Preparation, Purification, and Identification of a Monoclonal Antibody Against NRP2 b1b2 Domain

Abstract

First identified as a high-affinity kinase-deficient receptor for class-3 semaphorins and vascular endothelial growth factor (VEGF) families, Neuropilin2 (NRP2) is a transmembrane non-tyrosine-kinase glycoprotein that has a vital function in neuronal patterning. Furthermore, NRP2 expression is often upregulated in cancer tissues and correlated with poor prognosis. In the present study, we report the establishment of a monoclonal antibody specific for NRP2b1b2 domain (NRP2 MAb) through hybridoma method. NRP2 MAb is measured to have a titer of 5.12 × 10(5) against NRP2b1b2 in indirect ELISA. Western blotting, flow cytometry, and immunofluorescence analysis indicate that NRP2 MAb can combine full-length NRP2 in LoVo and SW480 cells. Besides helping further understand NRP2-related pathological mechanisms and cell-signaling pathways, NRP2 MAb may act as a therapeutic agent for cancer in the future.

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

“…Western blot analysis. According to the previously described methods (24), the expression of NRP-2 protein in A549, H1299 and 16HBE cell lines was determined by western blot analysis. Briefly, A549, h1299 and 16hBE cells were lysed in 1 ml RIPA solution (containing 1% Triton x-100, 1% deoxycholate, and 0.1% SDS; Beyotime Biotechnology, haimen, China) supplemented with a protease inhibitor (PMSF; Sangon, Shanghai, China) for 30 min at 4˚C, then centrifuged at 12,000 rpm for 30 min at 4˚C to obtain the total cell extracts.…”

“…Cellular immunofluorescence staining. Cellular immunof luorescence staining was performed as previously described (12,24). Briefly, the cell-seeded coverslips were washed and fixed.…”

“…Production and purification of anti-NRP-2 mAb. Anti-NRP-2 mAb was produced by hybridomas derived from mice immunized with a recombinant human NRP-2 b1b2 in our laboratory according to a method described earlier (24). Briefly, Six-week-old Balb/c mice were injected with hybridoma cells (2x10 5 -10 6 ).…”

“…In vivo imaging of tumor-receptor offers a more accurate and real-time assay of receptor expression both for patient stratification and monitoring expression-level changes in response to therapy, without such biopsy-associated pitfalls and the need of repetitive invasive biopsies (23). Our previous studies showed that a novel monoclonal antibody against NRP-2 b1b2 domain (anti-NRP-2 mAb), generated by our laboratory (24), can inhibit tumor proliferation, growth, and migration, such as bladder and colorectal cancer (unpublished data), suggesting that anti-NRP-2 mAb may be effective agents for NRP-2-targeted imaging and therapy. Anti-NRP-2 mAb specifically binds to NRP-2 b1b2 domain, but not NRP-1 b1b2 domain, consistent with a previous study (22).…”

Current drug design to target the Semaphorin/Neuropilin/Plexin complexes

“…2), yet this approach has not been further pursued for clinical translation. Moreover, despite development of a new anti-NRP2 antibody that targets the b1b2 domain 64 functional in vivo assays are missing. Function blocking antibodies were also raised against Plexins.…”