Development of Single-Chain Antibodies Specific to Lysophosphatidic Acid Receptor 2

Abstract

Lysophosphatidic acids (LPAs) are ubiquitous serum phospholipids that trigger diverse cellular responses such as cell proliferation, migration, cell survival, and calcium influx. LPA receptors belong to the G-protein coupled receptor (GPCR) family, and seven subtypes of LPA receptors have been identified. Among them, LPA receptor 2 (LPA(2)) is involved in the proliferation and metastasis of ovarian, cervical, and breast cancers. Hence, LPA(2)-specific antagonists or antibodies are considered as potential anticancer therapeutics. To develop antibodies against LPA(2), a recombinant LPA(2) was expressed in Escherichia coli, purified to homogeneity, and immobilized on a solid surface as an active conformation. An M13 phage library displaying single-chain variable fragments (scFvs) of human IgG containing randomized complementarity-determining regions was applied to select LPA(2)-specific scFv clones. After five rounds of biopanning, a few scFv clones which showed specific binding to LPA(2) were identified. Single-chain antibodies (scAbs), which contain the isolated scFvs and C domain, were constructed and expressed in E. coli. The purified scAbs showed specific binding to LPA(2) with K-D values of 200-600 nM.