Ross W. Cheloha

We describe a semisynthetic strategy that unites the versatility of chemical peptide synthesis with the high affinity and specificity of antibody fragments to create conjugates with remarkably potent anti-HIV activity. Using site-selective enzymatic ligation and click chemistry, we assembled nanobody-fusion inhibitor peptide (Nb-FI) conjugates that link synthetic HIV-1 fusion inhibitor peptides to nanobodies targeting host receptors. This modular approach enables variation of Nb-FI linkage geometry, including C-to-C terminal topologies inaccessible by genetic fusion. Selected semisynthetic conjugates exhibit antiviral potencies up to 10,000-fold better than comparator peptides and broad-spectrum activity across diverse HIV strains. Lead semisynthetic conjugates also outperform analogous genetic fusions. These findings underscore the power of combining chemical synthesis, protein engineering, and antibody delivery to leverage proximity effects in blocking viral infection.

Biodistribution of Al[18F]F-ssHN3 in mice bearing HepG2 xenografts at 1, 2, and 3 h after administration (n = 4-5 / time point).

Biodistribution of Al[18F]F-ssHN3 in mice bearing HepG2 orthotopic tumors at 1, 2, and 3 h after administration (n = 4 / time point).

Spearman’s plot suggesting correlation between SUVmean (A) and SUVmax (B) intensity and GPC3 staining of HepG2, Hep3B, and Huh7 tumors (n = 2).

Biolayer Interferometry graph of ssHN3-RESCA binding to human GPC3.

Tumor-to-tissue ratios of Al[18F]F-ssHN3 in orthotopic HepG2 liver cancer model calculated from ex vivo biodistribution (n = 4 / time point).

Fig. S5: Biodistribution of Al[18F]F-ssHN3 in mice bearing Hep3B xenografts at 1, 2, and 3 h after administration (n = 4 / time point).

Biodistribution of Al[18F]F-ssHN3 in mice bearing Huh7 xenografts at 1, 2, and 3 h after administration (n = 4 / time point).

Purpose:Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death and is characterized by poor survival rates and high recurrence after surgery. Glypican-3 (GPC3) is a proteoglycan highly expressed in HCC but absent in most normal tissue, making it an attractive diagnostic and therapeutic target. In this study, we introduce a second-generation GPC3-targeted single-domain antibody probe (ssHN3) bearing a positron-emitting isotope fluorine-18 (18F), ssHN3-Al[18F]F-RESCA (Al[18F]F-ssHN3), for HCC-selective PET imaging. In addition, we show its use as a functional imaging agent following focal tumor thermal ablation.Experimental Design:Site-specific conjugation was used to synthesize the nanobody-based PET (immunoPET) probe ssHN3-Al[18F]F-RESCA. Binding affinity was determined using biolayer interferometry, and in vivo PET/CT imaging and biodistribution studies were performed in three liver cancer models with varying GPC3 expression (HepG2 > Hep3B > Huh7). Mice inoculated with HepG2 orthotopic liver tumors were also imaged before and 1 week after thermal tumor ablation to assess response.Results:Our agent exhibited high purity (>98%), nanomolar affinity for GPC3, and tumor uptake corresponding to GPC3 expression on PET/CT and biodistribution studies. In orthotopic murine models of liver cancer, Al[18F]F-ssHN3 successfully distinguished between total versus subtotal thermal ablation, accurately identifying residual, viable disease.Conclusions:We successfully designed, engineered, and tested a GPC3-targeted 18F-labeled nanobody immunoPET agent, Al[18F]F-ssHN3, demonstrating that it can be used for same-day diagnostic imaging in murine models of liver cancer. Importantly, this agent could address the limitations of current imaging methods by detecting residual disease after thermal ablation and other locoregional therapies.

Biodistribution of Al[18F]F-ssHN3 in mice bearing HepG2-GPC3-/- xenografts at 1, 2, and 3 h after administration (n = 4-6 / time point).