Apparatus for the extracorporeal treatment of blood

Abstract

An apparatus for the extracorporeal treatment of blood comprising an extracorporeal blood circuit (2), a pump (6) configured to provide fluid displacement within the extracorporeal blood circuit, and a reaction chamber (8) connected to the extracorporeal blood circuit and configured to receive blood or plasma from the circuit and treat the blood or plasma. The reaction chamber comprises a protease enzyme immobilized to a support, in which the protease enzyme is specific for, and capable of irreversibly cleaving, a human C5a present in the blood or plasma, wherein the abundance of the human C5a in the treated blood or plasma is less than that in the untreated blood or plasma. The apparatus finds utility in the extracorporeal treatment of blood from patients with inflammatory conditions, especially auto-immune disease and sepsis.

1 . A method for the extracorporeal treatment of blood, the method comprising removing blood or a blood fraction from a patient and reacting the blood or blood fraction with a protease enzyme that is specific for and capable of irreversibly cleaving functional C5a, thereby reducing an abundance of functional C5a in the blood or blood fraction, wherein the protease enzyme is immobilised to a support. 
     
     
         2 . The method of  claim 1 , wherein the protease enzyme is a recombinant bacterial C5a protease comprising SEQ ID NO. 3, or a functional variant thereof having at least 90% sequence identity with SEQ ID NO: 3. 
     
     
         3 . The method of  claim 2 , wherein the functional variant of SEQ ID NO: 3 is SEQ ID NO: 4 or SEQ ID NO: 5. 
     
     
         4 . The method of  claim 1 , wherein the reacting step is carried out with an apparatus comprising:
 an extracorporeal blood circuit;   a pump configured to provide fluid displacement with the extracorporeal blood circuit; and   a reaction chamber connected to the extracorporeal blood circuit, the reaction chamber configured to receive the blood or blood fraction from the extracorporeal blood circuit and to treat the blood or blood fraction,   
       wherein the reaction chamber contains the protease enzyme immobilised to the support. 
     
     
         5 . The method of  claim 4 , wherein the protease enzyme is a recombinant bacterial C5a protease comprising SEQ ID NO. 3, or a functional variant thereof having at least 90% sequence identity with SEQ ID NO: 3. 
     
     
         6 . The method of  claim 5 , wherein the functional variant of SEQ ID NO: 3 is SEQ ID NO: 4 or SEQ ID NO: 5. 
     
     
         7 . The method of  claim 4 , wherein the apparatus further comprises separating means adapted to separate the blood or blood fraction into a C5a-containing fraction and a non-C5a containing fraction, wherein the reaction chamber receives the C5a-containing fraction. 
     
     
         8 . The method of  claim 7 , wherein the protease enzyme is a recombinant bacterial C5a protease comprising SEQ ID NO. 3, or a functional variant thereof having at least 90% sequence identity with SEQ ID NO: 3. 
     
     
         9 . The method of  claim 8 , wherein the functional variant of SEQ ID NO: 3 is SEQ ID NO: 4 or SEQ ID NO: 5. 
     
     
         10 . The method of  claim 7 , wherein the apparatus further comprises means configured to recombine the treated C5a-containing fraction with the non-C5a containing fraction. 
     
     
         11 . The method of  claim 1 , wherein the support includes a coordinated transition metal ion and one or more functional groups; the C-terminus of the protease enzyme comprises a first tag and a second tag located distally of the first tag, the second tag being separated from the first tag by a spacer; the first tag comprises a motif capable of covalently reacting with the one or more functional groups; and the second tag comprises a motif capable of interacting with the coordinated transition metal ion. 
     
     
         12 . The method of  claim 11 , wherein the first tag is poly-lysine, poly-glutamate, or poly-cysteine, and the second tag is poly-histidine. 
     
     
         13 . The method of  claim 12 , wherein the protease enzyme is a recombinant bacterial C5a protease comprising SEQ ID NO. 3, SEQ ID NO: 4, or SEQ ID NO: 5. 
     
     
         14 . The method of  claim 1 , wherein the support includes a silica material, a methacrylate, a polyacrylamide, a polypyrrole, or a polysaccharide. 
     
     
         15 . The method of  claim 14 , wherein the silica material is selected from the group consisting of mesoporous silica, a monodispersed mesoporous silicate, and a Ni2+-modified mesoporous silica. 
     
     
         16 . The method of  claim 1 , wherein the support comprises a multiplicity of beads and the protease enzyme is irreversibly immobilized to a surface of the beads. 
     
     
         17 . A method for treating sepsis, the method comprising removing blood or a blood fraction from a patient having sepsis and reacting the blood or blood fraction with a protease enzyme that is specific for and capable of irreversibly cleaving functional C5a, thereby reducing an abundance of functional C5a in the blood or blood fraction, wherein the protease enzyme is immobilised to a support. 
     
     
         18 . The method of  claim 17 , wherein the protease enzyme is a recombinant bacterial C5a protease comprising SEQ ID NO. 3, SEQ ID NO: 4, or SEQ ID NO: 5. 
     
     
         19 . A method for treating an inflammatory condition in a human, the method comprising removing blood or a blood fraction from a patient having an inflammatory condition and reacting the blood or blood fraction with a protease enzyme that is specific for and capable of irreversibly cleaving functional C5a, thereby reducing an abundance of functional C5a in the blood or blood fraction, wherein the protease enzyme is immobilised to a support. 
     
     
         20 . The method of  claim 19 , wherein the protease enzyme is a recombinant bacterial C5a protease comprising SEQ ID NO. 3, SEQ ID NO: 4, or SEQ ID NO: 5.