title: Improved tumor targeting of anti-epidermal growth factor receptor Nanobodies through albumin binding: taking advantage of modular Nanobody technology.
abstract: The approximately 15-kDa variable domains of camelid heavy-chain-only 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 (alphaEGFR-alphaEGFR) by fusion to a Nanobody unit binding to albumin (alphaAlb). Biodistributions of alphaEGFR-alphaEGFR, alphaEGFR-alphaEGFR-alphaAlb ( approximately 50 kDa), alphaTNF-alphaTNF-alphaAlb (control, binding tumor necrosis factor-alpha), and the approximately 150-kDa anti-EGFR antibody cetuximab were compared in A431 xenograft-bearing mice. The proteins were radiolabeled with (177)Lu to facilitate quantification. Tumor uptake of (177)Lu-alphaEGFR-alphaEGFR 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 alphaAlb. Blood levels of alphaEGFR-alphaEGFR-alphaAlb 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 alphaTNF-alphaTNF-alphaAlb. alphaEGFR-alphaEGFR-alphaAlb showed faster and deeper tumor penetration than cetuximab. These data show that simple fusion of alphaEGFR and alphaAlb building blocks results in a bifunctional Nanobody format, which seems more favorable for therapy as far as pharmacokinetics and tumor deposition are concerned.
authors: Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, De Boelelaan 1117, Amsterdam, The Netherlands.
authors: Bernard M
authors: BM
authors: Tijink
authors: Toon
authors: T
authors: Laeremans
authors: Marianne
authors: M
authors: Budde
authors: Marijke
authors: M
authors: Stigter-van Walsum
authors: Torsten
authors: T
authors: Dreier
authors: Hans J
authors: HJ
authors: de Haard
authors: C René
authors: CR
authors: Leemans
authors: Guus A M S
authors: GA
authors: van Dongen
mesh_terms: D000418:Albumins / Q000378:metabolism*; D000906:Antibodies / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000493:pharmacokinetics; D061067:Antibodies, Monoclonal, Humanized; D000068818:Cetuximab; D066246:ErbB Receptors / Q000276:immunology* / Q000378:metabolism; D036103:Nanotechnology; D011485:Protein Binding; D014018:Tissue Distribution
chemical_list: D000418:Albumins; D000906:Antibodies; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D066246:ErbB Receptors; D000068818:Cetuximab