Construction And Screening Of Humanized Anti - HER3 ScFv Gene Genes

Both HER3and HER2are important members of EGFR (epidermal growth factorreceptor) family, which belongs to a transmembrane receptor tyrosine kinase (RTK), andthey are related to the formation and development of a variety of human cancers. Theexpression level of HER3is highly correlated with HER2. HER3is an important cofactor forthe formation of HER3-HER2heterodimers, and it is also one of important factors for thedevelopment of cancer. It is reported that50%patients who suffer from breast cancer were found HER2positive, and41.8%patients were found HER3postive respectively. WhenHER3, HER2both are found positive in patients, trastuzumab, an antibody targeted breastcancer, has poor response.Thus, to suppress over-expression of EGFR or HER2by blockingthe HER3might be an important strategy for the cancer treatment.【Objective】In this study, a murine VH-CDR3(librare of CDR3of variable region of the murineheavy chain) library will be grafted onto a human scFv framework for the construction of ahumanized HER3scFv library. Then the library will be screened for obtaining anti-HER3scFv which may have potential applications in cancer treatment. On the other hand, thisstudy might provide a new approach for the rapid screening of humanized antibodies.【Methods】1. After immunized with Recombinant HER3antigen, serum of Balb/c mice wascollected for the detection of anti HER3serum titer. When titer reached1:200,000, totalRNA was extracted from the spleen cells of immunized mice and then the total cDNA wasgot by RT-PCR. A murine VH-CDR3library was amplified used cDNA as a template.Subsequently, the library was grafted on human scFv (VH3-VΚ1) framework for theconstruction of a humanized antibody gene library.2. Anti-HER3scFv templates were selected and enriched through ribosome displaytechonology. A humanized anti-HER3single chain antibody librarie was constructed byapplying mutilple cloning strategies such as adding expression related elements at the Nterminal of library for the efficient expression and2(His6) at the C terminal for theconvenience of purification.3. The target scFv library was transformed into E. coli BL21(DE3) for solubleexpression. The expressed soluble proteins were screened by ELISA assay. The final selectedtarget scFv was purified and its affinity was detected.【Results】A humanized anti-HER3ribosome display library which storage capacity is1012wasconstructed. A humanized antibody strains HER3-236scFv, affinity constant of3.021×10-8M, was successfμl obtained. 【Conclusions】This study provides a valuable antibody molecule for the development of anti-HER3antibodies. In addition, this study provides a new approach for rapid screening of humanizedantibodies.