PET/CT imaging of mice bearing PC-3-tumors.

<p>Animals were imaged at 4 hours, 24 hours and 48 hours after injection of the ⁶⁴Cu-MeCOSar radiotracer for shWTCH2 and IgG. Shown are the maximum-intensity projections. The color scale for the PET image data shows radiotracer uptake with white corresponding to the highest activity and blue to the lowest activity. T: tumor; K: kidney; L: liver.</p

PET/CT imaging of mice bearing PC-3-tumors.

<p>Animals were imaged at 4 hours, 24 hours and 48 hours after injection of the ⁶⁴Cu-MeCOSar radiotracer for B6 and B11. Shown are the maximum-intensity projections. The color scale for the PET image data shows radiotracer uptake with white corresponding to the highest activity and blue to the lowest activity. T: tumor; K: kidney; L: liver.</p

Loop library designs.

<p>Loop library designs.</p

SUV of radiolabeled constructs in the tissues of mice bearing PC-3 tumors at 4 h, 24 h and 48 h after tracer injection.

<p>Results are presented as mean (+/-SD).</p

Abdurin binding assays.

<p>Affinity matured binders to EphA2, selected by CIS display, were tested in end-point titration ELISA assays in 96-well plates coated with hEphA2-Fc (a), mEphA2-Fc (b) or strepavidin (c). The purified matured Abdurin clones D2, G7, B6 and B11 were tested alongside the purified non-matured parental EphA2 binding clones selected by phage display, E10par and H3par, as detected by anti-FLAG M2-HRP conjugated antibody. Binding of Abdurin binders and the truncated wild-type human CH2 (shWTCH2) to CHO cells transfected with human EphA2 (d) or to non-transfected cells (e), was assessed by FACS analysis.</p

Localization of B11 in early endosomes and lysosomes.

<p>A) B11 internalization into early endosomes was followed by co-localization experiments using an anti-FLAG antibody (green) and the EEA1 protein, a marker of early endosome compartment, and imaged with the anti-EEA1 antibody (red). As shown in the merged images the two proteins co-localized (yellow) within the early endosome vesicles at 30 minutes. B) B11 trafficking into the lysosomes was followed by co-localization experiments using the anti-FLAG antibody (green) and anti-LAMP1 (red). Co-localization can be observed in the merged view (yellow) at 60 minutes.</p

Amino acid sequences for Loop BC variants.

<p>Amino acid sequences for Loop BC variants.</p

Amino acid sequence of the Abdurin scaffold.

<p>Amino acid sequence of the Abdurin scaffold.</p

Specificity of B11 interaction and internalization.

<p>A) HEK293-hEphA2 cells were seeded in 96 well plates and either, not treated (top panel), or treated (bottom panel) with 100 nM doxycycline for 24 hours at 37°C to induce over-expression of the transfected EphA2 receptor. Cells were incubated with 1μg/well B11 for 30 minutes at 4°C after which time cells were fixed in 2% PFA. B11 binding was monitored by anti-FLAG antibody and anti-mouse AF488 secondary antibody. B) PC-3 cells seeded in 96 well plates were incubated with 1μg/well B11 and 10 μg/well of the anti-EphA2 monoclonal antibody (mAb) (molar ratio 1:1; bottom panel) or an unrelated mAb (top panel) for 30 minutes at 4°C followed by 1 hour incubation at 37°C to allow internalization. Cells were then fixed in 2% PFA and B11 binding monitored by anti-FLAG-AF488 antibody. Nuclei were stained with DAPI. Images were acquired at IN Cell 2000.</p

<i>K</i>_<i>D</i> measured using Biacore 3000.

<p><i>K</i>_<i>D</i> measured using Biacore 3000.</p