Isolation of Single Chain Antibodies Specific to Lysophosphatidic Acid Receptor 1 (LPA₁) from a M13 Phage Display Library Using Purified LPA₁ Stabilized in Nanodiscs

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 LPA₁ using a purified LPA₁ in nanodiscs and a library of M13 phages displaying human naïve single-chain variable fragment (scFv) sequences. The purified P9-LPA₁ was stabilized in native conformation in nanodiscs and attached to immobilized G_αi3 protein, and then M13 phages specific to LPA₁ were isolated after several rounds of biopanning. Two clones which specifically interacted with the immobilized LPA₁ 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 LPA₁ with K_D values of 300–400 nM. When LPA₁-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 LPA₁ agonist.