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 antibody, the method comprising the steps of:
i. selecting a wild-type antibody against an antigen;
ii. evolving the DNA which encodes the wild-type antibody using one or more evolutionary techniques to create mutant DNAs;
iii. expressing the mutant DNAs to obtain at least one mutant antibody;
iv. subjecting the at least one mutant antibody and the wild-type antibody to an assay under a normal physiological condition selected from the group consisting of temperature, pH, osmotic pressure, osmolality, oxidation and electrolyte concentration, and to an assay under an aberrant condition selected from the group consisting of temperature, pH, osmotic pressure, osmolality, oxidation and electrolyte concentration; and
v. selecting the conditionally active antibody from the at least one mutant antibody which exhibits both (a) a decrease in binding activity to the antigen in the assay at the normal physiological condition compared to the wild-type antibody, and (b) an increase in binding activity to the antigen in the assay under the aberrant condition compared to the wild-type antibody.
2. The method of claim 1 , wherein the normal physiological condition is temperature; and wherein the conditionally active antibody is inactive at the normal physiological temperature, and is active at an aberrant temperature less than the normal physiological temperature.
3. A method of preparing a conditionally active biological response modifier, the method comprising the steps of:
a. selecting an inflammatory response mediator;
b. identifying a wild-type antibody to the mediator;
c. evolving the wild-type antibody;
d. screening differentially for at least one mutant that exhibits decreased binding to the mediator relative to the wild-type antibody at a first condition selected from the group consisting of temperature, pH, osmotic pressure, osmolality, oxidation and electrolyte concentration, and exhibits increased binding affinity to the mediator relative to the wild-type antibody at a second condition selected from the group consisting of temperature, pH, osmotic pressure, osmolality, oxidation and electrolyte concentration to identify at least one up-mutant;
e. recombining the heavy chains and the light chains of the at least one up-mutant to create at least one recombined up-mutant; and
f. screening the at least one recombined up-mutant for at least one up-mutant that exhibits decreased binding to the mediator relative to the wild-type antibody at the first condition, and shows increased binding affinity to the mediator relative to the wild-type antibody at the second condition to identify the conditionally active biological response modifier.