Receptor-mediated Uptake Mechanism
The target-dependent uptake and toxicity of ADC can also be mediated by different receptors that recognize the Fc (fragment crystallization) region of the IgG part of ADC. The constant domain of IgG is highly conserved in structure, allowing it to interact with other components of the immune system through Fc receptors, thus initiating the effector immune function. Although the Fc-mediated effect function usually does not need to achieve the efficacy of ADC, the recognition and binding of Fc receptors to ADC antibody (IgG) components can mediate non-target internalization of normal cells.
Fc γ receptors (FC γ Rs): FC γ Rs play a key role in connecting cellular and humoral immune responses through antibody-mediated effector functions such as antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), phagocytic cytokines (IFN- γ and TNF- α) and other IgG immune complexes and the release of cytokines. These effects play an important role in regulating the efficacy of several therapeutic IgG antibodies. The effect function mediated by Fc γ R is very important for target-related efficacy, but it may contribute to off-target uptake and toxicity in normal cells.
Fc γ R-mediated IgG/ADC internalization: Fc γ Rs is not only an important molecule mediating the antibody effect of IgG, but also one of the most characteristic receptors on the surface of endocytosis cells, which plays a role in internalizing/scavenging IgG regulatory antigens in systemic circulation. The binding of Fc γ Rs to Fc region induces the aggregation/cross-linking of IgGs on cells and initiates downstream signal events, which leads to the phosphorylation and activation of PI3K, p70S6K and Akt kinases. These are directly involved in the recombination of actin cytoskeleton and the membrane remodeling formed by artificial feet and phagosomes. A similar mechanism may be applicable to Fc γ R-mediated ADC internalization, which contributes to the target-independent toxicity of normal cells.
ADC toxicity in normal cells is related to ADC uptake mediated by Fc γ Rs. Although the expression pattern of Fc γ Rs in normal healthy cells/tissues corresponds to several reported ADC target independent toxicity, the role of Fc γ Rs in mediating ADC miss toxicity is mainly considered to be the potential mechanism of hematotoxicity (hematotoxicity). Hematotoxicity is the most common off-target toxicity of ADCs containing MAME, MMAF, calcitamicin, and DM-1. In clinical studies, T-DM1-induced thrombocytopenia is a kind of DLT.
Neonatal Fc receptor (FcRn): FcRn is a member of MHCI glycoprotein, which binds to the FC domain and plays a key role. It is characterized by a long half-life (about 21 days). Different from other Fc receptors, FcRn interacts with ligands in a pH-dependent manner and binds with high affinity under slightly acidic pH (about 6.5). This pH dependence is considered to be the key to the mechanism by which FcRn prolongs the half-life of IgG/ADC.
FcRn binding and its potential role in ADC toxicity: ADC binds FcRn in the early acidic phase, mainly through endocytosis in the nonspecific fluid phase. Then, in the extracellular space or somatic circulation of neutral pH, the FcRn-ADC complex is divided into cyclic nuclear endosome transfer away from lysosome degradation and ADC release from the cell surface. In each endocytosis cycle, only ADC that is not bound to FcRn is transported to lysosomes for catabolism and payload release.
From a safety point of view, FcRn binding is also important for reducing the accumulation and catabolism of ADC in normal cells to release cytotoxic payloads. Therefore, in normal cells with significant expression of FcRn, the modification of FcRn binding may be a useful way to overcome adverse toxicity/adverse events.
C-type lectin receptor (CLR): CLR is a large, highly conservative, and well-characterized endocytosis receptor family. Type I CLR is calcium-dependent and has multiple (6–8) carbohydrate recognition domains (CRDs), as well as cysteine- and fibronectin-rich domains. Its members include macrophage mannose receptors (MR, MRC1, CD206), Endo180 (CD280, MRC2, uPAR-related proteins, uPARAP), DEC-205, PLA2R, and DCL-1. Type II CLRs contain a CRD that can be calcium dependent on Dectin 2, Mincle, CLECSF8, DCIR, DCAR, BDCA-2, DC-SIGN, MGL, and calcium independent of Dectin 1, CLEC5A, and DNGR-1 (CLEC9A).
The toxicity of ADC is related to CLR binding. Although there is no clear evidence that CLR plays a direct role in the off-target toxicity of ADC, mannose receptor (MR)-mediated uptake is considered to be the potential mechanism of ADC hepatotoxicity. LSECs are highly specialized endothelial cells. Compared with other hepatocytes, the level of lysosomal enzyme activity in LSECs is significantly increased, which may further lead to the high degradation of endocytosis ligands (including ADC) and release the degraded substance/cytotoxic payload to the surrounding chamber.
LSECs rely on MR-mediated uptake of lysosomal enzymes (glycoproteins) to maintain their high degradation ability. After the release of ligands in the early endosomes, the rapid internalization of MR-ligand complexes and the rapid circulation of MRs back to the cell surface may further help MR to express the high continuous endocytosis of cells. MR-mediated uptake is also an important mechanism for the clearance of endogenous and therapeutic glycoproteins and immunoglobulins in LSECs.
In addition, due to the existence of macropores (around 50–150nm) surrounded by microtubule (actin) filaments, without a diaphragm or basement membrane, LSECs are the most permeable endothelial cell type in vivo. Therefore, the diffusion of macromolecules (including ADC) into the scavenger receptors of LSECs through the membrane pores may also lead to hepatotoxicity.
It is worth noting that Kupffer cells also express MR and play an important role in the non-specific uptake and processing of ADC. Therefore, it can not be ruled out that MR-mediated ADC uptake and cytotoxic payload release from Kupffer cells will result in hepatotoxicity.