Antibody-drug conjugates (ADCs) are a new class of anticancer drugs, which consist of monoclonal antibodies, small-molecule cytotoxic drugs, and chemical linkers between them. ADCs can selectively deliver cytotoxic drugs to cancer cells, thereby reducing systemic exposure and expanding the therapeutic window. To date, 12 ADCs have been approved worldwide.
In addition to cancer cells, the target antigens of ADCs are usually expressed to some extent on extracellular vesicles (EVs). EVs are small particles surrounded by lipid membrane bilayers. They are generated from cells and have a variety contents. EVs can be carried over long distances in body fluids, and when they fuse with target cells, they can leave their bioactive contents behind in recipient cells, potentially altering their function. Since ADCs may bind to and be carried on EVs, ADC payloads may even be released far away from the tumor site, and EVs can affect the efficacy and safety of ADCs.
Introduction to EVs
EVs include exosomes (up to 150 nm in diameter; mainly derived from multivesicular bodies formed by the invagination of intracellular lysosomal particles, which are released into the extracellular matrix after fusion of the outer membrane of multivesicles with the cell membrane), microvesicles (up to 1000 nm in size and shed directly from the plasma membrane of cells), apoptotic bodies (1-2 μm in diameter), and other types of membrane vesicles with different sizes, functional properties, and biogenesis. All types of cells, including cancer cells, appear to secrete EVs.
EVs can be taken up by recipient cells through several mechanisms: 1) receptor-mediated endocytosis; 2) binding to target cell membranes; 3) macrophage phagocytosis. After ingestion, EVs delivers substances that can enter the cytoplasm or nucleus of recipient cells and alter cell biological functions (Figure 1).
Figure 1. The main mechanism of EVs uptake by recipient cells
Cancer cells secrete large amounts of EVs, which can be isolated from body fluids. EVs contain proteins, lipids, various RNA species, DNA, and metabolites. EVs can transfer their contents not only to cancer cells and tumor stromal cells, but also to distant non-malignant cells, where their contents remain active and affect the biological function of recipient cells. EVs are involved in the regulation of tumor growth and metastasis, tumor angiogenesis and anti-tumor immunity. It can also alter the effects of anticancer drugs in a variety of ways, sometimes leading to drug resistance. Let's take a look at how EVs affect the effects of the two main components of ADCs, small molecule anticancer drugs and mAbs.
Drug Resistance of EVS to Small Molecule Anticancer Drugs
Cancer cells can utilize EVs as an efflux mechanism for small-molecule anticancer drugs in at least two ways. First, cancer cells can aggregate small-molecule anticancer drugs into EVs, and then release the drug-loaded EVs into the extracellular space. Second, chemotherapy-resistant cancer cells may secrete EVs carrying ABC drug efflux transporters, which are the main cause of cancer multidrug resistance. Subsequently, chemotherapy-sensitive cancer cells may ingest EVs containing efflux transporters from extracellular space and accept functional efflux pump proteins, transforming drug-sensitive cancer cells into drug-resistant ones.
MicroRNAs of EVs can also promote drug resistance to small-molecule anticancer drugs by regulating gene expression. EVs can also deliver various other factors, such as PDGFR-β, TGF-β, or intermediate metabolites, that may contribute to the survival pathway of cancer cells.
EVs-mediated Drug Resistance to Anticancer Monoclonal Antibody Therapy
Rituximab is a CD20 mAb approved for the treatment of patients with CD20-positive non-Hodgkin lymphoma or chronic lymphocytic leukemia. Complement-dependent cytotoxicity plays a key role in the anticancer effect of rituximab. B-cell lymphoma cells secrete EVs carrying CD20, and rituximab binding to such EVs results in complement fixation on the EVs surface. This decoy effect mediated by CD20+ EVs leads to depletion of complement and free rituximab, thereby undermining the efficacy of rituximab against target cancer cells.
Trastuzumab, an anti-HER2 monoclonal antibody approved for the treatment of patients with HER2-positive breast cancer and gastric cancers. It has a direct inhibitory effect on HER2-positive cancer cells. Trastuzumab also recruits immune effector cells to kill cancer cells through antibody-dependent cellular cytotoxicity (ADCC). HER2-positive cancer cells secrete EVs carrying HER2, and the decoy effect mediated by trastuzumab binding to such EVs may attenuate the direct tumor growth inhibitory effect of trastuzumab as well as ADCC-related cytotoxicity.
Taken together, EVs may be a means by which cancer cells develop drug resistance to small-molecule anticancer drugs and antitumor antibodies, these are two key components of ADCs.
The Effect of EVs On The Efficacy of ADCs
After the ADC binds to the target on the EVs, the EVs deliver the ADC to the recipient cells. Since EVs can transport contents to nearby and distant cancerous and non-tumor cells, EVs may affect the efficacy of ADCs.
Bystander Effect of EVs and ADCs
Studies have shown that Brentuximab vedotin, an anti-CD30 ADC, binds to CD30-positive EVs secreted by lymphoma cells, and then CD30-negative lymphoma cells may absorb Brentuximab-bound EVs, leading to their death. Similarly, the anti-HER2 ADC, trastuzumab emtansine, bound to surface HER2-rich EVs may be carried to other cancer cells through EVs, leading to apoptotic death of other cancer cells. These observations suggest that EVs can deliver ADCs to adjacent cancer cells lacking the antibody target protein, resulting in a bystander effect that may increase the antitumor effect of ADCs (Figure 2).
Figure 2. EVs affects the efficacy and safety of ADC
Delivery of EVs to tumor stromal cells
In addition to cancer cells, non-malignant cells may also carry cancer-derived EVs. For example, endothelial cells accumulate EVs containing tissue factors from mesenchymal cancer cells and EVs containing epidermal growth factor receptor (EGFR), suggesting that cancer-derived EVs can transfer their contents to tumor stromal cells middle.
Therefore, EVs expressing ADC targets can deliver ADCs to non-malignant cells in the tumor microenvironment, which may have a role in promoting or inhibiting cancer growth, depending on the role of the receptor cells in cancer progression. For example, EVs-mediated damage of intratumoral macrophages and fibroblasts that promote tumor growth may inhibit tumor growth, while damage to intratumoral antitumor immune cells may promote tumor growth (Figure 2).
Drug Resistance of EVs to ADCs
For example, the effectiveness of trastuzumab-based anti-HER2 ADCs and other ADCs may be altered by EVS-mediated decoy mechanisms. HER2-positive gastric cancer cells resistant to trastuzumab emtansine leave trastuzumab emtansine in the extracellular space through EVs secretion, and since high concentrations of intracellular cytotoxic payloads are essential for their antitumor activity, the efflux mechanism of DM1 through EVs may also lead to the resistance of trastuzumab emtansine. Therefore, EVs can hinder the uptake of drugs into sensitive cancer cells and attenuate the anticancer efficacy of ADCs by effluxing the drugs.
The role of EVs-delivered ADCs in cancer metastasis
EVs were associated with the formation of pre-transfer niches (Fig. 3). For example, EVs derived from gastric cancer cells can transfer human epidermal growth factor receptor (EGFR) into liver stromal cells, resulting in altered regulation of the liver microenvironment, which in turn may promote the formation and growth of gastric cancer liver metastases. Likewise, cancer-derived EVs carrying integrin αvβ5 specifically bind to Kupffer cells in the liver and can promote pre-metastatic niche formation and liver metastasis in breast and pancreatic cancers.
Figure 3. Main mechanisms of EVs and pre-transfer niche formation
EVs and ADC Safety
Administration of ADCs is often associated with off-target adverse effects. ADCs can induce toxicity through their high-affinity binding to target antigens or nonspecific binding to Fc receptors. Additionally, if the linker is destabilized, early release of the payload in circulating or off-target tissues can lead to increased systemic exposure and adverse effects, which may lead to discontinuation of therapy.
EVs can also affect the safety of ADCs. Cancer-derived EVs found in blood and other bodily fluids, which can cause side effects. For example, cancer-derived EVs as described above translocate EGFR and integrin αv into hepatic stromal cells, which may lead to hepatotoxicity of anti-EGFR ADCs and anti-αv ADCs.
Conclusion
ADCs are one of the fastest growing cancer drug classes, with 12 drugs currently approved. Most ADC target antigens are known to be present on tumor-derived EVs. Since EVs can transfer their contents not only to cancer cells and tumor stromal cells, but also to distant non-malignant cells, they may mediate both the antitumor effects and side effects of ADCs. EVs may contribute to the success and failure of ADC therapy through several mechanisms, but some of these mechanisms are still hypothetical and need to be confirmed by subsequent further studies.
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References:
[1]. Extracellular vesicles as modifiers of antibody-drug conjugate efficacy.
[2]. Exosome-mediated metastasis: communication from a distance.
[3]. Shedding light on the cell biology of extracellular vesicles
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