Effect of Endocytosis Inhibitors on the Cytotoxicity and Antitumor Activity of Anti-GD2 ADCs.

Abstract

Cancer remains a critical public health challenge, and developing novel approaches to cancer therapy is highly relevant. Targeted tumor therapy using antibody-drug conjugates (ADCs) has already demonstrated its efficacy in multiple tumors. Ganglioside GD2 is a promising target for ADC development. However, its functional properties, which are important for the cytotoxicity of ADCs, remain poorly understood. This study focuses on the mechanisms of receptor-mediated endocytosis for the conjugate formed between the anti-GD2 antibody ch14.18 and monomethyl auristatin E (MMAE), as well as the approaches for modulating this process using endocytosis inhibitors. Our findings demonstrate that anti-GD2 ADCs are efficiently internalized into tumor cells through various endocytic pathways, including the clathrin- and caveolin-mediated pathways, as well as macropinocytosis. The efficiency of ADC accumulation directly correlates with the level of GD2 expression on the tumor cell surface and is determined by the properties of the parental antibody, independent of the cytotoxic payload. Endocytosis inhibitors can modulate the functional properties of anti-GD2 ADCs, either decreasing or increasing the cytotoxic effects of the conjugates in GD2-positive cells. Nystatin, a specific inhibitor of caveolin-mediated endocytosis, increased ADC accumulation in cells and reduced the IC₅₀ 1.5- to 2.5-fold depending on the cell line. Although administration of nystatin together with anti-GD2 ADCs did not enhance tumor growth inhibition compared to ADC monotherapy in the GD2- positive mouse tumor model, further optimization of combination therapy conditions aimed at enhancing receptor-mediated endocytosis is a promising strategy for potentiating the antitumor effects of anti-GD2 ADCs.