Discovering and producing conditionally active biologic proteins in the same eukaryotic cell production hosts

Abstract

A method of preparing a conditionally active biologic protein by selecting a wild-type biologic protein, evolving the DNA which encodes the wild-type biologic protein using one or more evolutionary techniques to create mutant DNAs, expressing the mutant DNAs in a eukaryotic cell production host to obtain a mutant protein, 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, selecting a conditionally active mutant protein which exhibits at least one of: (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; and producing the conditionally active biologic protein in the same eukaryotic cell production host used in the expression step.

1 . A method of producing a conditionally active biologic protein and optionally further modifying the conditionally active biologic protein, the method comprising:
 i. evolving a DNA which encodes a parent biologic protein using one or more evolutionary techniques to create mutant DNAs;   ii. expressing the mutant DNAs in a eukaryotic cell production host to obtain mutant proteins;   iii. subjecting the mutant proteins to an assay under a normal physiological condition and to an assay under an aberrant condition;   iv. selecting a conditionally active biologic protein from the mutant proteins expressed in step (iii) having an increased activity at the aberrant condition in comparison to activity at the normal physiological condition; and   vi. producing the conditionally active biologic protein in the same eukaryotic cell production host as used in step (ii),   wherein the assay under the normal physiological condition and the assay under the aberrant condition arc performed in a media containing a component selected from an inorganic compound, an ion and an organic molecule present in a bodily fluid of mammals or humans,   said inorganic compound is selected from boric acid, calcium chloride, calcium nitrate, di-ammonium phosphate, magnesium sulfate, mono-ammonium phosphate, mono-potassium phosphate, potassium chloride, potassium sulfate, copper sulfate, iron sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, calcium nitrate, chelates of calcium, chelates of copper, chelates of iron, chelates of manganese, chelates of zinc, ammonium molybdate, ammonium sulphate, calcium carbonate, magnesium phosphate, potassium bicarbonate, sodium bicarbonate, potassium nitrate, hydrochloric acid, carbon dioxide, sulfuric acid, phosphoric acid, carbonic acid, uric acid, hydrogen chloride, urea,   said ion is selected from phosphorus ions, sulfuric ions, chloride ions, magnesium ions, sodium ions, potassium ions, ammonium ions, iron ions, zinc ions and copper ions, and   said organic molecule is selected from citric acid, α-kctoglutaric acid, succinic acid, malic acid, fumaric acid, acetoacetic acid, β-hydroxybutyric acid, lactic acid, pyruvic acid, α-ketonic acid, acetic acid, and volatile fatty acids.   
     
     
         2 . The method of  claim 1  wherein the conditionally active biologic protein exhibits both (a) a decrease in activity in the assay at the normal physiological condition compared to the parent biologic protein, and (b) an increase in activity in the assay under the aberrant condition compared to the parent biologic protein. 
     
     
         3 . The method of  claim 1  wherein the parent biologic protein is selected from an antibody, a tissue plasminogen activator, streptokinase, urokinase, renin, hyaluronidase, a calcitonin gene-related peptide, substance P, neuropeptide Y, a vasoactive intestinal peptide, vasopressin, angiostatin, a protein that binds with a target protein on a target cell, and a DNA/RNA modifying protein. 
     
     
         4 - 5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the normal physiological condition is selected from one or more of temperature, pH, osmotic pressure, osmolality, oxidative stress and electrolyte concentration. 
     
     
         7 . The method of  claim 6 , wherein the normal physiological condition is a normal physiological 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. 
     
     
         8 . 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 any combination thereof. 
     
     
         9 . The method of  claim 1 , wherein the eukaryotic cell production host is selected from a fungal cell, an insect cell, a mammalian cell, an adenovirus, and a plant cell. 
     
     
         10 . The method of  claim 9 , wherein the eukaryotic cell production host is the fungal cell and is selected from cells of  Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Kluyveromyces lactis, Hansenula plymorpha , and  Aspergillus niger.    
     
     
         11 . The method of  claim 9 , wherein the eukaryotic cell production host is the insect cell and is selected from  Drosophila  S2 cells and  Spodoptera  Sf9 cells. 
     
     
         12 . The method of  claim 9 , wherein the eukaryotic cell production host is the mammalian cell and is selected from Bowes melanoma cells, COS-7 cells, C127 cells, HeLa cells, BHK cells, 3T3 mouse fibroblast cells, BHK21 Syrian hamster fibroblast cells, MDCK dog epithelial cells, PtK1 rat kangaroo epithelial cells, SP2/0 mouse plasma cells, NS0 mouse plasma cells, HEK 293 human embryonic kidney cells, COS monkey kidney cells, CHO cells, CHO—S, R1 mouse embryonic cells, E14.1 mouse embryonic cells, H1 human embryonic cells, H9 human embryonic cells, and PER C.6, human embryonic cells. 
     
     
         13 . The method of  claim 12 , wherein the mammalian cell is selected from CHO cells, NS0 mouse plasma cells, and HEK293 human embryonic kidney cells. 
     
     
         14 . The method of  claim 3 , wherein the parent biologic protein is the antibody, said method further comprising a step of conjugating the conditionally active antibody to a molecule selected from cytokines, interleukins, enzymes, hormones, growth factors, cytotoxic agents, chemotherapy drugs, radioactive particles and diagnostic agents. 
     
     
         15 . The method of  claim 14 , wherein the conjugating step comprises forming a covalent bond or a non-covalent bond between the conditionally active antibody and the molecule. 
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 14 , wherein the molecule is conjugated to the Fc region of the conditionally active antibody. 
     
     
         18 . The method of  claim 3 , wherein the parent biologic protein is the antibody, said method further comprising the step of engineering the conditionally active antibody to be multispecific. 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 1 , wherein the producing step comprises manufacturing the conditionally active biologic protein. 
     
     
         21 - 24 . (canceled) 
     
     
         25 . A conditionally active biologic protein prepared by the method of  claim 1 , wherein the conditionally active biologic protein is reversibly or irreversibly inactivated at the normal physiological condition. 
     
     
         26 . The conditionally active biologic protein of  claim 25 , wherein the conditionally active biologic protein comprises at least one non-natural amino acid. 
     
     
         27 . A chimeric antigen receptor comprising the conditionally active biologic protein of  claim 25 . 
     
     
         28 . A cytotoxic cell comprising the chimeric antigen receptor of  claim 27 .