Method of improving efficacy of biological response-modifying proteins and the exemplary muteins

Abstract

Disclosed is a protein variant which substitutes valine for phenylalanine residue in a binding domain having a biological response-modifying function by binding to a receptor, ligand or substrate. Also, the present invention discloses a DNA encoding the protein variant, a recombinant expression vector to which the DNA is operably linked, a host cell transformed or transfected with the recombinant expression vector, and a method of preparing the protein variant comprising cultivating the host cell and isolating the protein variant from the resulting culture. Further, the present invention discloses a pharmaceutical composition comprising the protein variant and a pharmaceutically acceptable carrier.

1 . A method of treating a patient having an anemia associated disease comprising administering to the patient a therapeutic effective amount of a pharmaceutical composition comprising a pharmaceutical acceptable carrier and a thrombopoietin (TPO) variant, wherein the variant comprises an amino acid substitution of a valine residue for a phenylalanine residue at position 141 of SEQ ID NO:25 thereby treating the anemia associated disease in the patient. 
     
     
         2 . The method of  claim 1 , wherein the anemia associated disease is selected from the group consisting of anemia, anemia in inflammatory bowel disease, anemia in progressive kidney disease, anemia of renal failure, anemia associated with HIV infection in zidovudine treated patients, anemia associated with cancer chemotherapy, anemia associated with Huntington's disease, anemia associated with sickle cell anemia, and anemia associated with Late Hyporegenerative anemia in neonates with Rh hemolytic disease after in-utero exchange transfusion. 
     
     
         3 . The method of  claim 1 , wherein the therapeutic effective amount of the variant is administered in a smaller amount than wild-type TPO. 
     
     
         4 . The method of  claim 3 , wherein the therapeutic effective amount of the variant is administered in an amount of 0.01-1000 μg/kg/day. 
     
     
         5 . The method of  claim 3 , wherein the therapeutic effective amount of the variant is administered in amount of 0.1-5000 μg/kg/day. 
     
     
         6 . The method of  claim 3 , wherein the therapeutic effective amount of the variant is administered in an amount of 1-100 μg/kg/day. 
     
     
         7 . The method of  claim 1 , wherein administering the therapeutic effective amount is selected from the group consisting of topically, orally, parenterally, intraocularly, transdermally, intrarectally and intraluminally. 
     
     
         8 . The method of  claim 1 , wherein the pharmaceutical composition is in a form selected from the group consisting of solutions, suspensions, tablets, pills, capsules and sustained released preparations. 
     
     
         9 . The method of  claim 1 , wherein the pharmaceutical acceptable carrier is selected from the group consisting of ion exchange, alumina, aluminum stearate, lecithin, serum proteins, buffering agents, water, salts, electrolytes, colloidal silica, magnesium, trisilicate, polyvinylpyrrolidone, cellulose-based substrates, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, waxes, polyethylene-polyoxypropylene-block copolymers, polyethelene glycol and wool fat.