Journal: Advanced Science
Article Title: A Complementarity‐Based Approach to De Novo Binder Design
doi: 10.1002/advs.202502015
Figure Lengend Snippet: The designs exhibit anti‐VEGF activity in vitro and in vivo. A) VEGF‐dependent survival of HUVEC primary cells was significantly reduced in a dose‐dependent manner by the designed binders. Yellow bars indicate the survival of the cells in an endothelial cell growth basal medium without VEGF, whereas green bars correspond to the survival of the cells in a basal medium with 30 nM of VEGF. Blue and red bars show results on cell survival in a basal medium with 30 n m of VEGF and increasing concentrations of Sam0.7 and Sima3.2, respectively. B) Treatment of U937 acute myeloid leukemia cell line with Sam0.7 and Sima3.2 proteins at low micromolar concentrations inhibited the cell growth. In contrast, the unmutated scaffold Sima_cntrl showed much weaker inhibitory activity, while Sam_cntrl did not inhibit proliferation at all. Error bars represent the standard deviations across nine replicates from three experiments. Statistical significance was calculated using Fisher's one‐sided t‐test ( * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 vs. the PBS group). C) Quantification of the engrafted LNZ308‐GFP glioma cells in zebrafish embryos that were injected with PBS, inactive protein (mvn_cntrl) as a negative control, bevacizumab as a positive control, Sam 0.7, or Sima 3.2. Each dot indicates one embryo. p ‐value was calculated by the Mann‐Whitney two‐tailed test. d – Cohen's d value. D) Representative zebrafish xenografts treated with PBS and Sam0.7. Arrowheads indicate transplanted LNZ308‐GFP cells in the brain. The scale bar is 200 µm. E) Schematic representation of in vitro microvasculature formation and analysis: iPSC‐derived endothelial cells (ECs) and pericytes (PCs) were co‐cultured in the fibrin gel with Sam0.7 or Sam_cntrl as negative control. The formed microvasculature was imaged at day 7, and images were analyzed to calculate microvasculature parameters. F) Quantitative analysis of microvasculature formation. Plots show the percentage of the area covered with blood vessels, and the blood vessel diameter. Statistical significance was calculated using the one‐way ANOVA test ( ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 treated Sam0.7 vs. Sam_cntrl group). G) Representative images showing in vitro microvasculature formation in the presence of Sam0.7 or Sam_cntrl at two working concentrations (50 and 100 µg mL −1 ). The scale bar is 500 µm.
Article Snippet: When the culture reached ≈80% confluency, cells were washed once with DPBS, and medium was changed to Endothelial Cell Basal Medium (EBM) (Cell Applications, Inc., 210–500) or EBM supplemented with 30 ng mL −1 rhVEGF, or with 30 ng mL −1 rhVEGF and different concentrations of VEGF inhibitors varying from 1 μg/mL to 100 μg/mL.
Techniques: Activity Assay, In Vitro, In Vivo, Injection, Negative Control, Positive Control, MANN-WHITNEY, Two Tailed Test, Derivative Assay, Cell Culture
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