Abstract
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.
Highlights
Programmed death-ligand 1 (PD-L1) is highly expressed in diverse cancers, and is regarded as a major contributor to the immune incompetency of immune cells in cancer microenvironments through the ligation between PD-L1 and programmed cell death protein 1 (PD-1) on T cells that generate strong suppressive signals in the activated T cell to make it anergic [1,2,3,4,5]
To isolate antibody clones that bind to the PD-L1 extracellular domain, a human naïve scFv antibody library was used for the panning against the PD-L1 antigen
93 monoclonal antibodies have been FDA approved for therapy, and a number of candidates are in the advanced phases of clinical trials [33,34]
Summary
Programmed death-ligand 1 (PD-L1) is highly expressed in diverse cancers, and is regarded as a major contributor to the immune incompetency of immune cells in cancer microenvironments through the ligation between PD-L1 and programmed cell death protein 1 (PD-1) on T cells that generate strong suppressive signals in the activated T cell to make it anergic [1,2,3,4,5]. Viruses 2020, 12, 684; doi:10.3390/v12060684 www.mdpi.com/journal/viruses “Phage display” was initially described by Smith in 1985 [10], in which the coating proteins of filamentous bacteriophages fused with exogenous peptides were expressed on their surface and used for the enrichment of specific phages. Adalimumab (Humira) was developed in 1990 by Winter as the first phage-display-derived therapeutic antibody to neutralize tumor necrosis factor-alpha (TNF-α) for immunological disease treatment. Antibody phage-display technologies were further developed by several other groups (Winter and McCafferty, Cambridge, UK; Lerner and Barbas, California, USA; and Breitling and Dübel, Heidelberg, Germany) [11,12,13,14,15,16]. In 2018, Smith and Winter were jointly awarded the Nobel Prize in chemistry for their contributions to the development of the phage-display technology
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