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

摘要

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 na?ve single‐chain variable fragment (scFv) sequences. The purified P9‐LPA1 was stabilized in native conformation in nanodiscs and attached to immobilized Gαi3 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 KD 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.