Selection methods for genetically-modified t cells

Abstract

In some aspects, isolated transgenic cells (e.g., transgenic T cells) are provided that comprise or express a transgene and DHFRFS and/or TYMSSS. Methods for selecting transgeneic cells are also provided.

1 - 74 . (canceled) 
     
     
         75 . An isolated engineered mammalian T cell expressing a first transgene and TYMS SS , wherein said T cell comprises (1) a nucleotide sequence encoding the first transgene and (2) a nucleotide sequence encoding TYMS SS . 
     
     
         76 . The isolated engineered mammalian T cell of  claim 75 , wherein the nucleotide sequence encoding the first transgene and the nucleotide sequence encoding TYMS SS  are operably linked. 
     
     
         77 . The isolated engineered mammalian T cell of  claim 75 , wherein the nucleotide sequence encoding the first transgene and the nucleotide sequence encoding TYMS SS , upon expression, are encoded on the same mRNA. 
     
     
         78 . The isolated engineered mammalian T cell of  claim 75 , wherein the nucleotide sequence encoding the first transgene and the nucleotide sequence encoding TYMS SS  are separated by an internal ribosomal entry site (IRES) or a ribosomal slip sequence. 
     
     
         79 . The isolated engineered mammalian T cell of  claim 75 , wherein nucleotide sequence encoding the first transgene is positioned 3′ relative to the nucleotide sequence encoding TYMS SS . 
     
     
         80 . The isolated engineered mammalian T cell of  claim 75 , wherein the first transgene is a chimeric antigen receptor (CAR) construct, a polypeptide hormone, a suicide gene, a T-cell receptor (TCR), a growth factor, or a cytokine. 
     
     
         81 . The isolated engineered mammalian T cell of  claim 80 , wherein the cytokine is IL-12 or IL-15. 
     
     
         82 . The isolated engineered mammalian T cell of  claim 75 , further comprising (3) a nucleotide sequence encoding DHFR FS . 
     
     
         83 . The isolated engineered mammalian T cell of  claim 82 , wherein the nucleotide sequence encoding DHFR FS  is operably linked to a nucleotide sequence encoding a second transgene. 
     
     
         84 . The isolated engineered mammalian T cell of  claim 83 , wherein the nucleotide sequence encoding the second transgene and the nucleotide sequence encoding DHFR FS , upon expression, are encoded on the same mRNA. 
     
     
         85 . The isolated engineered mammalian T cell of  claim 83 , wherein the nucleotide sequence encoding the second transgene and the nucleotide sequence encoding DHFR FS  are separated by an internal ribosomal entry site (IRES) or a ribosomal slip sequence. 
     
     
         86 . The isolated engineered mammalian T cell of  claim 83 , wherein the second transgene is a suicide gene, CAR, TCR, polypeptide hormone, cytokine, chemokine, or transcription factor. 
     
     
         87 . The isolated engineered mammalian T cell of  claim 75 , wherein the isolated engineered mammalian T cell is a T helper cell (TH cell), cytotoxic T cell (Tc cell or CTL), memory T cell (TCM cell), effector T cell (TEM cell), regulatory T cell (Treg cell; also known as suppressor T cell), natural killer T cell (NKT cell), mucosal associated invariant T cell, alpha-beta T cell (Tαβ cell), or gamma-delta T cell (Tγδ cell). 
     
     
         88 . A method of treating a patient with a cancer comprising to administering to the patient a therapeutically effective amount of the isolated engineered mammalian T cells of  claim 75 . 
     
     
         89 . A method of enriching for regulatory T cells in a population of T cells isolated from a mammal, the method comprising contacting the population of T cells with a thymidine synthesis inhibitor selected from the group consisting of methotrexate (MTX), 5-FU, Raltitrexed, and Pemetrexed, or a combination thereof, to selectively deplete effector T cells in the population. 
     
     
         90 . The method of  claim 89 , wherein the population of T cells isolated from a mammal is contacted with both MTX and 5-FU. 
     
     
         91 . The method of  claim 89 , wherein the T cells express one or both of DHFR FS  and TYMS SS . 
     
     
         92 . A method for selecting a T cell expressing a transgene of interest comprising:
 a) applying a thymidine synthesis inhibitor to a plurality of T cells that comprises a T cell expressing the transgene of interest and TYMS SS ; and   b) selecting for one or more T cells surviving after seven or more days of application of the thymidine synthesis inhibitor, wherein the one or more surviving T cell(s) expresses the transgene of interest and TYMS SS .   
     
     
         93 . The method of  claim 92 , wherein the T cell expressing the transgene of interest and TYMS SS  further expresses DHFR FS . 
     
     
         94 . The method of  claim 92 , wherein the thymidine synthesis inhibitor is selected from the group consisting of methotrexate (MTX), 5-FU, Raltitrexed, and Pemetrexed.