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Comparative binding, endocytosis, and biodistribution of antibodies and antibody-coated carriers for targeted delivery of lysosomal enzymes to ICAM-1 versus transferrin receptor

  • Original Article
  • Published:
Journal of Inherited Metabolic Disease

Abstract

Targeting lysosomal enzymes to receptors involved in transport into and across cells holds promise to enhance peripheral and brain delivery of enzyme replacement therapies (ERTs) for lysosomal storage disorders. Receptors being explored include those associated with clathrin-mediated pathways, yet other pathways seem also viable. Well characterized examples are that of transferrin receptor (TfR) and intercellular adhesion molecule 1 (ICAM-1), involved in iron transport and leukocyte extravasation, respectively. TfR and ICAM-1 support ERT delivery via clathrin- vs. cell adhesion molecule-mediated mechanisms, displaying different valency and size restrictions. To comparatively assess this, we used antibodies vs. larger multivalent antibody-coated carriers and evaluated TfR vs. ICAM-1 binding and endocytosis in endothelial cells, as well as in vivo biodistribution and delivery of a model lysosomal enzyme required in peripheral organs and brain: acid sphingomyelinase (ASM), deficient in types A-B Niemann Pick disease. We found similar binding of antibodies to both receptors under control conditions, with enhanced binding to activated endothelium for ICAM-1, yet only anti-TfR induced endocytosis efficiently. Contrarily, antibody-coated carriers showed enhanced binding, engulfment, and endocytosis for ICAM-1. In mice, anti-TfR enhanced brain targeting over anti-ICAM, with an opposite outcome in the lungs, while carriers enhanced ICAM-1 targeting over TfR in both organs. Both targeted carriers enhanced ASM delivery to the brain and lungs vs. free ASM, with greater enhancement for anti-ICAM carriers. Therefore, targeting TfR or ICAM-1 improves lysosomal enzyme delivery. Yet, TfR targeting may be more efficient for smaller conjugates or fusion proteins, while ICAM-1 targeting seems superior for multivalent carrier formulations.

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Acknowledgments

This study was funded by grants from the American Heart Association (09BGIA2450014) and National Institutes of Health (R01 HL098416) to SM.

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Authors and Affiliations

  1. Fischell Department of Bioengineering, School of Engineering, University of Maryland College Park, College Park, MD, 20742, USA

    Jason Papademetriou & Silvia Muro

  2. Department of Normal and Pathological Cytology and Histology, School of Medicine, University of Seville, Seville, 41009, Spain

    Carmen Garnacho

  3. Department of Cell Biology & Molecular Genetics and Biological Sciences Graduate Program, University of Maryland, College Park, MD, 20742, USA

    Daniel Serrano

  4. Institute for Bioscience and Biotechnology Research, University of Maryland, 5115 Plant Sciences Building, College Park, MD, 20742-4450, USA

    Tridib Bhowmick & Silvia Muro

  5. Department of Human Genetics, Mount Sinai School of Medicine, New York, NY, 10029, USA

    Edward H. Schuchman

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  1. Jason Papademetriou
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Correspondence to Silvia Muro.

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Communicated by: Frits Wijburg

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Supplementary Fig. 1

Fluorescence microscopy of antibody binding to ICAM-1 vs. TfR on endothelial cells. Control or TNFα-activated HUVECs were incubated with anti-ICAM or anti-TfR for 15 min at 37 °C. Cells were washed to remove unbound antibody, fixed, and stained with FITC-conjugated goat anti-mouse IgG. Phase-contrast images were used to delimit cell borders (dashed lines). Scale bar = 10 μm. (JPEG 43 kb)

High resolution image (TIFF 16563 kb)

Supplementary Fig. 2

Fluorescence microscopy of carrier binding to ICAM-1 vs. TfR on endothelial cells. Binding of ∼250 nm FITC-labeled anti-ICAM vs. anti-TfR carriers to control or TNFα-activated HUVECs after 1 h incubation. Cells were subsequently washed to remove unbound carriers. Phase-contrast images were used to delimit cell borders (dashed lines). Scale bar = 10 μm. (JPEG 36 kb)

High resolution image (TIFF 16569 kb)

Supplementary Fig. 3

Fluorescence microscopy of endocytosis of antibodies targeted to endothelial ICAM-1 vs. TfR. Uptake of anti-ICAM vs. anti-TfR by TNFα-activated HUVECs, assessed after 1 h incubation at 37 °C. Unbound antibodies were washed off and surface-bound antibodies were stained with a Texas-Red secondary IgG. Cells were then permeabilized and antibodies located both at the surface + internalized were stained with FITC-labeled secondary IgG. Internalized antibodies appear as FITC single-labeled in green, while surface-bound antibodies display FITC + Texas-Red double-labeled yellow color. Phase-contrast images were used to delimit cell borders (dashed lines). Scale bar = 10 μm. (JPEG 23 kb)

High resolution image (TIFF 7750 kb)

Supplementary Fig. 4

Fluorescence microscopy of endocytosis of carriers targeted to endothelial ICAM-1 vs. TfR. Uptake of ∼250 nm FITC-labeled anti-ICAM vs. anti-TfR carriers, assessed after 1 h incubation at 37 °C with control or TNFα-activated HUVECs. Unbound carriers were removed and surface-bound carriers were stained with a Texas-Red secondary IgG. Internalized carriers appear as FITC single-labeled in green, while surface-bound carriers display FITC + Texas-Red double-labeled yellow color. Phase-contrast images were used to delimit cell borders (dashed lines). Scale bar = 10 μm. (JPEG 53 kb)

High resolution image (TIFF 16565 kb)

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Papademetriou, J., Garnacho, C., Serrano, D. et al. Comparative binding, endocytosis, and biodistribution of antibodies and antibody-coated carriers for targeted delivery of lysosomal enzymes to ICAM-1 versus transferrin receptor. J Inherit Metab Dis 36, 467–477 (2013). https://doi.org/10.1007/s10545-012-9534-6

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