Development of a Specifically Labeled Zr Antibody for the Noninvasive Imaging of Tumors Overexpressing B7-H3.

B7-H3 has emerged as a promising target and potential biomarker for diagnosing tumors, evaluating treatment efficacy, and determining patient prognosis. Hu4G4 is a recombinant humanized antibody that selectively targets the extracellular domain of human B7-H3. In this study, we describe the radiolabeling of hu4G4 with the positron emission tomography (PET) emitter radionuclide zirconium 89 (Zr) and evaluate its potency as an immuno-PET tracer for B7-H3-targeted imaging by comparing it and to [Zr]Zr-DFO-DS-5573a using various models. The radiolabeled compound, [Zr]Zr-desferrioxamine-hu4G4 ([Zr]Zr-DFO-hu4G4), demonstrated a high radiochemical purity (RCP) of greater than 99% and a specific activity of 74 MBq/mg following purification. Additionally, it maintained stability in human serum albumin (HSA) and acetate buffer, preserving over 90% of its RCP after 7 days. Three cell lines targeting human B7-H3(U87/CT26/GL261) were used. Flow cytometry analysis indicated that the B7-H3-positive cells (U87/CT26/GL261) had a higher B7-H3 protein level with no expression in the B7-H3-negative cells (CT26/GL261) ( < 0.001). Moreover, the cellular uptake was 45.71 ± 3.78% for [Zr]Zr-DFO-hu4G4 in CT26 cells versus only 0.93 ± 0.47% in CT26 cells and 30.26 ± 0.70% when [Zr]Zr-DFO-hu4G4 in CT26 cells were blocked with 100× 8H9. The cellular uptake of [Zr]Zr-DFO-hu4G4 was akin to that observed with [Zr]Zr-DFO-DS-5573a with no significant differences (45.71 ± 3.78 % vs 47.07 ± 0.86 %) in CT26 cells. Similarly, the CT26 mouse model demonstrated markedly low organ uptake and elevated tumor uptake 48 h after [Zr]Zr-DFO-hu4G4 injection. PET/CT analysis showed that the tumor-to-muscle (T/M) ratios were substantially higher compared to other imaging groups: 27.65 ± 3.17 in CT26 mice versus 11.68 ± 4.19 in CT26 mice ( < 0.001) and 16.40 ± 0.78 when 100× 8H9 was used to block [Zr]Zr-DFO-hu4G4 in CT26 mice ( < 0.01) at 48 h post-injection. Additionally, the tracer showed markedly high accumulation in the tumor region (22.57 ± 3.03% ID/g), comparable to the uptake of [Zr]Zr-DFO-DS-5573a (24.76 ± 5.36% ID/g). A dosimetry estimation study revealed that the effective dose for [Zr]Zr-DFO-hu4G4 was 2.96 × 10 mSv/MBq, which falls within the acceptable range for further research in nuclear medicine. Collectively, these results indicated that [Zr]Zr-DFO-hu4G4 was successfully fabricated and applied in B7-H3-targeted tumor PET/CT imaging, which showed excellent imaging quality and tumor detection efficacy in tumor-bearing mice. It is a promising imaging agent for identifying tumors that overexpress B7-H3 for future clinical applications.