Selective Targeting of RalA with Intrabodies Impairs Triple Negative Breast Cancer Metastasis

This study describes the successful development and validation of isoform-specific intrabodies targeting the highly homologous Ral oncoproteins, key effectors in cancer progression. Phage display was used to isolate single-chain variable fragment (scFv) clones that recognize RalA (C1-A, G5-A), RalB (F6-B), or both (A12-AB). Using lentiviral transduction, these intrabodies were stably expressed as EGFP fusion proteins in murine 4T1 breast cancer cells. The anti-RalA C1-A and A12-AB clones clearly colocalized with RalA, confirming their binding activity inside the cell. Their activity in cells was confirmed by analyzing Ral-dependent pathways. All intrabodies targeting RalA (C1-A, G5-A, A12-AB), but not the one only against RalB, inhibited mitochondrial fission, a known RalA function. All clones, except A12-AB, altered the endolysosome pathway by decreasing lysosome number. Furthermore, the RalA-specific C1-A clone decreased lysosome size and strongly reduced extracellular vesicle secretion, highlighting its distinct inhibitory potential. In an orthotopic triple-negative breast cancer mouse model, the anti-RalA C1-A clone weakly, but significantly, reduced primary tumor growth, and exerted a powerful anti-metastatic effect, strongly decreasing lung metastases and leading to complete metastasis elimination in 2/5 mice.<br><br>In summary, these potent, isoform-specific anti-Ral intrabodies act as effective intracellular inhibitors and successfully modulate RalA-specific functions in cells, thus offering a promising therapeutic strategy for controlling tumor growth and metastasis in vivo.