| The present work describes two novel cancer targeting fusion proteins consisting of an engineered antibody fragment fused to Renilla luciferase or Gaussia luciferase for in vivo optical imaging of carcinoembryonic antigen (CEA). CEA is a cell surface target expressed in the majority of colorectal carcinomas and has been used as the target antigen for previous in vivo imaging studies using an engineered anti-CEA T84.66 diabody fragment (Db, a dimer of the single chain Fv) that has exhibited high level tumor targeting in biodistribution and microPET imaging studies.;Initially a fusion of the anti-CEA diabody and Renilla luciferase (RLuc) was created. Although the protein remained bifunctional, able to bind to the antigen and emit light in the presence of the substrate, the Db-RLuc was not stable enough for in vivo use. Ultimately, a fusion protein was created using RLuc8, which incorporates 8 amino acid substitutions, that exhibited enzymatic stability suitable for in vivo use. In vivo optical imaging of tumor bearing mice demonstrated specific targeting of DbRLuc8 fusion proteins to CEA-positive xenografts. Targeting and distribution were also evaluated by microPET imaging using 124I-diabody-RLuc8 fusion proteins to confirm that the optical signal was due to antibody-mediated localization of luciferase.;Gaussia luciferase was also explored as a fusion partner for the anti-CEA diabody due to its smaller size, brighter bioluminescent signal, and potentially greater stability. A fusion protein incorporating GLDelta15, a 15 amino acid N-terminal truncation of GLuc, remained bifunctional and was stable enough for in vivo use. Although in vitro characterization shows the Db-GLDelta5 fusion protein was brighter than Db-RLuc8, the Db-GLDelta15 was not as favorable for in vivo tumor targeting, potentially indicating the Db-GLDelta15 was getting clipped or dissociating into monomers in vivo.;The final chapter is a discussion of the various factors to be considered in the design of the fusion proteins including the components, linker length and composition, orientation, and expression method. The present work demonstrates the development of a novel optical imaging probe that can be used for detection of the endogenous expression of CEA after intravenous administration of the anti-CEA diabody-luciferase fusion protein. |
