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Evaluation of two internalizing carcinoembryonic antigen reporter genes for molecular imaging.

Barat B, Kenanova VE, Olafsen T, Wu AM - Mol Imaging Biol (2011)

Bottom Line: Surface expression of TR(1-99)-NA3 was lower than that of NA3-CD5.Specific targeting of TR(1-99)-NA3 or NA3-CD5 was shown by PET in xenografted mice.The in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.

View Article: PubMed Central - PubMed

Affiliation: Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1770, USA.

ABSTRACT

Purpose: The objective of this article is to develop internalizing positron emission tomography (PET) reporter genes for tracking genetically modified T cells in vivo.

Procedures: The 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.

Results: Surface 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.

Conclusions: The in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.

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Micro-PET imaging of CEA reporter proteins. a Coronal and transverse views of a nude mouse with TR(1–99)-NA3 transfected (+) and nontransfected (−) xenografts, injected with 140 μCi of 124I-labeled anti-CEA scFv-Fc H310/H435Q fragment at 24 h after injection. b Transverse view of a nude mouse with NA3-CD5 transfected (+) and nontransfected (-) xenografts, injected with 100 μCi of 64Cu-labeled anti-CEA scFv-Fc H310A fragment at 21 h postinjection.
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Fig5: Micro-PET imaging of CEA reporter proteins. a Coronal and transverse views of a nude mouse with TR(1–99)-NA3 transfected (+) and nontransfected (−) xenografts, injected with 140 μCi of 124I-labeled anti-CEA scFv-Fc H310/H435Q fragment at 24 h after injection. b Transverse view of a nude mouse with NA3-CD5 transfected (+) and nontransfected (-) xenografts, injected with 100 μCi of 64Cu-labeled anti-CEA scFv-Fc H310A fragment at 21 h postinjection.

Mentions: Micro-PET imaging was employed to evaluate the in vivo targeting of anti-CEA antibody fragments to the CEA reporter protein-positive tumors. For this purpose, nude mice (n = 3) carrying TR(1–99)-NA3 (CEA-positive) and parental Jurkat (CEA-negative) tumors were injected with the 124I-labeled anti-CEA scFv-Fc H310A/H435Q antibody fragment. Whole-body micro-PET scans were obtained at 24 and 48 h post tracer injection. Uptake in the CEA-positive tumor and low activity in the CEA-negative control tumor were seen at 24 h (Fig. 5a). At 48 h, no signal was detected in the positive tumor. The average tumor-to-background ratio at 24 h, based on ROI analyses, was 2.8. A biodistribution after the last scan (48 h) revealed that the TR(1–99)-NA3 expressing tumor had 0.95 ± 0.007% ID/g, which was significantly different (P = .030) from the uptake in the negative tumor (0.59 ± 0.06% ID/g), resulting in a positive to negative tumor ratio of 1.6. The radioactive uptake in the positive tumor was also significantly different from the activities in liver and kidneys (P < .05), but not significantly different from the activities in spleen and lungs (P > .05).Fig. 5


Evaluation of two internalizing carcinoembryonic antigen reporter genes for molecular imaging.

Barat B, Kenanova VE, Olafsen T, Wu AM - Mol Imaging Biol (2011)

Micro-PET imaging of CEA reporter proteins. a Coronal and transverse views of a nude mouse with TR(1–99)-NA3 transfected (+) and nontransfected (−) xenografts, injected with 140 μCi of 124I-labeled anti-CEA scFv-Fc H310/H435Q fragment at 24 h after injection. b Transverse view of a nude mouse with NA3-CD5 transfected (+) and nontransfected (-) xenografts, injected with 100 μCi of 64Cu-labeled anti-CEA scFv-Fc H310A fragment at 21 h postinjection.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2992604&req=5

Fig5: Micro-PET imaging of CEA reporter proteins. a Coronal and transverse views of a nude mouse with TR(1–99)-NA3 transfected (+) and nontransfected (−) xenografts, injected with 140 μCi of 124I-labeled anti-CEA scFv-Fc H310/H435Q fragment at 24 h after injection. b Transverse view of a nude mouse with NA3-CD5 transfected (+) and nontransfected (-) xenografts, injected with 100 μCi of 64Cu-labeled anti-CEA scFv-Fc H310A fragment at 21 h postinjection.
Mentions: Micro-PET imaging was employed to evaluate the in vivo targeting of anti-CEA antibody fragments to the CEA reporter protein-positive tumors. For this purpose, nude mice (n = 3) carrying TR(1–99)-NA3 (CEA-positive) and parental Jurkat (CEA-negative) tumors were injected with the 124I-labeled anti-CEA scFv-Fc H310A/H435Q antibody fragment. Whole-body micro-PET scans were obtained at 24 and 48 h post tracer injection. Uptake in the CEA-positive tumor and low activity in the CEA-negative control tumor were seen at 24 h (Fig. 5a). At 48 h, no signal was detected in the positive tumor. The average tumor-to-background ratio at 24 h, based on ROI analyses, was 2.8. A biodistribution after the last scan (48 h) revealed that the TR(1–99)-NA3 expressing tumor had 0.95 ± 0.007% ID/g, which was significantly different (P = .030) from the uptake in the negative tumor (0.59 ± 0.06% ID/g), resulting in a positive to negative tumor ratio of 1.6. The radioactive uptake in the positive tumor was also significantly different from the activities in liver and kidneys (P < .05), but not significantly different from the activities in spleen and lungs (P > .05).Fig. 5

Bottom Line: Surface expression of TR(1-99)-NA3 was lower than that of NA3-CD5.Specific targeting of TR(1-99)-NA3 or NA3-CD5 was shown by PET in xenografted mice.The in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.

View Article: PubMed Central - PubMed

Affiliation: Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1770, USA.

ABSTRACT

Purpose: The objective of this article is to develop internalizing positron emission tomography (PET) reporter genes for tracking genetically modified T cells in vivo.

Procedures: The 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.

Results: Surface 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.

Conclusions: The in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.

Show MeSH
Related in: MedlinePlus