Bispecific CAR T-cells for solid tumor targeting

1. A T cell comprising a first chimeric antigen receptor (CAR) encoded by a first polynucleotide, and a second CAR encoded by a second polynucleotide, wherein the first CAR is different from the second CAR, and the first CAR and the second CAR each comprise a 4-1BB signaling domain, a CD3-zeta signaling domain, a transmembrane domain, and an extracellular spacer comprising an IgG hinge spacer, and wherein:

the first polynucleotide comprises: a first nucleic acid encoding a leader sequence, and a second nucleic acid 3′ of the first nucleic acid, wherein the second nucleic acid encodes a first ligand binding domain which specifically binds to a B cell specific cell surface molecule; and

the second polynucleotide comprises: a third nucleic acid encoding a leader sequence, and a fourth nucleic acid 3′ of the third nucleic acid, wherein the nucleic acid encodes a second ligand binding domain which specifically binds to a cell surface tumor specific molecule; wherein:

(i) the B cell specific cell surface molecule is selected from CD19 or CD20, and wherein the first ligand binding domain is encoded by a nucleotide sequence having at least 95% identity with any one of SEQ ID NOs: 12 or 14; and

(ii) the cell surface tumor specific molecule is EGFR, and wherein the second ligand binding domain comprises the amino acid sequence of SEQ ID NO:19.

2. The T cell of claim 1 , wherein the first polynucleotide or the second polynucleotide, further comprises:

an inducible promoter, wherein the inducible promoter is 5′ of the first nucleic acid or the third nucleic acid; or

a suicide gene system.

3. The T cell of claim 1 , wherein the T cell is selected from:

a CD8+ T cytotoxic lymphocyte cell selected from a naïve CD8+ T-cell, a CD8+ memory T-cell, a central memory CD8+ T-cell, a regulatory CD8+ T-cell, an IPS derived CD8+ T-cell, an effector memory CD8+ T-cell, or a bulk CD8+ T-cell; or

a CD4+ T helper lymphocyte cell selected from a naïve CD4+ T-cell, a CD4+ memory T-cell, a central memory CD4+ T-cell, a regulatory CD4+ T-cell, an IPS derived CD4+ T-cell, an effector memory CD4+ T-cell, or a bulk CD4+ T-cell.

4. The T cell of claim 1 , wherein the B cell specific cell surface molecule is CD20, and the first ligand binding domain comprises the amino acid sequence of SEQ ID NO:13.

5. The T cell of claim 1 , wherein the B cell specific cell surface molecule is CD19, and the first ligand binding domain comprises the amino acid sequence of SEQ ID NO:11.

6. The T cell of claim 1 , wherein:

the first polynucleotide comprises: a fifth nucleic acid encoding a linker, wherein the fifth nucleic acid is 3′ of the nucleic acid encoding the first CAR, and a sixth nucleic acid encoding a marker domain, wherein the sixth nucleic acid sequence is 3′ of the fifth nucleic acid; and

the second polynucleotide comprises: a seventh nucleic acid encoding a linker, wherein the seventh nucleic acid is 3′ of the nucleic acid encoding the second CAR, and an eighth nucleic acid encoding a marker domain, wherein the eighth nucleic acid is 3′ of the seventh nucleic acid sequence.

7. The T cell of claim 6 , wherein:

the leader sequence of the first polynucleotide or the second polynucleotide comprises a granulocyte-macrophage colony-stimulating factor signal sequence;

the linker of the first polynucleotide or the second polynucleotide comprises a ribosome skip sequence or an internal ribosome entry sequence (IRES); and

the marker domain of the first polynucleotide or the second polynucleotide comprises a truncated HER2 polypeptide (Her2tG) or a truncated EGFR polypeptide (EGFRt).

8. The T cell of claim 7 , wherein:

the leader sequence of the first polynucleotide or the second polynucleotide comprises the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:31;

the extracellular spacer of the first CAR or the second CAR comprises the amino acid sequence selected from SEQ ID NO:01, SEQ ID NO:03, or SEQ ID NO:39;

the 4-1BB signaling domain comprises the amino acid sequence of SEQ ID NO:07, and the CD3-zeta signaling domain comprises the amino acid sequence of SEQ ID NO:09;

the linker of the first polynucleotide or the second polynucleotide comprises the nucleotide sequence of SEQ ID NO:34; and

the marker domain of the first polynucleotide or the second polynucleotide comprises the amino acid sequence of SEQ ID NO:35 or SEQ ID NO:37.

9. A pharmaceutical composition comprising the T cell of claim 1 and a pharmaceutically acceptable excipient.

10. A T cell comprising:

a first chimeric antigen receptor (CAR) comprising a first ligand biding domain capable of specifically binding to a B cell specific cell surface molecule; and

a second CAR comprising a second ligand biding domain capable of specifically binding to a cell surface tumor specific molecule,

wherein the first CAR is different from the second CAR, and wherein the first CAR and the second CAR each comprise a 41-BB signaling domain, CD3-zeta signaling domain, a transmembrane domain, and an extracellular spacer comprising an IgG hinge spacer; and wherein:

(i) the B cell specific cell surface molecule is selected from CD19 or CD20, and wherein the first ligand binding domain is encoded by a nucleotide sequence having at least 95% identity with any one of SEQ ID NOs: 12 or 14, and

(ii) the cell surface tumor specific molecule is EGFR, and wherein the second ligand binding domain comprises the amino acid sequence of SEQ ID NO:19.

11. The T cell of claim 10 , wherein the B cell specific cell surface molecule is CD20, and the first ligand binding domain comprises the amino acid sequence of SEQ ID NO:13.

12. The T cell of claim 11 , wherein the B cell specific cell surface molecule is CD19, and the first ligand binding domain comprises the amino acid sequence of SEQ ID NO:11.

13. The T cell of claim 10 , wherein the T cell is a CD4+ T cell.

14. The T cell of claim 10 , wherein the T cell is a CD8+ T cell.