Dean O. Campbell

Positron Emission Tomography Reporter Genes and Reporter Probes: Gene and Cell Therapy Applications

Positron emission tomography (PET) imaging reporter genes (IRGs) and PET reporter probes (PRPs) are amongst the most valuable tools for gene and cell therapy. PET IRGs/PRPs can be used to non-invasively monitor all aspects of the kinetics of therapeutic transgenes and cells in all types of living mammals. This technology is generalizable and can allow long-term kinetics monitoring. In gene therapy, PET IRGs/PRPs can be used for whole-body imaging of therapeutic transgene expression, monitoring variations in the magnitude of transgene expression over time. In cell or cellular gene therapy, PET IRGs/PRPs can be used for whole-body monitoring of therapeutic cell locations, quantity at all locations, survival and proliferation over time and also possibly changes in characteristics or function over time. In this review, we have classified PET IRGs/PRPs into two groups based on the source from which they were derived: human or non-human. This classification addresses the important concern of potential immunogenicity in humans, which is important for expansion of PET IRG imaging in clinical trials. We have then discussed the application of this technology in gene/cell therapy and described its use in these fields, including a summary of using PET IRGs/PRPs in gene and cell therapy clinical trials. This review concludes with a discussion of the future direction of PET IRGs/PRPs and recommends cell and gene therapists collaborate with molecular imaging experts early in their investigations to choose a PET IRG/PRP system suitable for progression into clinical trials.

Noninvasive prediction of tumor responses to gemcitabine using positron emission tomography

Gemcitabine (2′,2′-difluorodeoxycytidine, dFdC) and cytosine arabinoside (cytarabine, ara-C) represent a class of nucleoside analogs used in cancer chemotherapy. Administered as prodrugs, dFdC and ara-C are transported across cell membranes and are converted to cytotoxic derivatives through consecutive phosphorylation steps catalyzed by endogenous nucleoside kinases. Deoxycytidine kinase (DCK) controls the rate-limiting step in the activation cascade of dFdC and ara-C. DCK activity varies significantly among individuals and across different tumor types and is a critical determinant of tumor responses to these prodrugs. Current assays to measure DCK expression and activity require biopsy samples and are prone to sampling errors. Noninvasive methods that can detect DCK activity in tumor lesions throughout the body could circumvent these limitations. Here, we demonstrate an approach to detecting DCK activity in vivo by using positron emission tomography (PET) and 18F-labeled 1-(2′-deoxy-2′-fluoroarabinofuranosyl) cytosine] ([18F]FAC), a PET probe recently developed by our group. We show that [18F]FAC is a DCK substrate with an affinity similar to that of dFdC. In vitro, accumulation of [18F]FAC in murine and human leukemia cell lines is critically dependent on DCK activity and correlates with dFdC sensitivity. In mice, [18F]FAC accumulates selectively in DCK-positive vs. DCK-negative tumors, and [18F]FAC microPET scans can predict responses to dFdC. We suggest that [18F]FAC PET might be useful for guiding treatment decisions in certain cancers by enabling individualized chemotherapy.

Nucleoside salvage pathway kinases regulate hematopoiesis by linking nucleotide metabolism with replication stress

Endogenous thymidine plays a critical role in the induction of replication stress in thymocytes.

Novel PET Probes Specific for Deoxycytidine Kinase

Deoxycytidine kinase (dCK) is a rate-limiting enzyme in the deoxyribonucleoside salvage pathway and a critical determinant of therapeutic activity for several nucleoside analog prodrugs. We have previously reported the development of 1-(2′-deoxy-2′-18F-fluoro-β-d-arabinofuranosyl)cytosine (18F-FAC), a new probe for PET of dCK activity in immune disorders and certain cancers. The objective of the current study was to develop PET probes with improved metabolic stability and specificity for dCK. Toward this goal, several candidate PET probes were synthesized and evaluated in vitro and in vivo. Methods: High-pressure liquid chromatography was used to analyze the metabolic stability of 18F-FAC and several newly synthesized analogs with the natural d-enantiomeric sugar configuration or the corresponding unnatural l-configuration. In vitro kinase and uptake assays were used to determine the affinity of the 18F-FAC l-nucleoside analogs for dCK. The biodistribution of selected l-analogs in mice was determined by small-animal PET/CT. Results: Candidate PET probes were selected using the following criteria: low susceptibility to deamination, high affinity for purified recombinant dCK, high uptake in dCK-expressing cell lines, and biodistribution in mice reflective of the tissue-expression pattern of dCK. Among the 10 newly developed candidate probes, 1-(2′-deoxy-2′-18F-fluoro-β-l-arabinofuranosyl)cytosine (l-18F-FAC) and 1-(2′-deoxy-2′-18F-fluoro-β-l-arabinofuranosyl)-5-methylcytosine (l-18F-FMAC) most closely matched the selection criteria. The selection of l-18F-FAC and l-18F-FMAC was validated by showing that these two PET probes could be used to image animal models of leukemia and autoimmunity. Conclusion: Promising in vitro and in vivo data warrant biodistribution and dosimetry studies of l-18F-FAC and l-18F-FMAC in humans.

Structure-guided Engineering of Human Thymidine Kinase 2 as a Positron Emission Tomography Reporter Gene for Enhanced Phosphorylation of Non-natural Thymidine Analog Reporter Probe

Background: Humanized PET reporter gene (PRG) systems are needed to replace immunogenic, viral-derived systems. Results: Employing a structure-guided approach, we developed a highly sensitive humanized PRG characterized by reduced activity for its natural substrates. Conclusion: Sensitivity of PRGs can be improved by reducing their endogenous activities. Significance: Our method can be employed to rapidly develop highly sensitive humanized PRGs. Positron emission tomography (PET) reporter gene imaging can be used to non-invasively monitor cell-based therapies. Therapeutic cells engineered to express a PET reporter gene (PRG) specifically accumulate a PET reporter probe (PRP) and can be detected by PET imaging. Expanding the utility of this technology requires the development of new non-immunogenic PRGs. Here we describe a new PRG-PRP system that employs, as the PRG, a mutated form of human thymidine kinase 2 (TK2) and 2′-deoxy-2′-18F-5-methyl-1-β-l-arabinofuranosyluracil (l-18F-FMAU) as the PRP. We identified l-18F-FMAU as a candidate PRP and determined its biodistribution in mice and humans. Using structure-guided enzyme engineering, we generated a TK2 double mutant (TK2-N93D/L109F) that efficiently phosphorylates l-18F-FMAU. The N93D/L109F TK2 mutant has lower activity for the endogenous nucleosides thymidine and deoxycytidine than wild type TK2, and its ectopic expression in therapeutic cells is not expected to alter nucleotide metabolism. Imaging studies in mice indicate that the sensitivity of the new human TK2-N93D/L109F PRG is comparable with that of a widely used PRG based on the herpes simplex virus 1 thymidine kinase. These findings suggest that the TK2-N93D/L109F/l-18F-FMAU PRG-PRP system warrants further evaluation in preclinical and clinical applications of cell-based therapies.

Correlation of the Genotype of Paragangliomas and Pheochromocytomas with Their Metabolic Phenotype on 3,4-Dihydroxy-6-18F-Fluoro-l-Phenylalanin PET

Paragangliomas and pheochromocytomas are genetically heterogeneous diseases. The purpose of this study was to determine the sensitivity and specificity of PET with 3,4-dihydroxy-6-18F-fluoro-L-phenylalanin (18F-DOPA) for the detection and staging of pheochromocytomas/paragangliomas. Furthermore, we assessed whether the genotypes of pheochromocytomas and paragangliomas correlate with the uptake of 18F-DOPA. Methods: We retrospectively analyzed 101 consecutive patients who underwent 18F-DOPA PET or 18F-DOPA PET/CT for known or suspected pheochromocytomas or paragangliomas. Maximum 18F-DOPA tumor uptake was quantified relative to uptake in the liver. Results: Histopathology, cross-sectional imaging, and follow-up indicated the presence of paragangliomas and pheochromocytomas in 68 patients and the absence of a tumor in 33 patients. The average 18F-DOPA uptake by paragangliomas and pheochromocytomas, expressed as a tumor-to-liver ratio, was 5.9 ± 5.2. There was no significant difference in uptake among patients with von Hippel Lindau syndrome (VHL; n = 19), succinate dehydrogenase B–D mutation (n = 21), neurofibromatosis type 1 (n = 1), RET (n = 1), no germline mutation (n = 20), or unknown mutation status (n = 6) (P = 0.84). All 8 patients with an SDHD mutation were true-positive on 18F-DOPA PET. There were 2 cases of false-negative results each in the group with SDHB (2/12) and VHL mutations (2/19) and 1 false-negative result in the subgroup of patients with unknown mutation status (1/6). Overall, 18F-DOPA PET yielded a sensitivity of 93% and a specificity of 88% for the detection of paragangliomas and pheochromocytomas on a patient basis (positive and negative predictive value, 94% and 85%, respectively). Conclusion: 18F-DOPA PET is a sensitive and specific imaging modality for the detection and staging of pheochromocytomas and paragangliomas in different genotypes, including VHL-, SDHB-, and SDHD-mutation carriers, and in patients with no germline mutation.

Co-targeting of convergent nucleotide biosynthetic pathways for leukemia eradication

Co-targeting of both de novo and salvage pathways for dCTP biosynthesis shows efficacy in T-ALL and B-ALL.

Fast Metabolic Response to Drug Intervention Through Analysis on a Miniaturized, Highly Integrated Molecular Imaging System

We report on a radiopharmaceutical imaging platform designed to capture the kinetics of cellular responses to drugs. Methods: A portable in vitro molecular imaging system comprising a microchip and a β-particle imaging camera permitted routine cell-based radioassays of small numbers of either suspended or adherent cells. We investigated the kinetics of responses of model lymphoma and glioblastoma cancer cell lines to 18F-FDG uptake after drug exposure. Those responses were correlated with kinetic changes in the cell cycle or with changes in receptor tyrosine kinase signaling. Results: The platform enabled direct radioassays of multiple cell types and yielded results comparable to those from conventional approaches; however, the platform used smaller sample sizes, permitted a higher level of quantitation, and did not require cell lysis. Conclusion: The kinetic analysis enabled by the platform provided a rapid (∼1 h) drug screening assay.

Application of a Rapid, Simple, and Accurate Adenovirus-Based Method to Compare PET Reporter Gene/PET Reporter Probe Systems

PurposeThis study aims to use a simple, quantitative method to compare the HSV1sr39TK/18 F-FHBG PET reporter gene/PET reporter probe (PRG/PRP) system with PRGs derived from human nucleoside kinases.ProceduresThe same adenovirus vector is used to express alternative PRGs. Equal numbers of vectors are injected intravenously into mice. After PRP imaging, quantitative hepatic PET signals are normalized for transduction by measuring hepatic viral genomes.ResultsThe same adenovirus vector was used to express equivalent amounts of HSV1sr39TK, mutant human thymidine kinase 2 (TK2-DM), and mutant human deoxycytidine kinase (dCK-A100VTM) in mouse liver. HSV1sr39TK expression was measured with 18 F-FHBG, TK2-DM and dCK-A100VTM with 18 F-L-FMAU. TK2-DM/18 F-L-FMAU and HSV1sr39TK/18 F-FHBG had equivalent sensitivities; dCK-A100VTM/18 F-L-FMAU was twice as sensitive as HSV1sr39TK/18 F-FHBG.ConclusionsThe human PRG/PRP sensitivities are comparable and/or better than HSV1sr39TK/18 F-FHBG. However, for clinical use, identification of the best PRP substrate for each enzyme, characterization of probe distribution, and consequences of overexpressing nucleoside kinases must be evaluated.

Abstract SY03-02: Tumor metabolic phenotyping and treatment stratification by positron emission tomography

The ability to noninvasively measure key metabolic pathways in living subjects may assist the development of new, more efficacious therapies against cancer. Here we show that two new positron emission tomography (PET) probes, 1-(2′-deoxy-2′-18F-fluoro-β-D-arabinofuranosyl)cytosine (18F-FAC) and 1-(2′-deoxy-2′-18F-fluoro-β-L-arabinofuranosyl)-5-methylcytosine (L-18F-FMAC), detect distinct phenotypic signatures of tumor nucleoside metabolism. 18F-FAC and L-18F-FMAC accumulation in tumors measured in vivo by PET is indicative of the activities of deoxycytidine kinase (dCK) and cytidine deaminase (CDA), two key enzymes in nucleic acid metabolism. Metabolic subtypes detected by 18F-FAC and L-18F-FMAC were shown to correspond to the differential utilization by cancer cells of the nucleoside salvage pathway to generate and maintain deoxyribonucleotide pools required to sustain tumor growth. We also show that the metabolic subtypes defined by 18F-FAC and L-18F-FMAC PET measurements correlate with markedly distinct sensitivities to therapeutic interventions. These findings support the utility of PET-based phenotyping of tumor nucleoside metabolism for treatment stratification in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY03-02. doi:10.1158/1538-7445.AM2011-SY03-02