bc500499h_si_002.pdf (1.44 MB)
Chemoenzymatic Strategy for the Synthesis of Site-Specifically Labeled Immunoconjugates for Multimodal PET and Optical Imaging
journal contribution
posted on 2015-12-17, 06:25 authored by Brian M. Zeglis, Charles B. Davis, Dalya Abdel-Atti, Sean D. Carlin, Aimei Chen, Robert Aggeler, Brian J. Agnew, Jason S. LewisThe complementary nature of positron
emission tomography (PET) and optical imaging (OI) has fueled increasing
interest in the development of multimodal PET/OI probes that can be
employed during the diagnosis, staging, and surgical treatment of
cancer. Due to their high selectivity and affinity, antibodies have
emerged as promising platforms for the development of hybrid PET/OI
agents. However, the lack of specificity of many bioconjugation reactions
can threaten immunoreactivity and lead to poorly defined constructs.
To circumvent this issue, we have developed a chemoenzymatic strategy
for the construction of multimodal PET/OI immunoconjugates that have
been site-specifically labeled on the heavy chain glycans. The methodology
consists of four steps: (1) the enzymatic removal of the terminal
galactose residues on the heavy chain glycans; (2) the enzymatic incorporation
of azide-bearing galactose (GalNAz) residues into the heavy chain
glycans; (3) the strain-promoted click conjugation of chelator- and
fluorophore-modified dibenzocyclooctynes to the azide-modified sugars;
and (4) the radiolabeling of the immunoconjugate. For proof-of-concept,
a model system was created using the colorectal cancer-targeting antibody
huA33, the chelator desferrioxamine (DFO), the positron-emitting radiometal 89Zr, and the near-infrared fluorescent dye Alexa Fluor 680.
The bioconjugation strategy is robust and reproducible, reliably producing
well-defined and immunoreactive conjugates labeled with 89Zr, Alexa Fluor 680, or an easily and precisely tuned mixture of
the two reporters. In in vivo PET and fluorescence
imaging experiments, a hybrid 89Zr- and Alexa Fluor 680-labeled
huA33 conjugate displayed high levels of specific uptake (>45%
ID/g) in athymic nude mice bearing A33 antigen-expressing SW1222 colorectal
cancer xenografts.