Revealing the mechanism of Tilvestamab in treating cancer from a single-molecule perspective using atomic force microscopy.

[BACKGROUND] AXL receptor tyrosine kinase, activated by its ligand GAS6, is a key driver of tumor aggressiveness, metastasis, and therapeutic resistance in cancers. Therapeutic inhibition of AXL signaling has emerged as a promising anti-cancer strategy. Tilvestamab, a monoclonal antibody, competitively blocks GAS6-AXL binding to suppress oncogenic signaling. However, the biomechanical basis of tilvestamab's molecular interactions-including its binding kinetics, affinity relative to GAS6, and functional impact on cellular mechanics-remains uncharacterized at the single-molecule level. Addressing this gap is critical to elucidating its mechanism of action.

[RESULTS] Using atomic force microscopy, we systematically compared tilvestamab and GAS6 binding to AXL receptors on live lung cancer cells. Single-molecule force spectroscopy revealed that tilvestamab exhibits closely with its natural ligand GAS6 of binding affinity to AXL, and markedly higher binding frequency than GAS6. Kinetic analyses demonstrated tilvestamab forms more stable complexes with AXL, characterized by slower dissociation rates and shorter interaction times. Crucially, nano-indentation measurements showed tilvestamab treatment substantially increases cellular stiffness compared to control group of cells and GAS6-treated cells, counteracting malignancy-associated softening. Meanwhile, we treated two other AXL-expressing cancer cell lines:H1299 and MCF-7, similarly, an increase in overall stiffness was observed across the cell lines following Tilvestamab treatment. This mechanical rigidification was consistent across multiple cancer models and correlated with suppressed migratory capacity.

[SIGNIFICANCE] Our study reveals that tilvestamab's anti-cancer efficacy arises from superior kinetic stability in AXL binding and direct modulation of cellular biomechanics. By enhancing membrane rigidity, tilvestamab impairs cancer cell migration and proliferation-key drivers of metastasis. These findings provide the biomechanical rationale for tilvestamab's therapeutic activity, positioning it as a promising agent for targeting AXL-dependent cancers through mechanopharmacological mechanisms.