Pamela D. Garzone

Increasing Serum Half-life and Extending Cholesterol Lowering in Vivo by Engineering Antibody with pH-sensitive Binding to PCSK9

Background: An antagonistic anti-PCSK9 antibody exhibits target-mediated clearance, resulting in a dose-dependent PK. Results: Engineering of an antibody with pH-sensitive binding to PCSK9 decreases target-mediated clearance, resulting in increased PK and efficacy in vivo. Conclusion: pH-sensitive anti-PCSK9 antibodies are excellent candidates for therapeutic development. Significance: pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load. 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.

Safety and tolerability of LBR-101, a humanized monoclonal antibody that blocks the binding of CGRP to its receptor: Results of the Phase 1 program:

Background LBR-101 is a fully humanized monoclonal antibody that binds to calcitonin gene-related peptide. Objective The objective of this article is to characterize the safety and tolerability of LBR-101 when administered intravenously to healthy volunteers, by presenting the pooled results of the Phase 1 program. Methods LBR-101 was administered to 94 subjects, while 45 received placebo. Doses ranged from 0.2 mg to 2000 mg given once (Day 1), as a single IV infusion, or up to 300 mg given twice (Day 1 and Day 14). Results Subjects receiving placebo reported an average of 1.3 treatment-emerging adverse events vs 1.4 per subject among those receiving any dose of LBR-101, and 1.6 in those receiving 1000 mg or higher. Treatment-related adverse events occurred in 21.2% of subjects receiving LBR-101, compared to 17.7% in those receiving placebo. LBR-101 was not associated with any clinically relevant patterns of change in vital signs, ECG parameters, or laboratory findings. The only serious adverse event consisted of “thoracic aortic aneurysm” in a participant later found to have an unreported history of Ehlers-Danlos syndrome. Conclusion Single IV doses of LBR-101 ranging from 0.2 mg up to 2000 mg and multiple IV doses up to 300 mg were well tolerated. Overt safety concerns have not emerged. A maximally tolerated dose has not been identified.

A Phase I Randomized Study of a Specifically Engineered, pH‐Sensitive PCSK9 Inhibitor RN317 (PF‐05335810) in Hypercholesterolemic Subjects on Statin Therapy

This phase I study assessed the safety, tolerability, pharmacokinetics, and pharmacodynamics of RN317 (PF‐05335810), a specifically engineered, pH‐sensitive, humanized proprotein convertase subtilisin kexin type 9 (PCSK9) monoclonal antibody, in hypercholesterolemic subjects (low‐density lipoprotein cholesterol (LDL‐C) ≥ 80 mg/dl) 18–70 years old receiving statin therapy. Subjects were randomized to: single‐dose placebo, RN317 (subcutaneous (s.c.) 0.3, 1, 3, 6, or intravenous (i.v.) 1, 3, 6 mg/kg), or bococizumab (s.c. 1, 3, or i.v. 1 mg/kg); or multiple‐dose RN317 (s.c. 300 mg every 28 days; three doses). Of 133 subjects randomized, 127 completed the study. RN317 demonstrated a longer half‐life, greater exposure, and increased bioavailability vs. bococizumab. RN317 was well tolerated, with no subjects discontinuing because of treatment‐related adverse events. RN317 lowered LDL‐C by up to 52.5% (day 15) following a single s.c. dose of 3.0 mg/kg vs. a maximum of 70% with single‐dose bococizumab s.c. 3.0 mg/kg. Multiple dosing of RN317 produced LDL‐C reductions of ∼50%, sustained over an 85‐day dosing interval.

Effects of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibition with Bococizumab on Lipoprotein Particles in Hypercholesterolemic Subjects

PURPOSE Monoclonal antibody inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) elicit significant reductions in serum LDL-C levels. However, little is known about their effects on lipoprotein particles. The purpose of this analysis was to evaluate the effect of PCSK9 inhibition with bococizumab (RN316/PF-04950615), a humanized monoclonal antibody to PCSK9, on LDL, VLDL, and HDL particle concentration and size in hypercholesterolemic subjects. METHODS Data from 3 double-blind, placebo-controlled, randomized studies were analyzed. In study 1, a total of 67 hypercholesterolemic subjects received IV placebo or bococizumab 0.25, 0.5, 1, or 1.5 mg/kg weekly for 4 weeks. In studies 2 and 3, a total of 135 hypercholesterolemic subjects taking statins received IV placebo or bococizumab 0.25, 1, 3, or 6 mg/kg monthly for 12 weeks. Lipoprotein particle concentration and size were measured by using nuclear magnetic resonance spectroscopy. FINDINGS Overall, the majority of subjects were men (51.9%) aged >50 years of age and of white ethnic origin. In total, 189 subjects with both baseline and 2-week posttreatment data were included in the analysis. After PCSK9 inhibition with bococizumab 0.5, 1, 1.5, 3, and 6 mg/kg, concentrations of total LDL, total small LDL, and small VLDL particles decreased significantly versus baseline and placebo (P < 0.05), whereas concentrations of HDL particles increased (P < 0.05). The size of the LDL, VLDL, and HDL particles increased after PCSK9 inhibition. Reductions in LDL-C and total LDL particle concentrations were highly correlated. IMPLICATIONS The effect of inhibiting PCSK9 with bococizumab on lipoprotein particle concentration and size are consistent with the general mechanism of PCSK9 inhibitors in blocking PCSK9-mediated downregulation of LDL receptors. PCSK9 inhibition has the potential to provide a clinical benefit through the modulation of atherogenic lipoprotein particles in addition to LDL-C lowering, and this effect will likely be assessed in future analyses of data from cardiovascular outcomes trials of PCSK9 monoclonal antibodies that are currently being conducted. ClinicalTrials.gov identifiers: NCT01243151, NCT01342211, and NCT01350141.

EFFECTS OF RN316 (PF-04950615), A HUMANIZED IGG2ΔA MONOCLONAL ANTIBODY BINDING PROPROTEIN CONVERTASE SUBTILISIN KEXIN TYPE 9, ON LIPOPROTEIN PARTICLES IN HYPERCHOLESTEROLEMIC SUBJECTS

RN316 binds to Proprotein Convertase Subtilisin Kexin type 9 (PCSK9), preventing PCSK9-mediated down-regulation of the low density lipoprotein (LDL) receptor (R), improving LDL cholesterol (C) clearance from serum, and reducing LDL-C levels. High concentrations of small LDL particles contribute to

A Mechanism‐Based Pharmacokinetic/Pharmacodynamic Model for Bococizumab, a Humanized Monoclonal Antibody Against Proprotein Convertase Subtilisin/Kexin Type 9, and Its Application in Early Clinical Development

Bococizumab (RN316/PF‐04950615), a humanized monoclonal antibody, binds to secreted proprotein convertase subtilisin/kexin type 9 (PCSK9) and prevents its downregulation of low‐density lipoprotein receptor, leading to improved clearance and reduction of low‐density lipoprotein cholesterol (LDL‐C) in plasma. A mechanism‐based drug‐target binding model was developed, accounting for bococizumab, PCSK9, and LDL‐C concentrations and the effects of concomitant administration of statins. This model was utilized to better understand the pharmacokinetic/pharmacodynamic (PK/PD) data obtained from 3 phase 1 and 2 phase 2a clinical studies. First, simulations performed with this model demonstrated that the conventional method of the area‐under‐the‐curve ratio for bioavailability determination underestimated the subcutaneous bioavailability of bococizumab due to its target‐mediated disposition. Second, a covariate model component for statin effects on bococizumab PK/PD was characterized, including a description of the decreased baseline LDL‐C, increased baseline PCSK9, and increased LDL‐C lowering with concomitant use of statins. Last, the impact of the dosing regimens with and without a dose holiday on bococizumabs LDL‐C–lowering effectiveness was shown to be predictable due to the well‐characterized PK‐PD relationship.

Single and multiple ascending-dose study of glucagon-receptor antagonist RN909 in type 2 diabetes: a phase 1, randomized, double-blind, placebo-controlled trial

PurposeThis first-in-human study assessed safety, immunogenicity, pharmacokinetics, and pharmacodynamics of RN909, a monoclonal antibody antagonist of the glucagon receptor, in type 2 diabetes (T2DM) subjects.MethodsThis study enrolled 84 T2DM subjects receiving stable metformin regimens. Forty-four subjects were randomized to receive single escalating doses of RN909 (0.3 to 6 mg/kg subcutaneously (SC), or 1 mg/kg intravenously (IV)), or placebo; 40 subjects were randomized to receive multiple escalating doses (50 to 150 mg SC) or placebo every 4 weeks for 12 weeks.ResultsRN909 was well tolerated; treatment-related elevated liver function tests (LFTs) were observed in 4/33 (12.1%) and 5/32 (15.6%) subjects treated with single and multiple doses, respectively, versus 1/10 (10%) and 0 in the respective placebo groups. RN909 dose-normalized AUCinf increased more than dose-proportionally following single SC doses, and after multiple doses, accumulation ratios ranged from 1.3 to 3.4. The incidence of antidrug antibodies (ADA) was 33% after single doses and 50% after multiple doses. RN909 produced dose-dependent, durable fasting plasma glucose (FPG)-lowering at day 29 (mean change −20.6 to −97.5 mg/dL) and day 85 (mean change; −27.2 to −43.5 mg/dL) after single and multiple doses, respectively. HbA1c also was reduced after single (mean change −0.30% to −1.44%), and multiple doses (−0.83% to −1.56%).ConclusionRN909 was well tolerated after single and multiple doses in T2DM subjects, with diarrhea and elevated LFTs the most frequent adverse events. The appearance of ADA did not affect pharmacokinetics or efficacy. Robust lowering of FPG and HbA1c was observed.