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Clinical Trial
. 2012 Jan 2;287(1):446-454.
doi: 10.1074/jbc.M111.314666. Epub 2011 Nov 9.

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

Dean O Campbell  1 , Shahriar S Yaghoubi  2 , Ying Su  3 , Jason T Lee  1 , Martin S Auerbach  1 , Harvey Herschman  4 , Nagichettiar Satyamurthy  1 , Johannes Czernin  1 , Arnon Lavie  5 , Caius G Radu  6
Affiliations
Clinical Trial

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

Dean O Campbell et al. J Biol Chem. .

Abstract

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.

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Figures

FIGURE 1.
FIGURE 1.
Biodistribution of l-18F-FMAU and 18F-FHBG in mice. A, chemical structures of l-18F-FMAU and 18F-FHBG are shown. B, MicroPET/CT scans of C57/BL6 mice 3 h after injection of l-18F-FMAU (left) and 18F-FHBG (right) are shown. Images are co-registered displays of the separate microPET and microCT scans. Quantifications of the PET signals are listed in supplemental Table 1. B, bladder; GB, gallbladder. %ID/g, % injected dose/g.
FIGURE 2.
FIGURE 2.
Evaluation of the TK2-N93D mutant. A, model of WT TK2 bound with l-dT in both the closed (green) and open (pink) conformation of the enzyme. ADP is bound in the phosphate donor pocket shown in this model. The enzyme is active in the closed conformation, which is stabilized by bonds between residues Asn-93 and Glu-200. When asparagine 93 is mutated to a glutamine, the bonds are disrupted, and the enzyme is predicted to switch to an open (inactive) conformation. B, l-FMAU kinase assay using recombinant WT TK2 and TK2-N93D in the presence of increasing concentrations of dTTP is shown. C, l-18F-FMAU uptake assay using WT TK2- or TK2-N93D-expressing L1210 cells is shown. Probe uptake values are reported relative to a control L1210 cell line that expresses YFP. Results are for a representative experiment or n = 2 experiments. D, l-18F-FMAU microPET/CT scans of mice bearing L1210 tumors engineered to express various PRGs (TK2, L1210-TK2; N93D, L1210-TK2-N93D; YFP, L1210-YFP). E, shown is quantification of PET scans from panel D. %ID/g, % injected dose/g.
FIGURE 3.
FIGURE 3.
Evaluation of L109F and N93D/L109F TK2 mutants. A, a homology model of TK2 bound with l-dT (pink) and dT (green) is shown. The TK2 model (solid residues) is overlaid on a crystal structure of dCK (light colored residues) with bound substrates. The TK2 residue Leu-109 is highlighted in gold. B, a l-FMAU kinase assay using recombinant TK2-L109F and TK2-N93D/L109F in the presence of increasing dTTP concentrations is shown. C, shown is an in vitro l-18F-FMAU uptake assay using L1210 cells expressing either TK2-N93D, TK2-L109F, or TK2-N93D/L109F. The assay was done in either the presence or absence of 5 μm d-dT. Probe uptake values are reported relative to a control L1210 cell line that expresses YFP. Results are for a representative experiment or n = 2 experiments. p = 0.005 between N93D and N93D/L109F in the presence of 0 μm dT, and p = 0.0008 between N93D and N93D/L109F in the presence of 5 μm dT.
FIGURE 4.
FIGURE 4.
Comparison of ΔTK2/l-18F-FMAU and sr39tk/18F-FHBG PET reporter gene systems. A, shown are l-18F-FMAU microPET/CT scans of mice bearing L1210 tumors engineered to express various TK2-based PRGs. B, shown are 18F-FHBG microPET/CT scans of mice bearing L1210 tumors engineered to express sr39tk. C, shown is quantification of probe uptake in L1210 tumors from A and B. %ID/g, % injected dose/g.
FIGURE 5.
FIGURE 5.
Biodistribution of l-18F-FMAU and 18F-FHBG in humans. PET/CT scans of a healthy female (left) and a healthy male (right) volunteer 2 h after injection of l-18F-FMAU (A) and pretreatment glioma patient 2 h after injection of 18F-FHBG (B). B, bladder; GB, gallbladder; L, liver; M, myocardium. SUV, standard uptake value.
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