Car expression vector and car-expressing t cells

Abstract

An object of the present invention is to provide CAR-expressing T cells that coexpress a chimeric antigen receptor (CAR) and a T cell immune function-enhancing factor and have a high immunity-inducing effect and antitumor activity, and to provide a CAR expression vector for the preparation of the CAR-expressing T cells.A CAR expression vector comprises a nucleic acid encoding a chimeric antigen receptor (CAR) and a nucleic acid encoding a T cell immune function-enhancing factor, wherein the nucleic acid encoding an immune function-enhancing factor is a nucleic acid encoding interleukin-7 and a nucleic acid encoding CCL19, a nucleic acid encoding a dominant negative mutant of SHP-1, or a nucleic acid encoding a dominant negative mutant of SHP-2, or a CAR-expressing T cell introduced with the CAR expression vector are prepared.

1 . A CAR expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR), a nucleic acid encoding interleukin-7(IL-7), and a nucleic acid encoding CCL19. 
     
     
         2 . The CAR expression vector according to  claim 1 , wherein the nucleic acid encoding the CAR and the nucleic acids encoding IL-7 and CCL19 are linked via a sequence encoding a self-cleaving peptide. 
     
     
         3 . The CAR expression vector according to  claim 1 , wherein the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 are linked via a sequence encoding a self-cleaving peptide. 
     
     
         4 . The CAR expression vector according to  claim 1 , wherein the nucleic acid encoding the CAR contains a nucleic acid encoding a polypeptide of a CD8 transmembrane region. 
     
     
         5 . The CAR expression vector according to  claim 1 , wherein the nucleic acid encoding the CAR contains nucleic acids encoding one or more polypeptides selected from a CD28 intracellular region, a 4-1BB intracellular region, and a CD3 intracellular region. 
     
     
         6 . The CAR expression vector according to  claim 1 , comprising a nucleic acid encoding a suicide gene. 
     
     
         7 . An isolated CAR-expressing T cell, or a culture comprising the isolated CAR-expressing T cell, said isolated T cell prepared by introducing the CAR expression vector according to  claim 1 , and thereby expressing the CAR, IL-7, and CCL19. 
     
     
         8 . An isolated CAR-expressing T cell, or a culture comprising the isolated CAR-expressing T cell, said isolated T cell prepared by introducing the CAR expression vector according to  claim 2 , and thereby expressing the CAR, IL-7, and CCL19. 
     
     
         9 . An isolated CAR-expressing T cell, or a culture comprising the isolated CAR-expressing T cell, said isolated T cell prepared by introducing the CAR expression vector according to  claim 3 , and thereby expressing the CAR, IL-7 and CCL19. 
     
     
         10 . An isolated CAR-expressing T cell, or a culture comprising the isolated CAR-expressing T cell, said isolated T cell prepared by introducing the CAR expression vector according to  claim 6 , and thereby expressing the CAR, IL-7 and CCL19. 
     
     
         11 . An isolated CAR-expressing T cell, or a culture comprising the isolated CAR-expressing T cell, said isolated T cell prepared by introducing a first CAR expression vector that comprises a nucleic acid encoding a CAR and IL-7, and a second CAR expression vector that comprises a nucleic acid encoding a CAR and a nucleic acid encoding CCL19, and thereby expressing the CAR, IL-7, and CCL19. 
     
     
         12 . The isolated CAR-expressing T cell, or a culture comprising the isolated CAR-expressing T cell according to  claim 11 , said isolated T cell comprising a nucleic acid encoding a suicide gene in the first CAR expression vector, the second CAR expression vector, or both. 
     
     
         13 . An anticancer agent comprising the isolated CAR-expressing T cell or a culture comprising the isolated CAR-expressing T cell according to  claim 7  and a pharmaceutically acceptable additive. 
     
     
         14 . An anticancer agent comprising the isolated CAR-expressing T cell or a culture comprising the isolated CAR-expressing T cell according to  claim 8  and a pharmaceutically acceptable additive. 
     
     
         15 . An anticancer agent comprising the isolated CAR-expressing T cell or a culture comprising the isolated CAR-expressing T cell according to  claim 9  and a pharmaceutically acceptable additive. 
     
     
         16 . An anticancer agent comprising the isolated CAR-expressing T cell or a culture comprising the isolated CAR-expressing T cell according to  claim 10  and a pharmaceutically acceptable additive. 
     
     
         17 . An anticancer agent comprising the isolated CAR-expressing T cell or a culture comprising the isolated CAR-expressing T cell according to  claim 11  and a pharmaceutically acceptable additive. 
     
     
         18 . An anticancer agent comprising the isolated CAR-expressing T cell or a culture comprising the isolated CAR-expressing T cell according to  claim 12  and a pharmaceutically acceptable additive. 
     
     
         19 . The CAR expression vector of  claim 1 , wherein said IL-7 is human IL-7 and said CCL19 is human CCL19. 
     
     
         20 . The isolated CAR-expressing T cell or cell culture of  claim 7 , wherein said IL-7 is human IL-7 and said CCL19 is human CCL19. 
     
     
         21 . The isolated CAR-expressing T cell or cell culture of  claim 11 , wherein said IL-7 is human IL-7 and said CCL19 is human CCL19. 
     
     
         22 . An isolated T cell expressing a CAR, IL-7, and CCL19, or a culture comprising the isolated T cell, said isolated T cell comprising one or more recombinant constructs, wherein at least one recombinant construct encodes a CAR, at least one recombinant construct encodes IL-7 and at least one recombinant construct encodes CCL19. 
     
     
         23 . An anticancer agent comprising the isolated T cell or a culture comprising the isolated T cell according to  claim 22  and a pharmaceutically acceptable additive. 
     
     
         24 . A method for producing a T cell expressing a CAR, IL-7, and CCL19, comprising introducing a recombinant construct encoding the CAR, a recombinant construct encoding IL-7 and a recombinant construct encoding CCL19 into the T cell using a vector. 
     
     
         25 . An isolated T cell expressing a CAR from a recombinant construct encoding a CAR, IL-7 from a recombinant construct encoding IL-7, and CCL19 from a recombinant construct encoding CCL19. 
     
     
         26 . The CAR expression vector according to  claim 1 , wherein the vector is configured to integrate the nucleic acid encoding the CAR, the nucleic acid encoding IL-7, and the nucleic acid encoding CCL19 into a genome. 
     
     
         27 . The CAR expression vector according to  claim 26 , wherein the vector is a retrovirus vector, a lentivirus vector, an adeno-associated virus vector, or a transposon vector. 
     
     
         28 . The CAR expression vector according to  claim 1 , wherein the vector is configured for constant expression of the nucleic acid encoding the CAR, the nucleic acid encoding IL-7, and the nucleic acid encoding CCL19. 
     
     
         29 . The CAR expression vector according to  claim 1 , wherein the vector comprises control sequences selected from a group consisting of a promoter, a terminator, a drug resistance gene, and a reporter gene.