plxna1 rdc0967  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: A bispecific antibody designed to act as a NRP2/PLXNA1 agonist mimics anticancer activity of SEMA3F

doi: 10.1016/j.jbc.2025.111056

Figure Lengend Snippet: SEMA3F dimerizes surface NRP2 and PLXNA1, inhibits U-251 MG cell proliferation, and reduces p-AKT level and CCND1 gene expression . A , effects of SEMA3F on proliferation of various cancer cell lines. Cells were treated with full-length SEMA3F for 3 days, and cell viability was measured using CellTiter-Glo, normalized to untreated cells. B , schematic illustrates the full-length SEMA3F dimer, the furin processing site, and the resulting furin-processed SEMA3F (SEMA3F-p65). C , SEMA3F-p65 does not inhibit U-251 MG cell proliferation compared to nonprocessed SEMA3F dimer. D , the anti-NRP2 antibody (aNRP2-a2) blocks SEMA3F-mediated inhibition of U-251 MG cell proliferation. E , schematic illustrating the receptor dimerization assay. NanoLuc luciferase is split into Large BiT and Small BiT with low activity, and fused to the N termini of NRP2 and PLXNA1. SEMA3F induces NRP2-PLXNA1 dimerization, bringing Large BiT and Small BiT together to enhance luciferase activity through complementation. F , SEMA3F treatment induces NRP2-PLXNA1 dimerization. Left panel : Expi293F cells coexpressing Large_BiT-NRP2 and Small_BiT-PLXNA1 were treated with luciferase substrate and SEMA3F as indicated, and luminescence was recorded over time. Right panel : dimerization ratio changes following SEMA3F treatment. G , SEMA3F reduces p-AKT levels in U-251 MG cells in an NRP2-dependent manner. U-251 MG cells were treated as indicated for 30 min, and the p-AKT/AKT ratio was measured. H , SEM3F downregulates CCND1 expression in an NRP2-dependent manner. U-251 MG cells treated for 18 h. The expression of CCND1 was quantified using qPCR and normalized to untreated control cells. I , schematic representation of SEMA3F’s anti-proliferative mechanism in U-251 MG cells. NRP, neuropilin; aNRP2, anti-NRP2; PLXNA1, plexinA1; SEMA, semaphorin; p-AKT, phosphorylation of AKT; qPCR, quantitative PCR.

Article Snippet: Dimerization between cell surface NRP2 and PLXNA1 proteins were determined using a luciferase complementation assay as described previously ( ). cDNAs of NRP2 and PLXNA1 were obtained from OriGene and R&D Systems respectively (NRP2 C220706, PLXNA1 RDC0967).

Techniques: Gene Expression, Inhibition, Luciferase, Activity Assay, Expressing, Control, Phospho-proteomics, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: A bispecific antibody designed to act as a NRP2/PLXNA1 agonist mimics anticancer activity of SEMA3F

doi: 10.1016/j.jbc.2025.111056

Figure Lengend Snippet: Experimental workflow for the discovery of SEMA3F-mimetic bsAbs . MAb-targeting PLXNA1 (aPLXNA1) were generated and paired with anti-NRP2 antibodies to create bispecific antibodies (bsAbs). These bsAbs were initially evaluated in a PLXNA1-NRP2 dimerization assay to identify candidates that effectively promote PLXNA1–NRP2 interaction. Positive bsAbs were then assessed using a p-AKT assay, a qPCR assay for CCND1 expression, and a cell viability assay to determine which candidates mimic SEMA3F’s effects. Ultimately, one bsAb, P1943-Nb2cL, was identified as mimicking SEMA3F’s effects across all cell-based assays. aPLXNA1, anti-PLXNA1; NRP, neuropilin; PLXNA1, plexinA1; SEMA, semaphorin; p-AKT, phosphorylation of AKT; qPCR, quantitative PCR.

Article Snippet: Dimerization between cell surface NRP2 and PLXNA1 proteins were determined using a luciferase complementation assay as described previously ( ). cDNAs of NRP2 and PLXNA1 were obtained from OriGene and R&D Systems respectively (NRP2 C220706, PLXNA1 RDC0967).

Techniques: Generated, Expressing, Viability Assay, Phospho-proteomics, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: A bispecific antibody designed to act as a NRP2/PLXNA1 agonist mimics anticancer activity of SEMA3F

doi: 10.1016/j.jbc.2025.111056

Figure Lengend Snippet: Discovery of anti-PLXNA1 mAbs . A , SDS-PAGE analysis of purified PLXNA1-ECD and PLXNA1-LBD proteins used for mouse immunization. The gel displays nonreduced (without reducing agent in the loading buffer) and reduced (with DTT added to the loading buffer) conditions. B , schematic illustrating the mouse immunization schedule, detailing the timeline for immunization, serum collection, and cell harvest. C , ELISA-based quantification of serum antibody levels following booster immunizations. Data represents six mice immunized with either PLXNA1-ECD or PLXNA1-LBD proteins. D , analysis of germline heavy chain IGHV gene usage in anti-PLXNA1 antibodies. A total of 135 cloned antibodies were analyzed, revealing usage of 29 distinct heavy chain IGHV genes. The pie chart shows the distribution of antibodies based on their IGHV gene usage. ECD, extracellular domain; LBD, ligand-binding domain; PLXNA1, plexinA1; SEMA, semaphorin.

Article Snippet: Dimerization between cell surface NRP2 and PLXNA1 proteins were determined using a luciferase complementation assay as described previously ( ). cDNAs of NRP2 and PLXNA1 were obtained from OriGene and R&D Systems respectively (NRP2 C220706, PLXNA1 RDC0967).

Techniques: SDS Page, Purification, Enzyme-linked Immunosorbent Assay, Clone Assay, Ligand Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: A bispecific antibody designed to act as a NRP2/PLXNA1 agonist mimics anticancer activity of SEMA3F

doi: 10.1016/j.jbc.2025.111056

Figure Lengend Snippet: Identification of PLXNA1-NRP2 bsAb with SEMA3F-mimicking mechanism and activities . A , schematic illustrating structural format of PLXNA1-NRP2 bsAbs. The bsAbs are based on a human IGG4 backbone, with the light chain and heavy chain of the anti-NRP2 half linked by a 34 amino acid GS flexible peptide linker. The anti-NRP2 moiety contains a knob mutation, while the anti-PLXNA1 moiety features a hole mutation. B , screening for bsAbs that induce dimerization of cell surface PLXNA1 and NRP2. Each table cell represents a unique PLXNA1-NRP2 bsAb. bsAbs inducing a dimerization ratio change greater than 1.5 are considered positive hits and highlighted in orange . C , PLXNA1-NRP2 bsAbs that significantly reduced p-AKT level. D , PLXNA1-NRP2 bsAbs that significantly reduced CCND1 expressing in qPCR assay. E , PLXNA1-NRP2 bsAbs that significantly inhibited proliferation of U-251 MG cells. All data are presented as the mean ± SEM from three experiments. aPLXNA1, anti-PLXNA1; bsAb, bispecific antibody; NRP, neuropilin; p-AKT, phosphorylation of AKT; PLXNA1, plexinA1; qPCR, quantitative PCR; SEMA, semaphorin.

Article Snippet: Dimerization between cell surface NRP2 and PLXNA1 proteins were determined using a luciferase complementation assay as described previously ( ). cDNAs of NRP2 and PLXNA1 were obtained from OriGene and R&D Systems respectively (NRP2 C220706, PLXNA1 RDC0967).

Techniques: Mutagenesis, Expressing, Phospho-proteomics, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: A bispecific antibody designed to act as a NRP2/PLXNA1 agonist mimics anticancer activity of SEMA3F

doi: 10.1016/j.jbc.2025.111056

Figure Lengend Snippet: Characterization of the binding mechanism of aPLXNA1-19-43 Fab to PLXNA1 . A , epitope mapping of aPLXNA1-19-43 Fab to PLXNA1. ELISA was used to evaluate binding of aPLXNA1-19-43 Fab to various PLXNA1 fragments. Results are indicated by ELISA readings, with positive (+) binding shown in red for readings >0.1 and negative (−) binding for readings <0.1. B , determination of binding affinity of aPLXNA1-19-43 Fab to PLXNA1 ECD and LBD. Representative sensorgrams from triplicate experiments are shown. The Fab was immobilized using biosensor tips coated with anti-mouse kappa antibody and subsequently exposed to a concentration series of PLXNA1-ECD or PLXNA1-LBD ( black and gray lines ). Data were fitted to a 1:1 binding model ( red lines ) to calculate binding constants. C , cryo-EM density map of the aPLXNA1-19-43 Fab in complex with PLXNA1-LBD. The 2:2 dimeric complex is displayed, with one subunit colored as follows: PLXNA1-LBD in blue , Fab heavy chain in cyan , and Fab light chain in red . D , schematic binding model of aPLXNA1-19-43 Fab with PLXNA1. The critical residues involved in binding are enlarged in the inserted panels . E , identification of critical residues on PLXNA1 that are involved in the binding of aPLXNA1-19-43 Fab. The Fab is shown to bind to both the SEMA and PSI domains of PLXNA1, with key interactions highlighted. F , binding model of P1943-Nb2cL to the PLXNA1–NRP2–SEMA3F complex. G , schematic representation of the binding mechanism between P1943-Nb2cL and the PLXNA1–NRP2–SEMA3F complex, providing a simplified visual overview. aPLXNA1, anti-PLXNA1; ECD, extracellular domain; Fab, fragment of antigen binding; LBD, ligand-binding domain; NRP, neuropilin; PLXNA1, plexinA1; PSI, plexin-semaphorin-integrin; SEMA, semaphorin.

Article Snippet: Dimerization between cell surface NRP2 and PLXNA1 proteins were determined using a luciferase complementation assay as described previously ( ). cDNAs of NRP2 and PLXNA1 were obtained from OriGene and R&D Systems respectively (NRP2 C220706, PLXNA1 RDC0967).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cryo-EM Sample Prep, Ligand Binding Assay