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

• Published in: Molecular imaging and biology 2018-02, Vol.20 (1), p.47-54
• Main Authors: Gong, Li, Ding, Haiming, Long, Nicholas E., Sullivan, Brandon J., Martin, Edward W., Magliery, Thomas J., Tweedle, Michael F.
• Format: Article
• Language: English
• Subjects: Adenocarcinoma; Alkanesulfonic Acids - chemical synthesis; Alkanesulfonic Acids - chemistry; Animals; Antibodies, Monoclonal, Humanized - chemistry; Antigens, Neoplasm - metabolism; Binding; Cancer; Cell Line, Tumor; Colon; Colorectal cancer; Colorectal carcinoma; Colorectal Neoplasms - pathology; Competition; Female; Fluorescence; Glycoproteins - metabolism; High-performance liquid chromatography; Humans; Imaging; In vivo methods and tests; Indoles - chemical synthesis; Indoles - chemistry; Liquid chromatography; Lymphocytes; Lymphocytes T; Mass spectra; Medical personnel; Medicine; Medicine & Public Health; Mice, Inbred BALB C; Mice, Nude; Monoclonal antibodies; Optical Imaging; Purity; Radiology; Research Article; Surgery; Surgical instruments; Tumors; Xenograft Model Antitumor Assays
• ISSN: 1536-1632; 1860-2002
• DOI: 10.1007/s11307-017-1096-4

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.