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.

Desferrioxamine B (DFO) is the clinical standard chelator for preparing zirconium-89 labeled antibodies. In the current study, the stabilities of a zirconium-89 labeled panitumumab (PAN; Vectibix^®) with three different chelators (DFO, DFO*, and DOTA) were compared. PAN is an anti-HER1/EGFR monoclonal antibody approved by the FDA for the treatment of HER1-expressing colorectal cancers and was used as the model antibody for this study. DFO/DFO* conjugates of PAN were directly radiolabeled with zirconium-89 at room temperature to produce [⁸⁹Zr]Zr-DFO/DFO*-PAN conjugates following a well-established procedure. A zirconium-89 labeled DOTA-PAN conjugate was prepared by an indirect radiolabeling method. A cyclooctyne-linked DOTA chelator (BCN-DOTA-GA) was first radiolabeled with zirconium-89 at 90 °C under a two-step basic pH adjustment method followed by conjugation with PAN-tetrazene at 37 °C to produce a labeled conjugate, BCN-[⁸⁹Zr]Zr-DOTA-GA-PAN. High reproducibility of the radiolabeling was observed via this two-step basic pH adjustment. The overall radiochemical yield was 40–50% (n = 12, decay uncorrected) with a radiochemical purity of >95% in 2 h synthesis time. All three conjugates were stable in whole human serum for up to 7 days at 37 °C. The kinetic inertness of the conjugates was assessed against the EDTA challenge. BCN-[⁸⁹Zr]Zr-DOTA-GA-PAN exhibited excellent inertness followed by [⁸⁹Zr]Zr-DFO*-PAN. [⁸⁹Zr]Zr-DFO-PAN displayed the lowest level of inertness.

The use of radiolabeled glucose for PET imaging resulted in the most commonly used tracer in the clinic, 2-deoxy-2-[¹⁸F]fluoroglucose (FDG). More recently, other radiolabeled sugars have been reported for various applications, including imaging tumors and infections. Therefore, in this study, we developed a series of fluorine-18-labeled L-rhamnose derivatives as potential PET tracers of various fungal and bacterial strains. Acetyl-protected triflate precursors of rhamnose were prepared and radiolabeled with fluorine-18 followed by hydrolysis to produce L-deoxy [¹⁸F]fluororhamnose. The overall radiochemical yield was 7–27% in a 90 min synthesis time with a radiochemical purity of 95%. In vivo biodistribution of the ligands using PET imaging showed that 2-deoxy-2-[¹⁸F]fluoro-L-rhamnose is stable for at least up to 60 min in mice and eliminated via renal clearance. The tracer also exhibited minimal tissue or skeletal uptake in healthy mice resulting in a low background signal.

Small cell lung cancer (SCLC) remains one of the most lethal malignancies, with a 5-year survival of <10% despite advances in treatment. Seizure-related gene 6 (SEZ6), a type I transmembrane protein preferentially expressed in neuroendocrine tumors, has recently emerged as a promising therapeutic target, supported by clinical activity of SEZ6-targeted antibody–drug conjugates in relapsed SCLC. In this study, we developed and evaluated a humanized anti-SEZ6 monoclonal antibody for SEZ6-targeted positron emission tomography imaging and α-particle radioimmunotherapy. Immunohistochemistry of patient tissues confirmed strong, stage-independent SEZ6 expression. Cell-based studies demonstrated high SEZ6 receptor densities, supporting high-affinity antibody binding and rapid receptor-mediated internalization. [89Zr]Zr-DFO-SEZ6-Ab administered at low antibody mass exhibited rapid hepatosplenic clearance with minimal tumor uptake despite high intratumoral levels. This pattern was consistent with active ectodomain shedding and local accumulation of soluble antigen within the tumor microenvironment, creating an extracellular antigen sink and limiting effective target engagement. Increasing the antibody mass overcame this barrier, dramatically increasing tumor uptake in xenografts. Treatment with [225Ac]Ac-Macropa-SEZ6-Ab induced complete tumor regression, whereas coadministration of the ATR inhibitor berzosertib enhanced efficacy at lower radioactivity doses without added toxicity. Together, these findings identify tumor-localized SEZ6 shedding as a biological barrier to SEZ6-targeted therapy, define dosing strategies to overcome it, and demonstrate potent antitumor activity of SEZ6-directed α-particle radioimmunotherapy in SCLC. Significance: Dosing strategies that overcome antigen sink effects induced by a tumor-localized ectodomain shedding enable SEZ6-based imaging and theranostics in small cell lung cancer.

PET imaging of recurrent tumor using [89Zr]Zr-DFO-SEZ6-Ab at 90 days following radioligand therapy with 4.62 kBq [225Ac]Ac-Macropa-SEZ6-Ab

Characterization of SEZ6 expression, binding affinity, and cellular internalization in H524 cells