Obesity elevates circulating levels of A-FABP, which can directly target breast cancer (BC) cells to enhance mammary tumor initiation. We reasoned that inhibition of A-FABP activity by blocking monoclonal antibodies (mAb) reduced BC risk. We immunized mice with human A-FABP and identified multiple monoclonals which bound to both murine and human A-FABP antigens. Among these selected anti-A-FABP clones, we found that treatment with 12G2 clone, but not PBS or 6H10 clone, significantly inhibited E0771 mammary tumor growth in vivo. We synthesized chimeric mAb with variable (V) regions of 12G2 clone and human IgG1 constant (C) regions. Interestingly, treatment of SCID mice with this chimeric mAb dramatically inhibited human BC MCF-7 tumor growth in vivo. Given the success of mAb-based cancer immunotherapy (e.g. anti-PD-1/PD-L1), our data suggest that targeting A-FABP with blocking mAb represents a novel strategy for BC treatment.

Recently, we identified the complementarity determining regions (CDR) of the 12G2 antibody. Through the molecular modeling and in silico humanization processes, 4 humanized VH and VL sequences have been produced by grafting select CDR amino acids from 12G2 parental sequences onto the closest matching human acceptor germline sequences, which gives a possible 16 humanized antibodies. With this panel of purified variant antibodies, we will measure their antigen binding activity by Western blotting and ELISA. Variant antibodies with improved affinity will be selected for in vitro tests, which include BC cell spheroid invasion assay, stemness analysis (e.g. ALDH1 activity) and the limiting dilution assay. Variants which show effective activity of blocking A-FABP-mediated effects in vitro will be further tested in vivo. We will collect fresh BC tissues from the Department of Surgery, University of Iowa and establish patient-derived xenograft (PDX) models to verify the therapeutic efficacy of the select humanized antibodies in both lean and HFD-induced obese SCID mice. Using these clinical-relevant models, we will further determine the mechanisms of action of select antibodies by focusing on if antibody treatment 1) reduces circulating levels of A-FABP and its mediated metabolic effects on BC cells, and/or 2) blocks TAM infiltration and pro-tumor activities. These data will provide valuable information for future clinical trials of using anti-A-FABP antibodies for treatment of obesity-associated diseases, including breast cancer.