Fig. 9: Convertion of anti-Kras scFv-Y13-259 to high-affinity STAND.
From: An ultra-stable cytoplasmic antibody engineered for in vivo applications
a In-silico analysis of net charge (left axis) of Y13-259 fused with indicated peptide tags at cytoplasmic pH 6.6 (green), 7.03 (red), and 7.4 (blue), and pI (grey circles, right axis). b Intracellular stability comparison of scFv-Y13-259 fused with different peptide tags expressed in HeLa cells using immunocytochemistry; anti-Flag or anti-myc was used to detect scFv proteins. Arrows indicate cytoplasmic scFv aggregates. Arrowheads indicate stable expression of scFv proteins. Scale bar, 50 μm. c Percentage of scFv-expressing cells with aggregates in b (upper panel). Bottom panel shows total-cell and aggregated-cell numbers from 3 independent experiments. Statistical analysis: two-tailed one-way analysis of variance (ANOVA), F (2, 6) = 2110.0416, P < 0.00001, Tukey’s multiple comparison test, s3Flag-Y13-259-HA vs. s3Flag-Y13-259, P = 0.0141; s3Flag-Y13-259 vs. Myc-Y13-259, P < 0.00001; s3Flag-Y13-259-HA vs. Myc-Y13-259, P < 0.00001. d Western blot of cell lysate of HeLa cells transfected with vectors expressing STAND-Y13-259 under reduced (upper panel: 6% 2-ME, lower panel: 10 mM DTT) or non-reduced (2-ME, DTT-free) conditions. e Binding affinity of purified STAND-Y13-259 with GST-Kras measured by ELISA (n = 3 independent samples). f Immunoprecipitation analysis using 293 T cells co-transfected with Kras-expressing vector and indicated vectors; western blotting with an anti-HA antibody to detect scFv and anti-Kras antibodies. g Decreased interaction of Kras with the Ras-binding domain (RBD) of Raf1 by the expression of STAND-Y13-259; 293 T cells were transfected with Kras- and indicated STAND-expressing vectors. One day after transfection, the cell lysate was incubated with purified GST or GST-Raf1-RBD, then subjected to immunoprecipitation with anti-GST antibody. Immunoprecipitates were analysed using western blotting with an anti-Kras antibody. h Quantification of the relative amount of Kras protein co-immunoprecipitated with GST-Raf1-RBD (n = 5 independent samples). Precipitated Kras was normalised with the total Kras protein. Statistical analysis: two-tailed one-way ANOVA, F (2, 12) = 14.9438, P = 0.0005527, Tukey’s multiple comparison test, Mock vs. STAND-Y13-259, P = 0.009; Mock vs. STAND-A36, P = 0.8472; STAND-Y13-259 vs. STAND-A36, P = 0.0022. Error bars represent standard error of the mean. Source data are provided as a Source Data file.
