Chimeric antigen receptor (CAR) targeting multiple antigens, compositions and methods of use thereof

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 first polynucleotide encoding a first chimeric antigen receptor polypeptide (CAR), a second polynucleotide encoding a second chimeric antigen receptor polypeptide (CAR), a nucleotide encoding a viral self-cleavage peptide disposed between the first CAR and second CAR, under the transcriptional control of a single promoter, wherein:

• (i) the first CAR comprises a first signal peptide, a first antigen recognition domain, a first hinge region, a first transmembrane domain, a first 4-1BB or CD28 co-stimulatory domain, and a first CD3ζ signaling domain that form a first fusion protein;
• and
• (ii) the second CAR comprises a second signal peptide, a second antigen recognition domain, a second hinge region, a second transmembrane domain, a second 4-1BB or CD28 co-stimulatory domain, and a second CD3ζ signaling domain that form a second fusion protein;
• and
• wherein the first antigen recognition domain and the second antigen recognition domain are different and each bind to a different target, wherein when the target of one of the first antigen recognition domain or second antigen recognition domain is an scFv of CD33 or an scFv of CD123, then the target of the other antigen recognition domain is an scFv of C-type lectin-like molecule-1(CLL-1), 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).