ABSTRACT
OBJECTIVE: To investigate the inhibitory effects of bispecific antibody-drug conjugate Fv-LDM-NGR targeting EGFR and CD13 on human tumor cells and endothelial cells,and possible mechanisms. METHODS: Human breast cancer cells MCF-7, human lung adenocarcinoma cells A549 and human microvascular endothelial cells HMEC-1, were studied. MTT assay was applied to measure proliferative activity of tumor cells. The influence of Fv-LDM-NGR on tube formation of HMEC-1 was observed. Transwell assay was applied to measure migration and invasion capacity in tumor cells. Western blot was applied for analyzing intracellular signaling transduction pathways. Flow cytometry, Hochest stain and Annexin -FITC/PI were used to detect cell cycle and apoptosis. RESULTS: Fv-LDM-NGR could inhibit the proliferation of tumor cells and microvascular endothelial cells with IC50 values of 10-14-10-12mol•L-1. Fv-LDM-NGR prevented tube formation in microvascular endothelial cells, and suppressed migration and invasion in tumor cells. Fv-LDM-NGR interfered with the intracellular signaling transduction pathways, then caused G2/M and S phase arrest and induced apoptosis. CONCLUSION :Bispecific antibody-drug conjugate Fv-LDM-NGR could prevent cell invasion in tumor cells and tube formation in microvascular endothelial cells through blocking activity of CD13. And it could down-regulate the expression and the phosphorylation of EGFR, interfere with cellular signal pathways, induce cell cycle arrest and cell apoptosis, and inhibit cell proliferation and migration.
ABSTRACT
Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.