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T84.66 Anti-CEA diabody-GSTSGSGKPGSGEGSTSG-Renilla luciferase.

Authors

Zhang H1.

Source

Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.
2008 Sep 18 [updated 2008 Oct 23].

Author information

1
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, Email: micad@ncbi.nlm.nih.gov

Excerpt

Carcinoembryonic antigen (CEA) is a highly glycosylated protein with a molecular mass of 180 kDa that belongs to the immunoglobin supergene family (1). CEA contains ~60% carbohydrate and a protein portion of 668 amino acids. As a common human tumor antigen, CEA is overexpressed on >80% of colorectal, pancreatic, breast, non-small cell lung, and medullary cancers, and on >70% of gastric, invasive cervical, endometrial, urinary bladder, and head/neck cancers (2). A variety of antibodies have been developed to target CEA. For example, T84.66 is a murine monoclonal antibody with a molecular mass of 150 kDa with exceptionally high affinity (2.6 × 1010 M-1) for the α3β3 epitope on the CEA (2, 3). T84.66 has been used to quantify CEA expression in immunohistological staining and in in vivo imaging. The T84.66 diabody is a recombinant fragment of the antibody that comprises two identical single chain variable (scFv) regions (4). Each scFv consists of a VL domain of 107 amino acids, a peptide linker of 8 amino acids (GGGSGGGG), and a VH domain of 112 amino acids. The two scFvs associate asymmetrically to form a stable diabody (scFv dimer) with a molecular mass of 55 kDa. T84.66 diabody retains the high avidity of T84.66 via its bivalent binding. The small size of the diabody allows for rapid in vivo targeting in imaging and therapeutic applications. Renilla luciferase (Rluc) is a 36-kDa enzyme protein extracted from a bioluminescent soft coral (sea pansy (Renilla reniformis)) (5). Rluc can catalyze emission of light from substrates, i.e., the oxidation of coelenterazine to coelenteramide generates a green fluorescence (535–550 nm) (6). Unlike the luciferase obtained from the firefly (Fluc), the Rluc oxidation process does not depend on adenosine-5'-triphosphate (7). Thus, Rluc is suitable for use as an in vivo bioluminescent tag, i.e., Rluc has been reported as a marker of gene expression in various cells and as a reporter gene or cell tag for in vivo imaging (5). Rluc8 is a variant of Rluc that contains amino acid substitution at the following eight locations: A55T, C124A, S130A, K136R, A143M, M185V, M253L, and S287L (5). These substitutions enhance the enzymatic activity stability of the molecule (>200 h) and increase the light emission as much as four-fold. The small size of Rluc makes it suitable for fusion with other proteins to generate specifically labeled proteins for imaging applications. T84.66 Anti-CEA diabody-GSTSGSGKPGSGEGSTSG-Renilla luciferase (Db-18-Rluc8) is an optical agent used for imaging CEA (5). Db-18-Rluc8 contains three components: a T84.66 diabody for targeting CEA, an Rluc8 for catalyzing the coelenterazine substrate to emit light for optical detection, and a peptide linker of 18 amino acids to connect the diabody portion and the luciferase portion. The three components were assembled via protein fusion. Db-18-Rluc8 allows for localization of CEA-positive tumors in the living animal.

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