Light-enhanced cytotoxicity and intracellular trafficking of the PD-L1-targeting photoimmunoconjugate EITC-atezolizumab

Abstract

Background

Photodynamic therapy (PDT) uses light to generate reactive oxygen species (ROS) that initiate cytotoxic responses in cancer cells. Photoimmunotherapy (PIT) improves PDT specificity by linking photosensitizers (PS) to monoclonal antibodies. A promising monoclonal antibody (mAb) for immunoconjugates is atezolizumab (Tecentriq), an EMA/FDA-approved PD-L1 immune checkpoint inhibitor for treating non-small cell lung cancer (NSCLC).

Objective

Our goal was to: Optimize the cytotoxic efficacy of a photoimmunoconjugate of eosin-5-isothiocyanate and atezolizumab (EITC-atezolizumab) in NSCLC cells; Study the uptake and intracellular transport of atezolizumab; Evaluate EITC-atezolizumab as a candidate for photochemical internalization (PCI) of the ribosome-inactivating protein gelonin.

Methods

Cytotoxic efficacy of EITC-atezolizumab was evaluated in NSCLC cell lines H1975 (PD-L1-high) and A549 (PD-L1-low) using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Confocal microscopy was employed to observe the uptake and intracellular localization of AlexaFluor647-atezolizumab and its colocalization with either LysoTracker™Blue or an anti-CD63 antibody labelled with Alexa488.

Results

EITC-atezolizumab PIT significantly enhanced cytotoxicity, showing much higher sensitivity in H1975 than A549 NSCLC cells. PIT with EITC-atezolizumab did not enhance gelonin efficacy. Confocal microscopy of H1975 cells revealed near-plasmamembrane puncta fluorescence of Alexa647-atezolizumab, with fractions clustering in CD63⁺ organelles peaking at 6 h. In A549 cells, AlexaFluor647-atezolizumab showed sustained accumulation in LysoTracker™ Blue+ vesicles for up to 24 h.

Conclusion

To the best of our knowledge, this is the first documentation demonstrating that atezolizumab is transported to CD63-positive organelles, thereby enhancing our understanding of its intracellular trafficking. Our study also strengthens the concept of PIT and atezolizumab-based targeting of PD-L1⁺ NSCLCs.