Background:

ROS1 gene fusions, particularly CD74–ROS1 and EZR–ROS1, are clinically significant oncogenic drivers in a subset of non-small cell lung cancer (NSCLC) patients. These fusion proteins retain the constitutively active ROS1 kinase domain and represent attractive molecular targets for both therapeutic inhibition and diagnostic antibody development.

Objective:

This study aimed to construct and validate expression vectors encoding CD74–ROS1 and EZR–ROS1 fusion genes, assess their transient and stable expression in mammalian cell systems, purify the recombinant proteins, and predict linear B-cell epitopes as potential candidates for antibody generation.

Methods:

Codon-optimized CD74–ROS1 and EZR–ROS1 genes were cloned into pcDNA3.1(+) vectors with a His-tag and verified by sequencing. Transient transfection was performed in HEK293T, A549, and Calu-1 cells. Protein expression was analyzed by Western blotting, and His-tagged proteins were purified via Ni-NTA. Stable cell lines were selected with G418. B-cell epitopes near fusion junctions were predicted using BepiPred-2.0.

Results & Conclusion:

Both fusion genes were successfully cloned into pcDNA3.1 vectors, producing inserts of the expected sizes (CD74–ROS1: 2121 bp; EZR–ROS1: 2586 bp). Transient transfection in HEK293T cells resulted in detectable expression of the fusion proteins, with CD74–ROS1 displaying higher and more stable expression levels than EZR–ROS1. Notably, EZR–ROS1 proved more suitable for Ni-NTA affinity purification, although partial proteolytic degradation was observed. Attempts to establish stable expression of CD74–ROS1 in A549 and Calu-1 cell lines were unsuccessful, even under both standard and reduced concentrations of G418. Although plasmid integration was confirmed, no protein expression was detected in resistant clones, suggesting possible oncogene-induced cytotoxicity or epigenetic silencing, which likely contributed to either the inability to generate or the silencing of stable CD74–ROS1-expressing cell lines. B-cell epitope prediction identified two highly immunogenic linear epitopes at the fusion junctions: KPTDAPPKDDFWIPE (CD74–ROS1; residues 200–215) and KTKKAERDDFWIPETSFI (EZR–ROS1; residues 356–373).

Keywords:

CD74-ROS1; EZR-ROS1; NSCLC; ROS1-Fusion