Modified Caspase polypeptides and uses thereof
The technology relates in part to compositions comprising modified Caspase-9 polypeptides, compositions comprising nucleic acids coding for modified Caspase-9 polypeptides, chimeric modified Caspase-9 polypeptides, and methods of use thereof, including methods for cell therapy. Methods for cell therapy include modifying transfused cells to express an inducible modified Caspase-9 protein, with reduced basal activity in the absence of the inducer.
1. An isolated cell transfected or transduced with a nucleic acid comprising a nucleotide sequence that encodes a chimeric protein comprising a FKBP12 multimeric ligand binding region and a modified Caspase-9 polypeptide, wherein the modified Caspase-9 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9, and comprises the amino acid substitution N405Q, wherein the chimeric protein has a Caspase-9 basal activity less than 50% of the basal activity of a chimeric protein comprising a FKBP12 multimeric ligand binding region and a Caspase-9 polypeptide lacking the caspase recruitment domain (CARD) and having the amino acid sequence of SEQ ID NO: 9.
2. The isolated cell of claim 1 , wherein Caspase-9 activity is induced upon binding of a ligand to the FKBP12 multimeric ligand region of the chimeric protein.
3. The isolated cell of claim 2 , wherein the ligand is AP1903 or AP20187.
4. The isolated cell of claim 2 , wherein the FKBP12 region is a modified FKBP12 polypeptide comprising an amino acid substitution at position 36 that binds with higher affinity to AP1903 or AP20187 than the wild type FKBP12 polypeptide.
5. The isolated cell of claim 4 , wherein the FKBP12 region is a FKBP12v36 polypeptide.
6. The isolated cell of claim 1 , wherein the cell is selected from the group consisting of an inducible progenitor cell (iPS), an embryonic stem (ES) cell, a mesenchymal stem cell, a plasma (B) cell, a myocyte and a T cell.
7. The isolated cell of claim 6 , wherein the cell is a T cell.
8. The isolated cell of claim 1 , wherein the chimeric protein further comprises a marker polypeptide.
9. The isolated cell of claim 1 , wherein the cell is transduced or transfected with a second nucleic acid that encodes a heterologous protein.
10. The isolated cell of claim 9 , wherein the second nucleic acid encodes a chimeric antigen receptor.
11. The isolated cell of claim 1 , wherein the modified Caspase-9 polypeptide comprises the amino acid sequence of SEQ ID NO: 26.
12. The isolated cell of claim 1 , wherein the modified Caspase-9 polypeptide further comprises the substituted amino acid residue D330A.
13. The isolated cell of claim 1 , wherein the basal activity is measured using a secreted alkaline phosphatase (SEAP) reporter-based surrogate killing assay.