Tumor-specific IFNA secretion by car T-cells to reprogram the solid tumor microenvironment
The present application relates to fusion proteins, chimeric antigen bearing cells expressing fusion proteins and compositions comprising chimeric antigen bearing cells expressing fusion proteins. The application further relates to methods of using the fusion proteins, cells and compositions for modulating an immune response.
1. A nucleic acid encoding a fusion protein, the nucleic acid comprising:
a first polynucleotide encoding an extracellular domain of a mutant PD-1 protein selected from a PD-1 extracellular domain of a protein encoded by the nucleotide sequence of any one of SEQ ID NOs 14, 16 or 17;
a second polynucleotide encoding a spacer; and
a third polynucleotide encoding an interferon-α2 protein;
wherein the fusion protein comprising the extracellular domain of a mutant PD-1 protein is capable of specifically binding to a PD-L1 protein with an increased affinity compared to a fusion protein comprising an extracellular domain of a wild-type PD-1 protein.
2. The nucleic acid of claim 1 , wherein the extracellular domain of a mutant PD-1 protein comprises the amino acid sequence of SEQ ID NO:05, or SEQ ID NO:06.
3. The nucleic acid of claim 1 , wherein the interferon-α2 protein is IFNα2a.
4. The nucleic acid of claim 3 , wherein the IFNα2a is encoded by the nucleotide sequence of SEQ ID NO:01.
5. The nucleic acid of claim 1 , wherein the spacer comprises at least 3 consecutive glycine residues.
6. The nucleic acid of claim 5 , wherein the spacer comprises the amino acid sequence selected from the group consisting of SEQ ID NO:07, SEQ ID NO:08, and SEQ ID NO:09.
7. The nucleic acid of claim 1 , further comprising an elongation factor-1 (EF-1) promoter.
8. The nucleic acid of claim 1 , further comprising:
a polynucleotide encoding a marker selected from a truncated HER2 polypeptide (Her2tG) or a truncated epidermal growth factor receptor polypeptide (EGFRt); and
a polynucleotide encoding a T2A self-cleaving linker, wherein the polynucleotide encoding a T2A self-cleaving linker is located between the nucleic acid encoding the fusion protein and the polynucleotide encoding the marker.
9. A vector comprising the nucleic acid of claim 1 .
10. The vector of claim 9 , wherein the vector comprises a lentiviral vector.
11. A cell comprising the nucleic acid of claim 1 .
12. The cell of claim 11 , further comprising a nucleic acid encoding a chimeric antigen receptor (CAR).
13. The cell of claim 12 , wherein the CAR comprises an anti-CD19 CAR.
14. The cell of claim 11 , wherein the cell is selected from a lymphocyte, a NK cell, a precursor T-cell, a stem cell, a hematopoietic stem cell, a NK cell, a neuronal stem cell, or a B cell.
15. The cell of claim 11 , wherein the cell is a CD8+ T cell or a CD4+ T cell.
16. The nucleic acid of claim 1 , wherein the extracellular domain of a wild-type PD-1 protein comprises the amino acid sequence of SEQ ID NO:04.
17. The nucleic acid of claim 1 , wherein the extracellular domain of a mutant PD-1 protein comprises the amino acid sequence of SEQ ID NO:05.
18. The nucleic acid of claim 1 , wherein the extracellular domain of a mutant PD-1 protein is selected from a PD-1 extracellular domain of a protein encoded by the nucleotide sequence of SEQ ID NO:16, or SEQ ID NO:17.
19. A nucleic acid encoding a fusion protein, the nucleic acid comprising:
a first polynucleotide encoding an extracellular domain of a PD-1 comprising the amino acid sequence of SEQ ID NO:05 or SEQ ID NO:06;
a second polynucleotide encoding a spacer; and
a third polynucleotide encoding an interferon, wherein the interferon is encoded by the nucleotide sequence of SEQ ID NO:01.