131I-labeled monoclonal antibody targeting neuropilin receptor type-2 for tumor SPECT imaging.

As a co-receptor for vascular endothelial growth factor‑3 (VEGF‑3), neuropilin receptor type‑2 (NRP‑2) plays a central role in lymphangiogenesis and angiogenesis. Recently, mounting data of evidence show that NRP‑2 is overexpressed in several human cancers, and its overexpression is often associated with poor prognosis. Therefore, it is necessary for us to develop an affinity reagent for noninvasive imaging of NRP‑2 expression because it may be possible to provide early cancer diagnosis, more accurate prognosis, and better treatment planning. Due to their high affinity, and specificity, monoclonal antibodies (mAbs) have been considered attractive candidates for targeted cancer therapy and diagnostics. We recently generated and validated a monoclonal antibody that specifically binds NRP‑2 b1b2 domain with no cross‑reactivity to NRP‑1 b1b2 domain, also known to be overexpressed in a variety of cancers. Here, we developed a single photon emission computed tomography (SPECT) probe for imaging NRP‑2- positive tumors. Anti‑NRP‑2 monoclonal antibodies were prepared by hybridomas and were labeled with iodine‑131 by chloramine‑T method. The in vitro physicochemical properties of 131I‑anti‑NRP‑2 mAb was determined. Binding affinity and specificity of 131I‑anti‑NRP‑2 mAb to NRP‑2 were assessed using human lung adenocarcinoma A549 cells. Biodistribution and SPECT studies were performed in mice bearing A549 tumor xenografts to evaluate the in vivo performance of 131I‑anti‑NRP‑2 mAb. The preparation of anti‑NRP‑2 mAb was completed successfully by hybridoma with high purity (>95%) and specific for NRP‑2 b1b2 domain, but not NRP‑1 b1b2 domain. The radiosynthesis of 131I‑anti‑NRP‑2 mAb was completed successfully within 60 min with high labelling efficiency (94.69±3.63%), and radiochemical purity (98.56±0.48%). The resulting probe, 131I‑anti‑NRP‑2 mAb displayed excellent stability in PBS solution during 24-72 h. 131I‑anti‑NRP‑2 mAb showed high binding affinity with A549 cells (96.6±1.44 nM). In vivo biodistribution and SPECT studies demonstrated targeting of A549 glioma xenografts was NRP‑2 specific. The tumor uptake was 5.86±0.27% ID/g at 6 h, and kept at high level of 4.64±0.82% ID/g at 72 h‑post‑injection. The tumor to contralateral muscle ratio (T/NT) was 2.08±0.33 at 6 h, and reached the highest level of 3.83±0.18 at 72 h after injection. SPECT imaging studies revealed that 131I‑anti‑NRP‑2 mAb could clearly identify A549 tumors with good contrast, especially at 48‑72 h after injection. In conclusion, this study demonstrates that 131I‑anti‑NRP‑2 mAb exhibited highly selective uptake in NRP‑2‑expressing tumors, and may provide a promising SPECT probe for imaging NRP‑2 positive tumors.