Mucin1 (MUC1) is aberrantly glycosylated and overexpressed in various cancers, and it plays a crucial role in cancerogenesis. MUC1 is a type I membranous protein composed of α and β subunits. MUC1-α can be cleaved in cancers, exposing MUC1-β (MUC1-C). MUC1-C is involved with multiple cancer cellular functions, which makes it an attractive target for cancer treatment. However, its multifunctional mechanisms have not been fully elucidated and there has not been a successful therapeutic development against MUC1-C. Through a phage display process, we isolated the specific antibodies for the extracellular domain of MUC1-C. The relevant full IgG antibodies were produced successfully from mammalian cells and validated for their MUC1-C specificities through ELISA, dual FACS analysis, BLI assay, and confocal image analysis. In the comparison with reference antibody, elected antibodies showed characteristic bindings on target antigens. In the functionality assessment of high-ranking antibodies, SKM1-02, -13, and -20 antibodies highly inhibited invasion by triple-negative breast cancer (TNBC) cells and the SKM1-02 showed strong growth inhibition of cancer cells. Our results showed that these MUC1-C specific antibodies will be important tools for the understanding of MUC1 oncogenesis and are also highly effective therapeutic candidates against human breast cancers, especially TNBC cells.

The therapeutic functionality of the antibodies from phage display is verified after an initial screening. Several immunological assays such as ELISA, flow cytometry, the western blot, and surface plasmon resonance (SPR) assay are commonly used; the IgG-format antibody is usually preferred to verify the functionality of antibodies, which need elaborative mammalian expression and purification work. Here, we describe a biolayer interferometry (BLI)-based assay that can evaluate the inhibitory functions of antibodies at an earlier stage of screening. To develop a PD-L1-targeting antibody from phage display, we applied the BLI assay to the initial scFv antibody screening, in addition to common ELISA and fluorescence-activated cell sorting (FACS) assays, which showed high advantages and relevance with the in vitro cell-based PD-1/PD-L1 inhibition assay. The same assays for IgG-format antibodies showed high efficiency of the BLI assay in the functional characterization of antibodies, and one candidate selected from the BLI assay resulted in highly efficacious antitumor activity in an in vivo syngeneic mouse study. The BLI assay was also beneficial when searching for antibodies with diverse epitopes. These results demonstrated that the BLI-based inhibition assay is an excellent technique for high-throughput scFv antibody screening in earlier stages and can make phage-display antibody screening more efficient to develop therapeutic candidates.