AU2017200659A1

AU2017200659A1 - Mirac Proteins

Info

Publication number: AU2017200659A1
Application number: AU2017200659A
Authority: AU
Inventors: Hwai Wen Chang; Gerhard Frey; Gregory Frost; Jay M. Short
Original assignee: Bioalta LLC
Current assignee: Bioatla Inc
Priority date: 2009-03-09
Filing date: 2017-02-01
Publication date: 2017-02-16
Anticipated expiration: 2030-03-09
Also published as: AU2015204268B2; AU2017200659A2; AU2021202064B2; AU2021202064A1; AU2015204268A1; AU2018256476A1; AU2018256476B2; AU2017200659B2

Abstract

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.

Description

AUSTRALIA 2017200659 01 Feb 2017

Patents Act 1990

ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT

Invention title: MIRAC PROTEINS

The following statement is a full description of this invention, including the best method of performing it known to us: 1

MIRAC PROTEINS 2017200659 01 Feb 2017

RELATED APPLICATION INFORMATION

[0001] This application is a divisional of Australian Patent Application No 2015204268 which is a divisional of Australian Patent Application No 2010222818 which is the Australian national phase entry of PCT/US2010/026611, which claims priority to US provisional patent application No 61/209,489 filed 9 March 2009. Each of these applications is herein incorporated by reference in their entireties.

FIELD OF THE DISCLOSURE

[0002] This disclosure relates to the field of protein evolution and activity. Specifically, this disclosure relates to a method of generating conditionally active biologic proteins from wild type proteins, in particular therapeutic proteins, and 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.

BACKGROUND OF THE DISCLOSURE

[0003] There is a considerable body of literature describing the potential for evolving proteins for a variety of characteristics, especially enzymes for example, to be stabilized for operation at different conditions. For example, enzymes have been evolved to be stabilized at higher temperatures, with varying activity. In situations where there is an activity improvement at the high temperature, a substantial portion of the improvement can be attributed to the higher kinetic activity commonly described by the Q10 rule where it is estimated that in the case of an enzyme the turnover doubles for every increase of 10 degrees Celsius. In addition, there exist examples of natural mutations that destabilize proteins at their normal operating conditions, such as wild-type temperature activity of the molecule. For temperature mutants, these mutants can be active at the lower temperature, but typically are active at a reduced level compared to the wild type molecules (also typically described by a reduction in activity guided by the Q10 or similar rules). la

Claims

The claims defining the invention are as follows:

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 or 2, 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 first conditionally active polypeptide selected in step iii for an organism or cell.

5. The method of any one of claims 1 to 4, wherein the evolving step comprises a technique selected from the group consisting of non-stochastic polynucleotide chimerization and nonstochastic site-directed point mutagenesis.

6. The method of any one of claims 1 to 4, 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 any one of claims 1 to 4, 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 any one of claims 1 to 7, 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 any one of claims 1 to 8, further comprising a step of: iv. glycosylating the first conditionally active polypeptide.

10. The method of any one of claims 1 to 9, further comprising a step of converting the first conditionally active polypeptide to a pharmaceutically acceptable salt.

11. The method of any one of claims 1 to 10, further comprising a step of complexing the first conditionally active polypeptide.

12. The method of any one of claims 1 to 11, further comprising a step of packaging the first conditionally active polypeptide in a liposome.

13. The method of any one of claims 1 to 12, further comprising a step of formulating the first conditionally active polypeptide in a cream, ointment, solution or hydrogel.

14. The method of any one of claims 1 to 8, 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 any one of claims 1 to 14, wherein the first conditionally active polypeptide is inactive at the normal physiological condition.

16. The method of any one of claims 1 to 15, 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 any one of claims 1 to 17, 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 or a therapeutic polypeptide.

20. The method of any one of claims 1 to 19, 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 MES 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.

21. The method of claim 20, wherein the material is selected from the group consisting of a carbonate buffer, a bicarbonate buffer, a citrate buffer and a citric acid buffer.

22. The method of any one of claims 1 to 19, 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]-l]propanesulfonate, ethanol, streptavidin, histidine, dextran sulfate, Tris-HCl, N2PO and ethylenediaminetetraacetic acid.

23. The method of claim 22, wherein the compound is selected from the group consisting of sodium carbonate, sodium citrate, sodium bicarbonate, sodium citrate, citric acid and histidine.

24 The method of any one of claims 1 to 19, 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.

25. The method of any one of claims 1 to 24, wherein the condition is temperature, pH, osmotic pressure, osmolality, oxidation or electrolyte concentration.

26. A conditionally active polypeptide prepared by the method of any one of claims 1 to 25, wherein the conditionally active polypeptide is reversibly inactivated at the normal physiological condition.