A novel ¹⁸F-labeled two-helix scaffold protein for PET imaging of HER2-positive tumor

Two-helix scaffold proteins (~ 5 kDa) against human epidermal growth factor receptor type 2 (HER2) have been discovered in our previous work. In this research we aimed to develop an ¹⁸F-labeled two-helix scaffold protein for positron emission tomography (PET) imaging of HER2-positive tumors.

An aminooxy-functionalized two-helix peptide (AO-MUT-DS) with high HER2 binding affinity was synthesized through conventional solid phase peptide synthesis. The purified linear peptide was cyclized by I₂ oxidation to form a disulfide bridge. The cyclic peptide was then conjugated with a radiofluorination synthon, 4-¹⁸F-fluorobenzyl aldehyde (¹⁸F-FBA), through the aminooxy functional group at the peptide N terminus (30% yield, non-decay corrected). The binding affinities of the peptides were analyzed by Biacore analysis. Cell uptake assay of the resulting PET probe, ¹⁸F-FBO-MUT-DS, was performed at 37°C. ¹⁸F-FBO-MUT-DS with high specific activity (20–32 MBq/nmol, 88–140 μCi/μg, end of synthesis) was injected into mice xenograft model bearing SKOV3 tumor. MicroPET and biodistribution and metabolic stability studies were then conducted.

Cell uptake assays showed high and specific cell uptake (~12% applied activity at 1 h) by incubation of ¹⁸F-FBO-MUT-DS with HER2 high-expressing SKOV3 ovarian cancer cells. The affinities (K_D) of AO-MUT-DS and FBO-MUT-DS as tested by Biacore analysis were 2 and 1 nM, respectively. In vivo small animal PET demonstrated fast tumor targeting, high tumor accumulation, and good tumor to normal tissue contrast of ¹⁸F-FBO-MUT-DS. Biodistribution studies further revealed that the probe had excellent tumor uptake (6.9%ID/g at 1 h post-injection) and was cleared through both liver and kidneys. Co-injection of the probe with 500 μg of HER2 Affibody protein reduced the tumor uptake (6.9 vs 1.8%ID/g, p < 0.05).

F-FBO-MUT-DS displays excellent HER2 targeting ability and tumor PET imaging quality. The two-helix scaffold proteins are suitable for development of ¹⁸F-based PET probes.