Introduction
In addition to the need for ADCs to target antigens that are overexpressed in tumors, another important factor is the efficiency of endocytosis, which is necessary for the release of the drug’s active payload. In fact, the efficacy of ADCs depends on the efficiency of targeted-mediated internalization to deliver effective payloads within tumor cells.
The pathways and efficiency of ADC internalization are closely related to the efficacy and design of ADC drugs. This is because it is a crucial factor in selecting cleavable, non-cleavable, or pH/reduction-sensitive linkers, as well as whether the effective payload (or its active metabolites) can diffuse across the cell membrane to provide a “bystander effect,” and whether it enhances tumor kill rates or contributes to dose-limiting toxicity. Therefore, it is necessary to gain a deeper understanding of the endocytosis of ADCs and their mechanisms, as this is an extremely important first step for ADCs to exert their pharmacological effects in vivo.
Endocytic Pathways Related to ADCs
Generally, normal endocytosis can be divided into three stages: (1) budding formation, (2) membrane invagination and vesicle maturation, (3) membrane fission and release into the cytoplasm. Various endocytic pathways have overlapping aspects, thus making the general process of endocytosis highly flexible and complex.
Clathrin-Mediated Endocytosis
Clathrin-mediated endocytosis (CME) is conceptually a simple process that includes several consecutive and partially overlapping steps. CME can be initiated structurally by certain receptors on the plasma membrane or require the binding of ligands and/or antibodies. CME begins when endocytic coat proteins start to aggregate on the inner leaflet of the plasma membrane. The coat proteins continue to assemble and grow by recruiting additional proteins from the cytoplasm and interacting with them. Key adapter proteins cause the membrane to curve, concentrating the internalized receptor/ligand into a “clathrin-coated pit” (CCP). As CCP invagination increases, the neck of the CCP constricts and separates from the plasma membrane through a fission process. Actin polymerization helps pull the CCP into the cytoplasm until fission is complete, and the CCP is released to become a clathrin-coated vesicle (CCV). Finally, the CCV coat is disassembled, and the CCV fuses with endosomes to transport to specific subcellular locations or can be recycled back to the cell surface.
Clathrin is a key component of CME, composed of heavy and light chains. Three clathrin heavy chains and light chains form a trimer that interacts with other trimers to form a polygonal lattice around the emerging CCP. Adapter protein 2 (AP-2) is a heterotetrameric complex that mediates the constriction of the CCP neck. Dynamin is a GTPase that forms helical polymers at the neck of the mature vesicle. After GTP hydrolysis, dynamin induces the vesicle to pinch off from the plasma membrane.
Caveolae-Mediated Endocytosis
Endocytosis that does not rely on clathrin includes caveolae-mediated endocytosis, caveolin-independent carrier protein/GPI-enriched early endosomal compartments (CLIC/GEEC), and macropinocytosis.
Caveolae are flask-shaped invaginations of the plasma membrane characterized by high levels of cholesterol and sphingolipids, mediating endocytosis through a clathrin-independent pathway and present in most cell types. The main scaffold protein of caveolae is caveolin, a complete membrane protein that forms oligomers of 20-24 kDa. Caveolin shares common scaffold domains that mediate interactions with itself and other proteins containing caveolin-binding domains.
Although caveolae have a similar invagination morphology to CCPs, they are different. Simply put, the density of CCPs is constant, while the density of caveolae can vary significantly depending on the cell type. CCPs increase in size as budding endosomes mature, whereas caveolae vesicles maintain a constant size. Once inside the cell, caveolae form higher-order structures, rather than simple spherical endosomes formed by CCPs.
Another unique aspect of caveolin-mediated endocytosis is that only about 1% of caveolae are derived from budding off the plasma membrane. In a small fraction of internalized caveolae, it appears to follow a recycling pathway co-localized with Rab5 (a marker for early endosomes). This may pose a challenge for ADCs targeting receptors that utilize caveolin-mediated endocytosis.
CLIC/GEEC Endocytosis
CLIC/GEEC is an endocytic compartment that primarily occurs in ligand-activated cells, which may be triggered by growth factors, receptor crosslinking of antibodies, or bacterial toxins and viruses. Additionally, the cell membrane must be in a state of high fluidity, as CLIC/GEEC does not operate at sub-physiological temperatures or when the membrane is under higher tension.
CLIC increases at the leading edge of migrating cells. Other relevant parameters for recognizing the CLIC/GEEC pathway include dynamics-independent membrane fission, sensitivity to cholesterol depletion, acquisition of Rab5/early endosome fusion, placental alkaline phosphatase (PLAP), and GTPase regulatory factor associated with FAK (GRAF1).
Macropinocytosis
Macropinocytosis is a larger-scale form of endocytosis that typically involves regions/protrusions of the plasma membrane that are highly folded, which subsequently fuse with each other or with the plasma membrane. Membrane ruffles are a morphological characteristic of macropinocytosis.
Macropinocytosis depends on actin polymerization, Rac1 protein, and p21-activated kinase 1 (PAK1). PAK1 is a key regulatory factor as it interacts with Rac1, which activates phosphatidylinositol-3-kinase (PI3K), Ras, Src, and Hsp90 to promote macropinocytosis. Macropinocytosis is also cholesterol-dependent, which is necessary for recruiting Rac1. These components ultimately lead to an endocytosis with a larger uptake area than CME and caveolin-mediated endocytosis.
Endocytic Characteristics of ADC Target Antigens
CD33
CD33 is a 67 kDa transmembrane glycoprotein receptor typically expressed on normal myeloid cells and is a target for GO due to its preferential overexpression on AML cells. The intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) of CD33 regulates its endocytosis, which can be activated through CME. Regarding endocytic efficiency, there is no correlation between the expression level of CD33 in AML cells and its endocytic rate. CD33 is a slowly internalizing antigen, and additionally, crosslinking CD33 does not improve endocytosis. Non-responders to GO in AML patients may be related to dysfunctional endocytosis of the CD33 receptor.
CD30
CD30 is a 120 kDa transmembrane glycoprotein that belongs to the tumor necrosis factor receptor (TNFR) superfamily. Its extracellular portion consists of six extended conformations of cysteine-rich domains (CRD). CD30 is expressed on activated T cells and B cells, as well as various lymphomas (including Hodgkin lymphoma and ALCL).
CD30 does not exhibit endocytosis; rather, it sheds due to proteolytic cleavage, which is mediated by matrix metalloproteinases (MMPs). Shedding is a characteristic of CD30 biology, and high concentrations of circulating soluble CD30 can serve as a serum marker for monitoring tumor progression. Regarding the efficacy of ADCs, elevated circulating levels of CD30 seem to sequester injected ADCs, thereby reducing the number that can localize to CD30-positive tumor sites. Thus, the lack of endocytosis indicates that CD30 is not an ideal ADC target.
CD22
CD22 is a 140 kDa transmembrane glycoprotein, and like CD33, it is a member of the Siglec family and shares various structural features with that family. The key difference is that CD22 is much larger than CD33, as it has multiple Ig domains and ITIM/ITIM-like motifs. CD22 expression is limited to B cells, with elevated levels in most pro-B cells of various B cell malignancies (including ALL).
CD22 undergoes endocytosis via CME. Natural-like ligands rapidly internalize through the structural features of CD22, accumulating intracellularly. These ligands are classified and degraded in lysosomes, while CD22 recycles back to the cell surface. Furthermore, CD22 ligand-induced endocytosis activates intracellular pools, replenishing or increasing the surface expression levels of CD22. Therefore, CD22 has good endocytic characteristics for ADCs.
CD79b
CD79b is expressed only in immature and mature B cells and is overexpressed in ≥80% of malignant B cells. CD79a and CD79b are two non-covalently associated transmembrane proteins that mediate signaling and endocytosis. For the latter, the CD79a-CD79b heterodimer is the scaffold that controls BCR endocytosis. BCR endocytosis is primarily accomplished by CME and mediated by AP-2. Interestingly, CD79a directly interacts with the μ subunit of AP-2, which subsequently activates CD79b and leads to the endocytosis of the entire BCR complex.
Additionally, for ADCs, CD79a can be internalized as a monomer, but CD79b cannot. If the proximal membrane tyrosine (Y195) of CD79b is mutated, the binding of AP-2 to CD79a is blocked, and endocytosis is also inhibited. In 18% of activated B cell-like DLBCL specimens, Y195 is mutated. Overall, there is evidence that the endocytic activity of CD79b depends on the internalization of the entire BCR complex rather than as a monomer.
TROP-2
TROP-2 is a 46 kDa monomeric glycoprotein with selective overexpression, structural endocytosis, and lysosomal targeting characteristics, making it a very attractive target for ADCs. The internalization mechanism of TROP-2 is related to CME.
The observed robust endocytosis of TROP-2 may potentially be explained by significant TROP-2 clustering. Studies on the conformational dynamics of TROP-2 reveal that TROP-2 forms natural homodimers through interaction segments composed of amino acids “VVVVV” located in the transmembrane domain. The dimerization of TROP-2 can further recruit TROP-2 monomers to close proximity through other cell surface proteins. Therefore, TROP-2 clusters are likely formed by multiple dimers connected by lipid rafts and other membrane-associated proteins.
TROP-2 binds to various ligands, such as claudin-1, claudin-7, cyclin D1, and IGF1; however, none of these ligands have been shown to be internalized when binding or interacting with TROP-2. Thus, the endocytic activity of TROP-2 is more pronounced in tumor cells compared to normal cells, indicating that TROP-2 is a good target for ADCs.
BCMA
BCMA or CD269, also known as TNFR superfamily member 17, transmits signals that induce B cell survival and proliferation. BCMA has a molecular weight of only 20.2 kDa, with its extracellular region having a “chair-like” conformation composed of six CRDs. BCMA is expressed in many hematological malignancies, including multiple myeloma, Hodgkin lymphoma, and non-Hodgkin lymphoma.
However, little is known about the precise endocytic pathways utilized by BCMA. Regarding endocytosis, sialylation is a regulatory function that may induce BCMA to utilize CME for endocytosis.
HER2
HER2 is a 185 kDa transmembrane glycoprotein belonging to the EGFR family. Amplification of the HER2/neu gene is a known driver of human malignancies and metastasis. Due to the role of HER2 in cancer, it has been targeted for therapy for decades. HER2 has also been a target for ADCs, with T-DM1 and T-DXT approved for HER2-positive metastatic breast cancer patients.
HER2 endocytosis involves multiple mechanisms, primarily CME, as co-immunoprecipitation clearly shows that HER2 directly interacts with AP-2. Moreover, dynasore can completely block HER2 endocytosis in SKBR3 cells; secondly, the caveolin-binding motif φxφxxxxφ (where φ represents aromatic amino acids Trp, Phe, or Tyr) is commonly found in caveolin-associated proteins. Interestingly, the sequence WSYGVTIW has been identified in the intracellular kinase domain of HER2; additionally, studies have shown that HER2 can utilize the CLIC/GEEC endocytic pathway.
These different findings reveal important characteristics of HER2 endocytosis. Firstly, the endocytosis of HER2 is heterogeneous, and secondly, caveolin-mediated endocytosis seems to be more frequently utilized.
Nectin-4
Nectin-4 is a 66 kDa type I transmembrane protein primarily involved in promoting cell-cell contact. Nectin-4 is an attractive ADC target as studies have shown it is overexpressed in several tumor types but is nearly absent in normal adult tissues.
Currently, there is no information on the endocytosis of natural ligands or mAb/ADC complexes with nectin-4; however, research on nectin-4 binding to pathogens for endocytosis can provide insights. Nectin-4 is also a receptor for the measles virus, and studies have shown that the measles virus enters MCF7, HTB-20 breast cancer, and DLD-1 colorectal cancer cells via macropinocytosis. Virus entry requires PAK1, while the dynamin inhibitor dynasore does not affect virus entry. Additionally, cells expressing dominant-negative caveolin do not eliminate viral endocytosis.
Based on these indirect studies, nectin-4 exhibits strong endocytic activity required by viral receptors.
Conclusion
Determining the drug dose-exposure-effect relationship is a key part of ADC success, and endocytosis is a critical part of this relationship. Therefore, optimizing dosing regimens to maximize the therapeutic index is very important. However, despite the current booming ADC field, we still know very little about the endocytosis of target receptors. Moreover, many core components and key effectors of endocytosis are very important, but these proteins may undergo widespread mutations in cancer, which can also affect ADC endocytosis and efficacy. It is believed that with the development of this field and the accumulation of countless experiences, ADCs will eventually usher in a true spring.
References:
1. Impact of Endocytosis Mechanisms for the Receptors Targeted by the Currently Approved Antibody-Drug Conjugates (ADCs)—A Necessity for Future ADC Research and Development. Pharmaceuticals (Basel). 2021 Jul; 14(7): 674.
Source: Xiaoyao Shuo Yao 2024-03-27