Agonist antibody to guanylate cyclase receptor NPR1 regulates vascular tone

Abstract

Heart failure is a leading cause of morbidity and mortality^1,2. Elevated intracardiac pressures and myocyte stretch in heart failure trigger the release of counter-regulatory natriuretic peptides, which act through their receptor (NPR1) to affect vasodilation, diuresis and natriuresis, lowering venous pressures and relieving venous congestion^3-8. Recombinant natriuretic peptide infusions were developed to treat heart failure but have been limited by a short duration of effect^9,10. Here we report that in a human genetic analysis of over 700,000 individuals, lifelong exposure to coding variants of the NPR1 gene is associated with changes in blood pressure and risk of heart failure. We describe the development of REGN5381, an investigational monoclonal agonist antibody that targets the membrane-bound guanylate cyclase receptor NPR1. REGN5381, an allosteric agonist of NPR1, induces an active-like receptor conformation that results in haemodynamic effects preferentially on venous vasculature, including reductions in systolic blood pressure and venous pressure in animal models. In healthy human volunteers, REGN5381 produced the expected haemodynamic effects, reflecting reductions in venous pressures, without obvious changes in diuresis and natriuresis. These data support the development of REGN5381 for long-lasting and selective lowering of venous pressures that drive symptomatology in patients with heart failure.

Fig. 1. Protein-altering genetic variants in NPR1 and the associated effect on BP and NT-proBNP in 718,386 individuals.

a, We replicated three previously reported and functionally validated missense variants with a known effect on BP. Two reported LOF variants (NPR1:p.L1034F-rs116245325 and NPR1:p.G967K-rs35479618) were associated with increased BP and higher NT-proBNP, while a reported GOF variant (NPR1:p.G541S-rs61757359) was associated with lower BP and directionally lower NT-proBNP. We also found that individuals carrying a protein-truncating variant (presumed LOF) of NPR1 on average have higher BP and higher NT-proBNP than that of non-carriers. b, We defined any protein-altering variant associated with increased BP as presumed LOF (n = 9) and any BP-decreasing protein-altering variant as presumed GOF (n = 5). In an independent sample, we found that the combined collection of presumed LOF variants is associated with increased BP and higher NT-proBNP, while the collection of presumed GOF variants is associated with decreased BP and lower NT-proBNP. c, The collection of presumed LOF variants is associated with increased odds of HF, while the collection of presumed GOF variants is numerically associated with a decreased odds of HF, based on an additive genetic model performed in independent samples. Alt., alternative allele; CI, confidence intervals; freq., frequency; het., heterozygous allele; OR, odd ratio; ref., reference allele.

Fig. 2. The cryo-EM structure of antibody-bound NPR1 suggests that REGN5381 is an allosteric activator.

a, The extracellular domains of an inactive ANP-free NPR1 dimer (PDB: 1DP4) are shown in two different orientations as a molecular surface, one monomer in white and the other monomer in dark grey, with the principal axes of each monomer indicated as thick solid white or grey lines, respectively; the projection angle between the axes is indicated below the monomers. The active, ANP-bound NPR1 dimer (PDB: 1T34) is shown similarly to the diagram at left, as a blue surface for bound ANP. The same complexes are shown in schematic form (extracellular domain, transmembrane helix, ANP) on the right, including possible positions for the transmembrane helices. b, The NPR1–ANP–REGN5381 complex is shown similarly to a. c, The NPR1–REGN5381 complex is shown similarly to a, with the addition of REGN5381 Fab molecular surface in red.

Fig. 3. The NPR1 agonist antibody REGN5381 persistently reduces systolic BP in NPR1^hu/hu mice in the absence of diuresis, and acts as a direct vasodilator.

a, Administration of a single subcutaneous dose of 1, 5, 25 or 50 mg per kg REGN5381 in normotensive NPR1^hu/hu mice demonstrated marked reductions in systolic BP that were maintained for up to 28 days (black circles, REGN1945 (the immunoglobulin G4 isotype control), 25 mg per kg; pink squares, REGN5381, 1 mg per kg; blue triangles, REGN5381, 5 mg per kg; red triangles, REGN5381, 25 mg per kg; purple diamonds, REGN5381, 50 mg per kg). b, Post-dose assessments after administration of REGN5381 (red squares) or control monoclonal antibody (REGN1945; black circles) on urinary cGMP concentration and urine volume. c, Administration of REGN5381 induces greater venous dilation in precontracted ex vivo vessels from NPR1^hu/hu mice, as evaluated in an ex vivo vessel ring assay (grey bars, isotype control, vein; black bars, acetylcholine, vein; white bars, acetylcholine, artery; blue bars, REGN5381, artery; red bars, REGN5381, vein). d, NPR1 expression is significantly higher in the femoral vein than in the femoral artery.

Fig. 4. REGN5381 reduces systolic BP and CVP in a temporally dose-dependent manner.

a,b, Beagle canines were surgically implanted with a radiotelemetry transmitter. A cross-over design was used and animals each received a single intravenous bolus of saline (control) (n = 2; black circles) or REGN5381 25 mg per kg (n = 2; red circles). BP measurements were collected pre-dose (baseline measurements) and during a 48 h post-dose monitoring period. Post-dose systolic BP assessment after administration of REGN5381 (red circles) or control (saline, black circles) (a) and urinary cGMP concentration (b). PK, pharmacokinetics. c,d, Anaesthetized beagle canines were instrumented with venous and arterial catheters. Each canine received a single intravenous bolus of saline (control, n = 6) or 25 mg per kg REGN5381 (n = 6) and was monitored for acute haemodynamic changes. Post-dose CVP (c) and heart rate (d) change from baseline (subscripted ‘Δbaseline’) is shown. bpm, beats per minute. e–h, Cynomolgus monkeys were surgically implanted with a radiotelemetry transmitter. The animals each received a single intravenous bolus of saline/vehicle (n = 4; black circles) or REGN5381 subcutaneously (s.c.; n = 4, 1 mg per kg, pink line; n = 5, 5 mg per kg, pale yellow line; n = 5, 25 mg per kg, red line) or intravenously (n = 5, 5 mg per kg, light blue line; n = 5, 25 mg per kg, dark blue line). BP measurements were collected for each animal before dosing (baseline measurements) and until study day 56. The change from baseline over time was determined for each individual non-human primate (NHP) for systolic BP (e), heart rate (f) and pulse pressure (g). h, The baseline and 24 h post-dose cGMP levels in the urine of non-human primates. Each symbol represents one individual animal. Data are mean ± s.e.m. *P = 0.0178 (light blue group), **P = 0.0081 (dark blue group), ****P < 0.0001 (red group).

Fig. 5. REGN5381 reduces systolic BP and stroke volume, and increases pulse rate, in healthy volunteers.

a, The effect of intravenous REGN5381 100 mg (n = 6, blue line) or placebo (n = 12, grey line) on systolic BP, as measured by pulse-wave analysis (PWA), presented as the time-matched change from baseline. Pressure is presented as the average over 2 h. b, The time-matched mean change from baseline in heart rate, assessed by pulse-wave analysis, according to intravenous treatment group (pooled placebo, grey bar; REGN5381 10 mg, light blue bar; REGN5381 30 mg, blue bar; REGN5381 100 mg, dark blue bar). The mean change from baseline in heart rate and pulse pressure is shown as the average over 24 h. c, The time-matched mean change from baseline in pulse pressure assessed by pulse-wave analysis according to intravenous treatment group (pooled placebo, grey bar; REGN5381, 10 mg, light blue bar; REGN5381, 30 mg, blue bar; REGN5381, 100 mg, dark blue bar). d, The effect of intravenous REGN5381 100 mg (n = 6, blue line) or placebo (n = 12, grey line) on the stroke volume index as measured by pulse-wave analysis, shown as the time-matched change from baseline. The stroke volume index is shown as the average over 2 h. e, The change from baseline in urine cGMP levels after administration of study treatment (pooled placebo, grey bar; REGN5381, 10 mg, light blue bar; REGN5381, 30 mg, blue bar; REGN5381, 100 mg, dark blue bar). Data are mean ± s.e.m. f, The urine void volume was assessed before and after dosing according to treatment group (pooled placebo, grey bar; REGN5381, 10 mg, light blue bar; REGN5381, 30 mg, blue bar; REGN5381, 100 mg, dark blue bar).

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

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, IgG4^P; 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, IgG4^P; 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; IgG4^P, non-binding immunoglobulin G control; MFI, mean fluorescence intensity; NPR1, natriuretic peptide receptor 1; RFU, relative fluorescence units.

Extended Data Fig. 2. REGN5381 does not inhibit ANP binding to NPR1.

ANP binding in the presence of antibody was tested using Biacore S200. Human NPR1-mmh protein was captured over anti-His coated Biacore coupled chip for 10 min. NPR1 captured chip was first saturated with REGN5381 or IgG4 control antibody at 50 ug/ml or 200 nM of ANP for 4 min followed by testing with second injection of 200 nM ANP for 4 min over each complex NPR1 surface. The ANP binding response was recorded after the second injection and it showed that REGN5381 does not block ANP binding to NPR1-mmh. ANP, atrial natriuretic peptide; IgG4, immunoglobulin G; mab, monoclonal antibody; NPR1, natriuretic peptide receptor 1.

Extended Data Fig. 3. REGN5381 activated human NPR1-mediated cGMP production.

A, Schematic diagram of cGMP accumulation assay measuring hNPR1 activation-induced cGMP production. B, C, The activity of REGN5381 and ligand at hNPR1 was measured with HEK293/hNPR1 cells using different conditions due to the limit of quantitation for ligand at high concentrations. B, REGN5381 activated hNPR1 in the absence or presence of ligand (ANP or BNP) as measured by cGMP accumulation assays with HEK293/hNPR1 cells. Activation at higher ligand concentrations was seen, but cGMP level was not calculated due to the limit of quantitation. C, The full activity of ligand was evaluated with optimized conditions, where REGN5381 exhibited partial agonism compared to the ligand. Open symbols indicate assay conditions when no test article was added, and closed symbols indicate assay 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. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; cGMP, cyclic guanosine monophosphate; FRET, fluorescence resonance energy transfer; GTP, guanosine triphosphate; hNPR1, human NPR1; IgG4^P, non-binding immunoglobulin G control; NPR1, natriuretic peptide receptor 1.

Extended Data Fig. 4. Cryo-EM data processing.

A. Data processing workflow for the REGN5381 Fab + NPR1 ectodomain + ANP complex, including particle orientation distribution and FSC curve for the final reconstruction. B. Final map coloured by local resolution, from 2.5 to 5.5 Å. C. A sample of the EM density at the NPR1:REGN5381 interface, with selected residues labelled. D. A cutaway view of the EM density, sliced through the centre of the dimer to reveal the density at the peptide binding site. Density surface is coloured by molecule: REGN5381 Fab heavy chain – salmon; light chain – grey; NPR1 monomers – green and cyan (mostly hidden); ANP – purple; unassigned density – white. E. Data processing workflow for the REGN5381 Fab + NPR1 ectodomain (no ANP) complex. F. 3D FSC isosurface at FSC = 0.143, oriented with the lowest resolution along the z axis and the best resolution along the x axis, and coloured radially by resolution as indicated in the colour key. G. Local resolution map, oriented to show the absence of the ANP peptide in this complex. H. A sample of the EM density for this complex. I. A cutaway view of the EM density, sliced through the centre of the dimer to reveal the lack of density at the peptide binding site. Density surface is coloured by molecule: REGN5381 Fab heavy chain – salmon; light chain – grey; NPR1 monomers – green and cyan (mostly hidden); unassigned density – white. ANP, atrial natriuretic peptide; EM, electron microscopy; FSC, Fourier shell correlation; NPR1, natriuretic peptide receptor.

Extended Data Fig. 5. Hydrodynamic DNA delivery of ANP and BNP expression vectors in normotensive mice induced a sustained reduction in systolic blood pressure.

ANP and BNP overexpression resulted in systolic blood pressure reduction of over 30 mmHg. Key: black circles, control; red squares, ANP; purple triangles, BNP. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; HDD, hydrodynamic delivery.

Extended Data Fig. 6. REGN5381 reduces systolic blood pressure similar to SOC, with additive effects observed upon combination with some but not all SOC therapies.

The reduction in systolic blood pressure was assessed in telemetered normotensive NPR1^hu/hu mice administered a single subcutaneous dose of REGN5381 either alone or in combination with SOC agents for the treatment of HF and hypertension at therapeutically relevant doses (n = 5–7 per treatment group; pooled placebo n = 36). ARNi, angiotensin receptor/neprilysin inhibitor; HF, heart failure; NPR1, natriuretic peptide receptor; NPR1^hu/hu, NPR1 humanized; PDE, phosphodiesterase; RAAS, renin-angiotensin-aldosterone system; SGLT2, sodium-glucose cotransporter-2; SOC, standard-of-care.

Extended Data Fig. 7. Mean (+SD) total REGN5381 concentrations in serum vs time following a single subcutaneous or intravenous injection of REGN5381 in normotensive non-human primates.

Normotensive cynomolgus monkeys received a single intravenous bolus of saline/vehicle (n = 5; black circles) or REGN5381 subcutaneous (n = 5; 1 mg/kg [pink line]), (n = 5; 5 mg/kg [pale yellow line]), (n = 5; 25 mg/kg [red line]) or intravenous (n = 5; 5 mg/kg [light blue line]) or (n = 5; 25 mg/kg [dark blue line]). Concentrations of total REGN5381 (all drugs; without regard to binding site occupancy) in serum were determined using ELISA. ADA analysis was conducted using a generic immunoassay against anti-human IgG4 antibodies and 32% (8/25) of REGN5381—dosed animals showed a positive response. Nonetheless, all concentration data, regardless of whether potentially impacted by ADA, were included in this plot as the actual exposure would be that used for any correlation with PD effects. ADA, anti-drug antibodies; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; IV, intravenous; LLOQ, lower limit of quantification; PD, pharmacodynamics; SC, subcutaneous; SD, standard deviation.

Extended Data Fig. 8. REGN5381 leads to a reduction of NTproANP in normotensive non-human primates.

Normotensive cynomolgus monkeys received a single intravenous bolus of saline/vehicle (n = 5; black circles) or REGN5381 subcutaneous (n = 5; 1 mg/kg [pink line]), (n = 5; 5 mg/kg [pale yellow line]), (n = 5; 25 mg/kg [red line]) or intravenous (n = 5; 5 mg/kg [light blue line]) or (n = 5; 25 mg/kg [dark blue line]). Serum NTproANP levels were measured throughout the study. Data are mean ± SEM. Statistical Analysis: RM 2-Way ANOVA or 1-Way ANOVA; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. ANOVA, analysis of variance; NTproANP, N-terminal proatrial natriuretic peptide; RM, repeated measures; SEM, standard error of mean; Tx, treatment.

Extended Data Fig. 9. REGN5381-induced plasma cGMP production in healthy volunteers.

Mean ± standard error change from baseline in plasma cGMP levels following administration of study treatment (pooled placebo, grey bar; REGN5381 10 mg, light blue bar; REGN5381 30 mg, blue bar; REGN5381 100 mg, dark blue bar). cGMP, cyclic guanosine monophosphate; IV, intravenous; SE, standard error.