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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.

Agonistic antibodies, which bind specifically to death receptor 5 (DR5), can trigger apoptosis in tumor cells through the extrinsic pathway. In this present study, we describe the use of a phage display to isolate a novel fully human agonistic single chain fragment variable (scFv) antibody, which targets DR5. After five rounds of panning a large (1.2 × 108 clones) phage display library on DR5, a total of over 4000 scFv clones were screened by the phage ELISA. After screening for agonism in a cell-viability assay in vitro, a novel DR5-specific scFv antibody TR2-3 was isolated, which inhibited COLO205 and MDA-MB-231 tumor cell growth without any cross-linking agents. The activity of TR2-3 in inducing apoptosis in cancer cells was evaluated by using an Annexin V-PE apoptosis detection kit in combination with flow cytometry and the Hoechst 33342 and propidium iodide double staining analysis. In addition, the activation of caspase-dependent apoptosis was evaluated by Western blot assays. The results indicated that TR2-3 induced robust apoptosis of the COLO205 and MDA-MB-231 cells in a dose-dependent and time-dependent manner, while it remarkably upregulated the cleavage of caspase-3 and caspase-8. Furthermore, TR2-3 suppressed the tumor growth significantly in the xenograft model. Taken together, these data suggest that TR2-3 exhibited potent antitumor activity both in vitro and in vivo. This work provides a novel human antibody, which might be a promising candidate for cancer therapy by targeting DR5.