Immobilized tumor necrosis factor-alpha muteins for enhancing immune response in mammals

Abstract

An extracorporeal system for enhancing an immune response in a mammal to facilitate the elimination of a chronic pathology. The system includes an absorbent matrix capable of removing an immune system inhibitors such as soluble TNF receptor from the circulation of the mammal, thus, enabling a more vigorous immune response to the pathogenic agent. The removal of immune system inhibitors is accomplished by contacting biological fluids of a mammal with one or more binding partner(s) such as TNF-α muteins capable of binding to and, thus, depleting the targeted immune system inhibitor(s) from the biological fluids. The absorbent matrix may comprise an inert, biocompatible substrate joined covalently to a binding partner, such as a TNF-α mutein, capable of specifically binding to a targeted immune system inhibitor such as soluble TNF receptor.

1 . An absorbent matrix comprising:
 a plurality of tumor necrosis factor-alpha (TNF-α) immobilized on an extracorporeal substrate;   wherein the plurality of TNF-α muteins have at least one amino acid substitution relative to an unsubstituted native TNF-α; and   wherein each of the plurality of TNF-α muteins immobilized on the extracorporeal substrate have at least one binding site capable of selectively binding to a soluble TNF receptor with an affinity sufficient to deplete the soluble TNF receptor from a biological fluid.   
     
     
         2 . The absorbent matrix of  claim 1 , wherein the plurality of TNF-α muteins are selected from the group consisting of mutein 1 (SEQ ID NO:3), mutein 2 (SEQ ID NO:4), mutein 3 (SEQ ID NO:5), mutein 4 (SEQ ID NO:6), mutein 5 (SEQ ID NO:7), and mutein 6 (SEQ ID NO:8), or combinations thereof. 
     
     
         3 . The absorbent matrix of  claim 1 , wherein the plurality of TNF-α muteins comprise a conserved mutein sequence (SEQ ID NO:1). 
     
     
         4 . The absorbent matrix of  claim 1 , wherein the plurality of TNF-α muteins comprise the consensus mutein sequence (SEQ ID NO:9),
 wherein X 1  is an amino acid selected from Leu and Val; 
 wherein X 2  is a 2 or 3 amino acid peptide selected from GlnAsnSer, ArgAlaLeu, ArgThrPro, GlnAlaSer, and GlnThr; 
 wherein X 3  is an amino acid selected from Asp and Asn; 
 wherein X 4  is a 5 amino acid peptide selected from HisGlnValGluGlu, HisGlnAlaGluGlu, ProGlnValGluGly, ProGluAlaGluGly, LeuSerAlaProGly, IleSerAlaProGly, ProGlnAlaGluGly, IleAsnSerProGly, and ValLysAlaGluGly; 
 wherein X 5  is an amino acid selected from Glu, Gln and Arg; 
 wherein X 6  is a 4 amino acid peptide selected from LeuSerGlnArg, LeuSerArgArg, GlyAspSerTyr, LeuSerGlyArg, TrpAspSerTyr, GinSerGlyTyr, and LeuAsnArgArg; 
 wherein X 7  is an amino acid selected from Leu, Met, and Lys; 
 wherein X g  is a two amino acid peptide selected from MetAsp, MetLys, ValGlu, ValLys, and ValGln; 
 wherein X 9  is an amino acid selected from Lys, Thr, Glu, and Arg; 
 wherein X 10  is an amino acid selected from Val, Lys, and Ile; 
 wherein X 11  is a 2 amino acid peptide selected from AlaAsp, SerAsp, ThrAsp, LeuAsp, AlaGlu, and SerGlu; 
 wherein X 12  is an amino acid selected from Lys, Ser, Thr, and Arg; 
 wherein X 13  is an amino acid selected from Gln and His; 
 wherein X 14  is a 4 or 5 amino acid peptide selected from AspValValLeu, AspTyrValLeu, SerTyrValLeu, ProProProVal, SerThrHisValLeu, SerThrProLeuPhe, SerThrHisValLeu, and SerThrAsnValPhe; 
 wherein X 15  is an amino acid selected from Val and Ile; 
 wherein X 16  is an amino acid selected from Phe, Ile, and Leu; 
 wherein X 17  is an amino acid selected from Ile and Val; 
 wherein X 18  is a 2 amino acid peptide selected from GlnGlu, ProAsn, GlnThr, and ProSer; 
 wherein X 19  is an amino acid selected from Leu and Ile; 
 wherein X 20  is a 3 amino acid peptide selected from ProLysAsp, HisArgGlu, GlnArgGlu, and HisThrGlu; 
 wherein X 21  is an amino acid selected from Gly, Glu, Gln, and Trp or is absent; 
 wherein X 22  is an amino acid selected from Leu, Pro, and Ala; 
 wherein X 23  is an amino acid selected from Leu and Gln; 
 wherein X 24  is an amino acid selected from Gly and Asp; 
 wherein X 25  is an amino acid selected from Gln, Leu, and Arg; 
 wherein X 26  is an amino acid selected from Ala and Thr; 
 wherein X 27  is an amino acid selected from Val and Ile; 
 wherein X 28  is an amino acid selected from Leu, Gin, and Arg; 
 wherein X 29  is an amino acid selected from Lys, Glu, Ala, Asn, and Asp; 
 wherein X 30  is an amino acid selected from Phe, Ile, Leu and Tyr; and 
 wherein X 31  is an amino acid selected from Val and Ile. 
 
     
     
         5 . The absorbent matrix of  claim 1 , wherein the plurality of TNF-α muteins have an amino acid substitution in a region of TNF-α selected from region 1 amino acids 29-36, region 2 amino acids 84-91, and region 3 amino acids 143-149 of human TNF-α (SEQ ID NO:2) or an analogous position of TNF-α from another species. 
     
     
         6 . The absorbent matrix of  claim 1 , wherein the extracorporeal substrate is a biocompatible solid support. 
     
     
         7 . The absorbent matrix of  claim 6 , wherein the biocompatible solid support is in the form of a bead. 
     
     
         8 . The absorbent matrix of  claim 7 , wherein the bead is a macroporous bead. 
     
     
         9 . The absorbent matrix of  claim 8 , wherein the macroporous bead is chosen from agarose, cross-linked agarose, cellulose, controlled pore glass, polyacrylamide, azlactone, polymethacrylate and polystyrene. 
     
     
         10 . The aborbent matrix of  claim 1 , wherein the unsubstituted native TNF-α is a human TNF-α. 
     
     
         11 . The absorbent matrix of  claim 1 , wherein the soluble TNF receptor is a soluble tumor necrosis factor receptor Type I (sTNFRI) or a soluble tumor necrosis factor receptor Type II (sTNFRII). 
     
     
         12 . An extracorpeal system for reducing the amount of a targeted immune system inhibitor in blood of a donor mammal, the extracorpeal system comprising:
 an apheresis device in fluid communication with a blood source from the donor mammal that separates the whole blood component into a conduit containing a cellular component and a conduit containing an acellular component or a fraction of an acellular component, the acellular component or the fraction of the acellular component containing the targeted immune system inhibitor comprising a soluble TNF receptor;   an absorbent matrix in fluid communication with the apheresis device by the conduit containing the acellular component or the fraction of the acellular component, the absorbent matrix having a plurality of TNF-α muteins immobilized on an inert medium; the plurality of TNF-α muteins having at least one amino acid substitution relative to an unsubstituted native TNF-α; and each of the plurality of TNF-α muteins immobilized on the extracorporeal substrate having at least one binding site capable of selectively binding to a soluble TNF receptor with an affinity sufficient to deplete the soluble TNF receptor from the acellular component to produce an altered acellular component or an altered fraction of the acellular component contained in a conduit exiting the absorbent matrix; and   a volumetric pump in fluid communication with the apheresis device that provides a pressure differential to the conduit containing the cellular component and the conduit containing the altered acellular component or the altered fraction of the acellular component to combine the cellular component with the altered acellular component or the altered fraction of the acellular component to produce an altered whole blood source adapted to be administered to a recipient mammal.   
     
     
         13 . The extracorpeal system of  claim 12 , wherein the conduit containing the cellular component is a first conduit and the conduit containing the acellular component or the fraction of the acellular component is a second conduit. 
     
     
         14 . The extracorpeal system of  claim 12 , wherein the donor mammal of the blood source is the recipient mammal. 
     
     
         15 . The extracorpeal system of  claim 12 , further comprising a positive displacement blood pump that removes the blood source from the donor mammal. 
     
     
         16 . The extracorporeal system of  claim 12 , wherein the inert medium is selected from the group consisting of a hollow fiber, a macroporous bead, a cellulose-based fiber, a synthetic fiber, a flat membrane, a pleated membrane, and a silica-based particle. 
     
     
         17 . The extracorporeal system of  claim 12 , wherein the blood source is whole blood. 
     
     
         18 . The extracorporeal system of  claim 12 , wherein the plurality of TNF-α muteins are selected from the group consisting of mutein 1 (SEQ ID NO:3), mutein 2 (SEQ ID NO:4), mutein 3 (SEQ ID NO:5), mutein 4 (SEQ ID NO:6), mutein 5 (SEQ ID NO:7), and mutein 6 (SEQ ID NO:8), or combinations thereof. 
     
     
         19 . The extracorporeal system of  claim 12 , wherein the soluble TNF receptor is a soluble tumor necrosis factor receptor Type I (sTNFRI) or a soluble tumor necrosis factor receptor Type II (sTNFRII). 
     
     
         20 . The extracorporeal system of  claim 12 , wherein the plurality of TNF-α muteins have an amino acid substitution in a region of TNF-α selected from region 1 amino acids 29-36, region 2 amino acids 84-91, and region 3 amino acids 143-149 of human TNF-α (SEQ ID NO:2) or an analogous position of TNF-α from another species.