Dual car-t cells

An isolated polynucleotide comprising, from 5′ to 3′:

• a) a promoter (such as the EF1alpha promoter);
• b) a nucleic acid encoding a CAR20, wherein said CAR20 comprises:
• i) at least one extracellular domain comprising:
• an antigen binding domain specific for CD20 comprising a Variable Heavy chain (VH) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with SEQ ID NO:
• 15 and comprising the H-CDRs of amino acid sequences SEQ ID NO:
• 47, SEQ ID NO:
• 48, and SEQ ID NO:
• 49, and a Variable Light chain (VL) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with SEQ ID NO:
• 16 and comprising the L-CDRs of amino acid sequences SEQ ID NO:
• 50, SEQ ID NO:
• 51, and SEQ ID NO:
• 52, optionally a leader sequence,
• a hinge domain from CD8alpha,
• ii) a transmembrane domain from CD8alpha, and
• iii) an intracellular domain comprising a 4-1BB stimulatory domain and a CD3zeta signaling domain;
• c) a nucleic acid sequence encoding a self-cleaving peptide (such as P2A, T2A, E2A, or F2A);
• d) a nucleic acid encoding a CAR22, wherein said CAR22 comprises:
• i) at least one extracellular domain comprising:
• an antigen binding domain specific for CD22 comprising a Variable Heavy chain (VH) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with SEQ ID NO:
• 11 and comprising the H-CDRs of amino acid sequences SEQ ID NO:
• 41, SEQ ID NO:
• 42, and SEQ ID NO:
• 43, and a Variable Light chain (VL) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with SEQ ID NO:
• 12 and comprising the L-CDRs of amino acid sequences SEQ ID NO:
• 44, SEQ ID NO:
• 45, and SEQ ID NO:
• 46, optionally a leader sequence,
• a hinge domain from CD8alpha,
• ii) a transmembrane domain from CD8alpha, and
• iii) an intracellular domain comprising a 4-1BB stimulatory domain and a CD3zeta signaling domain;
• wherein the promoter of a) controls expression of said CAR20 and said CAR22.

A genetically engineered T cell expressing a Chimeric Antigen Receptor (CAR) that specifically binds to CD20 (CAR20) and a CAR that specifically binds to CD22 (CAR22) at its cell surface, wherein said CAR22 and said CAR20 are encoded by an exogenous nucleic acid incorporated in the genome of said T cell, and wherein said exogenous nucleic acid comprises, from 5′ to 3′:

• a) a promoter,
• b) a nucleic acid encoding said CAR20, wherein said CAR20 comprises the amino acid sequence of SEQ ID NO:
• 18, optionally without the leader sequence of SEQ ID NO:
• 1,
• c) a nucleic acid encoding a self-cleaving peptide, and
• d) a nucleic acid encoding said CAR22, wherein said CAR22 comprises the amino acid sequence of SEQ ID NO:
• 14, optionally without the leader sequence of SEQ ID NO:
• 1,
• wherein the promoter of a) controls expression of said CAR20 and said CAR22;
• wherein said genetically engineered T cell comprises at least one allele encoding TCR alpha, TCR beta, and/or CD3 that has been inactivated;
• and
• wherein said genetically engineered T cell has at least one inactivated CD52 allele.