Nanobodies are single-domain antigen-binding fragments derived from heavy-chain antibodies that are devoid of light chains and occur naturally in Camelidae. We have shown before that their small size and high affinity and specificity for their target antigen make Nanobodies ideal probes for in vivo tumor imaging. In the present study, we have evaluated the use of Nanobodies as a generic method for imaging the in vivo biodistribution of specific immune cell types, using myeloid cells as an example. METHODS: The cellular specificity of Nanobodies raised against murine bone marrow-derived dendritic cells was verified using flow cytometry on a range of myeloid and nonmyeloid cell types. The Nanobodies were then labeled with (99m)Tc and their biodistribution was analyzed using SPECT. The biodistribution was also assessed by measuring radioactivity in various organs and tissues. To verify whether the observed biodistribution was due to specific targeting through the antigen-binding loops, rather than retention in organs because of effects of the framework regions, we genetically grafted the antigen-binding loops of the Nanobodies onto the framework region of a Nanobody scaffold that by itself showed low background retention in the periphery. The cellular specificity and biodistribution of these grafted Nanobodies were determined as before. RESULTS: Nb-DC2.1, which recognizes a wide range of myeloid cells, targets most strongly to the liver, spleen, and lungs. Nb-DC1.8, which recognizes immature bone marrow-derived dendritic cells in vitro, gives a much smaller signal in the liver and spleen than does Nb-DC2.1 but mainly targets to the lungs and gives a pronounced signal in the skin. Grafting of the antigen-binding loops of Nb-DC1.8 or Nb-DC2.1 to the scaffold of Nb-BCII10 alters the observed biodistribution of the Nanobodies to resemble that of the Nanobody from which the antigen-binding loops have been derived. CONCLUSION: The observed in vivo biodistribution of the Nanobodies reflects the main in vivo locations of the cells recognized by the Nanobodies and is determined by the antigen-binding loops of the Nanobodies. Thus, Nanobodies represent elegant targeting probes for imaging the in vivo biodistribution of specific immune cell types.
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