Increasing serum half-life and extending cholesterol lowering in vivo by engineering antibody with pH-sensitive binding to PCSK9

Abstract

Target-mediated clearance and high antigen load can hamper the efficacy and dosage of many antibodies. We show for the first time that the mouse, cynomolgus, and human cross-reactive, antagonistic anti-proprotein convertase substilisin kexin type 9 (PCSK9) antibodies J10 and the affinity-matured and humanized J16 exhibit target-mediated clearance, resulting in dose-dependent pharmacokinetic profiles. These antibodies prevent the degradation of low density lipoprotein receptor, thus lowering serum levels of LDL-cholesterol and potently reducing serum cholesterol in mice, and selectively reduce LDL-cholesterol in cynomolgus monkeys. In order to increase the pharmacokinetic and efficacy of this promising therapeutic for hypercholesterolemia, we engineered pH-sensitive binding to mouse, cynomolgus, and human PCSK9 into J16, resulting in J17. This antibody shows prolonged half-life and increased duration of cholesterol lowering in two species in vivo by binding to endogenous PCSK9 in mice and cynomolgus monkeys, respectively. The proposed mechanism of this pH-sensitive antibody is that it binds with high affinity to PCSK9 in the plasma at pH 7.4, whereas the antibody-antigen complex dissociates at the endosomal pH of 5.5-6.0 in order to escape from target-mediated degradation. Additionally, this enables the antibody to bind to another PCSK9 and therefore increase the antigen-binding cycles. Furthermore, we show that this effect is dependent on the neonatal Fc receptor, which rescues the dissociated antibody in the endosome from degradation. Engineered pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load.

FIGURE 1.

Serum antibody concentrations after J16 or J10 treatment. A, total plasma J16 antibody concentration in cynomolgus monkey after a single dose of 0.1 (squares), 1 (triangles), 3 (circles), 10 (inverted triangles), and 100 (diamonds) mg/kg J16 over time. Results are expressed as mean ± S.E., n = 4/group. B and C, total serum J10 (circles) and an isotype control (Ctrl) (squares) antibody concentration in wild type (B) and PCSK9^−/− (C) mice after a single dose of 10 mg/kg antibodies over time. Antibodies were dosed as a bolus intravenous injection on day 0. Results are expressed as mean ± S.E. (error bars), n = 8–9/group.

FIGURE 2.

PK and PD of J17 in mice and non-human primates. A and B, serum antibody concentrations (A) and total cholesterol (B) in wild type mice dosed with a single dose of 1 (squares), 3 (triangles), and 10 (circles) mg/kg of J10 (blue), J16 (red), and isotype control (Ctrl) antibody (10 mg/kg only; black). Results are expressed as mean ± S.E. (error bars), n = 6–8/group. C and D, total antibody concentrations (C) and the percentage of base-line (day −2) LDL-C (D) and in cynomolgus monkey treated with a single intravenous injection of 1.5 mg/kg J16 (squares) and J17 (triangles) on day 0. Results are expressed as mean ± S.E., n = 4/group.

FIGURE 3.

J17, J16, and PCSK9 co-localization with LAMP2 in HepG2 cells. A, isotype control (IC; top row), J10 (middle row), and J17 (bottom row) antibodies are shown in red, and Lamp2 is shown in green. Images shown are maximum intensity projections from independent channels with the merged image in the middle right column. The far right column depicts a co-localization mask between the two channels. Scale bar, 10 μm. B, percentage of J10 and J17 puncta co-localized with Lamp2-positive puncta (left). Percentage of PCSK9 puncta co-localized with Lamp2-positive puncta in the presence of IC, J10, and J17 (right). Results are plotted as averages ± S.E. (error bars) of three independent experiments. *, p = 0.0166 by Student's t test.

FIGURE 4.

The effect of PCSK9 or FcRn knock-out on J17 serum half-life. A, antibody concentrations in PCSK9^−/− mice after intraperitoneal injection of 10 mg/kg J10 (blue circles), J17 (red squares), and an isotype control (Ctrl) antibody (black triangles) on day 0 and intravenous injection of 3 mg/kg recombinant human PCSK9 (huPCSK9) on day 15. n = 3/group. B, antibody concentrations in FCRN^−/− mice after intravenous injection of 10 mg/kg J10 (blue circles), J17 (red squares), and isotype control antibody (black triangles) on day 0. n = 7/group. Results are expressed as mean ± S.E. (error bars).