Figure 3 | Scientific Reports

Figure 3

From: Integrating SpyCatcher/SpyTag covalent fusion technology into phage display workflows for rapid antibody discovery

Figure 3

Characterization of anti-TEM1 clones isolated by dCI SpyC-antigen selection. (a) Binding of reformatted and purified scFv-Fc (hIgG1) clones (2 μg/ml) to human and murine cell lines. A673, human Ewing’s sarcoma; SK-N-AS, human neuroblastoma; 2H11, murine tumor vascular endothelium; TC1, murine lung adenocarcinoma; HEK293-6E, human embryonic kidney; HT-1080, human fibrosarcoma; MDA-MB-231, human mammary carcinoma; MS1, murine pancreatic islet endothelium. Additionally, binding of anti-TEM1 scFv-Fc clones to HEK293T cells transiently transfected with native FL-hTEM1 and an irrelevant surface protein (αhCD19:28ζ CAR) control is also shown. (b) SPR monovalent affinity determination of parental clone HS06 (left) and an affinity-matured variant, HS06mut (right). hTEM1-SpyC has been used as the immobilized ligand and monovalent HS06/HS06mut monovalent BiTE as the soluble analyte, with concentration ranges of 100, 50, 25, 12.5 and 0 nM for HS06, and 5, 2.5, 1.25, 0.625 and 0 nM for HS06mut. (c) Binding of HS06mut variant scFv-Fc (0.1 μg/ml) to endogenous human (A673) and murine (2H11) TEM1+ cell lines.

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