Receptor Crosslinking in Drug Delivery: Detour to the Lysosome?
Authors: Manfred Ogris, Haider Sami
Journal: Molecular Therapy (2015)

A universal method for delivery of bioactive molecules to lysosomes is highly desirable for a broad range of clinical applications. For instance, lysosomal targeting is important for delivery of ligand–drug conjugates and therapeutic strategies that involve degradation of growth-inducing oncogenic receptors. In this issue of Molecular Therapy, Moody and colleagues report a simple and generic method to achieve crosslinking of cell-surface receptors, which triggers internalization and subsequent redirected trafficking of the ligand–cargo complex to lysosomes.1Moody PR Sayers EJ Magnusson JP Alexander C Borri P Watson P et al.Receptor crosslinking: a general method to trigger internalization and lysosomal targeting of therapeutic receptor:ligand complexes.Mol Ther. 2015; 23: 1888-1898Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar Crosslinking was achieved by employing biotinylated ligands that were caused to form complexes using (multivalent) streptavidin after receptor binding. The authors show that this method is applicable to otherwise noninternalized ligand–receptor complexes, such as trastuzumab, the antibody specific to Her2 (used for therapy of Her2-positive breast cancer). Similarly, the method was also able to target transferrin (Tf) to lysosomes. Although the method has only limited in vivo applications, the results should spur the development of alternative crosslinking methods and agents that could facilitate the development of more efficient strategies of macromolecular and/or targeted drug delivery, immunotherapy of cancer, and receptor ablation therapy. Receptor-targeted delivery of drugs or genes involves linking of a therapeutic of choice to a specific ligand. This can mediate cell internalization of the complex after binding to a cognate ligand receptor on the target cell surface. In case of antibody–drug conjugates, specific activity within the target cell requires not only cell internalization but also subsequent trafficking to lysosomes. Although beneficial when delivering a ligand–drug conjugate, lysosomal targeting is to be avoided when delivering macromolecules such as nucleic acids, peptides, or proteins, which must be released intact into the cytoplasm. Otherwise, the low pH and lysosomal enzymes would easily degrade such payloads. Ligands binding to different receptors are internalized via different intracellular trafficking routes. Tf binds with very high affinity to the Tf receptor (TfR) and is internalized via clathrin-mediated endocytosis. After binding, clathrin directs the TfR:Tf complex into endosomes, followed by acidification through ATP-driven proton pumps. When its bound iron is released, the apo-Tf recycles back to the cell surface.2Daniels TR Delgado T Rodriguez JA Helguera G Penichet ML The transferrin receptor part I: biology and targeting with cytotoxic antibodies for the treatment of cancer.Clin Immunol. 2006; 121: 144-158Crossref PubMed Scopus (476) Google Scholar Tf has been extensively utilized for TfR-targeted delivery of nanoparticulate formulations.3Tortorella S Karagiannis TC Transferrin receptor–mediated endocytosis: a useful target for cancer therapy.J Membr Biol. 2014; 247: 291-307Crossref PubMed Scopus (214) Google Scholar Interestingly, recycling of such Tf-containing particles has not been observed. Instead, they remain in endosomal compartments for at least 48 hours4Spillmann CM Naciri J Algar WR Medintz IL Delehanty JB Multifunctional liquid crystal nanoparticles for intracellular fluorescent imaging and drug delivery.ACS Nano. 2014; 8: 6986-6997Crossref PubMed Scopus (49) Google Scholar or end up in lysosomal compartments.5Cotten M Langle-Rouault F Kirlappos H Wagner E Mechtler K Zenke M et al.Transferrin-polycation–mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels.Proc Natl Acad Sci USA. 1990; 87: 4033-4037Crossref PubMed Scopus (312) Google Scholar,6Ogris M Steinlein P Kursa M Mechtler K Kircheis R Wagner E The size of DNA/transferrin–PEI complexes is an important factor for gene expression in cultured cells.Gene Ther. 1998; 5: 1425-1433Crossref PubMed Scopus (568) Google Scholar Trastuzumab, a monoclonal antibody that interferes with the Her2/neu receptor, is mainly used to treat Her2-positive breast cancers. It is supposed to act via several routes: via the inhibition of the MAPK and PI3K/Akt pathways, by activation of antibody-dependent cellular cytotoxicity via its Fc part, and by triggering internalization of the Her2 receptor and its subsequent degradation.7Vu T Claret FX Trastuzumab: updated mechanisms of action and resistance in breast cancer.Frontiers Oncol. 2012; 2: 62Crossref PubMed Scopus (364) Google Scholar The degradation of oncogenic receptors can be a powerful strategy to interrupt the mitogenic effect of activation of growth factor receptors, which are often overexpressed in cancers, among them EGFR, VEGFR, PDGFR, and FGFR. Of course one should keep in mind that such an approach would only be successful for receptors, which are not constitutively active, i.e., independent of ligand activation. Moody et al. demonstrate enhanced delivery of exogenously administered proteins (targeting different receptors) to lysosomes by a simple and generic approach, irrespective of the natural trafficking route of these receptor–ligand complexes. This was achieved by employing biotinylated antibodies/ligands and inducing complex formation at the plasma membrane of cells by streptavidin. The approach was used to demonstrate lysosomal targeting of three model proteinaceous ligands: Tf, the anti-Her2 antibody trastuzumab, and an anti(MHC I) antibody. Crosslinking biotinylated Tf with streptavidin clearly altered its downstream processing, instead directing it to lysosomes and preventing recycling to the cell surface. Transcytosis is another highly relevant Tf transport pathway, which can be harnessed for drug delivery across the blood–brain barrier. Wiley et al. considered this aspect when designing Tf-decorated gold nanoparticles (NPs) for delivery across the blood–brain barrier8Wiley DT Webster P Gale A Davis ME Transcytosis and brain uptake of transferrin-containing nanoparticles by tuning avidity to transferrin receptor.Proc Natl Acad Sci USA. 2013; 110: 8662-8667Crossref PubMed Scopus (337) Google Scholar; particles containing a high density of ligands were highly avid but became stuck in the endothelial cells. In contrast, NPs with lower Tf density were successfully transcytosed. The lesson learned is that by tuning affinity and in particular avidity, TfR entry pathways can be modulated to meet the cargo's needs, potentially avoiding lysosomal targeting and payload degradation. Moody et al. also found that trastuzumab, which blocks Her2 signaling without being efficiently internalized, could also be biotinylated and crosslinked, leading to rapid internalization and temporary surface depletion and degradation of Her2. The biotin–streptavidin system described by Moody et al. has clear potential in vitro; however, its application for in vivo purposes is limited by potential immunogenic responses. Alternative crosslinking methods may be considered for in vivo applications. As mentioned in the study, the antibody–drug conjugate Kadcycla (trastuzumab–emtansine) was observed to internalize more efficiently when combined with pertuzumab, a trastuzumab-crosslinking antibody.9Hughes JB Rodland MS Hasmann M Madshus IH Stang E Pertuzumab increases 17-AAG–induced degradation of ErbB2, and this effect is further increased by combining pertuzumab with trastuzumab.Pharmaceuticals (Basel). 2012; 5: 674-689Crossref PubMed Scopus (13) Google Scholar It would be of interest for the field of oncology if other receptor-crosslinking agents could ablate oncogenic receptors from the cell surface without triggering mitogenic or other cell-activating signaling. These include multivalent biomolecules, such as antibodies (a naturally occurring example being IgM), synthetic peptides, and other, bi- or multivalent artificial ligands. Such agents might find use for controlled receptor ablation, not only in cancer but also in other diseases, such as chronic inflammation, allergic reactions, or autoimmune disease. Besides using whole antibodies, a minimal version combining the function of a paratope (i.e., the part of the antibody directly interacting with the binding site on the antigen) crosslinked by spacer molecules of varying length (as appropriate for the target) could be considered. Receptor crosslinking has already been described using particles exhibiting multiple receptor-binding ligands per particle. Via this mechanism, even large payloads can be delivered, for example bacteria expressing protein A on their surface, and subsequently decorated with trastuzumab or cetuximab (anti-EGFR1).10Heisig M Frentzen A Bergmann B Galmbacher K Gentschev I Hotz C et al.Specific antibody–receptor interactions trigger InlAB-independent uptake of Listeria monocytogenes into tumor cell lines.BMC Microbiol. 2011; 11: 163Crossref PubMed Scopus (6) Google Scholar This approach triggered internalization via trastuzumab in otherwise nonphagocytic cancer cells. NPs, which are usually decorated with many more than one ligand per particle, can also act as a "natural" crosslinker; for example, CD20-targeted, drug-loaded NPs are efficiently internalized and deliver their payload via the lysosomal route.11Wang T Kievit FM Veiseh O Arami H Stephen ZR Fang C et al.Targeted cell uptake of a noninternalizing antibody through conjugation to iron oxide nanoparticles in primary central nervous system lymphoma.World Neurosurg. 2013; 80: 134-141Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar In contrast, anti-CD20 antibodies (e.g., rituximab) do not internalize well. Within the ErbB receptor family, ErbB1 (epidermal growth factor receptor, EGFR) is the only receptor known to be lysosomally degraded after ligand-triggered internalization, although intracellular processing is affected by the type of bound ligand.12Tomas A Futter CE Eden ER EGF receptor trafficking: consequences for signaling and cancer.Trends Cell Biol. 2014; 24: 26-34Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar Binding of EGF triggers macropinocytosis, membrane ruffling, and downstream signaling,13Roepstorff K Grovdal L Grandal M Lerdrup M van Deurs B Endocytic downregulation of ErbB receptors: mechanisms and relevance in cancer.Histochem Cell Biol. 2008; 129: 563-578Crossref PubMed Scopus (150) Google Scholar whether the ligand is free or bound to a NP.14Mickler FM Mockl L Ruthardt N Ogris M Wagner E Brauchle C Tuning nanoparticle uptake: live-cell imaging reveals two distinct endocytosis mechanisms mediated by natural and artificial EGFR targeting ligand.Nano Lett. 2012; 12: 3417-3423Crossref PubMed Scopus (113) Google Scholar,15Schafer A Pahnke A Schaffert D van Weerden WM de Ridder CM Rodl W et al.Disconnecting the yin and yang relation of epidermal growth factor receptor (EGFR)–mediated delivery: a fully synthetic, EGFR-targeted gene transfer system avoiding receptor activation.Hum Gene Ther. 2011; 22: 1463-1473Crossref PubMed Scopus (59) Google Scholar Recently, we demonstrated that EGF-bound NPs carrying plasmid DNA completely deplete EGFR from the cell surface for at least 4 hours after a single exposure.15Schafer A Pahnke A Schaffert D van Weerden WM de Ridder CM Rodl W et al.Disconnecting the yin and yang relation of epidermal growth factor receptor (EGFR)–mediated delivery: a fully synthetic, EGFR-targeted gene transfer system avoiding receptor activation.Hum Gene Ther. 2011; 22: 1463-1473Crossref PubMed Scopus (59) Google Scholar Although transgene expression was very efficient with EGFR-targeted NPs, signaling downstream of EGFR (phosphorylation of Akt and Erk) was comparable to that observed with free EGF. Our alternative approach was to use a synthetic peptide (GE11) for receptor targeting in place of EGF, which avoided receptor activation but still mediated efficient gene delivery.15Schafer A Pahnke A Schaffert D van Weerden WM de Ridder CM Rodl W et al.Disconnecting the yin and yang relation of epidermal growth factor receptor (EGFR)–mediated delivery: a fully synthetic, EGFR-targeted gene transfer system avoiding receptor activation.Hum Gene Ther. 2011; 22: 1463-1473Crossref PubMed Scopus (59) Google Scholar,16Klutz K Schaffert D Willhauck MJ Grunwald GK Haase R Wunderlich N et al.Epidermal growth factor receptor–targeted (131)I-therapy of liver cancer following systemic delivery of the sodium iodide symporter gene.Mol Ther. 2011; 19: 676-685Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar Internalization was triggered by the constant recycling of EGFR via actin.14Mickler FM Mockl L Ruthardt N Ogris M Wagner E Brauchle C Tuning nanoparticle uptake: live-cell imaging reveals two distinct endocytosis mechanisms mediated by natural and artificial EGFR targeting ligand.Nano Lett. 2012; 12: 3417-3423Crossref PubMed Scopus (113) Google Scholar In this context, the approach used by Moody et al. represents a clever strategy to trigger internalization, leading to long-lasting receptor ablation in the absence of downstream signaling. For example, crosslinking of cetuximab might be useful to deplete the cell surface of EGFR. Cetuximab alone has little effect on receptor ablation when directly compared to free EGF (A. Vetter, M. Ogris, unpublished data). Cetuximab has already been used for the targeted delivery of recombinant adenovirus, suggesting that it can be internalized within the context of a nanoparticulate, multivalent virus.17Morrison J Briggs SS Green NK Thoma C Fisher KD Kehoe S et al.Cetuximab retargeting of adenovirus via the epidermal growth factor receptor for treatment of intraperitoneal ovarian cancer.Hum Gene Ther. 2009; 20: 239-251Crossref PubMed Scopus (38) Google Scholar Viruses are highly evolved natural nanomachines, with optimized uptake, intracellular processing, and activation. In case of some viruses, negative interference with uptake and intracellular processing could allow a direct antiviral treatment. In the case of virus-based gene therapy vectors, redirecting the viruses to uptake routes that are more productive for gene delivery is desirable. Nonenveloped viruses, such as adenovirus or rhinovirus, rely on acidification of the endosome, which triggers a conformational change of certain viral proteins, thereby destabilizing the endosomal membrane leading to cytoplasmic entry. For HIV, an enveloped virus, the opposite is observed: here, preventing acidification by adding lysosomotropic drugs promotes infectivity.18Fredericksen BL Wei BL Yao J Luo T Garcia JV Inhibition of endosomal/lysosomal degradation increases the infectivity of human immunodeficiency virus.J Virol. 2002; 76: 11440-11446Crossref PubMed Scopus (164) Google Scholar In turn, one could speculate that promoting lysosomal targeting, e.g., by HIV crosslinking, could represent an effective antiviral countermeasure. For the gene therapist using viral or nonviral vectors, this story has yet another implication: here, though aiming at avoiding the lysosomal pathway, such receptor crosslinking might be prevented when tuning the avidity of the targeted vectors in a way that still allows successful targeting. Taken together, these findings show that the desired application determines whether receptor crosslinking is beneficial or detrimental (see Table 1). Although advantageous for drug delivery via lysosomes, protein and nucleic acids payloads are at risk of being lysosomally degraded. Also in certain special delivery approaches, such as transcytosis, rather lower binding affinity and avidity will be necessary. For receptor ablation, directed and specific receptor crosslinking could become an important approach in oncology, but also in inflammatory diseases and other pathologies. Moody and colleagues have clearly reminded us of this important aspect in macromolecular drug delivery and regulation of signaling pathways.Table 1Pros and cons of receptor crosslinkingApplicationEffect of crosslinkingRemarkLysosomal delivery+Crosslinking leads to lysosomal accumulationReceptor ablation+Temporally controlled receptor ablationPromoting payload internalization+Utilizing otherwise noninternalizing receptorsGene and protein delivery+/-Promote uptake; countermeasures needed to avoid degradationTranscytosis-Reduce ligand density on nanoparticles, reduce avidity Open table in a new tab