A 3E8.scFv.Cys-IR800 Conjugate Targeting TAG-72 in an Orthotopic Colorectal Cancer Model

Abstract

Purpose Optical surgical navigation (OSN) will be a potent tool to help surgeons more accurately and efficiently remove tumors. The purpose of this study was to evaluate a novel humanized 3E8 antibody (3E8 MAb) fragment site-specifically conjugated with IR800, 3E8.scFv.Cys-IR800, as a potential OSN agent to target colorectal adenocarcinoma. Procedures An engineered single-chain variable fragment of 3E8 MAb (targeted to TAG-72), appending a C-terminal cysteine residue (3E8.scFv.Cys), was created and reacted with IRDye800-maleimide. 3E8.scFv.Cys-IR800 identity and purity were verified by MALDI-TOF mass spectra and 800 nm detected size exclusion column HPLC. In vitro human colon adenocarcinoma LS-174 T cells binding and competition assay validated biological functionality. We further evaluated the imaging ability and receptor-specific binding of 3E8.scFv.Cys-IR800 in an orthotopic LS-174 T mouse model. Results A 1:1 dye to protein conjugate was achieved at greater than 90 % HPLC purity. A 1 nmol dose of 3E8.scFv.Cys-IR800 via intraperitoneal injection administration was sufficient to produce high tumor to background fluorescence contrast. Blocking competition studies both in vitro and in vivo using a different blocking protein, 3E8ΔCH2, demonstrated 3E8.scFv.Cys-IR800 binding specificity for TAG-72 antigen. Conclusions 3E8.scFv.Cys-IR800 shows properties useful in a clinically viable OSN agent for colorectal cancer.

Fluorescence-Guided Surgery

“…The target-to-background ratio (TBR) achieved in this study (mean TBR of 5.2 in the highest dose range) improved the accuracy of surgical decision-making ( 95 ). Recently, cetuximab-IRDye800CW or bevacizumab-IRDye800CW (targeting vascular endothelial growth factor)-have also been in clinical trials targeting pancreatic adenocarcinoma, colon cancer, and breast cancer ( 102 , 126 ). It was noted that the conjugation of the IRDye800CW significantly shortened the circulating half-life of cetuximab despite a low antibody-to-dye conjugation ratio (approximately 1) ( 95 ).…”

Cyanine Conjugate‐Based Biomedical Imaging Probes

“…Combinatorial chemistry libraries, especially innovative dynamic combinatorial library and DNA-encoded library, commonly involved in target-based drug discovery are also applicable to ligand discovery as their underlying principles are interchangeable. [34] Additionally, biological screening platforms that rely on miscellaneous display systems, either cellular or acellular, to construct genetic mutant libraries www.advancedsciencenews.com www.advhealthmat.de (13)(14)(15) or its biomimetics (16)(17)(18)(19)(20) as targeting warheads. b) 3D image of mouse administered with conjugate 15 using fluorescence imaging tomography (FLIT) mode and confirmation of tumor cells in the primary tumor and four metastatic lymph nodes through H&E staining or cytokeratin immunostaining.…”

“…PET/NIRF dual-modal imaging probes are constructed by conjugating cyanine fluorophores with positron-emitting radionuclides, which typically have short half-lives, e.g., 68 Ga (t 1/2 = 67.7 min), 18 F (t 1/2 = 109.7 min), 64 Cu (t 1/2 = 12.7 h) (Figure 13a). 18 F-labeled fluorodeoxyglucose ( 18 F-FDG) is by far the most commonly used radiopharmaceuticals in oncological and neurological PET scans. A novel bioorthogonal aryl dioxaborolane decomposition process was introduced for rapid labeling of 18 F; take 67 for example, the conversion was completed in 1 h. [102] Alternatively, such 18 F-BF 3 group can be directly labeled by aqueous 18 F ions via isotopic exchange reaction.…”

“…18 F-labeled fluorodeoxyglucose ( 18 F-FDG) is by far the most commonly used radiopharmaceuticals in oncological and neurological PET scans. A novel bioorthogonal aryl dioxaborolane decomposition process was introduced for rapid labeling of 18 F; take 67 for example, the conversion was completed in 1 h. [102] Alternatively, such 18 F-BF 3 group can be directly labeled by aqueous 18 F ions via isotopic exchange reaction. [103] In another approach, 18 F was first incorporated as 1-azido-2-fluoroethane and then rapidly conjugated to an MMP inhibitor functionalized sulfo-Cy5.5 via click chemistry (68).…”

Linker engineering in anti-TAG-72 antibody fragments optimizes biophysical properties, serum half-life, and high-specificity tumor imaging

“…1A). For all V L -V H domain-oriented constructs, our original 3E8 construct (V L -205C-V H , manuscript in preparation) was used as the template (22). For the cloning of V H -V L orientation constructs, a gene containing a G 4 S linker (custom synthesis by Genewiz) was used as the template.…”