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

Abstract

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 ( 89 Zr, t1/ 2 ¼ 78 hours), administered to nude mice bearing U87 MG glioblastoma xenografts, and their biodistribution was assessed. In addition, their therapeutic effect was evaluated in the same animal model at doses of 10, 30, or 100 mg per mouse. The 89 Zr-Nanobodies showed similar biodistribution with selective tumor targeting. For example, 1E2-Alb8 showed decreased blood levels of 12.6%ID/g AE 0.6%ID/g, 7.2%ID/g AE 1.0%ID/g, 3.4%ID/g AE 0.3%ID/g, and 0.3%ID/g AE 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 AE 1.1%ID/g, 8.9%ID/g AE 1.0%ID/g, 8.7%ID/g AE 1.5%ID/g, and 7.2%ID/g AE1.6%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.

Llama-derived Single Variable Domains (Nanobodies) Directed against Chemokine Receptor CXCR7 Reduce Head and Neck Cancer Cell Growth in Vivo

“…Next, we engineered multivalent Nanobody constructs with repetitive GS sequences since this approach had previously led to a gain of function for the CXCR4 Nanobodies (19). In addition, CXCR7 Nanobodies were formatted by genetically linking them to a Nanobody binding serum albumin (referred to as Alb8) to increase their half-life for in vivo studies (28). Using the epitope data, we linked NB1 and NB3 as they bind to different epitopes and coupled them to Alb8, generating the formatted NB4.…”

“…Llama-derived immunoglobulin single variable domain antibodies have proven to be an excellent platform to use in cancer drug research, either as therapy or as a diagnostic tool (34). Therapeutic Nanobodies have been generated against cancer-specific drug targets such as the receptor tyrosine kinases EGFR/Erbb1 (35)(36)(37)(38), HER2 (39), c-Met (28), and VEGFR2 (40) and more recently against the chemokine receptor CXCR4 (19). Based on our experience with CXCR4 (19), we identified therapeutic and high affinity Nanobodies against the other CXCL12 receptor, i.e.…”

Human Epidermal Growth Factor Receptor 3–Specific Tumor Uptake and Biodistribution of89Zr-MSB0010853 Visualized by Real-Time and Noninvasive PET Imaging

“…This increase might be due to saturation of hepatic uptake of 89 Zr-MSB0010853, because hepatic uptake inversely correlated with blood levels. Increasing the protein dose of an albumin-binding antihepatocyte growth factor Nanobody construct (18) and an albumin-binding anti-HER2 Affibody construct (19) did not affect liver uptake. Therefore, saturation of 89 Zr-MSB0010853 liver uptake was not due to in vivo albumin binding.…”

“…Apart from the liver, saturation of 89 Zr-MSB0010853 was also observed in other organs that express HER3, such as the lung, stomach, small intestines, colon, and skin (18). Uptake of 89 Zr-MSB0010853 in these organs increased with increasing protein doses up to 25-100 mg.…”

89Zr-DFO-AMG102 Immuno-PET to Determine Local Hepatocyte Growth Factor Protein Levels in Tumors for Enhanced Patient Selection

“…To this end, we have developed a 89 Zr-based radioimmunoconjugate from the fully human antibody AMG102 to determine the local levels of HGF in tumors by PET imaging. Previous work toward molecular imaging of HGF is limited to an antibody fragment (Nanobody), which has been radiolabeled with 89 Zr and used to image HGF in U87MG glioblastoma xenografts with some success (26).…”