CPC C12N 5/0636 (2013.01) [C07K 14/7051 (2013.01); C07K 14/70517 (2013.01); C07K 14/70521 (2013.01); C07K 14/70578 (2013.01); C07K 16/289 (2013.01); C12N 5/0646 (2013.01); C07K 2319/00 (2013.01); C07K 2319/02 (2013.01); C07K 2319/03 (2013.01); C07K 2319/50 (2013.01); C12N 2510/00 (2013.01)] | 4 Claims |
1. An ex vivo engineered T cell or NK cell co-expressing two distinct chimeric antigen receptor (CAR) units at the cell surface, wherein the engineered T cell or NK cell comprises a nucleotide sequence comprising from 5′ to 3′ a single promoter selected from human elongation factor-1 alpha (EF-1α) or spleen focus forming virus (SFFV), a first polynucleotide encoding a first chimeric antigen receptor polypeptide (CAR), a nucleotide encoding a viral self-cleavage peptide, a second polynucleotide encoding a second chimeric antigen receptor polypeptide (CAR), and an enhancer selected from the group consisting of secreted IL-15/IL-15sushi, Il-15/Il-15 sushi anchor, PD-1, PD-L1, CSF1R, CTLA-4, TIM-3, TGFR-beta, IL-2, Il-7, IL-12, IL-15, IL-15RA, IL-21 or a functional fragment thereof, or a combination thereof, wherein said enhancer is separated from the first CAR and second CAR by a second cleavage site that flanks either end of the two distinct CAR units, wherein:
the first CAR comprises a first signal peptide, a first antibody binding domain, a first hinge region, a first transmembrane domain, a first co-stimulatory domain, and a first signaling domain; and
the second CAR comprises a second signal peptide, a second antibody binding domain, a second hinge region, a second transmembrane domain, a second co-stimulatory domain, and a second signaling domain; and
wherein the first antibody binding domain and the second antibody binding domain are different and each bind to a different target, wherein the targets of the first and second antibody binding domains irrespective of order are CD19 and CD20, or CD123 and CD33, or B cell maturation antigen (BCMA) (CD269) and CD19, or BCMA (CD269) and CD38, or and BCMA (CD269) and CS1, wherein the first and second co-stimulatory domains are intracellular, and wherein the cleavage site is selected from the group consisting of porcine teschovirus-1 2A (P2A), thoseaasigna virus 2A (T2A), equine rhinitis A virus (ERAV) 2A (E2A), and FMDV 2A (F2A).
|