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PurposeThe objective of this article is to develop internalizing positron emission tomography (PET) reporter genes for tracking genetically modified T cells in vivo.ProceduresThe transmembrane and cytoplasmic domains of the human transferrin receptor (TfR) and CD5 were each fused to the carcinoembryonic (CEA) minigene N-A3 and expressed in Jurkat T cells. Internalization was evaluated by confocal microscopy or by intracellular uptake of ¹²⁵I-labeled anti-CEA scFv-Fc. Reporter gene-transfected Jurkat xenografts in mice were analyzed by immunohistochemistry (IHC) and imaged by PET using ¹²⁴I- or ⁶⁴Cu-scFv-Fc as tracers.ResultsSurface expression of TR(1-99)-NA3 was lower than that of NA3-CD5. Both reporter genes were internalized following binding of the anti-CEA antibody fragment. IHC of tumors showed strong staining of NA3-CD5, whereas TR(1-99)-NA3 stained weakly. Specific targeting of TR(1-99)-NA3 or NA3-CD5 was shown by PET in xenografted mice.ConclusionsThe in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.

The long circulation persistence of human serum albumin (HSA) is enabled by its domain III (DIII) interaction with the neonatal Fc receptor (FcRn). A protein scaffold based on HSA DIII was designed. To modify the serum half life of the scaffold, residues H535, H510, and H464 were individually mutated to alanine. HSA DIII wild type (WT) and variants were fused to the anti-carcinoembryonic antigen (CEA) T84.66 diabody (Db), radiolabeled with 124I and injected into xenografted athymic mice for serial PET/CT imaging. All proteins targeted the CEA-positive tumor. The mean residence times (MRT) of the proteins, calculated by quantifying blood activity from the PET images, were: Db-DIII WT (56.7 h), H535A (25 h), H510A (20 h), H464A (17 h), compared with Db (2.9 h). Biodistribution confirmed the order of blood clearance from slow to fast: Db-DIII WT > H535A > H510A > H464A > Db with 4.0, 2.0, 1.8, 1.6 and 0.08 %ID/g of remaining blood activity at 51 h, respectively. This study demonstrates that attenuating the DIII-FcRn interaction provides a way of controlling the pharmacokinetics of the entire Db-DIII fusion protein without compromising tumor targeting. H464 appears to be most crucial for FcRn binding (greatest reduction in MRT), followed by H510 and H535. By mutating the DIII scaffold, we can dial serum kinetics for imaging or therapy applications. [ABSTRACT FROM AUTHOR]