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. 2015 Oct 29;5(3):e1091555. doi: 10.1080/2162402X.2015.1091555

Figure 1.

Figure 1.

6PHU3 binds selectively to CLDN6 and CD3. (A) Schematic overview of the bi-(scFv)2 6PHU3 sequence cassettes cloned into pcDNA3.1 mammalian expression vector. (B) SDS-PAGE analysis of IMAC-purified 6PHU3 and two different control bi-(scFv)2. (C) CLDN6 expression of human carcinoma cell lines PA-1(/luc), OV-90(/luc) and MDA-MB-231/luc as determined by qPCR. Fold expression of CLDN6 expression has been calculated from two independent experiments. Tissue samples from ovarian carcinoma and placenta served as positive controls, CLDN6– tissues heart and thymus as calibrators. (D) 6PHU3 target binding as measured by flow cytometry. Increasing concentrations of 6PHU3 were incubated with CLDN6+ cells (PA-1, OV-90) or CD3+ human T-cells. MDA-MB-231/luc cells and murine T cells served as negative controls. Bound proteins were detected via their 6xHis-tag. (E) Bispecificity of 6PHU3 as demonstrated by ELISA. 6PHU3 and a control bi-(scFv)2 were captured by a CD3-mimicking peptide. Detection was conducted by an antibody specific for the anti-CLDN6 scFv, and a 2nd detection antibody. ABS indicates absorption; APC, allophycocyanin; bi-(scFv)2, bispecific single chain variable fragment; CA, carcinoma; c, concentration; ctrl, control; H, 6xHis-tag; hu, human; LL, long linker (15–18 amino acids); mu, murine; Sec, secretion signal; SL, short linker (five amino acids); VL, variable light chain; VH, variable heavy chain; WB, western blot.