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Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery
Published on: April 26, 2024
Effect of Endocytosis Inhibitors on the Cytotoxicity and Antitumor Activity of Anti-GD2 ADCs
M M Titov1, I V Kholodenko2, D V Kalinovsky1
1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia.
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.
Insights
Antibody-drug conjugates (ADCs) targeting ganglioside GD2 are internalized via multiple pathways. Inhibiting specific endocytosis routes, like caveolin-mediated, can enhance ADC efficacy against GD2-positive tumors.
Area of Science:
- Oncology
- Immunology
- Cell Biology
Background:
- Antibody-drug conjugates (ADCs) represent a targeted cancer therapy approach.
- Ganglioside GD2 is a promising target for ADC development in various cancers.
- Understanding the internalization mechanisms of anti-GD2 ADCs is crucial for optimizing their efficacy.
Purpose of the Study:
- To investigate the receptor-mediated endocytosis mechanisms of anti-GD2 ADCs.
- To explore the modulation of ADC internalization using endocytosis inhibitors.
- To assess the impact of these modulations on ADC cytotoxicity and therapeutic potential.
Main Methods:
- Utilized the anti-GD2 antibody ch14.18 conjugated with monomethyl auristatin E (MMAE).
- Investigated internalization pathways including clathrin-mediated endocytosis, caveolin-mediated endocytosis, and macropinocytosis.
- Employed endocytosis inhibitors, specifically nystatin, to modulate cellular uptake.
- Assessed ADC accumulation, cytotoxicity (IC50), and in vivo efficacy in a GD2-positive mouse tumor model.
Main Results:
- Anti-GD2 ADCs are efficiently internalized through clathrin-mediated, caveolin-mediated, and macropinocytotic pathways.
- ADC internalization correlates with GD2 expression levels and is influenced by antibody properties, not the payload.
- Nystatin, a caveolin inhibitor, increased ADC accumulation and reduced IC50 values (1.5-2.5 fold).
- Combination therapy with nystatin did not improve tumor growth inhibition in vivo compared to ADC monotherapy.
Conclusions:
- Receptor-mediated endocytosis pathways significantly influence anti-GD2 ADC internalization and efficacy.
- Targeting specific endocytic pathways, such as caveolin-mediated endocytosis with nystatin, can enhance ADC potency in vitro.
- Further optimization of combination strategies involving endocytosis modulation holds promise for improving anti-GD2 ADC antitumor effects.
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