Modulating expression of polypeptides via new gene switch expression systems
Disclosed herein are polynucleotides encoding ligand-inducible gene switch polypeptides, and systems comprising gene switch polypeptides for modulating the expression of a heterologous gene and an interleukin in a host cell. The compositions, methods and systems described herein facilitate ligand dependent expression of polypeptides including but not limited to cytokines and antigen binding polypeptides.
1. A method for integrating heterologous gene(s) into the genome of a peripheral blood mononuclear cell (PMBC), the method comprising delivering to the cell:
(a) a non-naturally occurring polynucleotide comprising (i) an AttP or AttB recombination sequence and (ii) the heterologous gene(s); and
(b) a polynucleotide encoding a SF370, SPβc2, Bxb1, A118, or φRv1 serine recombinase;
wherein the heterologous gene(s) encode: (i) a chimeric antigen receptor; and (ii) a cell tag comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 190-195 and 197-200.
2. The method of claim 1 , wherein the PBMCs are T cells or NK cells.
3. The method of claim 1 , wherein the non-naturally occurring polynucleotide of (a) comprises an AttP recombination sequence.
4. The method of claim 1 , wherein the non-naturally occurring polynucleotide of (a) comprises an AttB recombination sequence.
5. The method of claim 1 , wherein the serine recombinase is an SF370 recombinase.
6. The method of claim 1 , wherein the serine recombinase is an SPβc2 recombinase.
7. The method of claim 1 , wherein the serine recombinase is a Bxb1 recombinase.
8. The method of claim 1 , wherein the serine recombinase is an A118 recombinase.
9. The method of claim 1 , wherein the serine recombinase is a φRv1 recombinase.
10. The method of claim 1 , wherein the delivery of the polynucleotides is by transfection, transformation, nucleofection, or transduction.
11. The method of claim 1 , wherein the delivery is by calcium phosphate precipitation, lipofection, particle bombardment, or microinjection.
12. The method of claim 1 , wherein the delivery is by nucleofection.
13. The method of claim 1 , wherein the non-naturally occurring polynucleotide and the polynucleotide encoding the serine recombinase are contained in a single vector.
14. The method of claim 1 , wherein the non-naturally occurring polynucleotide further encodes a cytokine and/or a cell tag.
15. The method of claim 14 , wherein the cytokine is IL-1, IL-2, IL-12, IL-15, or IL-21, or a functional variant thereof.
16. The method of claim 14 , wherein the cytokine is a membrane bound cytokine.
17. The method of claim 16 , wherein the membrane bound cytokine is membrane bound IL-15 (mbIL-15).
18. The method of claim 1 , wherein the non-naturally occurring polynucleotide further encodes a fusion protein comprising: (a) IL-15, or a functional variant thereof; and (b) IL-15Rα, or a functional variant thereof.
19. The method of claim 18 , wherein the non-naturally occurring polynucleotide further encodes a fusion protein comprising a sequence having at least 90% identity with SEQ ID NO: 205.
20. The method of claim 1 , wherein the cell tag comprises the amino acid sequence of any one of SEQ ID NOs: 190-195 and 197-200.
21. The method of claim 1 , wherein the cell tag comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 190.
22. The method of claim 1 , wherein the cell tag comprises the amino acid sequence of SEQ ID NO: 190.
23. The method of claim 1 , wherein the chimeric antigen receptor is capable of binding CD19, CD33, BCMA, CD44, α-Folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HERS, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFRvIII, CD123, and/or VEGF-R2.
24. The method of claim 23 , wherein the chimeric antigen receptor is capable of binding CD19, EGFRvIII, CD33, ROR1, and/or MUC-16.
25. The method of claim 24 , wherein the chimeric antigen receptor comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 210 or 211.
26. The method of claim 24 , wherein the chimeric antigen receptor comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 214 and an amino acid sequence having at least 90% identity with SEQ ID NO: 215.
27. The method of claim 24 , wherein the chimeric antigen receptor comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 216.
28. The method of claim 24 , wherein the chimeric antigen receptor comprises:
(a) an amino acid sequence having at least 90% identity with SEQ ID NO: 218 and an amino acid sequence having at least 90% identity with SEQ ID NO: 219;
(b) an amino acid sequence having at least 90% identity with SEQ ID NO: 220 and an amino acid sequence having at least 90% identity with SEQ ID NO: 221;
(c) an amino acid sequence having at least 90% identity with SEQ ID NO: 222 and an amino acid sequence having at least 90% identity with SEQ ID NO: 223;
(d) an amino acid sequence having at least 90% identity with SEQ ID NO: 224 and an amino acid sequence having at least 90% identity with SEQ ID NO: 225; or
(e) an amino acid sequence having at least 90% identity with SEQ ID NO: 226 and an amino acid sequence having at least 90% identity with SEQ ID NO: 227.
29. The method of claim 24 , wherein the chimeric antigen receptor comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 228-232.
30. The method of claim 24 , wherein the chimeric antigen receptor comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 233-244.