Mirac proteins

Abstract

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.

We claim: 
     
         1 . A method of preparing a conditionally active biologic protein, the method comprising:
 i. selecting a wild-type biologic protein;   ii. evolving the DNA which encodes the wild-type biologic protein using one or more evolutionary techniques to create a mutant DNA;   iii. expressing the mutant DNA to obtain a mutant protein;   iv. subjecting the mutant protein and the wild-type protein to an assay under a normal physiological condition and to an assay under an aberrant condition; and   v. selecting the conditionally active biologic protein from those mutant proteins which exhibit both (a) a decrease in activity in the assay at the normal physiological condition compared to the wild-type protein, and (b) an increase in activity in the assay under the aberrant condition compared to the wild-type protein.   
     
     
         2 . The method of  claim 1  wherein the wild-type biologic protein is an enzyme. 
     
     
         3 . The method of  claim 2  wherein the wild-type biologic protein is selected from the group consisting of tissue plasminogen activator, streptokinase, urokinase, renin, and hyaluronidase. 
     
     
         4 . The method of  claim 1  wherein the wild-type biologic protein is selected from calcitonin gene-related peptide, substance P, neuropeptide Y, vasoactive intestinal peptide, vasopressin, and angiostatin. 
     
     
         5 . The method of  claim 1 , wherein the normal physiological condition is selected from one or more of normal physiological temperature, pH, osmotic pressure, osmolality, oxidation and electrolyte concentration. 
     
     
         6 . The method of  claim 5 , wherein the normal physiological condition is temperature; and wherein the conditionally active biologic protein is substantially inactive at the normal physiological temperature, and is active at an aberrant temperature less than the normal physiological temperature. 
     
     
         7 . A conditionally active biologic protein prepared by the method of  claim 1 , wherein the protein is reversibly or irreversibly inactivated at the normal physiological condition. 
     
     
         8 . The conditionally active biologic protein of  claim 7 , wherein the protein is reversibly inactivated at the wild type normal physiological conditions. 
     
     
         9 . The conditionally active biologic protein of  claim 7 , wherein the wild-type biologic protein is selected from the group consisting of tissue plasminogen activator, streptokinase, urokinase, renin, and hyaluronidase. 
     
     
         10 . The conditionally active biologic protein of  claim 7 , wherein the wild-type biologic protein is selected from calcitonin gene-related peptide, substance P, neuropeptide Y, vasoactive intestinal peptide, vasopressin, and angiostatin. 
     
     
         11 . A pharmaceutical composition comprising an effective amount of the conditionally active biologic protein of  claim 7 , and a pharmaceutically acceptable carrier. 
     
     
         12 . The pharmaceutical composition of  claim 11 , wherein the conditionally active biologic protein is selected from the group consisting of a tissue plasminogen activator mutant, a streptokinase mutant, a urokinase mutant, a renin mutant, and a hyaluronidase mutant. 
     
     
         13 . The pharmaceutical composition of  claim 12 , wherein the conditionally active biologic protein is selected from the group consisting of a tissue plasminogen activator mutant, a streptokinase mutant, and a urokinase mutant. 
     
     
         14 . The pharmaceutical composition of  claim 11 , wherein the conditionally active biologic protein is selected from the group consisting of a calcitonin gene-related peptide mutant, a substance P mutant, a neuropeptide Y mutant, a vasoactive intestinal peptide mutant, a vasopressin mutant, and an angiostatin mutant. 
     
     
         15 . The pharmaceutical composition of  claim 14 , wherein the conditionally active biologic protein is selected from the group consisting of a calcitonin gene-related peptide mutant, a vasopressin mutant, and an angiostatin mutant. 
     
     
         16 . A method of preparing a conditionally active biological response modifier, the method comprising:
 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 mutants that exhibit decreased binding to the mediator relative to the wild-type antibody at a first condition, and exhibit increased binding affinity to the mediator at a second condition to identify up-mutants; and   e. recombining the heavy chains and the light chains of the up-mutants to create recombined up-mutants; and   f. screening the recombined up-mutants for mutants that exhibit decreased binding to the mediator relative to the wild-type antibody at the first condition, and show increased binding affinity to the mediator at the second condition to identify the conditionally active biological response modifier.   
     
     
         17 . The method of  claim 16  wherein the inflammatory response mediator is selected from IL-6, IL-6 receptor, TNF-alpha, IL-23 and IL-12. 
     
     
         18 . The method of  claim 16  wherein the first and second condition are each selected from conditions of pH, osmotic pressure, osmolality, oxidation and electrolyte concentration.