Methods for regulating protein function in cells in vivo using synthetic small molecules
Methods and compositions for the rapid and reversible destabilizing of specific proteins in vivo using cell-permeable, synthetic molecules are described.
1. An in vivo method for increasing the stability of IL-2 or TNF-α in a mammal comprising:
(a) introducing into one or more mammalian cells in vitro, a nucleic acid comprising a polynucleotide encoding a fusion protein, wherein the fusion protein comprises in an N-terminal to C-terminal direction:
i) a signal peptide, wherein the signal peptide facilitates secretion of the fusion protein,
(ii) a single-polypeptide chain, ligand-dependent, stability-affecting FKBP variant protein, comprising F36V and L106P amino acid substitutions fused to the signal peptide, and
(iii) IL-2 or TNF-α fused to the stability-affecting FKBP variant protein,
wherein said introducing generates one or more transformed mammalian cells expressing and secreting the fusion protein;
(b) implanting the one or more transformed cells from (a) into a mammal harboring a tumor, and
(c) administering a Shield1 ligand to the mammal from (b), wherein the Shield1 ligand binds to the stability-affecting FKBP variant protein and said binding increases stability of IL-2 or TNF-α by reducing degradation of the fusion protein, increases regulatory T-cell infiltration into tumor cells, and increases the survival rate of the mammal when compared to the same species of mammal constitutively expressing IL-2 or TNF-α.
2. The method of claim 1 , wherein the one or more mammal cells are stably transformed with the nucleic acid.
3. The method of claim 1 , wherein the nucleic acid sequence is in a viral vector.
4. The method of claim 3 , wherein the viral vector is a vaccinia virus.
5. The method of claim 1 , wherein the administering of the Shield1 ligand to the mammal is by injecting the ligand into the mammal intraperitoneally or intravenously.
6. The method of claim 1 , wherein the introducing of the nucleic acid sequence comprises transforming the one or more mammalian cells in culture with a plasmid comprising the nucleic acid to produce one or more stably transformed mammalian cells, and implanting the one or more transformed cells into a mouse.
7. The method of claim 1 , wherein administration of the Shield1 ligand to the mammal reduces the tumor burden of the mammal.
8. An in vivo method for reducing degradation of IL-2 or TNF-α in a mammal comprising:
(a) introducing into a mammal harboring a tumor, a vaccinia virus having a nucleic acid comprising a polynucleotide encoding a fusion protein, wherein the fusion protein comprises in an N-terminal to C-terminal direction:
i) a signal peptide, wherein the signal peptide facilitates secretion of the fusion protein,
(ii) a single-polypeptide chain, ligand-dependent, stability-affecting FKBP variant protein, comprising F36V and L106P amino acid substitutions fused to the signal peptide, and
(iii) IL-2 or TNF-α fused to the stability-affecting FKBP variant protein, and
wherein the IL-2 or TNF-α is expressed and secreted;
(b) administering a Shield1 ligand to the mammal from (a), wherein the Shield1 ligand binds to the stability-affecting FKBP variant protein and said binding increases stability of the IL-2 or TNF-α by reducing degradation of the fusion protein, increases regulatory T-cell infiltration into tumor cells, and increases the survival rate of the mammal when compared to the same species of mammal constitutively expressing IL-2 or TNF-α.
9. The method of claim 8 , wherein the administering of the Shield1 ligand to the mammal is by injecting the ligand into the mammal intraperitoneally or intravenously.
10. The method of claim 9 , wherein administration of the Shield1 ligand reduces the tumor burden of the mammal.
11. The method of claim 8 , wherein administration of the Shield1 ligand reduces the tumor burden of the mammal.
12. The method of claim 8 , wherein the fusion protein comprises in an N-terminal to C-terminal direction the signal peptide, the stability-affecting FKBP variant protein and IL-2 or TNF-α.
13. The method of claim 1 , wherein after the administering of the Shield1, ligand the level of IL-2 or TNF-α secreted by the transfected cells increases in a dose-dependent manner.
14. The method of claim 8 , wherein the level of IL-2 or TNF-α in the serum of the mammal administered the Shield1 ligand increases in a dose-dependent manner.