Development of a humanized anti-FABP4 monoclonal antibody for treatment of breast cancer

Abstract

BackgroundBreast cancer, lung cancer, and colorectal cancer are the primary contributors to newly diagnosed cases among women, with breast cancer representing the second highest proportion of the total. The treatment protocols vary depends on different stages of breast cancer, and numerous clinical trials are ongoing based on the data derived from laboratory. Our studies demonstrate that circulating adipose fatty acid binding protein (A-FABP, or FABP4) links obesity-induced dysregulated lipid metabolism and breast cancer risk, thus offering a new target for breast cancer treatment. MethodsWe immunized FABP4 knockout mice with recombinant human FABP4 and screened hybridoma clones with specific binding to FABP4. The potential effects of antibodies on breast cancer cells in vitro were evaluated using migration, invasion, and limit dilution assays. Tumor progression in vivo was evaluated in various types of tumorigenesis models including C57BL/6 mice, Balb/c mice, and SCID mice. The phenotype and function of immune cells in tumor microenvironment were characterized with multi-color flow cytometry. Tumor stemness was detected by ALDH assays. To characterize antigen-antibody binding capacity, we determined the dissociation constant of S-V9 against FABP4 via surface plasmon resonance. Further analyses in tumor tissue were performed using 10X Genomics Visium spatial single cell technology. ResultsHerein, we report the generation of humanized monoclonal antibodies blocking FABP4 activity for breast cancer treatment in mouse models. One clone, named 12G2, which significantly reduced circulating levels of FABP4 and inhibited mammary tumor growth, was selected for further characterization. After confirming the therapeutic efficacy of the chimeric 12G2 monoclonal antibody consisting of mouse variable regions and human IgG1 constant regions, 16 humanized 12G2 monoclonal antibody variants were generated by grafting its complementary determining regions to selected human germline sequences. Humanized V9 monoclonal antibody showed consistent results in inhibiting mammary tumor growth and metastasis by affecting tumor cell mitochondrial metabolism. ConclusionsOur current evidence suggest that targeting FABP4 with humanized monoclonal antibodies represents a novel strategy for the treatment of breast cancer and possibly other obesity- associated diseases.