Conditionally active biological proteins
Methods of generating conditionally active biologic proteins, in particular therapeutic or diagnostic proteins, which are more active at an aberrant condition than at a normal physiological condition. The methods include discovery methods using libraries of proteins and assays employing physiological concentrations of components of bodily fluids. The conditionally active biologic proteins may be further evolved, conjugated to other molecules, masked, reduced in activity by attaching a cleavable moiety. Criteria for selecting starting proteins for the discovery methods, as well as formats of the proteins are also disclosed.
1. A method of preparing a conditionally active biologic protein, wherein the conditionally active biologic protein is a protein which exhibits a decrease in activity in an assay at a normal physiological condition compared to a same activity in the assay under an aberrant condition, the method comprising steps of:
i. selecting a template polypeptide from a wild-type biologic protein, a mutated protein and a fragment of a wild-type protein with a desired property;
ii. evolving a DNA which encodes the template polypeptide using one or more evolutionary techniques to create mutant DNAs;
iii. expressing the mutant DNAs to obtain mutant proteins at least one of which exhibits a decrease in activity in the assay at the normal physiological condition compared to the same activity in the assay under the aberrant condition; and
iv. screening the mutant proteins to identify the conditionally active biologic protein that exhibits a decrease in activity in the assay at the normal physiological condition compared to the same activity in the assay under the aberrant condition by conducting assays to determine the activity at the normal physiological condition and at the aberrant condition,
wherein the assay under the normal physiological condition and the assay under the aberrant condition are performed in assay solutions containing at least one component at substantially the same concentration, said at least one component being selected from
a. an inorganic compound selected from boric acid, calcium chloride, di-ammonium phosphate, mono-ammonium phosphate, potassium sulfate, copper sulfate, iron sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, calcium nitrate, calcium chelate, copper chelate, iron chelate, manganese chelate, zinc chelate, ammonium molybdate, ammonium sulfate, calcium carbonate, magnesium phosphate, potassium nitrate, hydrochloric acid, sulfuric acid, phosphoric acid, uric acid, and hydrogen chloride;
b. an ion selected from a phosphorus ion, a sulfuric ion, an ammonium ion, an iron ion, a zinc ion, a copper ion, a sulfate ion, a bisulfate ion, a carbonate ion, a bicarbonate ion, a nitrate ion, a nitrite ion, a persulfate ion, a monopersulfate ion, and a borate ion; and
c. an organic molecule selected from amino acids, organic acids and sugars.
2. The method of claim 1 , wherein the at least one component has substantially the same concentration in the assay solutions for both the assay under the normal physiological condition and the assay under the aberrant condition.
3. The method of claim 1 , wherein the at least one component is a component that is present in a bodily fluid of a mammal at a normal physiologic concentration.
4. The method of claim 3 , wherein the at least one component is present in a concentration in a range of from about 5% to about 500% of the normal physiologic concentration in the bodily fluid.
5. The method of claim 1 , wherein the at least one component comprises a component selected from one or more of uric acid in concentration range of 2-7.0 mg/dL, calcium ion in a concentration range of 8.2-11.6 mg/dL, chloride ion in a concentration range of 355-381 mg/dL, iron ion in a concentration range of 0.028-0.210 mg/dL, potassium ion in a concentration range of 12.1-25.4 mg/dL, sodium ion in a concentration range of 300-330 mg/dL and carbonic acid in a concentration range of 15-30 mM.
6. The method of claim 1 , wherein the at least one component comprises the amino acid and the amino acide is selected from Histidine, Alanine, Isoleucine, Arginine, Leucine, Asparagine, Lysine, Aspartic acid, Methionine, Cysteine, Phenylalanine, Glutamic acid, Threonine, Glutamine, Tryptophan, Glycine, Valine, Pyrrolysine, Proline, Selenocysteine, Serine, and Tyrosine.
7. The method of claim 1 , wherein the at least one component comprises the organic acid and the organic acid is selected from citric acid, α-ketoglutaric acid, succinic acid, malic acid, fumaric acid, acetoacetic acid, β-hydroxybutyric acid, lactic acid, pyruvic acid, α-ketonic acid, acetic acid and carbonic acid.
8. The method of claim 1 , wherein the at least one component comprises the sugar and the sugar is selected from glucose, pentose, hexose, xylose, ribose, mannose, galactose, lactose, GlcNAcβ1-3Gal, Galα1-4Gal, Manα1-2Man, GalNAcβ1-3Gal and O—, N—, C—, and S-glycosides.
9. The method of claim 1 , wherein the condition is pH and the aberrant condition is a pH that is different from a normal physiological pH.
10. The method of claim 9 , wherein the ion has a pKa that is within about 1 pH unit from the pH of the aberrant condition.
11. The method of claim 1 , wherein the template polypeptide selected in step (i) is an antibody or an antibody fragment.
12. The method of claim 1 , wherein the desired property is selected from binding affinity, expression level and humanization.
13. The method of claim 1 , further comprising a step of evolving the conditionally active biologic protein selected in step (iv) to produce an improved conditionally active biologic protein with at least one improved property.
14. The method of claim 13 , wherein the at least one improved property is an increased ratio of the activity in the assay under the aberrant condition to the activity under the normal physiological condition, as compared to the conditionally active biologic protein selected in step (iv).
15. The method of claim 1 , wherein the conditionally active biologic protein is a conditionally active antibody, and said method further comprising a step of conjugating the conditionally active antibody to an agent selected from cytokines, interleukins, enzymes, hormones, growth factors, cytotoxic agents, chemotherapy drugs, radioactive particles and diagnostic agents.
16. The method of claim 1 , wherein the assay solutions for the assay under the normal physiological condition and the assay under the aberrant condition contain no serum.
17. The method of claim 1 , wherein the at least one component comprises a component selected from the group consisting of urea, potassium bicarbonate and carbon dioxide.