Pit-mediated endocytosis
Clathrin-independent endocytosis includes caveolin-mediated endocytosis, caveolin-independent carrier protein/GPI-enriched early inner compartment (CLIC/GEEC), and macropinocytosis.

The pit is a vial-like invagination of the plasma membrane, characterized by high levels of cholesterol and glycosphingolipids, which mediates endocytosis through a clathrin-independent pathway, and is present in most cell types. The main scaffold protein of caveolae is caveolin, a 20-24 kDa integral membrane protein that forms oligomers. Caveolins share a common scaffold domain that mediates interactions with itself and other proteins containing caveolin binding domains.

Although the pits have an invaginated morphology similar to CCPs, they are different. The density of CCP is constant, while the density of pits varies greatly depending on the cell type. CCPs increase as the germinated endosome matures. In contrast, fossa vesicles remain unchanged in size. Once inside the cell, the pit forms a higher-order structure instead of a simple spherical inner body formed by CCPs.

Another unique aspect of caveolin-mediated endocytosis is that only about 1% of caveolae germinate from the plasma membrane. In a small number of internalized pits, it seems to follow a cyclical pathway that co-localizes with Rab5 (a marker of early endoderm). This may pose challenges to ADCs that target receptors using caveolin-mediated endocytosis.

CLIC/GEEC endocytosis
CLIC/GEEC is an endocytic chamber, which mainly occurs in cells activated by ligands, which may be caused by growth factors, antibody receptor cross-linking, or bacterial toxins and viruses. In addition, the cell membrane must be in a state of high fluidity, because CLIC/GEEC does not work at lower than physiological temperature or when the membrane is under higher tension.

CLIC increases at the leading edge of migrating cells. Other relevant parameters that recognize the CLIC/GEEC pathway include kinetic-independent membrane rupture, sensitivity to cholesterol consumption, acquisition of Rab5/early endosome fusion, placental alkaline phosphatase (PLAP), and FAK-related GTPase regulators (GRAF1).

Macropinocytosis is a larger-scale form of internalization, usually involving highly folded areas/protrusions of the plasma membrane, which then fuse or with the plasma membrane. Membrane folds are the morphological feature 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 as required for the recruitment of Rac1. These components ultimately lead to endocytosis with a larger absorption area than CME and caveolin.
CD33 is a 67kda transmembrane glycoprotein receptor usually expressed on normal myeloid cells. As it is preferentially overexpressed on AML cells, it is the target of GO. The intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) of CD33 regulates the endocytosis of CD33, which can be activated by CME. Regarding endocytosis efficiency, there is no correlation between the expression level of CD33 in AML cells and its endocytosis rate. CD33 is a slowly internalized antigen. In addition, CD33 cross-linking does not improve endocytosis. AML patients who do not respond to GO may be related to the dysfunction of CD33 receptor endocytosis.

CD30 is a 120kda transmembrane glycoprotein belonging to the tumor necrosis factor receptor (TNFR) superfamily. The extracellular part consists of six cysteine-rich domains (CRDs) in an extended conformation. CD30 is expressed on activated T cells and B cells as well as various lymphoid tumors (including Hodgkin's lymphoma and ALCL).

CD30 does not have endocytosis, on the contrary, it is shed due to proteolytic cleavage, and the shedding of CD30 is mediated by matrix metalloproteinases (MMPs). Shedding is a biology characteristic of CD30, and high concentrations of circulating soluble CD30 can be used as a serum marker to monitor tumor progression. Regarding the efficacy of ADCs, elevated circulating levels of CD30 appear to isolate injected ADCs, thereby reducing the number of ADCs that can be localized to CD30-positive tumor sites. Therefore, the lack of endocytosis indicates that CD30 is not an ideal ADC target.

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