Recombinant endotoxin-neutralizing proteins

Abstract

In general, the invention features a recombinant endotoxin-neutralizing polypeptide (RENP) characterized by (i) an amino acid sequence, (ii) an amino acid sequence and structure that facilitates selective and specific binding to lipopolysaccharide and (iii) once bound to the lipopolysaccharide, provides endotoxin-neutralizing activity. Preferably, the RENP is composed of an amino acid sequence similar to, but not identical to, an amino acid sequence of BPI, LBP, or both. Preferably, the RENP contains an LPS-binding domain derived from the amino acid sequence of BPI, LBP, or both. Preferably, the RENPs are covalently bound to a molecule which enhances the half-life of the polypeptide. The RENPs of the invention can be used in pharmaceutical compositions for therapeutic and prophylactic regimens, as well as in various in vitro and in vivo diagnostic methods.

What is claimed is:  
     
         1 . A method of detecting a site of Gram-negative bacterial infection in a subject, said method comprising the steps of: 
 injecting into the patient's circulatory system an injectable formulation comprising an effective amount of a recombinant endotoxin-neutralizing polypeptide attached to a detectable label, wherein the polypeptide is characterized by (i) selective and specific binding to lipopolysaccharide and (ii) endotoxin-neutralizing activity, with the proviso that the amino acid sequence of the polypeptide is not identical to the amino acid sequences of BPI or LBP;    allowing the detectably labeled polypeptide sufficient time to circulate in the subject and bind to lipopolysaccharide in the patient; and    detecting a site of label binding in the patient, thereby detecting a site of Gram-negative bacterial infection.    
     
     
         2 . The method of  claim 1 , wherein the polypeptide is covalently bound to a molecule which enhances the half-life of the polypeptide.  
     
     
         3 . The method of  claim 1 , wherein the polypeptide contains an LPS binding domain of BPI, LBP, a BPI variant, or an LBP variant.  
     
     
         4 . The method of  claim 1 , wherein the detectable label is a radionucleotide.  
     
     
         5 . A method of detecting a Gram-negative bacterial infection in a subject, said method comprising the steps of: 
 obtaining a sample from a patient suspected of having a Gram-negative bacterial infection;    contacting said sample with a detectably labeled recombinant endotoxin-neutralizing polypeptide for a time sufficient for binding of the polypeptide to lipopolysaccharide in the sample; and    detecting formation of lipopolysaccharide-polypeptide complexes by detection of a detectable label bound to the polypeptide;    wherein detection of a level of detectable label in said sample significantly greater than a level of detectable label in a negative control sample is indicative of a Gram-negative bacterial infection in the subject.    
     
     
         6 . The method of  claim 5 , wherein the polypeptide contains an LPS binding domain of BPI, LBP, a BPI variant, or an LBP variant.  
     
     
         7 . The method of  claim 5 , wherein said detection is quantitative.  
     
     
         8 . The method of  claim 7 , wherein said quantitative detection is correlated with an Gram-negative bacterial infection load.  
     
     
         9 . A detectably labeled recombinant endotoxin-neutralizing polypeptide characterized by (i) selective and specific binding to lipopolysaccharide and (ii) endotoxin-neutralizing activity, with the proviso that the amino acid sequence of the polypeptide is not identical to the amino acid sequences of BPI or LBP.  
     
     
         10 . A polypeptide according to  claim 9 , wherein the polypeptide contains an LPS binding domain of BPI, LBP, a BPI variant, or an LBP variant.  
     
     
         11 . A detectably labeled polypeptide according to  claim 9 , wherein the polypeptide comprises a molecule which enhances the half-life of said polypeptide and is covalently bound to the polypeptide.  
     
     
         12 . A detectably labeled polypeptide according to  claim 11 , wherein said molecule is an immunoglobulin fragment, a half-life enhancing porion of LBP, a half-life enhancing portion of an LBP variant, or polyethylene glycol.  
     
     
         13 . A kit for detecting a site of Gram-negative bacterial infection in a subject, the kit comprising: 
 an injectable formulation comprising a detectably labeled recombinant endotoxin-neutralizing polypeptide characterized by (i) selective and specific binding to lipopolysaccharide and (ii) endotoxin-neutralizing activity, with the proviso that the amino acid sequence of the polypeptide is not identical to the amino acid sequences of BPI or LBP.    
     
     
         14 . A recombinant endotoxin-neutralizing polypeptide characterized by (i) selective and specific binding to lipopolysaccharide and (ii) endotoxin-neutralizing activity, with the proviso that the amino acid sequence of the polypeptide is not identical to the amino acid sequences of BPI or LBP.  
     
     
         15 . A recombinant endotoxin-neutralizing polypeptide according to  claim 14 , wherein said polypeptide is of the formula L 1-197 B 200-456  or a corresponding protein which (a) functions to bind lipopolysaccharide and (b) neutralizes endotoxin.  
     
     
         16 . A recombinant endotoxin-neutralizing polypeptide according to  claim 14 , wherein the polypeptide is BPI(S351→X), wherein X is any amino acid other than serine.  
     
     
         17 . A recombinant endotoxin-neutralizing polypeptide according to  claim 16 , wherein X is alanine.  
     
     
         18 . A recombinant endotoxin-neutralizing polypeptide according to  claim 14 , wherein the polypeptide contains the amino acid sequence of BPI having a cationic amino acid substituted with a neutral or anionic residue.  
     
     
         19 . A recombinant endotoxin-neutralizing polypeptide according to  claim 18 , wherein the cationic amino acid is at BPI amino acid residue positions 27, 30, 33, 42, 44, 48, 59, 77, 86, 90, 96, 118, 127, 148, 150, 160, 161, 167, 169, 177, 185, or 198.  
     
     
         20 . A recombinant endotoxin-neutralizing polypeptide according to  claim 19 , wherein the polypeptide contains neutral or anionic residues at BPI amino acid residue positions 27, 30, 33, 42, 44, 48, and 59.  
     
     
         21 . A recombinant endotoxin-neutralizing polypeptide according to  claim 19 , wherein the polypeptide contains neutral or anionic residues at BPI amino acid residue positions 77, 86, 90, 96, 118, and 127.  
     
     
         22 . A recombinant endotoxin-neutralizing polypeptide according to  claim 19 , wherein the polypeptide contains neutral or anionic residues at BPI amino acid residue positions 148, 150, 160, 161, 167, 169, 177, 185, and 198.  
     
     
         23 . A recombinant endotoxin-neutralizing polypeptide according to  claim 18 , wherein the polypeptide contains neutral or anionic residues at BPI amino acid residue positions 27, 30, 33, 42, 44, 48, 59, 77, 86, 90, 96, 118, 127, 148, 150, 160, 161, 167, 169, 177, 185, and 198.  
     
     
         24 . A recombinant endotoxin-neutralizing polypeptide according to  claim 14 , wherein the polypeptide contains the amino acid sequence of LBP having an amino acid substituted for an amino acid in a corresponding amino acid residue position of BPI.  
     
     
         25 . A recombinant endotoxin-neutralizing polypeptide according to  claim 24 , wherein the amino acid substituted is at LBP amino acid residue positions 77, 86, 96, 118, 126, 147, 148, 158, 159, 161, 165, 167, 175, 183, or 196.  
     
     
         26 . A recombinant endotoxin-neutralizing polypeptide according to  claim 14 , wherein the polypeptide contains the amino acid sequence of BPI having a cysteine residue substituted with an amino acid other than cysteine.  
     
     
         27 . A recombinant endotoxin-neutralizing polypeptide according to  claim 26 , wherein said cysteine residue is at BPI amino acid residue position 132, 135, or 175.  
     
     
         28 . A recombinant endotoxin-neutralizing polypeptide according to  claim 26 , wherein the cysteine residues of BPI at positions 132, 135, and 175 are substituted with an amino acid other than cysteine.  
     
     
         29 . A recombinant endotoxin-neutralizing polypeptide according to  claim 14 , wherein the polypeptide comprises a molecule which enhances the half-life of said polypeptide and is covalently bound to the polypeptide.  
     
     
         30 . A recombinant endotoxin-neutralizing polypeptide according to  claim 29 , wherein said polypeptide contains a lipopolysaccharide-binding domain of BPI, LBP, a BPI variant, or an LBP variant.  
     
     
         31 . A recombinant endotoxin-neutralizing polypeptide according to  claim 29 , wherein said molecule is an immunoglobulin fragment, a half-life enhancing portion of LBP, a half-life enhancing portion of an LBP variant, or polyethylene glycol.  
     
     
         32 . A recombinant endotoxin-neutralizing polypeptide according to  claim 29 , wherein the endotoxin-neutralizing polypeptide of (a) is a C-terminal fragment of BPI and the molecule of (b) is an N-terminal fragment of LBP.  
     
     
         33 . A recombinant endotoxin-neutralizing polypeptide according to  claim 32 , wherein said C-terminal fragment of BPI is a fragment having an amino acid sequence contained in BPI amino acid residues 60-456.  
     
     
         34 . A recombinant endotoxin-neutralizing polypeptide according to  claim 33 , wherein said C-terminal fragment of BPI is BPI amino acid residues 60-456, 136-456, 277- 456, 300-456, 200-456, 136 -361, 136-275, 200-275, or 200-361.  
     
     
         35 . A recombinant endotoxin-neutralizing polypeptide according to  claim 32 , wherein said N-terminal fragment of LBP is a fragment having an amino acid sequence contained in LBP amino acid residues 1-175.  
     
     
         36 . A recombinant endotoxin-neutralizing polypeptide according to  claim 21 , wherein said N-terminal fragment of LBP is LBP amino acid residues 1-59, 1-134, 1-164, 1-175, 1-274, 1-359, 1-134, or 1-197.  
     
     
         37 . A recombinant endotoxin-neutralizing polypeptide of  claim 18 , wherein the polypeptide further comprises a C-terminal fragment of LBP.  
     
     
         38 . A recombinant endotoxin-neutralizing polypeptide of  claim 23 , wherein the C-terminal fragment of LBP is LBP amino acid residues 360-456 or 274-456.  
     
     
         39 . An isolated DNA molecule encoding a recombinant endotoxin binding polypeptide according to  claim 14 .  
     
     
         40 . A vector comprising the DNA of  claim 39 .  
     
     
         41 . A transformed host cell comprising the DNA of  claim 39 .  
     
     
         42 . A method for producing a recombinant endotoxin-neutralizing polypeptide according to  claim 14 , said method comprising the steps of: 
 culturing a transformed host cell comprising DNA encoding a recombinant endotoxin binding polypeptide according to  claim 14 , said DNA being operably linked to a promoter for expression of the polypeptide encoded by the DNA, said culturing being under conditions allowing expression of said polypeptide; and    isolating the recombinant endotoxin binding polypeptide produced.    
     
     
         43 . A pharmaceutical composition comprising: 
 a therapeutically effective amount of a recombinant endotoxin-neutralizing polypeptide according to claim  14 ; and    a pharmaceutically acceptable carrier.    
     
     
         44 . A method of treating a subject suffering from an endotoxin-related disorder, said method comprising: 
 administering to a subject having an endotoxin-related disorder a therapeutically effective amount of a recombinant endotoxin-neutralizing polypeptide according to  claim 14 , wherein LPS-mediated stimulation of neutrophils and mononuclear cells is inhibited.    
     
     
         45 . A method of preventing an endotoxin-related disorder in a subject, said method comprising: 
 administering to a subject a prophylactically effective amount of a recombinant endotoxin-neutralizing polypeptide according to  claim 14 , wherein the endotoxin-related disorder is prevented.