Synthesis of the scFv fragment of anti-Frizzled-7 antibody and evaluation of its effects on triple-negative breast cancer in vitro study.

ABSTRACT

OBJECTIVES: Among the most promising antibody formats in terms of inhibiting carcinogenesis are single-stranded variable fragments, whose targeted binding to the Fzd7 receptor has been proven effective at suppressing tumorigenesis. In this study, we investigated the effectiveness of an anti-Fzd7 antibody fragment against both tumor growth and metastasis of breast cancer cells. METHODS: To develop anti-Fzd7 antibodies, bioinformatics approaches were used and the antibodies were expressed recombinantly in E. coli BL21 (DE3). The expression of anti-Fzd7 fragments was verified by Western blotting. Analysis of the antibody's binding capacity to Fzd7 was conducted by flow cytometry. Cell death and apoptosis were assessed by MTT and Annexin V/PI assays. The transwell migration and invasion assays, as well as the scratch method, were used to evaluate cell motility and invasiveness. RESULTS: The anti-Fzd7 antibody was expressed successfully as a single band of 31 kDa. It could bind to 21.5% of MDA-MB-231 cells, as opposed to only 0.54% of SKBR-3 cells as negative control. According to MTT assay, induced apoptosis was 73.7% in MDA-MB-231 cells, compared with 29.5% in SKBR-3 cells. Also, the antibody exerted a significant inhibitory effect of 76% and 58% on migration and invasion of MDA-MB-231 cells, respectively. CONCLUSION: The recombinantly developed anti-Fzd7 scFv of this study could exhibit significant antiproliferative and antimigratory properties, along with a high apoptosis-inducing potential, making it suitable for the immunotherapy of triple negative breast cancer.