Isolation And Identification Of Human Single Chain Fv Antibodies Against Breast Cancer From A Human Phage Display Library

Background:Breast cancer is one of the most common malignant tumor of women. Theincidence rate of breast cancer is about7%~10%of systemic malignancies,which causes about50million people to die every year. In addition to surgery,the current treatment methods of breast cancer mainly include radiationtherapy, chemotherapy and hormone therapy, etc. The first genetic targetedtherapy drug Herceptin comes out in1998. It mainly restrains cancer cellsproliferation through specifically blocking people epidermal growth factorreceptor2(HER2) downstream signaling pathways. This makes abreakthrough progress of the drug treatment of breast cancer. However, theapplication of herceptin also has many shortco mings, such as only effectivefor those over expressing of HER2genes, many side effects, and the highcosts, etc.Single chain fragment of variable region (scFv) is a recombinant protein which joins variable region of heavy chain (VH) and variable region of lightchain (VL) of traditional antibody with a connection peptide (linker) throughgenetic engineering method. ScFv is very suitable for tumor targeting therapyfor many advantages such as low molecular weight, strong penetration, weakimmunogenicity, positioning rapidly, and convenience to preparation andmodification. And the emergence of phage display technology in the1990’screated a simple, fast methods for preparation of genetic engineering antibodyas scFv.High capacity and preparation source are two important decision factorsaccess to efficient and broad-spectrum antibodies. The ideal antibody shouldbe screened from a library as large as possible and directly from the humanbody. But most related research is difficult to meet both the requirementsbecause of certain constraints.Objective:The aim of this study is to construct a human phage display scFv libraryagainst breast cancer by molecular cloning and phage display technology. Wehope to obtain the specific anti-mastocarcinoma antibodies from the librarywhich can be used further in the therapeutic research on breast cancer.Methods：1. Peripheral blood samples of breast cancer patients withoutchemotherapy were got from hospital. Using RT-PCR, we amplified thevariable region of heavy chain (VH) and variable region of light chain (VL)genes from peripheral blood lymphocytes (PBL). Then we obtained the scFvfragments through SOE-PCR, and the scFv fragments were cloned into thevector pCANTAB-5E and electroporated into competent E.coli TG1cells.Consequently, a primary scFv phage display library was constructed. 2. Screened with MCF-7cells, the recombinant phagemids were rescuedby reinfection of helper phage M13KO7. Recombinant phages specific forbreast cancer cells were enriched after four rounds.3. The antigen-positive clones were selected from the enriched clones byphage ELISA. Positive clones was used to infect E.coli HB2151to expresssoluble scFv antibody. The antigen binding activity of the soluble antibodieswere detected by Western blot.Results:1. The fragment of VH and VL were linked successfully in vitro to formscFv of about750bp. A recombinant phage display library with total of2.4×108was established. Randomized clones from unselected library digestedwith BstNI showed different patterns.2. The specific antibodies against MCF-7cells were enriched after fourrounds of affinity selection. Through the analysis of nucleic acid sequence andprotein sequence we confirmed that the obtained antibodies were consistentwith the characteristics of IgG antibodies.3. SDS-PAGE and Western blot results shown that there was a band at30kDa on the membrane, which indicated soluble antibodies were present.Through ELISA analysis, soluble antibodies had the affinity to a human breastcancer cell line MCF-7but not to other cancer cell line, which demonstratedscFv could react specifically with breast cancer cells.Conclusion:We constructed a scFv phage library against human breast cancer withhigh capacity by phage display technology. The scfv was demonstrated to besuccessfully expressed in E·coli HB2151and specific to breast cancer cells.Sequencing indicated that VH and VL genes were high homologous with the variable region of human IgG antibody. In the production of engineeringantibodies, this strategy may provide an alternative approach and a basis forfurther studies on diagnosis and therapy of breast cancer.