U.S. patent application number 09/962055 was filed with the patent office on 2002-05-02 for novel low density lipoprotein binding proteins and their use in diagnosing and treating atherosclerosis.
This patent application is currently assigned to Boston Heart Foundation, Inc., a Massachusetts corporation. Invention is credited to Arjona, Anibal A., Law, Simon W., Lees, Ann M., Lees, Robert S..
Application Number | 20020052033 09/962055 |
Document ID | / |
Family ID | 26707786 |
Filed Date | 2002-05-02 |
United States Patent
Application |
20020052033 |
Kind Code |
A1 |
Lees, Ann M. ; et
al. |
May 2, 2002 |
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.
Inventors: |
Lees, Ann M.; (Brookline,
MA) ; Lees, Robert S.; (Brookline, MA) ; Law,
Simon W.; (Lexington, MA) ; Arjona, Anibal A.;
(Boston, MA) |
Correspondence
Address: |
LOUIS MYERS
Fish & Richardson P.C.
225 Franklin Street
Boston
MA
02110-2804
US
|
Assignee: |
Boston Heart Foundation, Inc., a
Massachusetts corporation
|
Family ID: |
26707786 |
Appl. No.: |
09/962055 |
Filed: |
September 24, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09962055 |
Sep 24, 2001 |
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08979608 |
Nov 26, 1997 |
|
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60031930 |
Nov 27, 1996 |
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60048547 |
Jun 3, 1997 |
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Current U.S.
Class: |
435/196 ;
435/226; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61P 9/10 20180101; A61K
38/00 20130101; G01N 2800/323 20130101; C07K 14/705 20130101; G01N
33/6893 20130101; A61K 39/00 20130101; G01N 33/92 20130101; G01N
2800/044 20130101; C07K 14/47 20130101; A61K 49/0004 20130101 |
Class at
Publication: |
435/196 ;
435/226; 435/69.1; 435/325; 435/320.1; 536/23.2 |
International
Class: |
C12N 009/16; C12N
009/64; C07H 021/04; C12N 005/06; C12P 021/02 |
Claims
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.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/031,930 filed Nov. 27, 1996, and U.S.
Provisional Application No. 60/048,547 filed Jun. 3, 1997.
FIELD OF THE INVENTION
[0002] This invention relates to novel polypeptides (LBPs) which
bind to low density lipoprotein (LDL), polynucleotides which encode
these polypeptides, and treatments, diagnoses and therapeutic
agents for atherosclerosis.
BACKGROUND OF THE INVENTION
[0003] Atherosclerosis is the principal cause of heart attacks and
strokes. It has been reported that about 50% of all deaths in the
United States, Europe and Japan are due to atherosclerosis.
Atherosclerotic lesions in the arterial wall characterize
atherosclerosis. Cholesteryl esters (CE) are present in these
atherosclerotic lesions. Low density lipoprotein (LDL) has been
shown to be the major carrier of plasma CE, and has been implicated
as the agent by which CE enter the atherosclerotic lesions.
[0004] Scattered groups of lipid-filled macrophages, called foam
cells, are the first visible signs of atherosclerosis and are
described as type I lesions. These macrophages are reported to
contain CE derived from LDL. The macrophages recognize oxidized
LDL, but not native LDL, and become foam cells by phagocytosing
oxidized LDL. Larger, more organized collections of foam cells,
fatty streaks, represent type II lesions. These lesions further
develop into complex lesions called plaques, which can result in
impeding the flow of blood in the artery.
[0005] It is widely believed that accumulation of LDL in the artery
depends on the presence of functionally modified endothelial cells
in the arterial wall. It has been reported in animal models of
atherosclerosis that LDL, both native LDL and methylated LDL,
accumulates focally and irreversibly only at the edges of
regenerating endothelial islands in aortic lesions, where
functionally modified endothelial cells are present, but not in the
centers of these islands where endothelial regeneration is
completed. Similarly, LDL accumulates in human atherosclerotic
lesions. The mechanism by which the LDL accumulates focally and
irreversibly in arterial lesions has not heretofore been
understood.
SUMMARY OF THE INVENTION
[0006] It is an object of the invention to provide polypeptides
which bind to LDL.
[0007] It is yet another object of the invention to provide a
method for determining if an animal is at risk for
atherosclerosis.
[0008] It is yet another object of the invention to provide a
method for evaluating an agent for use in treating
atherosclerosis.
[0009] It is yet another object of the invention to provide a
method for treating atherosclerosis.
[0010] Still another object of the invention is to utilize an LBP
(low density lipoprotein binding protein) gene and/or polypeptide,
or fragments, analogs and variants thereof, to aid in the
treatment, diagnosis and/or identification of therapeutic agents
for atherosclerosis.
[0011] In one aspect, the invention features an isolated
polynucleotide comprising a polynucleotide encoding the polypeptide
comprising the amino acid sequence as set forth in SEQ ID NO:1, SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8 or SEQ ID NO:9; or a polynucleotide capable of
hybridizing to and which is at least about 95% identical to any of
the above polynucleotides and wherein the encoded polypeptide is
capable of binding to LDL; or a biologically active fragment of any
of the above polynucleotides wherein the encoded polypeptide is
capable of binding to LDL.
[0012] In certain embodiments, the polynucleotide comprises the
nucleic acid sequence as set forth in SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17 or SEQ ID NO:18.
[0013] Another aspect of the invention is an isolated polypeptide
comprising a polypeptide having the amino acid sequence as set
forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; or a
polypeptide which is at least about 95% identical to any of the
above polypeptides and wherein the polypeptide is capable of
binding to LDL; or a biologically active fragment of any of the
above polypeptides wherein the fragment is capable of binding to
LDL.
[0014] Another aspect of the invention is a method for determining
if an animal is at risk for atherosclerosis. An animal is provided.
An aspect of LBP metabolism or structure is evaluated in the
animal. An abnormality in the aspect of LBP metabolism or structure
is diagnostic of being at risk for atherosclerosis.
[0015] Another aspect of the invention is a method for evaluating
an agent for use in treating atherosclerosis. A test cell,
cell-free system or animal is provided. An agent is provided. The
agent is administered to the test cell, cell-free system or animal
in a therapeutically effective amount. The effect of the agent on
an aspect of LBP metabolism or structure is evaluated. A change in
the aspect of LBP metabolism or structure is indicative of the
usefulness of the agent in treating atherosclerosis.
[0016] Another aspect of the invention is a method for evaluating
an agent for the ability to alter the binding of LBP polypeptide to
a binding molecule, e.g., native LDL, modified LDL, e.g.,
methylated LDL or oxidized LDL, or an arterial extracellular matrix
structural component. An agent is provided. An LBP polypeptide is
provided. A binding molecule is provided. The agent, LBP
polypeptide and binding molecule are combined. The formation of a
complex comprising the LBP polypeptide and binding molecule is
detected. An alteration in the formation of the complex in the
presence of the agent as compared to in the absence of the agent is
indicative of the agent altering the binding of the LBP polypeptide
to the binding molecule.
[0017] Another aspect of the invention is a method for evaluating
an agent for the ability to bind to an LBP polypeptide. An agent is
provided. An LBP polypeptide is provided. The agent is contacted
with the LBP polypeptide. The ability of the agent to bind to the
LBP polypeptide is evaluated.
[0018] Another aspect of the invention is a method for evaluating
an agent for the ability to bind to a nucleic acid encoding an LBP
regulatory sequence. An agent is provided. A nucleic acid encoding
an LBP regulatory sequence is provided. The agent is contacted with
the nucleic acid. The ability of the agent to bind to the nucleic
acid is evaluated.
[0019] Another aspect of the invention is a method for treating
atherosclerosis in an animal. An animal in need of treatment for
atherosclerosis is provided. An agent capable of altering an aspect
of LBP structure or metabolism is provided. The agent is
administered to the animal in a therapeutically effective amount
such that treatment of the atherosclerosis occurs. In certain
embodiments, the agent is an LBP polypeptide, e.g., LBP-1, LBP-2 or
LBP-3, or a biologically active fragment or analog thereof. In
certain embodiments, the agent is a polypeptide of no more than
about 100, 50, 30, 20, 10, 5, 4, 3 or 2 amino acid residues in
length. In certain embodiments, the agent is a polypeptide having
an amino acid sequence that includes at least about 20%, 40%, 60%,
80%, 90%, 95% or 98% acidic amino acid residues.
[0020] Another aspect of the invention is a method for treating an
animal at risk for atherosclerosis. An animal at risk for
atherosclerosis is provided. An agent capable of altering an aspect
of LBP structure or metabolism is provided. The agent is
administered to the animal in a therapeutically effective amount
such that treatment of the animal occurs.
[0021] Another aspect of the invention is a method for treating a
cell having an abnormality in structure or metabolism of LBP. A
cell having an abnormality in structure or metabolism of LBP is
provided. An agent capable of altering an aspect of LBP structure
or metabolism is provided. The agent is administered to the cell in
a therapeutically effective amount such that treatment of the cell
occurs.
[0022] Another aspect of the invention is a pharmaceutical
composition for treating atherosclerosis in an animal comprising a
therapeutically effective amount of an agent, the agent being
capable of altering an aspect of LBP metabolism or structure in the
animal so as to result in treatment of the atherosclerosis, and a
pharmaceutically acceptable carrier.
[0023] Another aspect of the invention is a vaccine composition for
treating atherosclerosis in an animal comprising a therapeutically
effective amount of an agent, the agent being capable of altering
an aspect of LBP metabolism or structure in the animal so as to
result in treatment of the atherosclerosis, and a pharmaceutically
acceptable carrier.
[0024] Another aspect of the invention is a method for diagnosing
atherosclerotic lesions in an animal. An animal is provided. A
labeled agent capable of binding to LBP, e.g., LBP-1, LBP-2 or
LBP-3, present in atherosclerotic lesions is provided. The labeled
agent is administered to the animal under conditions which allow
the labeled agent to interact with the LBP so as to result in
labeled LBP. The localization or quantification of the labeled LBP
is determined by imaging so as to diagnose the presence of
atherosclerotic lesions in the animal.
[0025] Another aspect of the invention is a method for immunizing
an animal against an LBP, e.g., LBP-1, LBP-2 or LBP-3, or fragment
or analog thereof. An animal having LDL is provided. The LBP or
fragment or analog thereof is administered to the animal so as to
stimulate antibody production by the animal to the LBP or fragment
or analog thereof such that binding of the LBP to the LDL is
altered, e.g., decreased or increased.
[0026] Another aspect of the invention is a method of making a
fragment or analog of LBP polypeptide, the fragment or analog
having the ability to bind to native LDL and to modified LDL, e.g.,
methylated LDL, oxidized LDL, acetylated LDL, or
cyclohexanedione-treated LDL. An LBP polypeptide is provided. The
sequence of the LBP polypeptide is altered. The altered LBP
polypeptide is tested for the ability to bind to modified LDL and
native LDL.
[0027] Yet another aspect of the invention is a method for
isolating a cDNA encoding an LBP. A cDNA library is provided. The
cDNA library is screened for a cDNA encoding a polypeptide which
binds to native LDL and modified LDL, e.g., methylated LDL or
oxidized LDL. The cDNA which encodes the polypeptide is isolated,
the cDNA encoding an LBP.
[0028] The above and other features, objects and advantages of the
present invention will be better understood by a reading of the
following specification in conjunction with the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 depicts the amino acid sequence of rabbit LBP-1 (SEQ
ID NO:1). Differences in amino acids between rabbit and human LBP-1
are depicted in bold type.
[0030] FIG. 2 depicts the amino acid sequence of rabbit LBP-2 (SEQ
ID NO:2). Differences in amino acids between rabbit and human LBP-2
are depicted in bold type.
[0031] FIG. 3 depicts the amino acid sequence of amino acids 86 to
317 of rabbit LBP-2 (SEQ ID NO:3).
[0032] FIG. 4 depicts the amino acid sequence of amino acids 66 to
317 of rabbit LBP-2 (SEQ ID NO:4).
[0033] FIG. 5 depicts the amino acid sequence of rabbit LBP-3 (SEQ
ID NO:5). Differences in amino acids between rabbit and human LBP-3
are depicted in bold type.
[0034] FIG. 6 depicts the amino acid sequence of human LBP-1 (SEQ
ID NO:6). Differences in amino acids between rabbit and human LBP-1
are depicted in bold type.
[0035] FIG. 7 depicts the amino acid sequence of human LBP-2 (SEQ
ID NO:7). Differences in amino acids between rabbit and human LBP-2
are depicted in bold type.
[0036] FIG. 8 depicts the amino acid sequence of human LBP-3 (SEQ
ID NO:8). Differences in amino acids between rabbit and human LBP-3
are depicted in bold type.
[0037] FIG. 9 depicts the amino acid sequence of amino acids 14 to
33 of human or rabbit LBP-1, called EHF-1 (SEQ ID NO:9).
[0038] FIG. 10 depicts the cDNA sequence encoding rabbit LBP-1 (SEQ
ID NO:10) and the corresponding amino acid sequence. Differences in
amino acids between rabbit and human LBP-1 are depicted in bold
type.
[0039] FIG. 11 depicts the cDNA sequence encoding rabbit LBP-2 (SEQ
ID NO:11) and the corresponding amino acid sequence. Differences in
amino acids between rabbit and human LBP-2 are depicted in bold
type.
[0040] FIG. 12 depicts the cDNA sequence 256 to 1617 of rabbit
LBP-2 (SEQ ID NO:12) and the corresponding amino acid sequence.
[0041] FIG. 13 depicts the cDNA sequence 196 to 1617 of rabbit
LBP-2 (SEQ ID NO:13) and the corresponding amino acid sequence.
[0042] FIG. 14 depicts the cDNA sequence encoding rabbit LBP-3 (SEQ
ID NO:14) and the corresponding amino acid sequence. Differences in
amino acids between rabbit and human LBP-3 are depicted in bold
type.
[0043] FIG. 15 depicts the cDNA sequence encoding human LBP-1 (SEQ
ID NO:15) and the corresponding amino acid sequence. Differences in
amino acids between rabbit and human LBP-1 are depicted in bold
type.
[0044] FIG. 16 depicts the cDNA sequence encoding human LBP-2 (SEQ
ID NO:16) and the corresponding amino acid sequence. Differences in
amino acids between rabbit and human LBP-2 are depicted in bold
type.
[0045] FIG. 17 depicts the cDNA sequence encoding human LBP-3 (SEQ
ID NO:17) and the corresponding amino acid sequence. Differences in
amino acids between rabbit and human LBP-3 are depicted in bold
type.
[0046] FIG. 18 depicts the cDNA sequence encoding BHF-1 (SEQ ID
NO:18).
[0047] FIG. 19 corresponds to the amino acid sequence of rabbit
LBP-1 (top sequence) in alignment with the amino acid sequence of
human LBP-1 (bottom sequence).
[0048] FIG. 20 corresponds to the amino acid sequence of rabbit
LBP-2 (top sequence) in alignment with the amino acid sequence of
human LBP-2 (bottom sequence).
[0049] FIG. 21 corresponds to the amino acid sequence of rabbit
LBP-3 (top sequence) in alignment with the amino acid sequence of
human LBP-3 (bottom sequence).
DETAILED DESCRIPTION
[0050] In accordance with aspects of the present invention, there
are provided novel mature human and rabbit polypeptides, LBP-1,
LBP-2 and LBP-3, and biologically active analogs and fragments
thereof, and there are provided isolated polynucleotides which
encode such polypeptides. LBP is an abbreviation for low density
lipoprotein (LDL) binding protein. The terms polynucleotide,
nucleotide and oligonucleotide are used interchangeably herein, and
the terms polypeptides, proteins and peptides are used
interchangeably herein.
[0051] This invention provides for an isolated polynucleotide
comprising a polynucleotide encoding the polypeptide having the
amino acid sequence of rabbit LBP-1 as set forth in FIG. 1 (SEQ ID
NO:1); rabbit LBP-2 as set forth in FIG. 2 (SEQ ID NO:2); 86 to 317
of rabbit LBP-2 as set forth in FIG. 3 (SEQ ID NO:3); 66 to 317 of
rabbit LBP-2 as set forth in FIG. 4 (SEQ ID NO:4); rabbit LBP-3 as
set forth in FIG. 5 (SEQ ID NO:5); human LBP-1 as set forth in FIG.
6 (SEQ ID NO:6); human LBP-2 as set forth in FIG. 7 (SEQ ID NO:7);
human LBP-3 as set forth in FIG. 8 (SEQ ID NO:8); 14 to 33 of human
or rabbit LBP-1, called BHF-1, as set forth in FIG. 9 (SEQ ID
NO:9); a polynucleotide capable of hybridizing to and which is at
least about 80% identical, more preferably at least about 90%
identical, more preferably yet at least about 95% identical, and
most preferably at least about 98% identical to any of the above
polynucleotides, and wherein the encoded polypeptide is capable of
binding to LDL; or a biologically active fragment of any of the
above polynucleotides wherein the encoded polypeptide is capable of
binding to LDL.
[0052] This invention also includes an isolated polynucleotide
comprising a polynucleotide encoding the polypeptide having 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 set forth in
FIG. 7 (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 set forth in FIG.
1 (SEQ ID NO:1) and FIG. 6 (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 set forth in
FIG. 2 (SEQ ID NO:2); amino acid residues 96-110 (SEQ ID NO:29) of
rabbit LBP-3 as set forth in FIG. 5 (SEQ ID NO:5); amino acid
residues 53-59 (SEQ ID NO:41) of human LBP-3 as set forth in FIG. 8
(SEQ ID NO:8); a polynucleotide capable of hybridizing to and which
is at least about 80% identical, more preferably at least about 90%
identical, more preferably yet at least about 95% identical, and
most preferably at least about 98% identical to any of the above
polynucleotides, and wherein the encoded polypeptide is capable of
binding to LDL; or a biologically active fragment of any of the
above polynucleotides wherein the encoded polypeptide is capable of
binding to LDL.
[0053] By a polynucleotide encoding a polypeptide is meant a
polynucleotide which includes only coding sequence for the
polypeptide, as well as a polynucleotide which includes additional
coding and/or non-coding sequences. Thus, e.g., the polynucleotides
which encode for the mature polypeptides of FIGS. 1-9 (SEQ ID
NOS:1-9) may include only the coding sequence for the mature
polypeptide; the coding sequence for the mature polypeptide and
additional coding sequence such as a leader or secretory sequence
or a proprotein sequence; the coding sequence for the mature
polypeptide (and optionally additional coding sequence) and
non-coding sequence, such as introns or non-coding sequences 5'
and/or 3' of the coding sequence for the mature polypeptide. The
polynucleotides of the invention are also meant to include
polynucleotides in which the coding sequence for the mature
polypeptide is fused in the same reading frame to a polynucleotide
sequence which aids in expression and/or secretion of a polypeptide
from a host cell, e.g., a leader sequence. The polynucleotides are
also meant to include polynucleotides in which the coding sequence
is fused in frame to a marker sequence which, e.g., allows for
purification of the polypeptide.
[0054] The polynucleotides of the present invention may be in the
form of RNA, DNA or PNA, e.g., cRNA, cDNA, genomic DNA, or
synthetic DNA, RNA or PNA. The DNA may be double-stranded or single
stranded, and if single stranded may be the coding strand or
non-coding (anti-sense) strand.
[0055] In preferred embodiments, the polynucleotide comprises the
nucleic acid of rabbit LBP-1 as set forth in FIG. 10 (SEQ ID
NO:10); rabbit LBP-2 as set forth in FIG. 11 (SEQ ID NO:11);
nucleotide 256 to 1617 of rabbit LBP-2 as set forth in FIG. 12 (SEQ
ID NO:12); nucleotide 196 to 1617 of rabbit LBP-2 as set forth in
FIG. 13 (SEQ ID NO:13); rabbit LBP-3 as set forth in FIG. 14 (SEQ
ID NO:14); human LBP-1 as set forth in FIG. 15 (SEQ ID NO:15);
human LBP-2 as set forth in FIG. 16 (SEQ ID NO:16); human LBP-3 as
set forth in FIG. 17 (SEQ ID NO:17); or nucleotide 97 to 156 of
rabbit LBP-1 or nucleotide 157 to 216 of human LBP-1, (BHF-1), as
set forth in FIG. 18 (SEQ ID NO:18).
[0056] In other preferred embodiments, the polynucleotide comprises
the nucleic acid as set forth in SEQ ID NO:30, SEQ ID NO:31, SEQ ID
NO:32, SEQ ID NO:33 SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ
ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 or SEQ ID
NO:42.
[0057] The coding sequence which encodes the mature polypeptide may
be identical to the coding sequences shown in FIGS. 10-18 (SEQ ID
NOS:10-18) or SEQ ID NOS:30-40 or 42, or may be a different coding
sequence which coding sequence, as a result of the redundancy or
degeneracy of the genetic code, encodes the same mature
polypeptides as the DNA of FIGS. 10-18 (SEQ ID NOS:10-18) and SEQ
ID NOS: 30-40 and 42.
[0058] This invention also includes recombinant vectors comprising
the polynucleotides described above. The vector can be, e.g., a
plasmid, a viral particle or a phage. In certain embodiments, the
recombinant vector is an expression vector. The vectors may also
include various marker genes which are useful in identifying cells
containing such vectors.
[0059] This invention also includes a cell comprising such a
recombinant vector. The recombinant vectors described herein can be
introduced into a host cell, e.g., by transformation, transfection
or infection.
[0060] This invention also includes a method for producing an LBP
comprising culturing such a cell under conditions that permit
expression of the LBP.
[0061] This invention also includes an isolated polypeptide
comprising a polypeptide having the amino acid sequence as set
forth in FIG. 1 (SEQ ID NO:1); FIG. 2 (SEQ ID NO:2); FIG. 3 (SEQ ID
NO:3); FIG. 4 (SEQ ID NO:4); FIG. 5 (SEQ ID NO:5); FIG. 6 (SEQ ID
NO:6); FIG. 7 (SEQ ID NO:7); FIG. 8 (SEQ ID NO:8) or FIG. 9 (SEQ ID
NO:9); or a polypeptide which is at least about 80% identical, more
preferably at least about 90% identical, more preferably yet at
least about 95% identical, and most preferably at least about 98%
identical to the above polypeptides, and wherein said polypeptide
is capable of binding to LDL; or a biologically active fragment of
any of the above polypeptides wherein the fragment is capable of
binding to LDL. Differences in amino acids between the rabbit and
human LBP-1, LBP-2 and LBP-3 genes are depicted in bold type in the
figures. The differences in the amino acid sequences between rabbit
and human LBP-1, LBP-2 and LBP-3 are also specifically shown in
FIGS. 19, 20 and 21, respectively.
[0062] This invention also includes an isolated polypeptide
comprising a polypeptide having 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) as set forth in FIG. 7 (SEQ ID NO:7); amino acid residues
14-43 (SEQ ID NO:23) or 38-43 (SEQ ID NO:24) as set forth in FIG. 1
(SEQ ID NO:1) and FIG. 6 (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) as set forth in FIG. 2 (SEQ ID NO:2); amino
acid residues 96-110 (SEQ ID NO:29) as set forth in FIG. 5 (SEQ ID
NO:5); and amino acid residues 53-59 (SEQ ID NO:41) as set forth in
FIG. 8 (SEQ ID NO:8); or a polypeptide which is at least about 80%
identical, more preferably at least about 90% identical, more
preferably yet at least about 95% identical, and most preferably at
least about 98% identical to the above polypeptides, and wherein
said polypeptide is capable of binding to LDL; or a biologically
active fragment of any of the above polypeptides wherein the
fragment is capable of binding to LDL.
[0063] The polypeptides of the invention are meant to include,
e.g., a naturally purified product, a chemically synthesized
product, and a recombinantly derived product.
[0064] The polypeptides can be used, e.g., to bind to LDL, thereby
inhibiting formation of atherosclerotic plaques. The polypeptides
can also be used, e.g., in gene therapy, by expression of such
polypeptides in vivo. The polypeptides can also be used in
pharmaceutical or vaccine compositions. The polypeptides can also
be used as immunogens to produce antibodies thereto, which in turn,
can be used as antagonists to the LBP polypeptides.
[0065] Without being bound by any theory, it is believed that the
LBPs provide the mechanism by which atherosclerosis is promoted
through LDL oxidation. The LBPs are believed to be required in
order for focal, irreversible LDL binding to occur at the arterial
wall, and that such binding is a critical early event in
atherosclerosis because it allows the time necessary for LDL to be
changed from its native state to a fully oxidized state. Since
oxidized, but not native, LDL is a foreign protein, macrophages
ingest it, first becoming the foam cells of type I lesions, and
subsequently forming the fatty streaks of type II lesions.
[0066] This invention also includes a method for determining if an
animal is at risk for atherosclerosis. An animal is provided. An
aspect of LBP metabolism or structure is evaluated in the animal.
An abnormality in the aspect of LBP metabolism or structure is
diagnostic of being at risk for atherosclerosis.
[0067] By atherosclerosis is meant a disease or condition which
comprises several stages which blend imperceptibly into each other,
including irreversible binding of LDL, LDL oxidation, macrophage
recruitment, blockage of the artery and tissue death
(infarction).
[0068] By animal is meant human as well as non-human animals.
Non-human animals include, e.g., mammals, birds, reptiles,
amphibians, fish, insects and protozoa. Preferably, the non-human
animal is a mammal, e.g., a rabbit, a rodent, e.g., a mouse, rat or
guinea pig, a primate, e.g., a monkey, or a pig. An animal also
includes transgenic non-human animals. The term transgenic animal
is meant to include an animal that has gained new genetic
information from the introduction of foreign DNA, i.e., partly or
entirely heterologous DNA, into the DNA of its cells; or
introduction of a lesion, e.g., an in vitro induced mutation, e.g.,
a deletion or other chromosomal rearrangement into the DNA of its
cells; or introduction of homologous DNA into the DNA of its cells
in such a way as to alter the genome of the cell into which the DNA
is inserted, e.g., it is inserted at a location which differs from
that of the natural gene or its insertion results in a knockout or
replacement of the homologous host gene or results in altered
and/or regulatable expression and/or metabolism of the gene. The
animal may include a transgene in all of its cells including germ
line cells, or in only one or some of its cells. Transgenic animals
of the invention can serve as a model for studying atherosclerosis
or for evaluating agents to treat atherosclerosis.
[0069] In certain embodiments, the determination for being at risk
for atherosclerosis is done in a prenatal animal.
[0070] By LBP is meant a low density lipoprotein (LDL) binding
protein which is capable of binding LDL and methylated LDL. By
methylated LDL is meant that about 50% to about 90% of the lysine
residues of LDL have a methyl group chemically attached. Methylated
LDL is not recognized by previously reported cell surface
receptors. See, e.g., Weisgraber et al., J. Biol. Chem.
253:9053-9062 (1978) In certain embodiments, the LBP is also
capable of binding oxidized LDL. In certain preferred embodiments,
the binding of LDL to an LBP is irreversible. In certain preferred
embodiments, the LBP does not transport the LDL to any
intracellular compartment. Examples of LBPs are LBP-1, LBP-2 and
LBP-3 described herein.
[0071] By LBP metabolism is meant any aspect of the production,
release, expression, function, action, interaction or regulation of
LBP. The metabolism of LBP includes modifications, e.g., covalent
or non-covalent modifications, of LBP polypeptide. The metabolism
of LBP includes modifications, e.g., covalent or non-covalent
modifications, that LBP induces in other substances. The metabolism
of LBP also includes changes in the distribution of LBP
polypeptide, and changes LBP induces in the distribution of other
substances.
[0072] Any aspect of LBP metabolism can be evaluated. The methods
used are standard techniques known to those skilled in the art and
can be found in standard references, e.g., Ausubel et al., ed.,
Current Protocols in Mol. Biology, New York: John Wiley & Sons,
1990; Kriegler, M., ed., Gene Transfer and Expression, Stockton
Press, New York, N.Y., 1989; pDisplay gene expression system
(Invitrogen, Carlsbad, Calif.). Preferred examples of LBP
metabolism that can be evaluated include the binding activity of
LBP polypeptide to a binding molecule, e.g., LDL; the
transactivation activity of LBP polypeptide on a target gene; the
level of LBP protein; the level of LBP mRNA; the level of LBP
modifications, e.g., phosphorylation, glycosylation or acylation;
or the effect of LBP expression on transfected mammalian cell
binding of LDL.
[0073] By binding molecule is meant any molecule to which LBP can
bind, e.g., a nucleic acid, e.g., a DNA regulatory region, a
protein, e.g., LDL, a metabolite, a peptide mimetic, a non-peptide
mimetic, an antibody, or any other type of ligand. In certain
preferred embodiments, the aspect of LBP metabolism that is
evaluated is the ability of LBP to bind to native LDL and/or
methylated LDL and/or oxidized LDL. Binding to LDL can be shown,
e.g., by antibodies against LDL, affinity chromatography, affinity
coelectrophoresis (ACE) assays, or ELISA assays. See Examples. In
other embodiments, it is the ability of LBP to bind to an arterial
extracellular matrix stuctural component that is evaluated.
Examples of such components include proteoglycans, e.g.,
chondroitin sulfate proteoglycans and heparin sulfate
proteoglycans; elastin; collagen; fibronectin; vitronectin;
integrins; and related extracellular matrix molecules. Binding to
arterial extracellular matrix structural components can be shown by
standard methods known to those skilled in the art, e.g., by ELISA
assays. Primary antibodies to the LBP are then added, followed by
an enzyme-conjugated secondary antibody to the primary antibody,
which produces a stable color in the presence of an appropriate
substrate, and color development on the plates is measured in a
microtiter plate reader.
[0074] Transactivation of a target gene by LBP can be determined,
e.g., in a transient transfection assay in which the promoter of
the target gene is linked to a reporter gene, e.g.,
.beta.-galactosidase or luciferase, and co-transfected with an LBP
expression vector. Such evaluations can be done in vitro or in
vivo. Levels of LBP protein, mRNA or phosphorylation, can be
measured, e.g., in a sample, e.g., a tissue sample, e.g., arterial
wall, by standard methods known to those skilled in the art.
[0075] In certain embodiments, an aspect of LBP structure is
evaluated, e.g., LBP gene structure or LBP protein structure. For
example, primary, secondary or tertiary structures can be
evaluated. For example, the DNA sequence of the gene is determined
and/or the amino acid sequence of the protein is determined.
Standard cloning and sequencing methods can be used as are known to
those skilled in the art. In certain embodiments, the binding
activity of an antisense nucleic acid with the cellular LBP mRNA
and/or genomic DNA is determined using standard methods known to
those skilled in the art so as to detect the presence or absence of
the target mRNA or DNA sequences to which the antisense nucleic
acid would normally specifically bind.
[0076] The risk for atherosclerosis that is determined can be a
reduced risk or an increased risk as compared to a normal animal.
For example, an abnormality which would give a reduced risk is an
inactive LBP polypeptide. An abnormality which would give an
increased risk would be, e.g., an LBP polypeptide that has higher
activity, e.g., LDL binding activity, than native LBP
polypeptide.
[0077] The invention also includes a method for evaluating an agent
for use in treating atherosclerosis. A test cell, cell-free system
or animal is provided. An agent is provided. The agent is
administered to the test cell, cell-free system or animal in a
therapeutically effective amount. The effect of the agent on an
aspect of LBP metabolism or structure is evaluated. A change in the
aspect of LBP metabolism or structure is indicative of the
usefulness of the agent in treating atherosclerosis.
[0078] In certain embodiments, the method employs two phases for
evaluating an agent for use in treating atherosclerosis, an initial
in vitro phase and then an in vivo phase. The agent is administered
to the test cell or cell-free system in vitro, and if a change in
an aspect of LBP metabolism occurs, then the agent is further
administered to a test animal in a therapeutically effective amount
and evaluated in vivo for an effect of the agent on an aspect of
LBP metabolism.
[0079] By cell is meant a cell or a group of cells, or a cell that
is part of an animal. The cell can be a human or non-human cell.
Cell is also meant to include a transgenic cell. The cell can be
obtained, e.g., from a culture or from an animal. Animals are meant
to include, e.g., natural animals and non-human transgenic animals.
In certain embodiments, the transgenic cell or non-human transgenic
animal has an LBP transgene, or fragment or analog thereof. In
certain embodiments, the transgenic cell or non-human transgenic
animal has a knockout for the LBP gene.
[0080] The test cell, cell-free system or animal can have a wild
type pattern or a non-wild type pattern of LBP metabolism. A
non-wild type pattern of LBP metabolism can result, e.g., from
under-expression, over-expression, no expression, or a temporal,
site or distribution change. Such a non-wild type pattern can
result, e.g., from one or more mutations in the LBP gene, in a
binding molecule gene, a regulatory gene, or in any other gene
which directly or indirectly affects LBP metabolism. A mutation is
meant to include, e.g., an alteration, e.g., in gross or fine
structure, in a nucleic acid. Examples include single base pair
alterations, e.g., missense or nonsense mutations, frameshifts,
deletions, insertions and translocations. Mutations can be dominant
or recessive. Mutations can be homozygous or heterozygous.
Preferably, an aspect of LBP-1, LBP-2 or LBP-3 metabolism is
evaluated.
[0081] An agent is meant to include, e.g., any substance, e.g., an
anti-atherosclerosis drug. The agent of this invention preferably
can change an aspect of LBP metabolism. Such change can be the
result of any of a variety of events, including, e.g., preventing
or reducing interaction between LBP and a binding molecule, e.g.,
LDL or an arterial extracellular matrix structural component;
inactivating LBP and/or the binding molecule, e.g., by cleavage or
other modification; altering the affinity of LBP and the binding
molecule for each other; diluting out LBP and/or the binding
molecule; preventing expression of LBP and/or the binding molecule;
reducing synthesis of LBP and/or the binding molecule; synthesizing
an abnormal LBP and/or binding molecule; synthesizing an
alternatively spliced LBP and/or binding molecule; preventing or
reducing proper conformational folding of LBP and/or the binding
molecule; modulating the binding properties of LBP and/or the
binding molecule; interfering with signals that are required to
activate or deactivate LBP and/or the binding molecule; activating
or deactivating LBP and/or the binding molecule in such a way as to
prevent binding; or interfering with other receptors, ligands or
other molecules which are required for the normal synthesis or
functioning of LBP and/or the binding molecule. For example, the
agent can block the binding site on LDL for LBPs expressed focally
in the arterial wall extracellular matrix, or it could block the
binding site on an LBP for LDL, or it could be bifunctional, i.e.,
it could block both binding sites.
[0082] Examples of agents include LBP polypeptide, e.g., LBP-1,
LBP-2 or LBP-3, or a biologically active fragment or analog
thereof; a nucleic acid encoding LBP polypeptide or a biologically
active fragment or analog thereof; a nucleic acid encoding an LBP
regulatory sequence or a biologically active fragment or analog
thereof; a binding molecule for LBP polypeptide; a binding molecule
for LBP nucleic acid, the LBP nucleic acid being, e.g., a nucleic
acid comprising a regulatory region for LBP or a nucleic acid
comprising a structural region for LBP or a biologically active
fragment of LBP; an antisense nucleic acid; a mimetic of LBP or a
binding molecule; an antibody for LBP or a binding molecule; a
metabolite; or an inhibitory carbohydrate or glycoprotein. In
certain embodiments, the agent is an antagonist, agonist or super
agonist.
[0083] Knowledge of the existence of the sequence of the LBPs
allows a search for natural or artificial ligands to regulate LDL
levels in the treatment of atherosclerosis. In certain embodiments,
the agent is a natural ligand for LBP. In certain embodiments, the
agent is an artificial ligand for LBP.
[0084] By analog is meant a compound that differs from naturally
occurring LBP in amino acid sequence or in ways that do not involve
sequence, or both. Analogs of the invention generally exhibit at
least about 80% homology, preferably at least about 90% homology,
more preferably yet at least about 95% homology, and most
preferably at least about 98% homology, with substantially the
entire sequence of a naturally occurring LBP sequence, preferably
with a segment of about 100 amino acid residues, more preferably
with a segment of about 50 amino acid residues, more preferably yet
with a segment of about 30 amino acid residues, more preferably yet
with a segment of about 20 amino acid residues, more preferably yet
with a segment of about 10 amino acid residues, more preferably yet
with a segment of about 5 amino acid residues, more preferably yet
with a segment of about 4 amino acid residues, more preferably yet
with a segment of about 3 amino acid residues, and most preferably
with a segment of about 2 amino acid residues. Non-sequence
modifications include, e.g., in vivo or in vitro chemical
derivatizations of LBP. Non-sequence modifications include, e.g.,
changes in phosphorylation, acetylation, methylation,
carboxylation, or glycosylation. Methods for making such
modifications are known to those skilled in the art. For example,
phosphorylation can be modified by exposing LBP to
phosphorylation-altering enzymes, e.g., kinases or
phosphatases.
[0085] Preferred analogs include LBP or biologically active
fragments thereof whose sequences differ from the wild-type
sequence by one or more conservative amino acid substitutions or by
one or more non-conservative amino acid substitutions, deletions,
or insertions which do not abolish LBP biological activity.
Conservative substitutions typically include the substitution of
one amino acid for another with similar characteristics, e.g.,
substitutions within the following groups: valine, glycine;
glycine, alanine; valine, isoleucine, leucine; aspartic acid,
glutamic acid; asparagine, glutamine; serine, threonine; lysine,
arginine; and phenylalanine, tyrosine. Other examples of
conservative substitutions are shown in Table 1.
1TABLE 1 CONSERVATIVE AMINO ACID SUBSTITUTIONS For Amino Acid Code
Replace with any of Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys
Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile,
D-Met, D-Ile, Orn, D-Orn, L-NMMA, L-NAME Asparagine N D-Asn, Asp,
D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn,
Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr,
D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp
Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G
Ala, D-Ala, Pro, D-Pro, .beta.-Ala Asp Histidine H D-His Isoleucine
I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val,
D-Val, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg,
D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met,
S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe,
Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or 5-
phenylproline, cis-3,4, or 5- phenylproproline Proline P D-Pro,
L-I-thioazolidine-4-carboxylic acid, D-or
L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,
allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T
D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val,
D-Val Tryptophan W D-Trp, Phe, D-Phe, Tyr, D-Tyr Tyrosine Y D-Tyr,
Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile,
D-Ile, Met, D-Met
[0086] Amino acid sequence variants of a protein can be prepared by
any of a variety of methods known to those skilled in the art. For
example, random mutagenesis of DNA which encodes a protein or a
particular domain or region of a protein can be used, e.g., PCR
mutagenesis (using, e.g., reduced Taq polymerase fidelity to
introduce random mutations into a cloned fragment of DNA; Leung et
al., BioTechnique 1:11-15 (1989)), or saturation mutagenesis (by,
e.g., chemical treatment or irradiation of single-stranded DNA in
vitro, and synthesis of a complementary DNA strand; Mayers et al.,
Science 229:242 (1985)). Random mutagenesis can also be
accomplished by, e.g., degenerate oligonucleotide generation
(using, e.g., an automatic DNA synthesizer to chemically synthesize
degenerate sequences; Narang, Tetrahedron 39:3 (1983); Itakura et
al., Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules,
ed. A. G. Walton, Amsterdam: Elsevier, pp. 273-289 (1981)).
Non-random or directed mutagenesis can be used to provide specific
sequences or mutations in specific regions. These techniques can be
used to create variants which include, e.g., deletions, insertions,
or substitutions, of residues of the known amino acid sequence of a
protein. The sites for mutation can be modified individually or in
series, e.g., by (i) substituting first with conserved amino acids
and then with more radical choices depending upon results achieved,
(ii) deleting the target residue, (iii) inserting residues of the
same or a different class adjacent to the located site, or (iv)
combinations of the above. For example, analogs can be made by in
vitro DNA sequence modifications of the sequences of FIGS. 10-18
(SEQ ID NOS:10-18). For example, in vitro mutagenesis can be used
to convert any of these DNA sequences into a sequence which encodes
an analog in which one or more amino acid residues has undergone a
replacement, e.g., a conservative replacement as described in Table
1.
[0087] Methods for identifying desirable mutations include, e.g.,
alanine scanning mutagenesis (Cunningham and Wells, Science
244:1081-1085 (1989)), oligonucleotide-mediated mutagenesis
(Adelman et al., DNA 2:183 (1983)); cassette mutagenesis (Wells et
al., Gene 34:315 (1985)), combinatorial mutagenesis, and phage
display libraries (Ladner et al., PCT International Appln. No.
WO88/06630). The LBP analogs can be tested, e.g., for their ability
to bind to LDL and/or to an arterial extracellular matrix
component, as described herein.
[0088] Other analogs within the invention include, e.g., those with
modifications which increase peptide stability. Such analogs may
contain, e.g., one or more non-peptide bonds (which replace the
peptide bonds) in the peptide sequence. Also included are, e.g.:
analogs that include residues other than naturally occurring
L-amino acids, e.g., D-amino acids or non-naturally occurring or
synthetic amino acids, e.g., .beta. or .gamma. amino acids; and
cyclic analogs.
[0089] Analogs are also meant to include peptides in which
structural modifications have been introduced into the peptide
backbone so as to make the peptide non-hydrolyzable. Such peptides
are particularly useful for oral administration, as they are not
digested. Peptide backbone modifications include, e.g.,
modifications of the amide nitrogen, the .alpha.-carbon, the amide
carbonyl, or the amide bond, and modifications involving
extensions, deletions or backbone crosslinks. For example, the
backbone can be modified by substitution of a sulfoxide for the
carbonyl, by reversing the peptide bond, or by substituting a
methylene for the carbonyl group. Such modifications can be made by
standard procedures known to those skilled in the art. See, e.g.,
Spatola, A. F., "Peptide Backbone Modifications: A
Structure-Activity Analysis of Peptides Containing Amide Bond
Surrogates, Conformational Constraints, and Related Backbone
Replacements," in Chemistry and Biochemistry of Amino Acids,
Peptides and Proteins, Vol. 7, pp. 267-357, B. Weinstein (ed.),
Marcel Dekker, Inc., New York (1983).
[0090] An analog is also meant to include polypeptides in which one
or more of the amino acid residues include a substituent group, or
polypeptides which are fused with another compound, e.g., a
compound to increase the half-life of the polypeptide, e.g.,
polyethylene glycol.
[0091] By fragment is meant some portion of the naturally occurring
LBP polypeptide. Preferably, the fragment is at least about 100
amino acid residues, more preferably at least about 50 amino acid
residues, more preferably yet at least about 30 amino acid
residues, more preferably yet at least about 20 amino acid
residues, more preferably yet at least about 5 amino acid residues,
more preferably yet at least about 4 amino acid residues, more
preferably yet at least about 3 amino acid residues, and most
preferably at least about 2 amino acid residues in length.
Fragments include, e.g., truncated secreted forms, proteolytic
fragments, splicing fragments, other fragments, and chimeric
constructs between at least a portion of the relevant gene, e.g.,
LBP-1, LBP-2 or LBP-3, and another molecule. Fragments of LBP can
be generated by methods known to those skilled in the art. In
certain embodiments, the fragment is biologically active. The
ability of a candidate fragment to exhibit a biological activity of
LBP can be assessed by methods known to those skilled in the art.
For example, LBP fragments can be tested for their ability to bind
to LDL and/or to an arterial extracellular matrix structural
component, as described herein. Also included are LBP fragments
containing residues that are not required for biological activity
of the fragment or that result from alternative mRNA splicing or
alternative protein processing events.
[0092] Fragments of a protein can be produced by any of a variety
of methods known to those skilled in the art, e.g., recombinantly,
by proteolytic digestion, or by chemical synthesis. Internal or
terminal fragments of a polypeptide can be generated by removing
one or more nucleotides from one end (for a terminal fragment) or
both ends (for an internal fragment) of a nucleic acid which
encodes the polypeptide. Expression of the mutagenized DNA produces
polypeptide fragments. Digestion with "end-nibbling" endonucleases
can thus generate DNAs which encode an array of fragments. DNAs
which encode fragments of a protein can also be generated, e.g., by
random shearing, restriction digestion or a combination of the
above-discussed methods. For example, fragments of LBP can be made
by expressing LBP DNA which has been manipulated in vitro to encode
the desired fragment, e.g., by restriction digestion of any of the
DNA sequences of FIGS. 10-18 (SEQ ID NOS:10-18).
[0093] Fragments can also be chemically synthesized using
techniques known in the art, e.g., conventional Merrifield solid
phase f-Moc or t-Boc chemistry. For example, peptides of the
present invention can be arbitrarily divided into fragments of
desired length with no overlap of the fragments, or divided into
overlapping fragments of a desired length.
[0094] An LBP or a biologically active fragment or analog thereof,
or a binding molecule or a biologically active fragment or analog
thereof, can, e.g., compete with its cognate molecule for the
binding site on the complementary molecule, and thereby reduce or
eliminate binding between LBP and the cellular binding molecule.
LBP or a binding molecule can be obtained, e.g., from purification
or secretion of naturally occurring LBP or binding molecule, from
recombinant LBP or binding molecule, or from synthesized LBP or
binding molecule.
[0095] Therefore, methods for generating analogs and fragments and
testing them for activity are known to those skilled in the
art.
[0096] An agent can also be a nucleic acid used as an antisense
molecule. Antisense therapy is meant to include, e.g.,
administration or in situ generation of oligonucleotides or their
derivatives which specifically hybridize, e.g., bind, under
cellular conditions, with the cellular mRNA and/or genomic DNA
encoding an LBP polypeptide, or mutant thereof, so as to inhibit
expression of the encoded protein, e.g., by inhibiting
transcription and/or translation. The binding may be by
conventional base pair complementarity, or, for example, in the
case of binding to DNA duplexes, through specific interactions in
the major groove of the double helix.
[0097] In certain embodiments, the antisense construct binds to a
naturally-occurring sequence of an LBP gene which, e.g., is
involved in expression of the gene. These sequences include, e.g.,
promoter, start codons, stop codons, and RNA polymerase binding
sites.
[0098] In other embodiments, the antisense construct binds to a
nucleotide sequence which is not present in the wild type gene. For
example, the antisense construct can bind to a region of an LBP
gene which contains an insertion of an exogenous, non-wild type
sequence. Alternatively, the antisense construct can bind to a
region of an LBP gene which has undergone a deletion, thereby
bringing two regions of the gene together which are not normally
positioned together and which, together, create a non-wild type
sequence. When administered in vivo to a subject, antisense
constructs which bind to non-wild type sequences provide the
advantage of inhibiting the expression of a mutant LBP gene,
without inhibiting expression of any wild type LBP gene.
[0099] An antisense construct of the present invention can be
delivered, e.g., as an expression plasmid which, when transcribed
in the cell, produces RNA which is complementary to at least a
unique portion of the cellular mRNA which encodes an LBP
polypeptide. An alternative is that the antisense construct is an
oligonucleotide which is generated ex vivo and which, when
introduced into the cell causes inhibition of expression by
hybridizing with the mRNA (duplexing) and/or genomic sequences
(triplexing) of an LBP gene. Such oligonucleotides are preferably
modified oligonucleotides which are resistant to endogenous
nucleases, e.g. exonucleases and/or endonucleases, and are
therefore stable in vivo. Exemplary nucleic acid molecules for use
as antisense oligonucleotides are phosphoramidate, phosphothioate,
phosphorodithioates and methylphosphonate analogs of DNA and
peptide nucleic acids (PNA). (See also U.S. Pat. Nos. 5,176,996;
5,264,564; and 5,256,775). Additionally, general approaches to
constructing oligomers useful in antisense therapy have been
reviewed. (See, e.g., Van der Krol et al., Biotechniques 6:958-976,
(1988); Stein et al., Cancer Res. 48:2659-2668 (1988)).
[0100] By mimetic is meant a molecule which resembles in shape
and/or charge distribution LBP or a binding molecule. The mimetic
can be a peptide or a non-peptide. Mimetics can act as therapeutic
agents because they can, e.g., competitively inhibit binding of LBP
to a binding molecule. By employing, e.g., scanning mutagenesis,
e.g., alanine scanning mutagenesis, linker scanning mutagenesis or
saturation mutagenesis, to map the amino acid residues of a
particular LBP polypeptide involved in binding a binding molecule,
peptide mimetics, e.g., diazepine or isoquinoline derivatives, can
be generated which mimic those residues in binding to a binding
molecule, and which therefore can inhibit binding of the LBP to a
binding molecule and thereby interfere with the function of LBP.
Non-hydrolyzable peptide analogs of such residues can be generated
using, e.g., benzodiazepine (see, e.g., Freidinger et al., in
Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM
Publisher: Leiden, Netherlands (1988)); azepine (see, e.g., Huffman
et al., in Peptides: Chemistry and Biology, G. R. Marshall ed.,
ESCOM Publisher: Leiden, Netherlands (1988)); substituted gamma
lactam rings (see, e.g., Garvey et al., in Peptides: Chemistry and
Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands
(1988)); keto-methylene pseudopeptides (see, e.g., Ewenson et al.,
J. Med. Chem. 29:295 (1986); Ewenson et al., in Peptides: Structure
and Function (Proceedings of the 9th American Peptide Symposium)
Pierce Chemical Co. Rockland, Ill. (1985)); .beta.-turn dipeptide
cores (see, e.g., Nagai et al., Tetrahedron Lett. 26:647 (1985);
Sato et al., J. Chem. Soc. Perkin Trans. 1:1231 (1986)); or
.beta.-aminoalcohols (see, e.g., Gordon et al., Biochem. Biophys.
Res. Commun. 126:419 (1985); Dann et al., Biochem. Biophys. Res.
Commun. 134:71 (1986)).
[0101] Antibodies are meant to include antibodies against any
moiety that directly or indirectly affects LBP metabolism. The
antibodies can be directed against, e.g., LBP or a binding
molecule, or a subunit or fragment thereof. For example, antibodies
include anti-LBP-1, LBP-2 or LBP-3 antibodies; and anti-binding
molecule antibodies. Antibody fragments are meant to include, e.g.,
Fab fragments, Fab' fragments, F(ab').sub.2 fragments, F(v)
fragments, heavy chain monomers, heavy chain dimers, heavy chain
trimers, light chain monomers, light chain dimers, light chain
trimers, dimers consisting of one heavy and one light chain, and
peptides that mimic the activity of the anti-LBP or anti-binding
molecule antibodies. For example, Fab.sub.2' fragments of the
inhibitory antibody can be generated through, e.g., enzymatic
cleavage. Both polyclonal and monoclonal antibodies can be used in
this invention. Preferably, monoclonal antibodies are used. Natural
antibodies, recombinant antibodies or chimeric-antibodies, e.g.,
humanized antibodies, are included in this invention. Preferably,
humanized antibodies are used when the subject is a human. Most
preferably, the antibodies have a constant region derived from a
human antibody and a variable region derived from an inhibitory
mouse monoclonal antibody. Production of polyclonal antibodies to
LBP is described in Example 6. Monoclonal and humanized antibodies
are generated by standard methods known to those skilled in the
art. Monoclonal antibodies can be produced, e.g., by any technique
which provides antibodies produced by continuous cell lines
cultures. Examples include the hybridoma technique (Kohler and
Milstein, Nature 256:495-497 (1975), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., Immunology Today
4:72 (1983)), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole et al., in Monoclonal Antibodies and
Cancer Therapy, A.R. Liss, Inc., pp. 77-96 (1985)). Preferably,
humanized antibodies are raised through conventional production and
harvesting techniques (Berkower, I., Curr. Opin. Biotechnol.
7:622-628 (1996); Ramharayan and Skaletsky, Am. Biotechnol. Lab
13:26-28 (1995)). In certain preferred embodiments, the antibodies
are raised against the LBP, preferably the LDL-binding site, and
the Fab fragments produced. These antibodies, or fragments derived
therefrom, can be used, e.g., to block the LDL-binding sites on the
LBP molecules.
[0102] Agents also include inhibitors of a molecule that are
required for synthesis, post-translational modification, or
functioning of LBP and/or a binding molecule, or activators of a
molecule that inhibits the synthesis or functioning of LBP and/or
the binding molecule. Agents include, e.g., cytokines, chemokines,
growth factors, hormones, signaling components, kinases,
phosphatases, homeobox proteins, transcription factors, editing
factors, translation factors and post-translation factors or
enzymes. Agents are also meant to include ionizing radiation,
non-ionizing radiation, ultrasound and toxic agents which can,
e.g., at least partially inactivate or destroy LBP and/or the
binding molecule.
[0103] An agent is also meant to include an agent which is not
entirely LBP specific. For example, an agent may alter other genes
or proteins related to arterial plaque formation. Such overlapping
specificity may provide additional therapeutic advantage.
[0104] The invention also includes the agent so identified as being
useful in treating atherosclerosis.
[0105] The invention also includes a method for evaluating an agent
for the ability to alter the binding of LBP polypeptide to a
binding molecule. An agent is provided. An LBP polypeptide is
provided. A binding molecule is provided. The agent, LBP
polypeptide and binding molecule are combined. The formation of a
complex comprising the LBP polypeptide and binding molecule is
detected. An alteration in the formation of the complex in the
presence of the agent as compared to in the absence of the agent is
indicative of the agent altering the binding of the LBP polypeptide
to the binding molecule.
[0106] In preferred embodiments, the LBP polypeptide is LBP-1,
LBP-2 or LBP-3. Examples of a binding molecule include native LDL,
modified LDL, e.g., methylated LDL or oxidized LDL, and arterial
extracellular matrix structural components.
[0107] Altering the binding includes, e.g., inhibiting or promoting
the binding. The efficacy of the agent can be assessed, e.g., by
generating dose response curves from data obtained using various
concentrations of the agent. Methods for determining formation of a
complex are standard and are known to those skilled in the art,
e.g., affinity coelectrophoresis (ACE) assays or ELISA assays as
described herein.
[0108] The invention also includes the agent so identified as being
able to alter the binding of an LBP polypeptide to a binding
molecule.
[0109] The invention also includes a method for evaluating an agent
for the ability to bind to an LBP polypeptide. An agent is
provided. An LBP polypeptide is provided. The agent is contacted
with the LBP polypeptide. The ability of the agent to bind to the
LBP polypeptide is evaluated. Preferably, the LBP polypeptide is
LBP-1, LBP-2 or LBP-3. Binding can be determined, e.g., by
measuring formation of a complex by standard methods known to those
skilled in the art, e.g., affinity coelectrophoresis (ACE) assays
or ELISA assays as described herein.
[0110] The invention also includes the agent so identified as being
able to bind to LBP polypeptide.
[0111] The invention also includes a method for evaluating an agent
for the ability to bind to a nucleic acid encoding an LBP
regulatory sequence. An agent is provided. A nucleic acid encoding
an LBP regulatory sequence is provided. The agent is contacted with
the nucleic acid. The ability of the agent to bind to the nucleic
acid is evaluated. Preferably, the LBP regulatory sequence is an
LBP-1, LBP-2 or LBP-3 regulatory sequence. Binding can be
determined, e.g., by measuring formation of a complex by standard
methods known to those skilled in the art, e.g., DNA mobility shift
assays, DNase I footprint analysis (Ausubel et al., ed., Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
N.Y., (1989)).
[0112] The invention also includes the agent so identified as being
able to bind to a nucleic acid encoding an LBP regulatory
sequence.
[0113] The invention also includes a method for treating
atherosclerosis in an animal. An animal in need of treatment for
atherosclerosis is provided. An agent capable of altering an aspect
of LBP structure or metabolism is provided. The agent is
administered to the animal in a therapeutically effective amount
such that treatment of the atherosclerosis occurs.
[0114] In certain preferred embodiments, the agent is an LBP
polypeptide, e.g., LBP-1, LBP-2 or LBP-3, or a biologically active
fragment or analog thereof. The agent can be, e.g., the polypeptide
as set forth in SEQ ID NOS:1-9. Preferably, the agent is a
polypeptide of no more than about 100 amino acid residues in
length, more preferably of no more than about 50 amino acid
residues, more preferably yet of no more than about 30 amino acid
residues, more preferably yet of no more than about 20 amino acid
residues, more preferably yet of no more than about 10 amino acid
residues, more preferably yet of no more than about 5 amino acid
residues, more preferably yet of no more than about 4 amino acid
residues, more preferably yet of no more than about 3 amino acid
residues, and most preferably of no more than about 2 amino acid
residues. Preferably, the polypeptide includes at least about 20%
acidic amino acid residues, more preferably yet at least about 40%
acidic amino acid residues, more preferably yet at least about 60%
acidic amino acid residues, more preferably yet at least about 80%
acidic amino acid residues, more preferably yet at least about 90%
acidic amino acid residues, more preferably yet at least about 95%
acidic amino acid residues, and most preferably at least about 98%
acidic amino acid residues. Acidic amino acid residues include
aspartic acid and glutamic acid. An example of such an LBP
polypeptide is BHF-1, which is a 20 amino acid length fragment of
human or rabbit LBP-1 which contains amino acid residues 14 through
33. See FIG. 9 (SEQ ID NO:9). 45% of the amino acid residues of
BHF-1 are acidic. The invention also includes biologically active
fragments and analogs of BHF-1.
[0115] Other preferred acidic regions from the LBPs are amino acid
residues 8 through 22 (SEQ ID NO:19), 8 through 33 (SEQ ID NO:20),
23 through 33 (SEQ ID NO:21), and 208 through 217 (SEQ ID NO:22) of
human LBP-2 as depicted in FIG. 7 (SEQ. ID NO:7); amino acid
residues 14 through 43 (SEQ ID NO:23) and 38 through 43 (SEQ ID
NO:24) of rabbit or human LBP-1 as depicted in FIG. 1 (SEQ ID NO:1)
and FIG. 6 (SEQ ID NO:6); amino acid residues 105 through 120 (SEQ
ID NO:25), 105 through 132 (SEQ ID NO:26), 121 through 132 (SEQ ID
NO:27), and 211 through 220 (SEQ ID NO:28) of rabbit LBP-2 as
depicted in FIG. 2 (SEQ ID NO:2); amino acid residues 96 through
110 (SEQ ID NO:29) of rabbit LBP-3 as depicted in FIG. 5 (SEQ ID
NO:5); and amino acid residues 53-59 (SEQ ID NO:41) of human LBP-3
as depicted in FIG. 8 (SEQ ID NO:8). The invention is also meant to
include biologically active fragments and analogs of any of these
polypeptides.
[0116] Other examples of agents include homopolymers and
heteropolymers of any amino acid or amino acid analog. In certain
preferred embodiments, the agent is a homopolymer of an acidic
amino acid or analog thereof. In certain embodiments, the agent is
a heteropolymer of one or more acidic amino acids and one or more
other amino acids, or analogs thereof. For example, agents include
poly(glu), poly(asp), poly(glu asp), poly(glu N), poly(asp N) and
poly(glu asp N). By N is meant any amino acid, or analog thereof,
other than glu or asp. By poly(glu asp) is meant all permutations
of glu and asp for a given length peptide. A preferred peptide is
poly(glu) of no more than about 10 amino acids in length,
preferably about 7 amino acids in length.
[0117] In certain preferred embodiments, the agent is an LBP
nucleic acid or a biologically active fragment or analog thereof,
e.g., a nucleic acid encoding LBP-1, LBP-2 or LBP-3 polypeptide, or
a biologically active fragment or analog thereof. The agent can be,
e.g., a nucleic acid comprising a nucleotide sequence as set forth
in SEQ ID NOS:10-18. In other embodiments, the agent is an
antisense molecule, e.g., one which can bind to an LBP gene
sequence.
[0118] Treating is meant to include, e.g., preventing, treating,
reducing the symptoms of, or curing the atherosclerosis.
Administration of the agent can be accomplished by any method which
allows the agent to reach the target cells. These methods include,
e.g., injection, deposition, implantation, suppositories, oral
ingestion, inhalation, topical administration, or any other method
of administration where access to the target cells by the agent is
obtained. Injections can be, e.g., intravenous, intradermal,
subcutaneous, intramuscular or intraperitoneal. Implantation
includes inserting implantable drug delivery systems, e.g.,
microspheres, hydrogels, polymeric reservoirs, cholesterol
matrices, polymeric systems, e.g., matrix erosion and/or diffusion
systems and non-polymeric systems, e.g., compressed, fused or
partially fused pellets. Suppositories include glycerin
suppositories. Oral ingestion doses can be enterically coated.
Inhalation includes administering the agent with an aerosol in an
inhalator, either alone or attached to a carrier that can be
absorbed.
[0119] Administration of the agent can be alone or in combination
with other therapeutic agents. In certain embodiments, the agent
can be combined with a suitable carrier, incorporated into a
liposome, or incorporated into a polymer release system.
[0120] In certain embodiments of the invention, the administration
can be designed so as to result in sequential exposures to the
agent over some time period, e.g., hours, days, weeks, months or
years. This can be accomplished by repeated administrations of the
agent by one of the methods described above, or alternatively, by a
controlled release delivery system in which the agent is delivered
to the animal over a prolonged period without repeated
administrations. By a controlled release delivery system is meant
that total release of the agent does not occur immediately upon
administration, but rather is delayed for some time. Release can
occur in bursts or it can occur gradually and continuously.
Administration of such a system can be, e.g., by long acting oral
dosage forms, bolus injections, transdermal patches or subcutaneous
implants.
[0121] Examples of systems in which release occurs in bursts
include, e.g., systems in which the agent is entrapped in liposomes
which are encapsulated in a polymer matrix, the liposomes being
sensitive to a specific stimulus, e.g., temperature, pH, light,
magnetic field, or a degrading enzyme, and systems in which the
agent is encapsulated by an ionically-coated microcapsule with a
microcapsule core-degrading enzyme. Examples of systems in which
release of the agent is gradual and continuous include, e.g.,
erosional systems in which the agent is contained in a form within
a matrix, and diffusional systems in which the agent permeates at a
controlled rate, e.g., through a polymer. Such sustained release
systems can be, e.g., in the form of pellets or capsules.
[0122] The agent can be suspended in a liquid, e.g., in dissolved
form or colloidal form. The liquid can be a solvent, partial
solvent or non-solvent. In many cases water or an organic liquid
can be used.
[0123] The agent can be administered prior to or subsequent to the
appearance of atherosclerosis symptoms. In certain embodiments, the
agent is administered to patients with familial histories of
atherosclerosis, or who have phenotypes that may indicate a
predisposition to atherosclerosis, or who have been diagnosed as
having a genotype which predisposes the patient to atherosclerosis,
or who have other risk factors, e.g., hypercholesterolemia,
hypertension or smoking.
[0124] The agent is administered to the animal in a therapeutically
effective amount. By therapeutically effective amount is meant that
amount which is capable of at least partially preventing or
reversing atherosclerosis. A therapeutically effective amount can
be determined on an individual basis and will be based, at least in
part, on consideration of the species of animal, the animal's size,
the animal's age, the agent used, the type of delivery system used,
the time of administration relative to the onset of atherosclerosis
symptoms, and whether a single, multiple, or controlled release
dose regimen is employed. A therapeutically effective amount can be
determined by one of ordinary skill in the art employing such
factors and using no more than routine experimentation.
[0125] Preferably, the concentration of the agent is at a dose of
about 0.1 to about 1000 mg/kg body weight/day, more preferably at
about 0.1 to about 500 mg/kg/day, more preferably yet at about 0.1
to about 100 mg/kg/day, and most preferably at about 0.1 to about 5
mg/kg/day. The specific concentration partially depends upon the
particular agent used, as some are more effective than others. The
dosage concentration of the agent that is actually administered is
dependent at least in part upon the final concentration that is
desired at the site of action, the method of administration, the
efficacy of the particular agent, the longevity of the particular
agent, and the timing of administration relative to the onset of
the atherosclerosis symptoms. Preferably, the dosage form is such
that it does not substantially deleteriously affect the animal. The
dosage can be determined by one of ordinary skill in the art
employing such factors and using no more than routine
experimentation.
[0126] In certain embodiments, various gene constructs can be used
as part of a gene therapy protocol to deliver nucleic acids
encoding an agent, e.g., either an agonistic or antagonistic form
of an LBP polypeptide. For example, expression vectors can be used
for in vivo transfection and expression of an LBP polypeptide in
particular cell types so as to reconstitute the function of, or
alternatively, abrogate the function of, LBP polypeptide in a cell
in which non-wild type LBP is expressed. Expression constructs of
the LBP polypeptide, and mutants thereof, may be administered in
any biologically effective carrier, e.g. any formulation or
composition capable of effectively delivering the LBP gene to cells
in vivo. Approaches include, e.g., insertion of the subject gene in
viral vectors including, e.g., recombinant retroviruses,
adenovirus, adeno-associated virus, and herpes simplex virus-1, or
recombinant bacterial or eukaryotic plasmids. Viral vectors infect
or transduce cells directly; plasmid DNA can be delivered with the
help of, for example, cationic liposomes (lipofectin.TM. (Life
Technologies, Inc., Gaithersburg, Md.) or derivatized (e.g.
antibody conjugated), polylysine conjugates, gramacidin S,
artificial viral envelopes or other such intracellular carriers, as
well as direct injection of the gene construct or
Ca.sub.3(PO.sub.4).sub.2 precipitation carried out in vivo. The
above-described methods are known to those skilled in the art and
can be performed without undue experimentation. Since transduction
of appropriate target cells represents the critical first step in
gene therapy, choice of the particular gene delivery system will
depend on such factors as the phenotype of the intended target and
the route of administration, e.g., locally or systemically.
Administration can be directed to one or more cell types, and to
one or more cells within a cell type, so as to be therapeutically
effective, by methods that are known to those skilled in the art.
In a preferred embodiment, the agent is administered to arterial
wall cells of the animal. For example, a genetically engineered LBP
gene is administered to arterial wall cells. In certain
embodiments, administration is done in a prenatal animal or
embryonic cell. It will be recognized that the particular gene
construct provided for in in vivo transduction of LBP expression is
also useful for in vitro transduction of cells, such as for use in
the diagnostic assays described herein.
[0127] In certain embodiments, therapy of atherosclerosis is
performed with antisense nucleotide analogs of the genes which code
for the LBPs. Preferably, the antisense nucleotides have
non-hydrolyzable "backbones," e.g., phosphorothioates,
phosphorodithioates or methylphosphonates. The nucleoside base
sequence is complementary to the sequence of a portion of the gene
coding for, e.g., LBP-1, 2 or 3. Such a sequence might be, e.g.,
ATTGGC if the gene sequence for the LBP is TAACCG. One embodiment
of such therapy would be incorporation of an antisense analog of a
portion of one of the LBP genes in a slow-release medium, e.g.,
polyvinyl alcohol, which is administered, e.g., by subcutaneous
injection, so as to release the antisense nucleotide analog over a
period of weeks or months. In another embodiment, the antisense
analog is incorporated into a polymeric matrix, e.g., polyvinyl
alcohol, such that the gel can be applied locally to an injured
arterial wall to inhibit LBP synthesis and prevent LDL
accumulation, e.g., after angioplasty or atherectomy.
[0128] The invention also includes a method for treating an animal
at risk for atherosclerosis. An animal at risk for atherosclerosis
is provided. An agent capable of altering an aspect of LBP
structure or metabolism is provided. The agent is administered to
the animal in a therapeutically effective amount such that
treatment of the animal occurs. Being at risk for atherosclerosis
can result from, e.g., a family history of atherosclerosis, a
genotype which predisposes to atherosclerosis, or phenotypic
symptoms which predispose to atherosclerosis, e.g., having
hypercholesterolemia, hypertension or smoking.
[0129] The invention also includes a method for treating a cell
having an abnormality in structure or metabolism of LBP. A cell
having an abnormality in structure or metabolism of LBP is
provided. An agent capable of altering an aspect of LBP structure
or metabolism is provided. The agent is administered to the cell in
a therapeutically effective amount such that treatment of the cell
occurs.
[0130] In certain embodiments, the cell is obtained from a cell
culture or tissue culture or an embryo fibroblast. The cell can be,
e.g., part of an animal, e.g., a natural animal or a non-human
transgenic animal. Preferably, the LBP is LBP-1, LBP-2 or
LBP-3.
[0131] The invention also includes a pharmaceutical composition for
treating atherosclerosis in an animal comprising a therapeutically
effective amount of an agent, the agent being capable of altering
an aspect of LBP metabolism or structure in the animal so as to
result in treatment of the atherosclerosis, and a pharmaceutically
acceptable carrier. Pharmaceutically acceptable carriers include,
e.g., saline, liposomes and lipid emulsions.
[0132] In certain preferred embodiments, the agent of the
pharmaceutical composition is an LBP polypeptide, e.g., LBP-1,
LBP-2 or LBP-3, or a biologically active fragment or analog
thereof. The agent can be, e.g., the polypeptide as set forth in
SEQ ID NOS:1-9. Preferably, the agent is a polypeptide of no more
than about 100 amino acid residues in length, more preferably of no
more than about 50 amino acid residues, more preferably yet of no
more than about 30 amino acid residues, more preferably yet of no
more than about 20 amino acid residues, more preferably yet of no
more than about 10 amino acid residues, more preferably yet of no
more than about 5 amino acid residues, more preferably yet of no
more than about 4 amino acid residues, more preferably yet of no
more than about 3 amino acid residues, and most preferably of no
more than about 2 amino acid residues. Preferably, the polypeptide
includes at least about 20% acidic amino acid residues, more
preferably yet at least about 40% acidic amino acid residues, more
preferably yet at least about 60% acidic amino acid residues, more
preferably yet at least about 80% acidic amino acid residues, more
preferably yet at least about 90% acidic amino acid residues, more
preferably yet at least about 95% acidic amino acid residues, and
most preferably at least about 98% acidic amino acid residues.
[0133] In certain preferred embodiments, the agent is an LBP
nucleic acid, e.g., a nucleic acid encoding LBP-1, LBP-2 or LBP-3
polypeptide, or a biologically active fragment or analog thereof.
The agent can be, e.g., a nucleic acid comprising a nucleotide
sequence as set forth in SEQ ID NOS:10-18.
[0134] The invention also includes a vaccine composition for
treating atherosclerosis in an animal comprising a therapeutically
effective amount of an agent, the agent being capable of altering
an aspect of LBP metabolism or structure in the animal so as to
result in treatment of the atherosclerosis, and a pharmaceutically
acceptable carrier.
[0135] The invention also includes a method for diagnosing
atherosclerotic lesions in an animal. An animal is provided. A
labeled agent capable of binding to LBP present in atherosclerotic
lesions is provided. The labeled agent is administered to the
animal under conditions which allow the labeled agent to interact
with the LBP so as to result in labeled LBP. The localization or
quantification of the labeled LBP is determined by imaging so as to
diagnose the presence of atherosclerotic lesions in the animal.
[0136] Preferably, the LBP is LBP-1, LBP-2 or LBP-3. The imaging
can be performed by standard methods known to those skilled in the
art, including, e.g., magnetic resonance imaging, gamma camera
imaging, single photon emission computed tomographic (SPECT)
imaging, or positron emission tomography (PET).
[0137] Preferably, agents that bind tightly to LBPs in
atherosclerotic lesions are used for atherosclerotic imaging and
diagnosis. The agent is radiolabeled with, e.g., .sup.99mTc or
another isotope suitable for clinical imaging by gamma camera,
SPECT, PET scanning or other similar technology. Since LBPs occur
in very early lesions, such imaging is more sensitive than
angiography or ultrasound for locating very early lesions which do
not yet impinge on the arterial lumen to cause a visible bulge or
disturbed flow. In addition to locating both early and more
developed lesions, the imaging agents which bind to LBPs can also
be used to follow the progress of atherosclerosis, as a means of
evaluating the effectiveness of both dietary and pharmacological
treatments.
[0138] Thus, a diagnostic embodiment of the invention is the
adaptation of, e.g., a peptide complementary to one of the LBPs, by
radiolabeling it and using it as an injectable imaging agent for
detection of occult atherosclerosis. The peptide is selected from
those known to bind to LBPs, e.g., RRRRRRR or KKLKLXX, or any other
polycationic peptide which binds to the highly electronegative
domains of the LBPs. For extracorporeal detection with a gamma
scintillation (Anger) camera, technetium-binding ligands, e.g.,
CGC, GGCGC, or GGCGCF, can be incorporated into the peptides at the
N-terminus or C-terminus for .sup.99mTc labeling. For external
imaging by magnetic resonance imaging (MRI), e.g., the
gadolinium-binding chelator, diethylene triamine penta-acetic acid
(DTPA), is covalently bound to the N- or C-terminus of the
peptides. In yet other embodiments, the LBP-binding peptides are
covalently bound, e.g., to magnetic ion oxide particles by standard
methods known to those skilled in the art, e.g., conjugating the
peptides with activated polystyrene resin beads containing magnetic
ion oxide.
[0139] The invention also includes a method for immunizing an
animal against an LBP, e.g., LBP-1, LBP-2 or LBP-3, or fragment or
analog thereof. An animal having LDL is provided. An LBP or
fragment or analog thereof is provided. The LBP or fragment or
analog thereof is administered to the animal so as to stimiulate
antibody production by the animal to the LBP or fragment or analog
thereof such that binding of the LBP to the LDL is altered, e.g.,
decreased or increased.
[0140] The invention also includes a method of making a fragment or
analog of LBP polypeptide, the fragment or analog having the
ability to bind to modified LDL and native LDL. An LBP polypeptide
is provided. The sequence of the LBP polypeptide is altered. The
altered LBP polypeptide is tested for the ability to bind to
modified LDL, e.g., methylated LDL, oxidized LDL, acetylated LDL,
cyclohexanedione-treated LDL (CHD-LDL), and to native LDL.
[0141] The fragments or analogs can be generated and tested for
their ability to bind to these modified LDLs and to native LDL, by
methods known to those skilled in the art, e.g., as described
herein. Preferably, they are tested for their ability to bind to
methylated LDL and native LDL. The binding activity of the fragment
or analog can be greater or less than the binding activity of the
native LBP. Preferably, it is greater. In preferred embodiments,
the LBP is LBP-1, LBP-2 or LBP-3.
[0142] The invention also includes a method for isolating a cDNA
encoding an LBP. A cDNA library is provided. The cDNA library is
screened for a cDNA encoding a polypeptide which binds to native
LDL and modified LDL, e.g., methylated LDL or oxidized LDL. The
cDNA which encodes this polypeptide is isolated, the cDNA encoding
an LBP.
[0143] The following non-limiting examples further illustrate the
present invention.
EXAMPLES
Example 1
Construction of a Rabbit cDNA Library
[0144] This example illustrates the construction of a rabbit cDNA
library using mRNA from balloon-deendothelialized healing rabbit
abdominal aorta. Balloon-catheter deendothelialized rabbit aorta
has been shown to be a valid model for atherosclerosis (Minick et
al., Am. J. Pathol. 95:131-158 (1979).
[0145] The mRNA was obtained four weeks after ballooning to
maximize focal LDL binding in the ballooned rabbit aorta. First
strand cDNA synthesis was carried out in a 50 .mu.l reaction
mixture containing 4 .mu.g mRNA; 2 .mu.g oligo d(T) primer;
methylation dNTP mix (10 mM each); 10 mM DTT; 800 units superscript
II RT (Life Technologies, Gaithersburg, MD); 1.times. first strand
cDNA synthesis buffer (50 mM Tris-HCl, pH 8.3; 75 mM KCl; 5 mM
MgCl.sub.2), which was incubated for 1 hr at 37.degree. C. The
reaction mixture was then adjusted to 250 .mu.l through the
addition of 1.times. second strand buffer (30 mM Tris-HCl, pH 7.5;
105 mM KCl; 5.2 mM MgCl.sub.2); 0.1 mM DTT; methylation dNTP mix
(10 mM each); 50 units E. coli DNA polymerase I, 3 units RNase H;
15 units E. coli DNA ligase (all enzymes from Life Technologies),
which was incubated for an additional 2.5 hr at 15.degree. C. The
resulting double-stranded cDNAs (dscDNA) were then treated with 1.5
units T4 DNA polymerase (Novagen Inc., Madison, Wis.) for 20 min at
11.degree. C. to make blunt-ended dscDNA. These were then
concentrated by ethanol precipitation and EcoR1/Hind III linkers
were attached to the ends by T4 DNA ligase (Novagen Inc.). The
linker-ligated cDNAs were treated with EcoR1 and HindIII
restriction enzymes to produce EcoR1 and Hind III recognition
sequences at their 5' and 3' ends, respectively. After the removal
of linker DNA by gel exclusion chromatography, the dscDNAs were
inserted into .lambda.EXlox phage arms (Novagen Inc.) in a
unidirectional manner by T4 DNA ligase and packaged into phage
particles according to the manufacturer's protocol (Novagen Inc.).
A phage library of cDNAs containing 2.times.10.sup.6 independent
clones was established from 4 .mu.g of mRNA.
Example 2
Identification of Rabbit cDNAs Encoding LDL Binding Proteins
(LBPs)
[0146] This example illustrates a method of functionally screening
a rabbit cDNA library so as to identify cDNAs encoding LBPs which
bind to both native LDL and methyl LDL. Methyl LDL is not
recognized by previously reported cell surface receptors. See,
e.g., Weisgraber et al., J. Biol. Chem. 253:9053-9062 (1978).
[0147] A fresh overnight culture of E. coli ER1647 cells (Novagen
Inc.) was infected with the cDNA phage obtained from Example 1, and
plated at a density of 2.times.10.sup.4 plaque-forming units (pfu)
in 150 mm diameter plates containing 2.times. YT agar. A total of
50 plates, equivalent to 1.times.10.sup.6 phage, were plated and
incubated at 37.degree. C. until the plaques reached 1 mm in
diameter (5-6 hr). A dry nitrocellulose membrane, which had
previously been saturated with 10 mM IPTG solution, was layered on
top of each plate to induce the production of recombinant protein,
as well as to immobilize the proteins on the membranes. The plates
were incubated at 37.degree. C. for an additional 3-4 hr, and then
overnight at 4.degree. C.
[0148] The next day, the membranes were lifted from each plate and
processed as follows. Several brief rinses in TBST solution (10 mM
Tris-HCl, pH 8.0; 150 mM NaCl, 0.05% Tween 20); two 10-min rinses
with 6M guanidine-HCl in HBB (20 mM HEPES, pH 7.5; 5 mM MgCl.sub.2,
1 mM DTT, and 5 mM KCl); two 5-min rinses in 3M guanidine-HCl in
HBB; a final brief rinse in TBSEN (TBS, 1 mM EDTA, 0.02%
NaN.sub.3).
[0149] The membranes were then blocked for 30 min at room
temperature in a solution of TBSEN with 5% non-fat dry milk,
followed by 10 min in TBSEN with 1% non-fat dry milk. Following
blocking, the membranes were incubated with native human LDL
(obtained as described in Example 11 or methylated human LDL
(meLDL) (see Weisgraber et al., J. Biol. Chem. 253:9053-9062
(1978)), at a concentration of 4 .mu.g/ml, in a solution containing
1.times. TBSEN, 1% non-fat dry milk, 1 mM PMSF, 0.5.times. protease
inhibitor solution (1 mM .epsilon.-amino caproic acid/1 mM
benzamidine). Incubation was for 4 hr at room temperature in a
glass Petri dish with gentle stirring on a stirring table, followed
by overnight at 4.degree. C. with no stirring.
[0150] Specifically bound meLDL and native LDL were detected on the
nitrocellulose membranes by antibodies against human LDL. Sheep
anti-human LDL polyclonal antibodies (Boehringer Mannheim,
Indianapolis, Ind.) were adsorbed with E. coli plys E cell extracts
to abolish background. For adsorption, E. coli plys E cells were
grown to log phase, spun down and resuspended in PBS containing 1
mM PMSF, 2 mM e-amino caproic acid, and 1 mM benzamidine. The cell
suspension then underwent 8 freeze-thaw cycles via immersion in
liquid nitrogen and cold running tap water, respectively. The anti
LDL antibodies/cell extract solution were incubated with gentle
stirring for 1 hr at 4.degree. C. (1 ml of antibody solution/3 mg
crude cell extract). Following incubation, the mixture was
centrifuged (10,000.times. g; 10 min; 4.degree. C.) and the
supernatant was stored at 4.degree. C. in the presence of 0.02%
NaN.sub.3 until use. The membranes were processed for
immunoscreening as follows: (i) three 5-min washes at room
temperature in TBSEN containing 1% gelatin; (ii) 30 min incubation
in PBS, pH 7.4 with 1% gelatin; (iii) two-hr room temperature
incubation with gentle stirring in fresh PBS/gelatin solution
containing adsorbed sheep anti-human LDL antibodies (Boehringer
Manheim, Indianapolis, Ind.) (1:1000 dilution); (iv) three brief
washes in TBS, pH 7.4; (v) one-hr room temperature incubation with
gentle stirring in PBS/gelatin solution containing donkey antisheep
alkaline phosphatase-conjugated antibodies (Sigma, St. Louis, Mo.)
(1:10,000 dilution); (vi) three brief washes with TBS, pH 7.4.; and
(vii) development according to the manufacturer's instructions,
using an alkaline phosphatase substrate development kit (Novagen
Inc.). Phage plaques which produced LBPs appeared as blue-colored
"donuts" on the membranes.
[0151] The phage from Example 1 containing the LBP cDNAs were
plaque-purified and converted into plasmid subclones by following a
protocol called "Autosubcloning by Cre-mediated Plasmid Excision"
provided by Novagen Inc. DNA sequences were obtained by the
dideoxynucleotide chain-termination method (Sanger et al., Proc.
Natl. Acad. Sci., USA 74:5463-5467 (1977), and analyzed by an
Applied Biosystems automated sequencer. The open reading frame
(ORF) of each cDNA was determined from consensus sequences obtained
from both the sense and antisense strands of the cDNAs. Sequencing
confirmed that three previously unknown genes had been isolated.
Since the genes were selected by functional screening for LDL
binding, the proteins coded by these genes were termed LDL binding
proteins (LBPs), specifically, LBP-1, LBP-2 and LBP-3. The cDNA
sequences for rabbit LBP-1, LBP-2 and LBP-3 and the corresponding
proteins are set forth in SEQ ID NOS:10-14.
[0152] Based on their respective cDNA coding sequences, the sizes
of the recombinant proteins were determined to be 16.2 kDa for
LBP-1, 40 kDa for LBP-2, and 62.7 kDa for LBP-3.
Example 3
Northern Blot Analysis of Rabbit RNA Using LBP cDNA or cRNA
[0153] This example illustrates the size and tissue distribution of
LBP mRNAs. Total RNA was isolated from different rabbit tissues:
adrenals, thoracic aorta, abdominal aorta, ballooned and
reendothelialized abdominal aorta, heart, kidney, smooth muscle
cells, lung and liver, by Trizol reagent (Life Technologies) and
concentrated by ethanol precipitation. Gel electrophoresis of RNA
was carried out in 1.2% agarose gel containing 1.times. MOPS buffer
(0.2M MOPS, pH 7.0; 50 mM sodium acetate; 5 mM EDTA, pH 8.0) and
0.37M formaldehyde. Gels were loaded with 20 .mu.g total RNA from
each tissue examined and electrophoresed at 100 volts for 2 hr in
1.times. MOPS buffer. RNAs were blotted onto supported
nitrocellulose membranes (Schleicher & Schuell, Keene, N.H.)
and immobilized by baking at 80.degree. C. for 2 hr. Hybridization
to radiolabeled LBP-1, LBP-2 and LBP-3 cDNA or cRNA probes was
carried out by standard procedures known to those skilled in the
art (see, e.g., Ausubel et al., Current Protocols in Molecular
Biology; John Wiley & Sons (1989)); signals were detected by
autoradiography.
[0154] The results were as follows: the sizes of the mRNAs were
about 1.3 kb for LBP-1, about 2.3-2.5 kb for LBP-2, and about 4.7
kb for LBP-3. LBP-1, LBP-2 and LBP-3 mRNA were found in all tissues
tested, but the highest amount was in ballooned abdominal
aorta.
Example 4
Isolation of Human LBP cDNAs
[0155] This example illustrates isolation of human LBP cDNAs. Human
LBP cDNA clones were isolated from three cDNA libraries. A human
fetal brain cDNA library was obtained from Stratagene, LaJolla,
Calif., a human liver and a human aorta cDNA library were obtained
from Clontech, Palo Alto, Calif., and screened with a radiolabeled
cDNA probe derived from rabbit LBP-1, LBP-2 or LBP-3, according to
the method described in Law et al., Gene Expression 4:77-84 (1994).
Several strongly hybridizing clones were identified and
plaque-purified. Clones were confirmed to be human LBP-1, LBP-2 and
LBP-3, by DNA sequencing using the dideoxynucleotide
chain-termination method and analysis by an Applied Biosystems
automated sequencer. The cDNA sequences and the corresponding
proteins for human LBP-1, LBP-2 and LBP-3 are set forth in SEQ ID
NOS:15, 16 and 17, respectively. A comparison between the
corresponding LBP-1, LBP-2 and LBP-3 protein sequences for rabbit
and human are shown in FIGS. 19, 20 and 21.
Example 5
Isolation of Recombinant LBP-1, LBP-2 and LBP-3 Rabbit Proteins
from E. coli
[0156] LBP cDNA was isolated from the original pEXlox plasmids
obtained as described in Examples 1 and 2, and subcloned into the
PPROEX-HT vector (Life Technologies) for recombinant protein
expression. Induction of the recombinant protein by IPTG addition
to transformed E. coli DH10B cultures resulted in the expression of
recombinant protein containing a 6-histidine tag (N-terminal). This
tagged protein was then purified from whole cell proteins by
binding to Ni-NTA (nickel nitrilo-triacetic acid) as described in
the protocol provided by the manufacturer (Qiagen, Inc., Santa
Clara, Calif.). The preparation obtained after the chromatography
step was approximately 90% pure; preparative SDS-PAGE was performed
as the final purification step.
[0157] When required by the characterization procedure, iodination
of LBPs was carried out using Iodobeads (Pierce, Rockford, Ill.).
The Iodobeads were incubated with 500 .mu.Ci of Na.sup.125I
solution (17 Ci/mg) (New England Nuclear, Boston, Mass.) in a
capped microfuge tube for 5 min at room temperature. The protein
solution was added to the Iodobeads-Na.sup.125I microfuge tube and
incubated for 15 min at room temperature. At the end of this
incubation, aliquots were removed for the determination of total
soluble and TCA precipitable counts. The radiolabeled protein was
then precipitated with cold acetone (2.5 vol; -20.degree. C.; 2.5
hr). Following this incubation, precipitated protein was collected
by centrifugation (14,000 g; 1 hr; room temperature) and
resuspended in sample buffer (6 M urea/50 mM Tris, pH 8.0/2 mM
EDTA). Integrity of the protein preparation was assessed by
SDS-PAGE.
[0158] The identities of the recombinant LBPs were confirmed using
standard protein sequencing protocols known to those skilled in the
art. (A Practical Guide for Protein and Peptide Purification for
Microsequencing, Matsudaira, ed., Academic Press, Inc., 2d edition
(1993)). Analysis was performed using an Applied Biosystems Model
477A Protein Sequencer with on-line Model 120 PTH amino acid
analyzer.
Example 6
Production of Antibodies to LBP-1, LBP-2 and LBP-3
[0159] This example illustrates the production of polyclonal
antibodies to LBP-1, LBP-2 and LBP-3. A mixture of purified
recombinant LBP protein (0.5 ml; 200 .mu.g) and RIBI adjuvant (RIBI
ImmunoChem Research, Inc., Hamilton, Mont.) was injected
subcutaneously into male guinea pigs (Dunkin Hartley; Hazelton
Research Products, Inc., Denver, Pa.) at 3-5 sites along the dorsal
thoracic and abdominal regions of the guinea pig. Blood was
collected by venipuncture on days 1 (pre-immune bleeding), 28, 49
and 70. Booster injections were administered on days 21 (100 .mu.g;
SC), 42 (50 .mu.g; SC), and 63 (25 .mu.g; SC). The titer of the
guinea pig antiserum was evaluated by serial dilution "dot
blotting." Preimmune antiserum was evaluated at the same time.
After the third booster of LBP protein, the titer against the
recombinant protein reached a maximal level with a detectable
calorimetric response on a dot blot assay of 156 pg.
[0160] Specificity of the polyclonal antibody for recombinant
LBP-1, LBP-2 or LBP-3 was demonstrated using Western blot analysis.
(Towbin et al., Proc. Natl. Acad. Sci. USA 76:4350 (1979)). The
protein-antibody complex was visualized immunochemically with
alkaline phosphatase-conjugated goat anti-guinea pig IgG, followed
by staining with nitro blue tetrazolium (BioRad Laboratories,
Hercules, Calif.). Non-specific binding was blocked using 3%
non-fat dry milk in Tris buffered saline (100 mM Tris; 0.9% NaCl,
pH 7.4).
Example 7
Immunohistochemical Characterization
[0161] This example illustrates the presence of LBPs in or on
endothelial cells covering plaques, in or on adjacent smooth muscle
cells, and in the extracellular matrix. In addition,
co-localization of LDL and LBPs was demonostrated. These results
were obtained by examining ballooned rabbit arterial lesions and
human atherosclerotic plaques by immunohistochemical methods.
[0162] Ballooned deendothelialized aorta was obtained from rabbits
which had received a bolus injection of human LDL (3 mg; i.v.) 24
hr prior to tissue collection. Human aortas containing
atherosclerotic plaques were obtained from routine autopsy
specimens. Tissues were fixed in 10% buffered formalin (.ltoreq.24
hr) and imbedded in paraffin using an automated tissue-imbedding
machine. Tissue sections were cut (5-7.mu.) and mounted onto glass
slides by incubating for 1 hr at 60.degree. C. Sections were
deparaffinized. After a final wash with deionized H.sub.2O,
endogenous peroxidase activity was eliminated by incubating the
sections with 1% H.sub.2O.sub.2/H.sub.2O buffer for 5 min at room
temperature. Sections were rinsed with phosphate buffered saline
(PBS) for 5 min at room temperature and nonspecific binding was
blocked with 5% normal goat serum or 5% normal rabbit serum
depending on the source of the secondary antibody (Sigma, St.
Louis, Mo.) (1 hr; room temperature). Sections were then incubated
with a 1:50 dilution (in 5% normal goat serum/PBS) of a guinea pig
polyclonal antibody against the rabbit form of recombinant LBP-1,
LBP-2 or LBP-3. Controls included preimmune serum as well as
specific antisera to LBP-1, LBP-2, or LBP-3 in which the primary
antibody was completely adsorbed and removed by incubation with
recombinant LBP-1, LBP-2 or LBP-3 followed by centrifugation prior
to incubation with the tissue sections. An affinity purified rabbit
polyclonal antibody against human apolipoprotein B (Polysciences
Inc.; Warrington, Pa.) was used at a dilution of 1:100 (in 5%
normal rabbit serum/PBS). Sections were incubated for 2 hr at room
temperature in a humidified chamber. At the end of incubation,
sections were rinsed with PBS and incubated with a 1:200 dilution
(in 5% normal goat serum/PBS) of goat anti-guinea pig biotinylated
IgG conjugate (Vector Laboratories, Burlingame, Calif.) or a 1:250
dilution (in 5% normal rabbit serum/PBS) of rabbit anti-goat
biotinylated IgG conjugate (Vector Laboratories, Burlingame,
Calif.) for 1 hr at room temperature in a humidified chamber.
Sections were then rinsed with PBS and antigen-antibody signal
amplified using avidin/biotin HRP conjugate (Vectastain ABC kit;
Vector Laboratories, Burlingame, Calif.). Sections were developed
using DAB substrate (4-6 min; room temperature) and counterstained
with hematoxylin.
[0163] In the ballooned rabbit artery, immunohistochemistry with
the anti-LBP-1, LBP-2 and LBP-3 antibodies showed that LBP-1, LBP-2
and LBP-3 were located in or on functionally modified endothelial
cells at the edges of regenerating endothelial islands, the same
location in which irreversible LDL binding has been demonstrated
(Chang et al., Arteriosclerosis and Thrombosis 12:1088-1098
(1992)). LBP-1, LBP-2 and LBP-3 were also found in or on intimal
smooth muscle cells underneath the functionally modified
endothelial cells, and to a lesser extent, in extracellular matrix.
No LBP-1, LBP-2 or LBP-3 was detected in still deendothelialized
areas, where LDL binding had been shown to be reversible (Chang et
al., Arteriosclerosis and Thrombosis 12:1088-1098 (1992)).
Immunohistochemistry of ballooned rabbit aorta with anti-human
apolipoprotein B antibodies showed the presence of LDL at the same
locations as that found for LBP-1, LBP-2 and LBP-3.
[0164] In the human atherosclerotic plaques taken at routine
autopsies, immunohistochemistry with the anti-LBP-1, anti-LBP-2 and
anti-LBP-3 antibodies showed that LBP-1, LBP-2, and LBP-3 were also
found in or on endothelial cells covering plaques and in or on
adjacent smooth muscle cells. In the human tissue, there was
greater evidence of LBP-1, LBP-2 and LBP-3 in extracellular
matrix.
[0165] The results obtained with paraffin sections were identical
to those of frozen sections.
Example 8
Affinity Coelectrophoresis (ACE) Assays of LBPs and LDL or HDL
[0166] This example illustrates that binding occurs between LBP-1,
LBP-2 or LBP-3 and LDL, and that this binding is specific, as
illustrated by the fact that binding does not occur between LBP-1,
LBP-2 or LBP-3 and HDL (high density lipoprotein).
[0167] Analysis of the affinity and specificity of recombinant
rabbit LBP-1, LBP-2 or LBP-3 binding to LDL was carried out using
the principle of affinity electrophoresis (Lee and Lander, Proc.
Natl. Acad. Sci. USA 88:2768-2772 (1991)). Melted agarose (1%;
65.degree. C.) was prepared in 50 mM sodium MOPS, pH 7.0; 125 mM
sodium acetate, 0.5% CHAPS. A teflon comb consisting of nine
parallel bars (45.times.4.times.4 mm/3 mm spacing between bars) was
placed onto GelBond film (FMC Bioproducts, Rockland, Me.) fitted to
a plexiglass casting tray with the long axis of the bars parallel
to the long axis of the casting tray. A teflon strip
(66.times.1.times.1 mm) was placed on edge with the long axis
parallel to the short axis of the casting tray, at a distance of 4
mm from the edge of the teflon comb. Melted agarose (>65.degree.
C.) was then poured to achieve a height of approximately 4 mm.
Removal of the comb and strip resulted in a gel containing nine
45.times.4.times.4 mm rectangular wells adjacent to a 66.times.1 mm
slot. LDL or HDL samples were prepared in gel buffer (50 mM sodium
MOPS, pH 7.0, 125 mM sodium acetate) at twice the desired
concentration. Samples were then mixed with an equal volume of
melted agarose (in 50 mM MOPS, pH 7.0; 125 mM sodium acetate;
50.degree. C.), pipetted into the appropriate rectangular wells and
allowed to gel. The binding affinity and specificity of LBP-1 and
LBP-3 was tested using several concentrations of LDL (540 to 14 nM)
and HDL (2840-177 nM). A constant amount (0.003 nM-0.016 nM) of
.sup.125I-labeled LBP-1, LBP-2 or LBP-3 (suspended in 50 mM sodium
MOPS, pH 7.0; 125 mM sodium acetate; 0.5% bromphenol blue; 6%
(wt/vol) sucrose) was loaded into the slot. Gels were
electrophoresed at 70 v/2 hr/20.degree. C. At the end of the run,
the gels were air dried and retardation profiles were visualized by
exposure of X-ray films to the gels overnight at -70.degree. C.,
with intensifying screens).
[0168] LDL retarded LBP-1, LBP-2 and LBP-3 migration through the
gel in a concentration-dependent, saturable manner, indicating that
LBP-1, LBP-2 and LBP-3 binding to LDL was highly specific. This
conclusion is supported by the fact that HDL did not retard LBP-1,
LBP-2 or LBP-3. A binding curve generated from the affinity
coelectrophoresis assay indicated that LBP-1 binds to LDL with a
K.sub.d of 25.6 nM, that LBP-2 (rabbit clone 26) binds to LDL with
a K.sub.d of 100 nM, and that LBP-3 (80 kDa fragment) binds to LDL
with a K.sub.d of 333 nM.
[0169] In addition to testing affinity and specificity of LBP-1,
LBP-2 and LBP-3 binding to LDL, the ability of "cold" (i.e.,
non-radiolabeled) LBP-1, LBP-2 or LBP-3 to competitively inhibit
radiolabeled LBP-1, LBP-2 or LBP-3 binding to LDL, respectively,
was tested. Competition studies were carried out using fixed
concentrations of cold LDL and radiolabeled LBP-1 and increasing
amounts of cold recombinant LBP-1 (6-31 .mu.M). The ACE assay
samples and gel were prepared as described herein. Cold LBP-1
inhibited binding of radiolabeled LBP-1 to LDL in a
concentration-dependent manner, cold LBP-2 inhibited binding of
radiolabeled LBP-2 to LDL in a concentration-dependent manner, and
cold LBP-3 inhibited binding of radiolabeled LBP-3 to LDL in a
concentration-dependent manner.
[0170] Rabbit and human LBP-2 contain a long stretch of acidic
amino acids at the amino terminal (rabbit LBP-2 amino acid residues
105 through 132 and human LBP-2 amino acid residues 8 through 33).
The possibility that this segment of LBP-2 was the LDL binding
domain was tested by subcloning two rabbit LBP-2 clones which
differ from each other by the presence or absence of this acidic
region (clone 26 and clone 45, respectively) into expression
vectors, by standard methods known to those skilled in the art. ACE
assays were then conducted in order to assess the affinity and
specificity of the binding of these two clones to LDL. LDL retarded
clone 26 derived radiolabeled LBP-2 migration through the gel in a
concentration-dependent, saturable, manner while clone 45 derived
radiolabeled LBP-2 migration was not retarded.
[0171] Competition studies using fixed concentrations of cold LDL
and clone 26 derived radiolabeled LBP-2 and increasing
concentrations of cold recombinant LBP-2/clone 26 and LBP-2/clone
45 were carried out. Cold clone 26 derived LBP-2 inhibited binding
of clone 26 derived radiolabeled LBP-2 to LDL in a
concentration-dependent manner. Clone 45 derived LBP-2, on the
other hand, did not affect the binding of clone 26 derived
radiolabeled LBP-2 to LDL. These results indicate that the long
stretch of acidic amino acids contain a binding domain of LBP-2 to
LDL.
Example 9
Affinity Coelectrophoresis (ACE) Assays of LBP-1 or LBP-2 and LDL
in the Presence of Inhibitors
[0172] This example illustrates that binding between LBP-1 or LBP-2
and LDL is inhibited by polyglutamic acid or BHF-1. The ability of
a third compound to inhibit binding between two proteins previously
shown to interact was tested by a modification of the ACE assays
described in Example 8. The third compound was added to the top or
wells together with the radiolabeled protein. If the third compound
inhibited binding, the radiolabeled protein would run through the
gel. If the third compound did not inhibit binding, migration of
the radiolabeled protein was retarded by the protein cast into the
gel.
[0173] Inhibition of LBP-l/LDL or LBP-2/LDL binding by polyglutamic
acid (average MW about 7500, corresponding to about 7 monomers) was
shown by casting a constant amount of LDL (148 nM) in all the
rectangular lanes. A constant amount (1 .mu.l) of .sup.125I-labeled
LBP-1 or LBP-2 (0.003 nM-0.016 nM) was loaded in the wells at the
top of the gel, together with increasing concentrations of
polyglutamic acid (obtained from Sigma) (0-0.4 nM). The gel was
electrophoresed at 70 volts for 2 hr, dried and placed on X-ray
film, with intensifying screens, overnight at -70.degree. C. before
the film was developed to determine the retardation profile of
LBP-1 and LBP-2. As the concentration of polyglutamic acid
increased, retardation of radiolabeled LBP-1 and LBP-2 migration by
LDL decreased in a concentration-dependent manner, which showed
that polyglutamic acid inhibited binding between LBP-1, LBP-2 and
LDL.
[0174] Inhibition of LBP-1/LDL binding by BHF-1 was shown by
casting a constant amount of LDL (148 nM) in all the rectangular
lanes. A constant amount of .sup.125I-labeled LBP-1 (0.003 nM-0.016
nM) was loaded in the wells at the top of the gel, together with
increasing concentrations of BHF-1 (0-10 nM), obtained as described
in Example 15. The gel was electrophoresed at 70 volts for 2 hr,
dried and placed on X-ray film, with intensifying screens,
overnight at -70.degree. C. The film was then developed to
determine the retardation profile of .sup.125I-LBP-1. As the
concentration of BHF-i increased, retardation of LBP-1 by LDL
decreased in a concentration-dependent manner, which demonstrated
that BHF-1 inhibited binding between LBP-1 and LDL.
Example 10
Affinity Coelectrophoresis (ACE) Assays for Identifying Fragments,
Analogs and Mimetics of LBPs which Bind to LDL
[0175] This example illustrates a method for identifying fragments,
analogs or mimetics of LBPs which bind to LDL, and which thus can
be used as inhibitors of LDL binding to LBP in the arterial walls,
by occupying binding sites on LDL molecules, thereby rendering
these sites unavailable for binding to LBP in the arterial
wall.
[0176] Fragments of LBPs are generated by chemical cleavage or
synthesized from the known amino acid sequences. Samples of these
fragments are individually added (cold) to radiolabeled LBP as
described in Example 8, to assess the inhibitory potency of the
various fragments. By iterative application of this procedure on
progressively smaller portions of fragments identified as
inhibitory, the smallest active polypeptide fragment or fragments
are identified. In a similar manner, analogs of the LBPs are tested
to identify analogs which can act as inhibitors by binding to LDL.
And, similarly. mimetics of LBP (molecules which resemble the
conformation and/or charge distributions of the LDL-binding sites
on LBP molecules) are tested in a similar fashion to identify
molecules exhibiting affinities for the LDL-binding sites on
LBP.
[0177] The affinities of the inhibitors so identified are at least
as strong as the affinity of LDL itself for the LDL-binding sites
on LBP. The inhibitors bind at least competitively, and some
irreversibly and preferentially as well, to the LDL-binding sites,
thereby rendering such sites unavailable for binding to humoral
LDL.
Example 11
ELISA Assays
[0178] This example illustrates the use of an ELISA plate assay for
the quantification of a test compound's capacity to inhibit the
binding of LDL to a specific LBP.
[0179] The assay was carried out as follows: LDL was diluted in 50
mM Na.sub.2HCO.sub.3, pH 9.6/0.02% NaN.sub.3 and added to the wells
of a 96-well plate (ImmunoWare 96-Well Reacti-Bind EIA Polystyrene
Plates; Pierce (Rockford, Ill.)) to achieve a final concentration
ranging from 0.1 to 1 .mu.g/well. The plates were incubated for 6
hr at room temperature. At the end of the incubation period, the
wells were washed 3 times with Tris-buffered saline, pH 7.4 (TBS),
and blocked overnight with 200 .mu.l of 1% bovine serum albumin
(BSA) in TBS/0.02% NaN.sub.3 (Sigma; St. Louis Mo.) at room
temperature. The wells were then incubated with 200 .mu.l of LBP
protein (5-10 .mu.g/well) in TBS and varying concentrations of the
test compound. Plates were incubated for 1 hr at room temperature.
The wells were then washed three times with TBS and blocked for 2
hr with 200 .mu.l of 1% BSA in TBS/0.02% NaN.sub.3 at room
temperature. At the end of the incubation period, the wells were
washed 3 times with TBS and a 1:1000 dilution (in TBS/0.05% Tween
20) of the appropriate guinea pig anti-LBP protein polyclonal
antibody was added to the wells and incubated for 1 hr at room
temperature. The wells were then washed 3 times with TBS/0.05%
Tween 20; a 1:30,000 dilution of goat anti-guinea pig IgG alkaline
phophatase conjugate (Sigma) was added to each well. Plates were
incubated for 1 hr at room temperature. The wells were washed 3
times with TBS/0.05% Tween 20 and a calorimetric reaction was
carried out by adding 200 ml of p-nitrophenyl phosphate substrate
(Sigma; St. Louis Mo.) to the wells. The reaction was allowed to
proceed for 30 min at room temperature and stopped with 50 .mu.l of
3N NaOH. The absorbance was determined at 405 nm using an ELISA
plate reader. The test compound's effectiveness in blocking the
binding of LDL to the recombinant protein was assessed by comparing
the absorbance values of control and treated groups.
[0180] Alternatively, LBPs, rather than LDL, were bound to the
plate. Recombinant LBP protein binding to LDL and the effect of
varying concentration of the inhibitor on LBP-LDL binding was
determined through the use of antibodies against LDL. This
interaction was visualized through the use of a secondary antibody
conjugated to a reporter enzyme (e.g. alkaline phosphatase).
[0181] ELISA plate assays were used to screen agents which can
affect the binding of LBP proteins to LDL. For example, peptides
derived from LBP-1 and human LBP-3 protein sequences (BHF-1 and
BHF-2, respectively) were synthesized and have been shown to reduce
the binding of LDL to recombinant LBP-1 and LBP-2 in this format.
These results were in agreement with those obtained with the ACE
assays.
Example 12
Administration of Humanized Antibodies Against LBPs so as to Block
LDL-binding Sites on the LBPs
[0182] This example illustrates administration to patients of
humanized antibodies against LBP-1, LBP-2 or LBP-3 so as to block
LDL-binding sites on arterial LBP molecules. Mouse monoclonal
antibodies are humanized by recombinant DNA techniques and produced
by standard procedures known to those skilled in the art (Berkower,
I., Curr. Opin. Biotechnol. 7:622-628 (1996); Ramharayan and
Skaletsky, Am. Biotechnol. Lab 13:26-28 (1995)) against LBPs and/or
the LDL-binding sites on the LBPs. The corresponding Fab fragments
are also produced, as described in Goding, J. W., Monoclonal
Antibodies:Principles and Practice, Academic Press, New York, N.Y.
(1986). These antibodies are administered parenterally in
sufficient quantity so as to block LDL-binding sites on the LBP
molecules, i.e., 1-10 mg/kg daily. This prevents the irreversible
arterial uptake of LDL that is required to facilitate oxidation of
the LDL.
Example 13
Preparation of LDL
[0183] This example illustrates the preparation of LDL. LDL was
prepared from the plasma of normolipemic donors (Chang et al.,
Arterioscler. Thromb. 12:1088-1098 (1992)). 100 ml of whole blood
was placed into tubes containing 100 mM disodium EDTA. Plasma was
separated from red blood cells by low-speed centrifugation (2,000
g; 30 min; 4.degree. C.). Plasma density was adjusted to 1.025
gm/ml with a solution of KBr and centrifuged for 18-20 hr,
100,000.times. g, 12.degree. C. Very low density lipoproteins
(VLDL) were removed from the tops of the centrifuge tubes with a
Pasteur pipet. The density of the infranate was raised to 1.050
gm/ml with KBr solution and centrifuged for 22-24 hr,
100,000.times. g, 12.degree. C. LDL was removed from the tops of
the centrifuge tubes with a drawn out Pasteur pipet tip. Purity of
the LDL preparation was checked by Ouchterlony double
immunodiffusion against antibodies to human LDL, human HDL, human
immunoglobulins, and human albumin. KBr was removed from the LDL
solution by dialysis (1 L,.times.2,.apprxeq.16 hr) against 0.9%
saline, pH 9.0, containing 1 mM EDTA and 10 .mu.M butylated
hydroxytoluene (BHT), the latter to prevent oxidation of LDL.
Following dialysis, LDL protein was measured by the method of Lowry
(Lowry et al., J. Biol. Chem. 193:265-275 (1951)), and the LDL was
stored at 4.degree. C. until use. LDL preparations were kept for no
more than 4-6 weeks.
Example 14
Preparation of HDL
[0184] This example illustrates the preparation of HDL. HDL was
prepared from plasma of normolipemic donors. 100 ml of whole blood
was placed into tubes containing 100 mM disodium EDTA and plasma
was collected by centrifugation (2000 g; 30 min; 4.degree. C.).
Apolipoprotein B containing lipropoteins present in plasma were
then precipitated by the sequential addition of sodium heparin
(5,000 units/ml) and MnCl.sub.2 (1M) to achieve a final
concentration of 200 units/ml and 0.46 M, respectively (Warnick and
Albers, J. Lipid Res. 19:65-76 (1978)). Samples were then
centrifuged (2000 g; 1 hr; 4.degree. C.). The supernatant was
collected and density adjusted to 1.21 g/ml by the slow addition of
solid KBr. HDL was separated by ultracentrifugation (100,000 g;
>46 hr; 12.degree. C.). Purity of the HDL preparation was
assessed via Ouchterlony double immunodiffusion test using
antibodies against human HDL, human LDL, human immunoglobulins, and
human albumin. HDL samples were dialyzed against saline pH 9.0/1 mM
EDTA/10 .mu.M BHT (4 L; 24 hr/4.degree. C.) and total protein was
determined by the Lowry protein assay (Lowry et al., J. Biol. Chem.
193:265-275 (1951)). HDL was stored at 4.degree. C. until use. HDL
preparations were kept for no longer than 2 weeks.
Example 15
Synthesis of BHF-1
[0185] This example illustrates the synthesis of BHF-1, a fragment
of human or rabbit LBP-1 which contains amino acid residues 14
through 33. BHF-1 was synthesized using an Applied Biosystems Model
430A peptide synthesizer with standard T-Boc NMP chemistry cycles.
The sequence of BHF-1 is as follows:
[0186]
val-asp-val-asp-glu-tyr-asp-glu-asn-lys-phe-val-asp-glu-glu-asp-gly-
-gly-asp-gly (SEQ ID NO:9)
[0187] After synthesis, the peptide was cleaved with hydrofluoric
acid/anisole (10/1 v/v) for 30 min at -10.degree. C. and then
incubated for 30 min at 0.degree. C. BHF-1 was then precipitated
and washed three times with cold diethyl ether. Amino acid coupling
was monitored with the ninhydrin test (>99%).
[0188] The BHF-1 peptide was purified to homogeneity by high
performance liquid chromatography on a reverse phase Vydac C4
column (2.24.times.25 cm) using a linear gradient separation (2-98%
B in 60 min) with a flow rate of 9 ml/min. Buffer A consisted of
0.1% trifluoroacetic acid (TFA)/Milli Q water and Buffer B
consisted of 0.085% TFA/80% acetonitrile. The gradient was run at
room temperature and absorbance monitored at 210 and 277 nm.
[0189] Fast atom bombardment-mass spectrometry gave a protonated
molecular ion peak (M+H).sup.+ at m/z=2290.2, in good agreement
with the calculated value. On amino acid analysis, experimental
values for the relative abundance of each amino acid in the peptide
were in good agreement with theoretical values. The lyophilized
peptide was stored at -20.degree. C.
Example 16
In vitro Screening for Agents Which Inhibit Binding Between LDL and
LBPs
[0190] This example illustrates in vitro screening for agents which
inhibit binding between LDL and LBPs.
[0191] A candidate polypeptide for being an agent is chosen, e.g.,
LBP-1, LBP-2, LBP-3, BHF-1 or any other polypeptide. The shortest
fragment of the polypeptide that inhibits LDL binding to LBPs in
vitro is determined. Peptides are synthesized by standard
techniques described herein. Inhibition assays are performed using
standard ELISA techniques for screening, and affinity
coelectrophoresis (ACE) assays to confirm the ELISA results, as
described herein. Short peptides ranging, e.g., from dimers to
20-mers are constructed across sequences of the candidate
polypeptide whose chemical characteristics make them likely LDL
binding sites, e.g., acidic regions. The ability of shorter and
shorter lengths of the peptides to inhibit LDL binding in vitro and
to mammalian cells in culture is tested. For example, the effect of
the peptide on inhibiting LDL binding in mammalian cells
transfected to express an LBP gene is tested. Each of the peptides
so identified as an inhibitor is tested with each of LBP-1, LBP-2
and LBP-3, to determine whether a single inhibitor works against
all three LBPs.
[0192] Once the minimum active sequence is determined, the peptide
backbone is modified so as to inhibit proteolysis, as discussed
herein. For example, modification is accomplished by substitution
of a sulfoxide for the carbonyl, by reversing the peptide bond, by
substituting a methylene for the carbonyl group, or other similar
standard methodology. See Spatola, A. F., "Peptide Backbone
Modifications: A Structure-Activity Analysis of Peptides Containing
Amide Bond Surrogates, Conformational Constraints, and Related
Backbone Replacements," in Chemistry and Biochemistry of Amino
Acids, Peptides and Proteins, Vol. 7, pp. 267-357, B. Weinstein
(ed.), Marcel Dekker, Inc., New York (1983). The ability of these
analogs to inhibit LDL binding to the LBPs in vitro is tested by
ELISA and ACE assays in a similar manner as for the natural
peptides described above.
Example 17
In vitro Screening With Cultured Mammalian Cells for Agents Which
Inhibit Binding Between LDL and LPBs
[0193] This example illustrates cell-based in vitro screening of
agents which have been shown by in vitro tests such as ACE assay
and ELISA to be potential inhibitors of binding between LDL and
LBPs.
[0194] Mammalian cells, such as 293 cells, which are commonly used
for expression of recombinant gene constructs, are used to develop
cell lines which express LBPs on the cell surface. This is done by
subcloning LBP open reading frames (ORFs) into a mammalian
expression plasmid vector, pDisplay (Invitrogen, Carlsbad, Calif.),
which is designed to express the gene of interest on the cell
surface. The use of mammalian cells to produce LBPs allows for
their expression in a functionally active, native conformation.
Therefore, stably transfected mammalian cell lines with surface
expression of LBPs individually, or in combination, are
particularly suitable for assaying and screening inhibitors that
block LDL binding in cell culture, as well as to evaluate the
cytotoxicity of these compounds.
[0195] Specifically, LBP ORFs are amplified by PCR (Perkin Elmer,
Foster City, Calif.) from cDNA templates using Taq polymerase
(Perkin Elmer) and appropriate primers. The amplified LBP ORFs are
purified by agarose gel electrophoresis and extracted from gel
slices with the Bio-Rad DNA Purification kit (Bio-Rad, Hercules,
Calif.). The purified DNAs are then cut with the restriction
enzymes Bgl II and Sal I (New England Biolabs, Beverly, Mass.) to
generate cohesive ends, and purified again by agarose gel
electrophoresis and DNA extraction as described above. The LBP ORFs
are then subcloned into the Bgl II/Sal I sites in the mammalian
expression vector, pDisplay (Invitrogen) by ligation. Recombinant
plasmids are established by transformation in E.coli strains TOP10
(Invitrogen) or DH5.alpha. (Life Technologies, Grand Island, N.Y.).
Recombinant pDisplay/LBP plasmid DNA is isolated from overnight
E.coli cultures with the Bio-Rad Plasmid Miniprep kit, cut with Bgl
II/Sal I, and analyzed by agarose gel electrophoresis. LBP ORFs in
successfully transformed clones are verified by automated dideoxy
DNA sequencing. To transfect human kidney 293 cells, 1-2 .mu.g of
DNA is mixed with 6 .mu.l liofectamine reagent (Life Technologies)
and incubated with the cells as described in the Life Technologies
protocol. LBP expression in transfected cells is confirmed by
Western blot analysis of cell extracts obtained 48 hr after
transfection. To select for stably transfected 293 cells, the
antibiotic G418 (Life Technologies) is added to the growth medium
at a concentration of 800 .mu.g/ml. Colonies resistant to G418 are
tested for recombinant LBP expression by Western blot, and
recombinant clones expressing LBPs are expanded, assayed for LDL
binding and used to test compounds for their ability to inhibit LDL
binding.
Example 18
In vivo Screening for Agents Which Inhibit Binding Between LDL and
LBPs
[0196] This example illustrates in vivo screening of agents which
have been shown by in vitro tests to be promising candidate
inhibitors of binding between LDL and LBPs.
[0197] In vivo inhibitory activity is first tested in the healing
balloon-catheter deendothelialized rabbit aorta model of arterial
injury (Roberts et al., J. Lipid Res. 24:1160-1167 (1983); Chang et
al., Arterioscler. Thomb. 12:1088-1098 (1992)). This model was
shown to be an excellent analog for human atherosclerotic lesions.
Each candidate inhibitor is tested in five to ten ballooned
rabbits, while an equal number of rabbits receive a control
peptide, or placebo. Four weeks following aortic
deendothelialization, when reendothelialization (healing) is
partially complete, daily parenteral (intravenous or subcutaneous)
or intragastric administration of the peptides and the analogs
begins at an initial concentration of 10 mg/kg body weight, which
is varied down, or up to 100 mg/kg depending on results. 30 min
later, a bolus of intravenously injected .sup.125I (or .sup.99mTc-)
labeled LDL is given to zest the candidate inhibitor's ability in
short term studies to inhibit LDL sequestration in healing arterial
lesions. If .sup.125I-LDL is used, the animals are sacrificed 8-24
hr later, the aortas excised, washed and subjected to quantitative
autoradiography of excised aortas, as previously described (Roberts
et al., J. Lipid Res. 24:1160-1167 (1983); Chang et al.,
Arterioscler. Thomb. 12:1088-1098 (1992)). If .sup.99mTc-LDL is
used, analysis is by external gamma camera imaging of the live
anesthetized animal at 2-24 hr, as previously described (Lees and
Lees, Syndromes of Atherosclerosis, in Fuster, ed., Futura
Publishing Co., Armonk, N.Y., pp. 385-401 (1996)), followed by
sacrifice, excision and imaging of the excised aorta. Immediately
before the end of testing, the animals have standard toxicity
tests, including CBC, liver enzymes, and urinalysis.
[0198] The compounds which are most effective and least toxic are
then tested in short term studies of rabbits fed a 2% cholesterol
diet (Schwenke and Carew, Arteriosclerosis 9:895-907 (1989)). Each
candidate inhibitor is tested in five to ten rabbits, while an
equal number of rabbits receive a control peptide, or placebo.
Animals receive one or more doses per day of the candidate
inhibitor, or placebo, for up to two weeks. Daily frequency of
doses is determined by route of administration. If active drug or
placebo are administered parenterally, they are given 1-3 times
daily and the 2% cholesterol diet is continued. If drug or placebo
are given orally, they are mixed with the 2% cholesterol diet.
Schwenke and Carew (Arteriosclerosis 9:895-907 (1989)) have shown
that the LDL concentration in lesion-prone areas of the rabbit
aorta is increased 22-fold above normal in rabbits fed a 2%
cholesterol diet for 16 days, and that the increased LDL content
precedes the histological evidence of early atherosclerosis.
Therefore, analysis of the effect of the candidate inhibitors is
tested two weeks after the start of cholesterol feeding by
injecting .sup.125I-LDL, allowing it to circulate for 8-24 hr, and
then performing quantitative autoradiography on the excised aortas
of both test and control animals. If appropriate, quantitation of
aortic cholesterol content is also carried out (Schwenke and Carew,
Arteriosclerosis 9:895-907 (1989); Schwenke and Carew,
Arteriosclerosis 9:908-918 (1989).
[0199] The above procedures identify the most promising candidate
inhibitors, as well as the best route and frequency of their
administration. Inhibitors so identified are then tested in
long-term studies of cholesterol-fed rabbits. These tests are
carried out in the same way as the short-term cholesterol feeding
studies, except that inhibitor effectiveness is tested by injection
of .sup.125I-LDL at longer intervals following the initiation of
cholesterol feeding, and lesion-prone areas of the aorta are
examined histologically for evidence of atherosclerosis. Testing
times are at two, four, and six months. Major arteries are examined
grossly and histologically for evidence and extent of
atherosclerosis. If necessary, other accepted animal models, such
as atherosclerosis-susceptible primates (Williams et al.,
Arterioscler. Thromb. Vasc. Biol. 15:827-836 (1995) and/or Watanabe
rabbits are tested with short- and long-term cholesterol
feeding.
Example 19
In vivo Inhibition of Radiolabeled LDL Accumulation in the
Ballooned Deendothelialized Rabbit Aorta via Induction of Active
Immunity Against LBP Protein
[0200] This example illustrates the effect that induction of
immunity against LBP protein has on the accumulation of
radiolabeled LDL in the ballooned deendothelialized rabbit aorta
model of atherosclerosis.
[0201] Immunity was induced in male New Zealand White rabbits
(Hazelton Research Products, Denver, Pa.) as follows: A mixture of
purified human recombinant LBP-2 or BHF-1 peptide (1 ml; 1 mg) and
RIBI adjuvant (RIBI ImmunoChem Research, Inc., Hamilton, Mont.) was
injected subcutanously at 2-5 sites along the dorsal thoracic and
abdominal regions of the rabbits. Blood was collected by
venipuncture on days 1 (preimmune bleeding), 35, 63, and 91.
Booster injections were administered on days 28 (500 .mu.g; SC), 56
(250 .mu.g; SC), and 84 (125 .mu.g; SC).
[0202] The titer of the rabbits was evaluated by serial dilution
using an ELISA plate format. Preimmune serum was evaluated at the
same time. After the third booster of LBP protein or peptide, the
titer reached a maximal level with a detectable calorimetric
response on an ELISA plate of 156 pg. Titer is defined as the
maximum dilution of antibody which generates an absorbance reading
of 0.5 above control in 30 min. Specificity of the polyclonal
antibodies was demonstrated using Western blot analysis as
described in Example 6.
[0203] On day 93, the abdominal aorta of immunized and control
rabbits was deendothelialized using a Fogarty number 4 embolectomy
catheter (Chang et al., Arteriosclerosis and Thrombosis
12:1088-1098 (1992)). Four weeks after ballooning, rabbits received
a bolus injection of .sup.125I-labeled LDL (1 ml; i.v.). Blood
samples were collected at 1 hr intervals for 8 hr, and 24 hr post
injection. Blood samples were centrifuged for 30 min at 2000 rpm
(40.degree. C.) and total activity present in the serum was
determined using a Gamma counter. Total TCA precipitable counts
were determinined by addition of TCA to the serum to a final
concentration of 10% followed by incubation for 10 min at 4.degree.
C. Serum samples were then centrifuged (2000 rpm; 30 min;
40.degree. C.) and total activity present in the supernate was
determined. TCA precipitable counts were calculated by substration:
total soluble counts minus counts present in the supernate after
TCA precipitation. Blood samples for the determination of antibody
titers were collected prior to the injection of the radiolabeled
LDL.
[0204] After 24 hr, the rabbits were injected intravenously with 5%
Evan's blue dye which was allowed to circulate for 15 min. Areas of
the aorta in which the endothelial covering is absent stain blue
while those areas covered by endothelium remain unstained. At the
end of the incubation period, the rabbits were euthanized and the
abdominal and thoracic aorta were dissected out, rinsed, and fixed
overnight in 10% TCA at room temperature. The aortas were then
rinsed exhaustively with physiological saline, weighed, counted,
blotted dry and placed onto X-ray film in order to visualize the
pattern of radiolabeled LDL accumulation in the deendothelialized
rabbit abdominal aorta.
[0205] Immunization of rabbits against recombinant human LBP-2 or
BHF-1 peptide altered the pattern of radiolabeled LDL accumulation
in the ballooned deendothelialized abdominal aorta. When corrected
for dosage, and percent reendothelialization, immunized-ballooned
rabbits had lower accumulation of radiolabeled LDL compared to
nonimmune-ballooned rabbits. These results indicate that active
immunization against LBP provides an effective means by which the
accumulation of LDL in the injured arterial wall can be
modified.
Example 20
Screening Agents in Humans Which Inhibit Binding Between LDL and
LBPs
[0206] Human studies are carried out according to standard FDA
protocols for testing of new drugs for safety (Phase I), efficacy
(Phase II), and efficacy compared to other treatments (Phase III).
Subjects, who are enrolled into studies after giving informed
consent, are between the ages of 18 and 70. Women who are pregnant,
or likely to become pregnant, or subjects with diseases other than
primary atherosclerosis, such as cancer, liver disease, or
diabetes, are excluded. Subjects selected for study in FDA Phase II
and Phase III trials have atherosclerotic disease previously
documented by standard techniques, such as ultrasound and/or
angiography, or are known to be at high risk of atherosclerosis by
virtue of having at least one first degree relative with documented
atherosclerosis. Subjects themselves have normal or abnormal plasma
lipids. Initial testing includes 20-50 subjects on active drug and
20-50 subjects, matched for age, sex, and atherosclerotic status,
on placebo. The number of subjects is pre-determined by the number
needed for statistical significance. Endpoints for inhibitor
efficacy includes ultrasound measurements of carotid artery
thickness in high risk subjects, as well as in subjects with known
carotid or coronary disease; atherosclerotic events;
atherosclerotic deaths; and all-cause deaths in all subjects.
Non-invasive analysis (carotid artery thickness by ultrasound) as
per Stadler (Med. and Biol. 22:25-34 (1996)) are carried out at 6-
to 12-month intervals for 3 years. Atherosclerotic events and
deaths, as well as all-cause deaths are tabulated at 3 years.
[0207] Oral dosage of drug in FDA Phase I trials ranges from 0.01
to 10 gm/day, and is determined by results of animal studies,
extrapolated on a per kg basis. Based on data obtained from Phase I
studies, the dose range and frequency are narrowed in Phase II and
III trials. If parenteral administration of drug is determined by
animal studies to be the only effective method, parenteral
administration in human subjects is tested by injection, as well as
by the transdermal and nasal insufflation routes. Testing of
parenteral drug follows the same outline as that for oral
administration.
[0208] The optimal treatment schedule and dosage for humans is thus
established.
Example 21
Treating an Individual Having Atherosclerosis with BHF-1
[0209] This example illustrates a method for treating an individual
having atherosclerosis with an LBP fragment, e.g., BHF-1, so as to
decrease the levels of arterially bound LDL in the individual.
BHF-1 is obtained as described herein. The BHF-1 is administered to
the mammal intravenously as a bolus or as an injection at a
concentration of 0.5-10 mg/kg body weight. Such administrations are
repeated indefinitely in order to prevent the development or
progression of symptomatic atherosclerosis, just as is done
currently with cholesterol-lowering drugs. Stable subjects are
examined twice yearly to evaluate the extent of any atherosclerotic
disease by physical exam and non-invasive studies, such as carotid
artery thickness, ultrasound, and/or gamma camera imaging of the
major arteries, to determine if atherosclerotic lesions are
present, and, if previously present, have regressed or progressed.
Such a regimen results in treatment of the atherosclerosis.
[0210] Those skilled in the art will be able to ascertain using no
more than routine experimentation, many equivalents of the specific
embodiments of the invention described herein. These and all other
equivalents are intended to be encompassed by the following
claims.
* * * * *