Discovery and in vitro characterization of a human anti-CD36 scFv

Figures

• 

  Figure 1. Identification of the scFv anti-CD36 D11 (A) Strategy of panning of the ALTHEA Gold Libraries™ for identification of anti-CD36 scFvs. (B) Phage ELISA for 90 randomly selected clones from the third round of panning. Graph shows the signals generated by binding to rhCD36 (red) or protein L (ProL; brown). As control, we detected rhCD36 with the commercial antibody JC63.1 (blue). (C) Determination of structural integrity of purified scFv D11 by denaturing SDS-PAGE. R, reducing condition; NR, non-reducing condition; MW, molecular weight. (D, E) ELISA assay evaluating the binding of purified D11 (green) to protein L (D) or rhCD36 (E). JC63.1 (blue) was used as a positive control and specificity was assessed by using the unrelated scFv P5E1A6 (black). (F) Representative competition ELISA (from two performed) evaluating the binding to rhCD36 captured by the reference antibody JC63.1 in presence of D11 (green) or the specificity control P5E1A6 (black). The signal generated in the absence of D11 is shown in red. (G) Number of clones identified at each stage of the discovery process. (G) was created with Biorender.com.

• 

  Figure 2. Binding of D11 to membrane CD36. (A) CD36 expression in macrophage-like THP-1 cells, as detected with the anti-CD36 FA6-152 (pink). The fluorescence generated by staining with the secondary antibody alone is shown in gray. (B) Concentration-response curve evaluating the binding of D11 to CD36+ macrophage-like THP-1 cells. Data from two independent experiments. Representative histograms are shown on the right. (C) CD36 expression in HepG2 cells. (D) Binding of D11 (green) to HepG2 cells and representative histograms (right). Data are representative of two independent experiments, each one including duplicates. (B, D) show the signals from the secondary antibody anti-His-tag PE (gray) and the unrelated scFv P5E1A6 (black).

• 

  Figure 3. Effect of D11 in the uptake of CD36 ligands. (A) Analysis of oxLDL-DyLigth uptake in macrophage-like THP-1 cells. Cells were treated with D11 (green), JC63.1 (blue), or the unrelated P5E1A6 (black). Response was normalized against the MFI of untreated cells. Data from three independent experiments. (B) Representative histograms of the effect of the different treatments (100 µg/mL) on the uptake of oxLDL. (C) Effect of the listed antibodies (100 µg/mL) on the uptake of BODIPY-palmitate in HepG2 cells. Bars in the graph show the percentage of cells with BODIPY high fluorescence from three independent experiments (mean ± SEM). *p < 0.05; **p < 0.01, ns: not significant, Bonferroni’s multiple comparisons test. (D) Representative dot plots of the experiments reported in (C).

• 

  Figure 4. Effect of D11 on the oxLDL-induced foam cell phenotype. (A) Effect of treatments on LD accumulation in macrophage-like THP-1 cells exposed to oxLDL (50 μg/ml) for 16-18 h. Graph shows the quantification (mean ± SEM) of oil red staining from two independent experiments. **p < 0.01; ****p < 0.0001, Bonferroni´s multiple comparisons test. (B) Representative micrographs of the experiments reported in (A). (C-E) Effect of treatments on the relative expression of CD36 (C), SRA1 (D), and ACAT (E). Graphs show the geometric mean ± geometric SD from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, ns: not significant, Bonferroni´s multiple comparisons test.

• 

  Figure 5. D11 impairs LD accumulation and clonogenicity on HepG2 cells. (A) Experimental protocol for the analysis of phenotypic changes induced by CD36 blockage. (B) Effect of treatments on LD accumulation. The bars in the graph (mean ± SEM) show the oil red-stained area from two independent experiments. **p < 0.01; ***p < 0.001; ****p < 0.0001, ns: not significant, Bonferroni´s multiple comparisons test. (C) Representative micrographs of the results presented in (B). (D) Effect of treatments (100 µg/mL) on CD36 mRNA expression. Graph shows the geometric mean ± geometric SD from two independent experiments. *p < 0.05; **p < 0.01, ns: not significant, Bonferroni´s multiple comparisons test. (E) Experimental protocol for the evaluation of the effect of scFv D11 on the clonogenicity of HepG2 cells. (F) Effect of treatments on sphere formation efficiency (SFE). Data (mean ± SEM) from three independent experiments. *p < 0.05; ****p < 0.0001, ns: not significant, Bonferroni´s multiple comparisons test. (G) Representative micrographs of the results presented in (F), with insets showing enlarged images of tumorspheres (scale=100 µm). (A, E) were created with Biorender.com.