scFv-Fcs as a Radiotheranostic Platform for Targeting TRA-1-60 in Pancreatic Cancers

Abstract

Abstract Purpose: TRA-1-60 (TRA) is an established cancer stemness marker implicated in tumor initiation, therapeutic resistance, and disease relapse. It is minimally expressed in normal differentiated tissues, making it an appealing biomarker for immunopositron emission tomography (immunoPET) imaging and radiopharmaceutical therapy (RPT). Full-length monoclonal antibodies (mAbs) often exhibit prolonged circulation, which can lead to increased background signal and potential off-target radiation exposure to normal tissues, thereby raising concerns of blood-associated toxicity. To address these limitations, we developed a smaller TRA-targeted scFv-Fc antibody fragment and evaluated its potential as a unified immuno-theranostic platform for both noninvasive molecular imaging and targeted RPT of TRA⁺ tumor subpopulations. Experimental design: A TRA-specific scFv-Fc was conjugated with DFO-NCS and radiolabeled with zirconium-89 (t₁/₂ ≈ 3.27 days) for immunoPET imaging. In vitro uptake of [⁸⁹Zr]Zr-DFO-anti-TRA scFv-Fc demonstrates membrane-bound and internalized accumulation in high TRA-expressing NCCIT embryonal carcinoma cells. In vivo imaging was performed in BxPC-3 and AsPC-1 pancreatic xenografts with high and low TRA expression, respectively. Time activity curves were generated to determine the optimum timepoint for imaging. Tissue distribution was investigated in BxPC-3-bearing mice to define the pharmacokinetics of the new imaging agent. For treatment, the scFv-Fc was conjugated to CHX-A″-DTPA and radiolabeled with Lutetium-177 (t 1/2 ~ 6.67 d). SPECT imaging, biodistribution, and receptor-blocking studies were performed to evaluate the RPT agent’s specificity and accumulation. Therapeutic efficacy in BxPC3 tumor-bearing mice was evaluated using escalating activities (300-750 mCi). Tumor growth was monitored. Toxicity was evaluated through blood chemistry studies and histopathologic analyses of liver, kidney and spleen. TRA expression post-treatment was probed via immunofluorescence while tumor response was further characterized by immunohistochemical (IHC) analysis of Ki-67. Analyses of macrophage recruitment was probed via IHC to establish involvement of innate immunity post-TRA RPT. Results: [⁸⁹Zr]Zr-DFO-anti-TRA scFv-Fc was produced with high radiochemical purity and stability. Tumor accumulation peaked as early as 24 h p.i. with enhanced contrast achieved between 24-48 h p.i. Minimal uptake was observed in gastrointestinal tissues especially in the pancreas and stomach. Importantly, blood pool residency was significantly decreased at 120 h p.i. ImmunoPET enabled sensitive and specific visualization of TRA + expression with the radiotracer displaying tumor retention up to 120 h p.i. Competitive blocking studies attenuated the uptake of the radiotracer in BxPC-3 tumors, demonstrating specificity. The radiolabeling of the RPT agent [¹⁷⁷Lu]Lu-DTPA-anti-TRA scFv-Fc exhibited high radiochemical purity. Specific and sustained tumor uptake was confirmed via SPECT imaging and biodistribution. Blocking studies significantly reduced tumor uptake of TRA RPT, confirming specificity. Treatment resulted in dose-dependent tumor growth suppression without significant hematologic or organ toxicity. TRA expression was reduced across all three treated cohorts compared to the untreated group. Histological analyses revealed marked reductions in tumor burden based on Ki-67-defined proliferative activity. Increased infiltration of macrophages was observed in the periphery in treated versus untreated groups, suggesting that the RPT agent elicited an immune response. Conclusions: We successfully established a TRA-targeted radiotheranostic platform which integrated immunoPET imaging and RPT using an scFv-Fc scaffold. This approach enables non-invasive diagnostic mapping of tumor-specific TRA expression and supports targeted delivery of therapeutic radiation with minimal organ toxicity while effectively suppressing both proliferative tumor subpop