We are developing assays for noninvasive, quantitative imaging of reporter genes with positron emission tomography (PET), for application both in animal models and in human gene therapy. We report here a method to improve the detection of lower levels of PET reporter gene expression by utilizing a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) as a PET reporter gene. Several approaches are being developed to image reporter gene expression in living animals. These include methods that rely on charge-coupled device camera imaging and bioluminescent reporter genes (1), single-photon emission computed tomography using the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene (2), approaches that use magnetic resonance imaging (reviewed in ref.3), methods based on the HSV1-tk reporter gene and positron emission tomography (PET) (4, 5), and the use of the dopamine type 2 receptor (D2R) as a reporter gene for PET (6). The use of reporter genes that can be imaged in vivo will permit many different applications, including monitoring of both somatic gene transfer and transgenic͞knock-in reporter gene expression (7).PET provides repeated, noninvasive imaging of biological processes in living subjects (8, 9). PET utilizes molecular probes labeled with positron-emitting radioisotopes (e.g., fluorine-18, with a half-life of 110 min). PET probes typically are either positron-labeled ligands for receptors or positron-labeled substrates for intracellular enzymes. Tracer quantities of PET probes yield a tomographic image after their retention, as a consequence of either binding of positron-labeled ligand to a receptor or conversion of positron-labeled substrate to ''trapped'' metabolic product(s). PET is particularly well suited for application to human studies. PET reporter gene imaging in humans will allow monitoring of the location(s), magnitude, and duration of therapeutic͞suicide gene expression, by using vectors for DNA delivery in which the reporter gene and therapeutic gene are expressed from a common transcript (10,11 HSV1-tk refers to the gene, HSV1-TK refers to the enzyme.) HSV1-TK phosphorylates a range of substrates, including acycloguanosines (e.g., acyclovir, ganciclovir, penciclovir) and uracil derivatives [e.g., 2Ј-f luoro-2Ј-deoxy-1-␤-arabinofuranosyl-5-iodouracil (FIAU)]. In contrast, mammalian thymidine kinases phosphorylate acycloguanosines only minimally, making these substrates advantageous as reporter gene imaging probes (11). Acycloguanosine derivatives are currently used extensively both as cytotoxic pharmaceuticals to treat herpes infections and for HSV1-tk suicide gene therapy (13).Improvements in sensitivity of the HSV1-tk reporter gene imaging assay can be achieved either (i) by identifying substrates that exhibit higher V max ͞K m for HSV1-TK or (ii) by engineering TK enzyme(s) with improved V max ͞K m for a particular reporter substrate. Decreased V max ͞K m of HSV1-TK for thymidine (an endogenous competitor) also should improve HSV1-tk reporter gene assay sensiti...

We are developing quantitative assays to repeatedly and noninvasively image expression of reporter genes in living animals, using positron emission tomography (PET). We synthesized positron-emitting 8

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Human epidermal growth factor receptor type 2 (HER2) is a wellestablished tumor biomarker that is overexpressed in a wide variety of cancers and that serves as a molecular target for therapeutic intervention. HER2 also serves as a prognostic indicator of patient survival and as a predictive marker of the response to antineoplastic therapy. The development of 18 F-labeled biomolecules for PET imaging of HER2 (HER2 PET ) is very important because it may provide a powerful tool for the early detection of HER2-positive tumor recurrence and for the monitoring of HER2-based tumor treatment. Methods: In this study, anti-HER2 monomeric and dimeric protein scaffold molecules [Z HER2:477 and (Z HER2:477 ) 2 , respectively] were radiofluorinated at a reasonable radiochemical yield (13%-18%) by use of site-specific oxime chemistry. The resulting radiofluorinated protein scaffold molecules were then evaluated as potential molecular probes for small-animal HER2 PET by use of a SKOV3 tumor-bearing mouse model. Results: The 4-18 F-fluorobenzaldehyde conjugated aminooxy-protein scaffolds [ 18 F-N-(4-fluorobenzylidene)oxime (FBO)-Z HER2:477 and 18 F-FBO-(Z HER2:477 ) 2 ] both displayed specific HER2-binding ability in vitro. Biodistribution and small-animal PET imaging studies further revealed that 18 F-FBO-Z HER2:477 showed rapid and high SKOV3 tumor accumulation and quick clearance from normal tissues, whereas 18 F-FBO-(Z HER2:477 ) 2 showed poor in vivo performance (low tumor uptake and tumorto-normal tissue ratios). The specificity of 18 F-FBO-Z HER2:477 for SKOV3 tumors was confirmed by its lower uptake on pretreatment of tumor-bearing mice with the HER2-targeting agents Z HER2 and trastuzumab. Moreover, small-animal PET imaging studies revealed that 18 F-FBO-Z HER2:477 produced higher-quality tumor imaging than 18 F-FBO-(Z HER2:477 ) 2 . 18 F-FBO-Z HER2:477 could clearly identify HER2-positive tumors with good contrast. Conclusion: Overall, these data demonstrate that 18 F-FBO-Z HER2:477 is a promising PET probe for imaging HER2 expression in living mice. It has a high potential for translation to clinical applications. The radiofluorination method developed can also be used as a general strategy for the site-specific labeling of other proteins with 18 F. The protein scaffold molecules used here are attractive for the further development of PET probes for other molecular targets.

A major barrier to successful use of allogeneic hematopoietic cell transplantation is acute graft-versus-host disease (aGVHD), a devastating condition that arises when donor T cells attack host tissues. With current technologies, aGVHD diagnosis is typically made after end-organ injury and often requires invasive tests and tissue biopsies. This impacts patient prognosis as treatments are dramatically less effective at late disease stages. Here we show that a novel positron emission tomography (PET) radiotracer, 2′-deoxy-2′-[18F]fluoro-9-β-D-arabinofuranosylguanine ([18F]F-AraG), targeted towards two salvage kinase pathways preferentially accumulates in activated primary T cells. [18F]F-AraG PET imaging of a murine aGVHD model enabled visualization of secondary lymphoid organs harboring activated donor T cells prior to clinical symptoms. Tracer biodistribution in healthy humans showed favorable kinetics. This new PET strategy has great potential for early aGVHD diagnosis, enabling timely treatments and improved patient outcomes. [18F]F-AraG may be useful for imaging activated T cells in various biomedical applications.