Albumin-binding Fab derived from Herceptin improves penetration into HER2 breast cancer cells

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Albumin-binding Fab4D5 (AB.Fab) derived from Herceptin is a bi-functional molecule that can simultaneously bind albumin and the tumor antigen HER2 (erbB2). The small antibody fragment may increase its ability to penetrate into tumors compared to the full length IgG (Herceptin) while its constant association with albumin substantially reduces clearance relative to Fab4D5 (Fab). This study compared the uptake and distribution of Herceptin, AB.Fab and Fab using FITC conjugates in a dorsal skin window model. After anesthesia, two symmetrical titanium frames were used to sandwich the extended double layer of skin in athymic nude mice. One layer of skin was removed in a circular area approx 15 mm in diameter, and the remaining layer was covered with a glass overslip incorporated into one of the frames. Two days later, a piece (1 mm in diameter) of HER2-F2-1282 tumor was implanted into the center of the window. When tumors reached the desired size, the mice were randomized to receive 10 mg/kg FITC-Herceptin, 20 mg/kg FITC-Fab, or 20 mg/kg FITC-AB.Fab (n=3 in each group) by iv injection. Tumors were observed and recorded using a confocal laser scanning microscope at 15 and 45 min, 2, 6, and 24 hours, 2, 3, 4, and 5 days. The time-course study indicated that the uptake of fluorescence in tumor cells was maximal at 6 hours for FITC-Fab or 24 hours for FITC-AB.Fab and FITC-Herceptin after injection. Utilizing these times of maximal staining in a separate study, the tumor was imaged by intavital microscopy, and then removed, embedded in OCT compound and frozen at -80° C for IHC studies (n=4-5 in each group). Slides, applied with rat anti-mouse CD31 and Cy3 conjugated goat anti-rat IgG, were mounted with Vectashied mounting media with DAPI nuclear stain. Quantitative analysis on histological sections was performed using Image J software. The tumor vasculature was visualized at 15 min after injection for each of the 3 molecules. Penetration into tumor cells was initiated at 45 min by Fab, 2 hours by AB.Fab, and 6 hours by Herceptin, while tumor deposition was sustained for only 6 hours by Fab but up to 5 days by AB.Fab or Herceptin. Both intravital microscopic and histological imaging showed that Herceptin penetrated only the outer-layers of F2-1282 tumor cells, while the penetrated area of both Fab and AB.Fab was increased compared to that of Herceptin. Quantitative analysis revealed that the penetrated area of Fab or AB.Fab was significantly larger than that of Herceptin (P<0.01) while the penetration of AB.Fab was better than that of Fab4D5 (P<0.05). Similarly, the ratio of penetrated area to total cell area was significantly higher for Fab or AB.Fab compared to Herceptin (P<0.01) and the ratio was even greater for AB.Fab than Fab (P<0.05). These data demonstrate that compared to Herceptin, AB.Fab exhibits rapid targeting and improved tumor cell penetration with retaining sustained tumor deposition, suggesting its potential for imaging and therapy.