ANGPTL3 blockade with a human monoclonal antibody reduces plasma lipids in dyslipidemic mice and monkeys

Abstract

Angiopoietin-like protein 3 (ANGPTL3) is a circulating protein synthesized exclusively in the liver that inhibits LPL and endothelial lipase (EL), enzymes that hydrolyze TGs and phospholipids in plasma lipoproteins. Here we describe the development and testing of a fully human monoclonal antibody (REGN1500) that binds ANGPTL3 with high affinity. REGN1500 reversed ANGPTL3-induced inhibition of LPL activity in vitro. Intravenous administration of REGN1500 to normolipidemic C57Bl/6 mice increased LPL activity and decreased plasma TG levels by ≥50%. Chronic administration of REGN1500 to dyslipidemic C57Bl/6 mice for 8 weeks reduced circulating plasma levels of TG, LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C) without any changes in liver, adipose, or heart TG contents. Studies in EL knockout mice revealed that REGN1500 reduced serum HDL-C through an EL-dependent mechanism. Finally, administration of a single dose of REGN1500 to dyslipidemic cynomolgus monkeys caused a rapid and pronounced decrease in plasma TG, nonHDL-C, and HDL-C. REGN1500 normalized plasma TG levels even in monkeys with a baseline plasma TG greater than 400 mg/dl. Collectively, these data demonstrate that neutralization of ANGPTL3 using REGN1500 reduces plasma lipids in dyslipidemic mice and monkeys, and thus provides a potential therapeutic agent for treatment of patients with hyperlipidemia.

Keywords: angiopoietin-like protein 3; cholesterol; dyslipidemia; endothelial lipase; hyperlipidemia; lipoprotein lipase; triglycerides.

Fig. 1.

REGN1500 reduces serum lipid levels in chow-fed C57Bl/6 mice. Serum samples were collected after a 4 h fast from male C57Bl/6 mice (n = 6 per group) 7 days before (Prebleed) and at the indicated days following a single subcutaneous injection of REGN1500 or isotype control antibody (Control Ab) (10 mg/kg). Serum TG (A) and cholesterol (B) were measured enzymatically. C: Plasma levels of mouse ANGPTL3 were measured by ELISA. Serum (20 μl) from each mouse collected 7 days after treatment with REGN1500 or Control Ab (10 mg/kg) were size-fractionated by HPLC. TG (D) and cholesterol (E) were measured in each fraction. Values shown are mean ± SEM, except for the chromatograms where only mean values are shown. Statistical analysis was conducted by two-way ANOVA with Bonferroni correction posttest. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2.

REGN1500 increases postheparin plasma LPL and improves lipid tolerance. A: Postheparin plasma LPL and HL activity of chow-fed C57Bl/6 mice treated with REGN1500 or control antibody (Control Ab) (10 mg/kg, n = 5 per group). Postheparin plasma was pooled and fractionated on a heparin column to separate HL and LPL, and TG hydrolase activity was measured as described in the Materials and Methods. B: Effect of REGN1500 treatment on plasma TG levels following a lipid tolerance test. Male C57Bl/6 mice were treated with a single dose of REGN1500 or control antibody (10 mg/kg, n = 5 per group) 4 days prior to the intraperitoneal administration of Intralipid (10 μl/g of 20% Intralipid). Values are mean ± SEM. Statistical analysis was conducted by Student’s t-test comparing the groups at each time point. *P < 0.05; **P < 0.01.

Fig. 3.

Weekly administration of REGN1500 reduces serum lipid levels in mice maintained on a high-fat high-cholesterol diet for 8 weeks. Serum samples (Prebleed) were collected from male C57Bl/6 mice (n = 10 per group, 8 weeks old) after a 4 h fast. After 5 days, the mice were placed on a high-fat high-cholesterol diet. Three days later, the mice started receiving a weekly injection of with REGN1500 or isotype control antibody (Control Ab) (25 mg/kg). Serum samples were collected 6 days after each injection following a 4 h fast. Changes in TG (A), TC (B), and LDL-C (C) serum levels are shown. D: Body weights were monitored weekly. E: Liver, epididymal white adipose tissue (epiWAT), and hearts were collected at the end of the study and weighed. F: TG content of liver, epiWAT, and hearts. All values are mean ± SEM. Statistical analysis was conducted by two-way ANOVA with Bonferroni correction posttest. **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 4.

REGN1500 reduces HDL-C in WT but not in EL-deficient (Lipg^−/−) mice. Serum samples were collected after a 4 h fast from male chow-fed Lipg^−/− mice and their WT littermates (n = 7 per group) before (Prebleed; 7 days) and 4 days after a single administration of REGN1500 or isotype control antibody (Control Ab) at 10 mg/kg. Serum was analyzed for TG (A) and HDL-C (B). C: Chromatogram showing cholesterol distribution among lipoprotein subclasses after REGN1500 or control antibody administration in WT and Lipg^−/− mice. All values are mean ± SEM, except chromatograms where only mean values are shown. Statistical analysis was by two-way ANOVA with Bonferroni correction posttest. *P < 0.05; **P < 0.01.

Fig. 5.

REGN1500 reduces circulating TG, nonHDL-C, and HDL-C levels in dyslipidemic cynomolgus monkeys. Baseline serum samples were collected at days −15, −7, −2, and 0 following a 16 h fast. Seventeen monkeys were divided into three groups and were administered vehicle or REGN1500 (3 or 10 mg/kg) on day 0 of the study. Serum samples were collected on multiple days and analyzed for TG (A), nonHDL-C (C), LDL-C (D), and HDL-C (E), and are represented as a percent change from baseline. All values are mean ± SEM. Panel (B) shows average changes in absolute TG values before and following treatment with REGN1500 (10 mg/kg). For each parameter, Student’s t-test (two tails, paired) was performed in each group comparing the mean value at each time point to the baseline value. *P < 0.05; **P < 0.01.