Preclinical Development Nanobodies Targeting the Hepatocyte Growth Factor: Potential New Drugs for Molecular Cancer Therapy

Hepatocyte growth factor (HGF) and its receptor c-Met are associated with increased aggressiveness of tumors and poor prognostic outcome of patients with cancer. Here, we report the development and characterization of therapeutic anti-HGF (aHGF)-Nanobodies and their potential for positron emission tomographic (PET) imaging to assess HGF expression in vivo. Two aHGF-Nanobodies designated 1E2 and 6E10 were identified, characterized, and molecularly fused to an albumin-binding Nanobody unit (Alb8) to obtain serum half-life extension. The resulting Nanobody formats were radiolabeled with the positron emitter zirconium-89 (Zr, t1/21⁄4 78 hours), administered to nudemice bearing U87MG glioblastoma xenografts, and their biodistributionwas assessed. In addition, their therapeutic effectwas evaluated in the same animalmodel at doses of 10, 30, or 100 mg per mouse. The Zr-Nanobodies showed similar biodistribution with selective tumor targeting. For example, 1E2-Alb8 showeddecreasedblood levels of 12.6%ID/g 0.6%ID/g, 7.2%ID/g 1.0%ID/g, 3.4%ID/g 0.3%ID/g, and 0.3%ID/g 0.1%ID/g at 1, 2, 3, and 7 days after injection, whereas tumor uptake levels remained relatively stable at these time points: 7.8%ID/g 1.1%ID/g, 8.9%ID/g 1.0%ID/g, 8.7%ID/g 1.5%ID/g, and 7.2%ID/g 1.6%ID/g. Uptake in normal tissues was lower than in tumor, except for kidneys. In a therapy study, all Nanobody-treated mice showed tumor growth delay compared with the control saline group. In the 100-mg group, four of six mice were cured after treatment with 1E2-Alb8 and 73 days follow-up, and three of six mice when treated with 6E10-Alb8. These results provide evidence that Nanobodies 1E2-Alb8 and 6E10-Alb8 have potential for therapy and PET imaging of HGFexpressing tumors. Mol Cancer Ther; 11(4); 1017–25. 2012 AACR.