Polynucleotides encoding bispecific chimeric antigen receptors

1. A polynucleotide, which encodes a bispecific chimeric antigen receptor, comprising

a. a nucleic acid sequence encoding at least two antigen-specific targeting regions, each antigen-specific targeting region comprising a single-domain antibody;

b. a nucleic acid sequence encoding an extracellular spacer domain;

c. a nucleic acid sequence encoding a transmembrane domain;

d. a nucleic acid sequence encoding at least one co-stimulatory domain; and

e. a nucleic acid sequence encoding an intracellular signaling domain.

2. The polynucleotide of claim 1 , wherein the nucleic acid sequence encoding at least two antigen-specific targeting regions encodes at least two antigen-specific targeting regions which bind (i) CD19 and CD20, (ii) CD20 and L1-CAM, (iii) L1-CAM and GD2, (iv) EGFR and L1-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viii) C-MET and HER2, or (ix) EGFR and ROR1;

the nucleic acid sequence encoding an extracellular spacer domain encodes an extracellular space that comprises any one or more of an Fc fragment of an antibody, a hinge region of an antibody, a constant domain of heavy chain CH2 region of an antibody, a CH3 region of an antibody, and combinations thereof;

the nucleic acid sequence encoding a transmembrane domain encodes a transmembrane domain that comprises any one or more of a transmembrane domain of a zeta chain of a T cell receptor complex, CD28, CD8a, and combinations thereof;

the nucleic acid sequence encoding at least one co-stimulatory domain encodes at least one co-stimulatory domain that comprises a signaling domain from any one or more of CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, intercellular adhesion molecule 1 (ICAM-1), lymphocyte function-associated antigen 1 (LFA-1), Lck, tumor necrosis factor receptor type I (TNFR-I), TNFR-II, Fas, CD30, CD40 and combinations thereof;

and the nucleic acid sequence encoding intracellular signaling domain encodes an intracellular signaling domain that comprises a signaling domain of one or more of a human CD3 zeta chain, FcγRIII, FcεRI, a cytoplasmic tail of a Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, and combinations thereof.

3. The polynucleotide of claim 1 , wherein the polynucleotide further comprises a nucleic acid sequence encoding a therapeutic control, and the therapeutic control comprises any one or more of: truncated epidermal growth factor receptor (EGFRt), thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyl transferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase/linamarin/glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP)/indole-3-acetic (IAA), gamma-glutamylcysteine synthetase, CD20/alphaCD20, CD34/thymidine kinase chimera, dox-depedent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903/Fas system, a chimeric cytokine receptor (CCR), and a selection marker.

4. A vector comprising the polynucleotide of claim 1 .

5. A virus comprising the polynucleotide of claim 1 .

6. The virus of claim 5 , wherein the virus is an RNA virus.

7. The virus of claim 5 , wherein the virus is a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, a pox virus or a herpes virus.

8. A genetically engineered cell, comprising the polynucleotide of claim 1 , a chimeric antigen receptor encoded by the polynucleotide of claim 1 , or a combination of a bispecific chimeric antigen receptor and a therapeutic control as encoded by the polynucleotide of claim 1 .

9. The genetically engineered cell of claim 8 , wherein the cell is a T-lymphocyte (T-cell).

10. The genetically engineered cell of claim 9 , wherein the cell is a nave T-cell, a central memory T-cell, an effector memory T-cell, or a combination thereof.

11. The genetically engineered cell of claim 8 , wherein the cell is a natural killer (NK) cell, a hematopoietic stem cell (HSC), an embryonic stem cell, or a pluripotent stem cell.

12. A polynucleotide, which encodes a bispecific chimeric antigen receptor, comprising:

a. a nucleic acid sequence encoding at least two antigen-specific targeting regions, each antigen-specific targeting region comprising a single-domain antibody and binding a different antigen;

b. a nucleic acid sequence encoding an extracellular spacer domain;

c. a nucleic acid sequence encoding a transmembrane domain;

d. a nucleic acid sequence encoding at least one co-stimulatory domain; and

e. a nucleic acid sequence encoding an intracellular signaling domain.

13. A vector comprising the polynucleotide of claim 12 .

14. A virus comprising the polynucleotide of claim 12 .

15. The virus of claim 14 , wherein the virus is an RNA virus.

16. The virus of claim 14 , wherein the virus is a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, a pox virus or a herpes virus.

17. A genetically engineered cell, comprising the polynucleotide of claim 12 or a chimeric antigen receptor encoded by the polynucleotide of claim 12 .

18. The genetically engineered cell of claim 17 , wherein the cell is a T-lymphocyte (T-cell).

19. The genetically engineered cell of claim 18 , wherein the cell is a nave T-cell, a central memory T-cell, an effector memory T-cell, or a combination thereof.

20. The genetically engineered cell of claim 17 , wherein the cell is a natural killer (NK) cell, a hematopoietic stem cell (HSC), an embryonic stem cell, or a pluripotent stem cell.

21. The polynucleotide of claim 12 , wherein the polynucleotide further comprises a nucleic acid sequence encoding truncated epidermal growth factor receptor (EGFRt).

22. The genetically engineered cell of claim 17 , wherein the cell further comprises truncated epidermal growth factor receptor (EGFRt) or a nucleic acid sequence encoding the EGFRt.