RESUMO
The bioluminescent protein Gaussia luciferase (GLuc) was fused to an anti-carcinoembryonic antigen (CEA) antibody fragment, the diabody, for in vivo optical tumor imaging. A 15-amino acid N-terminal truncation (GLDelta15) resulted in a brighter protein. Fusions of the anti-CEA diabody to full-length GLuc and GLDelta15 retained high affinity for the antigen, emitted light, and exhibited excellent enzymatic stability. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-GLDelta15 to CEA-positive xenografts, with a tumor/background ratio of 3.8 +/- 0.4 at four hours after tail-vein injection, compared to antigen-negative tumors at 1.3 +/- 0.1 (p = 0.001). MicroPET imaging using (124)I-diabody-GLDelta15 demonstrated specific uptake in the CEA-positive tumor (2.6% ID [injected dose]/g) compared to the CEA-negative tumor (0.4% ID/g) at 21 hours. Although further optimization of this fusion protein may be needed to improve in vivo performance, the diabody-GLDelta15 is a promising optical imaging probe for tumor detection in vivo.
Assuntos
Anticorpos Antineoplásicos , Antígeno Carcinoembrionário/imunologia , Luciferases , Neoplasias Experimentais/diagnóstico , Sequência de Aminoácidos , Animais , Anticorpos Antineoplásicos/genética , Sequência de Bases , Linhagem Celular Tumoral , Copépodes/enzimologia , Copépodes/genética , Primers do DNA/genética , Feminino , Humanos , Fragmentos de Imunoglobulinas/genética , Radioisótopos do Iodo , Luciferases/genética , Medições Luminescentes , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Transplante de Neoplasias , Neoplasias Experimentais/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Engenharia de Proteínas , Compostos Radiofarmacêuticos , Ratos , Proteínas Recombinantes de Fusão/genética , Transplante HeterólogoRESUMO
An anti-carcinoembryonic antigen (CEA) antibody fragment, the anti-CEA diabody, was fused to the bioluminescence enzyme Renilla luciferase (RLuc) to generate a novel optical imaging probe. Native RLuc or one of two stabilized variants (RLucC124A, RLuc8) was used as the bioluminescent moiety. A bioluminescence ELISA showed that diabody-luciferase could simultaneously bind to CEA and emit light. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-RLuc8 to CEA-positive xenografts, with a tumor:background ratio of 6.0 +/- 0.8 at 6 h after intravenous injection, compared with antigen-negative tumors at 1.0 +/- 0.1 (P = 0.05). Targeting and distribution was also evaluated by microPET imaging using (124)I-diabody-RLuc8 and confirmed that the optical signal was due to antibody-mediated localization of luciferase. Renilla luciferase, fused to biospecific sequences such as engineered antibodies, can be administered systemically to provide a novel, sensitive method for optical imaging based on expression of cell surface receptors in living organisms.