title: 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.
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.
authors.affiliation: Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, California, 90095; Ahmanson Translational Imaging Division, UCLA, Los Angeles, California, 90095.
authors.forename: Dean O
authors.initials: DO
authors.lastname: Campbell
authors.affiliation: Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, California, 90095; Ahmanson Translational Imaging Division, UCLA, Los Angeles, California, 90095; CellSight Technologies, San Francisco, California 94107.
authors.forename: Shahriar S
authors.initials: SS
authors.lastname: Yaghoubi
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607.
authors.forename: Ying
authors.initials: Y
authors.lastname: Su
authors.affiliation: Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, California, 90095; Ahmanson Translational Imaging Division, UCLA, Los Angeles, California, 90095.
authors.forename: Jason T
authors.initials: JT
authors.lastname: Lee
authors.affiliation: Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, California, 90095; Ahmanson Translational Imaging Division, UCLA, Los Angeles, California, 90095.
authors.forename: Martin S
authors.initials: MS
authors.lastname: Auerbach
authors.affiliation: Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, California, 90095; Department of Biological Chemistry, UCLA, Los Angeles, California 90095.
authors.forename: Harvey
authors.initials: H
authors.lastname: Herschman
authors.affiliation: Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, California, 90095; Ahmanson Translational Imaging Division, UCLA, Los Angeles, California, 90095.
authors.forename: Nagichettiar
authors.initials: N
authors.lastname: Satyamurthy
authors.affiliation: Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, California, 90095; Ahmanson Translational Imaging Division, UCLA, Los Angeles, California, 90095.
authors.forename: Johannes
authors.initials: J
authors.lastname: Czernin
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607. Electronic address: Lavie@uic.edu.
authors.forename: Arnon
authors.initials: A
authors.lastname: Lavie
authors.affiliation: Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, California, 90095; Ahmanson Translational Imaging Division, UCLA, Los Angeles, California, 90095. Electronic address: CRadu@mednet.ucla.edu.
authors.forename: Caius G
authors.initials: CG
authors.lastname: Radu
mesh_terms: D000328:Adult; D000818:Animals; D001086:Arabinofuranosyluracil / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D005260:Female; D005462:Fluorine Radioisotopes; D017930:Genes, Reporter / Q000235:genetics*; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008875:Middle Aged; D008958:Models, Molecular; D010766:Phosphorylation; D049268:Positron-Emission Tomography / Q000379:methods*; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D013936:Thymidine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000493:pharmacokinetics; D013937:Thymidine Kinase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: C113308:9-(4-fluoro-3-hydroxymethylbutyl)guanine; D005462:Fluorine Radioisotopes; D001086:Arabinofuranosyluracil; D006147:Guanine; C105339:thymidine kinase 2; D013937:Thymidine Kinase; C034935:clevudine; D013936:Thymidine
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