Development of a Novel, Fully Human, Anti-PCSK9 Antibody with Potent Hypolipidemic Activity by Utilizing Phage Display-Based Strategy

Abstract

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) levels by facilitating the degradation of the LDL receptor (LDLR) and is an attractive therapeutic target for hypercholesterolemia intervention. Herein, we generated a novel fully human antibody with favorable druggability by utilizing phage display-based strategy. Methods: A potent single-chain variable fragment (scFv) named AP2M21 was obtained by screening a fully human phage display library with hPCSK9, and performing twoin vitro affinity maturation processes including CDR-targeted tailored mutagenesis and cross-cloning. Thereafter, it was transformed to a full-length Fc-silenced anti-PCSK9 antibody FAP2M21 by fusing to a modified human IgG1 Fc fragment with L234A/L235A/N297G mutations and C-terminal lysine deletion, thus eliminating its immune effector functions and mitigating mAb heterogeneity. Findings: Our data showed that the generated full-length anti-PCSK9 antibody FAP2M21 binds to hPCSK9 with a KD as low as 1.42 nM, and a dramatically slow dissociation rate (koff, 4.68 × 10−6s-1), which could be attributed to its lower binding energy (-47.51 kcal/mol) than its parent counterpart FAP2 (-30.39 kcal/mol). We verified that FAP2M21 potently inhibited PCSK9-induced reduction of LDL-C uptake in HepG2 cells, with an EC50 of 43.56 nM. Further, in hPCSK9 overexpressed C57BL/6 mice, a single tail i.v. injection of FAP2M21 at 1, 3 and 10 mg/kg, dose-dependently up-regulated hepatic LDLR levels, and concomitantly reduced serum LDL-C by 3.3% (P=0.658), 30.2% (P=0.002) and 37.2% (P=0.002), respectively. Interpretation: FAP2M21 with potent inhibitory effect on PCSK9 may serve as a promising therapeutic agent for treating hypercholesterolemia and associated cardiovascular diseases. Funding Statement: This work was funded by National Fund for Major Projects of China (2009ZX09103-653; 2013ZX09301303-006; 2018ZX09301035), The Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), National Fund for Fostering Talents of Basic Science (NFFTBS, 3050040016), China Pharmaceutical University “Double First-Class” project (CPU2018GY15). Declaration of Interests: The authors declare no conflict of interest concerning this article. Ethics Approval Statement: The experimental procedures for the in vivo studies were approved by the Animal Ethics Committees of China Pharmaceutical University (No. 201601179, 19 October 2016) and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.