title: Sortase A-mediated site-specific labeling of camelid single-domain antibody-fragments: a versatile strategy for multiple molecular imaging modalities.
abstract: A generic site-specific conjugation method that generates a homogeneous product is of utmost importance in tracer development for molecular imaging and therapy. We explored the protein-ligation capacity of the enzyme Sortase A to label camelid single-domain antibody-fragments, also known as nanobodies. The versatility of the approach was demonstrated by conjugating independently three different imaging probes: the chelating agents CHX-A"-DTPA and NOTA for single-photon emission computed tomography (SPECT) with indium-111 and positron emission tomography (PET) with gallium-68, respectively, and the fluorescent dye Cy5 for fluorescence reflectance imaging (FRI). After a straightforward purification process, homogeneous single-conjugated tracer populations were obtained in high yield (30-50%). The enzymatic conjugation did not affect the affinity of the tracers, nor the radiolabeling efficiency or spectral characteristics. In vivo, the tracers enabled the visualization of human epidermal growth factor receptor 2 (HER2) expressing BT474M1-tumors with high contrast and specificity as soon as 1 h post injection in all three imaging modalities. These data demonstrate Sortase A-mediated conjugation as a valuable strategy for the development of site-specifically labeled camelid single-domain antibody-fragments for use in multiple molecular imaging modalities. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: In vivo Cellular and Molecular Imaging laboratory, Vrije Universiteit Brussel (VUB), 1090, Brussels, Belgium.|Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), 1050, Brussels, Belgium.
authors.forename: Sam
authors.initials: S
authors.lastname: Massa
authors.affiliation: Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), 1050, Brussels, Belgium.
authors.forename: Niravkumar
authors.initials: N
authors.lastname: Vikani
authors.affiliation: Laboratory of Organic Chemistry, Vrije Universiteit Brussel (VUB), 1050, Brussels, Belgium.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Betti
authors.affiliation: Laboratory of Organic Chemistry, Vrije Universiteit Brussel (VUB), 1050, Brussels, Belgium.
authors.forename: Steven
authors.initials: S
authors.lastname: Ballet
authors.affiliation: Structural Biology Brussels, Vrije Universiteit Brussel (VUB), 1050, Brussels, Belgium.|Structural Biology Research Center, VIB, 1050, Brussels, Belgium.
authors.forename: Saskia
authors.initials: S
authors.lastname: Vanderhaegen
authors.affiliation: Structural Biology Brussels, Vrije Universiteit Brussel (VUB), 1050, Brussels, Belgium.|Structural Biology Research Center, VIB, 1050, Brussels, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.affiliation: Infinity-MEDISIP-iMinds Medical IT, Department of Electronics and Information Systems, Ghent University, 9000, Ghent, Belgium.
authors.forename: Benedicte
authors.initials: B
authors.lastname: Descamps
authors.affiliation: Infinity-MEDISIP-iMinds Medical IT, Department of Electronics and Information Systems, Ghent University, 9000, Ghent, Belgium.
authors.forename: Christian
authors.initials: C
authors.lastname: Vanhove
authors.affiliation: Interventional Molecular Imaging Laboratory, Department of Radiology, Leiden University Medical Center, 2333ZA, Leiden, The Netherlands.
authors.forename: Anton
authors.initials: A
authors.lastname: Bunschoten
authors.affiliation: Interventional Molecular Imaging Laboratory, Department of Radiology, Leiden University Medical Center, 2333ZA, Leiden, The Netherlands.
authors.forename: Fijs W B
authors.initials: FW
authors.lastname: van Leeuwen
authors.affiliation: In vivo Cellular and Molecular Imaging laboratory, Vrije Universiteit Brussel (VUB), 1090, Brussels, Belgium.
authors.forename: Sophie
authors.initials: S
authors.lastname: Hernot
authors.affiliation: In vivo Cellular and Molecular Imaging laboratory, Vrije Universiteit Brussel (VUB), 1090, Brussels, Belgium.|Nuclear Medicine Department, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB), 1090, Brussels, Belgium.
authors.forename: Vicky
authors.initials: V
authors.lastname: Caveliers
authors.affiliation: In vivo Cellular and Molecular Imaging laboratory, Vrije Universiteit Brussel (VUB), 1090, Brussels, Belgium.|Nuclear Medicine Department, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB), 1090, Brussels, Belgium.
authors.forename: Tony
authors.initials: T
authors.lastname: Lahoutte
authors.affiliation: Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), 1050, Brussels, Belgium.
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.affiliation: In vivo Cellular and Molecular Imaging laboratory, Vrije Universiteit Brussel (VUB), 1090, Brussels, Belgium. cxavier@vub.ac.be.
authors.forename: Catarina
authors.initials: C
authors.lastname: Xavier
authors.affiliation: In vivo Cellular and Molecular Imaging laboratory, Vrije Universiteit Brussel (VUB), 1090, Brussels, Belgium. ndevoogd@vub.ac.be.|Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), 1050, Brussels, Belgium. ndevoogd@vub.ac.be.
authors.forename: Nick
authors.initials: N
authors.lastname: Devoogdt
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D045744:Cell Line, Tumor; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D064593:Heterografts; D006801:Humans; D051379:Mice; D064847:Multimodal Imaging / Q000379:methods*; D009369:Neoplasms / Q000000981:diagnostic imaging; D061848:Optical Imaging / Q000379:methods; D049268:Positron-Emission Tomography / Q000379:methods; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000276:immunology; D013194:Staining and Labeling / Q000379:methods*; D015899:Tomography, Emission-Computed, Single-Photon / Q000379:methods
chemical_list: D019881:Aminoacyltransferases; D001426:Bacterial Proteins; D003546:Cysteine Endopeptidases; D018719:Erb-b2 Receptor Tyrosine Kinases; D061905:Single-Domain Antibodies; C120542:sortase A; C508053:ERBB2 protein, human
keywords: HER2; Sortase A; chemoenzymatic ligation; multimodal; nanobody; sdAb; site-specific conjugation