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A Dual-Modality Linker Enables Site-Specific Conjugation of Antibody Fragments for 18F-Immuno-PET and Fluorescence Imaging.

ABSTRACT: Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue-bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with 18F-labeled trans-cyclooctene (18F-TCO). To generate a dual-modality antibody fragment-based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with 18F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a DML for conjugation of a fluorescent dye and 18F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with 18F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after injection. Rapid renal clearance of the 50-kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. Conclusion: The DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level biodistribution and fluorescence image-guided identification of tumor margins during surgery.

SUBMITTER: Zettlitz KA

PROVIDER: S-EPMC6785789 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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A Dual-Modality Linker Enables Site-Specific Conjugation of Antibody Fragments for <sup>18</sup>F-Immuno-PET and Fluorescence Imaging.

Zettlitz Kirstin A KA Waldmann Christopher M CM Tsai Wen-Ting K WK Tavaré Richard R Collins Jeffrey J Murphy Jennifer M JM Wu Anna M AM

Journal of nuclear medicine : official publication, Society of Nuclear Medicine 20190315 10

Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. <b>Methods:</b> We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue-bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prostheti ...[more]

PMID: 30877181

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Project description:Pancreatic cancer has a high mortality rate due to late diagnosis and the tendency to invade surrounding tissues and metastasize at an early stage. A molecular imaging agent that enables both presurgery antigen-specific PET (immuno-PET) and intraoperative near-infrared fluorescence (NIRF) guidance might benefit diagnosis of pancreatic cancer, staging, and surgical resection, which remains the only curative treatment. Methods: We developed a dual-labeled probe based on A2 cys-diabody (A2cDb) targeting the cell-surface prostate stem cell antigen (PSCA), which is expressed in most pancreatic cancers. Maleimide-IRDye800CW was site-specifically conjugated to the C-terminal cys-tag (A2cDb-800) without impairing integrity or affinity (half-maximal binding, 4.3 nM). Direct radioiodination with 124I (124I-A2cDb-800) yielded a specific activity of 159 ± 48 MBq/mg with a radiochemical purity exceeding 99% and 65% ± 4.5% immunoreactivity (n = 3). In vivo specificity for PSCA-expressing tumor cells and biodistribution of the dual-modality tracer were evaluated in a prostate cancer xenograft model and compared with single-labeled 124I-A2cDb. Patient-derived pancreatic ductal adenocarcinoma xenografts (PDX-PDACs) were grown subcutaneously in NSG mice and screened for PSCA expression by immuno-PET. Small-animal PET/CT scans of PDX-PDAC-bearing mice were obtained using the dual-modality 124I-A2cDb-800 followed by postmortem NIRF imaging with the skin removed. Tumors and organs were analyzed ex vivo to compare the relative fluorescent signals without obstruction by other organs. Results: Specific uptake in PSCA-positive tumors and low nonspecific background activity resulted in high-contrast immuno-PET images. Concurrent with the PET studies, fluorescent signal was observed in the PSCA-positive tumors of mice injected with the dual-tracer 124I-A2cDb-800, with low background uptake or autofluorescence in the surrounding tissue. Ex vivo biodistribution confirmed comparable tumor uptake of both 124I-A2cDb-800 and 124I-A2cDb. Conclusion: Dual-modality imaging using the anti-PSCA cys-diabody resulted in high-contrast immuno-PET/NIRF images of PDX-PDACs, suggesting that this imaging agent might offer both noninvasive whole-body imaging to localize PSCA-positive pancreatic cancer and fluorescence image-guided identification of tumor margins during surgery.

Project description:PurposeRANKL expression in the tumor microenvironment has been identified as a biomarker of immune suppression, negating the effect of some cancer immunotherapies. Previously we had developed a radiotracer based on the FDA-approved RANKL-specific antibody denosumab, [89Zr]Zr-DFO-denosumab, enabling successful immuno-PET imaging. Radiolabeled denosumab, however, showed long blood circulation and delayed tumor uptake, potentially limiting its applications. Here we aimed to develop a smaller radiolabeled denosumab fragment, [64Cu]Cu-NOTA-denos-Fab, that would ideally show faster tumor accumulation and better diffusion into the tumor for the visualization of RANKL.Experimental designFab fragments were prepared from denosumab using papain and conjugated to a NOTA chelator for radiolabeling with 64Cu. The bioconjugates were characterized in vitro using SDS-PAGE analysis, and the binding affinity was assessed using a radiotracer cell binding assay. Small animal PET imaging evaluated tumor targeting and biodistribution in transduced RANKL-ME-180 xenografts.ResultsThe radiolabeling yield of [64Cu]Cu-NOTA-denos-Fab was 58 ± 9.2%, with a specific activity of 0.79 ± 0.11 MBq/µg (n = 3). A radiotracer binding assay proved specific targeting of RANKL in vitro. PET imaging showed fast blood clearance and high tumor accumulation as early as 1 h p.i. (2.14 ± 0.21% ID/mL), which peaked at 5 h p.i. (2.72 ± 0.61% ID/mL). In contrast, [64Cu]Cu-NOTA-denosumab reached its highest tumor uptake at 24 h p.i. (6.88 ± 1.12% ID/mL). [64Cu]Cu-NOTA-denos-Fab specifically targeted human RANKL in transduced ME-180 xenografts compared with the blocking group and negative ME-180 xenograft model. Histological analysis confirmed RANKL expression in RANKL-ME-180 xenografts.ConclusionsHere, we report on a novel RANKL PET imaging agent, [64Cu]Cu-NOTA-denos-Fab, that allows for fast tumor imaging with improved imaging contrast when compared with its antibody counterpart, showing promise as a potential PET RANKL imaging tool for future clinical applications.

Project description:BACKGROUND:A long dynamic scanning protocol may be required to accurately measure longitudinal changes in amyloid load. However, such a protocol results in a lower patient comfort and scanning efficiency compared to static scans. A compromise can be achieved by implementing dual-time-window protocols. This study aimed to optimize these protocols for quantitative [18F]flutemetamol and [18F]florbetaben studies. METHODS:Rate constants for subjects across the Alzheimer's disease spectrum (i.e., non-displaceable binding potential (BPND) in the range 0.02-0.77 and 0.02-1.04 for [18F]flutemetamol and [18F]florbetaben, respectively) were established based on clinical [18F]flutemetamol (N?=?6) and [18F]florbetaben (N?=?20) data, and used to simulate tissue time-activity curves (TACs) of 110?min using a reference tissue and plasma input model. Next, noise was added (N?=?50) and data points corresponding to different intervals were removed from the TACs, ranging from 0 (i.e., 90-90?=?full-kinetic curve) to 80 (i.e., 10-90) minutes, creating a dual-time-window. Resulting TACs were fitted using the simplified reference tissue method (SRTM) to estimate the BPND, outliers (??1.5?×?BPND max) were removed and the bias was assessed using the distribution volume ratio (DVR?=?BPND?+?1). To this end, acceptability curves, which display the fraction of data below a certain bias threshold, were generated and the area under those curves were calculated. RESULTS:[18F]Flutemetamol and [18F]florbetaben data demonstrated an increased bias in amyloid estimate for larger intervals and higher noise levels. An acceptable bias (??3.1%) in DVR could be obtained with all except the 10-90 and 20-90-min intervals. Furthermore, a reduced fraction of acceptable data and most outliers were present for these two largest intervals (maximum percentage outliers 48 and 32 for [18F]flutemetamol and [18F]florbetaben, respectively). CONCLUSIONS:The length of the interval inversely correlates with the accuracy of the BPND estimates. Consequently, a dual-time-window protocol of 0-30 and 90-110?min (=maximum of 60?min interval) allows for accurate estimation of BPND values for both tracers. [18F]flutemetamol: EudraCT 2007-000784-19, registered 8 February 2007, [18F]florbetaben: EudraCT 2006-003882-15, registered 2006.

Dataset Information

A Dual-Modality Linker Enables Site-Specific Conjugation of Antibody Fragments for 18F-Immuno-PET and Fluorescence Imaging.

Publications

A Dual-Modality Linker Enables Site-Specific Conjugation of Antibody Fragments for <sup>18</sup>F-Immuno-PET and Fluorescence Imaging.

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