ImmunoPET and Near-Infrared Fluorescence Imaging of Pancreatic Cancer with a Dual-Labeled Bispecific Antibody Fragment

Abstract

Dual-targeted imaging agents have shown improved targeting efficiencies in comparison to single-targeted entities. The purpose of this study was to quantitatively assess the tumor accumulation of a dual-labeled heterobifunctional imaging agent, targeting two overexpressed biomarkers in pancreatic cancer, using positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging modalities. A bispecific immunoconjugate (heterodimer) of CD105 and tissue factor (TF) Fab′ antibody fragments was developed using click chemistry. The heterodimer was dual-labeled with a radionuclide (64Cu) and fluorescent dye. PET/NIRF imaging and biodistribution studies were performed in four-to-five week old nude athymic mice bearing BxPC-3 (CD105/TF+/+) or PANC-1 (CD105/TF−/−) tumors xenografts. A blocking study was conducted to investigate the specificity of the tracer. Ex vivo tissue staining was performed to compare TF/CD105 expression in tissues with PET tracer uptake to validate in vivo results. PET imaging of 64Cu-NOTA-heterodimer-ZW800 in BxPC-3 tumor xenografts revealed enhanced tumor uptake (21.0 ± 3.4 %ID/g; n = 4) compared to the homodimer of TRC-105 (9.6 ± 2.0 %ID/g; n=4; p < 0.01) and ALT-836 (7.6 ± 3.7 %ID/g; n=4; p < 0.01) at 24 h post-injection. Blocking studies revealed that tracer uptake in BxPC-3 tumors could be decreased by four-fold with TF blocking and two-fold with CD105 blocking. In the negative model (PANC-1), heterodimer uptake was significantly lower than that found in the BxPC-3 model (3.5 ± 1.1 %ID/g; n=4; p < 0.01). The specificity was confirmed by the successful blocking of CD105 or TF, which demonstrated the dual targeting with 64Cu-NOTA-heterodimer-ZW800 provided an improvement in overall tumor accumulation. Also, fluorescence imaging validated the PET imaging, allowing for clear delineation of the xenograft tumors. Dual-labeled heterodimeric imaging agents, like 64Cu-NOTA-heterodimer-ZW800, may increase the overall tumor accumulation in comparison to single-targeted homodimers, leading to improved imaging of cancer and other related diseases.

Antibody Fragment and Affibody ImmunoPET Imaging Agents: Radiolabelling Strategies and Applications

“…[63,64] For these reasons there has been as hift toward using NOTAa st he chelator of choice for 64 Cu-labelling of fragments. Cai and co-workers [65] recently reported the 64 Cu-labelling of ab ispecific fragment using aN ODA chelator and used it for imaging CD105 and tissue factor (TF) in mice bearing pancreatic cancerx enographs. The same fragment was also labelled with af luorescent tag, to generate ad ual labelledp robe, that also enabled near-infrared fluorescence( NIRF) imaging.…”

Interactions Between Tumor Biology and Targeted Nanoplatforms for Imaging Applications

“…PET and NIRF imaging allowed for clear delineation of the cancer. However, the NIRF signal was significantly weaker than the PET signal 289…”

“…CD105 and TF are two biomarkers which are both over‐expressed in pancreatic cancer. Luo et al289 designed a dual receptor‐targeted construct consisting of a bi‐specific heterodimer of Fab' antibody fragments recognizing CD105 and tissue factor, using a click chemistry approach. It was dual‐labeled with NIRF and PET imaging reporters ( 64 Cu‐NOTA‐heterodimer‐ZW800) and used for the imaging of pancreatic tumors ( Figure ).…”

New Developments in Dual-Labeled Molecular Imaging Agents

“…Bispecific antibody fragments have been used to target 2 different antigens and increase overall tumor uptake (12). Luo et al applied this concept toward the development of a dual-labeled heterobifunctional immunoconjugate for pancreatic cancer (13). Using biorthogonal click-chemistry, tetrazine functionalized CD105 and transcyclooctene (TCO)-functionalized tissue factor Fab9 antibody fragments were conjugated together, followed by dual labeling with 64 Cu and the zwitterionic NIRF dye ZW800 (Ex/Em, 772/788 nm) (14).…”