Department of Cell and Molecular Biology, School of Life Science and Technology, State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, China Pharmaceutical University, Nanjing 210009, P.R. China.
Department of Cell and Molecular Biology, School of Life Science and Technology, State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, China Pharmaceutical University, Nanjing 210009, P.R. China; Vaccine Engineering Center of China Medical City, Taizhou 225300, Jiangsu Province, P.R. China.
EBioMedicine. 2021 Mar;65:103250. doi: 10.1016/j.ebiom.2021.103250. Epub 2021 Feb 26.
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 favourable druggability by utilizing phage display-based strategy.
A potent single-chain variable fragment (scFv) named AP2M21 was obtained by screening a fully human scFv phage display library with hPCSK9, and performing two in 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.
Our data showed that the generated full-length anti-PCSK9 antibody FAP2M21 binds to hPCSK9 with a K as low as 1.42 nM, and a dramatically slow dissociation rate (k, 4.68 × 10 s), 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 EC 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, unpaired Student's t-test), 30.2% (P = 0.002, Mann-Whitney U-test) and 37.2% (P = 0.002, Mann-Whitney U-test), respectively.
FAP2M21 with potent inhibitory effect on PCSK9 may serve as a promising therapeutic agent for treating hypercholesterolemia and associated cardiovascular diseases.
前蛋白转化酶枯草溶菌素 9（PCSK9）通过促进 LDL 受体（LDLR）的降解来调节血清 LDL 胆固醇（LDL-C）水平，是治疗高胆固醇血症的一个有吸引力的治疗靶点。在此，我们通过噬菌体展示技术策略生成了一种具有良好成药性的新型全人源抗体。
通过用 hPCSK9 筛选全人 scFv 噬菌体展示文库，获得了一种有效的单链可变片段（scFv），命名为 AP2M21，并进行了两次体外亲和力成熟过程，包括 CDR 靶向定制诱变和交叉克隆。然后，通过与 L234A/L235A/N297G 突变和 C 末端赖氨酸缺失的修饰人 IgG1 Fc 片段融合，将其转化为全长 Fc 沉默抗 PCSK9 抗体 FAP2M21，从而消除其免疫效应功能并减轻 mAb 异质性。
我们的数据表明，生成的全长抗 PCSK9 抗体 FAP2M21 以低至 1.42 nM 的 K 结合 hPCSK9，解离速率（k）极慢（4.68×10 s），这归因于其结合能（-47.51 kcal/mol）低于其亲本 FAP2（-30.39 kcal/mol）。我们验证了 FAP2M21 能够有效抑制 PCSK9 诱导的 HepG2 细胞中 LDL-C 摄取减少，其 EC 为 43.56 nM。此外，在 hPCSK9 过表达的 C57BL/6 小鼠中，尾静脉注射 FAP2M21 1、3 和 10 mg/kg，剂量依赖性地上调肝脏 LDLR 水平，同时血清 LDL-C 分别降低 3.3%（P=0.658，未配对学生 t 检验）、30.2%（P=0.002，曼-惠特尼 U 检验）和 37.2%（P=0.002，曼-惠特尼 U 检验）。
FAP2M21 对 PCSK9 具有强大的抑制作用，可能成为治疗高胆固醇血症和相关心血管疾病的有前途的治疗药物。
