases with increasing nanobody concentration.It has the same antitumor activity as the monoclonal antibody EGFR Dimer 5G9.Nanobodies targeting the EGFR dimerization interface have a good prospect for treating tumor diseases.Conclusion:Four EGFR dimer interface-targeted humanized nanobodies were screened from commercially available humanized nanobody libraries using?-loop polypeptide EGFR^237-267 as antigen which is directly targeting the EGFR dimerization interface.Highly soluble expression of two nanobodies,EGFR dimer Nb77 and EGFR dimer Nb96,was achieved in the host E.coli ShuffleT7-B.The highly purified nanobody EGFR dimer Nb77 specifically binds to cell surface EGFR and significantly inhibits EGFR over-expressing the growth of tumor cells.