title: Efficient selection of genetically modified human T cells using methotrexate-resistant human dihydrofolate reductase.
abstract: Genetic modification of human T cells to express transgene-encoded polypeptides, such as tumor targeting chimeric antigen receptors, is an emerging therapeutic modality showing promise in clinical trials. The development of simple and efficient techniques for purifying transgene(+) T cells is needed to facilitate the derivation of cell products with uniform potency and purity. Unlike selection platforms that utilize physical methods (immunomagnetic or sorting) that are technically cumbersome and limited by the expense and availability of clinical-grade components, we focused on designing a selection system on the basis of the pharmaceutical drug methotrexate (MTX), a potent allosteric inhibitor of human dihydrofolate reductase (DHFR). Here, we describe the development of self inactivating (SIN) lentiviral vectors that direct the coordinated expression of a CD19-specific chimeric antigen receptor (CAR), the human EGFRt tracking/suicide construct, and a methotrexate-resistant human DHFR mutein (huDHFR(FS); L22F, F31S). Our results demonstrate that huDHFR(FS) expression renders lentivirally transduced primary human CD45RO(+)CD62L(+) central memory T cells resistant to lymphotoxic concentrations of MTX up to 0.1 μM. Our modular complementary DNA (cDNA) design insures that selected MTX-resistant T cells co-express functionally relevant levels of the CD19-specific CAR and EGFRt. This selection system on the basis of huDHFR(FS) and MTX has considerable potential utility in the manufacturing of clinical-grade T cell products.
authors.affiliation: Departments of Cancer Immunotherapeutics and Tumor Immunology, and Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA.
authors.forename: M
authors.initials: M
authors.lastname: Jonnalagadda
authors.forename: C E
authors.initials: CE
authors.lastname: Brown
authors.forename: W C
authors.initials: WC
authors.lastname: Chang
authors.forename: J R
authors.initials: JR
authors.lastname: Ostberg
authors.forename: S J
authors.initials: SJ
authors.lastname: Forman
authors.forename: M C
authors.initials: MC
authors.lastname: Jensen
mesh_terms: D018941:Antigens, CD19 / Q000235:genetics; D060846:Cell Engineering; D005493:Folic Acid Antagonists / Q000494:pharmacology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008213:Lymphocyte Activation / Q000187:drug effects / Q000235:genetics; D008727:Methotrexate / Q000494:pharmacology; D013601:T-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
chemical_list: D018941:Antigens, CD19; D005493:Folic Acid Antagonists; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate