Endocytosis Properties of GD2-Specific Antibodies in Tumor Cells

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Alina O. Makarova^1,2, Matvey M. Titov^1,2, Daniel V. Kalinovsky¹, Irina V. Kholodenko³, Alexey V. Kibardin⁴, Sergey S. Larin⁴, Elena V. Svirshchevskaya¹, Sergey M. Deyev^1,5,6, Roman V. Kholodenko^1,7,a*

¹Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia

²Lomonosov Moscow State University, 119991 Moscow, Russia

³Orekhovich Institute of Biomedical Chemistry, 119121 Moscow, Russia

⁴Rogachev Federal Research Center of Pediatric Hematology, Oncology and Immunology, 117997 Moscow, Russia

⁵Sechenov First Moscow State Medical University, 119992 Moscow, Russia

⁶National Research Center “Kurchatov Institute”, 123182 Moscow, Russia

⁷LLC “Real Target”, 108841 Moscow, Russia

^* To whom correspondence should be addressed.

Received: February 16, 2025; Revised: February 16, 2025; Accepted: March 11, 2025

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Antibody–drug conjugates (ADCs) represent one of the most promising classes of monoclonal antibody-based (mAb) targeted cancer therapies. To date, 15 drugs of this class have received clinical approval, with numerous others under investigation in more than 100 clinical trials. Similarly to unconjugated antibodies, ADCs target various tumor markers including carbohydrate antigens such as glycosphingolipids. Among these targets, ganglioside GD2 is considered the most promising marker. Recent studies have demonstrated significant potential for the anti-GD2 ADCs in clinical application. Internalization of the antigen-antibody complexes and their subsequent transport to cellular lysosomes are critical factors that substantially influence ADC efficacy. However, internalization capacity of the GD2-specific antibodies and mechanisms of endocytosis of their complexes with GD2 have been insufficiently characterized. This study investigated internalization mechanisms of the ganglioside GD2 complexes with several of the most relevant GD2-specific antibody formats, namely full-length antibodies, minibodies, and scFv fragments. It was demonstrated that all used antibody variants successfully internalize into the GD2-positive tumor cells and enter their lysosomal compartments. Full-length antibodies and minibodies exhibited high endocytosis efficiency in the GD2-positive cells, occurring through several pathways, primarily macropinocytosis and caveolae-mediated endocytosis. These findings may be of interest for the development of more effective targeted therapeutics for GD2-positive tumors.

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KEY WORDS: internalization, endocytosis, ganglioside GD2, monoclonal antibodies, antibody fragments, antibody–drug conjugates, cancer immunotherapy

DOI: 10.1134/S0006297925600395

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