Improved tumor targeting of anti-epidermal growth factor receptor Nanobodies through albumin binding: taking advantage of modular Nanobody technology

The similar to 15-kDa variable domains of camelid heavy-chainonly antibodies (called Nanobodies) can easily be formatted as multivalent or multispecific single-chain proteins. Because of fast excretion, however, they are less suitable for therapy of cancer. In this study, we aimed for improved tumor targeting of a bivalent anti-epidermal growth factor receptor (EGFR) Nanobody (alpha EGFR-alpha EGFR) by fusion to a Nanobody unit binding to albumin (alpha Alb). Biodistributions of alpha EGFR-alpha EGFR, alpha EGFR-alpha EGFR-alpha Alb (similar to 50 kDa), (alpha TNF-alpha TNF-alpha Alb (control, binding tumor necrosis factor-alpha), and the similar to 150-kDa anti-EGFR antibody cetuximab were compared in A431 xenograft-bearing mice. The proteins were radiolabeled with Lu-177 to facilitate quantification. Tumor uptake of Lu-177-alpha EGFR-alpha EGFR decreased from 5.0 +/- 1.4 to 1.1 +/- 0.1 %ID/g between 6 and 72 h after injection. Due to its rapid blood clearance, tumor-to-blood ratios >80 were obtained within 6 h after injection. Blood clearance became dramatically slower and tumor uptake became significantly higher by introduction of alpha Alb. Blood levels of alpha EGFR-alpha EGFR-alpha Alb were 21.2 +/- 2.5, 11.9 +/- 0.6, and 4.0 +/- 1.4 and tumor levels were 19.4 +/- 5.5, 35.2 +/- 7.5, and 28.0 +/- 6.8 %ID/g at 6, 24, and 72 h after injection, respectively. Tumor uptake was at least as high as for cetuximab (15.5 +/- 3.9, 27.1 +/- 7.9, and 25.6 +/- 6.1 %ID/g) and significantly higher than for alpha TNF-alpha TNF- alpha Alb. alpha EGFR-alpha EGFR-alpha Alb showed faster and deeper tumor penetration than cetuximab. These data show that simple fusion of alpha EGFR and alpha Alb building blocks results in a bifunctional Nanobody format, which seems more favorable for therapy as far as pharmacokinetics and tumor deposition are concerned.