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Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis for Favorably Treating Hypercholesterolemia
- Source :
- Biomedicines; Volume 9; Issue 12; Pages: 1783, Biomedicines, Vol 9, Iss 1783, p 1783 (2021), Biomedicines
- Publication Year :
- 2021
- Publisher :
- Multidisciplinary Digital Publishing Institute, 2021.
-
Abstract
- Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has become an attractive therapeutic strategy for lowering low-density lipoprotein cholesterol (LDL-C). In this study, a novel high affinity humanized IgG1 mAb (named h5E12-L230G) targeting the catalytic domain of human PCSK9 (hPCSK9) was generated by using CDR-grafting, alanine-scanning mutagenesis, and saturated site-directed mutagenesis. The heavy-chain constant region of h5E12-L230G was modified to eliminate the cytotoxic effector functions and mitigate the heterogeneity. The biolayer interferometry (BLI) binding assay and molecular docking study revealed that h5E12-L230G binds to the catalytic domain of hPCSK9 with nanomolar affinity (KD = 1.72 nM) and an extremely slow dissociation rate (koff, 4.84 × 10ā5 sā1), which interprets its quite low binding energy (ā54.97 kcal/mol) with hPCSK9. Additionally, h5E12-L230G elevated the levels of LDLR and enhanced the LDL-C uptake in HepG2 cells, as well as reducing the serum LDL-C and total cholesterol (TC) levels in hyperlipidemic mouse model with high potency comparable to the positive control alirocumab. Our data indicate that h5E12-L230G is a high-affinity anti-PCSK9 antibody candidate with an extremely slow dissociation rate for favorably treating hypercholesterolemia and relevant cardiovascular diseases.
- Subjects :
- QH301-705.5
Chemistry
PCSK9
Ligand binding assay
Mutagenesis
single-chain variable fragment (scFv)
alanine scanning
saturated site-directed mutagenesis
molecular docking
slow dissociation rate
catalytic domain
Subtilisin
Medicine (miscellaneous)
Alanine scanning
Proprotein convertase
Article
General Biochemistry, Genetics and Molecular Biology
Biophysics
Kexin
Biology (General)
Site-directed mutagenesis
Subjects
Details
- Language :
- English
- ISSN :
- 22279059
- Database :
- OpenAIRE
- Journal :
- Biomedicines; Volume 9; Issue 12; Pages: 1783
- Accession number :
- edsair.doi.dedup.....9091315f8020328d8ede6971fb1d5a5d
- Full Text :
- https://doi.org/10.3390/biomedicines9121783