Sortase Aa??mediated sitea??specific labeling of camelid singlea??domain antibodya??fragments: a versatile strategy for multiple molecular imaging modalities

摘要

A generic sitea??specific conjugation method that generates a homogeneous product is of utmost importance in tracer development for molecular imaging and therapy. We explored the proteina??ligation capacity of the enzyme Sortase A to label camelid singlea??domain antibodya??fragments, also known as nanobodies. The versatility of the approach was demonstrated by conjugating independently three different imaging probes: the chelating agents CHXa??A"a??DTPA and NOTA for singlea??photon emission computed tomography (SPECT) with indiuma??111 and positron emission tomography (PET) with galliuma??68, respectively, and the fluorescent dye Cy5 for fluorescence reflectance imaging (FRI). After a straightforward purification process, homogeneous singlea??conjugated tracer populations were obtained in high yield (30a??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 BT474M1a??tumors with high contrast and specificity as soon as 1a??h post injection in all three imaging modalities. These data demonstrate Sortase Aa??mediated conjugation as a valuable strategy for the development of sitea??specifically labeled camelid singlea??domain antibodya??fragments for use in multiple molecular imaging modalities. Copyright ?? 2016 John Wiley & Sons, Ltd.