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. Author manuscript; available in PMC: 2021 May 19.
Published in final edited form as: Nat Biomed Eng. 2020 Oct 12:10.1038/s41551-020-00625-5. doi: 10.1038/s41551-020-00625-5

Fig. 2. Specific and high-affinity binding of YG1 scFv–Fc to the AIQ–HLA-A2 complex on AML cells.

Fig. 2

a, Schematic of the switchable yeast display/secretion vector for expressing the scFv–Fc fusion protein. In this switchable system, scFv–Fc can be secreted or displayed on the yeast cells depending on whether or not OmeY is added to the culture[20]. b, SDS-PAGE analysis of purified scFv–Fc proteins (lane 1: protein ladder; lane 2: non-reduced scFv–Fc protein (1 μg); lane 3: reduced scFv–Fc protein (1 μg)). Gel was stained using Coomassie Blue. Shown are the representative data from three separate experiments. The image of the full scan (with the boundaries of the crops outlined) is provided in the Supplementary Information. c, Flow cytometry analysis of HLA-A2 expression by OCI-AML3, T2, GMB and PC-3 cells. Red histograms show staining with anti-HLA-A2, whereas cyan histograms show staining with isotype control antibody. Representative data from technical triplicates are shown. d, Flow cytometry analysis of AIQ–HLA-A2 expression by OCI-AML3, T2, GMB and PC-3 cells. Red histograms show staining with YG1 scFv–Fc and anti-haemagglutinin, whereas cyan histograms show staining with BSA followed by anti-haemagglutinin. Representative data from three separate experiments with technical triplicates are shown. e, Kinetic analysis of the interactions between scFv–Fc and AIQ–HLA-A2, SLL–HLA-A2 or HLA-A2 by biolayer interferometry. The streptavidin biosensor tips of the FortéBio Octet RED96 instrument were coated with biotinylated scFv–Fc protein. The tips were dipped in increasing concentrations (indicated at the bottom of binding curve) of AIQ–HLA-A2, SLL–HLA-A2 or HLA-A2 to measure their binding to scFv–Fc (association) and subsequently moved to wells containing buffer to measure the dissociation rate (dissociation). Shown are representative data from three separate experiments.