Novel low density lipoprotein binding proteins and their use in diagnosing and treating atherosclerosis

Abstract

Isolated polynucleotides encoding novel polypeptides which are capable of binding to native and methylated LDL (low density lipoprotein), the isolated polypeptides, called LBPs (LDL binding proteins), and biologically active fragments and analogs thereof, are described. Also described are methods for determining if an animal is at risk for atherosclerosis, methods for evaluating an agent for use in treating atherosclerosis, methods for treating atherosclerosis, and methods for treating a cell having an abnormality in structure or metabolism of LBP. Pharmaceutical compositions and vaccine compositions are also provided.

What is claimed is:  
     
         1 . An isolated polynucleotide comprising a member selected from the group consisting of: 
 (a) a polynucleotide encoding the polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:1;    (b) a polynucleotide encoding the polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:2;    (c) a polynucleotide encoding the polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:3;    (d) a polynucleotide encoding the polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:4;    (e) a polynucleotide encoding the polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:5;    (f) a polynucleotide encoding the polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:6;    (g) a polynucleotide encoding the polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:7;    (h) a polynucleotide encoding the polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:8;    (i) a polynucleotide encoding the polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:9;    (j) a polynucleotide capable of hybridizing to and which is at least about 95% identical to the polynucleotide of (a)-(h) or (i) wherein the encoded polypeptide is capable of binding to LDL; and    (k) a biologically active fragment of polynucleotide (a)-(i) or (j) wherein the encoded polypeptide is capable of binding to LDL.    
     
     
         2 . An isolated polynucleotide of  claim 1  wherein said member is selected from the group consisting of: 
 (a) a polynucleotide encoding the polypeptide comprising the amino acid residues 8-22 (SEQ ID NO:19), 8-33 (SEQ ID NO:20), 23-33 (SEQ ID NO:21) or 208-217 (SEQ ID NO:22) of the amino acid sequence as set forth in SEQ ID NO:7;  
 (b) a polynucleotide encoding the polypeptide comprising the amino acid residues 14-43 (SEQ ID NO:23) or 38-43 (SEQ ID NO:24) of the amino acid sequence as set forth in SEQ ID NO:1 and SEQ ID NO:6;  
 (c) a polynucleotide encoding the polypeptide comprising the amino acid residues 105-120 (SEQ ID NO:25), 105-132 (SEQ ID NO:26), 121-132 (SEQ ID NO:27) or 211-220 (SEQ ID NO:28) of the amino acid sequence as set forth in SEQ ID NO:2;  
 (d) a polynucleotide encoding the polypeptide comprising the amino acid residues 96-110 (SEQ ID NO:29) of the amino acid sequence as set forth in SEQ ID NO:5;  
 (e) a polynucleotide encoding the polypeptide comprising the amino acid residues 53-59 (SEQ ID NO:41) of the amino acid sequence as set forth in SEQ ID NO:8;  
 (f) a polynucleotide capable of hybridizing to and which is at least about 95% identical to the polynucleotide of (a)-(d) or (e) wherein the encoded polypeptide is capable of binding to LDL; and  
 (g) a biologically active fragment of polynucleotide (a)-(e) or (f) wherein the encoded polypeptide is capable of binding to LDL.  
 
     
     
         3 . The polynucleotide of  claim 1  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:10.  
     
     
         4 . The polynucleotide of  claim 1  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:11.  
     
     
         5 . The polynucleotide of  claim 1  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:12.  
     
     
         6 . The polynucleotide of  claim 1  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:13.  
     
     
         7 . The polynucleotide of  claim 1  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:14.  
     
     
         8 . The polynucleotide of  claim 1  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:15.  
     
     
         9 . The polynucleotide of  claim 1  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:16.  
     
     
         10 . The polynucleotide of  claim 1  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:17.  
     
     
         11 . The polynucleotide of  claim 1  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:18.  
     
     
         12 . The polynucleotide of  claim 2  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:30.  
     
     
         13 . The polynucleotide of  claim 2  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:31.  
     
     
         14 . The polynucleotide of  claim 2  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:32.  
     
     
         15 . The polynucleotide of  claim 2  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:33.  
     
     
         16 . The polynucleotide of  claim 2  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:34.  
     
     
         17 . The polynucleotide of  claim 2  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:35.  
     
     
         18 . The polynucleotide of  claim 2  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:36.  
     
     
         19 . The polynucleotide of  claim 2  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:37.  
     
     
         20 . The polynucleotide of  claim 2  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:38.  
     
     
         21 . The polynucleotide of  claim 2  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:39.  
     
     
         22 . The polynucleotide of  claim 2  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:40.  
     
     
         23 . The polypeptide of  claim 2  wherein said polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:42.  
     
     
         24 . The polynucleotide of  claim 1  wherein said polynucleotide is selected from the group consisting of DNA and RNA.  
     
     
         25 . The polynucleotide of  claim 1  wherein said polynucleotide is genomic DNA.  
     
     
         26 . A recombinant vector comprising said polynucleotide of  claim 1 .  
     
     
         27 . A cell comprising said recombinant vector of  claim 26 .  
     
     
         28 . A method for producing an LDL binding protein comprising culturing a cell of  claim 27  under conditions that permit expression of said LDL binding protein.  
     
     
         29 . An isolated polypeptide comprising a member selected from the group consisting of: 
 (a) a polypeptide having the amino acid sequence as set forth in SEQ ID NO:1;    (b) a polypeptide having the amino acid sequence as set forth in SEQ ID NO:2;    (c) a polypeptide having the amino acid sequence as set forth in SEQ ID NO:3;    (d) a polypeptide having the amino acid sequence as set forth in SEQ ID NO:4;    (e) a polypeptide having the amino acid sequence as set forth in SEQ ID NO:5;    (f) a polypeptide having the amino acid sequence as set forth in SEQ ID NO:6;    (g) a polypeptide having the amino acid sequence as set forth in SEQ ID NO:7;    (h) a polypeptide having the amino acid sequence as set forth in SEQ ID NO:8;    (i) a polypeptide having the amino acid sequence as set forth in SEQ ID NO:9;    (j) a polypeptide which is at least about 95% identical to the polypeptide of (a)-(h) or (i) wherein said polypeptide is capable of binding to LDL; and    (k) a biologically active fragment of polypeptide (a)-(i) or (j) wherein said fragment is capable of binding to LDL.    
     
     
         30 . An isolated polypeptide of  claim 29  wherein said member is selected from the group consisting of: 
 (a) a polypeptide having the amino acid residues 8-22 (SEQ ID NO:19), 8-33 (SEQ ID NO:20), 23-33 (SEQ ID NO:21) or 208-217 (SEQ ID NO:22) of the amino acid sequence as set forth in SEQ ID NO:7;  
 (b) a polypeptide having the amino acid residues 14-43 (SEQ ID NO:23) or 38-43 (SEQ ID NO:24) of the amino acid sequence as set forth in SEQ ID NO:1 and SEQ ID NO:6;  
 (c) a polypeptide having the amino acid residues 105-120 (SEQ ID NO:25), 105-132 (SEQ ID NO:26), 121-132 (SEQ ID NO:27) or 211-220 (SEQ ID NO:28) of the amino acid sequence as set forth in SEQ ID NO:2;  
 (d) a polypeptide having the amino acid residues 96-110 (SEQ ID NO:29) of the amino acid sequence as set forth in SEQ ID NO:5;  
 (e) a polypeptide having the amino acid residues 53-59 (SSEQ ID NO:41) of the amino acid sequence as set forth in SEQ ID NO:8;  
 (f) a polypeptide which is at least about 95% identical to the polypeptide of (a)-(d) or (e) wherein said polypeptide is capable of binding to LDL; and  
 (g) a biologically active fragment of polypeptide (a)-(e) or (f) wherein said fragment is capable of binding to LDL.  
 
     
     
         31 . A method for determining if an animal is at risk for atherosclerosis, comprising: 
 providing an animal; and    evaluating an aspect of LBP metabolism or structure in said animal, an abnormality in said aspect of LBP metabolism or structure being diagnostic of being at risk for atherosclerosis.    
     
     
         32 . The method of  claim 31  wherein said LBP is selected from the group consisting of LBP-1, LBP-2 and LBP-3.  
     
     
         33 . The method of  claim 31  wherein said aspect of LBP metabolism is the ability of said LBP to bind to LDL.  
     
     
         34 . The method of  claim 31  wherein said aspect of LBP metabolism is the ability of said LBP to bind to an arterial extracellular matrix structural component.  
     
     
         35 . The method of  claim 34  wherein said component is selected from the group consisting of proteoglycans, elastin, collagen, fibronectin, vitronectin and integrins.  
     
     
         36 . The method of  claim 31  wherein said risk is a reduced risk as compared to a normal animal.  
     
     
         37 . The method of  claim 36  wherein said abnormality results in an inactive LBP polypeptide.  
     
     
         38 . The method of  claim 31  wherein said risk is an increased risk as compared to a normal animal.  
     
     
         39 . The method of  claim 38  wherein said abnormality results in an LBP polypeptide that has higher activity than native LBP polypeptide.  
     
     
         40 . The method of  claim 31  wherein said animal is a prenatal animal.  
     
     
         41 . A method for evaluating an agent for use in treating atherosclerosis, comprising: 
 providing a test cell, cell-free system or animal;    providing an agent;    administering said agent to said test cell, cell-free system or animal in a therapeutically effective amount; and    evaluating the effect of said agent on an aspect of LBP metabolism or structure, a change in said aspect of LBP metabolism or structure being indicative of the usefulness of said agent in treating atherosclerosis.    
     
     
         42 . The method of  claim 41  wherein said test cell, cell-free system or animal has a wild type pattern of LBP metabolism.  
     
     
         43 . The method of  claim 41  wherein said test cell, cell-free system or animal has a non-wild type pattern of LBP metabolism.  
     
     
         44 . The method of  claim 41  wherein said LBP is selected from the group consisting of LBP-1, LBP-2 and LBP-3.  
     
     
         45 . The method of  claim 41  wherein said agent comprises LBP-1, LBP-2 or LBP-3 polypeptide or a biologically active fragment or analog thereof.  
     
     
         46 . The method of  claim 41  wherein said agent is selected from the group consisting of a polypeptide comprising an amino acid sequence as set forth in FIGS.  1 - 8  and  9  (SEQ ID NOS:1-9).  
     
     
         47 . The method of  claim 41  wherein said agent comprises a nucleic acid encoding LBP-1, LBP-2 or LBP-3 polypeptide or a biologically active fragment or analog thereof.  
     
     
         48 . The method of  claim 41  wherein said agent is selected from the group consisting of a nucleic acid comprising a nucleotide sequence as set forth in FIGS.  10 - 17  and  18  (SEQ ID NOS:10-18).  
     
     
         49 . The method of  claim 41  wherein said agent comprises a nucleic acid encoding an LBP regulatory sequence or a biologically active fragment thereof.  
     
     
         50 . The method of  claim 41  wherein said agent is selected from the group consisting of a binding molecule for said LBP polypeptide and a binding molecule for said LBP nucleic acid.  
     
     
         51 . The method of  claim 41  wherein said agent is an antisense nucleic acid or analog thereof.  
     
     
         52 . The method of  claim 41  wherein said agent is selected from the group consisting of a mimetic of said LBP and a mimetic of a binding molecule of said LBP.  
     
     
         53 . The method of  claim 41  wherein said agent is a polyclonal or monoclonal antibody, or fragment thereof, that can immunoreact with an LBP polypeptide.  
     
     
         54 . The method of  claim 41  wherein said agent is selected from the group consisting of a natural antibody, a recombinant antibody, a chimeric antibody and a humanized antibody that can immunoreact with an LBP polypeptide.  
     
     
         55 . The method of  claim 41  wherein said agent is a natural ligand for said LBP.  
     
     
         56 . The method of  claim 41  wherein said agent is an artificial ligand for said LBP.  
     
     
         57 . The method of  claim 41  wherein said agent is selected from the group consisting of an antagonist, an agonist and a super agonist.  
     
     
         58 . The method of  claim 41  wherein said agent is administered to a member selected from the group consisting of a transgenic cell and a transgenic animal.  
     
     
         59 . The method of  claim 41  wherein said agent is administered to said test cell or cell-free system in vitro, and if said change in said aspect of said LBP metabolism occurs, then further administering said agent to a test animal in a therapeutically effective amount and evaluating the in vivo effect of said agent on an aspect of LBP metabolism.  
     
     
         60 . The agent identified in  claim 41 .  
     
     
         61 . A method for evaluating an agent for the ability to alter the binding of LBP polypeptide to a binding molecule, comprising: 
 providing an agent;    providing LBP polypeptide;    providing a binding molecule;    combining said agent, said LBP polypeptide and said binding molecule; and    detecting the formation of a complex comprising said LBP polypeptide and said binding molecule, an alteration in the formation of said complex in the presence of said agent as compared to in the absence of said agent being indicative of said agent altering the binding of said LBP polypeptide to said binding molecule.    
     
     
         62 . The method of  claim 61  wherein said LBP polypeptide is selected from the group consisting of LBP-1, LBP-2 and LBP-3 polypeptide.  
     
     
         63 . The method of  claim 61  wherein the altering of the binding of said LBP polypeptide to said binding molecule is inhibiting the binding.  
     
     
         64 . The method of  claim 61  wherein the altering of the binding of said LBP polypeptide to said binding molecule is promoting the binding.  
     
     
         65 . The method of  claim 61  wherein said binding molecule is selected from the group consisting of native LDL and modified LDL.  
     
     
         66 . The method of  claim 61  wherein said binding molecule is an arterial extracellular matrix structural component.  
     
     
         67 . The agent identified in  claim 61 .  
     
     
         68 . A method for evaluating an agent for the ability to bind to an LBP polypeptide, comprising: 
 providing an agent;    providing an LBP polypeptide;    contacting said agent with said LBP polypeptide; and    evaluating the ability of said agent to bind to said LBP polypeptide.    
     
     
         69 . The method of  claim 68  wherein said LBP polypeptide is selected from the group consisting of LBP-1, LBP-2 and LBP-3 polypeptide.  
     
     
         70 . The agent identified in  claim 68 .  
     
     
         71 . A method for evaluating an agent for the ability to bind to a nucleic acid encoding an LBP regulatory sequence, comprising: 
 providing an agent;    providing a nucleic acid encoding an LBP regulatory sequence;    contacting said agent with said nucleic acid; and    evaluating the ability of said agent to bind to said nucleic acid.    
     
     
         72 . The method of  claim 71  wherein said LBP regulatory sequence is selected from the group consisting of LBP-1, LBP-2 and LBP-3.  
     
     
         73 . The agent identified in  claim 71 .  
     
     
         74 . A method for treating atherosclerosis in an animal, comprising: 
 providing an animal in need of treatment for atherosclerosis;    providing an agent capable of altering an aspect of LBP structure or metabolism;    administering said agent to said animal in a therapeutically effective amount such that treatment of said atherosclerosis occurs.    
     
     
         75 . The method of  claim 74  wherein said agent is an LBP polypeptide.  
     
     
         76 . The method of  claim 75  wherein said LBP polypeptide is LBP-1, LBP-2 or LBP-3 polypeptide or a biologically active fragment or analog thereof.  
     
     
         77 . The method of  claim 76  wherein said agent is selected from the group consisting of a polypeptide comprising an amino acid sequence as set forth in SEQ ID NOS:1-8 and 9.  
     
     
         78 . The method of  claim 76  wherein said agent is selected from the group consisting of a polypeptide comprising amino acid residues 8-22 (SEQ ID NO:19), 8-33 (SEQ ID NO:20), 23-33 (SEQ ID NO:21) or 208-217 (SEQ ID NO:22) of human LBP-2 as depicted in SEQ ID NO:7; amino acid residues 14-43 (SEQ ID NO:23) or 38-43 (SEQ ID NO:24) of rabbit or human LBP-1 as depicted in SEQ ID NO:1 and SEQ ID NO:6; amino acid residues 105-120 (SEQ ID NO:25), 105-132 (SEQ ID NO:26), 121-132 (SEQ ID NO:27) or 211-220 (SEQ ID NO:28) of rabbit LBP-2 as depicted in SEQ ID NO:2; amino acid residues 96-110 (SEQ ID NO:29) of rabbit LBP-3 as depicted in SEQ ID NO:5; and amino acid residues 53-59 (SEQ ID NO:41) as set forth in SEQ ID NO:8.  
     
     
         79 . The method of  claim 74  wherein said agent is a polypeptide of no more than about 50 amino acid residues in length.  
     
     
         80 . The method of  claim 74  wherein said agent is a polypeptide having an amino acid sequence that includes at least about 20% acidic amino acid residues.  
     
     
         81 . The method of  claim 74  wherein said agent is selected from the group consisting of a homopolymer of an acidic amino acid or analog thereof, and a heteropolymer of one or more acidic amino acids and one or more other amino acids or analogs thereof.  
     
     
         82 . The method of  claim 74  wherein said agent is selected from the group consisting of poly(glu), poly(asp) and poly(glu asp).  
     
     
         83 . The method of  claim 74  wherein said agent is selected from the group consisting of poly(glu N), poly(asp N) and poly(glu asp N).  
     
     
         84 . The method of  claim 74  wherein said agent is poly(glu) of no more than about 10 amino acid residues in length.  
     
     
         85 . The method of  claim 74  wherein said agent is an LBP nucleic acid or a biologically active fragment or analog thereof.  
     
     
         86 . The method of  claim 85  wherein said LBP nucleic acid comprises a nucleic acid encoding LBP-1, LBP-2 or LBP-3 polypeptide or a biologically active fragment or analog thereof.  
     
     
         87 . The method of  claim 86  wherein said agent is selected from the group consisting of a nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NOS:10-17 and 18.  
     
     
         88 . The method of  claim 74  wherein said agent is an antisense nucleic acid or analog thereof.  
     
     
         89 . A method for treating an animal at risk for atherosclerosis, comprising: 
 providing an animal at risk for atherosclerosis;    providing an agent capable of altering an aspect of LBP structure or metabolism; and    administering said agent to said animal in a therapeutically effective amount such that treatment of said animal occurs.    
     
     
         90 . A method for treating a cell having an abnormality in structure or metabolism of LBP, comprising: 
 providing a cell having an abnormality in structure or metabolism of LBP;    providing an agent capable of altering an aspect of LBP structure or metabolism; and    administering said agent to said cell in a therapeutically effective amount such that treatment of said cell occurs.    
     
     
         91 . The method of  claim 90  wherein said LBP is selected from the group consisting of LBP-1, LBP-2 and LBP-3.  
     
     
         92 . The method of  claim 90  wherein said cell is obtained from a cell culture or tissue culture.  
     
     
         93 . The method of  claim 90  wherein said cell is obtained from an embryo fibroblast.  
     
     
         94 . The method of  claim 90  wherein said cell is part of an animal.  
     
     
         95 . The method of  claim 94  wherein said animal is a non-human transgenic animal.  
     
     
         96 . A pharmaceutical composition for treating atherosclerosis in an animal, comprising: 
 a therapeutically effective amount of an agent, said agent being capable of altering an aspect of LBP metabolism or structure in said animal so as to result in treatment of said atherosclerosis; and    a pharmaceutically acceptable carrier.    
     
     
         97 . The pharmaceutical composition of  claim 96  wherein said agent is an LBP polypeptide or nucleic acid, or biologically active fragment or analog thereof.  
     
     
         98 . The pharmaceutical composition of  claim 96  wherein said agent is a polypeptide of no more than about 50 amino acid residues in length.  
     
     
         99 . The pharmaceutical composition of  claim 96  wherein said agent is a polypeptide having an amino acid sequence that includes at least about 20% acidic amino acid residues.  
     
     
         100 . A vaccine composition for treating atherosclerosis in an animal, comprising: 
 a therapeutically effective amount of an agent, said agent being capable of altering an aspect of LBP metabolism or structure in said animal so as to result in treatment of said atherosclerosis; and    a pharmaceutically acceptable carrier.    
     
     
         101 . A method for diagnosing atherosclerotic lesions in an animal, comprising: 
 providing an animal;    providing a labeled agent capable of binding to LBP present in atherosclerotic lesions;    administering said labeled agent to said animal under conditions which allow said labeled agent to interact with said LBP so as to result in labeled LBP; and    determining the localization or quantification of said labeled LBP by imaging so as to diagnose the presence of atherosclerotic lesions in said animal.    
     
     
         102 . The method of claim  101  wherein said LBP is selected from the group consisting of LBP-1, LBP-2 and LBP-3.  
     
     
         103 . The method of claim  101  wherein said imaging is selected from the group consisting of magnetic resonance imaging, gamma camera imaging, single photon emission computed tomographic (SPECT) imaging and positron emission tomography (PET).  
     
     
         104 . A method for immunizing an animal against an LBP or fragment or analog thereof, comprising: 
 providing an animal having LDL;    providing an LBP or fragment or analog thereof;    administering said LBP or fragment or analog thereof to said animal so as to stimulate antibody production by said animal to said LBP or fragment or analog thereof such that binding of said LBP to said LDL is altered.    
     
     
         105 . The method of claim  104  wherein binding of said LBP to said LDL is decreased.  
     
     
         106 . A method of making a fragment or analog of LBP polypeptide, said fragment or analog having the ability to bind to modified LDL and native LDL, comprising: 
 providing an LBP polypeptide;    altering the sequence of said LBP polypeptide; and    testing said altered LBP polypeptide for the ability to bind to modified LDL and native LDL.    
     
     
         107 . The method of claim  106  wherein said LBP is selected from the group consisting of LBP-1, LBP-2 and LBP-3.  
     
     
         108 . The method of claim  106  wherein said altered LBP polypeptide is selected from the group consisting of an antagonist, an agonist and a super agonist.  
     
     
         109 . A method for isolating a cDNA encoding an LBP, comprising: 
 providing a cDNA library;    screening said cDNA library for a cDNA encoding a polypeptide which binds to native LDL and modified LDL; and    isolating said cDNA which encodes said polypeptide, said cDNA encoding an LBP.