Antibodies to cell surface proteins redirect intracellular trafficking pathways

https://doi.org/10.1016/j.yexmp.2011.05.011Get rights and content

Abstract

Antibody-mediated intracellular delivery of therapeutic agents has been considered for treatment of a variety of diseases. These approaches involve targeting cell-surface receptor proteins expressed by tumors or viral proteins expressed on infected cells. We examined the intracellular trafficking of a viral cell-surface-expressed protein, rabies G, with or without binding a specific antibody, ARG1. We found that antibody binding shifts the native intracellular trafficking pathway of rabies G in an Fc-independent manner. Kinetic studies indicate that the ARG1/rabies G complex progressively co-localized with clathrin, early endosomes, late endosomes, and lysosomes after addition to cells. This pathway was different from that taken by rabies G without addition of antibody, which localized with recycling endosomes. Findings were recapitulated using a cellular receptor with a well-defined endogenous recycling pathway. We conclude that antibody binding to cell-surface proteins induces redirection of intracellular trafficking of unbound or ligand bound receptors to a specific degradation pathway. These findings have broad implications for future developments of antibody-based therapeutics.

Highlights

► Comparison of rabies virus glycoprotein internalization alone or bound to antibody. ► Internalization of the transferrin receptor bound to ligand or antibody. ► Bound antibodies mediate localization to degradation endosomes. ► Antibodies can redirect endogenous internalization pathways of receptor proteins.

Introduction

The use of antibodies to target specific cells or cell types has become an increasingly desirable method of treatment for a variety of infections and diseases. Several publications have indicated the benefit to using monoclonal antibodies to specifically deliver drugs, including the targeted delivery of cytokines and siRNAs to tumor cells (Kamizuru et al., 2001, Kaspar et al., 2007, Marecos et al., 1998, Peer et al., 2007, Polson et al., 2007, Trachsel et al., 2007). It has also been shown that glycoprotein-specific antibodies can mediate internalization of viral glycoproteins (Favoreel et al., 1999, Favoreel et al., 2004, Sarmiento et al., 2007, Van de Walle et al., 2001).

Rabies virus infections occur in over 100 countries and territories and are fatal once symptoms develop (WHO, 2007). To prevent development of the disease, treatment of exposed individuals includes administration of the rabies vaccine and human rabies immune globulin, which helps to neutralize the virus. There are several ways that rabies G-specific antibodies have been shown to mediate inhibition of the virus. Neutralizing antibodies can bind to the virion-expressed glycoprotein to either block infection of target cells or to inhibit escape of the virus from endosomal compartments following entry (Dietzschold et al., 1987), an important step in viral uncoating. Antibodies can also bind to rabies G expressed on the surface of infected cells to inhibit cell-to-cell spread (Lodmell and Ewalt, 1987). Virus-specific antibodies have also been exploited to target virus-infected cells and deliver antiviral agents, while sparing uninfected cells, by targeting cell surface-expressed viral proteins (Song et al., 2005, Wen et al., 2007). Using mouse neuroblastoma (MNA) cells that express rabies G on the cell surface to mimic an infected cell state (Wiktor and Koprowski, 1978), we examined the ability of a rabies G specific antibody to induce internalization and localization of the rabies G protein to degradative endosomal compartments as a model of antibody conjugates.

Due to the broad-range of cell surface proteins that have been successfully utilized for antibody-based internalization or delivery, we hypothesized that the internalization pathway seen when an antibody is bound to a cell surface protein may remain the same, regardless of the natural internalization pathway of the membrane protein to which the antibody is specific. In other words, we theorized that there might be a common antibody-mediated internalization pathway that exists when an antibody is bound to a cell surface protein. In order to examine this, we studied the internalization of a surface expressed viral protein, with and without antibody. We also analyzed the internalization of an endogenous cell surface protein, the transferrin receptor, when bound to an antibody (αCD71) and compared this to the well-known, recycling pathway observed when the receptor is bound to its ligand, transferrin.

Section snippets

Cells and cell culture

Mouse neuroblastoma (MNA) cells and HEK293T (ATCC) cells were grown in complete medium (Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin, 1% l-glutamine, and 1% sodium pyruvate) at 37 °C with 10% CO2. For localization experiments, 60–80% confluent cells were transfected using GeneJuice reagent (Novagen) according to the manufacturer's protocol.

Plasmids

The codon-optimized rabies G glycoprotein expression plasmid was generated from the

Initial binding and internalization of the ARG1/rabies G complex

We examined the internalization and fate of a viral cell surface glycoprotein, rabies G, by using a fluorescent rabies-specific antibody, ARG1, which specifically binds to and internalizes in mouse neuroblastoma cells expressing rabies G (MNAG) on the surface (Fig. S1). The endosomal localization of rabies G has not been studied. We therefore performed an in-depth analysis of the internalization and endocytic pathway of ARG1-bound rabies G, and compared this to the endosomal pathway of

Discussion

Studies have shown that the use of antibodies to target cellular proteins can be beneficial for the treatment of various diseases and cancers. The presence of antibodies specific for the β-amyloid peptide on the surface of neuronal cells, which can mediate internalization and degradation of the protein (Tampellini et al., 2007), have been shown to slow cognitive deterioration and reduce plaque burden in mice and Alzheimer's patients (Hock et al., 2003, Masliah et al., 2005, Solomon and Frenkel,

Conflict of interest

The authors express no conflicts of interest for this work.

Acknowledgments

We thank the following individuals at UMass: E. Latz for the XFP-Rab expression plasmids, D. Lambright for the GFP-Rab5a expression plasmid, and the Biomedical Imaging Group, especially Clive Standley and Karl Bellve, for assistance with TIRF image collection and analysis. This work was supported by NIH grants R01AI64349 to RWF and P01AI083215-01 to RWF and EKJ, NIH/NIAID grant U54AI057159 to RWF, and JDRF grant to RWF. Core resources supported by the Diabetes Endocrinology Research Center

References (49)

  • A.G. Polson et al.

    Antibody-drug conjugates targeted to CD79 for the treatment of non-Hodgkin lymphoma

    Blood

    (2007)
  • R.A. Roth et al.

    Regulation of the insulin receptor by a monoclonal anti-receptor antibody. Evidence that receptor down regulation can be independent of insulin action

    J. Biol. Chem.

    (1983)
  • A.L. Schwartz et al.

    Antibody-induced receptor loss. Different fates for asialoglycoproteins and the asialoglycoprotein receptor in HepG2 cells

    J. Biol. Chem.

    (1986)
  • D. Tampellini et al.

    Internalized antibodies to the Abeta domain of APP reduce neuronal Abeta and protect against synaptic alterations

    J. Biol. Chem.

    (2007)
  • G.R. Van de Walle et al.

    Involvement of cellular cytoskeleton components in antibody-induced internalization of viral glycoproteins in pseudorabies virus-infected monocytes

    Virology

    (2001)
  • G.R. Van de Walle et al.

    Antibody-induced internalization of viral glycoproteins in pseudorabies virus-infected monocytes and role of the cytoskeleton: a confocal study

    Vet. Microbiol.

    (2002)
  • D. Axelrod

    Total internal reflection fluorescence microscopy in cell biology

    Traffic

    (2001)
  • B. Chesebro et al.

    Antibody-induced modulation of Friend virus cell surface antigens decreases virus production by persistent erythroleukemia cells: influence of the Rfv-3 gene

    Proc. Natl. Acad. Sci. U. S. A.

    (1979)
  • W.R. Dowdle et al.

    Inhibition of virus release by antibodies to surface antigens of influenza viruses

    J. Virol.

    (1974)
  • H.W. Favoreel et al.

    Antibody-induced endocytosis of viral glycoproteins and major histocompatibility complex class I on pseudorabies virus-infected monocytes

    J. Gen. Virol.

    (1999)
  • H.W. Favoreel et al.

    Copatching and lipid raft association of different viral glycoproteins expressed on the surfaces of pseudorabies virus-infected cells

    J. Virol.

    (2004)
  • I. Gaidarov et al.

    Spatial control of coated-pit dynamics in living cells

    Nat. Cell Biol.

    (1999)
  • C. Harding et al.

    Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes

    J. Cell Biol.

    (1983)
  • C.R. Hopkins et al.

    Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells

    J. Cell Biol.

    (1983)
  • Cited by (29)

    • Early events in rabies virus infection—Attachment, entry, and intracellular trafficking

      2019, Virus Research
      Citation Excerpt :

      The endogenous RABV G protein colocalizes with Rab5a (EEs) and Rab11a (REs) but not with Rab9 (LEs). However, when RABV G binds to a monoclonal specific anti-RABV G antibody, its colocalization shifts to Rab5a (EEs), Rab9a (LEs), and low pH lysosomes (Pierre et al., 2011). This relocalization initiates the degradation pathway, which is different from that of endogenous RABV G. Live cell imaging also shows that after the glycoprotein binds to p75NTR with high affinity, RABV particles hijack p75NTR to undergo clathrin-dependent endocytosis and fuse with p75NTR-positive endosomes for a faster migration route (Deinhardt et al., 2010).

    • Biorecognition: A key to drug-free macromolecular therapeutics

      2019, Biomaterials
      Citation Excerpt :

      This was proven on a number of different cell lines [52 and references therein]. Crosslinking of receptors was induced by different methods, including SA [52,58,59] and bivalent antibodies [54]. In addition, receptor crosslinking may have an impact on transcytosis of cargo via the blood brain barrier (BBB).

    • Receptor Crosslinking: A General Method to Trigger Internalization and Lysosomal Targeting of Therapeutic Receptor:Ligand Complexes

      2015, Molecular Therapy
      Citation Excerpt :

      In order to evaluate delivery of exogenous proteins to lysosomes within the context of ADCs, we sought to exploit the enhanced trafficking to lysosomes that many receptors perform when clustered or crosslinked into “supramultivalent” interactions. This enhanced and sometimes aberrant lysosomal delivery has been observed for many receptors,12 including rabies G protein,13 ErbB family receptors such as epidermal growth factor receptor,14,15 acetylcholine receptors,16,17 and FcRn receptors.18 These findings were demonstrated in a range of cell types, including hamster kidney,12 mouse neuroblastoma,13 human kidney,14 human epidermal,15 rat muscle,16 Xenopus muscle,17 and human endothelial cells.18

    • Rabies virus envelope glycoprotein targets lentiviral vectors to the axonal retrograde pathway in motor neurons

      2014, Journal of Biological Chemistry
      Citation Excerpt :

      This might be related to the ability of the intact virus to block degradation in the cell. A recent study indicated that RV-G bound to an antibody transfected in a neuroblastoma cell line is targeted via early and late endosomes to lysosomes (58). In addition, lentiviral vector transduction of dendritic cells involved endosomal trafficking, and its efficiency was enhanced by suppressing autophagy (59).

    View all citing articles on Scopus
    View full text