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BackgroundSensitive antibody-based tumor targeting has the potential not only to image metastatic and micrometastatic disease, but also to be the basis of targeted therapy. The vast majority of pancreas cancers express carcinoembryonic antigen (CEA). Thus, we sought to evaluate the potential of CEA as a pancreatic cancer target utilizing a rapidly clearing engineered anti-CEA scFv-Fc antibody fragment with a mutation in the Fc region [anti-CEA scFv-Fc H310A].MethodsImmunohistochemistry (IHC) with the antibody fragment was used to confirm expression of CEA on human pancreas cancer specimens. In vivo tumor targeting was evaluated by tail vein injection of I124-labeled anti-CEA scFv-Fc(H310A) into mice harboring CEA-positive and -negative xenografts. MicroPET/CT imaging was performed at successive time intervals. Radioactivity in blood and tumor was measured after the last time point. Additionally, unlabeled anti-CEA scFv-Fc(H310A) was injected into CEA-positive tumor bearing mice and ex vivo IHC was performed to identify the presence of the antibody to define the microscopic intratumoral pattern of targeting.ResultsModerate to strong staining by IHC was noted on 84% of our human pancreatic cancer specimens and was comparable to staining of our xenografts. Pancreas xenograft imaging with the radiolabeled anti-CEA scFv-Fc(H310A) antibody demonstrated average tumor/blood ratios of 4.0. Immunolocalization demonstrated peripheral antibody fragment penetration of one to five cell diameters (0.75 to 1.5 μm).ConclusionsWe characterized a preclinical xenograft model with respect to CEA expression that was comparable to human cases. We demonstrated that the anti-CEA scFv-Fc(H310A) antibody exhibited antigen-specific tumor targeting and shows promise as an imaging and potentially therapeutic agent.

Introduction. Sensitive and specific imaging of pancreas cancer are necessary for accurate diagnosis, staging, and treatment. The vast majority of pancreas cancers express the carbohydrate tumor antigen CA19-9. The goal of this study was to determine the potential to target CA19-9 with a radiolabeled anti-CA19-9 antibody for imaging pancreas cancer. Methods. CA19-9 was quantified using flow cytometry on human pancreas cancer cell lines. An intact murine anti-CA19-9 monoclonal antibody was labeled with a positron emitting radionuclide (Iodine-124) and injected into mice harboring antigen positive and negative xenografts. MicroPET/CT were performed at successive time intervals (72 hours, 96 hours, 120 hours) after injection. Radioactivity was measured in blood and tumor to provide objective confirmation of the images. Results. Antigen expression by flow cytometry revealed approximately 1.3 × 10(6) CA19-9 antigens for the positive cell line and no expression in the negative cell line. Pancreas xenograft imaging with Iodine-124-labeled anti-CA19-9 mAb demonstrated an average tumor to blood ratio of 5 and positive to negative tumor ratio of 20. Conclusion. We show in vivo targeting of our antigen positive xenograft with a radiolabeled anti-CA19-9 antibody. These data demonstrate the potential to achieve anti-CA19-9 antibody based positron emission tomography of pancreas cancer.

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]