U.S. patent application number 10/437706 was filed with the patent office on 2003-10-23 for assays for beta-amyloid processing.
Invention is credited to Cordell, Barbara, Huggins, John, Mischak, Ronald P., Pruss, Rebecca, Rautmann, Guy, Scardina, Jan Marian.
Application Number | 20030199094 10/437706 |
Document ID | / |
Family ID | 29219856 |
Filed Date | 2003-10-23 |
United States Patent
Application |
20030199094 |
Kind Code |
A1 |
Pruss, Rebecca ; et
al. |
October 23, 2003 |
Assays for beta-amyloid processing
Abstract
The invention relates to the design, construction and use of
eukaryotic cell lines useful in the identification of inhibitors of
.beta.-amyloid processing. More specifically, the invention relates
to in vitro assays capable of identifying or quantifying a 4.2 kDa
.beta.-amyloid protein. The present invention also provides for DNA
and protein molecules for the design, construction and use of
eukaryotic cell lines useful in the identification of inhibitors of
.beta.-amyloid processing.
Inventors: |
Pruss, Rebecca; (Strasbourg,
FR) ; Huggins, John; (Kent, GB) ; Rautmann,
Guy; (Strasbourg, FR) ; Cordell, Barbara;
(Palo Alto, CA) ; Scardina, Jan Marian; (San
Carlos, CA) ; Mischak, Ronald P.; (Palo Alto,
CA) |
Correspondence
Address: |
ROSS J. OEHLER
AVENTIS PHARMACEUTICALS INC.
ROUTE 202-206
MAIL CODE: D303A
BRIDGEWATER
NJ
08807
US
|
Family ID: |
29219856 |
Appl. No.: |
10/437706 |
Filed: |
May 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10437706 |
May 14, 2003 |
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09027258 |
Feb 20, 1998 |
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60067389 |
Feb 24, 1997 |
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60067390 |
Apr 2, 1997 |
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60072099 |
Jul 31, 1997 |
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Current U.S.
Class: |
435/456 ;
435/358; 536/23.2 |
Current CPC
Class: |
G01N 2500/04 20130101;
G01N 2500/10 20130101; G01N 33/6896 20130101 |
Class at
Publication: |
435/456 ;
435/358; 536/23.2 |
International
Class: |
C12N 005/06; C12N
015/86; C07H 021/04 |
Claims
We claim:
1. An eukaryotic cell line having a exogenous gene construction,
said exogenous gene construction comprising a cytomegalovirus
promoter encoding sequence, a strong ribosome binding site encoding
sequence, a amyloid precursor protein encoding sequence, a
selectable marker encoding sequence, and a poly-adenylation signal
encoding sequence.
2. The eukaryotic cell line according to claim 1, wherein said
exogenous gene construction is capable of stably expressing a
.beta.-amyloid precursor protein molecule.
3. The eukaryotic cell line according to claim 1, wherein said
exogenous gene construction further comprises an internal ribosome
entry site encoding sequence.
4. The eukaryotic cell line according to claim 1, wherein said
.beta.-amyloid precursor protein encoding sequence encodes the 695
amino acid isoform of human .beta.-amyloid precursor protein
molecule.
5. The eukaryotic cell line according to claim 1, wherein said
.beta.-amyloid precursor protein encoding sequence encodes the 751
amino acid isoform of human .beta.-amyloid precursor protein
molecule.
6. The eukaryotic cell line according to claim 1, wherein said
eukaryotic cell line is selected from the group consisting of
chinese hamster ovary cell line, dihydrofolate reductase deficient
hamster cell line, human kidney cell line, rat neuroglimo cell
line, human neuroglimo cell line, and rat neuroblastoma cell
line.
7. The eukaryotic cell line according to claim 6, wherein said
eukaryotic cell line is chinese hamster ovary cell line.
8. The eukaryotic cell line according to claim 7, wherein said
eukaryotic cell line is chinese hamster ovary cell line K1.
9. The eukaryotic cell line according to claim 1, wherein the
selectable marker encoding sequence is selected from the group
consisting of: neomycin phosphotransferase encoding sequence,
dihydrofolate reductase encoding sequence, xanthine-guanine
phosphoribosyltransferase encoding sequence, aspartate
transcarbamoylase encoding sequence, adenosine deaminase encoding
sequence, adenylate deaminase encoding sequence, UMP synthetase
encoding sequence, glutamine synthetase encoding sequence,
asparagine synthetase encoding sequence ornithine decarboxylase
encoding sequence, the thymidine kinase encoding sequence; the
aminoglycosidase phosphotransferase encoding sequence; hygromycin B
phosphotransferase-encoding sequence; and the CAD encoding
sequence.
10. The eukaryotic cell line according to claim 9, wherein the
selectable marker encoding sequence is bacterial neomycin
phosphotransferase encoding sequence.
11. The eukaryotic cell line according to claim 3, wherein said
internal ribosome site encoding sequence is an
encephalomycocarditis virus internal ribosome entry site encoding
sequence linked to said .beta.-amyloid precursor protein encoding
sequence.
12. The eukaryotic cell line according to claim 11, wherein said
.beta.-amyloid precursor protein encoding sequence encodes the 695
amino acid isoform of human .beta.-amyloid precursor protein
molecule.
13. The eukaryotic cell line according to claim 11, wherein said
.beta.-amyloid precursor protein encoding sequence encodes the 751
amino acid isoform of human .beta.-amyloid precursor protein
molecule.
14. The eukaryotic cell line according to claim 11, wherein the
selectable marker encoding sequence is selected from the group
consisting of: neomycin phosphotransferase encoding sequence,
dihydrofolate reductase encoding sequence, xanthine-guanine
phosphoribosyltransferase encoding sequence, aspartate
transcarbamoylase encoding sequence, adenosine deaminase encoding
sequence, adenylate deaminase encoding sequence, UMP synthetase
encoding sequence, glutamine synthetase encoding sequence,
asparagine synthetase encoding sequence ornithine decarboxylase
encoding sequence, the thymidine kinase encoding sequence; the
aminoglycosidase phosphotransferase encoding sequence; hygromycin B
phosphotransferase encoding sequence; and the CAD encoding
sequence.
15. The eukaryotic cell line according to claim 14, wherein the
selectable marker encoding sequence is bacterial neomycin
phosphotransferase encoding sequence.
16. The eukaryotic cell line according to claim 1, wherein said
strong ribosome binding site encoding sequence is linked to said
.beta.-amyloid precursor protein encoding sequence and said
cytomegalovirus promoter encoding sequence to provide efficient
expression of said .beta.-amyloid precursor protein encoding
sequence.
17. The eukaryotic cell line according to claim 16, wherein said
.beta.-amyloid precursor protein encoding sequence encodes the 695
amino acid isoform of human .beta.-amyloid precursor protein
molecule.
18. The eukaryotic cell line according to claim 16, wherein said
.beta.-amyloid precursor protein encoding sequence encodes the 751
amino acid isoform of human .beta.-amyloid precursor protein
molecule.
19. A eukaryotic cell line selected from the group consisting of
21-N-1, 21 -N-2, 21-N-3, 21-N-4, 21-N-5, 21-N-6, 21-N-7 21-N-8, and
21-N-9 or a derivative thereof.
20. The eukaryotic cell line according to claim 19, wherein said
eukaryotic cell line is selected from the group consisting of
21-N-9 and 21-N-3.
21. The eukaryotic cell line according to claim 20, wherein said
eukaryotic cell line is 21-N-9.
22. The eukaryotic cell line according to claim 1, wherein said
eukaryotic cell line produces a 4.2 kDa .beta.-amyloid protein
molecule to a greater extent than a CP-6 cell line.
23. An eukaryotic cell line capable of producing greater than 30 ng
4.2 kDa .beta.-amyloid protein molecule per mg protein.
24. The eukaryotic cell line of claim 23, wherein said eukaryotic
cell line is capable of producing greater than 50 ng 4.2 kDa
.beta.-amyloid protein molecule per mg protein.
25. The eukaryotic cell line of claim 24, wherein said eukaryotic
cell line is capable of producing greater than 60 ng 4.2 kDa
.beta.-amyloid protein molecule per mg protein.
26. The eukaryotic cell line of claim 25, wherein said eukaryotic
cell line is capable of producing greater than 65 ng 4.2 kDa
.beta.-amyloid protein molecule per mg protein.
27. The eukaryotic cell line of claim 25, wherein said eukaryotic
cell line is capable of producing greater than 70 ng 4.2 kDa
.beta.-amyloid protein molecule per mg protein.
28. The eukaryotic cell line according to claim 23, wherein said
eukaryotic cell line produces a 4.2 kDa .beta.-amyloid protein
molecule to a greater extent than a CP-7 cell line.
29. The eukaryotic cell line according to claim 28, wherein said
eukaryotic cell line produces greater than three fold more said 4.2
kDa .beta.-amyloid protein molecule than said CP-7 cell line.
30. The eukaryotic cell line according to claim 29, wherein said
eukaryotic cell line produces greater than six fold more said 4.2
kDa .beta.-amyloid protein molecule than said CP-7 cell line.
31. The eukaryotic cell line according to claim 30, wherein said
eukaryotic cell line produces greater than eight fold more said 4.2
kDa .beta.-amyloid protein molecule than said CP-7 cell line.
32. The eukaryotic cell line according to claim 31, wherein said
eukaryotic cell line produces greater than eight fold more said 4.2
kDa .beta.-amyloid protein molecule than said CP-7 cell line.
33. The eukaryotic cell line according to claim 23, wherein said
eukaryotic cell line produces a A.beta.1-40 peptide molecule to a
greater extent than a CP-7 cell line.
34. The eukaryotic cell line according to claim 33, wherein said
eukaryotic cell line produces greater than two fold more said
A.beta.1-40 peptide molecule than said CP-7 cell line.
35. The eukaryotic cell line according to claim 34, wherein said
eukaryotic cell line produces greater than three fold more said
A.beta.1-40 peptide molecule than said CP-7 cell line.
36. The eukaryotic cell line according to claim 35, wherein said
eukaryotic cell line produces greater than 3.5 fold more said
A.beta.1-40 peptide molecule than said CP-7 cell line.
37. The eukaryotic cell line according to claim 23, wherein said
eukaryotic cell line produces a A.beta.1-42 peptide molecule to a
greater extent than a CP-7 cell line.
38. The eukaryotic cell line according to claim 37, wherein said
eukaryotic cell line produces greater than two fold more said
A.beta.1-42 peptide molecule than said CP-7 cell line.
39. The eukaryotic cell line according to claim 38, wherein said
eukaryotic cell line produces greater than four fold more said
A.beta.1-42 peptide molecule than said CP-7 cell line.
40. The eukaryotic cell line according to claim 39, wherein said
eukaryotic cell line produces greater than six fold more said
A.beta.1-42 peptide molecule than said CP-7 cell line.
41. An eukaryotic cell line having a exogenous gene construction,
said exogenous gene construction comprising a cytomegalovirus
promoter encoding sequence, a ribosome binding site encoding
sequence, a .beta.-amyloid precursor protein encoding sequence, a
selectable marker encoding sequence, and a poly-adenylation signal
encoding sequence.
42. A substantially purified nucleic acid molecule that encodes, in
order: a cytomegalovirus promoter; a ribosome binding site; a
.beta.-amyloid precursor protein molecule; a selectable marker; and
a poly-adenylation signal.
43. A substantially purified nucleic acid molecule that encodes, in
order: a cytomegalovirus promoter; a strong ribosome binding site;
a .beta.-amyloid precursor protein molecule; a selectable marker;
and a poly-adenylation signal.
44. A nucleic acid molecule comprising
pCMV-IRES-.beta.APP.sub.695.
45. A method for identifying an inhibitor of .beta.-amyloid
processing pathway comprising: administering said inhibitor to a
eukaryotic cell line having a exogenous gene construction, said
exogenous gene construction comprising a cytomegalovirus promoter
encoding sequence, a strong ribosome binding site encoding
sequence, a .beta.-amyloid precursor protein encoding sequence, a
selectable marker encoding sequence, and a poly-adenylation signal
encoding sequence; and quantifying a protein molecule that is
indicative of inhibition of .beta.-amyloid processing pathway.
46. The method for identifying an inhibitor of .beta.-amyloid
processing according to claim 45, further comprising incubating
said eukaryotic cell line with said inhibitor.
47. A method for identifying an inhibitor of .beta.-amyloid
processing according to claim 46, further comprising an internal
ribosome site encoding sequence linked to said .beta.-amyloid
precursor protein encoding sequence to facilitate translation of
said .beta.-amyloid precursor protein encoding sequence and said
selectable marker from a single messenger RNA transcript.
48. A method for identifying an inhibitor of .beta.-amyloid
processing according to claim 46, wherein said strong ribosome
encoding sequence is linked to said .beta.-amyloid precursor
protein encoding sequence and said cytomegalovirus promoter
encoding sequence to provide efficient expression of said
.beta.-amyloid precursor protein encoding sequence.
49. The method for identifying an inhibitor of .beta.-amyloid
processing according to claim 46, wherein said cell line selected
from the group consisting of chinese hamster ovary cell line,
dihydrofolate reductase deficient hamster cell line, human kidney
cell line, rat neuroglimo cell line, human neuroglimo cell line,
and rat neuroblastoma cell line.
50. The method for identifying an inhibitor of .beta.-amyloid
processing according to claim 49, wherein said eukaryotic cell line
is chinese hamster ovary cell line.
51. The method for identifying an inhibitor of .beta.-amyloid
processing according to claim 50, wherein said eukaryotic cell line
is chinese hamster ovary cell line K1.
52. The method for identifying an inhibitor of .beta.-amyloid
processing according to claim 46, wherein said protein molecule is
encoded by said .beta.-amyloid precursor protein or a derivative
thereof encoding sequence.
53. The method for identifying an inhibitor of .beta.-amyloid
processing according to claim 46, wherein said protein molecule is
selected from the group consisting of 4.2 kDa .beta.-amyloid
protein molecule, A.beta.1-39 peptide molecule, A.beta.1-40 peptide
molecule, A.beta.1-41 peptide molecule, A.beta.1-42 peptide
molecule, and A.beta.1-43 peptide molecule.
54. The method for identifying an inhibitor of .beta.-amyloid
processing according to claim 52, wherein said protein molecule is
a 4.2 kDa .beta.-amyloid protein molecule.
55. The method for identifying an inhibitor of .beta.-amyloid
processing according to claim 54, wherein determining said level of
a said 4.2 kDa .beta.-amyloid protein molecule is by
immunodisplacement of said 4.2 kDa .beta.-amyloid protein molecule
using an antibody.
56. The method for identifying an inhibitor of .beta.-amyloid
processing according to claim 55, wherein said antibody is
.beta.-amyloid 1101.1 4.2 kDa-specific monoclonal antibody.
57. A composition comprising a eukaryotic cell line and an
inhibitor of a .beta.-amyloid processing pathway, said eukaryotic
cell line having a exogenous gene construction, said exogenous gene
construction comprising a cytomegalovirus promoter encoding
sequence, a strong ribosome binding site encoding sequence, a
.beta.-amyloid precursor protein encoding sequence, a selectable
marker encoding sequence, and a poly-adenylation signal encoding
sequence.
58. A composition comprising a media having a protein molecule that
is indicative of inhibition of a .beta.-amyloid processing pathway,
said media incubated with a eukaryotic cell line having a exogenous
gene construction, said exogenous gene construction comprising a
cytomegalovirus promoter encoding sequence, a strong ribosome
binding site encoding sequence, a .beta.-amyloid precursor protein
encoding sequence, a selectable marker encoding sequence, and a
poly-adenylation signal encoding sequence.
59. The composition of matter according to claim 58, wherein said
media having a protein molecule that is indicative of inhibition of
.beta.-amyloid processing pathway is essentially free of said
eukaryotic cell line.
60. A composition comprising a media having a protein molecule that
is indicative of inhibition of .beta.-amyloid processing pathway,
said media supplemented with biotinylated 4.2 kDa .beta.-amyloid
protein molecule, said media incubated with a eukaryotic cell line
and an inhibitor of .beta.-amyloid processing pathway, said
eukaryotic cell line having a exogenous gene construction, said
exogenous gene construction comprising a cytomegalovirus promoter
encoding sequence, a strong ribosome binding site encoding
sequence, a .beta.-amyloid precursor protein encoding sequence, a
selectable marker encoding sequence, and a poly-adenylation signal
encoding sequence.
61. The composition according to claim 60, wherein said composition
of matter further comprises an antibody capable of specifically
binding to a 4.2 kDa .beta.-amyloid protein molecule.
62. The composition according to claim 61, wherein said antibody is
.beta.-amyloid 1 101.1 4.2 kda-specific monoclonal antibody.
63. The composition of matter according to claims 61 or 62, wherein
said antibody is attached to a solid phase.
64. A method for identifying an inhibitor of .beta.-amyloid
processing pathway comprising, (a) incubating an inhibitor of
.beta.-amyloid processing and a eukaryotic cell line to produce a
protein molecule that is indicative of inhibition of .beta.-amyloid
processing pathway in a medium containing a labeled amino acid,
said eukaryotic cell line having a exogenous gene construction,
said exogenous gene construction comprising a cytomegalovirus
promoter encoding sequence, a strong ribosome binding site encoding
sequence, a .beta.-amyloid precursor protein encoding sequence, a
selectable marker encoding sequence, and a poly-adenylation signal
encoding sequence, wherein said exogenous gene construction is
capable of stably expressing a .beta.-amyloid precursor protein
molecule; (b) separating said eukaryotic cell line and said medium;
(c) separating said protein molecule from said eukaryotic cell line
and said medium; and (d) quantifying said protein molecule.
65. The method for identifying an inhibitor of .beta.-amyloid
processing pathway according to claim 64, wherein said protein
molecule is separated by SDS polyacrylamide gel
electrophoresis.
66. The method for identifying an inhibitor of .beta.-amyloid
processing pathway according to claim 65, wherein said labeled
amino acid is selected from the group consisting of
.sup.35S-methonine, .sup.35S-cysteine and a mixture of
.sup.35S-methonine and .sup.35S-Cysteine
67. The method for identifying an inhibitor of .beta.-amyloid
processing pathway according to claim 65, wherein said protein
molecule is immunoprecipitated with an antibody specific for
.beta.-amyloid precursor protein.
68. The method for identifying an inhibitor of .beta.-amyloid
processing pathway according to claim 65, wherein said protein
molecule from said eukaryotic cell line is immunoprecipitated by a
antibody specific for an epitope diagnostic for the C-terminal
region of human .beta.-amyloid precursor protein.
69. The method for identifying an inhibitor of .beta.-amyloid
processing pathway according to claim 67, wherein said protein
molecule from said medium is immunoprecipitated by a antibody
specific for human .beta.-amyloid precursor protein.
70. The method for identifying an inhibitor of .beta.-amyloid
processing pathway according to claim 65, wherein said identifying
is by phosphorimager.
71. The method for identifying an inhibitor of .beta.-amyloid
processing pathway according to claim 66, wherein said identifying
is by autoradiography.
72. A high throughput assay for identification of an inhibitor of
.beta.-amyloid processing comprising: (a) incubating an inhibitor
of .beta.-amyloid processing and a eukaryotic cell line to produce
a protein molecule that is indicative of inhibition of
.beta.-amyloid processing pathway, said eukaryotic cell line having
a exogenous gene construction, said exogenous gene construction
comprising a cytomegalovirus promoter encoding sequence, a strong
ribosome binding site encoding sequence, a .beta.-amyloid precursor
protein encoding sequence, a selectable marker encoding sequence,
and a poly-adenylation signal encoding sequence; and (b)
quantifying said protein molecule produced by the incubation
step.
73. The high throughput assay for identification of an inhibitor of
.beta.-amyloid processing according to claim 72, wherein said
incubation is undertaken in a volume of less than about 300
.mu.l.
74. The high throughput assay for identification of an inhibitor of
.beta.-amyloid processing according to claim 73, wherein said
incubation is undertaken in a volume of less than about 250
.mu.l.
75. The high throughput assay for identification of an inhibitor of
.beta.-amyloid processing according to claim 74, wherein said
incubation is undertaken in a volume of about 200 .mu.l.
76. The high throughput assay for identification of an inhibitor of
.beta.-amyloid processing according to claim 72, wherein said
incubation is conducted in a 96 well microtitre plate.
77. The high throughput assay for identification of an inhibitor of
.beta.-amyloid processing according to claim 72, wherein said
incubation is carried out in contact with a surface area of less
than about 0.6 cm.sup.-2.
78. The high throughput assay for identification of an inhibitor of
.beta.-amyloid processing according to claim 77, wherein said
incubation is carried out in contact with a surface area of less
than about 0.4 cm.sup.-2.
79. The high throughput assay for identification of an inhibitor of
.beta.-amyloid processing according to claim 78, wherein said
protein molecule is encoded by said .beta.-amyloid precursor
protein encoding sequence or encoded by a fragment of said
.beta.-amyloid precursor protein encoding sequence.
80. The high throughput assay for identification of an inhibitor of
.beta.-amyloid processing according to claim 72, wherein said
protein molecule is selected from the group consisting of 4.2 kDa
.beta.-amyloid protein molecule, A.beta.1-39 peptide molecule
molecule, A.beta.1-40 peptide molecule, A.beta.1-41 peptide
molecule, A.beta.1-42 peptide molecule, and A.beta.1-43 peptide
molecule.
81. The high throughput assay for identification of an inhibitor of
.beta.-amyloid processing according to claim 80, wherein said
protein molecule is a 4.2 kDa .beta.-amyloid protein molecule.
82. The high throughput assay for identification of an inhibitor of
.beta.-amyloid processing according to claim 81, wherein
determining said level of said 4.2 kDa .beta.-amyloid protein
molecule is by inmunodisplacement of said 4.2 kDa .beta.-amyloid
protein molecule using an antibody.
83. The high throughput assay for identification of an inhibitor of
.beta.-amyloid processing according to claim 82, wherein said
antibody is .beta.-amyloid 1101.1 4.2 kDa-specific monoclonal
antibody.
84. A composition comprising, a .beta.-amyloid 108.1 42 specific
monoclonal antibody, a .beta.-amyloid 1101.1 4.2 kDa-specific
monoclonal antibody and a media having a protein molecule that is
indicative of inhibition of .beta.-amyloid processing pathway, said
media incubated with a eukaryotic cell line and an inhibitor of
.beta.-amyloid processing pathway, said eukaryotic cell line having
a exogenous gene construction, said exogenous gene construction
comprising a cytomegalovirus promoter encoding sequence, a strong
ribosome binding site encoding sequence, a .beta.-amyloid precursor
protein encoding sequence, a selectable marker encoding sequence,
and a poly-adenylation signal encoding sequence, wherein said
.beta.-amyloid 108.1 42 specific monoclonal antibody and said
.beta.-amyloid 1101.1 4.2 kDa-specific monoclonal antibody
specifically bind said protein molecule that is indicative of
inhibition of .beta.-amyloid processing pathway.
85. A eukaryotic cell line ATCC No CRL 12329 or a derivative
thereof.
86. A monoclonal antibody selected from the group consisting of
monoclonal antibody 108.1 or a derivative thereof, monoclonal
antibody 1101.1 or a derivative thereof, and monoclonal antibody
1702.1 or a derivative thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Application Serial No. 60/067,389, filed
Feb. 24, 1997, which was converted from application Ser. No.
08/804,971 on Sep. 30, 1997. This application also claims the
benefit under 35 U.S.C. .sctn. 119(e) of U.S. Provisional
Application Serial No. 60/067/390, filed Apr. 2, 1997, which was
converted from application Ser. No. 08/825,737 on Sep. 30, 1997,
which is a continuation-in-part of U.S. patent application Ser. No.
08/804,971, filed Feb. 24, 1997. This application also claims the
benefit under 35 U.S.C. .sctn. 119(e) of U.S. Provisional
Application Serial No. To Be Assigned, filed Jul. 31, 1997, which
was converted from Application Ser. No. 08/904,296 on Dec. 19,
1997, which is a continuation-in-part of U.S. patent application
Ser. No. 08/825,737, filed Apr. 2, 1997, which is a
continuation-in-part of U.S. patent application Ser. No.
08/804,971, filed Feb. 24, 1997.
FIELD OF THE INVENTION
[0002] The invention relates to the design, construction and use of
eukaryotic cell lines useful in the identification of inhibitors of
.beta.-amyloid processing. More specifically, the invention relates
to in vitro assays capable of identifying or quantifying 4.2 kDa
.beta.-amyloid protein. The present invention also provides for DNA
and protein molecules for the design, construction and use of
eukaryotic cell lines and in vitro assays useful in the
identification of inhibitors of .beta.-amyloid processing.
BACKGROUND OF THE INVENTION
[0003] Alzheimer's disease has emerged as a significant health
problem. It is estimated that over 5% of the U.S. population and
over 15% of the U.S. population over 85 are beset with some form of
Alzheimer's disease (Cross, Eur. J. Pharmacol. 82: 77-80 (1982);
Terry, et al., Ann. Neurol. 14:497-506 (1983)). Currently, there is
no remission in the progression of the disease, nor is there any
truly effective pharmaceutical intervention or method for
diagnosing the disease (Schehr, Bio/Technology 12: 140-144 (1994);
Cordell, Ann. Rev. Pharmacol. Toxicol. 34: 69-89 (1994), herein
incorporated by reference). A patient with Alzheimers progresses
toward increasing mental and physical incapacitation.
[0004] A characteristic feature of Alzheimer's disease is the
formation or deposit of .beta.-amyloid plaques in affected
individuals. Mature .beta.-amyloid plaques are often associated
with degenerating neuronal processes. .beta.-amyloid deposits are
not solely associated with persons suffering from Alzheimer's
disease but are also associated with persons suffering from other
amyloidoses, for example, brain trauma or Downs syndrome (Cordell,
Ann. Rev. Pharmacol. Toxicol. 34: 69-89 (1994); Pierce et al.,
Journal of Neuroscience, 16: 1083-1090 (1996); Roberts, Lancet 338:
1422-1423 (1991); Motte and Williams, Acta Neuropathol 77: 535-546
(1989); Mann and Esiri, Journal of the Neurological Sciences 89:
169-179 (1989); Masters, Proc. Natl. Acad. Sci., 82: 4245-4249
(1985)).
[0005] The .beta.-amyloid proteins isolated from neuritic plaques
are self aggregating moieties termed the 4.2 kDa .beta.-amyloid
protein or alternatively termed one of the following: the A4
protein (Ponte et al., Nature 311: 525-527 (1988), herein
incorporated by reference); the .beta.-amyloid peptide (Schehr,
Bio/Technology 12: 140-144 (1994), herein incorporated by
reference); 4.2 kDa .beta.-amyloid polypeptide (Neve et al., Neuron
1: 669-677 (1988), herein incorporated by reference); 4K peptide or
A.beta. (Haas et al., Nature 359: 322-325 (1992), herein
incorporated by reference); amyloid B-protein or amyloid A4 (Tanzi
et al., Nature 331: 528-530 (1988), herein incorporated by
reference); 4 kD amyloid .beta.protein, .beta.AP or 4 kD protein
(Shoji et al., Science 258: 126-129 (1992), herein incorporated by
reference); the 4.2-4.5 kd amyloid protein subunit (Wolf et al.,
EMBO 9.7: 2079-2084 (1990), herein incorporated by reference);
.beta.protein (Weidmann et al., Cell 57: 115-126 (1989); the
beta-amyloid core protein (Cordell, U.S. Pat. No. 5,221,607, herein
incorporated by reference); a composition of peptides individually
referred to as A.beta.1-39 peptide, A.beta.1-40 peptide,
A.beta.1-41 peptide and A.beta.1-42 peptide (described in Glenner
and Wong, Biochem. Biophys. Res. Comm. 120: 885-890 (1984), herein
incorporated by reference; (Schehr, Nature Biotechnology 15: 19-20
(1997), herein incorporated by reference) and A.beta.1-43 peptide
(Shoji et al., Science 258: 126-129 (1992); Selkoe, Science 275:
630-631 (1997), herein incorporated by reference). A.beta.1-39
peptide, A.beta.1-40 peptide, A.beta.1-41 peptide, A.beta.1-42
peptide and A.beta.1-43 peptide comprise 39, 40, 41, 42 and 43
amino acids respectively.
[0006] Characterization of cDNA encoding the 4.2 kDa .beta.-amyloid
protein showed that the 4.2 kDa .beta.-amyloid protein was
transcribed as part of a larger precursor protein, .beta.-amyloid
precursor protein (APP)(Lemaire et al., Nucleic Acid Research 17:
517-22 (1989)). Three major APP protein isoforms, APP.sub.695,
APP.sub.751 and APP.sub.770, have been characterized (Cordell, Ann.
Rev. Pharmacol. Toxicol. 34: 69-89 (1994)). The isoforms
APP.sub.695, APP.sub.751 and APP.sub.770, contain respectively,
695, 751 and 770 amino acids (Weidmann et al., Cell 57: 115-126
(1989), herein incorporated by reference). These isoforms are the
result of differential splicing of the primary APP RNA transcript.
The APP.sub.695 isoform differs from the longer isoforms in not
containing the Kunitz proteinase inhibitor domain (Lemaire, et al.,
Nucleic Acids Research 172: 517-522 (1989); herein incorporated by
reference); Mullan, U.S. Pat. No. 5,455,169, herein incorporated by
reference); Ponte et al., Nature 331: 525-527 (1988), herein
incorporated by reference). The mRNA corresponding to the
APP.sub.695 isoform also differs from the longer isoforms in that
it has been shown to be preferentially expressed in the brain (Neve
et al., Neuron 1: 669-677 (1988)).
[0007] Certain APP mutations have been reported (Ashall and Goate,
Trends in Biochemical Sciences, 19: 42-46 (1994), herein
incorporated by reference); Hardy and Allsop, Trends in
Pharmological Sciences 12: 383-388 (1991), herein incorporated by
reference). Examples of characterized APP mutants include: the
"Swedish FAD double mutant" (Mullan et al., Nature Genetics 1:
345-347 (1992), herein incorporated by reference), the "London
mutant" (Van Broeckhoven, et al., Science 248: 1120-1123 (1990),
herein incorporated by reference); Levy, Science 24: 1124-1126
(1990), herein incorporated by reference;
valine.sup.717.fwdarw.isoleucine mutant (Goate et al., Nature 349:
704-706 (1991), herein incorporated by reference); Hardy et al.,
Lancet 337: 1342-1343 (1991), herein incorporated by reference);
valine.sup.717.fwdarw.glycine mutant (Harlan et al., Nature 353:
844-846 (1991); and valine.sup.717.fwdarw.phenylalanine mutant
(Murrell et al., Science 254: 97-99 (1991), herein incorporated by
reference).
[0008] In each of the APP isoforms, the 4.2 kDa .beta.-amyloid
protein corresponds to an internal region that begins 99 residues
from the carboxyl terminal end of the APP isoforms (Shoji et al.,
Science 258: 126-129 (1992)). Two major pathways of APP processing
have been reported (See Higaki et al., Neuron 14: 651-659 (1995),
herein incorporated by reference). One pathway involves proteolytic
cleavages that result in the formation of truncated carboxyl
terminal 8-12 kD remnants (Caporaso et al., Proc. Natl. Acad. Sci.
USA 89: 2252-2256 (1992), herein incorporated by reference). It has
been reported that carboxyl terminal 8-12 kD remnants are
nonamyloidgenic (Seubert et al., Nature 361:260-263 (1993), herein
incorporated by reference). It is suggested that the proteolytic
processing of APP in this pathway occurs at a number of
intracellular and membrane locations (See Higaki et al., Neuron 14:
651-659 (1995)). The carboxyl terminal 8-12 kD remnants have been
reported to remain cell-associated, possibly due to retention of a
transmembrane domain within the carboxyl terminal 8-12 kD remnants
(Cordell, Ann. Rev. Pharmacol. Toxicol. 34: 69-89 (1994)). The
second processing pathway has been reported to be associated with
the endosomal/lysosomal system and it is this pathway that is
reportedly responsible for the production of the 4.2 kDa
.beta.-amyloid protein (See Higaki et al., Neuron 14: 651-659
(1995)).
[0009] Cell lines capable of producing a 4.2 kDa .beta.-amyloid
protein have been reported (Naidu et al., Journal of Biological
Chemistry 270: 1369-1374 (1995), herein incorporated by reference;
Higaki et al., Neuron 14: 651-669 (1995), herein incorporated by
reference). Naidu et al. and Higaki et al. report the establishment
of the CP-6 cell line by stably transfecting a human cDNA encoding
the 695-amino acid isoform of .beta.-amyloid precursor protein,
driven by a .beta.-actin promoter, into Chinese hamster ovary
fibroblasts. Busciglio et al. report transient transfection of a
COS cell line (a monkey cell line containing part of the SV 40
early promoter) with an expression plasmid under the control of a
cytomegalovirus promoter, which overexpressed the 695-amino acid
isoform of .beta.-amyloid precursor protein (Busciglio et al.,
Proc. Natl. Acad Sci. 90: 2092-2096 (1993), herein incorporated by
reference). In addition, Haass et al. report stable transfection of
kidney cell lines which overexpressed the 695-amino acid isoform of
.beta.-amyloid precursor protein (Haass et al., Nature 359: 322-325
(1992)).
[0010] Cordell, et al., U.S. Pat. No. 5,221,607, reports stably
expressing two different proteins in chinese hamster cells. These
proteins are 99 and 42 amino acids respectively corresponding to an
amyloid protein of 99 amino acids and the beta-amyloid core
protein. In both cases, protein expression was facilitated by the
use of a .beta.-actin promoter. Again, in both cases, the
selectable marker (the bacterial neomycin gene) was genetically
linked to a second, different promoter (SV40 early promoter).
[0011] Cell lines transfected with a human cDNA encoding the
695-amino acid isoform of .beta.-amyloid precursor protein have
been used to screen putative inhibitors of the .beta.-amyloid
processing pathway (Higaki et al., Neuron 14: 651-669 (1995)).
Higaki et al. report a inhibition assay carried out in 10 cm dishes
(Higaki et al., Neuron 14: 651-669 (1995)). The CP-6 cells utilized
by Higaki et al. in their inhibitor assay were propagated in 10 cm
dishes containing a 1:1 mixture of growth media supplemented with
200 .mu.l inhibitor, 200 .mu.l leupeptin, 200 .mu.l E64, 100 .mu.l
chloroquine and 30 mM NH.sub.4Cl, 30 mM NH.sub.4-acetate, 30 mM
methylamine, 10 .mu.M monesin and 10 .mu.M brefeldin A (Higaki et
al., Neuron 14: 651-669 (1995)).
[0012] In another inhibition assay, described in Patent Application
PCT/US93/01014, herein incorporated by reference, a human embryonic
cell line, which had been stably transfected with a vector
containing the cDNA encoding for the APP.sub.751 isoform. This
transfected cell line was utilized to screen potential inhibitors
of a 22 kDa pre-amyloid intermediate.
[0013] Antibodies or antiserum specific to epitopes located within
a 4.2 kDa .beta.-amyloid protein have been reported (Ponte and
Cordell, U.S. Pat. No. 5,220,013, herein incorporated by reference;
Majocha et al., U.S. Pat. No. 5,231,000; amyloid beta antibodies
cat. nos. 0490-1916, 0490-1858, 0490-1857, ANAWA Biomedical
Services & Products, Wangen Switzerland; mouse monoclonal
anti-.beta.-amyloid peptide (1-28), Zymed Laboratories, South San
Francisco, Calif.; mouse anti-beta amyloid monoclonal cat no.
RDI-BAMYLOID, Research Diagnostics, Inc. Flanders, N.J.). For
example, Naidu et al. report antiserum reactive to epitopes located
within the carboxyl terminus of a 4.2 kDa .beta.-amyloid protein
and Busciglio et al. report serum containing antibodies raised
against a short peptide region of a 4.2 kDa .beta.-amyloid protein
(amino acids 28-40) (Naidu et al., Journal of Biological Chemistry
270: 1369-1374 (1995); and Busciglio et al., Proc. Natl. Acad Sci.
90: 2092-2096 (1993)). In addition, monoclonal antibodies reactive
to epitopes located within a 4.2 kDa .beta.-amyloid protein have
been reported (Haass et al., Nature 359: 322-325 (1992)). Haass et
al. report a monoclonal antibody, 6C6, which is reactive against an
epitope contained within a short peptide region of a 4.2 kDa
.beta.-amyloid protein (amino acids 1-16) and a monoclonal
antibody, 266, which is reactive against a different epitope
contained within a short peptide region of a 4.2 kDa .beta.-amyloid
protein (Haass et al., Nature 359: 322-325 (1992)). In addition,
Patent Application PCT/US93/01014 describes a number of antibodies
directed against a variety of regions found within the APP.sub.751
isoform.
[0014] A significant problem associated with cell lines, such as
CP-6 (Naidu et al., Journal of Biological Chemistry 270: 1369-1374
(1995); and Higaki et al., Neuron 14: 651-669 (1995)), is that the
level of the 4.2 kDa .beta.-amyloid protein produced by such cell
lines is lower than is necessary for automated measurement. Due to
the insufficient level of expression in cell lines, such as CP-6,
the volume of cells necessary for accurate and routine measurement
is not suitable for routine high throughput screening.
[0015] The present invention provides cell lines that express an
APP protein at a level sufficient for high throughput screening.
High throughput screening of inhibitors is typically carried out
using 96 well microtitre dishes, which can contain cells in a
volume of about 250 .mu.l. The volume and surface area constraints
associated with 96 well microtitre plates, place a premium on cell
lines capable of expressing the desired protein at high levels.
SUMMARY OF THE INVENTION
[0016] The invention relates to the design, construction and use of
eukaryotic cell lines useful in the identification of inhibitors of
.beta.-amyloid processing. More specifically, the invention relates
to in vitro assays capable of identifying or quantifying a 4.2 kDa
.beta.-amyloid protein. The present invention also provides for DNA
and protein molecules for the design, construction and use of
eukaryotic cell lines and in vitro assays useful in the
identification of inhibitors of .beta.-amyloid processing.
[0017] An object of the present invention is an eukaryotic cell
line having a exogenous gene construction, the exogenous gene
construction comprising a cytomegalovirus promoter encoding
sequence, a strong ribosome binding site encoding sequence, a
.beta.-amyloid precursor protein encoding sequence, a selectable
marker encoding sequence, and a poly-adenylation signal encoding
sequence.
[0018] Another object of the present invention is an eukaryotic
cell line selected from the group consisting of 21-N-1, 21-N-2,
21-N-3, 21-N-4, 21-N-5, 21-N-6, 21-N-7, 21-N-8, and 21-N-9 or a
derivative thereof.
[0019] Another object of the present invention is an eukaryotic
cell line capable of producing greater than 30 ng 4.2 kDa
.beta.-amyloid protein molecule per mg protein.
[0020] Another object of the present invention is an eukaryotic
cell line that produces a 4.2 kDa .beta.-amyloid protein molecule
to a greater extent than a CP-7 cell line.
[0021] Another object of the present invention is an eukaryotic
cell line that produces a 4.2 kDa .beta.-amyloid protein molecule
to a greater extent than a CP-6 cell line
[0022] Another object of the present invention is an eukaryotic
cell line that produces a A.beta.1-40 peptide molecule to a greater
extent than a CP-7 cell line.
[0023] Another object of the present invention is an eukaryotic
cell line that produces a A.beta.1-42 peptide molecule to a greater
extent than a CP-7 cell line.
[0024] Another object of the present invention is an eukaryotic
cell line having a exogenous gene construction, the exogenous gene
construction comprising a cytomegalovirus promoter encoding
sequence, a ribosome binding site encoding sequence, a
.beta.-amyloid precursor protein encoding sequence, a selectable
marker encoding sequence, and a poly-adenylation signal encoding
sequence.
[0025] Another object of the present invention is a substantially
purified nucleic acid molecule that encodes, in order: a
cytomegalovirus promoter; a ribosome binding site; a .beta.-amyloid
precursor protein molecule; a selectable marker; and a
poly-adenylation signal. A further object of the present invention
is a substantially purified nucleic acid molecule that encodes, in
order: a cytomegalovirus promoter; a strong ribosome binding site;
.beta.-amyloid precursor protein molecule; a selectable marker; and
a poly-adenylation signal.
[0026] Another object of the present invention is a method for
identifying an inhibitor of .beta.-amyloid processing pathway
comprising: administering the inhibitor to a eukaryotic cell line
having an exogenous gene construction, the exogenous gene
construction comprising a cytomegalovirus promoter encoding
sequence, a strong ribosome binding site encoding sequence, a
.beta.-amyloid precursor protein encoding sequence, a selectable
marker encoding sequence, and a poly-adenylation signal encoding
sequence; and quantifying a protein molecule that is indicative of
inhibition of .beta.-amyloid processing pathway.
[0027] Another object of the present invention is a method for
identifying an inhibitor of .beta.-amyloid processing pathway
comprising, (a) incubating an inhibitor of .beta.-amyloid
processing and a eukaryotic cell line to produce a protein molecule
that is indicative of inhibition of .beta.-amyloid processing
pathway in a medium containing a labeled amino acid, the eukaryotic
cell line having a exogenous gene construction, the exogenous gene
construction comprising a cytomegalovirus promoter encoding
sequence, a strong ribosome binding site encoding sequence, a
.beta.-amyloid precursor protein encoding sequence, a selectable
marker encoding sequence, and a poly-adenylation signal encoding
sequence, where the exogenous gene construction is capable of
stably expressing a .beta.-amyloid precursor protein molecule; (b)
separating the eukaryotic cell line and the medium; (c) separating
the protein molecule from the eukaryotic cell line and the medium;
and (d) quantifying the protein molecule.
[0028] A further object of the present invention is a high
throughput assay for identification of an inhibitor of
.beta.-amyloid processing comprising: (a) incubating an inhibitor
of .beta.-amyloid processing and a eukaryotic cell line to produce
a protein molecule that is indicative of inhibition of
.beta.-amyloid processing pathway, the eukaryotic cell line having
a exogenous gene construction, the exogenous gene construction
comprising a cytomegalovirus promoter encoding sequence, a strong
ribosome binding site encoding sequence, a .beta.-amyloid precursor
protein encoding sequence, a selectable marker encoding sequence,
and a poly-adenylation signal encoding sequence (b) quantifying the
protein molecule produced by the incubation step.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1 shows a diagrammatic representation of a region of
selected plasmids between the immediate early gene transcription
enhancer/promoter from cytomegalovirus and a polyadenylation signal
from SV40 virus.
[0030] FIG. 2 shows a diagrammatic representation of a construction
strategy of pCMV-IRES-.beta.APP.sub.695.
[0031] FIG. 3 shows a typical standard curve using RIA.
[0032] FIG. 4 shows an expected pattern of APP products (SEQ ID NO:
1).
[0033] FIG. 5 diagrammatically represents the epitopes recognized
by antibodies BA#1, 108.1, 1702.1 and 1101.1 (SEQ ID NO: 2).
[0034] FIG. 6 depicts standard curves of the competition ELISA for
total 4.2 kDa .beta.-amyloid protein, the sandwich ELISA for
A.beta.1-40 peptide and the sandwich ELISA for A.beta.1-42
peptide.
[0035] FIG. 7 illustrates the stability of 4.2 kDa .beta.-amyloid
protein in cell lines 21-N-3 (N3) and 21-N-9 (N9).
[0036] FIG. 8 illustrates the effect of inhibitor compounds on 4.2
kDa .beta.-amyloid protein production by 21-N-9 cells.
DETAILED DESCRIPTION OF THE INVENTION
[0037] I. Overview of the Invention
[0038] The invention relates to the design, construction and use of
eukaryotic cell lines useful in the identification of inhibitors of
.beta.-amyloid processing. More specifically, the invention relates
to in vitro assays capable of identifying or quantifying a 4.2 kDa
.beta.-amyloid protein. The present invention also provides for DNA
and protein molecules for the design, construction and use of
eukaryotic cell lines and in vitro assays useful in the
identification of inhibitors of .beta.-amyloid processing.
[0039] II. Agents and Definitions
[0040] As used herein, an agent be it naturally occurring molecule
or otherwise may be "substantially purified," if desired, such that
one or more molecules that is or may be present in a naturally
occurring preparation containing that molecule will have been
removed or will be present at a lower concentration than that at
which it would normally be found.
[0041] The agents of the present invention will preferably be
"biologically active" with respect to either a structural
attribute, such as the capacity of a nucleic acid to hybridize to
another nucleic acid molecule, or the ability of a protein to be
bound by antibody (or to compete with another molecule for such
binding). Alternatively, such an attribute may be catalytic, and
thus involve the capacity of the agent to mediate a chemical
reaction or response.
[0042] The agents of the present invention comprise cell lines,
nucleic acid molecules, proteins, and organic molecules.
[0043] As used herein, the term "4.2 kDa .beta.-amyloid protein"
refers, without limitation, to all of following: the A4 protein;
the .beta.-amyloid peptide; 4.2 kDa .beta.-amyloid polypeptide; 4K
peptide or A.beta.; amyloid B-protein; amyloid A4; 4 kD amyloid
.beta. protein; .beta.AP; 4 kD protein; the 4.2-4.5 kd amyloid
protein subunit; .beta. protein; and the beta-amyloid core protein.
In addition, as used herein, the term refers to an approximately 4
kDa protein or peptide identified by Wong et al., Proc. Nat. Acad.
Sci. 82: 8729-8732 (1985), herein incorporated by reference; Master
et al., Proc. Nat. 4245-4249 (1985), herein incorporated by
reference, which is defined by amino acid sequence analysis as a
mixture of four peptides with slightly different amino termini, the
amino termini of the three smaller peptides being internal to the
largest peptide.
[0044] As used herein the terms "A.beta.1-39 peptide", "A.beta.1-40
peptide", "A.beta.1-41 peptide", "A.beta.1-42 peptide" and
"A.beta.1-43 peptide" refer to certain peptide constituents of the
4.2 kDa .beta.-amyloid protein. The A.beta.1-39 peptide consists of
39 amino acids, the A.beta.1-40 peptide consists of 40 amino acids,
A.beta.1-41 peptide consists of 41 amino acids, A.beta.1-42 peptide
consists of 42 amino acids and A.beta.1-43 peptide consists of 43
amino acids. These peptides can be heterogenous at their N
termini.
[0045] As used herein, the term "protein molecule" or "peptide
molecule" includes any molecule that comprises five or more amino
acids. It is well know in the art that proteins may undergo
modification, including post-translational modifications, such as,
but not limited to, disulfide bond formation, glycosylation,
phosphorylation, or oligomerization. Thus, as used herein, the term
"protein molecule" or "peptide molecule" includes any protein
molecule that is modified by any biological or non-biological
process. The terms "amino acid" and "amino acids" refer to all
naturally occurring L-amino acids. This definition is meant to
include norleucine, ornithine, and homocysteine.
[0046] As used herein, the term "exogenous gene construction" is
any DNA, whether naturally occurring or otherwise, from any source,
that is capable of being inserted into any organism.
[0047] As used herein, "transfection" or "transformation" refers to
any process for altering the DNA content of a eukaryotic cell. This
includes, without limitation, calcium phosphate or
DEAE-dextran-mediated transfection, polybrene transfection,
protoplast fusion transfection, electroporation transfection,
liposome transfection, and direct microinjection transfection or
such other means for effecting controlled DNA uptake as are known
in the art (See Sambrook, et al., Molecular Cloning 3: 16.30-16.31
(1989), herein incorporated by reference).
[0048] As used herein, "stably expressing" refers to any eukaryotic
cell or any eukaryotic cell line capable of producing a desired
protein molecule over a desired time frame.
[0049] As used herein, "efficient expression of .beta.-amyloid
precursor protein" refers to the expression level of .beta.-amyloid
precursor protein that is greater than cell lines CP-6 or CP-7.
[0050] As used herein, "stably transformed" or "stably transfected"
with reference to any eukaryotic cell or any eukaryotic cell line
refers to any eukaryotic cell or any eukaryotic cell line having an
exogenous gene construction, which is capable of generating progeny
comprising the same exogenous gene construction.
[0051] As used herein, the terms ".beta.-amyloid precursor protein"
or "APP protein" refers to isoforms APP.sub.695, APP.sub.751 and
APP.sub.770 or mutant thereof. Mutants of APP include, without
limitation,: the Swedish FAD double mutant; the "London mutant; the
valine.sup.717.fwdarw.isoleucine mutant;
valine.sup.717.fwdarw.glycine mutant; and the
valine.sup.717.fwdarw.phenylalanine mutant.
[0052] As used herein, a ".beta.-amyloid precursor protein or
derivative thereof" is any .beta.-amyloid precursor protein or
protein or peptide fragment that corresponds to any protein or
peptide fragment that is greater than five amino acids that is
identical to any contiguous amino acid sequence located within the
.beta.-amyloid precursor protein.
[0053] As used herein the terms ".beta.-amyloid precursor encoding
sequence mRNA" or "APP mRNA" refers to any messenger RNA that is
capable of specifically hybridizing, in whole or in part to any
part of any .beta.-amyloid precursor encoding sequence.
[0054] As used herein the terms ".beta.-amyloid precursor encoding
sequence" or "APP encoding sequence" refer to any DNA molecule that
encodes a APP.sub.695, APP.sub.751 and APP.sub.770 or mutant
thereof. Mutants of APP include, without limitation, the Swedish
FAD double mutant; the "London mutant; the
valine.sup.717.fwdarw.isoleucine mutant;
valine.sup.717.fwdarw.glycine mutant; and the
valine.sup.717.fwdarw.pheny- lalanine mutant.
[0055] As used herein, the term "internal ribosome entry site
encoding sequence" or "IRES encoding sequence" is any nucleic acid
sequence that can help facilitate the translation of more than one
protein encoding sequence from a single promoter.
[0056] As used herein, the term "encoding sequence" refers to a
sequence having a specified characteristic. This "encoding
sequence" can, without limitation, be non-transcribed, transcribed,
non-translated or translated.
[0057] As used herein, the term "inhibitor" refer to any molecule
that effects the processing of .beta.-amyloid precursor
protein.
[0058] As used herein, the term ".beta.-amyloid precursor pathway"
is any biological process or a step within a biological process
that modifies .beta.-amyloid precursor protein.
[0059] As used herein, the term "media" or "medium" is any
composition capable of sustaining the desired cells.
[0060] As used herein a "strong ribosome binding site encoding
sequence" or a "strong Kozak encoding sequence" is any ribosome
binding site encoding sequence or Kozak encoding sequence that has
a purine at position -3 (relative the ATG initiation codon) and a
guanine at position +4 (relative to the ATG initiation codon).
[0061] As used herein, a "weak ribosome binding site encoding
sequence" or a "weak Kozak encoding sequence" is any ribosome
binding site encoding sequence or Kozak encoding sequence that
lacks a purine at position -3 (relative the ATG initiation codon)
or lacks a guanine at position +4 (relative to the ATG initiation
codon).
[0062] As used herein, a "high throughput assay" is any assay
capable of being carried out in its entirety in a reaction volume
less than about 500 .mu.l.
[0063] As used herein, two nucleic acid molecules are said to be
capable of specifically hybridizing to one another if the two
molecules are capable of forming an anti-parallel or reverse
complementary, double-stranded nucleic acid structure.
[0064] (A) Cell Lines
[0065] An embodiment of the present invention is an eukaryotic cell
line having an exogenous gene construction, the exogenous gene
construction comprising a cytomegalovirus promoter encoding
sequence, a strong ribosome binding site encoding sequence, a
.beta.-amyloid precursor protein encoding sequence, a selectable
marker encoding sequence, and a poly-adenylation signal encoding
sequence.
[0066] Another embodiment of the present invention is an eukaryotic
cell line having a exogenous gene construction, the exogenous gene
construction comprising a cytomegalovirus promoter encoding
sequence, a ribosome binding site encoding sequence, a
.beta.-amyloid precursor protein encoding sequence, a selectable
marker encoding sequence, and a poly-adenylation signal encoding
sequence.
[0067] The eukaryotic cell line of the present invention may be any
cell line having a exogenous gene construction capable of
expressing a .beta.-amyloid precursor protein. In a preferred
embodiment of the present invention, the eukaryotic cell line is
selected from the group consisting of chinese hamster ovary cell
line, chinese hamster ovary cell line K1, dihydrofolate reductase
deficient hamster cell line, human kidney cell line, rat neuroglimo
cell line, human neuroglimo cell line, and rat neuroblastoma cell
line. In a more preferred embodiment of the present invention, the
eukaryotic cell line is a chinese hamster ovary cell line. In an
even more preferred embodiment of the present invention, the
eukaryotic cell line is the chinese hamster ovary cell line K1.
[0068] The eukaryotic cell line having an exogenous gene
construction of the present invention may be any cell line
transformed or transfected with an exogenous gene construction. In
a preferred embodiment of the present invention, the eukaryotic
cell line having an exogenous gene construction of the present
invention may be any cell line stably transformed or stably
transfected with an exogenous gene construction.
[0069] In an embodiment, exogenous gene construction is any DNA,
whether naturally occurring or otherwise, from any source, that is
capable of being inserted into any organism. Preferably, exogenous
gene construction is any DNA, whether naturally occurring or
otherwise, from any source that is capable of being stably
introduced into eukaryotic cells.
[0070] The .beta.-amyloid precursor protein encoding sequence may
encode any APP protein or derivative. In a preferred embodiment,
the .beta.-amyloid precursor protein may encode an APP protein
selected from the group consisting of three major APP protein
isoforms, APP.sub.695, APP.sub.751 and APP.sub.770, and the
"Swedish FAD double mutant", the "London mutant", the
valine.sup.717.fwdarw.isoleucine mutant, the
valine.sup.717.fwdarw.glycine mutant, the
valine.sup.717.fwdarw.phenylala- nine mutant. In an even more
preferred embodiment the .beta.-amyloid precursor protein encoding
sequence may encode the APP.sub.695 protein isoform.
[0071] The selectable marker encoding sequence may be any sequence
that encodes a protein which facilitates the identification of
cells that have an exogenous gene construction. In a preferred
embodiment, the selectable marker encoding sequence is selected
from the group consisting of: neomycin phosphotransferase encoding
sequence; dihydrofolate reductase encoding sequence;
xanthine-guanine phosphoribosyltransferase encoding sequence;
aspartate transcarbamoylase encoding sequence; adenosine deaminase
encoding sequence; adenylate deaminase encoding sequence; UMP
synthetase encoding sequence; glutamine synthetase encoding
sequence; asparagine synthetase encoding sequence; ornithine
decarboxylase encoding sequence; the thymidine kinase encoding
sequence; the aminoglycosidase phosphotransferase encoding
sequence; hygromycin B phosphotransferase encoding sequence; and
the CAD encoding sequence (see, for example, Sambrook et al., In
Molecular Cloning: A Laboratory Manual, 16.8-16.15, Cold Spring
Harbor Press (1989), herein incorporated by reference; Old and
Primrose, In Principles of Gene Manipulation, 307-310 (1994),
herein incorporated by reference). In an even more preferred
embodiment the selectable marker is encoded by the neomycin
phosphotransferase gene.
[0072] The promoter encoding sequence of the present invention is
any promoter including requisite transcription enhancer sequences
capable of stably expressing high levels of .beta.-amyloid
precursor encoding sequence mRNA or 4.2 kDa .beta.-amyloid protein
in a transfected eukaryotic cell line. In a preferred embodiment,
the promoter encoding sequence is the immediate early gene human
cytomegalovirus promoter and associated transcription enhancer
elements (Boshart et al., Cell 41: 521-530 (1985), herein
incorporated by reference). In an even more preferred embodiment of
the present invention, the promoter encoding sequence corresponding
to -601 to -14 as set forth in FIG. 3 of Boshart et al., Cell 41:
521-530 (1985).
[0073] During the expression of eukaryotic genes, RNA polymerase II
transcribes through the site where a termination signal is present.
The sequence elements of the polyadenylation site is recognized and
polyadenylation will then occur. Consequently, the 3' terminus of
the mature mRNA is formed by site specific cleavage and
polyadenylation. Two distinct elements are often required for
accurate and efficient polyadenylation: (1) GU or U-rich sequences
located downstream from the polyadenylation site and (2) a highly
conserved sequence of six nucleotides as described by Wickens and
Stephenson, Science 226: 1045-1051 (1984), herein incorporated by
reference and Sambrook et al., 16:6, In Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press (1989), herein
incorporated by reference.
[0074] The poly-adenylation signal encoding sequence of the present
invention is any encoding sequence that is required for accurate
and efficient polyadenylation. In a preferred embodiment of the
polyadenylation signal encoding sequence is derived from SV40. In
an even more preferred embodiment, the poly-adenylation signal
encoding sequence of the present invention is as described by
Wickens and Stephenson, Science 226: 1045-1051 (1984). In a further
preferred embodiment, the polyadenylation signal comprises a
transcription termination signal.
[0075] The .beta.-amyloid precursor protein of the present
invention is any .beta.-amyloid precursor protein or derivative
thereof. In a preferred embodiment of the present invention the
.beta.-amyloid precursor protein or derivative thereof is selected
from the group consisting of isoforms APP.sub.695, APP.sub.751 and
APP.sub.770 or a mutant thereof. Potential mutants of
.beta.-amyloid precursor protein include, without limitation, the
Swedish FAD double mutant; the London mutant; the
valine.sup.717.fwdarw.isoleucine mutant;
valine.sup.717.fwdarw.glycine mutant; and-the
valine.sup.717.fwdarw.pheny- lalanine mutant. In an even more
preferred embodiment of the .beta.-amyloid precursor protein of the
present invention is APP.sub.695.
[0076] An embodiment of the present invention is to provide
eukaryotic cell lines which produce a 4.2 kDa .beta.-amyloid
protein to a greater extent than eukaryotic cell line CP-6 or
CP-7.
[0077] An embodiment of the present invention is to provide
eukaryotic cell lines which produce a 4.2 kDa .beta.-amyloid
protein to a greater extent than eukaryotic cell line CP-7. In a
preferred embodiment, the eukaryotic cell lines of the present
invention produce greater than two fold more 4.2 kDa .beta.-amyloid
protein than eukaryotic cell line CP-7. In a more preferred
embodiment, the eukaryotic cell lines of the present invention
produce greater than four fold more 4.2 kDa .beta.-amyloid protein
than eukaryotic cell line CP-7. In an even more preferred
embodiment, the eukaryotic cell lines of the present invention
produce greater than six fold more 4.2 kDa .beta.-amyloid protein
than eukaryotic cell line CP-7.
[0078] In particularly preferred embodiments, the eukaryotic cell
lines of the present invention produce (i) greater than eight fold
more 4.2 kDa .beta.-amyloid protein than eukaryotic cell line CP-7
or (ii) produce greater than ten fold more 4.2 kDa .beta.-amyloid
protein than eukaryotic cell line CP-7 or (iii) be suitable for use
in a high throughput assay.
[0079] An embodiment of the present invention is to provide
eukaryotic cell lines which produce a A.beta.1-40 peptide to a
greater extent than eukaryotic cell line CP-7. In a preferred
embodiment, the eukaryotic cell lines of the present invention
produce greater than two fold more A.beta.1-40 peptide than
eukaryotic cell line CP-7. In a more preferred embodiment, the
eukaryotic cell lines of the present invention produce greater than
three fold more A.beta.1-40 peptide than eukaryotic cell line CP-7.
In an even more preferred embodiment, the eukaryotic cell lines of
the present invention produce greater than 3.5 fold more
A.beta.1-40 peptide than eukaryotic cell line CP-7.
[0080] An embodiment of the present invention is to provide
eukaryotic cell lines which produce a A.beta.1-42 peptide to a
greater extent than eukaryotic cell line CP-7. In a preferred
embodiment, the eukaryotic cell lines of the present invention
produce greater than two fold more A.beta.1-42 peptide than
eukaryotic cell line CP-7. In a more preferred embodiment, the
eukaryotic cell lines of the present invention produce greater than
three fold more A.beta.1-42 peptide than eukaryotic cell line CP-7.
In an even more preferred embodiment, the eukaryotic cell lines of
the present invention produce greater than four fold more
A.beta.1-42 peptide than eukaryotic cell line CP-7. In particularly
preferred embodiment, the eukaryotic cell lines of the present
invention produce greater than six fold more A.beta.1-42 peptide
than eukaryotic cell line CP-7.
[0081] An embodiment of the present invention is to provide a
eukaryotic cell line that produces greater than about 30 ng 4.2 kDa
.beta.-amyloid protein/mg protein as measured or quantified by RIA.
In a more preferred embodiment of the present invention, the
eukaryotic cell line of present invention produces greater than
about 40 ng 4.2 kDa .beta.-amyloid protein/mg protein as measured
or quantified by RIA. In an even more preferred embodiment of the
present invention, the eukaryotic cell line of present invention
produces greater than about 50 ng 4.2 kDa .beta.-amyloid protein/mg
protein as measured or quantified by RIA. In a particularly
preferred embodiment of the present invention, the eukaryotic cell
line of the present invention produces greater than about 60 ng 4.2
kDa .beta.-amyloid protein/mg protein as measured or quantified by
RIA. In an even more preferred embodiment of the present invention,
the eukaryotic cell line of the present invention produces greater
than about 65 ng 4.2 kDa .beta.-amyloid protein/mg protein as
measured or quantified by RIA. In a particularly preferred
embodiment of the present invention, the eukaryotic cell line of
the present invention produces greater than about 70 ng 4.2 kDa
.beta.-amyloid protein/mg protein as measured or quantified by
RIA.
[0082] An embodiment of the present invention is to provide
eukaryotic cell lines which produce a higher relative amount of APP
mRNA, as detected by RT-PCR, than the cell line designated
CP-7.
[0083] In a preferred embodiment, the secretion rate for the
eukaryotic cell lines of the present invention is estimated at
about 4 ng of 4.2 kDa .beta.-amyloid protein (total) per 100 .mu.l
medium during 4 hours when cells are cultured at density of
0.5-1.times.10.sup.5 cells per well in 96 well plates (surface area
-0.4 cm.sup.2). In a preferred embodiment, the secretion rate for
the eukaryotic cell lines of the present invention is estimated at
about 5 ng of 4.2 kDa .beta.-amyloid protein (total) per 100 .mu.l
medium during 4 hours when cells are cultured at density of
0.5-1.times.10.sup.5 cells per well in 96 well plates (surface area
-0.4 cm.sup.2).
[0084] The consensus sequence for initiation of translation by
eukaryotic ribosomes is GCC GCC A.sup.-3/GCC A.sup.1UGG.sup.4 (SEQ
ID NO: 3), Kozak, M. J. Cell. Biol. 108: 229-241 (1989), herein
incorporated by reference; Sambrook et al., 16.16, In Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989),
herein incorporated by reference. It has reported that, for
practical purposes, an initiation of translation by eukaryotic
ribosomes is considered "strong" or "weak" by considering positions
labeled -3 and +4. As long as there is a purine (A or G) at
position -3 and a guanine at +4. An illustration of a weak ribosome
binding site encoding sequence or a weak Kozak encoding sequence
can be found between the .beta.-actin promoter and an APP.sub.695
encoding sequence present in CP-6 cells as utilized by Higaki et
al., ((5'-CCC CGA TGC-3' (SEQ ID NO: 4)(Higaki et al., Neuron 14:
651-669 (1995)).
[0085] The ribosome binding site encoding sequence of the present
invention is any sequence that initiates translation by eukaryotic
ribosomes. In a preferred embodiment of the present invention, the
ribosome encoding sequence of the present invention is a strong
ribosome binding site encoding sequence. In an even more preferred
embodiment the ribosome binding site encoding sequence of the
present invention is found within the following sequence: 5'-TTT
TCA AAG CTT ACC ATG CTG CCC GGT TTG CAC TG -3' (NO: 5).
[0086] The IRES encoding sequence of the present invention is any
sequence that facilitates the translation of a .beta.-amyloid
precursor protein or derivative thereof encoding sequence and a
selectable marker encoding sequence from a single messenger RNA
transcript and single promoter. In a preferred embodiment of the
present invention, the IRES encoding sequence facilitates the
translation of the 695 isoform of human .beta.-amyloid precursor
protein and the encoding sequence for bacterial neomycin
transferase. In a even more preferred embodiment of the IRES
encoding sequence of the present invention is an
encephalomycarditis virus IRES (Ghattas et al., Molecular and
Cellular Biology 11.12: 5848-5859 (1991), herein incorporated by
reference).
[0087] In a particularly preferred embodiment of the present
invention, a single cytomegalovirus promoter encoding sequence is
capable of co-expressing a .beta.-amyloid precursor protein
encoding sequence and a selectable marker encoding sequence.
[0088] (B) Nucleic Acid Molecules
[0089] An object of the present invention is a substantially
purified nucleic acid molecule that encodes, in order: a
cytomegalovirus promoter; a ribosome binding site; a .beta.-amyloid
precursor protein; a selectable marker; and a poly-adenylation
signal.
[0090] Another object of the present invention is a substantially
purified nucleic acid molecule that encodes, in order: a
cytomegalovirus promoter; a strong ribosome binding site; a
.beta.-amyloid precursor protein; a selectable marker; and a
poly-adenylation signal.
[0091] Another object of the present invention is a substantially
purified nucleic acid molecule that encodes, in order: a
cytomegalovirus promoter; a ribosome binding site; a .beta.-amyloid
precursor protein; internal ribosome entry site; a selectable
marker; and a poly-adenylation signal.
[0092] Another object of the present invention is a substantially
purified nucleic acid molecule that encodes, in order: a
cytomegalovirus promoter; a strong ribosome binding site; a
.beta.-amyloid precursor protein; internal ribosome entry site; a
selectable marker; and a poly-adenylation signal.
[0093] It is also understood that any of the exogenous gene
constructions of the present invention described above are nucleic
acid molecules of the present invention. It is further understood
that any of the nucleic acid molecules of the present invention can
be substantially purified and/or be biologically active.
[0094] Practitioners are familiar with the standard resource
materials which describe specific conditions and procedures for the
construction, manipulation and isolation of macromolecules (e.g.,
DNA molecules, plasmids, etc.), generation of recombinant organisms
and the screening and isolating of clones, (see for example,
Sambrook et al., In Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Press (1989), herein incorporated by reference.
[0095] (C) Antibodies
[0096] One aspect of the present invention concerns antibodies,
single-chain antigen binding molecules, or other proteins that
specifically bind to any .beta.-amyloid precursor protein or
derivative thereof and their analogs, fusions or fragments. As used
herein, an antibody or peptide is said to "specifically bind" to
any .beta.-amyloid precursor protein or derivative thereof and
their analogs, fusions or fragments if such binding is not
competitively inhibited by the presence of non-.beta.-amyloid
precursor protein or derivative thereof and its analogs, fusions or
fragments.
[0097] Practitioners are familiar with the standard resource
materials which describe specific conditions and procedures for the
construction, manipulation and isolation of antibodies (see, for
example, Harlow and Lane, In Antibodies: A Laboratory Manual Cold
Spring Harbor Press (1988), herein incorporated by reference).
Antibodies which specifically bind to any .beta.-amyloid precursor
protein or derivative thereof and its analogs, fusions or fragments
may be prepared using such techniques.
[0098] Preferred examples of antibodies or antisera that
specifically bind .beta.-amyloid precursor protein or derivative
thereof and its analogs, fusions or fragments comprise the
following: monoclonal antibodies 1101.1 (1101.1 was deposited with
the American Tissue Type Collection, Rockville, Md. on Apr. 25,
1997 and assigned ATCC No HB12347), 1702.1 (1702.1 was deposited
with the American Tissue Type Collection, Rockville, Md. on Jun. 3,
1997 and assigned ATCC No HB12363) and 108.1 (108.1 was deposited
with the American Tissue Type Collection, Rockville, Md. on Jun. 3,
1997 and assigned ATCC No HB12362) and antisera BA#1, BA#2, amyloid
beta antibodies cat. nos. 0490-1916, 0490-1858, 0490-1857 (ANAWA
Biomedical Services & Products, Wangen Switzerland); mouse
monoclonal anti-.beta.-amyloid peptide (1-28) (Zymed Laboratories,
South San Francisco, Calif.); mouse anti-beta amyloid monoclonal
cat no. RDI-BAMYLOID, Research Diagnostics, Inc. Flanders, N.J.
Particularly preferred examples of antibodies or antisera that
specifically bind .beta.-amyloid precursor protein or derivative
thereof and its analogs, fusions or fragments are selected from the
following: monoclonal antibodies 1101.1, 1702.1 and 108.1 and
antisera BA#1 and BA#2.
[0099] (D) Indicative Protein Molecules
[0100] One aspect of the present invention concerns indicative
protein molecules. In an embodiment of the present invention, the
protein molecule that is indicative of inhibition of .beta.-amyloid
processing pathway is any protein molecule. In a preferred
embodiment of the present invention, the protein molecule that is
indicative of inhibition of .beta.-amyloid processing pathway is
any .beta.-amyloid precursor protein or derivative thereof. In a
more preferred embodiment of the present invention the protein
molecule that is indicative of inhibition of .beta.-amyloid
processing pathway is selected from the group consisting of 4.2 kDa
.beta.-amyloid protein, A.beta.1-39 peptide, A.beta.31-40 peptide,
A.beta.1-41 peptide, A.beta.1-42 peptide, A.beta.1-43 peptide,
truncated carboxyl terminal 8-12 kD remnants, 99 amino acid protein
as described by Cordell, et al., U.S. Pat. No. 5,221,607, a 22 kDa
pre-amyloid intermediate as described in PCT/US93/01014, and the 3
kDa peptides including the reported product of .alpha.- and
.gamma.-secretase and 3B, which includes peptides 12-39, 12-40,
12-42 and 12-43 as described herein.
[0101] In an even more preferred embodiment of the present
invention the protein molecule that is indicative of inhibition of
.beta.-amyloid processing pathway is selected from the group
consisting of 4.2 kDa .beta.-amyloid protein, A.beta.1-39 peptide,
A.beta.1-40 peptide, A.beta.1-41 peptide, A.beta.1-42 peptide, and
A.beta.1-43 peptide. In another even more preferred embodiment of
the present invention the protein molecule that is indicative of
inhibition of .beta.-amyloid processing pathway is selected from
the group consisting of 4.2 kDa .beta.-amyloid protein, A.beta.1-40
peptide, and A.beta.1-42 peptide. In another even more preferred
embodiment of the present invention the protein molecule that is
indicative of inhibition of .beta.-amyloid processing pathway is
the 4.2 kDa .beta.-amyloid protein. Variation in the composition or
pattern of APP products can be indicative of inhibitors of
.alpha.-, .beta., .gamma.-secretase.
[0102] III. Uses of the Agents of the Invention
[0103] An embodiment of the present invention is a method for
identifying an inhibitor of .beta.-amyloid processing pathway
comprising: administering the inhibitor to a eukaryotic cell line
having a exogenous gene construction, the exogenous gene
construction comprising a cytomegalovirus promoter encoding
sequence, a strong ribosome binding site encoding sequence, a
.beta.-amyloid precursor protein encoding sequence, a selectable
marker encoding sequence, and a poly-adenylation signal encoding
sequence; and quantifying a protein molecule that is indicative of
inhibition of .beta.-amyloid processing pathway.
[0104] Another embodiment of the present invention is a method for
identifying an inhibitor of .beta.-amyloid processing pathway
comprising, (a) incubating an inhibitor of .beta.-amyloid
processing and a eukaryotic cell line to produce a protein molecule
that is indicative of inhibition of .beta.-amyloid processing
pathway in a medium containing a labeled amino acid, the eukaryotic
cell line having a exogenous gene construction, the exogenous gene
construction comprising a cytomegalovirus promoter encoding
sequence, a strong ribosome binding site encoding sequence, a
.beta.-amyloid precursor protein encoding sequence, a selectable
marker encoding sequence, and a poly-adenylation signal encoding
sequence, where the exogenous gene construction is capable of
stably expressing a .beta.-amyloid precursor protein molecule; (b)
separating the eukaryotic cell line and the medium; (c) separating
protein molecule from the eukaryotic cell line and the medium; and
(d) quantifying the protein molecule.
[0105] A further embodiment of the present invention is a high
throughput assay for identification of an inhibitor of
.beta.-amyloid processing comprising: (a) incubating an inhibitor
of .beta.-amyloid processing and a eukaryotic cell line to produce
a protein molecule that is indicative of inhibition of
.beta.-amyloid processing pathway, the eukaryotic cell line having
a exogenous gene construction, the exogenous gene construction
comprising a cytomegalovirus promoter encoding sequence, a strong
ribosome binding site encoding sequence, a .beta.-amyloid precursor
protein encoding sequence, a selectable marker encoding sequence,
and a poly-adenylation signal encoding sequence; and (b)
quantifying the protein molecule produced by the incubation
step.
[0106] In another particularly preferred embodiment, the high
throughput assay of the present invention may be carried out or
undertaken in a volume of less than about 300 .mu.l. In another
more particularly preferred embodiment, the high throughput assay
of the present invention may be carried out or undertaken in a
volume of less than about 250 .mu.l. In another even more
particularly preferred embodiment, the high throughput assay of the
present invention may be carried out or undertaken in a volume of
about 200 .mu.l. It is understood that the volume of the high
throughput assay of the present invention may vary during the high
throughput assay. As used herein, the terms "be carried out" in a
specified volume or "undertaken" in a specified volume means that
the entire high throughput assay, including all reagents, cell line
or other biological materials or chemicals utilized never at, any
temporal stage, during the assay exceed the specified volume.
However, it is understood that the total volume of reagents, cell
lines or other biological materials or chemicals utilized by the
high throughput assay of the present invention may exceed the
specified volume
[0107] In another particularly preferred embodiment, the high
throughput assay of the present invention may be carried out on a
surface area of less than about 1 cm.sup.-2. In another more
particularly preferred embodiment, the high throughput assay of the
present invention may be carried out on a surface area less than
about 0.6 cm.sup.-2. In another even more particularly preferred
embodiment, the high throughput assay of the present invention may
be carried out on a surface area of less than about 0.4 cm.sup.-2.
As used herein, the "surface area" refers to the area in physical
contact with a eukaryotic cell line.
[0108] Incubation of the eukaryotic cells in the absence of an
inhibitor of the present invention may be for any period of time
and under any appropriate conditions for cell growth or
maintenance. Incubation of the eukaryotic cells with an inhibitor
of .beta.-amyloid processing of the present invention may be for
any period of time and under any appropriate conditions for cell
growth or maintenance.
[0109] In a preferred embodiment of the high throughput assay of
the present invention, the incubation of an inhibitor of
.beta.-amyloid processing and a eukaryotic cell line to produce a
protein molecule that is indicative of inhibition of .beta.-amyloid
processing pathway may be carried out for between about 1 hour and
about 20 hours. In an even more preferred embodiment of the high
throughput assay of the present invention, the incubation of an
inhibitor of .beta.-amyloid processing and a eukaryotic cell line
to produce a protein molecule that is indicative of inhibition of
.beta.-amyloid processing pathway may be carried out for between
about 2 hours and about 10 hours. In a particularly preferred
embodiment of the high throughput assay of the present invention,
the incubation of an inhibitor of .beta.-amyloid processing and a
eukaryotic cell line to produce a protein molecule that is
indicative of inhibition of .beta.-amyloid processing pathway may
be carried out for between about 3 hours and about 5 hours.
[0110] Any conventional 96-well polystyrene microtiter dishes used
in diagnostic laboratories and in tissue culture may be used with
this invention. Methods for synthesizing polystyrene are known in
the art; such methods are disclosed in, for example, treatises on
plastics and polymers such as Byrdson, J. A. , Plastics Materials,
Fifth Edition, Butterworth Heinemann, London (1991), herein
incorporated by reference; Maxisorb plate (Nunc, Rochester,
N.Y.).
[0111] The level of a protein molecule indicative of inhibition of
.beta.-amyloid processing pathway may be measured or quantified
using any of the techniques known in the art. For example, any of a
wide array of immunoassays formats may be used for this purpose
(Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Press (1988), herein incorporated by reference; Fackrell, J.
Clin. Immunoassay 8:213-219 (1985), herein incorporated by
reference; Yolken, R. H., Rev. Infect. Dis. 4:35 (1982), herein
incorporated by reference; Collins, W. P., In Alternative
Immunoassays, John Wiley & Sons, NY (1985), herein incorporated
by reference; Ngo, T. T. et al., In Enzyme Mediated Immunoassay,
Plenum Press, NY (1985), herein incorporated by reference).
[0112] The simplest immunoassay involves merely incubating an
antibody that is capable of binding to a predetermined target
molecule with a sample suspected to contain the target molecule.
The presence of the target molecule is determined by the presence,
and proportional to the concentration, of any antibody bound to the
target molecule. In order to facilitate the separation of
target-bound antibody from the unbound antibody initially present,
a solid phase is typically employed. Thus, for example, the sample
can be passively bound to a solid support, and, after incubation
with the antibody, the support can be washed to remove any unbound
antibody.
[0113] In more sophisticated immunoassays, the concentration of the
target molecule is determined by binding the antibody to a support,
and then permitting the support to be in contact with a sample
suspected of containing the target molecule. Target molecules that
have become bound to the immobilized antibody can be detected in
any of a variety of ways. For example, the support can be incubated
in the presence of a labeled, second antibody that is capable of
binding to a second epitope of the target molecule. Immobilization
of the labeled antibody on the support thus requires the presence
of the target, and is proportional to the concentration of the
target in the sample. In an alternative assay, the target is
incubated with the sample and with a known amount of labeled
target. The presence of target molecule in the sample competes with
the labeled target molecules for antibody binding sites. Thus, the
amount of labeled target molecules that are able to bind the
antibody is inversely proportional to the concentration of target
molecule in the sample.
[0114] In general, immunoassay formats employ either radioactive
labels ("RIAs") or enzyme labels ("ELISAs"). RIAs have the
advantages of simplicity, sensitivity, and ease of use. Radioactive
labels are of relatively small atomic dimension, and do not
normally affect reaction kinetics. Such assays suffer, however,
from the disadvantages that, due to radioisotopic decay, the
reagents have a short shelf-life, require special handling and
disposal, and entail the use of complex and expensive analytical
equipment. RIAs are described in Laboratory Techniques and
Biochemistry in Molecular Biology, by Work, T. S., et al., North
Holland Publishing Company, N.Y. (1978), with particular reference
to the chapter entitled "An Introduction to Radioimmune Assay and
Related Techniques" by Chard, T., incorporated by reference herein.
ELISAs have the advantage that they can be conducted using
inexpensive equipment, and with a myriad of different enzymes, such
that a large number of detection strategies--colorimetric, pH, gas
evolution, etc.--can be used to quantitate the assay. In addition,
the enzyme reagents have relatively long shelf-lives, and lack the
risk of radiation contamination that attends to RIA use. ELISAs are
described in ELISA and Other Solid Phase Immunoassays (Kemeny, D.
M. et al., Eds.), John Wiley & Sons, N.Y. (1988), incorporated
by reference herein.
[0115] It is further understood that the level (i.e., the
concentration of a protein molecule that is indicative of
inhibition of .beta.-amyloid processing pathway, etc.) or pattern
(i.e., the kinetics of expression, rate of decomposition, stability
profile of a protein molecule that is indicative of inhibition of
.beta.-amyloid processing pathway, etc.) may be measured or
quantified.
[0116] The following examples are presented to illustrate the
present invention and to assist one of ordinary skill in making and
using the same. The examples are not intended in any way to
otherwise limit the scope of the invention.
EXAMPLE 1
Cloning of Human APP.sub.695
[0117] The DNA region encoding the human APP.sub.695 isoform is
amplified using 5' and 3' primers. The sequence of the 5' primer is
as follows: 5'-TTT TCA AAG CTT ACC ATG CTG CCC GGT TTG CAC TG -3'
(SEQ ID NO: 5). The 5' primer contains a HindIll, restriction
endonuclease site (underlined) and, for efficient ribosome binding,
a purine (adenosine) at the -3 position (italics) relative to the
initiation ATG (underlined/italics). The sequence of the 3' primer
is as follows: 5'-A GGC TGC TCT AGA GGG GGT CTA GTT CTG CA T
-3'(SEQ ID NO: 6). Within the 3' primer is an XbaI restriction
endonuclease site (underlined) separated by five nucleotides from a
stop codon. The stop sequence corresponds to stop sequence located
within the cDNA (underlined/italics). A polymerase chain reaction
(PCR) reaction is carried out using the 5' and 3' primer in
combination with the plasmid .beta.-actin-.beta.-APP.sub.695 (Scios
Nova, Mountain View, Calif.) as the template. The PCR product is
digested with HindIII and XbaI and ligated to pBluescript-SK
(Stratagene, La Jolla, Calif.), which is precut with HindIII and
XbaI. The insert is confirmed by sequencing.
EXAMPLE 2
Construction of an Expression Vector Capable of Expressing High
Levels of APP.sub.695
[0118] Using standard molecular cloning methods, a DNA vector to
transform cells is constructed, which contains a bacterial origin
of replication and a .beta.-lactamase gene derived from pUC19
Yanisch-Peron et al.,Gene 33:103-119 (1985), herein incorporated by
reference), the immediate early gene transcription
enhancer/promoter from cytomegalovirus (Boshart, M, et al., Cell
41:521-530 (1985), herein incorporated by reference), the coding
sequence for human APP.sub.695 modified to include a Kozak
consensus sequence at the 5' end for efficient ribosome binding
(Kozak, Nature 308:241-246. (1984), herein incorporated by
reference), an internal ribosome entry site (IRES) from
encephalolmyocarditis virus (Jang, et al., J. Virology 63:1651-1660
(1989), herein incorporated by reference) the E. coli gene for
aminoglycoside phosphotransferase (neo)(Blazquez, et al., Mol.
Microbiol. 5:1511-1518 (1991), herein incorporated by reference);
and a polyadenylation signal from SV40 virus (Wickens and
Stephenson, Science 226:1045-1051 (1984), herein incorporated by
reference). A diagrammatic representation of the region of the
plasmid between the immediate early gene transcription
enhancer/promoter from cytomegalovirus and a polyadenylation signal
from SV40 virus is set out in FIG. 1.
[0119] The cloning steps are essentially as follows: The human
APP.sub.695 insert is removed from pBluescript-SK by digesting with
SalI followed by a fill-in reaction to produce blunt ends. The
resulting DNA is then digested with XbaI. The insert is isolated by
agarose gel electrophoresis followed by purification of the DNA
from the excised gel band. The plasmid pCMV-IRES is digested with
NotI. The digested DNA is then filled-in to produce blunt ends. The
filled-in DNA is then digested with XbaI. The human APP.sub.695
insert (described above) is ligated to the digested plasmid to
produce pCMV-IRES-.beta.APP.sub.695. The construction strategy is
diagramatically illustrated in FIG. 2.
EXAMPLE 3
Stable Transfection of Chinese Hamster Ovary Cells
[0120] Chinese hamster ovary cells are transfected by
liposome-mediated DNA transfection with either 16 or 32 .mu.g of
pCMV-IRES-.beta.APP.sub.69- 5 and selected for stable integration
of the human APP.sub.695 and neo transcription unit as follows.
CHO-K1 cells (American Type Culture Collection, 12301 Parklawn
Drive, Rockville, Md. 20852, USA) are routinely cultured in the
following medium: DMEM/NUT MIX F-12 (Gibco 041-01331M,
Gaithersburg, Md.); 1% Non-essential amino acids; 2.5 mM
L-glutamine; 0.5 mM Pyruvate; 1% Penicillin, 1% Streptomycin; 10%
Fetal calf serum (Gibco 011-06290 (Australia)). Cells are
subcultured onto 150 cm.sup.2 plates four days prior to
transfection to obtain an expected density of 70% confluence by the
day of transfection. DNA is mixed with lipofectamine reagent
(GIBCO/BRL, Gaithersburg, Md.). The cells are then transfected
according to the manufacturer's directions (GIBCO/BRL,
Gaithersburg, Md.), herein incorporated by reference, overnight and
refed with fresh medium the following day. Three days after
transfection, cells are placed in medium containing 1 mg/ml
geneticin and allowed to grow for three to four weeks. Colonies
arising from surviving cells are collected by mild trypsinization,
transferred to individual wells of a 96 well dish and cultured in
medium without geneticin. Cells are expanded and evaluated for the
expression of APPmRNA.
EXAMPLE 4
Analysis of APP mRNA Expression
[0121] RNA is extracted from cells rising from individual isolates
using RNA Now (Biogenex, San Ramon, Calif.) and analyzed for the
presence of human APP transcripts by reverse
transcriptase-polymerase chain reaction (RT-PCR). The reverse
transcriptase reaction is primed with random hexamers and the PCR
amplification is performed using primers designed to amplify a -600
bp fragment of human APP mRNA (5' primer: 5'-GGTGGAAGAAGAAGAAGCC-3'
(SEQ ID NO:7); 3' primer: 5'-GTGACGAGGCCGAGGAGGAAC-3' (SED ID
NO:8). The colonies are scored for the presence and relative amount
of APP compared to CP-6 cells (See Table 1). CP-6 is a CHO cell
line expressing human APP.sub.695 under control of the .beta.-actin
promoter (Higaki et al., Neuron 14:651-669 (1995)).
1 TABLE 1 Clone DNA .mu.g PCR RIA .eta.M 21-N-A 16 1+ 0.51 21-N-B
16 1+ 0.66 21-N-C 16 3+ 2.64 21-N-D 16 3+ 0.47 21-N-E 16 3+ 0.21
21-N-F 16 0 ND 21-N-G 16 0 ND 21-N-H 16 0 ND 21-N-I 16 3+ 0.46
21-N-I 16 3+ 0.66 21-N-K 16 1+ 0.71 21-N-L 16 1+ 1.01 21-N-M 16 1+
1.01 21-N-N 16 2+ 0.37 21-N-O 16 3+ 5.40 21-N-P 16 1+ 0.84 22-N-A
32 1+ 1.13 22-N-B 32 0 ND 22-N-C 32 1+ 0.88 22-N-D 32 1+ 0.71
22-N-E 32 1+ 0.92 22-N-F 32 2+ 0.37 22-N-G 32 1+ 0.80 22-N-H 32 2+
0.37 CP-6 -- 2+ ND CP-7 -- -- 0.85 -In Table 1, PCR results are
expressed as the relative amount of APP mRNA detected by RT-PCR and
ND refers to values not determined.
EXAMPLE 5
Radioimmunoassay Analysis of the 4.2 kDa .beta.-amyloid Protein
[0122] Cell lines are assayed for the production of 4.2 kDa
.beta.-amyloid protein by radioimmunoassay (RIA) following reverse
phase chromatography to concentrate 4.2 kDa .beta.-amyloid protein
from assay medium. Rabbit BA#1 antiserum and iodinated A.beta.1-40
peptide are used in the RIA. The cells are incubated for 5 hours in
RIA assay medium (EMEM (Gibco 041-01090M, Gaithersburg, Md.), 1%
non-essential amino acids, 2 mM L-glutamine, 1% Penicillin, 1%
Streptomycin) containing 1% FCS and A.beta. peptide concentrate.
After incubation, RIA assay medium is removed from cells,
centrifuged at 1,500 rpm for 10 minutes then stored frozen at
-20.degree. C. 1.5-2 ml of medium is passed through a using
Lichrolut cartridges (Merck, N.J.) or Sepak C18 cartridges (Waters
Corp., Mass.), which are then washed first with 2 ml 5% CH.sub.3CN
in 0.1% TFA and then washed with 2 ml 25% CH.sub.3CN in 0.1% TFA.
The 4.2 kDa .beta.-amyloid protein is eluted with 2 ml 50%
CH.sub.3CN in 0.1% TFA, dried by speedvac centrifugation and then
stored frozen at -20.degree. C. Concentrated peptides are
re-dissolved in 250 .mu.l of 0.1% Triton-X 100+0.1% BSA in
water.
[0123] The RIA is carried out as follows: A.beta. 1-40 peptide is
labeled with .sup.125I using the chloramine T method (Amersham,
Arlington Heights, Ill.), and purified by reverse phase HPLC on a
C18 column (Vydac, Hesperia, Calif.) using a linear gradient from
20% to 50% CH.sub.3CN in 0.1% TFA. The labeled peptide is eluted at
40% CH.sub.3CN, and stored at -20.degree. C.
[0124] To glass 12.times.75 mm tubes are added in order; 100 .mu.l
BA#1 antiserum diluted 1:150 in Buffer A (0.1 M sodium phosphate,
pH 7.4+0.1% BSA+0.1% Triton-X 100) or 1% bovine serum albumin in
PBS, 2.times. the concentration necessary to bind 30% of the
labeled peptide in the absence of competing ligand, 50 .mu.l
unknown samples, peptide standards (A.beta.1-40 peptide, Bachem,
King of Prussia, Pa.) diluted in Buffer A or 1% bovine serum
albumin in PBS to concentrations ranging from 0.4-100 nM (resulting
in final concentrations of 0.1-25 nM--standards are prepared from a
1 mg/ml stock solution dissolved in 20% isopropanol and stored at
-20.degree. C.)), total binding (Buffer A or 1% bovine serum
albumin in PBS is used for total binding) or non-specific binding
(10 .mu.M A.beta.1-40 peptide in Buffer A or 1% bovine serum
albumin in PBS is used to measure total displacement/nonspecific
binding) and 50 .mu.l HPLC purified .sup.125I-A.beta.1-40 peptide
diluted in Buffer C to give between 8,000-10,000 cpm/tube. The
solution is made up to a total volume 200 .mu.l.
[0125] The solution is then vortexed and incubated overnight at
4.degree. C. After incubation, 50 .mu.l normal rabbit serum is
added to the sample followed by 800 .mu.l 15.8% polyethylene glycol
(MW 6,000-8,000) dissolved in Buffer A or 1% bovine serum albumin
in PBS. The sample is then incubated for 10 minutes at 4.degree. C.
Following incubation, the sample is centrifuged for 20 minutes at
3,200 rpm (Sorvall T600B). After pelleting the sample, the
supernatant is aspirated and radioactivity of the pellets is
measured in a gamma counter.
[0126] A typical standard curve using the RIA is shown in FIG. 3.
Results obtained from cells generated from these two transfections
are summarized in Table 1 and is the average of two media samples,
each assayed in duplicate. The five most positive clones are
further evaluated for the effect of different media on 4.2 kDa
.beta.-amyloid protein secretion and stability. These results are
summarized in Table 2. Certain cell line derived from transfection
termed 21, N-O are estimated to produce 10 times more 4.2 kDa
.beta.-amyloid protein than CP-7 cells and is chosen for
subcloning.
[0127] Table 2 illustrates the effect of incubation medium on 4.2
kDa .beta.-amyloid protein levels in selected CHO-K1 cell clones
transfected with pCMV-IRES-.beta.APP.sub.695.
2TABLE 2 RIA, ng A.beta.* Cell line Medium mg protein RIA, nM per
mg protein CP-7 1% FCS 0.53 0.85 7.0 0.2% BSA 0.52 0.92 7.7 SFM
0.52 0.51 4.2 21-N-C 1% FCS 0.97 1.2 5.4 0.2% BSA 0.91 2.75 13.1
SFM 0.92 0.75 3.5 21-N-M 1% FCS 0.72 1.2 7.2 0.2% BSA 0.71 1.3 7.9
SFM 0.70 1.15 7.1 21-N-O 1% FCS 0.70 9.2 57.1 0.2% BSA 0.63 9.35
64.2 SFM 0.72 7.9 47.4 22-N-C 1% FCS 0.79 0.9 4.9 0.2% BSA 0.70
1.35 8.3 SFM 0.74 0.95 5.6 22-N-E 1% FCS 0.79 0.7 3.8 0.2% BSA 0.77
0.65 3.6 SFM 0.81 0.4 2.1 *A.beta. = 4.2 kDa .beta.-amyloid
protein
[0128] For the comparison of media, cells are cultured in 6 well
plates (9.5 cm.sup.2/well) until confluent. Medium is then replaced
with 1.5 ml RIA assay medium containing either 1% fetal calf serum
(FCS), 0.2% bovine serum albumin (BSA) or no additional material
(SFM). After 5 hours medium is collected and 4.2 kDa .beta.-amyloid
protein concentrated for RIA determination (as described above).
Cell protein is determined after solubilization overnight in 0.5 N
NaOH.
[0129] Further, the 4.2 kDa .beta.-amyloid protein levels produced
by nine subclones of 21-N-O are evaluated by RIA and the data are
shown in Table 3.
3TABLE 3 RIA, ng A.beta. per Clone Protein RIA, nM RIA, ng mg
protein 21-N-1 1.18 3.58 15.5 39.2 21-N-2 1.09 5.23 22.6 62.4
21-N-3 1.27 6.39 27.7 65.4 21-N-4 1.28 4.93 21.4 50.0 21-N-S 1.47
6.36 27.5 56.2 21-N-6 1.48 5.23 22.6 45.9 21-N-7 1.53 4.67 20.2
37.2 21-N-8 1.14 6.22 26.9 71.1 21-N-9 1.3 7.32 31.7 73.2 *A.beta.
= 4.2 kDa .beta.-amyloid protein
[0130] Level of the 4.2 kDa .beta.-amyloid protein produced by
subclones of 21-N-O cells evaluated by RIA is set out in Table 3.
The analysis is carried out as follows: Confluent 25 cm.sup.2
flasks of cells are incubated with 4 ml RIA assay medium containing
1% FCS for 5 hours as described above. The 4.2 kDa .beta.-amyloid
protein are concentrated from 1.5 ml of the medium prior to
performing the RIA. Cell protein level is determined after
solubilizing the cells in 0.5 N NaOH overnight. Values presented in
Table 3 are the means of duplicate determinations.
EXAMPLE 6
Analysis of APP Processing by Pulse-labeling and
Immunoprecipitation
[0131] The level and pattern of APP processing to 4.2 kDa
.beta.-amyloid protein and C-terminal fragments is studied by
pulse-labeling and immunoprecipitation of the four selected
subclones and results compared to those seen for the parent 21-N-O
cell line and CP-7 cells.
[0132] APP production, its processing pattern and the level of 4.2
kDa .beta.-amyloid protein secretion are monitored by
pulse-labeling and immunoprecipitation using protocols described by
Higaki et al., Neuron 14:651-659 (1995). Essentially, cells are
first starved of cysteine and methionine then incubated for 4 hours
with medium containing .sup.35S-methionine and .sup.35S-cysteine.
4.2 kDa .beta.-amyloid protein secreted into the culture medium is
immunoprecipitated using BA#2 rabbit antiserum. Cell associated APP
peptides are immunoprecipitated from cell lysates using BA#2 rabbit
antiserum which recognizes an epitope near the C-terminal of human
APP. The pattern of APP products expected is illustrated in FIG. 4.
Peptides present in the media include 4 kDa and 3 kDa products. The
4 kDa products include A.beta.1-39 peptide, A.beta.1-40 peptide,
A.beta.1-41 peptide, A.beta.1-42 peptide. The 3 kDa peptides
include 3A, a reported product of .alpha.- and .gamma.-secretase
and 3B, which includes peptides 12-39, 12-40, 12-42 and 12-43.
C-terminal fragments include portions of the include portions of
the membrane and intracellular 100 amino acids C-terminal tail of
APP. Variation in the composition or pattern of APP products is
indicative of inhibitors of .alpha.-, .beta., .gamma.-secretase
[0133] The solubilized immunoprecipitates are separated by SDS
polyacrylamide gel electrophoresis and radioactive proteins
quantified using a phosphorimager. The areas under each peak are
normalized to the amount of cell protein and the labeling patterns
compared to those obtained from a control cell line, CP-7 (Scios
Nova, Mountain View, Calif.).
[0134] The level of 4.2 kDa .beta.-amyloid protein and C-terminal
fragments produced by selected subclones of 21-N-O cells are
evaluated by pulse labeling and immunoprecipitation. These results
are summarized in Table 4 and are used to select two subclones,
21-N-3 and 21-N-9 (21-N-9 was deposited with the American Tissue
Type Collection, Rockville Md. on Mar. 26, 1997 and assigned ATCC
No CRL 12329), for expansion and further characterization of 4.2
kDa .beta.-amyloid protein production.
4 TABLE 4 C-terminal fragments Cell Fragment Fragment Fragment
Fragment Line A.beta. 14 15A 15B 16 CP-7 0.4 1.09 0.38 0.26 0.45
21-N-O 0.59 8.08 2.47 2.57 3.98 21-N-2 2.72 13.4 2.64 2.98 4.59
21-N-3 9.27 15.47 3.24 3.11 5.22 21-N-8 2.44 13.72 1.77 2.39 4.02
21-N-9 2.22 14.68 2.04 2.29 3.83 * A.beta. = 4.2 kDa .beta.-amyloid
protein
[0135] The immunoprecipitable radioactivity corresponding to 4.2
kDa .beta.-amyloid protein and C-terminal fragments set forth in
Table 4 is quantified using a phosphorimager and normalized to the
amount of cell protein. Units in Table 4 are in area
(mm.sup.2)/.mu.g protein.
EXAMPLE 7
Enzyme-linked Immunoassays (ELISA) Analysis of Total 4.2 kDa
.beta.-amyloid Protein
[0136] Total 4.2 kDa .beta.-amyloid protein is measured with a
competitive ELISA using biotinylated A.beta.1-28 as a tracer and
monoclonal antibody designated 1101.1 which binds to an epitope
located between amino acids 13 and 22 of the 4.2 kDa .beta.-amyloid
protein (See FIG. 5).
[0137] The total 4.2 kDa .beta.-amyloid protein is measured as
follows: A 96 well microtitre Maxisorb plate (Nunc, Rochester,
N.Y.) is coated with 200 .mu.l/well goat anti-mouse lgG Fc specific
(Sigma, M-3534, St. Louis, Mo.) at 2.4 .mu.g/ml diluted in 0.1 M
sodium bicarbonate buffer and incubated for 2 hours at 37.degree.
C. The wells are then washed with phosphate buffered saline (PBS)
or PBS/0.05% TWEEN poloxyethyleneysoribtan-20 (4.times.250 .mu.l).
After washing the wells, 200 .mu.l PBS, 0.05% TWEEN-20, 1% BSA
(PBS/TWEEN/BSA) or 1% bovine serum albumin in PBS is added to each
well and incubated for 1 hour at 37.degree. C.
[0138] The solution is removed from the plate and to the plates is
added 100 .mu.l per well of the monoclonal antibody designated
1101.1 (concentration of 2.5 ng/ml in PBS/TWEEN/BSA). To each well
is added either 100 .mu.l/well of cell supernatant (with or without
samples) or 100 .mu.l/well of A.beta.1-40 peptide or A.beta.1-42
peptide (Bachem, King of Prussia, Pa.) diluted in Buffer A to
concentrations ranging from 0.4-100 nM (resulting in final
concentrations of 0.1-25 nM--standards are prepared from a 1 mg/ml
stock solution dissolved in 20% isopropanol and stored at
-20.degree. C.). The samples are then diluted in ELISA assay medium
(DMEM (Gibco 041-01965M), 1% non-essential amino acids, 2.5 mM
L-glutamine, 0.5 mM Pyruvate, 1% Penicillin, 1% Streptomycin, 0.2%
Bovine serum albumin (BSA), Sigma A3296, protease free or
Calbiochem 126609)) and incubated overnight at 4.degree. C.
[0139] To each sample is added 50 .mu.l/well of biotinylated
A.beta.1-28 tracer (Neosystems) at a concentration of 8-12 ng/ml in
PBS/TWEEN/BSA and the sample is incubated for 1 hour at 4.degree.
C. The microtitre dish containing the samples is then inverted and
drained on paper towels. 200 .mu.l/well horseradish
peroxidase-coupled streptavidin (Zymed, South San Francisco, Calif.
43-4323) diluted 1:3,000 in PBS/TWEEN/BSA is added to the drained
microtitre dish. The samples are then incubated for 1 hour at
4.degree. C. After the samples are incubated, the wells are washed
with PBS+0.05% TWEEN-20 (PBS/TWEEN) (5.times.200 .mu.l). Following
the washing, 200 .mu.l/well of tetramethylbenzidine (TMB) (Sigma
T-5525) is added to the wells. The microtitre dishes are then
incubated for between 15 minutes and 1 hour at room temperature.
Incubation of the samples is terminated by stopping the reaction
with 100 .mu.l/well 2.5 M H.sub.2SO.sub.4. The absorbance .sub.450
of the samples is measured.
EXAMPLE 8
Enzyme-linked Immunoassays (ELISA) Analysis of A.beta.1-40 peptide
and A.beta.1-42 Peptide
[0140] A.beta.1-40 peptide and A.beta.1-42 peptide are measured by
sandwich ELISAs using mAb 1101.1 as a capture antibody and either
rabbit antiserum BA#1 or mAb 1702.1 which binds to the C-terminus
of A.beta.1-40 peptide or mAb 108.1 which binds to the C-terminus
of A.beta.1-42 peptide. The epitopes of A.beta. peptides recognized
by the antibodies used for the RIA and ELISAs are shown in FIG.
5.
[0141] 1. Sandwich ELISA for A.beta.1-40 Peptide
[0142] To each well is added either 100 .mu.l/well of cell
supernatant to be analyzed or 100 .mu.l A.beta.1-40 peptide or
A1-42 peptide (Bachem, King of Prussia, Pa.) at concentrations
ranging from 0.0137 ng/well to 10 ng/well. The standards are
prepared in the ELISA assay meduim (DMEM (Gibco 041-01965M), 1%
non-essential amino acids, 2.5 mM L-glutamine, 0.5 mM pyruvate, 1%
penicillin, 1% streptomycin, 0.2% bovine serum albumin (Sigma A3296
protease free or Calbiochem 126609)) which is the same as the cell
culture meduim. The wells are incubated overnight at 4.degree. C.
After the overnight incubation the plates are washed with PBS/TWEEN
(3.times.250 .mu.l per well) and 100 .mu.l/well of BA#1 rabbit
antiserum diluted 1:2,000 or 1:4,000 in PBS/TWEEN+0.1% BSA is added
to each well. The microtitre plates are then incubated for 2 hours
at 37.degree. C. After incubation, the wells are washed with
PBS/TWEEN (3.times.250 .mu.l) and 100 .mu.l/well of horseradish
peroxidase-conjugated donkey or goat anti-rabbit lgG, pre-adsorbed
with mouse serum proteins, diluted 1:3,000 or 1:5,000 in
PBS/TWEEN/BSA is added. The microtitre plates are then incubated
for 2 hours at 37.degree. C. and washed with PBS/TWEEN (3.times.250
.mu.l per well). To the wells is added 100 .mu.l/well of freshly
prepared or pre-made TMB substrate (Sigma T-5525, St. Louis,
Mo.)(prepared by dissolving one 1 mg tablet in 1 ml DMSO and then
adding 9 ml of phosphate-citrate buffer (0.2 M dibasic sodium
phosphate, 0.1 M citric acid, pH 5.0); add 2 .mu.l of fresh 30%
H.sub.2O.sub.2 for 10 ml of substrate solution immediately prior to
use. After approximately 7 minutes (as needed for color to
develop), 100 .mu.l/well of 2.5 M H.sub.2SO.sub.4 is added to stop
the reaction and absorbance at 450 nm is read.
[0143] 2. Sandwich ELISA for A.beta.1-42 Peptide
[0144] 96 well microtitre plates are incubated overnight at
4.degree. C. containing 100 .mu.l/well of a 4 .mu.g/ml solution of
purified mAb 110.1.1 diluted in PBS. After this incubation, the
microtitre plates are washed with PBS/TWEEN (3.times.250 .mu.l per
well) and the wells are blocked with 125 .mu.l/well of 0.5% BSA in
PBS for 1 hour at 37.degree. C.
[0145] The blocked wells are then washed with PBS/TWEEN
(3.times.250 .mu.l per well) and 100 .mu.l/well of medium or
peptide standards (A.beta.-42 peptide, Bachem, King of Prussia,
Pa.) diluted in assay medium is added to each well. The plates are
covered then incubated overnight at 4.degree. C. After the
overnight incubation, the microtitre plates are washed with
PBS/TWEEN (3.times.250 .mu.l per well) and 100 .mu.l/well of
biotinylated mAb 108.1 diluted from 0.5 mg/ml aliquots to 0.75
.mu.g/ml in PBS/TWEEN+0.1% BSA is added to each well. The plates
are then incubated for 2 hours at 37.degree. C. or 4 hours at room
temperature. After this incubation, the microtitre dishes are
washed with PBS/TWEEN (3.times.250 .mu.l per well) and 100
.mu.l/well of horseradish peroxidase-conjugated streptavidin
diluted 1:10,000 or 1:20,000 in PBS/TWEEN/BSA is added.
[0146] The samples are then incubated for 15 or 30 minutes at room
temperature prior to being washed with PBS/TWEEN (3.times.250 .mu.l
per well). To the washed wells is added 100 .mu.l/well of freshly
prepared TMB substrate prepared by dissolving one 1 mg tablet in 1
ml DMSO to which is added 9 ml of phosphate-citrate buffer (0.2 M
dibasic sodium phosphate, 0.1 M citric acid, pH 5.0) and 2 .mu.l of
fresh 30% H.sub.2O.sub.2 for 10 ml of substrate solution
immediately prior to use. After approximately 7 minutes (as needed
for color to develop) 100 .mu.l/well of 2.5 M H.sub.2SO.sub.4 is
added to stop the reaction and absorbance at 450 nm is read.
[0147] The various forms of 4.2 kDa .beta.-amyloid protein produced
by 21-N-3 and 21-N-9 cells are quantified using selective ELISAs
for total 4.2 kDa .beta.-amyloid protein, A.beta.1-40 peptide and
A.beta.1-42 peptide. Typical standard curves for these three assays
are shown in FIG. 6. 4.2 kDa .beta.-amyloid protein are collected
in an assay medium using DMEM and 0.2% BSA and assayed directly
without a concentration step. The level of the peptides measured in
the different cell lines is shown in Table 5. Both 21-N-3 and
21-N-9 secreted 10-15 times more 4.2 kDa .beta.-amyloid protein
than CP-7 cells. The secretion rate for these cell lines is
estimated at about 5 ng of 4.2 kDa .beta.-amyloid protein (total)
per 100 .mu.l medium during 4 hours when cells are cultured at
density of 0.5-1.times.10.sup.5 cells per well in 96 well plates
(surface area -0.4 cm.sup.2). The two cell lines are analyzed for
their stability by measuring the amount of 4.2 kDa .beta.-amyloid
protein secreted with time in culture (FIG. 7) following
re-establishment of these cell lines from frozen stocks. The use of
these cell lines for identifying inhibitors of 4.2 kDa
.beta.-amyloid protein production is validated using compounds
previously identified using the CP-6 cell line (FIG. 8).
[0148] Table 5 sets forth estimates of the different forms of 4.2
kDa .beta.-amyloid protein secreted from cell lines.
5 TABLE 5 Cell line Total * A.beta., ng A.beta.1-40, ng
A.beta.1-42, ng CP-7 0.68 0.18 0.02 21-N-3 8.83 0.66 0.14 21-N-9
7.9 0.68 0.12 * A.beta. = 4.2 kDa .beta.-amyloid protein
[0149] To obtain the data in Table 5, cells are cultured in 6 well
dishes at a density of 5.times.10.sup.5 cells/well. The following
day culture medium is replaced with 2 ml DMEM assay medium
containing 0.2% BSA. After 4 hours medium is collected and stored
at -20.degree. C. until assayed for 4.2 kDa .beta.-amyloid protein
using the three ELISAs described above. FIG. 5 diagramatically
illustrates the epitopes of 4.2 kDa .beta.-amyloid protein
recognized by the antibodies, 1101.1, BA#1 and 108.1. By
substituting mAb 1702.1 (1.75 .mu.g/ml) for mAb 108.1 in the
foregoing protocal, a sandwich ELISA fs carried out for A.beta.1-40
peptide.
EXAMPLE 9
The Effect of Inhibitors on 4.2 kDa .beta.-amyloid Protein
Formation
[0150] Putative inhibitors are dissolved in DMSO and diluted in RIA
assay medium containing 0.2% BSA. 21-N-9 cells are incubated for 5
hours in the presence of putative inhibitors. After five hours,
medium is collected and the 4.2 kDa .beta.-amyloid protein levels
are determined by RIA without prior concentration as described
above. The results are set forth in FIG. 8. The data presented is
the mean of duplicate determinations.
EXAMPLE 10
High Throughput Assay
[0151] 21-N-9 cells (200 .mu.l of a stock at an approximate
concentration of 4.times.10.sup.5 cells/ml) are seeded in a 96 well
microtitre plate (Costar) in culture medium (DMEM/NUT MIX F-12
(Gibco,/BRL, Gaithersburg, Md.). The microtitre plates are
incubated overnight at 37.degree. C. in 5% CO.sub.2. The cells are
then washed with PBS (1.times.200 .mu.l). To the wells is added 10
.mu.l of the 20.times. inhibitor solution (dissolved in DMSO) and
190 .mu.l assay medium (MEM (Gibco/BRL, Gaithersburg, Md.), 1%
non-essential amino acids, 2.5 mM L-glutamine, 0.5 mM Pyruvate, 1%
penicillin, 1% streptomycin and 0.2 bovine serum albumin (BSA,
Calbiochem, La Lolla, Calif.). The microtitre plate is then
incubated at 37.degree. C., 5% CO.sub.2 for 4 hours. The level of
4.2 kDa .beta.-amyloid protein is estimated using the procedure set
forth in Example 7.
Sequence CWU 1
1
* * * * *