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. A composition comprising a nucleic acid comprising a nucleotide sequence that encodes a chimeric protein comprising a FKBP 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
a) the chimeric protein has a basal activity less than 50% of the basal activity of a chimeric protein comprising a FKBP 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; and
b) Caspase-9 activity is induced upon binding of a ligand to the FKBP multimeric ligand binding region of the chimeric protein.
2. The composition of claim 1 , wherein the modified Caspase-9 polypeptide comprises the amino acid sequence of SEQ ID NO: 26.
3. The composition of claim 1 , wherein the FKBP multimeric ligand binding region comprises a F v′ , F vis amino acid sequence.
4. The composition of claim 1 , wherein the ligand is dimeric.
5. The composition of claim 3 , wherein the ligand is dimeric FK506, or a dimeric FK506-like analog.
6. The composition of claim 5 , wherein the ligand is AP1903 or AP20187.
7. The composition of claim 1 , wherein the basal activity is measured using a secreted alkaline phosphatase (SEAP) reporter-based surrogate killing assay.
8. The composition of claim 1 , wherein the modified Caspase-9 polypeptide further comprises the amino acid substitution D330A.
9. The composition of claim 8 , wherein the modified Caspase-9 polypeptide comprises the amino acid sequence of SEQ ID NO: 28.