Production of an anti-ROS1 scFv antibody with enhanced specificity by a point mutation in the HCDR3

Abstract

The proto oncogene tyrosine kinase ROS1, which is located at 6p22 on chromosome 6, plays a key role in carcinogenesis through gene rearrangements. Gene rearrangements result in the expression of fusion proteins from partner genes. ROS1 fusion proteins consist of the C-terminal intracellular region of ROS1 and the N-terminal extracellular region of other partners. However, the full-length wild type ROS1 has rarely been reported. The possibility of full-length ROS1 expression has been proposed to be under epigenetic regulation. Here, the 3B20 scFv antibody, which is reactive to the N-terminal region of ROS1, was generated for the detection of full-length ROS1 in cancerous tissues. Unfortunately, in testing the reactivity of the 3B20 scFv antibody, cross-reactivity to the heat shock protein (Hsp) 70 family was also observed during immunoblot and immunoprecipitation analyses. Sequence homology indicated that the Asp, Leu, Gly, and Thr (DLGT) amino acid sequence was shared between ROS1 and Hsp70s. In addition, the epitope was confirmed to harbor this amino acid sequence by alanine scanning mutagenesis of ROS1. To modify the idiotope, which is the unique antigenic determinant of an antibody, random mutations were introduced into the heavy chain complementary determining region 3 (HCDR3) of the 3B20 scFv antibody. Consequently, a site-direct mutagenesis HCDR3 library was constructed specifically to eradicate cross-reactivity of the 3B20 scFv antibody while maintaining its binding affinity to ROS1. Finally, a new anti-ROS1 clone, 3B20-H1-13 scFv, was successfully generated through point mutation. The 3B20-H1-13 scFv antibody was shown to only react with ROS1 in enzyme-linked immunosorbent assay (ELISA), immunoblot, and immune-precipitation analyses. Immunohistochemistry (IHC) using 3B20-H1-13 scFv showed that ROS1 was absent in non-neoplastic lung tissues, and was overexpressed in a case of lung adenocarcinoma.