Dataset Information

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 favourable druggability by utilizing phage display-based strategy.

Methods

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.

Findings

Our data showed that the generated full-length anti-PCSK9 antibody FAP2M21 binds to hPCSK9 with a K_D as low as 1.42 nM, and a dramatically slow dissociation rate (k_off, 4.68 × 10^-6 s^-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, 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.

Interpretation

FAP2M21 with potent inhibitory effect on PCSK9 may serve as a promising therapeutic agent for treating hypercholesterolemia and associated cardiovascular diseases.

SUBMITTER: Xu M

PROVIDER: S-EPMC7921758 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A large human natural single‑chain fragment variable (scFv) phage library was constructed based on Cre‑LoxP recombination, and used to successfully identify antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9). The library was derived from 400 blood samples, 30 bone marrow samples, and 10 cord blood samples from healthy donors. Lymphocytes were isolated from each sample and cDNA was synthesized using reverse transcription‑quantitative PCR. Two‑step overlap PCR was then used for scFv synthesis using a LoxP peptide as the linker. The scFv gene was inserted into the phagemid vector pDF by enzymatic digestion and ligation, and then transformed into Escherichia coli (E. coli) SS320 to establish a primary antibody library in the form of scFvs. A primary antibody library consisting of 5x107 peripheral blood and umbilical cord blood sources, as well as a primary antibody library of 5x107 bone marrow samples were obtained. By optimizing the recombination conditions, the primary phage library was used to infect E. coli BS1365 strain (which expresses the Cre enzyme), and a human scFv recombinant library with a size of 1x1011 was obtained through Cre‑LoxP enzyme‑mediated heavy and light chain replacement and recombination. This constructed recombinant library was employed to screen for antibodies against recombinant PCSK9. After four rounds of selection, a fully human antibody (3D2) was identified with a binding affinity of 1.96±1.56ⅹ10‑10 M towards PCSK9. In vitro, the PCSK9/low‑density lipoprotein receptor (LDLR) pathway of Hep‑G2 cells was inhibited by 3D2 treatment, thereby increasing LDL uptake in these cells. In addition, combination treatment with 3D2 and statin was more effective at increasing LDLR levels than treatment with 3D2 or statin alone. Furthermore, 3D2 resulted in a 3‑fold increase in hepatic LDLR levels, and lowered total serum cholesterol by up to 61.5% in vivo. Taken together, these results suggest that the constructed human Cre‑LoxP scFv phage display library can be used to screen fully human scFv, and that 3D2 may serve as a candidate hypolipidemic therapy.