Nonspecific endocytosis
Endocytosis is an important process in cell uptake of nutrients, regulation of transmembrane kinetics, synaptic vesicle recycling, and so on. Endocytosis can also play an important role in the uptake and distribution of macromolecules, including IgG/ADC in normal cells. Endocytosis is roughly divided into phagocytosis (granule internalization) and endocytosis (soluble molecular internalization, also known as liquid phase endocytosis). In addition, endocytosis is divided into macroscopic and microscopic endocytosis according to the size of endocytosis vesicles.
The endocytosis of “large scale” includes phagocytosis and macroendocytosis, which involve the internalization of large particles and large volume particles, respectively. Phagocytosis involves the absorption of larger particles, resulting in deformation of the cell membrane (rearrangement of local actin). This process may also absorb immune complexes containing ADC or ADC aggregates.
Similar to phagocytosis, macrocytophagy is also an actin-dependent process that involves the wrinkling and extension of the plasma membrane around a relatively large amount of extracellular fluid (rather than particles) to mediate endocytosis. Microscale endocytosis includes microsomal phagocytosis of less than 200 nm.
These processes usually require special coat proteins, such as gridding proteins or vesicles. The binding of ligands to specific membrane receptors initiates a series of signal events that lead to the recruitment of specific adapter proteins to achieve the formation of grid protein-coated vesicles. These newly formed vesicles are cut off by kinetin (a GTPase enzyme) and released for further intracellular transport.
Vesicular protein-mediated endocytosis involves a bottle-shaped structure (vesicles) formed by membrane-coated protein vesicles, which also rely on dynamic proteins to cut off vesicles. Vesicular protein-mediated uptake plays a major transport role in many types of cells, especially endothelial cells.
In general, the above macroscopic and microscopic endocytosis processes may help ADC enter normal cells. With regard to the toxicity of ADC to normal cells, non-specific endocytosis mechanisms such as vesicle-dependent endocytosis, giant endocytosis, and phagocytosis are potentially important mechanisms. It is also clear that the endocytosis mechanism and the total endocytosis rate are different in normal tissues and cell types.
Many specialized immune cells (including macrophages and dendritic cells) have higher endocytosis rates. For example, Kupffer cells (resident macrophages in the liver) play a major role in the non-specific uptake and clearance of immune conjugates, including ADC. Because endothelial cells are located at the interface between blood vessels and interstitial chambers, they also have high macromolecular endocytosis rates. Understanding the endocytosis rates of different normal cells/tissues is valuable for understanding the role of nonspecific endocytosis as a potential mechanism of ADC uptake and toxicity.
Factors affecting the non-specific endocytosis of IgG/ADC: the physical and chemical properties of macromolecules may affect endocytosis in normal cells/tissues. The molecular charge on the surface of IgG/ADC is one of the important parameters that affect the tissue distribution and competition of antibodies. Positively charged molecules are attracted to negatively charged groups in mammalian cell membranes and the extracellular matrix (heparin sulfate proteoglycans). The local concentration of ADC was increased, resulting in more non-specific endocytosis in normal tissues/cells.
Generally speaking, the increase in the net positive charge of IgG antibodies leads to an increase in tissue distribution and plasma clearance, while the decrease of the net positive charge leads to a decrease of tissue distribution. Importantly, changes in the isoelectric point (pI) of at least one or more units are sufficient to produce measurable changes in organizational distribution and competition. These conclusions may also be applicable to ADC and support the hypothesis that ADC charge may affect the non-specific endocytosis of normal cells. Therefore, charge modification—by reducing the positive charge or balancing the overall surface charge distribution—is a method considered in the design of ADC.
However, it is worth noting that, similar to normal tissues, charge modification may also affect the target antigen-dependent ADC uptake required for tumor cell efficacy. Optimizing the surface charge of ADC and reducing the uptake of normal cells while retaining target-mediated uptake in tumor cells can improve TI.
The hydrophobicity of ADC may also play a role in its non-specific uptake by normal cells. Many drug linker combinations used in ADC are hydrophobic, making antibodies significantly hydrophobic, especially for ADC with high DAR. The increased hydrophobicity of high DAR can promote the aggregation of ADC and accelerate non-specific clearance. Like hepatocytes, ADC with high DAR may be rapidly cleared by other normal cells with high non-specific endocytosis, resulting in off-target toxicity.
Nonspecific endocytosis (especially giant endocytosis) is considered to be the pathway of ADC uptake by normal corneal epithelial cells and megakaryocytes, which leads to ocular toxicity and thrombocytopenia, respectively. Similarly, endocytosis-mediated internalization can reduce the toxicity of ADC (AGS-16C3F) to megakaryocytes (thrombocytopenia).