Isolation of Single Chain Antibodies Specific to Lysophosphatidic Acid Receptor 1 (LPAx) from a M13 Phage Display Library Using Purified LPAX Stabilized in Nanodiscs

摘要

Received February 26, 2019, Accepted April 13, 2019, Published online May 8, 2019 G-protein coupled receptors (GPCRs) comprise the largest membrane protein family and are involved in various kinds of physiological phenomena. LPA1 belongs to the rhodopsin-type GPCR family and mediates various biological functions, such as cell proliferation, platelet aggregation, smooth muscle contraction, and tumor cell invasion. Hence, LPA1 -specific antibodies have the potential to be used as therapeutic agents against cancer or ophthalmic disease. In this study, we identified single-chain antibodies specific to LPAi using a purified LPAi in nanodiscs and a library of M13 phages displaying human naive single-chain variable fragment (scFv) sequences. The purified P9-LPAi was stabilized in native conformation in nanodiscs and attached to immobilized Gai3 protein, and then Ml3 phages specific to LPAi were isolated after several rounds of biopanning. Two clones which specifically interacted with the immobilized LPAi were isolated, and single-chain antibody fragments (scAbs) that contained the isolated scFv fragment and a human kappa light chain constant domain were constructed and expressed in E. coli. The two purified scAbs (B8 and D4) showed specific binding to LPAi with KD values of 300-400 nM. When LPAr overexpressing HT29 cells were treated with a scAb (D4) and lysophosphatidic acid, an increase in the cytosolic calcium level was observed relative to cells treated only with lysophosphatidic acid, indicating that the isolated single chain antibody (D4) acts as a functional LPAi agonist.