A mobile endocytic network connects clathrin\independent receptor endocytosis to recycling and promotes T cell activation

A mobile endocytic network connects clathrin\independent receptor endocytosis to recycling and promotes T cell activation. Rabbit Polyclonal to PLA2G4C OKT3 (1 ng mL?1, 10 ng mL?1, 100 ng mL1) in the presence of dynasore (40 M, grey bars) or DMSO (white bars). Each bar represents a single determination, using blood from one donor. BPH-177-2696-s001.pdf (739K) GUID:?72DE6EE6-4735-45B0-B5DF-BEA00D27C169 Abstract Background and Purpose Antibodies targeting cell surface receptors are considered Butein to enable highly selective therapeutic interventions for immune disorders and cancer. Their Butein biological profiles are found, generally, to represent the net effects of antibodyCtarget interactions. The former therapeutic anti\integrin L2 antibody efalizumab seems to defeat this paradigm by eliciting, via mechanisms currently unknown, much broader effects than would be predicted based on its target specificity. Experimental Approach To elucidate the mechanisms behind these broad effects, we investigated in primary human lymphocytes in vitro the effects of anti\L2 antibodies around the expression of L2 as well as unrelated 4 integrins, in comparison to Fab fragments and small\molecule inhibitors. Important Results We demonstrate that anti\L2 mAbs directly induce the internalization of 4 integrins. The endocytotic phenomenon is a direct result of their antibody nature. Butein It is inhibited when monovalent Fab fragments or small\molecule inhibitors are used. It is impartial of crosslinking via anti\Fc mAbs and of L2 activation. The cross\modulatory effect is usually unidirectional and not observed in a similar fashion with the 4 integrin antibody natalizumab. Conclusion and Implications The present study identifies endocytotic cross\modulation as a hitherto unknown non\canonical functionality of anti\L2 antibodies. This cross\modulation has the potential to fundamentally alter an antibody’s benefit risk profile, as obvious with efalizumab. The newly explained phenomenon may be of relevance to other therapeutic antibodies targeting cluster\forming receptors. Thus, pharmacologists should be cognizant of this action when investigating such antibodies. Abstract AbbreviationsAPCallophycocyaninCDcluster of differentiationCy7cyanine\7Fabantigen\binding fragmentFcfragment crystallizableFITCfluorescein isothiocyanateICAM\1intercellular adhesion molecule\1LFA\1leukocyte\function associated antigen\1mAbmonoclonal antibodyPBMCperipheral blood mononuclear cellPEphycoerythrinPerCPperidinin\chlorophyll\proteinPMAphorbol 12\myristate 13\acetate What is already known Efalizumab unexpectedly reduces the expression of major immune receptors on patients circulating T cells. The mechanism/s are unknown; altered T\cell trafficking remains a potential explanation. What this study adds This study clarifies the mechanism by which anti\L2 mAbs, including efalizumab, directly down\regulate 4 Butein integrins. The study explains endocytotic cross\modulation as a novel non\canonical antibody functionality. What is the clinical significance Endocytotic cross\modulation has the potential to fundamentally alter the effect profiles of therapeutic antibodies. Pharmacologists should be aware of this when developing therapeutic antibodies targeting cluster\forming receptors. 1.?INTRODUCTION Antibodies targeting cell surface receptors are generally considered to enable highly selective therapeutic interventions. Their biological profiles are expected and generally found to represent the net effect of antibody binding to the target via their antigen\binding fragment (Fab) regions, resulting in target Butein inhibition or activation. Additional functionalities of therapeutic antibodies may derive from their interaction with the immune system through their fragment crystallizable (Fc) portion. The interaction of the Fc part with complement triggers match\dependent cytotoxicity. Further, by binding of the Fc region to Fc receptors, antibodies can induce antibody\dependent cell\mediated cytotoxicity, phagocytosis and Fc receptor\mediated trogocytosis. The recruitment of these effectors is dependent around the antibody’s isotype and its ability to interact with match or effector cells (Chames, Van Regenmortel, Weiss, & Baty, 2009; Smith, 2015; Taylor et al., 2015). Against this background, an antibody whose effect profile does not.