Hepatic Sensing Loop Regulates PCSK9 Secretion in Response to Inhibitory Antibodies

Abstract

Background: Monoclonal antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9i) lower LDL-C by up to 60% and increase plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) levels by 10-fold.

Objectives: The authors studied the reasons behind the robust increase in plasma PCSK9 levels by testing the hypothesis that mechanisms beyond clearance via the low-density lipoprotein receptor (LDLR) contribute to the regulation of cholesterol homeostasis.

Methods: In clinical cohorts, animal models, and cell-based studies, we measured kinetic changes in PCSK9 production and clearance in response to PCSK9i.

Results: In a patient cohort receiving PCSK9i therapy, plasma PCSK9 levels rose 11-fold during the first 3 months and then plateaued for 15 months. In a cohort of healthy volunteers, a single injection of PCSK9i increased plasma PCSK9 levels within 12 hours; the rise continued for 9 days until it plateaued at 10-fold above baseline. We recapitulated the rapid rise in PCSK9 levels in a mouse model, but only in the presence of LDLR. In vivo turnover and in vitro pulse-chase studies identified 2 mechanisms contributing to the rapid increase in plasma PCSK9 levels in response to PCSK9i: 1) the expected delayed clearance of the antibody-bound PCSK9; and 2) the unexpected post-translational increase in PCSK9 secretion.

Conclusions: PCSK9 re-entry to the liver via LDLR triggers a sensing loop regulating PCSK9 secretion. PCSK9i therapy enhances the secretion of PCSK9, an effect that contributes to the increased plasma PCSK9 levels in treated subjects.

Keywords: LDL cholesterol; LDL receptor; PCSK9; cholesterol homeostasis; monoclonal antibodies; turnover studies.

FIGURE 1. Long-Term Effects of PCSK9i Therapy on Plasma PCSK9 and LDL-C Levels

Patients eligible for antibody-based proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition therapy received 140 mg evolocumab or 75 mg alirocumab every 2 weeks. Plasma was collected before initiation of therapy (time 0) and during the 18 months of treatment (A). (B and D) Plasma LDL-C levels, and (C and E) plasma PCSK9 levels. Changes from baseline (red dotted line) are plotted (mean ± SE) (B and C) and absolute values are presented in the inset tables (mean ± SE, and 95% CI) (D and E). Using absolute values, changes from baseline were analyzed by linear mixed-effects model (P < 0.001 for PCSK9 and LDL-C) followed by Dunnet’s post hoc test (P < 0.001 for all time points). CI = confidence interval; LDL-C = low-density lipoprotein cholesterol; PCSK9i = proprotein convertase subtilisin/kexin type 9 inhibitory therapy with monoclonal antibodies.

FIGURE 2. Short-Term Response to PCSK9i in a Cohort of Healthy Volunteers

Participants received a single dose of either evolocumab (140 mg, n = 4) or alirocumab (75 mg, n = 3). Blood samples were collected 30 minutes prior to antibody injection, and then 3, 12, 24, 72, 216, and 504 hours after injection (A). Plasma PCSK9 (B and D) and low-density lipoprotein cholesterol (LDL-C) (C and E) levels are presented as changes from baseline (mean ± SE) and absolute values are presented in the inset tables (mean ± SE, and 95% CI) (D and E). Using absolute values, changes from baseline were analyzed by linear mixed-effects model (P < 0.001 for PCSK9 and LDL-C) followed by Dunnet’s post hoc test (PCSK9: 0.5 and 3–21 days; P < 0.05; LDL-C: 3–21 days; P < 0.01). Abbreviations as in Figure 1.

FIGURE 3. Short-Term Effects of PCSK9i in Mice

Wild type (WT) and low-density lipoprotein receptor deficient (Ldlr^−/−) mice were given a single peritoneal injection of PCSK9i antibody (10 mg/kg) or vehicle (phosphate-buffered saline [PBS]). Blood samples were collected 24 hours before injection, and then 2, 4, 8, 16, and 24 hours after injection (A). PCSK9 plasma levels were quantified in WT (B) and Ldlr^−/− (C) mice over time and plotted relative to baseline. Total cholesterol (TC) was quantified at the endpoint (D). Values are mean ± SE (n = 6–22). Using absolute values, changes over time were analyzed by linear mixed-effects model (P < 0.001 for WT; P > 0.05 for Ldlr^−/−) followed by Dunnet’s post hoc test (vehicle injected: all time points P > 0.05; PCSK9i injected: all time points; P < 0.001). Changes in total cholesterol were evaluated for significance by 2-tailed unpaired Student’s t-test. #P < 0.08; ***P < 0.001. IP = intraperitoneal injections; other abbreviations as in Figure 1.

FIGURE 4. Effect of PCSK9i on PCSK9 Clearance

The clearance of free PCSK9 was compared with PCSK9 bound to PCSK9i. 5 μg of human PCSK9, either alone (n = 3) or after preincubation with 0.105 mg anti-PCSK9 antibody (n = 3) was administered to WT mice via retro-orbital injection. Blood samples were collected 5, 30, 60, 120, and 240 minutes after injection (A). Human PCSK9 plasma levels were quantified and plotted relative to baseline (B). Murine PCSK9 plasma levels were quantified and plotted relative to baseline (C). Values are plotted as the mean ± SE, n = 3. Changes in circulating PCSK9 overtime and bound or not to the antibody were assessed for significance by 2-way analysis of variance (B: P < 0.01; C: P < 0.001). hPCSK9 = human PCSK9; mPCSK9 = murine PCSK9; other abbreviations as in Figures 1 and 3.

FIGURE 5. Short-Term Effects of PCSK9i in Murine Livers

WT and Ldlr ^−/− mice were given a single peritoneal injection of PCSK9i antibody (10 mg/kg) or vehicle (PBS). Livers were collected 24 hours after injection (A). The effects of the antibody on hepatic LDLR protein (B), mRNA (C), PCSK9 protein (D), and mRNA (E) were quantified. Values are plotted as the mean ± SE (mRNA expression is relative to the WT control), n = 6–22. Changes were evaluated for significance by 2-tailed unpaired Student’s t-test. #P < 0.08; ***P < 0.001. Abbreviations as in Figures 1 and 3.

FIGURE 6. Kinetics of PCSK9 Production and Secretion In Vitro

Storage phosphor screen scans show the appearance and accumulation of PCSK9 over time in cell extracts (A) and media (B) from cells incubated with vehicle (1× PBS) or PCSK9i (PCSK9i, 8 μg/mL). Values are plotted as the mean ± SE (n = 4 independent experiments with 1 replicate in each experiment) of the quantified and normalized band intensity compared with control at time 0 or 2 hours. Changes in PCSK9, with or without antibody, were assessed for significance by 2-way analysis of variance (B: P < 0.05). Abbreviations as in Figure 1 and 3.

CENTRAL ILLUSTRATION. A Sensing Loop Regulates PCSK9 Secretion Upon Inhibitory Therapy

Two mechanisms contribute to the rapid increase in plasma PCSK9 levels in response to PCSK9 inhibition: the expected delayed clearance of the antibody-bound PCSK9; and an unexpected post-translational increase in PCSK9 secretion. Our results uncover a sensing loop for PCSK9 returning to the liver via low-density lipoprotein receptors and suggest that hepatic re-entry of plasma PCSK9 is a central contributor to trafficking of both PCSK9 and low-density lipoprotein. LDL= low-density lipoprotein; LDLR = low-density lipoprotein receptor; PCSK9 = proprotein convertase subtilisin/kexin type 9.