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, 27 (5), 1390-1399

New Dioxaborolane Chemistry Enables [(18)F]-Positron-Emitting, Fluorescent [(18)F]-Multimodality Biomolecule Generation From the Solid Phase

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New Dioxaborolane Chemistry Enables [(18)F]-Positron-Emitting, Fluorescent [(18)F]-Multimodality Biomolecule Generation From the Solid Phase

Erik A Rodriguez et al. Bioconjug Chem.

Abstract

New protecting group chemistry is used to greatly simplify imaging probe production. Temperature and organic solvent-sensitive biomolecules are covalently attached to a biotin-bearing dioxaborolane, which facilitates antibody immobilization on a streptavidin-agarose solid-phase support. Treatment with aqueous fluoride triggers fluoride-labeled antibody release from the solid phase, separated from unlabeled antibody, and creates [(18)F]-trifluoroborate-antibody for positron emission tomography and near-infrared fluorescent (PET/NIRF) multimodality imaging. This dioxaborolane-fluoride reaction is bioorthogonal, does not inhibit antigen binding, and increases [(18)F]-specific activity relative to solution-based radiosyntheses. Two applications are investigated: an anti-epithelial cell adhesion molecule (EpCAM) monoclonal antibody (mAb) that labels prostate tumors and Cetuximab, an anti-epidermal growth factor receptor (EGFR) mAb (FDA approved) that labels lung adenocarcinoma tumors. Colocalized, tumor-specific NIRF and PET imaging confirm utility of the new technology. The described chemistry should allow labeling of many commercial systems, diabodies, nanoparticles, and small molecules for dual modality imaging of many diseases.

Figures

Figure 1
Figure 1
Chemical structure of the 18F-PET/NIRF probe. The amide reactive 18F-PET/NIRF precursor, 1, the maleimide precursor, Mal-1, and the monoclonal antibody conjugate, mAb-1 (EpCAM) or Cetuximab-1 as R groups (left panel). A fluoride-labile, mAb-1-bearing, solid support is generated when the N-hydroxy succinimide ester (NHS)/maleimide precursor is reacted with mAb and then exposed to a streptavidin bearing support. mAb that is not covalently attached to 1 cannot be retained by the support and is removed with washing. Treatment of the solid support with aqueous fluoride achieves conversion of 1 into a 18/19F labeled trifluoroborate 2, a species useful for PET/NIRF multimodality imaging and simultaneous release of 18F-PET/NIRF labeled mAb-2 from the solid support. Unreacted mAb-1 remains bound to the support through the biotin handle on the solid-phase support (Scheme S1).
Figure 2
Figure 2
Validation of solid-phase, fluoride triggered mAb-2 elution. (a) Brightfield (i) and fluorescent (ii) images of the solid-phase generator. A spin column loaded with a streptavidin-agarose that binds biotin on mAb-1. (b) SDS-PAGE gel of eluent generated by treating the mAb-1-streptavidin-agarose (a) with aqueous fluoride. (Lane 1) Control with only mAb-1 (no streptavidin-agarose or HF). mAb-1-streptavidin-agarose was treated with 0.40, 1.2, 3.6, or 11 mM fluoride for 1 h (Lanes 2–5, respectively). mAb-2 was removed by centrifugation and analyzed by SDS-PAGE. Untreated mAb-1 (Lane 1) has a similar MW to HF treated samples (Lanes 2–5), and lacks lower MW species. Increasing quantities of mAb-2 are released with higher concentrations of fluoride.
Figure 3
Figure 3
Radiolabeling of [18F]-mAb-2. (a) Radioactive, SEC HPLC of [18F]-mAb-2 generated by solution fluoridation of mAb-1 (1 h, [18F]-hydrogen fluoride, pH 3). No streptavidin-agarose was utilized in this synthesis. (b) Radioactive, SEC HPLC [18F]-mAb-2 fluoride triggered elution from mAb-1-streptavidin-agarose. Note the 13-fold enhancement of specific activity. Elution of [18F]-mAb-2 takes 5–7 min, and is free of [18F]-fluoride ion. (c) Phosphorimaging (i) and Cy7 fluorescence imaging (ii) of fractions verify SEC HPLC data. Fractions 5–7 min contain [18F]-mAb-2 and are both radioactive (i) and fluorescent (ii), indicating the successful synthesis of a dual modality, PET/NIRF imaging mAb-2.
Figure 4
Figure 4
Chemical attachment of mAb to 1, solid-support immobilization, and reaction with [19F]-fluoride does not affect mAb-2 EpCAM-antigen binding. (a) Fluorescence of mAb-2 bound to PC3 cells (30 min incubation) shows membrane localization. (b) (Control 1) Membrane bound mAb-2 (a) was challenged with 100-fold excess of unlabeled mAb for 1 h. Membrane bound fluorescence is diminished, illustrating that antigen binding is required for membrane labeling. Endocytosed mAb-2 fluorescence remains visible inside the cells. (c) (Control 2) mAb-2 was reduced to heavy and light chains with TCEP. Equal fluorescent quantities of the reduced mAb-2 (mAb-2 + TCEP), as in (a), was incubated with PC3 cells (30 min incubation). The lack of fluorescence demonstrates that mAb-2 antigen binding is essential for both membrane and endocytosed fluorescence. mAb-2 + TCEP is not nonspecifically binding and/or endocytosed in PC3 cells. (d) mAbs (used in (a) and (c)) show equivalent Cy7 fluorescence. (e) mAb-2 labeled PC3 cells were incubated for 5 h to promote endocytosis. (f) E2-Crimson fluorescence in the cytoplasm and nuclei of PC3 cells. (g) Overlay of mAb-2 (e) and E2-Crimson (f) fluorescence. (h) DiO (membrane stain) delineating PC3 cell membranes. (i) Overlay of mAb-2 (e) and membrane fluorescence (h), confirming increased incubation time confers greater endocytosis of mAb-2. Scale bar = 50 μm.
Figure 5
Figure 5
mAb-2 fluorescence imaging in mice bearing PC3-DsRed2 tumor xenografts, which target both primary and metastatic tumors. (a) Brightfield image of mouse with primary PC3-DsRed2 tumor and stomach metastases (i) without skin and (ii) removed organs. (b) DsRed2 fluorescence of PC3-DsRed2 tumors in mice (i) 6 and (ii) 48 h post mAb-2 injection, and (iii) ex vivo imaging of organs (48 h). Note in (iii), DsRed2 fluorescence was overexposed to visualize stomach metastases. (c) mAb-2 (600 pmol) fluorescence imaging at (i) 6 and (ii) 48 h post injection, and (iii) ex vivo imaging of organs (48 h). DsRed2 and mAb-2 fluorescence colocalize in the primary and metastatic tumors. DsRed2 and Cy7 fluorescence is not visible in the stomach of mice without PC3-DsRed2 tumors or mAb-2 (see control organ in bottom left (b,iii) and (c,iii)).
Figure 6
Figure 6
[18F]-Cetuximab-2 imaging in mice bearing orthotopic A549 tumor xenografts expressing luciferase and GFP. (a) Bioluminescent imaging of A549 orthotopic lung tumor xenograft shows primary tumor above the lung. (b,c) Axial, coronal, and sagittal [18F]-Cetuximab-2 CT (b) and PET (c) cross-sectional images of the primary tumor (green circle) 4.5 h after i.v. injection. (d) Excised organs following PET imaging. Scale bar = 1 cm. (e) Fluorescent imaging of primary and lung metastatic tumors expressing GFP. (f) NIRF imaging of Cy7 labeled Cetuximab-2, verifying colocalization of GFP and Cetuximab-2 to primary and lung metastatic tumors. (g) Scintillated biodistribution (obtained after ex vivo imaging) at 2.5 (n = 3) and 6 h (n = 4). Error bars = SEM (h) Secondary Ab-phycoerythrin labeling of Cetuximab-1 and Cetuximab-2. Flow cytometry demonstrates that Cetuximab-1 treated with [18F]-fluoride binds EGFR expressed on A549 cells. (i,j) Fluorescent imaging of A549 cells expressing GFP in tissue slices from (e) showing a heterogeneous GFP expression (i) and metastatic invasion into the lung (j). GFP is green, DAPI (nuclear stain) is blue, and scale bar = 400 μm.

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