CD33
CD33, a 67kda transmembrane glycoprotein receptor, is usually expressed in normal myeloid cells and it is the target of GO because it is preferentially overexpressed in AML cells. The intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) of CD33 regulates the endocytosis of CD33, which can be activated by CME. As for the endocytosis efficiency, there was no correlation between the expression level of CD33 in AML cells and the endocytosis rate. CD33 is a slowly internalized antigen. In addition, CD33 cross-linking can not improve endocytosis. AML patients who do not respond to GO may be related to the low function of CD33 swallowing in vivo.
CD30
CD30 is a 120kda transmembrane glycoprotein, which belongs to the tumor necrosis factor receptor (TNFR) superfamily. Its extracellular part consists of six extended conformational cysteine-rich domains (CRDs). CD30 is expressed on activated T and B cells as well as various lymphoid tumors, including Hodgkin’s lymphoma and ALCL.
CD30 has no endocytosis. On the contrary, it falls off due to proteolysis, and the exfoliation of CD30 is mediated by matrix metalloproteinases (MMPs). Exfoliation is a characteristic of CD30 biology. High concentrations of circulating soluble CD30 can be used as a serum marker for monitoring tumor progression. For the efficacy of ADC, elevated CD30 circulation levels seem to isolate injected ADC, thereby reducing the number of ADC that can be located in CD30-positive tumors. Therefore, the lack of endocytosis indicates that CD30 is not an ideal target for ADC.
CD22
CD22 is a 140 kDa transmembrane glycoprotein. Like CD33, it is a member of the Siglec family and shares a variety of structural features. The key difference is that CD22 is much larger than CD33 because it has multiple Ig domains and ITIM/ITIM-like motifs. The expression of CD22 is limited to B cells, and the expression of CD22 is increased in most mother cells of all kinds of B-cell malignant tumors (including ALL).
CD22 endocytosis occurs through CME. Natural-like ligands accumulate in cells through the rapid structural endocytosis of CD22. These ligands are classified and degraded in lysosomes, while CD22 is recycled back to the cell surface. In addition, endocytosis induced by the CD22 ligand activates the intracellular cistern, which complements or increases the expression of CD22 on the cell surface. Therefore, CD22 has good endocytosis toward ADC.
CD79b
CD79b is expressed only in immature and mature B cells and overexpressed in the B cells of malignant tumors ≥ 80%. CD79a and CD79b are two non-covalently bound transmembrane proteins that mediate signal transduction and endocytosis. For the latter, the CD79a-CD79b heterodimer is the scaffold that controls BCR endocytosis. BCR endocytosis is mainly accomplished by CME and mediated by AP-2. Interestingly, CD79a directly interacts with the μ subunit of AP-2, which activates CD79b and leads to the endocytosis of the whole BCR complex.
In addition, for ADC, CD79a can be internalized as a monomer, but CD79b cannot. If tyrosine (Y195) in the proximal membrane of CD79b is mutated, the binding of AP-2 to CD79a is blocked and endocytosis is blocked. In 18% of the activated B cell-like DLBCL samples, Y195 was mutated. In conclusion, there is evidence that the endocytosis activity of CD79b depends on the internalization of the whole BCR complex, not as a monomer.
TROP-2
Trop-2 is a monomer glycoprotein of 46 kDa that has the characteristics of selective overexpression, structural endocytosis, and targeting lysosomes, which makes it a very attractive target for ADC. The internalization mechanism of Trop2 is related to CME.
One potential explanation for the observed strong endocytosis of Trop-2 may be due to significant Trop-2 aggregation. The conformational kinetics of Trop-2 was studied. It was found that Trop-2 formed a natural homodimer through the interaction fragment of the amino acid “VVVVV” located in the transmembrane domain. The dimerization of Trop-2 can further recruit Trop-2 monomers to closer positions through other cell surface proteins. Therefore, the Trop-2 cluster is likely to be formed by multiple dimers linked by lipid rafts and other membrane-binding proteins.
Trop-2 binds to a variety of ligands, such as claudin-1, claudin-7, cyclin D1, and IGF1. However, none of these ligands has been proven to be internalized when binding to or interacting with Trop-2. Therefore, compared with normal cells, Trop-2 has stronger endocytosis in tumor cells, which indicates that Trop-2 is a good target for ADC.
BCMA
BCMA or CD269, also known as member 17 of the TNFR superfamily, transduces signals that induce B cell survival and proliferation. The molecular weight of BCMA is only 20.2 kDa. The extracellular domain bound by its ligand has an “armchair” conformation and consists of six CRDs. In addition to multiple myeloma, BCMA is also expressed in many hematological malignancies, such as Hodgkin’s lymphoma and non-Hodgkin’s lymphoma.
However, little is known about the precise endocytosis pathway used by BCMA. Related to endocytosis, sialylation is a regulatory function, which may induce BCMA to endocytosis by CME.
HER2
HER2 is a 185kda transmembrane glycoprotein belonging to the EGFR family. The amplification of the HER2/neu gene is the known driving factor of human malignant tumors and metastasis. HER2 has been used as a therapeutic target for decades because of its role in cancer. HER2 has also been the target of ADCs. Both T-DM1 and T-DXT have been approved for HER2-positive metastatic breast cancer.
There are many mechanisms for HER2 endocytosis. First is the CME. Co-immunoprecipitation clearly shows that HER2 binds directly to AP-2, in addition, dynasore can completely block the HER2 endocytosis of SKBR3 cells. Secondly, the caveolin binding motif φ x φ xxxx φ (φ represents aromatic amino acids Trp, Phe, or Tyr) usually exists on caveolins. Interestingly, the sequence WSYGVTIW has been identified in the intracellular kinase domain of HER2. In addition, studies have shown that HER2 can take advantage of the endocytosis pathway of CLIC/GEEC.
These different findings reveal the important characteristics of HER2 endocytosis. First of all, the endocytosis of HER2 is mixed. Secondly, the caveolae-mediated endocytosis pathway seems to be more frequently used.
Nectin-4
Nectin-4 is a 66 kDa type I transmembrane protein whose main function is to promote cell-to-cell contact. Nectin-4 is an attractive target for ADC because studies have shown that it is overexpressed in several tumor types but almost non-existent in normal adult tissues.
At present, no information about the endocytosis of natural ligands or complexes of mAb/ADC and nectin-4 has been found, but we can learn from the study of the endocytosis of nectin-4 binding pathogens. Nectin-4 is also the receptor of the measles virus. Studies have shown that the measles virus enters MCF7, HTB-20 breast cancer cells, and DLD-1 colorectal cancer cells through macropinocytosis. Virus entry requires PAK1. On the contrary, dynamin inhibitor Dynasore has no effect on virus entry. In addition, cells expressing dominant negative caveolin could not eliminate the endocytosis of the virus.