Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5

Scientific Reports
Yosuke HashimotoHiroyuki Takeda

Abstract

The production of antibodies against the extracellular regions (ECR) of multispanning membrane proteins is notoriously difficult because of the low productivity and immunogenicity of membrane proteins due to their complex structure and highly conserved sequences among species. Here, we introduce a new method to generate ECR-binding antibodies utilizing engineered liposomal immunogen prepared using a wheat cell-free protein synthesis system. We used claudin-5 (CLDN-5) as the target antigen, which is a notoriously difficult to produce and poorly immunogenic membrane protein with two highly conserved extracellular loops. We drastically improved the productivity of CLDN-5 in the cell-free system after suppressing and normalizing mRNA GC content. To overcome its low immunogenicity, two engineered antigens were designed and synthesized as proteoliposomes: a human/mouse chimeric CLDN-5, and a CLDN-5-based artificial membrane protein consisting of symmetrically arranged ECRs. Intraperitoneal immunization of both engineered CLDN-5 ECR antigens induced ECR-binding antibodies in mice with a high success rate. We isolated five monoclonal antibodies that specifically recognized CLDN-5 ECR. Antibody clone 2B12 showed high affinity (<10 nM) a...Continue Reading

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Citations

Dec 12, 2018·The Journal of Pharmacology and Experimental Therapeutics·Yosuke HashimotoMasuo Kondoh
Aug 21, 2019·International Journal of Molecular Sciences·Yosuke HashimotoMasuo Kondoh
Mar 22, 2020·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Srujan Kumar DondapatiStefan Kubick
May 1, 2020·Biochimica Et Biophysica Acta. Biomembranes·Yosuke Hashimoto, Matthew Campbell
May 12, 2021·Life Science Alliance·Géraldine MayeuxJean-Luc Lenormand
Jun 13, 2021·Journal of Controlled Release : Official Journal of the Controlled Release Society·Keisuke TachibanaMasuo Kondoh
May 23, 2020·Drug Discovery Today·Yosuke HashimotoMasuo Kondoh

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Methods Mentioned

BETA
electrophoresis
flow cytometry
ELISA
glycosylation

Software Mentioned

GraphPad
GraphPad Prism
GeneOptimizer
GeneArt GeneOptimizer
GeneArt

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