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. 2024 Sep 11;633(8030):654–661. doi: 10.1038/s41586-024-07903-1

Extended Data Fig. 1. REGN5381 binds and activates NPR1 in the presence or absence of natriuretic peptide ligands.

Extended Data Fig. 1

A–C, Specific binding of REGN5381 to human and cynomolgus monkey NPR1 was tested with A, HEK293, B, HEK293/hNPR1, and C, HEK293/MfNPR1 cells by flow cytometry. REGN5381 binds to B, hNPR1 with EC50 values of 4.58 nM (no ligand), 4.56 nM (with 100 nM ANP) and 5.69 nM (with 100 nM BNP) and C, mfNPR1 EC50 values of 2.27 nM (no ligand), 2.42 nM (100 nM ANP) and 3.01 nM (with 100 nM BNP). Key: brown solid triangles, REGN5381; dark grey solid diamonds, REGN5381 + 100 nM ANP; grey solid circles, REGN5381 + 100 nM BNP; blue solid triangle, IgG4P; blue open circle, antibody control. D, schematic diagram of calcium flux assay measuring calcium mobilization through CNGA2 channel upon ligand or REGN5381-induced NPR1 activation. E, REGN5381 activated hNPR1 with EC50 values of 46.8 nM (no ligand), 68.1 nM (40 pM ANP) and 42.5 nM (150 pM BNP) as measured by calcium flux assay with and HEK293/CNGA2/hNPR1. F, mfNPR1 with EC50 values of 82.2 nM (no ligand), 81.7 nM (40pM ANP) and 57.5 nM (150 pM BNP) with HEK293/CNGA2/mfNPR1 cells. Open symbols indicate conditions when no test article was added, and closed symbols indicate conditions when the test article was added in a range of concentrations. Key: blue solid circles, ANP; light brown solid square, BNP; brown solid triangle, REGN5381; dark grey solid diamond, REGN5381 + 40 pM ANP; grey solid circle, REGN5381 + 150 pM BNP; blue solid triangle, IgG4P; dark grey open diamond, 40 pM ANP; grey open circle, 150 pM BNP; blue open circle, dilution buffer control. REGN5381 binds to cells expressing human NPR1 (hNPR1) and monkey NPR1 (mfNPR1) in the absence of ligand, and binding is enhanced in the presence of ANP and BNP. ANP, atrial natriuretic peptide; AUC, area under the curve; BNP, brain natriuretic peptide; CNGA2, cyclic-nucleotide-gated calcium channels; GTP, guanosine triphosphate; IgG4P, non-binding immunoglobulin G control; MFI, mean fluorescence intensity; NPR1, natriuretic peptide receptor 1; RFU, relative fluorescence units.