Intracellular Dynamics and Fate of a Humanized Anti–Interleukin-6 Receptor Monoclonal Antibody, Tocilizumab

Abstract

Tocilizumab (TCZ), a humanized anti-interleukin-6 (IL-6) receptor (IL-6R) monoclonal antibody, abrogates signal transducer protein gp130-mediated IL-6 signaling by competitively inhibiting the binding of IL-6 to the receptor, and shows clinical efficacy in autoimmune and inflammatory diseases. Despite accumulating evidence for therapeutic efficacy, the behavior and fate of TCZ at the cellular level remain largely unknown. To address this, we evaluated the endocytosis and intracellular trafficking of IL-6R in HeLa cells. The results of our study provide evidence that IL-6R is constitutively internalized from the cell surface by ligand or TCZ binding and the expression of gp130 in an independent manner and is targeted via endosomes without being significantly directed to the recycling pathway to, and degraded in, lysosomes. Furthermore, the cytoplasmic tail of IL-6R is required for constitutive endocytosis of the receptor, which is mediated by the clathrin and AP-2 complex. We further demonstrate that FcRn, whose function is to regulate the serum persistence of IgG, is confined primarily to early/recycling endosomes and rapidly transits between these compartments and late endosomes/ lysosomes without being degraded. Importantly, the expression of FcRn induces the segregation of TCZ from IL-6R, resulting in extensive colocalization of TCZ and FcRn in IL-6R-depleted endosomal compartments. Collectively, our results suggest that FcRn can accelerate the retrieval of the internalized TCZ, not only from endosomes but also from lysosomes. Our findings provide new insight into the mechanism by which the antibody internalized into cells is rescued from lysosomal degradation and into how its serum levels are maintained.

SorLA in Interleukin-6 Signaling and Turnover

“…In contrast, endocytosis, the subcellular localization, the turnover (half-life), and the shedding of the IL-6R proved unaffected by coexpression with differentially sorted SorLA constructs. Thus, in agreement with previous reports addressing IL-6R sorting and endocytosis (54, 55, 58), we find that IL-6R trafficking relies exclusively on sorting motifs within its own cytoplasmic domain. Likewise, SorLA trafficking was unaffected by IL-6R and, in a broader perspective, it is tempting to question the very concept that a receptor-A can “take over” the sorting of a receptor B (and why not vice versa?)…”

CYTL1 inhibits tumor metastasis with decreasing STAT3 phosphorylation

“…IL-6 exerts its function by binding IL-6Rα (IL-6 receptor-α) and activates downstream signals through gp130 (glycoprotein 130 or IL-6Rβ). 32,33 CYTL1 likely impacts the binding of ligands and receptors or downstream signal transduction, thus inhibiting STAT3. However, STAT3 also has some negative regulation mechanisms.…”

Transmembrane TNF-dependent uptake of anti-TNF antibodies

“…However, if the antibody molecule fails to interact with FcRn in a productive manner, the antibody molecule follows the degradation pathway in the lysosomes 43 . In the case of antibodies that are internalized upon binding to their cell surface targets, the fate of the antibody molecule is determined by the biology of the target receptor and the nature of antibody/target interaction 44 . For example, certain receptors re-cycle efficiently and, if the antibody remains bound to the target at low pH in the endosomes, it may re-cycle back to the surface with the receptor 45 .…”