PET of VEGFR expression with ⁶¹Cu-labeled lysine-tagged VEGF₁₂₁

Objectives Overexpression of vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) indicates poor prognosis in many tumor types. Our goal is to develop a VEGFR-targeted positron emission tomography (PET) tracer based on VEGF₁₂₁, with three lysine residues fused to the N-terminus (denoted as K₃-VEGF₁₂₁), which can facilitate radiolabeling without affecting its VEGFR binding affinity. ⁶¹Cu (t_1/2: 3.3 h; 62% β⁺) was used as the radiolabel to match its uptake kinetics and minimize radiation dosimetry.

Methods Purified K₃-VEGF₁₂₁ was conjugated to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and labeled with ⁶¹Cu. Cell binding assay was performed to compare the VEGFR-2 binding affinity of K₃-VEGF₁₂₁ and NOTA-K₃-VEGF₁₂₁. PET imaging, biodistribution, blocking, and histology studies were carried out in 4T1 murine breast tumor-bearing mice to evaluate the tumor targeting efficacy and in vivo specificity of ⁶¹Cu-NOTA-K₃-VEGF₁₂₁. Radiation dosimetry was extrapolated to humans as well.

Results Based on cell binding assay using ¹²⁵I-VEGF₁₆₅ as the radioligand, the IC₅₀ values of NOTA-K₃-VEGF₁₂₁ and K₃-VEGF₁₂₁ were comparable to VEGF₁₂₁ (~ 1 nM). ⁶¹Cu labeling was achieved with good yield (> 50 %) and specific activity. Serial PET imaging showed that the 4T1 tumor uptake of ⁶¹Cu-NOTA-K₃-VEGF₁₂₁ was 3.4±0.5, 4.9±1.0, 5.2±1.0, and 4.8±0.8 %ID/g (n = 4) at 0.5, 2, 4, and 8 h post-injection respectively, consistent with biodistribution data measured by γ counting. Blocking and histology experiments confirmed VEGFR specificity of ⁶¹Cu-NOTA-K₃-VEGF₁₂₁. Dosimetry calculation showed that the radiation dose was much lower than other VEGF-based tracers.

Conclusions Successful PET imaging of VEGFR expression was achieved with ⁶¹Cu-NOTA-K₃-VEGF₁₂₁, which retained high affinity to VEGFR and exhibited desirable radiation dosimetry. The use of ⁶¹Cu is optimal for small proteins like K₃-VEGF₁₂₁, which has higher β⁺ branching ratio than ⁶⁴Cu (62% vs. 17%). Thus, it can offer stronger signal intensity and require lower tracer dose for PET imaging than ⁶⁴Cu, which is ideal for clinical translation.