ADC is a complex that conjugates cytotoxic small molecule drugs to monoclonal antibodies through a rationally constructed linker, which can selectively deliver effective cytotoxic drugs into tumors. Currently, the FDA has approved 14 ADC drugs for marketing, and hundreds more are in clinical trials.
In addition to overexpression in tumors, another important factor in the selection of ADC drug targets is the efficiency of endocytosis, which is necessary for drug release activity. The effectiveness of an ADC depends on the efficiency with which targeted-mediated internalization delivers payloads within tumor cells. The pathway and efficiency of ADC internalization are also closely related to the efficacy and design of ADC drugs. Because it is a vital factor in ADC linker selection for cleavable, non-cleavable, or pH/reduction-sensitive types, as well as whether the payload (or its active metabolite) can diffuse across cell membranes to provide a bystander effect, and whether it increases tumor killing-rates or contributes to dose-limiting toxicity. Therefore, it is crucial to understand the endocytosis and mechanism of ADC. Multiple endocytosis pathways have overlapping aspects, so the general process of endocytosis is highly flexible, complex, and can be divided into three stages：(1) bud formation, (2) membrane curvature and vesicle maturation, (3) scission and release of the membrane into the cytoplasm.

ADC Endocytosis Pathways
CLIC/GEEC Endocytosis
CLIC/GEEC is an endocytosis chamber that occurs mainly in ligand-activated cells, which may be caused by receptor crosslinking of growth factors, antibodies, or bacterial toxins and viruses. In addition, the membrane must be in a highly fluid state because CLIC/GEEC does not work below physiological temperature or at higher membrane tension.
CLIC increased at the anterior edge of migrating cells. Other relevant parameters for identifying the CLIC/GEEC pathway include dynamic independent membrane rupture, sensitivity to cholesterol consumption, acquisition of Rab5 with early internal body fusion, placental alkaline phosphatase (PLAP), and FAK-related GTPase regulator (GRAF1).

Mediated Endocytosis
Clathrin-mediated endocytosis (CME) is conceptually a simple process consisting of several successive and partially overlapping steps. CMEs can be initiated by certain receptor structures on the plasma membrane or by the binding of ligands and/or antibodies. CME begins when endocytic capsid proteins in the cytoplasm begin to aggregate in the inner lobules of the plasma membrane. Capsid proteins continue to assemble and grow by recruiting from the cytoplasm and interacting with additional protein adapters. Key adaptor proteins bend the membrane, thus concentrating the internalized receptor/ligand into a “clathrin encapsulation pit (CCP)”. As CCP invagination increases, the CCP neck shrinks and separates from the plasma membrane through a fracture process. Actin polymerization helps to pull CCP inward into the cytoplasm until the rupture is complete and CCP is released as a clathrin-coated vesicle (CCV). Finally, the CCV shell is broken down, and the CCV fuses with the endosome for transport to specific subcellular sites or can be returned to the cell surface.

Macropinocytosis
Macropinocytosis is a form of endocytosis on a larger scale, usually involving highly wrinkled regions/projections of the plasma membrane that subsequently fuse or with the plasma membrane. The membrane folds are morphological features of macropinocytosis. Macropinocytosis depends on actin polymerization, Rac1 protein, and P21-activated kinase 1 (PAK1). PAK1 is a key regulator because it interacts with Rac1, which activates phosphatidylinositol-3-kinase (PI3K), Ras, Src, and Hsp90 to promote macropinocytosis. Macropinocytosis is also cholesterol-dependent, which is required for Rac1 recruitment. These components eventually lead to endocytosis with a larger absorption area than CME and fossa proteins.

Caveolae-Mediated Endocytosis
The caveolae is a vial invagination of the plasma membrane characterized by high levels of cholesterol and glycosphingolipids mediated by clathrin-independent endocytosis and is present in most cell types. The main scaffold protein of the caveolae is the caveolins, which is a complete membrane protein of 20-24 kDa oligomer. Caveolins share common scaffold domains that mediate interactions with themselves and other proteins containing caveolin-binding domains.
A unique aspect of caveolin-mediated endocytosis is that only about 1% of the caveolae germinate from the plasma membrane. In a small number of internalized pits, it appears to follow a circular pathway of co-localization with Rab5, a marker of early endosomes. This may pose a challenge for ADCs that target receptors that utilize caveolin-mediated endocytosis.