Mirac proteins
This disclosure relates to a method of generating conditionally active biologic proteins from wild type proteins, in particular therapeutic proteins, which are reversibly or irreversibly inactivated at the wild type normal physiological conditions. For example, evolved proteins are virtually inactive at body temperature, but are active at lower temperatures.
1. A method of preparing a conditionally active polypeptide, the method comprising the steps of:
i. evolving DNA which encodes a template polypeptide using one or more evolutionary techniques to create a first set of mutant DNAs;
ii. expressing and assaying the first set of mutant DNAs to obtain at least one first mutant polypeptide which exhibits both:
(a) a decrease in activity in an assay at a normal physiological condition that is within a normal range of the physiological condition at a site of administration of the first mutant polypeptide to a subject, or at a tissue or organ at a site of action of the first mutant polypeptide of a subject, when compared to the activity of the template polypeptide in the assay at the same normal physiological condition, and
(b) an increase in activity in an assay under an aberrant condition that deviates from the normal range of the physiological condition at the site of administration of the first mutant polypeptide, or at the tissue or organ at the site of action of the first mutant polypeptide, when compared to the activity of the template polypeptide in the assay at the same aberrant condition; and
iii. selecting a first conditionally active polypeptide from the at least one first mutant polypeptide which exhibits both:
(a) the decrease in the activity in the assay at the normal physiological condition when compared to the activity of the template polypeptide in the assay at the same normal physiological condition, and
(b) the increase in the activity in the assay under the aberrant condition when compared to the activity of the template polypeptide in the assay at the same aberrant condition; and
wherein the normal physiological condition and aberrant condition are the same condition selected from the group consisting of temperature, pH, osmotic pressure, oxidation, osmolality, and electrolyte concentration.
2. The method of claim 1 , wherein the first conditionally active polypeptide is a fragment of a protein and the first conditionally active polypeptide is at least 10 amino acids in length.
3. The method of claim 1 , wherein the first conditionally active polypeptide is codon optimized for an organism or cell.
4. The method of claim 3 , further comprising a step of:
iv. codon optimizing the firstconditionally active polypeptide selected in step iii for an organism or cell.
5. The method of claim 1 , wherein the evolving step comprises a technique selected from the group consisting of non-stochastic polynucleotide chimerization and non-stochastic site-directed point mutagenesis.
6. The method of claim 1 , wherein the evolving step comprises a technique selected from the group consisting of substituting one or more nucleotides for a different nucleotide, deleting one or more nucleotides, adding one or more nucleotides and combinations thereof.
7. The method of claim 1 , wherein the evolving step comprises a technique selected from the group consisting of PCR, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis, in vitro mutagenesis, ligase chain reaction, oligonucleotide synthesis and combination thereof.
8. The method of claim 1 , wherein the expressing step is performed in a cell line selected from the group consisting of a COS-7 cell line, a C127 cell line, a 3T3 cell line, a CHO cell line, a HeLa cell line and a BHK cell line.
9. The method of claim 1 , further comprising a step of:
iv. glycosylating the first conditionally active polypeptide.
10. The method of claim 1 , further comprising a step of converting the first conditionally active polypeptide to a pharmaceutically acceptable salt.
11. The method of claim 1 , further comprising a step of complexing the first conditionally active polypeptide.
12. The method of claim 1 , further comprising a step of packaging the first conditionally active polypeptide in a liposome.
13. The method of claim 1 , further comprising a step of formulating the first conditionally active polypeptide in a cream, ointment, solution or hydrogel.
14. The method of claim 1 , further comprising steps of
iv. evolving DNA which encodes the first conditionally active polypeptide obtained in step iii using one or more evolutionary techniques to create a second set of mutant DNAs;
v. expressing and assaying the second set of mutant DNAs from step iv to obtain at least one second mutant polypeptide which exhibits both:
(a) a decrease in activity in the assay at the normal physiological condition, when compared to the activity of the first conditionally active polypeptide obtained in step iii in the assay at the same normal physiological condition, and
(b) an increase in activity in the assay under the aberrant condition, when compared to the activity of the first conditionally active polypeptide obtained in step iii in the assay at the same aberrant condition; and
vi. selecting a second mutant conditionally active polypeptide from the at least one second mutant polypeptide which exhibits both:
(a) the decrease in the activity in the assay at the normal physiological condition when compared to the activity of the first conditionally active polypeptide obtained in step iii in the assay at the same normal physiological condition, and
(b) the increase in the activity in the assay under the aberrant condition when compared to the activity of the first conditionally active polypeptide obtained in step iii in the assay at the same aberrant condition.
15. The method of claim 1 , wherein the first conditionally active polypeptide is inactive at the normal physiological condition.
16. The method of claim 1 , wherein the first conditionally active polypeptide is a mimetic that comprises at least one non-natural amino acid.
17. The method of claim 16 , wherein the at least one non-natural amino acid is introduced into the mimetic by one of a protein chemical synthesis technique and a recombinant technique.
18. The method of claim 1 , wherein the template polypeptide is obtained by mutagenesis of a parental polynucleotide.
19. The method of claim 18 , wherein the parental polynucleotide is a wild-type polypeptide.
20. The method of claim 18 , wherein the parental polynucleotide is a therapeutic polypeptide.
21. The method of claim 1 , wherein the assays are carried out in a same assay media comprising a material selected from the group consisting of phosphate buffered saline, a 1-methyl imidazole buffer, a carbonate buffer, a triethyl ammonium buffer, a trimethyl ammonium buffer, a polysorbate buffer, a sodium dodecyl sulfate buffer, a sodium citrate buffer, a phosphate buffered saline-ethylenediaminetetraacetic acid buffer, an acetate buffer, a tris(hydroxymethyl)aminomethane buffer, an IVIES buffer, a citrate buffer, a citric acid buffer, a sodium chloride-citric acid buffer, a borate buffer, a sodium bicarbonate buffer, a sodium carbonate buffer and a sodium dodecyl sulfate buffer.
22. The method of claim 21 , wherein the material is selected from the group consisting of a carbonate buffer, a bicarbonate buffer, a citrate buffer and a citric acid buffer.
23. The method of claim 1 , wherein the assays are carried out in a same assay media comprising a compound selected from the group consisting of sodium carbonate, sodium citrate, sodium bicarbonate, sodium dodecyl sulfate, sodium citrate, sodium, phosphonate, sodium sulfate, sodium chloride, citric acid, transferrin, haptoglobin, goat antihuman globulin, horseradish peroxidase, t-butyl phenyl carbonate, potassium carbonate, N-{2-hyddroxyethyl} piperazine-N′-[2-ethanesulfonic acid], sucrose, 3-[3[(cholamidopropyl)dimethylammonio]-1]propanesulfonate, ethanol, streptavidin, histidine, dextran sulfate, Tris-HCl, N 2 PO and ethylenediaminetetraacetic acid.
24. The method of claim 23 , wherein the compound is selected from the group consisting of sodium carbonate, sodium citrate, sodium bicarbonate, sodium citrate, citric acid and histidine.
25. The method of claim 1 , wherein the assays are carried out in a same assay media comprising a compound selected from the group consisting of human serum albumin and bovine serum albumin.
26. The method of claim 1 , wherein the condition is temperature.
27. The method of claim 1 , wherein the condition is pH.
28. The method of claim 1 , wherein the condition is osmotic pressure.
29. The method of claim 1 , wherein the condition is osmolality.
30. The method of claim 1 , wherein the condition is oxidation.
31. The method of claim 1 , wherein the condition is electrolyte concentration.
32. A conditionally active polypeptide prepared by the method of claim 1 , wherein the conditionally active polypeptide is reversibly inactivated at the normal physiological condition.