Modification of CAR-T cells
Modified immune cells are provided, the modified immune cells expressing a heterologous polynucleotide comprising a nucleotide sequence encoding a function (e.g., at least one of persistence, proliferation, or cytotoxicity) booster, e.g., an apoptosis inhibitor. In one aspect, the modified T cells further comprise a chimeric antigen receptor. Methods, kits, and components for making and using the modified immune cells are also provided.
1. A modified T cell expressing a heterologous polynucleotide, the heterologous polynucleotide comprising:
(A) a first gene comprising a nucleotide sequence encoding a protein selected from the group consisting of Bcl-xL, Survivin, or CD28-D124E/T195P, wherein the first gene is operably linked to a first heterologous regulatory sequence effective for expression of the protein within a T cell, thereby enhancing survival of the T cell; and
(B) a second gene comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain that specifically binds to a CD19 antigen, and wherein the second gene is operably linked to a second heterologous regulatory sequence effective for expression of the CAR in the T cell,
wherein the first heterologous regulatory sequence and the second heterologous regulatory sequence are separate regulatory sequences, and
wherein the heterologous polynucleotide is integrated into the genome of the modified T cell.
2. The modified immune cell of claim 1 , wherein the CAR further comprises a costimulatory polypeptide selected from CD28 or 4-1BB.
3. A pharmaceutical composition comprising the modified T cell of claim 1 .
4. A method for preparing a modified T cell,
wherein the method comprises:
introducing a heterologous polynucleotide into a T cell ex-vivo, the heterologous polynucleotide comprising:
(A) a first gene comprising a nucleotide sequence encoding a protein selected from the group consisting of Bcl-xL, Survivin, or CD28-D124E/T195P, wherein the first gene is operably linked to a first heterologous regulatory sequence effective for expression of the protein within the T cell, thereby enhancing survival of the T cell; and
(B) a second gene comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain that specifically binds to a CD19 antigen, and wherein the second gene is operably linked to a second heterologous regulatory sequence effective for expression of the CAR in the T cell,
wherein the first heterologous regulatory sequence and the second heterologous regulatory sequence are separate regulatory sequences.
5. The method of claim 4 , wherein the heterologous polynucleotide comprises a transposon.
6. The method of claim 5 , wherein:
the heterologous polynucleotide is flanked by a pair of transposon ends,
wherein a corresponding transposase is introduced into the T cell, and
wherein the heterologous polynucleotide is transposable by the transposase.
7. The method of claim 6 , wherein the transposase is introduced as a nucleic acid encoding the transposase.
8. The method of claim 7 , wherein the nucleic acid encoding the transposase is an mRNA.
9. The method of claim 4 , wherein the heterologous polynucleotide comprises a lentivirus.
10. The modified T cell of claim 1 , wherein the protein is Bcl-xL.
11. The modified T cell of claim 1 , wherein the protein is Survivin.
12. The modified T cell of claim 1 , wherein the protein is CD28-D124E/T195P.
13. The modified T cell of claim 2 , wherein the costimulatory polypeptide is CD28.
14. The modified T cell of claim 2 , wherein the costimulatory polypeptide is 4-1BB.
15. A polynucleotide, comprising:
(A) a first gene comprising a nucleotide sequence encoding a protein selected from the group consisting of Bcl-xL, Survivin, or CD28-D124E/T195P, wherein the first gene is operably linked to a first regulatory sequence effective for expression of the protein within a T cell, thereby enhancing survival of the T cell; and
(B) a second gene comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain that specifically binds to a CD19 antigen, and wherein the second gene is operably linked to a second regulatory sequence effective for expression of the CAR in the T cell,
wherein the first regulatory sequence and the second regulatory sequence are separate sequences.
16. The polynucleotide of claim 15 , wherein the CAR further comprises a costimulatory polypeptide selected from CD28 or 4-1BB.
17. The polynucleotide of claim 16 , wherein the costimulatory polypeptide is CD28.
18. The polynucleotide of claim 16 , wherein the costimulatory polypeptide is 4-1BB.
19. A transposon comprising the polynucleotide according to claim 15 .
20. A lentiviral vector comprising the polynucleotide according to claim 15 .